No title

Series Editor Gerald P. Schatten Director, PITTSBURGH DEVELOPMENTAL CENTER Deputy Director, Magee-Women’s Research Institute Professor and Vice-Chair of Ob-Gyn-Reproductive Sci. & Cell Biol.-Physiology University of Pittsburgh School of Medicine Pittsburgh, Pennsylvania 15213

Editorial Board Peter Gru¨ss Max-Planck-Institute of Biophysical Chemistry Go¨ttingen, Germany

Philip Ingham University of Sheffield, United Kingdom

Mary Lou King University of Miami, Florida

Story C. Landis National Institutes of Health National Institute of Neurological Disorders and Stroke Bethesda, Maryland

David R. McClay Duke University, Durham, North Carolina

Yoshitaka Nagahama National Institute for Basic Biology, Okazaki, Japan

Susan Strome Indiana University, Bloomington, Indiana

Virginia Walbot Stanford University, Palo Alto, California

Founding Editors A. A. Moscona Alberto Monroy

Contributors Numbers in parentheses indicate the pages on which the authors’ contributions begin.

Marina P. Antoch (173), Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195 Kermit L. Carraway III (1), UC Davis Cancer Center, Sacramento, California 95817 George H. Caughey (23), Cardiovascular Research Institute and Department of Medicine, University of California at San Francisco, San Francisco, California; San Francisco Veterans AVairs Medical Center, San Francisco, California; Northern California Institute for Research and Education, San Francisco, California Hanson K. Fong (47), Department of Materials Science and Engineering, University of Washington, Seattle, Washington 98195 Brian L. Foster (47), Department of Periodontics, School of Dentistry, University of Washington, Seattle, Washington 98195 Melanie Funes (1), UC Davis Cancer Center, Sacramento, California 95817 I. R. Garrett (127), OsteoScreen, San Antonio, Texas 78229 Victoria Y. Gorbacheva (173), Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195 Devrim Gozuacik (217), Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel Adi Kimchi (217), Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel Roman V. Kondratov (173), Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195 Carole Plane`s (23), INSERM U773, Centre de Recherche Biome´dicale BichatBeaujon (CRB3), Universite´ Paris 7, 75018 Paris, France; Department of Physiology, UFR de Me´decine Paris Ile de France Ouest, Universite´ de Versailles-Saint Quentin, 78000 Versailles, France Tracy E. Popowics (47), Department of Oral Biology, School of Dentistry, University of Washington, Seattle, Washington 98195 Martha J. Somerman (47), Department of Periodontics, School of Dentistry, University of Washington, Seattle, Washington 98195; Department of ix

x

Contributors

Oral Biology, School of Dentistry, University of Washington, Seattle, Washington 98195 Colleen Sweeney (1), UC Davis Cancer Center, Sacramento, California 95817 Heather C. Workman (1), UC Davis Cancer Center, Sacramento, California 95817

Contents

Contributors

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1 Contribution of Membrane Mucins to Tumor Progression Through Modulation of Cellular Growth Signaling Pathways Kermit L. Carraway III, Melanie Funes, Heather C. Workman, and Colleen Sweeney I. II. III. IV. V.

Mucin Structure, Function, and Involvement in Tumor Progression MUC1 Contributions to Tumor Cell Growth Signaling 5 MUC4 Contributions to Tumor Cell Growth Signaling 10 Inhibition of Signaling by Mucins 14 Perspectives 15 References 16

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2 Regulation of the Epithelial Naþ Channel by Peptidases Carole Plane`s and George H. Caughey I. Introduction 24 II. ENaC Regulation by Peptidases: In Vitro and Biochemical Evidence III. ENaC Regulation by Peptidases: In Vivo Evidence 31 References 41

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3 Advances in Defining Regulators of Cementum Development and Periodontal Regeneration Brian L. Foster, Tracy E. Popowics, Hanson K. Fong, and Martha J. Somerman I. Introduction 48 II. Question 1. What Are the Unknowns That Must Be Considered in Order to Replicate the Enamel (Crown) and How Do the Proteins Involved in Crown Development Relate to Root Development? 51 v

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Contents III. Question 2. What Do We Know About the Cells Required for Periodontal Development and Regeneration? 63 IV. Question 3. What Genes and Associated Proteins Are Important for Root/Periodontal Tissue Formation? 76 V. Conclusions and Future Directions 101 Acknowledgments 103 References 103

4 Anabolic Agents and the Bone Morphogenetic Protein Pathway I. R. Garrett I. II. III. IV.

Introduction 128 Bone Metabolism 131 The BMP Pathway and Bone Anabolic Therapies Conclusions and Future Directions 153 References 154

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5 The Role of Mammalian Circadian Proteins in Normal Physiology and Genotoxic Stress Responses Roman V. Kondratov, Victoria Y. Gorbacheva, and Marina P. Antoch I. Introduction 174 II. Circadian Rhythms and the Organization of the Mammalian Circadian System 175 III. Molecular Organization of the Circadian Oscillator in Mammals IV. Human Disorders Associated with Altered Function of the Circadian System 185 V. Pathologies and Developmental Defects in Circadian Mutant Mice VI. Circadian Control of the Organism’s Response to Genotoxic Stress VII. Circadian Proteins as Targets for Therapeutic Intervention 207 Acknowledgments 207 References 207

6 Autophagy and Cell Death Devrim Gozuacik and Adi Kimchi I. Introduction 218 II. Description of Programed Cell Death Morphologies

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176 190 200

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Contents III. IV. V. VI. VII.

Autophagic Cell Death 219 Autophagy and Autophagic Cell Death Regulatory Mechanisms Autophagy–Apoptosis Crosstalks 234 Survival Versus Death Aspects of Autophagy 236 Conclusions 237 Acknowledgments 238 References 238

Index 247 Contents of Previous Volumes

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Contribution of Membrane Mucins to Tumor Progression Through Modulation of Cellular Growth Signaling Pathways Kermit L. Carraway III, Melanie Funes, Heather C. Workman, and Colleen Sweeney UC Davis Cancer Center, Sacramento, California 95817

I. Mucin Structure, Function, and Involvement in Tumor Progression A. Mucin Structure B. Membrane Mucins and Tumor Progression II. MUC1 Contributions to Tumor Cell Growth Signaling A. Signaling by ErbB RTKs B. MUC1 Interaction with EGFR C. MUC1 Interaction with
‐Catenin D. Other MUC1 Growth Regulatory Mechanisms E. Mouse Models of MUC1 Action III. MUC4 Contributions to Tumor Cell Growth Signaling A. Alteration of ErbB2 Localization by MUC4 Interaction B. MUC4‐Mediated Tumor Growth and Metastasis IV. Inhibition of Signaling by Mucins V. Perspectives References

Mucins are large, heavily O‐glycosylated proteins expressed by epithelial tissues. The canonical function of membrane mucins is to provide protection to vulnerable epithelia by forming a steric barrier against assault, and by contributing to the formation of protective extracellular mucin gels. The aberrant overexpression of mucins is thought to contribute to tumor progression by allowing tumor cells to evade immune recognition, and by aiding in the breakdown of cell–cell and cell–matrix contacts to facilitate migration and metastasis. Recent evidence suggests that we should now modify our thinking about mucin function by considering their roles in signaling pathways leading to cellular growth control. Here we review the markedly divergent mechanisms by which membrane mucins, specifically MUC1 and MUC4, influence pathways contributing to cellular proliferation and survival. The cytoplasmic domain of MUC1 serves as a scaVold for the assembly of a variety of signaling proteins, while MUC4 influences the traYcking and localization of growth factor receptors, and hence their responses to external stimuli. We also discuss how tumor cells exploit these mechanisms to promote their own growth and metastasis. ß 2007, Elsevier Inc. Current Topics in Developmental Biology, Vol. 78 Copyright 2007, Elsevier Inc. All rights reserved.

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0070-2153/07 $35.00 DOI: 10.1016/S0070-2153(06)78001-2

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I. Mucin Structure, Function, and Involvement in Tumor Progression Mucins and mucin‐like proteins comprise a family of large transmembrane or secreted glycoproteins that are commonly associated with epithelial tissues (Strous and Dekker, 1992), but are also present at the surfaces of other selected cell types. Mucins are the major glycoprotein components of the mucous layer coating the cells lining the respiratory, digestive, and urogenital tracts, and are also prominently expressed by the ocular epithelium. Mucins possess specific domains that promote their oligomerization into viscous solutions or gels. In addition, their extensive O‐glycosylation contributes to a high negative charge and a rigid and extended protein conformation, resulting in steric and charge repulsion of cells or other molecules (Jentoft, 1990). These properties allow mucins to function in protecting epithelial tissues from infection, dehydration, and physical or chemical assault, and to serve as a lubricant for tissues exposed to mechanical stresses (Perez‐Vilar and Hill, 1999). The observed expression of mucins by vascular endothelial cells (Zhang et al., 2005) suggests that they may contribute to the formation of poorly adhesive surfaces in the lumen of blood vessels, augmenting blood flow by suppressing the counterproductive binding of blood components. On the other hand, some mucin glycosyl groups can serve as ligands for selectins and other cell adhesion molecules, promoting cell–cell interactions. In this context, mucin expression on the leading edge of activated T cells (Correa et al., 2003) suggests that mucins could also play a role in extravasation during inflammation. A. Mucin Structure The unifying structural feature of all mucins and mucin‐like proteins is that they express O‐linked glycans in serine‐ and threonine (Ser/Thr)‐rich clusters in their extracellular regions. A major subset of the mucins, a subfamily of proteins encoded by at least 20 distinct genes (designated MUC1–MUC20), contain Ser/Thr‐rich tandem repeats of 8 to 59 amino acid residues in length, depending on the identity of the mucin (Baldus et al., 2004a). Repeats are heavily O‐glycosylated, such that typically greater than 50% of mucin mass is O‐linked oligosaccharide. N‐linked glycans are also often present in mucins but to a much lesser degree. The number of Ser/Thr‐rich repeats encoded by a particular mucin gene can vary markedly among individuals due to genetic polymorphism, and variable number tandem repetition (VNTR) polymorphisms in mucins are associated with susceptibility to various diseases (Fowler et al., 2001). Moreover, mucin glycosylation patterns are tissue and cell type dependent, and can be altered with cellular diVerentiation state or with

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neoplastic transformation. Aberrant mucin glycosylation occurs in essentially all types of human cancers and is associated with tumorigenecity and metastasis (Moniaux et al., 2004). Many glycosyl epitopes are tumor‐associated antigens and have been used in diagnosis and immunotherapy. Mucins are divided into three subgroups: the gel‐forming, soluble, and transmembrane mucins. The gel‐forming mucins, composed of MUC subfamily proteins MUC2, MUC5AC, MUC5B, MUC6, and MUC19, are characterized by the presence of several cysteine‐rich domains in their extracellular regions (Chen et al., 2004). A single C‐terminal cysteine‐knot (CTCK) domain may be involved in the dimerization of these mucins. Trypsin inhibitor‐like (TIL) domains and von Willebrand factor C (VWC) or D (VWD) domains are commonly found in a variety of extracellular proteins and may be involved in specific protein–protein interactions that contribute to the gel‐forming properties of these mucins. Gel‐forming mucins are typically expressed by specialized glands or goblet cells and are major contributors to the viscoelastic properties of mucus secretion. For example, MUC5AC and MUC5B mucins are the predominant gel‐forming glycoproteins in airway mucus (Thornton and Sheehan, 2004). The soluble mucin MUC7 is significantly smaller than the gel‐forming mucins and has no recognizable domains in its extracellular region. It is most abundantly expressed in the less viscous secretions of salivary (Bobek et al., 1993) and lachrymal glands (Gipson, 2004). Membrane mucins, including MUC1, MUC3A, MUC3B, MUC4, MUC11, MUC12, MUC13, MUC17, and MUC20 are defined by the presence of a single hydrophobic transmembrane domain that secures them to the cell surface. With the exception of MUC4, all membrane mucins contain a SEA (Sea urchin sperm protein, Enterokinase, and Agrin) domain in their extracellular regions that may be involved in binding carbohydrate moieties. Several of the membrane mucins also contain EGF‐like domains that may be involved in protein– protein interactions. Membrane mucins are usually found at the apical surface of epithelial cells, providing protection to the epithelia. Membrane mucins can also be found as soluble molecules as a result of either splice removal of exons encoding the transmembrane and intracellular domains exons (Moniaux et al., 2000; Williams et al., 1999), or by proteolytic removal of the mucin from its transmembrane tether (Boshell et al., 1992; Wang et al., 2002). The soluble forms of membrane mucins can be found in fluids such as milk, tears, and saliva, or may remain loosely bound to the cell surfaces as part of the protective mucin barrier. While their functional roles remain to be fully elucidated, it is possible that soluble forms of membrane mucins could aid in the protection of epithelia from microbes. For example, it has been suggested that the presence of soluble membrane mucins in mothers’ milk could provide the newborn with protection from enteric pathogens (Newburg et al., 2005; Ruvoen‐Clouet et al., 2006).

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B. Membrane Mucins and Tumor Progression Intense interest has developed in understanding the role of aberrantly expressed mucins in the genesis and progression of tumors. MUC1 is highly expressed in the vast majority (>90%) of human breast tumors, and mislocalization of MUC1 (nonapical localization) predicts poor patient outcome (Rakha et al., 2005). MUC1 is overexpressed and diVerentially glycosylated in a number of other human tumors as well, including pancreatic, gastrointestinal, and lung, where it is associated with metastatic and invasive potential and has been used as a diagnostic and prognostic factor (Taylor‐Papadimitriou et al., 1999). Several assays to detect tumor‐associated antigens in serum samples are based on antibodies directed against circulating MUC1 antigens (Baldus et al., 2004a), and are used in the postoperative monitoring of breast and ovarian carcinoma patients. MUC4 overexpression may also eventually serve as a diagnostic and prognostic marker for numerous cancers, including pancreatic tumors (Swartz et al., 2002), where its expression is associated with metastatic phenotype (Singh et al., 2004), lung adenocarcinomas (Llinares et al., 2004), and mass‐forming type intrahepatic cholangiocarcinoma, where its coexpression with ErbB2 correlates with short survival time (Shibahara et al., 2004). MUC4 expression was shown to be increased during the pathologic process of squamous dysplastic transformation of exocervical epithelium, and could possibly be used as a marker in this process (Lopez‐Ferrer et al., 2001). Of the membrane mucins, human MUC1 and rat MUC4 have been studied in the greatest detail functionally and biochemically. Both are present at the cell surface as heterodimers formed from high molecular weight precursors, proteolytically cleaved early in their transit to the cell surface (Ligtenberg et al., 1992; Sheng et al., 1990). Both provide protection to the cell surface by virtue of their rigid and extended mucin subunit (Hilkens et al., 1992). Mucin expression confers on the cell potent antiadhesive properties, leading to the loss of cell–cell and cell–matrix interactions. Antiadhesiveness in turn confers antirecognition properties, leading to evasion from immune surveillance. The antiadhesive function of MUC4 is dependent on the number of tandem repeats present, suggesting that the eVects may be due to steric hindrance at the cell surface (Carraway et al., 2001; Wesseling et al., 1996). Overexpression of MUC1 and MUC4 is not compatible with the maintenance of polarized epithelia, and their expression must be tightly regulated in such cells. Hence, they are most often found at high levels in nonpolarized tumor cells where their expression may actively contribute to the loss of polarity. Paradoxically, the mucin‐mediated disruption of adhesion does not initiate anoikis, a type of apoptosis that normally occurs in response to a loss of cell–cell and cell–matrix interactions. Muc4‐expressing tumor cells are viable in suspension (M. Funes, unpublished observations),

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raising the possibility that membrane mucins might contribute to metastasis by inhibiting the apoptotic processes that occur when tumor cells escape their environment. Expression of both MUC1 and MUC4 has also been implicated in anticancer drug resistance of tumor cells. MUC4 overexpression has been shown to render melanoma cells resistant to cytotoxic agents such as taxol, doxorubicin, vinblastine, rhodamine 123, and 2‐deoxyglucose (Hu et al., 2003). MUC4 overexpression also reduces the binding of the therapeutic antibody Herceptin to tumor cells overexpressing ErbB2, presumably through steric interference (Nagy et al., 2005; Price‐Schiavi et al., 2002). Likewise, overexpression and knockdown of MUC1 suppresses and augments, respectively, the response of lung and breast tumor cells to the genotoxic agent cisplatin (Ren et al., 2004). It should be noted that while many of the classic protective properties of the membrane mucins may be attributed to their heavy glycosylation, splice variations in both MUC1 and MUC4 exist that lack the glycosylated tandem repeats. For example, nearly two dozen distinct splice variants of human MUC4 gene have been identified (Choudhury et al., 2000; Escande et al., 2002), generated by mechanisms including alternative use of cassette exons, exon skipping, and cryptic splice donor/acceptor sites. These MUC4 splice variants encode an assortment of secreted and membrane‐bound forms of the protein. Of particular interest is the existence of membrane‐bound forms lacking the O‐glycosylated tandem repeat region. At least two such forms exist for both MUC1 and MUC4, called MUC1/X and MUC1/Y (Zrihan‐Licht et al., 1994a), and MUC4/X and MUC4/Y (Choudhury et al., 2000). While the biological roles of membrane mucin splice variants remain to be determined, the existence of splice variants raises the possibility that membrane mucins have activities beyond the protective and antiadhesive functions generally ascribed to them. Indeed, the observation that MUC1 protein can become phosphorylated on tyrosine residues (Zrihan‐Licht et al., 1994b) originally raised the possibility that membrane mucins are involved in the signal transduction processes associated with cellular growth control.

II. MUC1 Contributions to Tumor Cell Growth Signaling The membrane mucins MUC1, MUC4, and MUC20 have each been implicated in the regulation of cellular growth signaling through their interactions with growth factor receptor tyrosine kinases (RTKs). RTKs receive signals in the form of polypeptide growth factor hormones derived from nearby cells or from endocrine sources, eliciting a cellular growth response such as proliferation, diVerentiation, migration, or survival.

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A. Signaling by ErbB RTKs There are 60 RTKs encoded by the human genome, which fall into about 20 RTK subfamilies. RTKs share an overall domain structure, with an extracellular ligand binding region that can vary markedly among the receptor subfamilies, a single transmembrane domain, and a cytoplasmic region containing a protein tyrosine kinase domain. The ErbB subfamily, which includes ErbB1/epidermal growth factor receptor (EGFR), ErbB2/Her2/ Neu, ErbB3, and ErbB4, is widely studied as a model for understanding the mechanisms by which RTKs transmit growth factor signals to the cell interior. The EGF‐like growth factor family of ligands binds the extracellular domain of the ErbB receptors leading to the formation of both homo‐ and heterodimers (Riese and Stern, 1998). Ligand‐induced dimer formation is followed by cross‐phosphorylation of specific tyrosine residues in the cytoplasmic tail, which then act as docking sites for the activation of intracellular signaling proteins, initiating a variety of signaling cascades that disseminate signals to the nucleus and cytoskeleton (Schlessinger, 2000). The major downstream pathways triggered by activated ErbB receptors, the phosphatidylinositol 30 ‐kinase (PI3K) and mitogen‐activated protein kinase (MAPK) pathways, can lead to a variety of outcomes depending on cellular context and duration of pathway activation. These outcomes include cellular proliferation, diVerentiation, migration, and survival. As such, ErbB receptors play critical roles both in development and tissue maintenance (Yarden and Sliwkowski, 2001). Overexpression and aberrant activation of ErbB receptor tyrosine kinases have been implicated in the genesis and progression of a variety of human tumors (Holbro et al., 2003a). In particular, overexpression of ErbB2 has been observed in a number of diVerent tumor types, including colorectal (Ross and McKenna, 2001), prostate (Ratan et al., 2003), breast and ovarian (Wang and Hung, 2001), and lung, pancreatic, bladder, and Wilms’ tumor (Menard et al., 2001). The most is known about ErbB2’s role in breast cancer, where its overexpression has been correlated with poor patient prognosis and decreased survival time as well as therapeutic resistance (Slamon et al., 1987). Because of their prominent overexpression and intimate involvement in tumor progression, ErbB receptors are attractive therapeutic targets, and drugs targeting EGFR and ErbB2 are currently in clinical use (Normanno et al., 2003; Shelton et al., 2005; Wakeling, 2005). Although no soluble ligand exists for ErbB2, it is the preferred heterodimeric partner of the other members of the ErbB family, making it an important coreceptor and activator of many signaling cascades (Marmor et al., 2004; Olayioye et al., 2000). ErbB3, the favored dimeric partner of ErbB2, is overexpressed in several types of human tumors. These include clear cell carcinoma of soft tissue where it may serve as a diagnostic marker (Schaefer et al., 2004), oral squamous cell carcinoma where it correlates with lymph node metastases (Shintani et al., 1995), and breast tumors where

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it is commonly found cooverexpressed with ErbB2 (Naidu et al., 1998) and correlates with shorter relapse‐free survival (Bieche et al., 2003). ErbB3 is the catalytically deficient member of the ErbB family (Guy et al., 1994) and therefore must heterodimerize with other ErbB receptors in order to signal (Carraway and Cantley, 1994). The C‐terminal domain of ErbB3 contains six binding sites for the p85 subunit of PI‐3 kinase, thus making ErbB3 the most potent activator of this pathway (Hellyer et al., 2001). It was reported that ErbB2 overexpression and activation alone are insuYcient to promote breast tumor cell proliferation. ErbB3 coupling of activated ErbB2 to the PI‐3 kinase pathway is required for proliferation of ErbB2 overexpressing cells, suggesting that the ErbB2/ErbB3 complex functions as an ‘‘oncogenic unit’’ which promotes breast tumor cell proliferation (Holbro et al., 2003b). B. MUC1 Interaction with EGFR MUC1 overexpression is suYcient to induce anchorage‐independent growth and tumorigenicity, the hallmarks of oncogene products (Huang et al., 2003; Li et al., 2003; Ren et al., 2002; Schroeder et al., 2004). MUC1 contributes to the dysregulation of cellular growth control in part by acting as a substrate for several kinases such as the Ser/Thr kinases GSK3
(Li et al., 2001a) and PKC (Ren et al., 2002), the cytosolic tyrosine kinase src (Li et al., 2001a), and ErbB subfamily RTKs (Li et al., 2001b). The 72 residue MUC1 cytoplasmic domain contains seven tyrosine residues, several of which are capable of becoming phosphorylated (Wang et al., 2003). As in RTKs, phosphorylation of specific tyrosine residues leads to the context‐dependent recruitment and activation of intracellular molecules that stimulate intracellular signaling pathways. MUC1 physically associates with all four ErbB receptors at the plasma membrane in a growth factor‐independent manner (Li et al., 2001b; Schroeder et al., 2001). EGF stimulation leads to the phosphorylation of Y60 of the intracellular domain, allowing binding of the adapter protein Grb2 and the associated ras guanine nucleotide exchange protein SOS (Pandey et al., 1995). (It should be noted that because of the variable number of repeats in the extracellular region, the numbering of amino acids in mucins cannot be conventionally carried out from the N‐terminus. Here we are numbering residues from N‐terminus of the cytoplasmic domain). This interaction provides an activation mechanism for Ras and its downstream eVectors such as MAPK. Consistent with this, activated MAPK is elevated when wild‐type MUC1 is overexpressed in the mammary glands of transgenic mice (Schroeder et al., 2001). C. MUC1 Interaction with b‐Catenin MUC1 is also able to influence cellular signaling through its interaction with
‐catenin.
‐Catenin is a key eVector of the Wnt signaling pathway (Bienz, 2005; Moon et al., 2004). Binding of the Wnt mitogen to its cell surface

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receptor leads to the cytosolic stabilization of
‐catenin and its accumulation in the nucleus. Here
‐catenin acts as a transcriptional coactivator to augment the expression of cellular growth control genes such as cyclin‐D1 and c‐myc (Smalley and Dale, 2001). Activity of the Ser/Thr kinase GSK3
facilitates
‐catenin degradation in the absence of Wnt signaling and is suppressed on Wnt stimulation. In tumors, the deregulation of
‐catenin activity leads to uncontrolled cell proliferation and neoplastic progression via the constitutive expression of Wnt target genes (Moon et al., 2004).
‐Catenin also associates with the transmembrane cell adhesion molecule E‐cadherin at adherens junctions. Binding of the E‐cadherin extracellular domain to cadherins on neighboring cells mediates lateral interactions between epithelial cells.
‐Catenin couples E‐cadherin to ‐catenin, which in turn is coupled to the cytoskeleton. By coupling the cell surface to the cytoskeleton,
‐catenin plays a central role in tethering adjacent epithelial cells together to form epithelial sheets. Phosphorylation of
‐catenin on specific tyrosine residues, either by Src overexpression or EGFR activation, suppresses E‐cadherin‐mediated adhesion by disrupting
‐catenin interaction with E‐cadherin and ‐catenin (Lilien and Balsamo, 2005). MUC1 and
‐catenin do not eYciently interact in normal polarized epithelia because MUC1 resides on the apical surface while
‐catenin resides on the lateral surface. However, loss of cell polarity during transformation creates a permissive environment for MUC1 and
‐catenin interaction. Under these conditions,
‐catenin can bind directly to the amino acid sequence 50‐SAGNGGSSL‐59 of the MUC1 cytoplasmic domain (Yamamoto et al., 1997). Similar amino acid motifs are responsible for
‐catenin binding by E‐cadherin and adenomatous polyposis coli (APC) gene product. The MUC1/
‐catenin interaction is tightly regulated by oncogenic kinases; phosphorylation of the nearby T41 by the Ser/Thr kinase PKC (Ren et al., 2002) or Y46 by src or EGFR (Li et al., 2001a) enhances MUC1/
‐catenin interaction, while phosphorylation of S44 by GSK3
inhibits the interaction (Li et al., 2001a). MUC1 binding can aVect several aspects of
‐catenin function. Disruption of the
‐catenin binding site in MUC1 suppresses its ability to induce anchorage‐dependent and ‐independent growth, indicating
‐catenin binding to MUC1 is a critical component of its tumorigenic activity. The underlying mechanism appears to involve the MUC1‐mediated protection of
‐catenin from GSK3
‐mediated proteasomal degradation (Huang et al., 2005). Surprisingly, MUC1 colocalizes with
‐catenin in the nucleus (Baldus et al., 2004b; Wen et al., 2003) and coactivates transcription of Wnt target genes (Huang et al., 2003). Similarly, other studies have shown that MUC1 binds directly to p53 and regulates transcription of p53‐target genes in response to genotoxic stress (Wei et al., 2005). MUC1 binding to
‐catenin also suppresses its ability to interact with E‐cadherin at adherens junctions, leading to a breakdown in cell–cell interactions, and GSK3
‐mediated disruption of

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the complex restores the E‐cadherin/
‐catenin interaction (Li et al., 1998). Hence, MUC1‐induced abrogation of cell–cell interactions may be mediated both by steric hindrance by its mucinous extracellular domain and by the interaction of its intracellular domain with
‐catenin.

D. Other MUC1 Growth Regulatory Mechanisms MUC1 suppresses apoptosis induced by genotoxic agents at least in part by suppressing the release of apoptogenic factors from mitochondria (Ren et al., 2004). Together with the ability of MUC1 extracellular domain to interfere with tumor cell recognition, these observations suggest that MUC1 overexpression in tumors could potently contribute to tumor resistance by a variety of mechanisms. Interestingly, the antiapoptotic eVect of MUC1 may occur as a result of the translocation of the MUC1 intracellular domain to mitochondria in tumor cells. The growth factor neuregulin‐1 (NRG1), which acts through the ErbB3 or ErbB4 RTKs, stimulates MUC1 mitochondrial localization. Sequential src phosphorylation of Y46 and binding of the chaperone Hsp90 are required for translocation (Ren et al., 2006). It has been observed that the intracellular domain of MUC1 interacts with the DNA binding domain of the estrogen receptor (ER), and that this association is enhanced by estrogen stimulation (Wei et al., 2006). The functional consequence of the interaction is to augment cellular ER levels by suppressing its ubiquitination and degradation. As a result, MUC1 enhances ER‐mediated transcription and stimulates the hormone‐mediated growth and survival of breast cancer cells. This oVers a mechanism by which MUC1 can contribute to the growth and progression of hormone‐dependent tumors.

E. Mouse Models of MUC1 Action Studies with genetically modified mice have provided strong support for the biochemical and signaling studies described above. Mammary expression of polyoma virus middle T antigen (PyMT) results in the formation of multifocal mammary tumors with a high frequency of metastasis (Guy et al., 1992). This PyMT model has proven invaluable in elucidating the signaling pathways that impact mammary gland transformation. PyMT exerts its oncogenic action by interacting with and modulating the activities of multiple signaling proteins such as src, PI3K, protein phosphatase 2A, shc, and PLC. A role for MUC1 in PyMT action was demonstrated by analysis of PyMT‐induced mammary tumorigenesis in wild‐type mice versus MUC1 knockout animals. While MUC1 knockouts exhibited no overt phenotype,

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the growth rate of primary mammary tumors induced by polyoma middle T (PyMT) antigen was significantly slower in MUC1 deficient mice than in the wild‐type background (Spicer et al., 1995). A more extensive examination of these tumors suggests that MUC1 promotes src signaling by enhancing its association with PI‐3 kinase and
‐catenin (Al Masri and Gendler, 2005). Multiparous transgenic mice overexpressing MUC1 in the mammary gland develop unifocal tumors after a long latency, whereas similar transgenics expressing MUC1 lacking its intracellular domain do not (Schroeder et al., 2004). In this instance, tumor formation may be related to the ability of Muc1 to inhibit postlactational involution through the inhibition of apoptosis. These observations underscore a potential role for MUC1 in mammary tumor formation and progression, and suggest that signaling through the MUC1 intracellular domain is essential for these processes. Consistent with this, Wnt‐1‐induced tumors develop more slowly in MUC1 null mice than in a wild‐type background, suggesting that MUC1 interaction with
‐catenin facilitates tumor progression (Schroeder et al., 2003). This study also demonstrated that MUC1 binds to
‐catenin only in tumors, and binding promotes the redistribution of
‐catenin to the invading margins of tumors. Analysis of human breast tumors confirmed that MUC1/
‐catenin interactions occur in patients, and that the interaction is increased in metastatic lesions compared with primary tumors. These observations then raise the possibility that MUC1 also contributes to invasive tumorigenesis in the breast through the modulation of
‐catenin localization and subsequent cytoskeletal dynamics (Schroeder et al., 2003).

III. MUC4 Contributions to Tumor Cell Growth Signaling The vast majority of biochemical studies on MUC4 have been carried out using the rat version. Rat MUC4 is a noncovalently associated heterodimeric complex composed of two subunits, ASGP1 and ASGP2, arising from a single transcript (Carraway et al., 2001; Rossi et al., 1996). The translated precursor is cleaved into the two subunits during transit to the cell surface. ASGP1 is heavily O‐glycosylated and contains three Ser/Thr‐rich regions, including a 50‐amino acid N‐terminal domain, the variable tandem repeat domain typical of mucins, and a third 609 amino acid Ser/Thr‐rich domain. Immediately adjacent to one another at the C‐terminus of ASGP1 is a nidogen domain and an AMOP (adhesion‐ associated domain present in MUC4 and other proteins) domain. While the function of neither domain is known, both are found in the extracellular regions of proteins thought to be involved in cell adhesion. ASGP2 is an N‐glycosylated protein containing a VWD domain, common in mucins, three EGF or EGF‐ like domains, a single transmembrane domain, and a very short cytoplasmic domain of 17 amino acids. While there are significant diVerences between rat

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and human MUC4 in the ASGP1 mucin portion of the protein (called MUC4 in human), the two share 60–70% amino acid identity in ASGP2 (MUC4
).

A. Alteration of ErbB2 Localization by MUC4 Interaction MUC4 can be coimmunoprecipitated with ErbB2 from a variety of tissues and cell lines, and rat MUC4 stably associates with ErbB2 only when the two proteins are coexpressed in the same cell (Carraway et al., 1999). If ASGP2 and ErbB2 extracellular domains are individually expressed and then mixed, no complex formation occurs. ASGP2/ErbB2 complex assembly occurs within the cell prior to transit to the cell surface (Ramsauer et al., 2006). Deletion analysis indicates that one of the EGF‐like domains of ASGP2 is necessary for ASGP2/ErbB2 complex formation. While the original studies suggested ASGP2 selectively binds ErbB2 (Carraway et al., 1999), more recent studies with improved reagents indicate that other ErbB family members can also associate with ASGP2 (Funes, unpublished observations). The MUC4/ErbB2 complex has been observed in many of the tissues where MUC4 is prominently expressed, including epithelia of the ocular surface (Swan et al., 2002), lachrymal gland (Arango et al., 2001), female reproductive tract, and the mammary gland (Price‐Schiavi et al., 2005). MUC4 and ErbB2 are also commonly overexpressed and interact in highly aggressive human breast tumors (Price‐Schiavi et al., 2005). These data suggest that MUC4 may modulate ErbB signaling via its interaction with ErbB2, in both normal epithelia and in tumors. In contrast to MUC1, transgenic mice overexpressing MUC4 in the mammary gland do not develop tumors more frequently than wild‐type animals. Interestingly, MUC4 transgenics exhibit a ‘‘bifurcated’’ mammary development pattern where mammary ducts grow along blood vessels outside of the mammary fat pad (Price‐Schiavi et al., 2005). This phenotype has also been observed in mice conditionally expressing activated ErbB2 under its endogenous promoter (Andrechek et al., 2003), suggesting that MUC4 enhances ErbB2 activity or signals through similar cellular growth pathways as does ErbB2. The observations that MUC4 associates with ErbB2 and that its overexpression mimics the phenotype of activated ErbB2 in the mammary gland has led to the proposal that MUC4 acts as an unconventional membrane‐associated ligand for the growth factor receptor (Ramsauer et al., 2006). In this model, ErbB2 and MUC4 associate early in their traYcking to the cell surface, and the interaction promotes the constitutive growth factor‐independent tyrosine phosphorylation of the receptor. The evidence supporting this model is equivocal. In some cell lines, ErbB2 tyrosine phosphorylation is mildly augmented on MUC4 expression (Jepson et al., 2002), while in other cell lines this is not the

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case (Ramsauer et al., 2006; Funes et al., 2006). MUC4 expression does not stimulate MAPK (Erk) or PI3K signaling under many circumstances, as might be expected for an activating ligand. The lack of a uniform response of ErbB2 to MUC4, together with the unresolved question of why a cell would need to signal itself via a ligand that cannot act as a diVusible factor, calls into question the model that MUC4 acts as an activating ligand for ErbB2. It is clear, however, that MUC4 expression results in the potentiation of NRG1‐stimulated tyrosine phosphorylation of both ErbB2 and ErbB3 under all circumstances examined (Funes et al., 2006), with accompanying potentiation of the PI3K pathway, cellular proliferation, and survival. MUC4 potentiates ErbB2/ErbB3 receptor tyrosine phosphorylation and signaling by making more receptors available at the plasma membrane for interaction with the NRG1 ligand, without aVecting the total quantity of receptors expressed by the cell. In contrast with the EGF receptor, under normal conditions, significant quantities of ErbB2 and ErbB3 can accumulate within internal compartments in many cell types in the absence of added growth factor. MUC4 expression induces the translocation of these receptors to the cell surface, markedly augmenting the number of receptors available for signaling (Funes et al., 2006). MUC4 also enhances the recruitment of PI3K to activated ErbB3 and augments ErbB2/ErbB3 signaling through the PI3K pathway. PI3K activation occurs most eYciently at the plasma membrane because receptors in intracellular compartments have poor access to the lipid substrates required for PI3K signaling (Haugh and Meyer, 2002). Hence, the ability of MUC4 to trap receptors at the plasma membrane likely underlies PI3K potentiation. The PI3K pathway is an important regulator of cell proliferation and survival, and it has been demonstrated that PI3K is essential for ErbB2/ErbB3‐mediated breast tumor cell proliferation (Holbro et al., 2003b). Hence, MUC4‐potentiated ErbB2/ErbB3 signaling through PI3K may play a prominent role in breast cancer progression. Much focus has been placed on understanding the mechanisms underlying the growth and progression of the 20–30% of breast tumors that overexpress ErbB receptors. ErbB2 and ErbB3 overexpression has been correlated with poor patient prognosis and decreased survival time, and has been implicated in the tumor progression of various cancers. The observations outlined above may oVer insight into the growth properties of the remaining majority of tumors that express modest ErbB2 and ErbB3 levels. Since MUC4 is highly expressed in 90% of human breast tumors (Rakha et al., 2005), enhanced localization of receptors to the plasma membrane in such cells could be synonymous with receptor overexpression in terms of outcome. In addition to its ability to elicit the translocation of ErbB2 from intracellular compartments to the surface of tumor cells, MUC4 can also translocate ErbB2 from the basolateral surface to the apical surface of polarized epithelial

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cells (Ramsauer et al., 2003). Hence, it appears that the primary impact of MUC4 on ErbB receptors is to mediate their relocalization to plasma membrane subdomains. In normal epithelial cells, relocalization of ErbBs from the basolateral surface to the apical surface would expose the receptors to apical activating ligands, signaling in response to apical cues. In this regard it is interesting that ErbB receptors may be transactivated by a variety of heterologous stimuli (Carraway and Sweeney, 2002). Activating ligands for cytokine receptors, G‐protein–coupled receptors, and even other growth factor receptors have been demonstrated to activate ErbB receptors in cultured cells. Mechanisms mediating these events include the activation of src activity, the elevation of intracellular reactive oxygen species, and the induction of ErbB ligand shedding from the cell surface by proteases, all of which elevate ErbB receptor activity. Hence, the physiological purpose of mucin‐ mediated ErbB translocation could be to allow these receptors to mediate signaling by apical heterologous ligands, facilitating cellular sensing of the apical environment. In nonpolar tumor cells, the outcome of this mechanism would be to augment surface levels of ErbB receptors leading to their hyperactivation. This model could also explain the inconsistencies observed with MUC4‐induced ErbB2 phosphorylation. The elevation of cell surface ErbB2 by MUC4 would augment ErbB2 tyrosine phosphorylation only in cell lines that also express significant amounts of a receptor for serum heterologous ligands, for example, lysophosphatidic acid. The molecular mechanisms underlying MUC4‐dependent ErbB localization is an active area of pursuit.

B. MUC4‐Mediated Tumor Growth and Metastasis MUC4 could potentially facilitate tumor development and progression through multiple mechanisms, including its antiadhesive, antirecognition, and survival‐promoting properties (Carraway et al., 2001, 2003). In xenograft models, MUC4 antiapoptotic properties dominate in contributing to the growth rate of primary tumors. Subcutaneous injection of human melanoma cells in nude mice demonstrated that MUC4 overexpression enhances primary tumor growth rate relative to cells that do not express MUC4 by three‐ to fivefold (Komatsu et al., 2001). Analysis of the tumors revealed that the diVerence in growth rate was due to a suppression of apoptosis rather than an enhancement of cellular proliferation. In cultured cells, MUC4 overexpression causes the release of cells from the plate (Komatsu et al., 1997), undoubtedly due to mucin steric hindrance with cell contacts with the culture dish. In cells lacking MUC4 expression, inhibition of adhesion potently induces apoptosis; however, nonadhesive cells overexpressing MUC4 are viable (Funes, unpublished observations). The mechanism by which MUC4 suppresses apoptosis is unclear. Since MUC4 can augment ErbB2 activity, it is tempting to speculate that this RTK is

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involved, however this has not been demonstrated. Interestingly, MUC4 overexpression was shown to upregulate p27kip (Jepson et al., 2002), a cell cycle inhibitor that can suppress apoptosis in some contexts (Eymin et al., 1999; Sgambato et al., 2000). However, no evidence thus far points to a role for p27kip in the MUC4‐mediated survival. The mechanism underlying the antiapoptotic eVect of MUC4 remains a key question to fully understanding MUC4 influence on cellular growth control and contribution to tumor progression. Metastasis involves several steps: dissociation from primary tumor, escape from immune cell recognition, extravasation, and growth at a secondary tumor site. The ability of MUC4 to suppress apoptosis and promote primary tumor growth, coupled with its potent antiadhesive and antirecognition properties, suggests that it may play a role in several of the steps involved in metastasis. MUC4 overexpression markedly augments lung metastases of melanoma cells injected into the tail vein or the foot pads of nude mice fold relative to control cells (Komatsu et al., 2000). Consistent with these observations, MUC4 knockdown in a highly aggressive and metastatic pancreatic tumor cell line that overexpresses the mucin results in decreased cell motility, adhesion and aggregation in vitro, and suppressed tumor growth and metastasis rates in nude mice (Singh et al., 2004). While MUC4 overexpression promotes loss of cell adhesion in primary tumor cells, it also contains the E‐selectin ligand, sialyl Lewis X (Slex) (Lasky, 1994; Park et al., 2003). Extravasation of leukocytes from the circulatory system into surrounding tissues is dependent on vascular endothelial cell‐expressed selectins, suggesting that MUC4 Slex might promote metastasis (Kannagi, 1997). MUC4 was found prominently expressed in several cultured endothelial cell lines and on the luminal surface of blood vessels (Price‐Schiavi et al., 2005). While its role there is not clear, it is probable that it serves as a protective, nonadhesive barrier for the luminal surface of blood vessel endothelium. However, it may also be of importance in the selective adhesion required for extravasation of tumor cells.

IV. Inhibition of Signaling by Mucins The membrane mucin MUC20 is highly expressed in kidney, where signaling by the Met RTK in response to hepatocyte growth factor (HGF) plays a key role in tubule formation and diVerentiation. MUC20 and Met physically interact independent of HGF stimulation. In contrast with MUC4, which interacts with ErbB receptors to potentiate growth factor signaling, MUC20 interaction with Met selectively suppresses HGF‐stimulated MAPK (Erk) activation. While overall Met tyrosine phosphorylation is not aVected, recruitment of the Grb2 adaptor to the activated receptor is potently suppressed (Higuchi et al., 2004). Interestingly, many of the hallmark cellular

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responses to HGF such as increased cell motility and survival are not influenced by MUC20 expression, probably because these processes are heavily dependent on the PI3K pathway. MAPK‐dependent processes such as renal cell proliferation and metalloprotease production are specifically suppressed by MUC20 expression. While far less is known about MUC20 function in normal tumors, these observations raise the possibility that mucins might also influence signaling pathway usage by RTKs, selectively augmenting or suppressing their range of function according to the growth requirements of the cell. A similar mechanism has been observed in the yeast Saccharomyces cerevisiae (Clevers, 2004; Cullen et al., 2004). The membrane mucin Mbs2 was identified in a screen for factors that contribute to filamentous growth, a yeast growth response to adverse conditions. Mbs2 has six tandem Ser/Thr‐rich repeats in its extracellular region, the hallmark of mucin proteins, and localizes to polarized sites at the cell surface. Mbs2 interacts with two proteins also known to be involved in filamentous growth: the transmembrane protein Sho1 and the Cdc42 GTP‐binding protein. Interestingly, deletion of the tandem repeat domain results in hyperactive filamentous growth, suggesting that the highly glycosylated mucin portion suppresses signaling leading to filamentous growth. This then leads to the suggestion that expression of splice variants of mammalian mucins that lack the tandem repeat domains, such as MUC1/Y and MUC4/Y, might confer a growth advantage to cells by relieving suppressive eVects on growth signaling pathways. Biochemical characterization of these forms awaits further study.

V. Perspectives It is becoming clear that membrane mucins have evolved functions beyond simple protection of epithelial surfaces. The diVerent membrane mucins employ a variety of mechanisms to either augment or suppress signaling pathways involved in cellular growth control. Moreover, mucin modulation of growth signaling is a theme that extends evolutionarily back to the simplest eukaryotes. While it is also clear that tumor cells can exploit these mechanisms to their advantage, so far there is no clear indication as to whether or not these mechanisms contribute to normal biological processes. For example, a role for MUC4 in the relocalization of ErbB receptors to diVerent plasma membrane subdomains has been established, presumably to contribute to diVerent signaling processes. However, the nature of the alternate signaling events is not known, and may vary considerably from one epithelial tissue to the next. In addition, MUC1 has been observed to bind to
‐catenin only in depolarized tumor cells, and not in normal polarized epithelial cells. However, the
‐catenin binding site in MUC1 is conserved across species, suggesting that it might serve a useful biological function. Could the MUC1/
‐catenin

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interaction play a role in development, prior to the establishment of cell polarity? MUC1 knockout animals show no overt developmental abnormalities, but other processes could make up for MUC1 loss. Hence, a key question concerns the biological significance of mucin signaling modulation during the processes of normal development and tissue maintenance. While the transmembrane mucin family is composed of several members, including MUCs 1, 3, 4, 11, 12, 13, and 20, only MUC1 and 4 have been extensively studied. Though less well characterized, MUCs 3, 11, 12, and 13 all contain EGF‐like domains. Since one of the EGF‐like domains of MUC4 is required for its interaction with ErbB2, it is of interest to determine whether other mucins in this subfamily can associate with receptors to modulate signaling. Interestingly, increased expression of MUC3 has been reported in invasive ductal adenocarcinoma of the pancreas (Park et al., 2003), renal clear cell carcinoma (Leroy et al., 2002), esophageal cancers (Packer et al., 2004), and breast carcinomas (Cao et al., 1997), suggesting that MUC3 modulation of signaling pathways might also contribute to the growth of some tumor types. A number of questions remain concerning the functional consequences of the MUC4/ErbB2 interaction. As suggested above, it remains to be determined whether MUC4‐mediated cellular survival is ErbB2‐dependent or whether MUC4 independently activates an unknown pathway that promotes cell survival. Likewise, it is not clear whether the physical interaction between MUC4 and ErbB receptors is responsible for ErbB relocalization, or whether MUC4 independently impacts receptor traYcking and localization. Future studies aimed at disrupting the interaction between MUC4 and ErbB2 will clarify the contribution of each to such processes. If the interaction between MUC4 and ErbB2 is required for processes that promote tumor progression, it could be useful to target this interaction therapeutically.

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Regulation of the Epithelial Naþ Channel by Peptidases Carole Plane`s*,{ and George H. Caughey {,§,k *INSERM U773, Centre de Recherche Biome´dicale Bichat‐Beaujon (CRB3) Universite´ Paris 7, 75018 Paris, France { Department of Physiology, UFR de Me´decine Paris Ile de France Ouest Universite´ de Versailles‐Saint Quentin, 78000 Versailles, France { Cardiovascular Research Institute and Department of Medicine University of California at San Francisco, San Francisco, California § San Francisco Veterans AVairs Medical Center, San Francisco, California k Northern California Institute for Research and Education San Francisco, California

I. Introduction II. ENaC Regulation by Peptidases: In Vitro and Biochemical Evidence A. Inhibition of Naþ Transport in Epithelial Monolayers by Antipeptidases B. Peptidases in ENaC Maturation and Disposal C. CAPs in Mammalian Epithelia D. Candidate Physiological Inhibitors of ENaC‐Activating Peptidases III. ENaC Regulation by Peptidases: In Vivo Evidence A. Regulation of Alveolar Naþ and Water Transport by Serine Peptidases B. Regulation of Colonic Prostasin Expression by Aldosterone and Dietary Naþ C. Regulation of Prostasin by Aldosterone in the Kidney D. Potential Role of Prostasin in Regulating Aldosterone Production and Hypertension E. Role of CAPs in Maintaining the Epidermal Permeability Barrier F. ENaC Dysregulation in the Pathophysiology of Cystic Fibrosis: CAPs as Potential Drug Targets References

Recent investigations point to an important role for peptidases in regulating transcellular ion transport by the epithelial Naþ channel, ENaC. Several peptidases, including furins and proteasomal hydrolases, modulate ENaC maturation and disposal. More idiosyncratically, apical Naþ transport by ENaC in polarized epithelia of kidney, airway, and gut is stimulated constitutively by one or more trypsin‐family serine peptidases, as revealed by inhibition of amiloride‐sensitive Naþ transport by broad‐spectrum antipeptidases, including aprotinin and bikunin/SPINT2. In vitro, the transporting activity of aprotinin‐suppressed ENaC can be restored by exposure to trypsin. Current Topics in Developmental Biology, Vol. 78 Copyright 2007, Elsevier Inc. All rights reserved.

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0070-2153/07 $35.00 DOI: 10.1016/S0070-2153(06)78002-4

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The prototypical channel‐activating peptidase (CAP) is a type 1 membrane‐ anchored tryptic peptidase first identified in Xenopus kidney cells. Frog CAP1 strongly upregulates Naþ transport when coexpressed with ENaC in oocytes. The amphibian enzyme’s apparent mammalian orthologue is prostasin, otherwise known as CAP1, which is coexpressed with ENaC in a variety of epithelia. In airway cells, prostasin is the major basal regulator of ENaC activity, as suggested by inhibition and knockdown experiments. Other candidate regulators of mature ENaC include CAP2/TMPRSS4 and CAP3/matriptase (also known as membrane‐type serine protease 1/ST14). Mammalian CAPs are potential targets for treatment of ENaC‐mediated Naþ hyperabsorption by the airway in cystic fibrosis (CF) and by the kidney in hypertension. CAPs can be important for mammalian development, as indicated by embryonic lethality in mice with null mutations of CAP1/prostasin. Mice with selectively knocked out expression of CAP1/prostasin in the epidermis and mice with globally knocked out expression of CAP3/matriptase exhibit phenotypically similar defects in skin barrier function and neonatal death from dehydration. In rats, transgenic overexpression of human prostasin disturbs salt balance and causes hypertension. Thus, several converging lines of evidence indicate that ENaC function is regulated by peptidases, and that such regulation is critical for embryonic development and adult function of organs such as skin, kidney, and lung. ß 2007, Elsevier Inc.

I. Introduction Many epithelia form high‐resistance barriers to Naþ and other ionic solutes. Control and modulation of the passage of Naþ across these barriers is critical for development and proper functioning of a variety of organs before, during, and after birth. Such epithelia use transcellular flux of Naþ to control the movement of water, which migrates passively across and between cells to equalize inequalities in concentrations of Naþ and other solutes. Movement of water in response to modulation of transepithelial Naþ flux is important in processes as diverse as removing liquid from neonatal mammalian lung on exiting the womb, maintaining blood pressure and volume, preventing excessive electrolyte loss via sweating, and maintaining optimal hydration of the luminal surface of wet and ciliated mucosa. Failure to regulate Naþ flux can cause or contribute to a variety of serious diseases, including respiratory distress syndrome, high or low blood pressure, and cystic fibrosis (CF). Although several Naþ channels have been described, probably the most important for regulating Naþ and water flux across high‐resistance epithelia of airway, alveoli, bladder, kidney, and distal colon is the apical epithelial Naþ channel, ENaC, which allows Naþ to pass into the cell following electrochemical gradients established by extrusion of Naþ by a basolateral membrane pump (Naþ, Kþ‐ATPase). Mutations in ENaC genes cause

2. ENaC Control by Peptidases

25

diseases associated with channel overactivity [e.g., Liddle’s syndrome (Shimkets et al., 1994)] as well as with loss of function [e.g., pseudo‐hypoaldosteronism (Chang et al., 1996)]. Given the critical contributions of ENaC to organ homeostasis and the ever‐changing dynamics of water and salt intake and loss, it is no surprise that ENaC function is subject to regulation by a variety of ‘‘accessory factors’’ originating from epithelial cells (Huang et al., 2004; Snyder, 2005; Stutts et al., 1995; Thomas and Itani, 2004; Voilley et al., 1997; Warnock, 1999). In the past decade, investigators in several laboratories recognized and explored an initially surprising but potent modulation of ENaC function by peptidases, which is the focus of this review.

II. ENaC Regulation by Peptidases: In Vitro and Biochemical Evidence A. Inhibition of Naþ Transport in Epithelial Monolayers by Antipeptidases A seminal observation suggesting that ENaC is regulated by endogenous serine peptidases was made in a frog line of kidney cells (Xenopus A6) (Vallet et al., 1997). Amiloride‐sensitive Naþ uptake in these cells was partially blocked by apical exposure to the broad‐spectrum inhibitor of serine peptidases, aprotinin, and was restored in aprotinin‐treated cells by application of trypsin. This suggested the presence of an ENaC‐activating serine peptidase in the apical membrane. Vallet and colleagues then used an expression‐cloning strategy to identify a candidate ‘‘channel‐activating peptidase 1’’ (CAP1), which augmented ENaC activity when coexpressed with ENaC in oocytes. Similar responses to apical aprotinin and trypsin were noted in M‐1 cells derived from mouse cortical collecting duct (Nakhoul et al., 1998), in human nasal airway cells in primary culture (Donaldson et al., 2002), and in JME/CF15 airway cells derived from an individual with CF (Tong et al., 2004), suggesting similar upregulation of ENaC by endogenous peptidases in mammalian epithelia. Further studies (Bridges et al., 2001) suggested another layer of regulation by revealing inhibition of ENaC in cultured airway cells by a recombinant serine peptidase inhibitor (BAY 39–9437) derived from human placental bikunin, which may be an endogenous modulator of CAP activity. The exact mechanism of the eVects of trypsin remains to be fully established. However, the overall eVect on rat ENaC is to increase channel open probability of previously near‐silent channels by lengthening the time spent open and diminishing time spent closed—without aVecting open channel conductance to Naþ (Caldwell et al., 2004). In other informative studies, aprotinin blocked ENaC‐mediated Naþ transport across primary rat alveolar type II cell monolayers by 70% without

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modifying ‐ and ‐ENaC cell surface expression, whereas exogenous trypsin had little or no eVect (Planes et al., 2005). This suggested that aprotinin‐ sensitive epithelial serine peptidases activate ENaC in alveolar epithelium mostly by increasing channel open probability, and that this activation is maximal or near‐maximal under usual culture conditions. Interestingly, preincubation of alveolar cells with aprotinin also completely abolished stimulation of Naþ transport by 2‐adrenergic agonists. Inhibition of Naþ transport by apically applied aprotinin also occurs in cultured mouse bronchioloalveolar epithelial cells, although to a lesser extent than in rat cells (Planes et al., 2005). This variance may be due to interspecies diVerences in expression or aprotinin‐sensitivity of endogenous peptidases, as shown between mouse and amphibian kidney cells (Vuagniaux et al., 2002). In vitro, cAMP agonists augment ENaC activity and transepithelial Naþ transport in various cell types, including native alveolar epithelial cells. This occurs mostly by increasing turnover and promoting insertion of ENaC subunits at the cell surface (Planes et al., 2002; Snyder, 2000). Serine peptidases and 2‐adrenergic agonists therefore could act synergistically, with peptidases activating Naþ channels newly recruited to apical membrane under the influence of 2‐agonists. A similar synergism was reported between membrane‐bound CAPs and serum‐ and glucocorticosteroid‐regulated kinase in frog oocytes (Vuagniaux et al., 2002).

B. Peptidases in ENaC Maturation and Disposal ENaC is a heterotetrameric membrane protein built from products of homologous genes encoding ‐, ‐, and ‐subunits. Mature ENaC is thought to be composed of two ‐, one ‐, and one ‐subunits (Fig. 1). Each subunit has two transmembrane domains bridged by a large extracellular loop. After initial translation‐coupled embedding in the endoplasmic reticulum membrane, subunits mature prior to insertion into the plasma membrane by addition and processing of N‐linked sugars (Hughey et al., 2004b). Some subunits also are processed by nicking of  at two sites and at one site by furin (Hughey et al., 2003), a membrane‐anchored subtilisin‐type peptidase thought to reside principally in the lumen of the trans‐Golgi network. Nicking of the ‐subunit activates channel Naþ‐transporting activity (Hughey et al., 2004a), apparently by dissociating an inhibitory domain in the extracellular loop (Carattino et al., 2006). Furin‐mediated hydrolysis of ENaC  subunits also relieves ‘‘self‐inhibition’’ by Naþ (Sheng et al., 2006), thereby increasing Naþ transport. The importance of furin cleavage sites and furin itself has been suggested by loss of activity in channels with mutated furin cleavage sites and by reduced activity in furin‐deficient cells (Hughey et al., 2004a). At the cell surface, ENaC function appears to be further upregulated

27

2. ENaC Control by Peptidases Free serine peptidase (trypsin)

3

+ C

+ a

g b a

4

6

a

g

ba

SS

Na+

+

Type I transmembrane serine peptidase (prostasin)

Type II transmembrane serine peptidase (TMPRSS3)

N 2

5

Furin

?

a

1

g ba

ENaC

Figure 1 Proteolytic regulation of ENaC. The ENaC  heterotetramer is thought to be assembled initially in the endoplasmic reticulum. The N‐terminal and C‐terminal ends of each subunit reside in the cytosol. (Step 1) Portions of the extracellular domains of ‐ and ‐subunits are nicked by the transmembrane peptidase furin, probably in the lumen of trans‐Golgi network. These intracellular processing events may increase ion transport activity when ENaC is inserted into the plasma membrane (Step 2). As shown in Step 3, trypsin‐family ‘‘CAPs’’ upregulate Naþ transport via ENaC. These interactions probably are extracellular, as depicted. Known ENaC‐ activating peptidases include the type I transmembrane peptidase prostasin, shown here attached to the plasma membrane via a GPI lipid anchor. Certain type II transmembrane peptidases, including TMPRSS3 shown here, also activate ENaC. In addition, some free trypsin‐like serine peptidases, notably trypsin itself, can increase ENaC‐mediated Naþ transport. The target of these peptidases is not known; it may be ENaC or a protein that regulates ENaC. The amount of functional ENaC on the cell surface can be decreased by endocytic uptake (Step 4 ). A portion of endocytosed ENaC may be subject to further processing and recycled to the membrane (Step 5). Otherwise, ENaC in endosomes is likely to be ubiquinated, thereby tagging it for denaturation and total destruction by the peptidases and other proteins associated with the cytosolic proteasome (Step 6).

by one or more extracellular or plasma membrane‐associated trypsin‐like peptidases, as further reviewed below. Not all channels reaching the plasma membrane have been proteolytically processed (Hughey et al., 2004b), suggesting that there is a population of channels available for proteolytic activation at the cell surface. Presently, there is no direct evidence that extracellular serine peptidases increase Naþ‐transporting activity at the cell surface by cleaving ENaC at or near the unprocessed furin‐sensitive sites. However, combinatorial peptide substrate profiling of one of the potential

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activators, prostasin, does suggest the potential to do so (Shipway et al., 2004). Although Naþ transport across epithelia can be increased by ‘‘exocytotic’’ insertion of ENaC from an intracellular, subapical pool [some of which may be recycled (Butterworth et al., 2005)], this does not seem to be the mechanism by which extracellular serine peptidases increase ENaC activity (Caldwell et al., 2004). An alternative fate of ENaC endocytosed from the cell surface is ubiquitination by one or more ubiquitin ligases and subsequent translocation to the cytosol followed by destruction by the proteasome. This is an important means of regulating surface expression of ENaC and the magnitude of transepithelial transport of Naþ, as revealed by the hypertension and volume expansion in Liddle’s syndrome, which is associated with genetic defects in parts of ENaC involved in binding to Nedd4 and related ubiquitin ligases (Shimkets et al., 1994).

C. CAPs in Mammalian Epithelia A number of peptidases are candidate endogenous activators of ENaC in mammalian epithelia. Some features of those peptidases for which there is biochemical or other functional support are listed in Table I. The mammalian peptidase that is the most likely orthologue of frog CAP1 is CAP1/prostasin. Like Xenopus CAP, mammalian prostasins are known or predicted to be type I tryptic serine peptidases with a C‐terminal membrane anchor (Verghese et al., 2004; Yu et al., 1995). Furthermore, phylogenetic analysis suggests that prostasins are Xenopus CAP1’s closest mammalian relatives (Caughey et al., 2000; Verghese et al., 2004; Vuagniaux et al., 2002). Recombinant mouse CAP1/ prostasin, when coexpressed with rat ENaC subunits in frog oocytes, augments amiloride‐sensitive transport of Naþ (Vuagniaux et al., 2000, 2002). Patterns of expression of human and mouse prostasins are similar to those of ENaC itself, as one would expect if one protein were regulating the other (Donaldson et al., 2002; Verghese et al., 2004). Human prostasin is inhibited by aprotinin, which also inhibits ENaC‐mediated epithelial Naþ transport. Also, in rats, transgenic expression of human prostasin causes hypertension (Wang et al., 2003), as one would predict of an enzyme augmenting Naþ absorption. Direct evidence of a role for prostasin in modulating ENaC‐mediated Naþ currents was provided by results of siRNA‐mediated knockdown of prostasin expression in CF epithelial cells (Tong et al., 2004). Although the above collection of circumstantial and direct evidence builds a case favoring a prominent role for prostasin in regulating ENaC, such a role was not initially entertained for this enzyme, which was discovered as a soluble tryptic peptidase in secretions from the prostate gland (Yu et al., 1994), where it is highly expressed. Although human prostasin exists free in solution in seminal fluid, it is synthesized

Table I

Candidate Endogenous Serine Peptidase Activators of ENaC

Peptidase (Human) Prostasin

Transmembrane protease, serine 4 Matriptase

Transmembrane protease, serine 3 Furin

Aliases CAP1 (frog, mouse)

Gene PRSS8

Location

Membrane Anchor

Inhibitors

16p11.2

Type I; peptide or lipid/GPI

SPINT2/bikunin

CAP2 (mouse)

TMPRSS4

11q23.3

Type II; peptide

SPINT1/HAI‐1B PN1 ?

MT‐SP1 ST14 Epithin/CAP3 (mouse)

ST14

11q24

Type II; peptide

SPINT1/HAI‐1B

TMPRSS3

21q22.3

Type II; peptide

FUR

15q25.6

Type I; peptide

PACE

Serpin PI8

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initially with a C‐terminal peptide anchor. The role of the peptide anchor is unclear, but in several cell types the peptide anchor is exchanged intracellularly for a lipid anchor, glycosylphosphatidyl inositol (GPI) (Chen et al., 2001). In Xenopus CAP1, the GPI anchor consensus attachment motif and surface expression appear to be important for retention of ENaC stimulating activity (Vallet et al., 2002; Verghese et al., 2006). Intriguingly, mutation of Xenopus CAP1 to a catalytically incompetent form diminishes but does not abolish ENaC‐stimulating activity, raising the possibility that nonenzymatic binding events upregulate Naþ transport. Other candidate endogenous regulators of human ENaC include TMPRSS4 (transmembrane protease, serine 4; also known as CAP2), matriptase (also known as membrane‐type serine protease 1, ST14, and CAP3), and TMPRSS3. Although these peptidases, like Xenopus CAP1 and prostasin, feature trypsin‐ like catalytic domains, they diVer in the mode of membrane anchoring (Fig. 1), being type II rather than type I transmembrane peptidases. For CAP3/matriptase, the actual mode of membrane association of the mature enzyme is not clear. Nonetheless, both CAP2/TMPRSS4 and CAP3/matriptase are coexpressed with ENaC in some epithelia and possess the potential to activate ENaC, as revealed by coexpression of recombinant versions of the peptidases and ENaC in frog oocytes (Vuagniaux et al., 2002). Mutations of TMPRSS3 have an intriguing connection with congenital deafness (Guipponi et al., 2002; Lee et al., 2003). Although ENaC is expressed in the developing inner ear, a proposed link between defects in peptidase function and ENaC regulation presently is a matter of speculation.

D. Candidate Physiological Inhibitors of ENaC‐Activating Peptidases Some possible endogenous inhibitors of human CAPs are listed in Table I. Furin can be inhibited by the largely intracellular serpin PI8 (Dahlen et al., 1998). However, it is not known whether PI8 modulates furin processing of ENaC. Prostasin can be inactivated by the kunitz‐type inhibitors placental bikunin/SPINT2 [a recombinant version of which depresses primate ENaC‐ mediated airway epithelial cell transport in vitro (Bridges et al., 2001) and in vivo (Bowden et al., 2006)] and by hepatocyte growth factor activator (HGFA) inhibitor‐1B, also known as SPINT1 (Fan et al., 2005). Prostasin also can be inhibited by a serpin‐type inhibitor, protease nexin‐1 (Chen et al., 2004). Matriptase is likewise susceptible to SPINT1, but little is known about inhibitor susceptibilities of TMPRSS3 and TMPRSS4.

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III. ENaC Regulation by Peptidases: In Vivo Evidence A. Regulation of Alveolar Naþ and Water Transport by Serine Peptidases In mammals, the three ENaC subunits are expressed in Naþ‐transporting epithelia in the collecting ducts of the kidney, the mucosal surface of distal colon, ducts of salivary and sweat glands, and airway and alveolar epithelium (Duc et al., 1994). Surprisingly, ENaC subunits also are expressed in nontransporting epithelia, such as epidermis (Brouard et al., 1999). In distal nephron and colon, ENaC controls Naþ balance, extracellular fluid volume and blood pressure, and is regulated principally by aldosterone. In airways and alveoli, ENaC helps to control epithelial lining fluid volume and is regulated mostly by glucocorticosteroids. Although activation of ENaC by various serine peptidases is seen in aldosterone‐ and glucocorticosteroid‐ responsive epithelial cells, the in vivo importance of this activation remains to be fully established. Nonetheless, recent investigations suggest that plasma membrane‐associated or secreted ENaC‐activating peptidases are likely to be physiologically and pathologically significant in several organs. Active transcellular Naþ transport by alveolar epithelial cells is a driving force for reabsorption of fluid from the alveolar space (accounting for the lung’s remarkable ability to remove alveolar fluid at the time of birth) and is the main mechanism for resolution of alveolar edema (Basset et al., 1987a, b; Matthay et al., 1982, 2002). ENaC in the apical membrane of types I and II alveolar epithelial cells is rate‐limiting for Naþ absorption (Johnson et al., 2002). Indeed, newborn mice with inactivated ‐ENaC develop respiratory distress and die within 40 h of birth from failure to clear lungs of fluid (Hummler et al., 1996). In fetal and adult alveolar epithelium, ENaC is regulated by glucocorticosteroids, which increase ENaC expression by transcriptional and posttranscriptional mechanisms. Alveolar ENaC also is regulated by ‐adrenergic agonists, which increase the number of Naþ channels at the cell surface and the probability of being open (Matthay et al., 2002). Recent emergence of the concept of autocrine or paracrine regulation of ENaC by extracellular serine peptidases owes a great deal to recent studies of Naþ and water transport across alveolar epithelium. The potential role of serine peptidases in regulating ENaC was examined in rodent lung by measuring Naþ‐driven alveolar fluid clearance (AFC) in the presence of broad‐spectrum inhibitors or exogenous proteases, such as trypsin. Surprisingly, studies in a rat fluid‐filled lung model (Swystun et al., 2005) showed that addition of aprotinin to the instillate did not modify Naþ‐driven AFC measured over 30 min. An in situ nonventilated mouse lung model (Planes et al., 2005) also failed to find an acute inhibitory eVect of intra‐alveolar

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aprotinin on AFC measured over 15 min. Aprotinin’s failure to change baseline AFC in these models, although it inhibits ENaC‐mediated alveolar epithelial cell Naþ transport in vitro, has several potential explanations, including the presence of endogenous bikunin, which is a native inhibitor of prostasin expressed in lung and detected in bronchial mucus. However, this explanation seems unlikely since baseline AFC was not increased by excess trypsin, which should circumvent potential AFC inhibition by endogenous inhibitors. Considering the long inhibitor incubation time (
75 min) needed to inhibit ENaC maximally in vitro, these shorter term measurements may be inappropriate for detecting delayed inhibition by aprotinin in vivo. In in situ mouse lung, aprotinin also had the interesting eVect of abolishing terbutaline‐stimulated AFC, consistent with in vitro findings in rat alveolar epithelial cells (Planes et al., 2005). This finding suggested that serine peptidase‐mediated activation of ENaC optimizes in vivo eYcacy of 2‐adrenergic agonists, which markedly increase mammalian alveolar Naþ transport and fluid clearance in vivo (Matthay et al., 2002). Among several serine peptidases with the potential to regulate Naþ transport across murine alveolar epithelium, CAP1/prostasin is a prime candidate inasmuch as mRNA and protein corresponding to this peptidase is abundant in extracts of cultured or freshly isolated alveolar epithelial type II cells and in sections of alveolar epithelium (Planes et al., 2005; Verghese et al., 2004). Prostasin is expressed at the apical cell surface of cultured rat type II cells (Verghese et al., 2004). In lung and kidney epithelial cells, prostasin is shed by endogenous GPI‐specific phospholipase D1 (Verghese et al., 2006). In mouse lung, mCAP1/prostasin is secreted into epithelial lining fluid. The presence of mCAP1/prostasin mRNA and protein in alveolar macrophages (Planes et al., 2005) suggests that sources other than epithelial cells may contribute to prostasin in epithelial lining fluid. Whatever the cells of origin, if secreted CAP1/ prostasin is enzymatically active, as is true of human prostasin in seminal fluid (Chen et al., 2001), then it may act not only as an autocrine but also a paracrine modulator of Naþ absorption—able, for instance, to activate ENaC in type I cells, which cover most of the alveolar surface. In addition to CAP1/prostasin, CAP2 (mouse orthologue of human transmembrane serine protease TMPRSS4) and CAP3 (mouse orthologue of human matriptase MT‐SP1) are expressed, at least at the mRNA level, in mouse alveolar epithelial cells but not in alveolar macrophages (Planes et al., 2005). CAP2 and CAP3 potentially could regulate ENaC independently or via an activation cascade involving CAP1/prostasin (Rossier, 2004). Other serine peptidases secreted into alveolar fluid by epithelial and inflammatory cells also could modulate ENaC activity and alveolar Naþ and water transport in some conditions. In this connection, a soluble, trypsin‐like protease (20–28 kDa on zymograms) in rat lung bronchoalveolar lavage fluid (Swystun et al., 2005) may be important. This peptidase was proposed to

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facilitate lung liquid clearance because intra‐alveolar addition of soybean trypsin inhibitor or 1‐antitrypsin decreased Naþ‐driven AFC. Furthermore, addition of an excess of trypsin abolished the inhibition. The role of nonepithelial serine peptidases in alveolar Naþ transport remains to be established, but the concept that pathologically activated alveolar inflammatory cells release paracrine regulators of ENaC deserves further investigation.

B. Regulation of Colonic Prostasin Expression by Aldosterone and Dietary Naþ ENaC genes have been cloned from the distal colon of Naþ‐deprived rats (Canessa et al., 1993, 1994). Transcripts and protein corresponding to the three ENaC subunits are expressed by surface epithelial cells of the distal colon, but not by crypt cells (Duc et al., 1994). Surface epithelium of the distal colon is typically responsive to aldosterone, which fine‐tunes dietary absorption of Naþ. Hyperaldosteronism arising from depletion of dietary Naþ or infusion of aldosterone increases amiloride‐sensitive Naþ transport and ‐ and ‐ENaC mRNA expression in distal colon (Greig et al., 2002; Halevy et al., 1986). Several serine peptidases shown to activate ENaC in vitro also are expressed in the gastrointestinal tract, with a tissue distribution broader than that of ENaC itself. CAP1/prostasin mRNA is present in stomach and colon in rats and in stomach, small intestine and distal colon in mice (Adachi et al., 2001; Vuagniaux et al., 2000). Mouse CAP2 and CAP3 mRNAs also are present in mouse stomach, small intestine, and colon (Vuagniaux et al., 2002). Because CAPs are coexpressed with ENaC in distal colon, they may influence Naþ reabsorption by modulating ENaC activity and may modulate physiological responses to aldosterone in vivo. Consequences of altering plasma levels of aldosterone include reported changes in prostasin and ENaC mRNA expression in colonic epithelium (Fukushima et al., 2004). In these studies, adult rats were given normal or Naþ‐depleted diets, and continuous infusion of vehicle or aldosterone for 2 or 4 weeks. Prostasin and ENaC subunit mRNAs in extracts of colonic epithelial cells were quantitated by PCR or mRNA blot analysis. Dietary Naþ depletion and aldosterone infusion both dramatically increased plasma levels of aldosterone, accompanied by a marked increase in arginine vasopressin. Expression of ‐ENaC mRNA was detected in epithelial cells from proximal and distal colon under basal conditions, but was induced at 1, 2, and 4 weeks in both models only in the distal colon. ‐ENaC mRNA was absent or barely detected under basal conditions, but was also clearly enhanced in distal colon in the context of prolonged hyperaldosteronism (2 and 4 weeks).

‐ENaC mRNA, quantified by real time RT‐PCR because of low basal expression, also increased at 2 weeks in distal (but not proximal) colonic

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epithelium from Na ‐depleted and aldosterone‐infused rats. Prostasin mRNA increased
3‐fold at 2 and 4 weeks in distal colonic epithelial cells from Naþ‐ depleted and aldosterone‐infused rats. Prostasin mRNA tended to increase in proximal colonic epithelial cells from aldosterone‐infused rats, but not significantly. These data show that a rise in plasma levels of aldosterone enhances prostasin and ENaC subunit mRNA expression in distal colonic epithelium in vivo. It remains to be established whether prostasin and ENaC are coexpressed in the same colonic cells and whether prostasin induction in the aldosterone response is physiologically important. An interesting question raised by the above study was the potential role of arginine‐vasopressin on prostasin expression in the distal colon, since the stimuli used in both models dramatically increased plasma levels of arginine‐ vasopressin, together with the expected increase in aldosterone. The authors (Fukushima et al., 2004) evaluated the eVect of arginine‐vasopressin on prostasin expression in vitro by exposing mineralocorticoid receptor‐expressing T84 colon cancer cells to aldosterone or arginine‐vasopressin alone, or to the combination of aldosterone and arginine‐vasopressin. Surprisingly, although neither aldosterone nor arginine‐vasopressin alone had much eVect, together they markedly stimulated expression of prostasin mRNA, suggesting synergism. Additional studies are needed to evaluate whether arginine‐vasopressin exerts eVects on ENaC or prostasin complementary to those of aldosterone, as reported for ENaC in the kidney (Ecelbarger et al., 2000).

C. Regulation of Prostasin by Aldosterone in the Kidney Although ENaC‐activating serine peptidases such as CAP1/prostasin, CAP2, or CAP3/MT‐SP1 matriptase were initially cloned from kidney cell lines (Vallet et al., 1997; Vuagniaux et al., 2000, 2002), direct evidence that these peptidases regulate renal Naþ transport in vivo is lacking. However, reports that prostasin expression in rat and human kidney is regulated by aldosterone suggest that CAPs could participate in stimulation of Naþ transport by aldosterone (Narikiyo et al., 2002; Olivieri et al., 2005). Fine regulation of Naþ excretion in the distal nephron occurs principally in the collecting duct and is mediated chiefly by aldosterone, via eVects on ENaC. In kidney, the ENaC subunits are specifically expressed in epithelial cells from the aldosterone‐sensitive distal nephron (ASDN) (Duc et al., 1994; Masilamani et al., 1999), where ENaC expression and activity at the cell surface is generally rate‐limiting. The mechanism whereby aldosterone stimulates ENaC activity and Naþ transport in distal nephron is complex, tissue‐ specific, and not completely understood. Aldosterone eVects are mainly mediated via mineralocorticoid receptor‐induced changes in expression of

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various gene products, such as serum glucocorticoid‐induced kinase 1, which are elements of a network that controls function of the Naþ transport machinery and expression of ENaC itself (LoYng et al., 2001). In rats, elevation of aldosterone levels in plasma rapidly and selectively increased the abundance of ENaC mRNA and protein along the entire ASDN, and induced apical translocation of ENaC subunits in the initial portion of the ASDN (Masilamani et al., 1999). Interestingly, aldosterone also induced a shift in size of ‐ENaC from 85 to 70 kDa. The authors proposed that this shift, the functional consequences of which are not known, could result from cleavage of the extracellular loop by peptidases. Several studies using kidney epithelial cell lines showed that exposure to apical aprotinin induced a substantial decrease in ENaC‐mediated transepithelial Naþ transport. CAP1/prostasin (and CAP2 and CAP3), coexpressed with ENaC in these cells, was proposed to be targets of aprotinin and physiological activators of ENaC (Liu et al., 2002; Vallet et al., 1997; Vuagniaux et al., 2000, 2002). A few studies subsequently evaluated aldosterone’s eVects on prostasin expression in vitro, with conflicting results. One set of studies (Liu et al., 2002) failed to detect an eVect of aldosterone treatment on the magnitude either of aprotinin‐induced decrease in Naþ current or of prostasin/CAP1 mRNA expression in M‐1 cortical collecting duct cells, even though cells were exposed for 48 h to a high concentration (1 mM) of aldosterone. In contrast, exposing the same cell line to the same hormone concentration for 6–48 h, other investigators (Narikiyo et al., 2002) found that aldosterone progressively increased CAP1/prostasin mRNA and protein expression. Surprisingly, prostasin was detected neither in the cytosolic nor in the membrane fraction; rather, it was found in cell culture medium in a soluble, secreted form, which increased following cell exposure to aldosterone. Prostasin induction was associated with an increase in the magnitude of aprotinin inhibition of amiloride‐sensitive 22Na uptake, suggesting that prostasin in part mediates stimulation of Naþ transport by aldosterone. To assess the in vivo relevance of the findings, these investigators exposed rats for 7 days to continuous aldosterone (100 g/100 g body weight/day) or vehicle infusion, before evaluating renal expression and urinary excretion of prostasin (Narikiyo et al., 2002). Rats treated with aldosterone developed systemic hypertension and metabolic alkalosis. Prostasin protein could not be detected in cytosolic or membrane fractions of renal cortex or medulla with or without aldosterone. However, soluble prostasin was detected in rat urine under basal conditions, suggesting massive secretion of the protein by kidney tubular cells. Indeed, urinary excretion of prostasin increased fourfold in aldosterone‐treated rats. This study also examined urinary prostasin excretion in patients with primary aldosteronism before and 7 days after adrenalectomy, and in patients undergoing surgery for another disease. Interestingly, urinary prostasin excretion was high

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in subjects with primary aldosteronism as compared with controls, and adrenalectomy normalized plasma aldosterone concentration and decreased urinary prostasin excretion. Of note, urinary prostasin excretion before and after adrenalectomy correlated well with the urinary Naþ/Kþ ratio, reflecting the eVect of aldosterone on kidney cells. These data suggesting a relationship between urinary prostasin and aldosterone production were confirmed recently in normotensive and hypertensive subjects with inappropriate aldosterone secretion. This study first evaluated the changes in urinary prostasin in healthy normotensive individuals after spironolactone‐induced blockade of mineralocorticoid receptor or acute volume expansion (Olivieri et al., 2005). The investigators detected prostasin in urine from all normotensive subjects tested, regardless of gender and dietary Naþ intake. Treatment with spironolactone induced an expected 30% increase in urinary Naþ/Kþ ratio and at the same time decreased urinary prostasin in normotensive subjects in whom the renin/aldosterone axis was activated by a low Naþ intake, but was ineVective in normotensive subjects with high Naþ intake. Saline infusion decreased both plasma aldosterone levels and urinary prostasin excretion in all normotensive subjects investigated. In contrast, urinary prostasin paradoxically increased after saline infusion in hypertensive patients with primary aldosteronism. Two‐dimensional immunostaining revealed a complex pattern of expression with up to seven distinct bands with diVerent glycosylation patterns. Some of these isoforms were paradoxically overexpressed following volume expansion in patients with primary aldosteronism. The authors concluded that some prostasin is excreted independently of Naþ balance and plasma levels of aldosterone, and that additional prostasin is excreted under the influence of aldosterone. Taken together, the above studies strongly support the concept that prostasin expression and urinary excretion are regulated by aldosterone. Although urinary prostasin is a possible marker of ENaC activation in the kidney, several issues relating to its roles and importance in vivo remain to be addressed, starting with its cellular origin. In this regard, it should be noted that expression of prostasin, unlike that of ENaC, is not restricted to the distal nephron, for prostasin/CAP1 mRNA is highly expressed in mouse proximal tubule, a segment of the nephron that is aldosterone‐unresponsive (Vuagniaux et al., 2000). This ‘‘proximal’’ source of urinary prostasin may contribute to aldosterone‐independent basal secretion. It also remains to be established if any of the several soluble forms of urinary prostasin are enzymatically active. If this is so, then urinary prostasin may exert a paracrine eVect on renal tubular cells and activate apically expressed ENaC. The functional importance of aldosterone‐induced prostasin expression to renal Naþ reabsorption also remains to be answered. Tissue‐specific inactivation of prostasin/CAP1 gene should help to answer these questions.

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D. Potential Role of Prostasin in Regulating Aldosterone Production and Hypertension The physiological links between prostasin, aldosterone, and ENaC function might be more complex than prostasin being an aldosterone‐induced protein that activates ENaC. Indeed, a study suggested that prostasin could influence the processing of aldosterone itself (Wang et al., 2003) based on eVects of adenovirus‐mediated human prostasin gene delivery on blood pressure and electrolyte homeostasis in rats. Animals receiving a single injection of adenovirus achieved high‐level expression of human prostasin mRNA in adrenal gland and liver and low‐level expression in kidney, lung, heart, and aorta. Immunoreactive prostasin was detected in blood and urine for almost 3 weeks. Prostasin gene transfer induced a marked and prolonged (3–4 week) increase in blood pressure. Interestingly, this increase was preceded by an increase in plasma aldosterone and a moderate decrease in plasma renin. Because human prostasin mRNA was expressed in adrenal gland (the principal site of aldosterone generation), as well as in vessel walls [where aldosterone also may be produced (Takeda et al., 1995)], the authors suggested that prostasin could be involved in aldosterone maturation, processing, or secretion. In this regard, it is uncertain whether endogenous prostasin normally is expressed in rat adrenal gland and vessel walls. Previous studies failed to identify prostasin mRNA and protein expression in rat and human aorta, vein, and artery (Yu et al., 1994, 1995). It is possible that prostasin‐induced aldosterone production in this study is a nonphysiological consequence of unregulated, ectopic production of human prostasin. The mechanism whereby prostasin gene transfer‐induced aldosterone production elevated systemic blood pressure also remains to be fully explained. Although plasma aldosterone levels increased in rats receiving prostasin‐ adenoviral construct, urinary Naþ and Kþ excretion did not change at Day 3, 7, or 21 postinjection, and body weight curves did not diVer from those of control rats. Paradoxically, increased urinary Naþ excretion with concomitantly decreased Kþ excretion was seen at Day 14 in treated animals. The authors proposed that this could be explained by escape from the aldosterone eVect, perhaps mediated by upregulation of the renal kallikrein–kinin system. Possibly, the change in Naþ balance is due to eVects of elevated aldosterone on vascular resistance. Whatever the mechanism, these intriguing data emphasize the complexity of interactions between prostasin and aldosterone.

E. Role of CAPs in Maintaining the Epidermal Permeability Barrier In humans and rodents, the skin is thought not to be involved in ion or fluid absorption, but surprisingly, ENaC is expressed in the epidermis, the outermost layer of the skin (Brouard et al., 1999; Roudier‐Pujol et al., 1996). The epidermis

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is a self‐renewing epithelium, consisting mostly of keratinocytes. Basal keratinocytes in the inner layer of epidermis proliferate and migrate upward while terminally diVerentiating, leading to the generation of flattened, anucleated corneocytes, which are interconnected by desmosomes and embedded in a lipid matrix. This structure constitutes the watertight stratum corneum, which prevents excessive loss of body fluids, and protects against mechanical, chemical, or biological insults. Human keratinocytes express the three ENaC subunits (Brouard et al., 1999). However, ENaC’s role in the skin is unclear. Previous in vitro studies found that expression of ENaC, especially of ‐subunit, increased dramatically during cell growth and diVerentiation of human cultured keratinocytes, suggesting that ENaC may play a role in terminal diVerentiation (Brouard et al., 1999; Oda et al., 1999). Patch‐clamp experiments in keratinocytes showed a channel similar to ENaC in a small proportion or recorded cells, but no transepithelial Naþ transport was identified. Interestingly, there is an epidermal Ca2þ gradient, with more Ca2þ in upper than lower epidermis. This gradient appears to regulate keratinocyte growth and diVerentiation (Dotto, 1999). The fact that ENaC subunit expression exhibits a similar vertical gradient suggests a possible interrelationship between Naþ and Ca2þ flux in these cells (Guitard et al., 2004). Whatever the mechanism, ENaC’s importance in skin diVerentiation and development was highlighted by findings in ‐ENaC‐deficient mice, in which newborns exhibited epidermal thickening, disordered diVerentiation, premature lipid secretion in the upper epidermis, and abnormal keratohyalin granules (Mauro et al., 2002). Numerous peptidases are expressed in the epidermis. Some of them such as profilaggrin endopeptidase 1 (Resing et al., 1995), calpain‐1, cathepsin L (Benavides et al., 2002), or stratum corneum chymotryptic enzyme (Ekholm and Egelrud, 1998) are implicated in processing and maturation of profilaggrin to filaggrin filaments, and in epidermal diVerentiation and desquamation. Among the peptidases expressed in mouse skin are ENaC‐activating CAP1/prostasin (Vuagniaux et al., 2000) and CAP3/MT‐SP1 matriptase (Vuagniaux et al., 2002). CAP1/prostasin was detected in the top layers of the stratum granulosum and at the transition between stratum granulosum and stratum corneum (Leyvraz et al., 2005). In vitro, CAP1/prostasin and ENaC are coexpressed in keratinocytes with similar patterns of expression, that is, with mRNA levels increasing gradually as cells diVerentiate. Interestingly, two recent studies revealed that inactivation of either CAP3/MT‐ SP1 matriptase or CAP1/prostasin in mice induced a lethal phenotype with deficient epidermal barrier function. In the first study, introduction of a null mutation into the CAP3/MT‐SP1 matriptase gene (List et al., 2002) results in newborns with dry, red, shiny, and wrinkled skin. Epidermal barrier function was compromised, as demonstrated by increased toluidine blue penetration and transepidermal water loss, which led to rapid and fatal dehydration. In the second study, mice lacking epidermal CAP1/prostasin

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(Leyvraz et al., 2005) were generated by crossing mice with a conditional ‘‘floxed’’ CAP1/prostasin allele with mice expressing Cre recombinase controlled by the keratin 14 promoter, which is active in basal keratinocytes. Newborn pups lacking epidermal CAP1/prostasin had low body weight and died within 60 h of birth. Their stratum corneum was severely malformed with abnormal lipid composition, corneocyte morphogenesis, and profilaggrin processing. As in CAP3/MT‐SP1 matriptase‐deficient pups, early death was due to impaired skin barrier function and dehydration. Among the most interesting findings was the complete absence of occludin in tight junctions of CAP1/prostasin‐deficient epidermis. This was associated with increased permeability of tight junctions, as evidenced by failure to prevent diVusion of the subcutaneously injected biotin (600 kDa) to the skin surface. This implied involvement of CAP1/prostasin in maintaining tight junction integrity is consistent with prior studies in which exposure of renal and alveolar epithelial cell monolayers to apical aprotinin (an inhibitor of CAP1/prostasin) reduced transepithelial resistance (Liu et al., 2002; Planes et al., 2005), whereas treatment with exogenous trypsin increased it (Swystun et al., 2005). Collectively, these data strongly suggest that CAP1/prostasin can modulate tight junctions and paracellular permeability in various epithelia, possibly through regulation of occludin expression or function. It is possible that occludin is cleaved by prostasin, though it lacks preferred polybasic cleavage sites (Leyvraz et al., 2005). Another issue to be resolved is the relationship between ENaC and CAP3/MT‐SP1 matriptase or CAP1/prostasin in the skin. Presently, there is no evidence that ENaC dysfunction plays a role in skin abnormalities observed in CAP1/prostasin‐ or CAP3/MT‐SP1 matriptase‐deficient mice. Nonetheless, these studies suggest a potential role of CAPs in regulating paracellular permeability beyond an eVect on function of ENaC.

F. ENaC Dysregulation in the Pathophysiology of Cystic Fibrosis: CAPs as Potential Drug Targets The mucosa of proximal and distal airways is covered by airway surface liquid (ASL), which is a first line of defense against inhaled pathogens. The ASL is composed of the watery periciliary liquid layer (PCL) surrounding the cilia on the apical face of ciliated cells, and of the mucin‐rich mucous layer, which resides atop of the PCL. Maintenance of proper ASL volume/depth and ionic composition is critical to optimize ciliary beating and mucus clearance (Matsui et al., 1998). Fine regulation of ASL volume is mainly achieved by active transepithelial transport of salt, leading to liquid absorption or secretion. Airway epithelial cells have the capacity to actively reabsorb Naþ and to secrete Cl (Boucher, 2003;

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Tarran et al., 2001). In the basal state, the dominant ion transport across the airway epithelium is amiloride‐sensitive Naþ absorption through ENaC, which induces passive Cl absorption. ENaC subunits are expressed throughout the respiratory tract (with a large predominance of ‐ and ‐ over ‐subunits in proximal airways), and are upregulated by glucocorticoids (Farman et al., 1997; Pitkanen et al., 2001; Renard et al., 1995). Active Cl secretion also can occur following stimulation by agents such as cAMP agonists and ATP. Cl secretion mostly occurs through the apical Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), and possibly through other channels such as the Ca2þ‐ activated Cl channel. CFTR is a protein kinase A‐activated Cl channel in the apical membrane of secretory epithelia, but also regulates other ion transport systems. In particular, CFTR influences ENaC activity. In various epithelial cells and overexpressing recombinant cells, activation of CFTR inhibits ENaC currents, most likely by increasing intracellular concentration of Cl (Konig et al., 2001; Mall et al., 1998). Attention has focused on airway epithelial ion transport processes because CF is caused by mutations in the CFTR gene (Rowe et al., 2005). CF is characterized by a generalized exocrine dysfunction, but lung disease is the leading cause of death. Respiratory manifestations of CF include airway obstruction by hyperviscous secretions leading to iterative bacterial infections, chronic airway inflammation, and bronchiectasis. Although it is clear that CF lung disease reflects a defect in the innate defense of airway surfaces against bacterial infection, the basis of this defect and its relationship with CFTR dysfunction are not well understood. One of the prevailing explanations, called the ‘‘low volume hypothesis,’’ emphasizes the role of the CFTR defect in both Cl secretion and ENaC regulation. According to this hypothesis, absence of CFTR leads to lack of Cl (and water) secretion, but also to enhanced Naþ (and water) absorption because tonic ENaC inhibition by CFTR is lost. This Naþ hyperabsorption decreases ASL volume, impairs clearance of mucus, and promotes accumulation of hyperviscous mucus, thereby favoring bacterial infection (Knowles et al., 1986; Matsui et al., 1998, 2000). Recent animal and human studies underscore the potential importance of ENaC in the pathogenesis of CF. Increased airway epithelial Naþ absorption caused by airway‐specific overexpression of ‐ENaC induced a severe CF‐like lung disease in mice, including mucus obstruction, goblet cell metaplasia, neutrophilic inflammation, and decreased bacterial clearance (Mall et al., 2004). ENaC hyperactivity in the airways was associated with ASL volume depletion, increased mucus concentration, delayed mucus transport, and mucus adhesion to cell surfaces. Furthermore, an association study searching for genetic modifiers identified SCNN1B and SCNN1G (genes encoding ‐ and ‐ENaC, respectively) as potential modulators of CF severity (Stanke et al., 2006). Pharmacological inhibitors of ENaC are being actively investigated as treatment of CF lung disease (Hirsh et al., 2004). Physiological activators of ENaC in the airways—including CAPs—also can be regarded as candidates

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for therapeutic inhibition. Prostasin is abundantly expressed in normal and CF nasal epithelial cells (Bridges et al., 2001; Donaldson et al., 2002; Tong et al., 2004), and silencing prostasin expression in CF cells using siRNA decreases ENaC‐mediated Naþ transport by 75% (Tong et al., 2004). As noted, the kunitz‐type inhibitor aprotinin markedly decreased ENaC‐mediated Naþ transport across primary human nasal and bronchial epithelial cells from individuals with and without CF (Bridges et al., 2001; Donaldson et al., 2002). Interestingly, Bridges and colleagues reported that BAY 39‐9437, a recombinant inhibitor derived from the human kunitz‐type inhibitor bikunin, also inhibited ENaC in normal and CF bronchial epithelial cells. In contrast, two broad‐spectrum non‐kunitz inhibitors (soybean trypsin inhibitor and 1‐antitrypsin) were ineVective (Bridges et al., 2001). It is therefore tempting to conclude that kunitz‐type inhibitors targeting prostasin‐like peptidases possess therapeutic potential for reversing Naþ hyperabsorption in CF. In this regard, the report that recombinant‐truncated bikunin/SPINT2 produces long‐ lasting changes in nasal potential diVerence in monkeys in vivo is encouraging (Bowden et al., 2006). The eVects of chronic inflammation on expression and activity of prostasin‐like peptidases in the airways are unknown. Of note, exposure of mouse cortical collecting duct M‐1 cells to transforming growth factor (TGF)‐ 1 markedly downregulated expression of CAP1/prostasin (Tuyen do et al., 2005). This raised the possibility that proinflammatory cytokines released in CF airways influence expression and activity of prostasin or other epithelial CAPs. Also, there are most likely some physiological endogenous inhibitors of prostasin‐like proteases (such as bikunin and HGFA inhibitor‐1B) in ASL. Their regulation and potential role under inflammatory conditions are currently unknown. Finally, epithelial CAPs may not be the sole activators of ENaC in the airways; other peptidases released in bronchial mucus by inflammatory cells could also influence ENaC activity under pathological conditions. For instance, human neutrophil elastase (hNE), a serine protease, activates ENaC and transepithelial Naþ transport in a human bronchial cell line (Caldwell et al., 2005). Under normal conditions, extracellular airway hNE is inactive and bound to an endogenous inhibitor (e.g., 1‐antitrypsin or secretory leukoprotease inhibitor), but can be present and active in CF airways because the large number of disintegrating neutrophils overwhelms the antiprotease shield. Thus, hNE could participate in ENaC activation in CF airways. Accordingly, development of pharmacological inhibitors of prostasin‐like proteases and hNE may be useful for treatment of CF.

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Advances in Defining Regulators of Cementum Development and Periodontal Regeneration Brian L. Foster,* Tracy E. Popowics,{ Hanson K. Fong,{ and Martha J. Somerman*,{ *Department of Periodontics, School of Dentistry University of Washington, Seattle, Washington 98195 { Department of Oral Biology, School of Dentistry University of Washington, Seattle, Washington 98195 { Department of Materials Science and Engineering University of Washington, Seattle, Washington 98195

I. Introduction II. Question 1. What Are the Unknowns That Must Be Considered in Order to Replicate the Enamel (Crown) and How Do the Proteins Involved in Crown Development Relate to Root Development? A. Enamel Structure B. Enamel Biomineralization: Role of Proteins C. Future Prospects for Enamel Regeneration III. Question 2. What Do We Know About the Cells Required for Periodontal Development and Regeneration? A. Developmental Cells B. Derivation of Cementum: Competing Theories of Cementoblast Origin C. DiVerences Between Cementoblasts and Osteoblasts D. Tooth Stem Cell Populations IV. Question 3. What Genes and Associated Proteins Are Important for Root/Periodontal Tissue Formation? A. Factors Associated with the Putative Epithelial Niche (HERS and ERM) and Surrounding Mesenchyme B. Bone Morphogenetic Proteins C. Periostin and Nuclear Factor I‐C/CAAT Box Transcription Factor D. Regulators of Phosphate and Pyrophosphate Metabolism E. Factors Known to Regulate Osteoprogenitor Cells and Osteoblasts F. Emerging and Other Factors to Consider V. Conclusions and Future Directions Acknowledgments References

Substantial advancements have been made in defining the cells and molecular signals that guide tooth crown morphogenesis and development. As a result, very encouraging progress has been made in regenerating crown tissues by using Current Topics in Developmental Biology, Vol. 78 Copyright 2007, Elsevier Inc. All rights reserved.

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dental stem cells and recombining epithelial and mesenchymal tissues of specific developmental ages. To date, attempts to regenerate a complete tooth, including the critical periodontal tissues of the tooth root, have not been successful. This may be in part due to a lesser degree of understanding of the events leading to the initiation and development of root and periodontal tissues. Controversies still exist regarding the formation of periodontal tissues, including the origins and contributions of cells, the cues that direct root development, and the potential of these factors to direct regeneration of periodontal tissues when they are lost to disease. In recent years, great strides have been made in beginning to identify and characterize factors contributing to formation of the root and surrounding tissues, that is, cementum, periodontal ligament, and alveolar bone. This review focuses on the most exciting and important developments over the last 5 years toward defining the regulators of tooth root and periodontal tissue development, with special focus on cementogenesis and the potential for applying this knowledge toward developing regenerative therapies. Cells, genes, and proteins regulating root development are reviewed in a question‐ answer format in order to highlight areas of progress as well as areas of remaining uncertainty that warrant further study. ß 2007, Elsevier Inc.

I. Introduction During the last decade, we have gained substantial insights into the mechanisms and factors controlling formation of many organs and tissues and with this, new ideas on how to regenerate tissues lost as a consequence of pathologies, injuries, and genetic disorders. These insights, based on new technologies and on the exponential growth in defining the factors/genes/proteins regulating tissue and organ development, have allowed us to enjoy more rapid discoveries than in the past. Technological advances have resulted in increased eVorts to develop improvements in existing therapies targeted at replacement of lost tissues/organs/ body parts. An area of focus has been the oral cavity, with improvements seen in (1) materials used to restore decayed, damaged tooth structure; (2) prosthetic devices to replace missing teeth—full dentures, partial dentures, bridges, and implants; and (3) materials/agents used to regenerate periodontal tissues—for example, root cementum, periodontal ligament attachment, and alveolar bone. In fact, with regard to developing clinical products that promote and/or protect against bone loss, some of the newer products (on the market within the last 3 years) were first appreciated for their role in regulating key events during formation and diVerentiation of hard tissues, including teeth. These include bone morphogenetic protein [(rhBMP‐2): INFUSE; Medtronic Sofamor Danek, Minneapolis, MN)] approved for clinical use in open fracture of long bones, nonunions and vertebral arthrodesis, and parathyroid hormone (Fortio;

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teriparatide (rhDNA origin) injection contains human PTH (1‐34), Eli Lilly & Co.) given intermittently to promote bone formation in individuals with severe osteoporosis and in whom antiresorptive therapies have proven insuYcient. To date, attempts to regenerate a complete tooth—crown, root, PDL, bone—have not been successful; however, progress has been made in regenerating crown tissues. Because of the parallel of epithelial‐mesenchymal (E‐M) signaling in crown formation, that is, ameloblasts‐enamel; odontoblasts‐ dentin, with E‐M signaling in other tissues during development, rodent models of tooth crown development have been studied extensively, resulting in a wealth of information as to the cells/factors and events controlling crown development (Chai and Slavkin, 2003; Fong et al., 2005; ThesleV, 2003; ThesleV and Mikkola, 2002; Zhang et al., 2005). Yet, there is still more information needed in order to mimic enamel‐dental formation as a way to restore lost tissue structure. What is known and was first realized decades ago is that appropriately timed mixing of cells obtained from tooth epithelium with tooth mesenchyme simulates cell diVerentiation toward ameloblasts and odontoblasts with subsequent crown formation (the development of tooth germs in tissue culture, 1965; Duailibi et al., 2004; Harada et al., 1999; Huggins et al., 1934; Kollar and Baird, 1969, 1970a,b; Kollar and Fisher, 1980; Mina and Kollar, 1987; Nieminen et al., 1998; Ohazama et al., 2004b; Tucker and Sharpe, 2004; Young et al., 2002). In contrast, the events/factors leading to formation of the root and surrounding tissues, that is, cementum, alveolar bone and a functional periodontal ligament (PDL) are just beginning to unfold. This review focuses on the most exciting developments over the last 5 years toward defining the regulators of root development, that is, cementogenesis and the significance of this knowledge toward developing therapies targeted at regeneration of a whole tooth and surrounding support structures. We recognize that modulators of PDL and bone formation are key for root formation and thus at times address these tissues, but the emphasis for this review is on cementum. Further, based on the recent emphasis on defining the possible role of epithelial‐derived factors during root development, a discussion on the enamel‐associated factors produced by ameloblasts, and their known and putative roles in formation of enamel and cementum, are discussed. As the knowledge of factors that influence development of the periodontium increases in coming years, developing an eVective platform for the delivery of these known factors will become increasingly important for purposes of tissue regeneration. In the areas of drug delivery and tissue engineering, advances have been made in the development of materials that can serve as a vehicle to deliver proteins/genes/cells in vivo. Agents identified with regenerative potential may then be partnered with delivery systems to localize and regulate release of cells and factors at sites of repair/regeneration of lost periodontal structures. Much exciting work has been done to advance the design and fabrication of delivery systems, and several excellent reviews

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Foster et al. Cells Enamel (#2) biomineralization (#1)

Genes/ proteins (#3)

Development Healthy Delivery system

Diseased Figure 1 Progression of root development and regeneration. The tooth root develops as a result of complex interactions of cells, signals, and matrix proteins, now just beginning to be understood. The question‐answer format used in this review addresses recent progress in defining key modulators of root development, and defines areas warranting further study. Therapies targeted at regenerating the whole tooth will necessarily incorporate factors relating to crown development and possibilities for enamel regeneration (Question 1), as well as cells (Question 2) and genes/proteins (Question 3) that regulate periodontal development. Lastly, cells and factors to be used in regenerative therapies should be partnered with eVective delivery systems that serve as a scaVold for cells and/or function in controlled release of bioactive factors to the local area.

and primary publications may be consulted for detailed information on this work (Abukawa et al., 2006; Bartold et al., 2006b; Jin et al., 2003; Nakahara, 2006; Taba et al., 2005). A question‐answer format has been used to address progress in ascertaining the key modulators of root development over the past 5 years, as well as to recognize areas of remaining uncertainty that warrant further study. A model visualizing the progression, from the stage of initiation of root development and from a diseased periodontal state to a functional tooth is shown in Fig. 1 to visually demonstrate the questions being posed. 1. What are the unknowns that must be considered in order to replicate the enamel (crown) and how do the proteins involved in crown development relate to root development? 2. What is known about the cells required for development and regeneration of cementum? 3. What are the genes and associated proteins required for root development and regeneration?

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II. Question 1. What Are the Unknowns That Must Be Considered in Order to Replicate the Enamel (Crown) and How Do the Proteins Involved in Crown Development Relate to Root Development? Enamel is the hardest biological tissue in the body. Although the primary component of enamel, hydroxyapatite (HAP), does not compare favorably with most known structural ceramics in terms of mechanical properties, it exhibits remarkable durability. The key to enamel’s durability despite repeated attrition in the bacteria‐laden environment of the oral cavity lies in its intricate microstructure; hence, regenerative strategies with the aim of successful replication of the crown must involve not only the chemical makeup of enamel, but the structural makeup as well. Current materials engineering technology has yet to find a way to fabricate the complex 3‐D enamel structure. To complicate matters, mature enamel is a nonliving tissue, as the ameloblasts that synthesize enamel matrix are lost on tooth eruption. A key to enhancing progress toward regenerating enamel includes understanding the cellular and molecular mechanisms regulating formation of this tissue. The following discussion focuses on our current understanding of enamel structure as it relates to mechanical functions, and on the genes/proteins regulating enamel biomineralization. Also discussed are future approaches to consider for designing regenerative enamel.

A. Enamel Structure The organization of enamel can be imagined as a hierarchical structure, starting at the smallest scale with HAP crystals approximately 50‐nm wide. These crystals are bundled into a few micrometers wide which are referred to as enamel rods or prisms, representing the next scale of hierarchy. The interweaving of enamel rods builds the bulk of enamel tissue. The crystal rod/interrod organization has been investigated carefully, and beautiful, illustrative images can be found in textbooks such as Ten Cate’s Oral Histology (Nanci, 2003). Increasing evidence suggests that orientation and decussation of enamel rods are important properties for preserving the mechanical integrity of mature enamel (Marshall et al., 2001; Xu et al., 1998). For example, rod organization is important for preserving the overall enamel structural integrity by directing microcracks traveling through the dentin‐enamel junction (DEJ) into the dentin, where they are then arrested (Imbeni et al., 2005). Despite the extensive body of data characterizing the enamel structure, several questions remain regarding key structural details

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that must be elucidated in order to understand the development and regeneration potential for enamel. Two of these critical questions include: (1) What are the directional changes in enamel rods extending from the DEJ to enamel surface, and (2) How are interrods structurally related to enamel rods? Answering these questions is essential to achieve an adequate understanding of the mechanical properties of enamel, as well as to gain insight toward regeneration of a functional crown.

B. Enamel Biomineralization: Role of Proteins 1. Amelogenin Amelogenin is the most abundant and best characterized protein in developing enamel. The amino acid sequence of amelogenin protein is highly conserved across many species, suggesting physiologic relevance and common functional properties across species (Paine and Snead, 2005). Amelogenin’s eVect on enamel development has been aptly studied in both in vivo and in vitro systems. Several lines of evidence support proper self‐assembly of amelogenin proteins is essential for facilitating directional nucleation of hydroxyapatite minerals. Human phenotypes for the condition amelogenesis imperfecta (AI) demonstrate lack of or altered amelogenin, resulting in inferior enamel characterized by hypoplasticity or hypomineralization, and often associated with disorganized enamel rods (Gibson et al., 2001b, 2005; Wright et al., 2003). Likewise, a severe form of AI, similar to humans with amelogenin defects, was observed in amelogenin knockout (KO) mice (Gibson et al., 2001a). The current understanding of amelogenin’s role in enamel mineralization has come to light through characterization of enamel in situ and isolated recombinant amelogenin, in normal and defective forms. The first indication of amelogenin’s ability to self‐assemble and dictate mineral organization came from investigations focused on analyzing developing mouse enamel where arrays of nanospheres were observed to approximate the sides of needle‐like HAP crystallites (Moradian‐Oldak et al., 1995; Robinson et al., 1981). Later, atomic force microscopy (AFM) and dynamic light scattering measurements on M‐180 (mouse full‐length) amelogenin revealed that the protein assembled into 20 nm nanospheres (Moradian‐Oldak et al., 2000). Furthermore, dynamic light scattering measurements performed on M‐180 with altered C‐terminal and N‐terminal domains indicated disruption of self‐ assembly, resulting in smaller nanospheres with a wider size distribution (Moradian‐Oldak et al., 2000). These findings revealed that C‐ and N‐terminal domains were essential for proper amelogenin self‐assembly.

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When transgenic mice were developed to express the same altered C‐ or N‐ terminal domains of amelogenin, similar disruption in the nanosphere self‐ assembly was observed, resulting in disruption in crystal organization of the mineral phase during the secretory stage of enamel formation (Paine et al., 2001). The resulting mature enamel was found to be hypomineralized with disorganized rods, a direct eVect of altered C‐ and N‐terminal domains manifested in a disorganized mineral phase in the nucleation stage of enamel formation (Paine et al., 2001). Details of the interactions between amelogenin and mineral are still emerging, however the evidence to date indicates that amelogenin has a strong binding aYnity to HAP via the hydrophilic C‐terminal domain. Several investigators have demonstrated controlled HAP growth in the presence of amelogenin using in vitro systems (Beniash et al., 2005; Iijima et al., 2002). In one example, HAP crystals with a long ribbonlike morphology resulted from growth in the presence of amelogenin (Iijima et al., 2002). Another solution precipitation experiment showed formation of needle‐like HAP crystals when amelogenin was introduced into the system (Beniash et al., 2005). In both of these examples, the long axis of the crystals was the crystallographic [001] direction (normal to the crystallographic (001) plane), similar to that found in physiological enamel HAP, suggesting amelogenins bind to the crystal surface(s) perpendicular to the (001) plane, limiting the growth direction in [001] only. Additional binding studies by Hablitz et al. showed that when amelogenins were introduced to a composite that exposed fluoroapatite and glass, both having hydrophilic surfaces, amelogenins bound only to fluoroapatite (Habelitz et al., 2004). NMR studies further revealed that the binding site was through the C‐terminal domain of amelogenin (Shaw et al., 2004).

2. Non‐amelogenin Proteins Although present in minor amounts relative to amelogenin, additional enamel matrix proteins (EMPs) identified in developing enamel have been shown to play a role in regulation of crystal growth. These non‐amelogenin EMPs include enamelin, ameloblastin, tuftelin, biglycan, decorin, and amelotin. The specific roles of these proteins in influencing biomineralization of enamel are not fully understood and are currently under active investigation (Table I). In both humans and mice, an AI‐like phenotype is the result of mutation or KO of genes associated with some of these proteins, suggesting that they play an important role in biomineralization. Mutations in the enamelin gene in humans and mice result in AI characterized by hypoplastic enamel (Hu and Yamakoshi, 2003; Kim et al., 2005;

Table I Factors Found in Developing Enamel Factor

Cells/Tissues

Function/Putative Function

Amelogenin

Ameloblasts, HERS, odontoblasts, periodontal tissues (see also Table II)

Directs hydroxyapatite crystal habit during developmental stage of enamel formation by assembling into extracellular protein matrix in which mineral nucleates. Amelogenin is also considered a potential signaling molecule in dentin and cementum development (Table II)

Leucine‐rich amelogenin peptide (LRAP)

Ameloblasts—alternative splice product of amelogenin

Suggested functions include: responsible for binding to hydroxyapatite, and implicated as a signaling molecule in periodontal tissue formation

Models

References

Human X‐linked amelogenesis imperfecta (AI) (reduction or elimination of amelogenin expression by X‐chromosome): partially hypomineralized enamel Transgenic mice: altered A domain (AA 1‐42) and altered B domain (AA 157‐173) of M‐180 amelogenin—hypomineralization of enamel in both cases Amelogenin KO mice: hypomineralized enamel (Table II regarding root resorption) In vitro mineralization: mineral morphology control—short needlelike or long ribbonlike crystal shapes depending on mineralization condition Transgenic LRAP: expressed in amelogenin null mice did not rescue hypomineralized enamel LRAP overexpression in mice: enamel pitting; in vitro, aVects genes associated with PDL cells and cementoblasts, and some studies suggest proliferative eVects

Beniash et al. (2005); Gibson et al. (2001a,b), (2005); Iijima et al. (2002); Lagerstrom‐Fermer and Landegren (1995); Paine et al. (2001); Wright et al. (2003)

Boabaid et al. (2004b); Chen et al. (2003)

Tyrosine‐rich amelogenin peptide (TRAP)

Ameloblasts—cleavage product of amelogenin

Byproduct of amelogenin generated by protease; no known eVect on enamel; Implicated as signaling molecule in periodontal tissue formation Regulates mineralization of enamel, but its role has not been determined

Enamelin

Ameloblasts

Ameloblastin

Ameloblasts, ERM (see also Table II)

Acts as a repressor of amelogenin, limits ameloblast proliferation, may regulate crystal nucleation (see also Table II)

Tuftelin

May contribute to amelogenesis

Amelotin Biglycan

Found concentrated in dentin‐enamel junction (DEJ) Ameloblasts Bone, dentin, enamel

Unknown Repressor of amelogenin

Decorin

Bone, dentin, enamel

Unknown

TRAP overexpression in mice: no eVect on enamel, regulates cementoblast behavior in vitro

Paine et al. (2004); Swanson et al. (2006)

Enamelin mutation in humans: hypoplastic enamel Enamelin mutation in mice: hypoplastic enamel Ameloblastin KO mice: Amelogenesis imperfecta Ameloblastin overexpression in mice: partially disrupted enamel rod structure

Kim et al. (2005); Masuya et al. (2005); Rajpar et al. (2001)

Tuftelin overexpression in mice: disrupted rod/interrod morphology Not reported Biglycan KO mice: transient eVect included increased enamel formation, interrod as primary enamel structure while rod structure was minimally affected in newborns. Adult teeth appeared normal Decorin KO mice: transient eVect included decreased enamel formation and disorganized rod structure in newborns. Adult teeth appeared normal

Luo et al. (2004)

Fukumoto et al. (2004, 2005); Paine et al. (2003)

Iwasaki et al. (2005) Goldberg et al. (2002, 2005)

Goldberg et al. (2005)

(Continued )

Table I Continued Factor Dentin sialophosphoprotein (DSPP) Enamelysin (MMP‐20)

Kallikrein‐4 (KLK4)

Cells/Tissues

Function/Putative Function

Models

References

Pre‐secretory ameloblasts, DEJ

AVects DEJ morphology via regulation of predentin formation

DSPP KO mice: irregular DEJ

Sreenath et al. (2003)

Dentin, enamel

Proteolytically breaks down enamel proteins, e.g., amelogenin, in order to facilitate mineral growth. MMP‐20 is expressed in secretory and transition stages of enamel development

MMP‐20 KO mice: disrupted rod pattern, hypoplastic enamel MMP‐20 mutation in mice: heavily pigmented, hypoplastic enamel

Bartlett et al. (2004); Bartlett et al. (2006); Caterina et al. (2002); Hu et al. (2002)

Odontoblasts, ameloblasts, prostate

Proteolytically breaks down enamel proteins, e.g., amelogenin, in order to facilitate mineral growth. KLK4 is expressed in transition and maturation stages of enamel development

KLK4 mutation in human: yellow‐brown discoloration, lower mineral content in enamel

Hart et al. (2004); Hu et al. (2002)

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Masuya et al., 2005; Rajpar et al., 2001). However, how enamelin interacts with HAP mineral and/or the major matrix protein, amelogenin, remains largely unknown. Similarly, ameloblastin, whose expression by ameloblasts decreases from secretory stage to maturation stage of amelogenesis, has been found to aVect mineralization. Ameloblastin KO mice were reported to develop hypocalcified enamel with no recognizable rod structure (Fukumoto et al., 2004, 2005). In transgenic mice overexpressing (O/E) ameloblastin, Paine et al observed disruption of the rod/interrod structure in localized regions of enamel (Paine et al., 2003). Studies on ameloblastin null mice have also suggested that the protein functions as a cell adhesion molecule and regulator of cell growth (Fukumoto et al., 2004, 2005), however the specific role and mechanism for ameloblastin in mineral formation remains unclear. Tuftelin, another enamel protein with reported self‐assembly properties, may be an important protein in enamel mineralization, but its physiological function has yet to be well characterized (Deutsch et al., 1998, 2002; Paine et al., 1996, 1998). Tuftelin O/E in mice resulted in disruption of the enamel rod/ interrod structure, and the loss of the characteristic ribbonlike enamel crystallite morphology within rods (Luo et al., 2004). Other nonamelogenin proteins identified with enamel formation include biglycan, decorin, and amelotin. Biglycan and decorin are leucine‐rich proteoglycans, implicated in regulation of mineralized tissues. Loss of biglycan or decorin expression in KO mice resulted in an increase or decrease in enamel tissue formation, respectively (Goldberg et al., 2002). In both cases, the mineral structure was initially disrupted, but recovered with maturation of enamel. The reader is directed to Section IV.F for further information on the proteoglycans decorin and biglycan, and their role in regulating mineralized tissues of the tooth. Amelotin, discovered and reported to be an ameloblast‐specific gene, has been identified in humans and mice (Iwasaki et al., 2005). Expression of amelotin mRNA was restricted to maturation stage ameloblasts in mice. Amelotin’s potential role in enamel development is under investigation. 3. Proteases While amelogenins are critical in the nucleation step of enamel biomineralization, degradation of amelogenins is important for providing the space for HAP mineral crystals to expand during the growth stage of amelogenesis. Two proteolytic enzymes, matrix metalloproteinase‐20 (MMP‐20) and kallikrein‐4 (KLK‐4), have been identified as enzymes required for breaking down amelogenins (Bartlett et al., 1998; Fukae et al., 1998; Simmer et al., 1998). MMP‐20, secreted into the enamel extracellular matrix by ameloblasts during the secretory stage, is responsible for the proteolytic cleavage

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of amelogenin protein into smaller fragments. A study by Ryu and coworkers showed that incubation of MMP‐20 with recombinant porcine amelogenin (rP172) produced the same cleaved amelogenin fragments that are found in vivo (Ryu et al., 1999). KLK‐4, secreted during early maturation stage, functions to further digest matrix proteins and cleavage products incompletely digested by MMP‐20, facilitating nearly complete removal of proteins from mature enamel (Simmer and Hu, 2002). The latest data using in vitro models support the notion of diVerences in the way MMP‐20 and KLK‐4 digest 32‐kDa enamelin. While MMP‐20 cleaved enamelin only after it was deglycosylated, KLK‐4 readily cleaved enamelin into nine cleavage products (Yamakoshi et al., 2006). Human phenotypes carrying mutated MMP‐20 or KLK‐4 exhibit autosomal recessive hypomaturation AI (Hart et al., 2004; Kim et al., 2005; Ozdemir et al., 2005). Likewise, hypoplastic enamel with a disrupted rod pattern was reported in MMP‐20 null mice (Caterina et al., 2002). The MMP‐20 null mice demonstrated that complete elimination of EMPs from the enamel space failed to occur in the absence of this proteolytic enzyme, resulting in limited space required for expansion of the mineral phase.

C. Future Prospects for Enamel Regeneration The knowledge accumulated to date on the structure and biomineralization of enamel is abundant. However, there is much more to learn about these two aspects of enamel before we can fully describe the structure–function relationship of mature enamel and the processes involved in enamel formation. In terms of structure–function relationships, there is yet to be a clear model describing the true 3‐D rod architecture throughout the tissue. The ability to precisely describe directional changes of rods and their decussation pattern from the DEJ to the crown surface is critical for understanding the ability of the crown to distribute masticatory loads. From observation of biological models, the building of the enamel structure has proven to be a complex process. It requires an orchestration of protein–protein and protein–mineral interactions that occur in a temporally and spatially coordinated manner. Proper assembly and elimination of amelogenin has been shown to be critical in nucleation and growth of the mineral phase during formation of enamel. However, much is unknown with regard to the specific functions of other proteins in the enamel biomineralization process. Furthermore, emerging data have revealed that enamel proteins may serve a critical role as signaling molecules in tooth root development (see Section IV.A and Table II for details on the potential roles of enamel matrix proteins in root formation). Continued investigations targeted at understanding the detailed structure of enamel and the functions of individual EMPs, in enamel

Table II

Factors Associated with the Putative Epithelial Niche (HERS and ERM) and Surrounding Mesenchyme

Factor Msx2 (homeobox containing transcription factor known to play a role in crown formation)

BMP‐2 and 4 (bone morphogenetic protein 2 and 4)

FGF‐10 (fibroblast growth factor 10)

Cells/Tissues HERS (not in apical mesenchyme), and present in many other cell types including dental pulp and PDL (limited) Apical mesenchyme/ follicle region (not HERS)

Apical mesenchyme/ follicle region (not HERS)

Function/Putative Function

Models

References

Products from the mesenchymal cells in the local region, e.g., BMPs, may regulate HERS production of Msx2 and/or other factors. One outcome of this interaction may be control of root patterning

Msx2 KO: presence of irregularly shaped molar roots and increased expression of periostin

Satokata et al. (2000); Yamamoto et al. (2004a)

BMP‐2/4 and possibly BMP‐3, also found in high concentrations in the follicle region, regulate products produced by the HERS cells; this interaction controls growth and morphogenesis of the root sheath, and thus root patterning Continuous FGF‐10 expression by apical mesenchyme maintains epithelial stem cell population (as in continuously erupting rodent incisors). Cessation of FGF‐10 expression necessary for transition to root formation in teeth of limited eruption (e.g., rodent molars)

BMP‐4 KO: arrested at earlier stages of tooth development, therefore specific root defects are unknown

Yamashiro et al. (2003); Yamamoto et al. (2004a)

Mouse, FGF‐10 deficiency and overexpression: Deficiency: defect of epithelial stem cell (apical bud) compartment.

Harada et al. (1999), (2002a,b); Yokohama‐Tamaki et al. (2006)

Overexpression: formation of apical bud in mouse molars, inhibiting HERS formation and root development (Continued )

Table II Continued Factor

Cells/Tissues

Ameloblastin

Ameloblasts, HERS, cementocytes (low levels)

Amelogenina

Ameloblasts, HERS region, odontoblasts, PDL/cementum, and possibly in other tissues

Shh (sonic hedgehog)

HERS, dental mesenchyme, inner enamel epithelium, enamel knot

Function/Putative Function A known product of ameloblasts thought to regulate enamel crystal size Ameloblastin produced by HERS cells is hypothesized by some to promote acellular cementum formation A major protein of developing enamel and a known product of ameloblasts involved in regulating crystal structure. Suggested functions in non‐epithelial tissues include acting as a signaling molecule to regulate diVerentiation of odontoblasts and cementoblastsb. Hatakeyama et al. (2003, 2006) suggest that amelogenin acts to protect the root from osteoclast‐mediated root resorption Involved in epithelial‐mesenchymal interactions during tooth morphogenesis. May contribute to root elongation through signaling with Ptc1 and Gli1 genes and proliferation of dental mesenchyme

Models

References

Ameloblastin KO: exhibits an enamel phenotype but no root deformities have been reported

Simmer and Fincham (1995); Zeichner‐David et al. (2003)

Amelogenin KO: exhibits defective, chalky enamel similar to that observed in humans with amelogenesis imperfecta (Gibson et al., 2001a). After root formation is completed, root resorption is enhanced in association with osteoclasts and cementicles in the periodontal region Shh null mice: not viable Ptc1 mes mutants: reduced proliferation of mesenchyme adjacent to HERS and shorter roots

Boabaid et al. (2004b); Bosshardt and Nanci (2004); Bosshardt (2005); Gibson et al. (2001b); Hatakeyama et al. (2003); Hatakeyama et al. (2006); Nebgen et al. (1999); Shimizu et al. (2005); Veis et al. (2000); Viswanathan et al. (2003) Nakatomi et al. (2006)

IGF‐1 (insulin‐like growth factor‐I)

HERS

OPN, BMP‐2, ameloblastin

Epithelial cell rests of Malassez (ERM)

a

May contribute to elongation of the HERS, IGF receptors are present in vivo, and elongation of HERS/increased cell proliferation occurred in the outer epithelial layer when IGF was added in vitro May assist in repair of cementum by increasing cell proliferation; alternatively, may be vestigial products of HERS with no function in the mature tissues of the periodontium

IGF‐1 localized to HERS in 5‐day‐old mice. In vitro experiments supported a role for IGF‐1 in regulating mitotic activity in HERS cells No animal models with defective ERM have been developed

Fujiwara et al. (2005)

Hasegawa et al. (2003); Yamashiro et al. (2003)

Note: Amelogenin has several isoforms (Bartlett et al., 2006):
LRAP (6.9 kDa); also called [A‐4]/M59 and suggested to be a signaling molecule for odontoblasts and cementoblasts (Tompkins and Veis, 2002). LRAP KO and LRAP overexpression in mice do not appear to have a root phenotype.
[Aþ4]/M73 (8.1 kDa) has also been proposed as a signaling molecule (Tompkins and Veis, 2002).
TRAP: Similarly, suggested as a signaling molecule (Swanson et al., 2006). b Studies by Wang et al. (2005a) and Tompkins et al. (2006) have identified LAMPs as possible regulators of amelogenins, either involved in assisting with breakdown of amelogenins (LAMP‐3, Wang et al.) or possibly serving as cell surface receptors (LRAP‐LAMP‐1, Tompkins et al.). Further, Tompkins et al. reported that A‐4/LRAP binds to murine LAMP‐1, a lysosomal associated membrane protein, also present on cell surfaces, in a saturable fashion in murine myoblasts (C2C12 cells).

Origins of cementoblasts and cementum Precursor cells

Differentiation

Hypothesized activities

Ectomesenchymal Induction of cementogenesis (Alatli-Kut et al., 1994; Takano et al., 2003)

Papilla Pulp/Odontoblast Cementoblast

Follicle-derived cementoblasts secrete acellular and cellular cementum (Cho and Garant, 2000; Diekwisch, 2001; Luan et al., 2006)

PDL Cell Follicle Osteoblast Epithelial

Follicle-derived cementoblasts secrete (only) cellular cementum (Chai et al., 2000; ZeichnerDavid et al., 2003)

HERS cells e-m transform and secrete cellular cementum (Bosshardt, 2005; Bosshardt and Nanci, 2004; Lezot et al., 2000; Thomas, 1995)

IEE HERS

HERS secretes acellular cementum (Bosshardt, 2005; Zeichner-David et al., 2003) HERS secretes proteins that induce cementogenesis (Fong and Hammarstrom, 2000; Fukae et al., 2001; Gestrelius et al., 2000; Hu et al., 2001)

OEE

HERS dislocates from developing root surface and forms ERM (Cho and Garant, 2000; Diekwisch, 2001; Luan et al., 2006)

Generally established

Proposed hypothesis

ERM

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as well as in root development, will require concentrated research eVorts in the next decade. Knowledge built from these research findings will enhance our basis for regenerating the crown as well as the ‘‘whole’’ tooth.

III. Question 2. What Do We Know About the Cells Required for Periodontal Development and Regeneration? While the origins for cells and tissues of the tooth crown have been fairly well established, much remains unclear about cells involved with forming the periodontium, and this has been a subject of speculation for at least five decades, arguably with no authoritative statement yet made. The following section will discuss the cells involved in periodontal development, their potential contribution to regeneration, as well as controversies regarding their origins.

A. Developmental Cells Odontogenesis is characterized by sequential, reciprocal, reiterative signaling between tissues of the epithelium (dental lamina) and mesenchyme (ectomesenchyme derived from cranial neural crest) and ultimately, both epithelial and ectomesenchymal cells are involved in periodontal tissue formation (Fig. 2). Tooth development continues with ectomesenchymal cells developing into the dental follicle and surrounding the epithelial enamel organ and the mesenchymal dental papilla (Nanci and Somerman, 2003). Cells within the follicle region have been proposed to be the origin for tissues of the periodontium, namely cementum, periodontal ligament (PDL) and alveolar bone. But this hypothesis has not been accepted without challenge, as will be discussed in some detail below. The exact origin of cementum and cementoblasts remains a matter of debate; current hypotheses are summarized in Fig. 2 and described in the following text.

Figure 2 Origins of cementoblasts and cementum. This figure reviews competing hypotheses on origins of cementoblasts and cementum tissue by considering possible fates for cells of ectomesenchymal (top panel) and epithelial (bottom panel) origin, and hypothesized roles in tooth root formation. The primary division lies between a proposed ‘‘classical’’ mesenchymal origin (represented in top panel) and an ‘‘alternative’’ epithelial origin (represented in bottom panel). However, several variations exist within each hypothesis, and these diVerences need not be mutually exclusive. IEE ¼ inner enamel epithelium; OEE ¼ outer enamel epithelium; HERS ¼ Hertwig’s epithelial root sheath; ERM ¼ epithelial cell rests of Malassez; PDL ¼ periodontal ligament; e‐m ¼ epithelial‐mesenchymal transformation.

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1. Ectomesenchymally Derived Cells During the cap stage of tooth development, the epithelial enamel organ takes on a concave form and is bordered by two ectomesenchymal tissues, papilla and follicle, descended from cranial neural crest (CNC) cells. The dental papilla is composed of densely packed cells that during the subsequent bell stage become increasingly sequestered within the developing enamel organ, eventually giving rise to the pulp and dentin tissue. The mesenchymal cells surrounding the developing enamel organ and papilla compose the dental follicle (sometimes called the dental sac), a collagenous tissue separating the nascent tooth bud from surrounding oral tissues. Dental follicle has been proposed to be the common origin for supportive tissues of the tooth (i.e., the periodontium), including cementum, PDL, and alveolar bone (Cho and Garant, 2000; Nanci and Somerman, 2003; Saygin et al., 2000). Cells within the follicle region are also essential for signaling associated with tooth eruption, through regulation of osteoclasts in the coronal portion of the bony crypt via CSF‐1, RANKL, and OPG expression, signaled in turn by PTHrP and other factors still to be identified (Liu et al., 2005a; Wise et al., 2002, 2005). During tooth eruption and root elongation, the formative dental follicle gives rise to the mature structure of the PDL, a highly vascular and innervated region that provides attachment of the tooth to the surrounding alveolar bone via collagen fibers. The PDL is also home to a heterogeneous population of cells, including stem cells with potential for regeneration of periodontal tissues, which will be discussed in more detail at the end of this section (Cho and Garant, 2000; Nanci and Somerman, 2003; Seo et al., 2004).

2. Hertwig’s Epithelial Root Sheath Cells Root initiation begins after the crown dentin and enamel have formed, and before tooth eruption. The cervical loop, the most apical extension of the enamel organ, extends into the bilayered Hertwig’s epithelial root sheath (HERS), composed of the outer enamel epithelium (OEE) and inner enamel epithelium (IEE). The HERS layers proliferate and extend apically, outlining the future shape of the nascent tooth root (Luan et al., 2006). In mammalian root formation, dislocation from the root and disintegration of the double‐ layered HERS is considered a key event, allowing access of the underlying dentinal surface to cementum‐forming cells (Cho and Garant, 1988; Diekwisch, 2001). This general sequence of events has been further supported by in vitro tissue recombination experiments (MacNeil and Thomas, 1993). As root formation continues, the dislocated HERS cells break up into epithelial ‘‘nests’’ and ‘‘cords,’’ which may be subsequently reduced to epithelial cells rests of Malassez (ERM) (Wentz et al., 1950). In addition to the possibility

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of HERS cells migrating away from the root surface to contribute to ERM, it has also been documented that some HERS cells undergo apoptosis (Kaneko et al., 1999) or become incorporated into the cellular cementum (Lezot et al., 2000). Developmental studies, as well as a review of evolutionary evidence (Luan et al., 2006), provide information indicative of a role for ERM in regulating PDL homeostasis, protecting against resorption and ankylosis, and perhaps contributing to cementum repair (Hasegawa et al., 2003). It has been proposed that HERS plays an active role in induction or secretion of acellular and/or cellular cementum, and this hypothesis is described in detail below (under the ‘‘alternative’’ epithelial hypothesis of cementogenesis). Potential signals of HERS that may stimulate cementum formation are discussed under Section III and listed in Table II.

B. Derivation of Cementum: Competing Theories of Cementoblast Origin 1. Acellular Versus Cellular Cementum Acellular cementum covers approximately two‐thirds of the root, and around the time the tooth comes into occlusion, cementum development shifts from acellular to cellular. Acellular cementum (acellular extrinsic fiber cementum, AEFC) forms first on the coronal and mid‐portion of the root at a slow rate, while cellular cementum (cellular intrinsic fiber cementum, CIFC), a more bone‐like tissue, forms more apically and more rapidly, incorporating cells into the mineralized matrix that become cementocytes. Acellular cementum seems to be more dependent on alkaline phosphatase activity (Jayawardena et al., 2002), as it may be more severely aVected than cellular cementum in hypophosphatasia (Beertsen et al., 1999; van den Bos et al., 2005). The cause or mechanism of the shift from acellular to cellular cementum is not well understood, though hypotheses to explain this transition include the possibilities that occlusal mechanical forces somehow cue the shift, cells producing each type of cementum are from diVerent populations, or diVerent extracellular factor(s) regulate(s) acellular versus cellular cementum. Potential regulators that have been considered include the dentin matrix of the root, enamel matrix proteins, and other components of the ECM. In experiments modeled after those performed by Hammarstro¨m (Alatli‐ Kut et al., 1994; Hammarstro¨m et al., 1996), Takano et al. treated rats and guinea pigs with bisphosphonate to delay dentin matrix mineralization, and observed that acellular cementum was precluded by formation of a cellular type of cementum on the nonmineralized dentin along the entire surface of the root (Takano et al., 2003). While dentin sialoprotein (DSP) was localized

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to the border between dentin and cellular cementum (but not acellular cementum) in untreated rats, in the bisphosphonate‐treated rats DSP penetrated the soft dentin matrix along much of the root, even diVusing into surrounding tissues. The authors hypothesize that the timing of mineralization of mantle dentin in conjunction with dentin matrix proteins influences the type of cementum forms. 2. The ‘‘Classical’’ Mesenchymal Hypothesis The ‘‘classical’’ hypothesis, nearly 50 years old (Paynter and Pudy, 1958), proposes cementoblasts are cells descended from the dental follicle that migrate to the developing root surface and are triggered to diVerentiate into cementum matrix‐secreting cells, that is, cementoblasts (Bosshardt and Selvig, 1997; Cho and Garant, 2000; Diekwisch, 2001; Luan et al., 2006; Saygin et al., 2000). This hypothesis fits into an overarching proposition of a common developmental origin (i.e., the dental follicle) for the three formative cell populations of the periodontium, namely cementoblasts, PDL cells, and alveolar osteoblasts (Melcher, 1985; Ten Cate, 1997). During rat molar root development, mesenchymal cells of the follicle were reported to migrate to the HERS, disrupt the epithelial structure, and begin to lay down cementum matrix via cellular processes, as interpreted from studies employing light and electron microscopy (Cho and Garant, 1988, 2000). Similar observations of mesenchymal cells accessing the developing root surface were reported in the mouse molar, with the exception that HERS cells may themselves contribute to the disruption of the HERS structure prior to root formation, while the first matrix secreting cells in cementum formation were the migrating mesenchymal (follicle) cells (Diekwisch, 2001; Luan et al., 2006; Ten Cate, 1997). Migratory capabilities of dental follicle cells were supported in mouse molar organ culture, where fluorescently tagged follicle cells migrated apically and were found in PDL and alveolar bone (Diekwisch, 2002). Human and porcine specimens in the extensive Bernard Gottlieb collection (Baylor College of Dentistry, Dallas, TX) yielded similar observations that HERS cells departed the root surface prior to initiation of cementum, forming a loose network of cells that subsequently disintegrated, with some presumably contributing to the population of ERM, islands of epithelial‐derived cells that remain in the PDL into adulthood with uncertain function (Diekwisch, 2001). In support of the ‘‘classical’’ hypothesis, in a well‐executed developmental study in mice, Chai et al. were able to track cells of cranial neural crest (CNC) origin, from embryogenesis to 6 weeks of age, using a two‐component (Wnt1‐Cre, R26R) genetic system for cell lineage tracking through development (Chai et al., 2000). In this way, CNC progeny were identified by ‐galactosidase activity (only present in cells expressing Wnt1 and constitutive

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R26R, but marked indelibly, even when Wnt1 expression is shut oV). CNC‐ derived cells contributed to formation of cementum and periodontal ligament, as well as to condensed dental mesenchyme, dental papilla, odontoblasts, and other tissues. While cementum showed strong lacZ expression, indicating a CNC origin, these results need not preclude an epithelial contribution. Some species‐specific diVerences in cementum development are worth noting, one being that in rodents the sequence of events is muddied by HERS initially covering the entire root surface and remaining in close proximity as cementum is formed, as opposed to humans where HERS is more completely divorced from the developing root prior to any observable cementum. Supposing the classical hypothesis of common origin for cellular and acellular cementum, PDL, and alveolar bone, the question naturally arises, ‘‘What factors direct a common precursor cell to become a cementoblast, osteoblast, or PDL cell?’’ This is a valid question worthy of future study, with some potential regulators discussed under Question 3 and presented in Tables II–V.

3. The ‘‘Alternative’’ Epithelial Hypothesis An alternative hypothesis that has been proposed (Slavkin and Boyde, 1975) questions the evidence for a mesenchymal origin and instead considers an epithelial contribution from HERS to cementogenesis (Bosshardt, 2005; Bosshardt and Nanci, 1997, 2004; Bosshardt and Schroeder, 1996; MacNeil and Somerman, 1999; Thomas, 1995; Zeichner‐David, 2006; Zeichner‐David et al., 2003). Under this proposal, cementoblasts are thought to be derived from an epithelial‐mesenchymal transformation of HERS cells, which then secrete cementum matrix proteins. DiVerences of opinion exist regarding origins of acellular and cellular cementum, as delineated below. In a careful observation of cementogenesis in pigs using light microscopy and TEM with immunogold labeling, Bosshardt and Nanci found a lack of compelling evidence for a mesenchymal migration of follicle cells, but rather observed a potential phenotypic epithelial‐mesenchymal transformation of outer enamel epithelium (OEE) cells to a secretory, connective tissue cell‐like morphology in the vicinity of initiation of cementogenesis (Bosshardt and Nanci, 2004). Studies using a Dlx‐2/LacZ reporter construct in transgenic mice localized Dlx‐2 expression to root epithelium (HERS) during root development, and also to a limited population of cementoblasts during acellular and cellular cementum formation, but failed to detect Dlx‐2 in dental follicle and papilla (Lezot et al., 2000). During acellular cementum formation, Dlx‐2 was identified in diVerentiated cementoblasts, and during cellular cementum formation in innermost cementoblasts and cementocytes.

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Some of the Dlx‐2 positive cementoblasts also stained positive for ameloblastin. The authors concluded a complex origin for cementum‐forming cells, in other words, suggesting that a select population of cementoblasts were derived from the HERS. Another interpretation of these results could be that HERS cells are passively incorporated within the forming cementum matrix being synthesized by mesenchymally derived cementoblasts. Evidence for an epithelial origin for acellular cementum also lies in the demonstration that these cells can produce proteins characteristic of mesenchymal cells, and cementum in particular (Bosshardt and Nanci, 1997; Mouri et al., 2003; Zeichner‐David, 2006; Zeichner‐David et al., 2003). If HERS cells transform to contribute to acellular cementum formation, the possibility of cellular cementum derived from HERS may also be considered. A hypothesis based on morphological examinations in human and porcine teeth proposes that HERS is the origin not only for both types of cementum, but also for subpopulations of periodontal ligament fibroblasts (Bosshardt, 2005). This hypothesis would explain the diVerent phenotypes of cementoblast versus osteoblast, and the heterogeneity of cells populating the PDL region. While strides are being made toward describing the origins of cementum, and a great dialogue of diVering viewpoints has been cultivated in the literature, the origin of cementum is still under debate. 4. Involvement of Epithelial‐Derived Products in Cementum Formation Apart from ideas about the transformation of HERS to cementoblasts, it has been suggested that HERS may induce cementogenesis by secretion of enamel matrix proteins (EMPs) (e.g., amelogenin, ameloblastin, and enamelin) or other proteins that influence cell migration, attachment, and/or matrix secretion leading to cementogenesis (Gestrelius et al., 2000; Hammarstro¨m et al., 1996; Slavkin, 1976; Zeichner‐David, 2001, 2006). Evidence supporting a role for EMPs in cementogenesis has been accumulating from investigations employing immunohistochemistry, in situ hybridization, and in vitro assays, all supporting EMP expression by HERS cells in several species (Bosshardt and Nanci, 1998; Fong and Hammarstrom, 2000; Fukae et al., 2001; Hamamoto et al., 1996; Hammarstro¨m, 1997; Hu et al., 2001; Luo et al., 1991; Slavkin et al., 1989a,b). However, serious discrepancies in these collective reports remain unresolved. Reports conflict with one another on several points, including: which EMPs are or are not expressed, how much protein is present and if levels are suYcient to play an important role in root formation, the region of localization on the root, and what cells produce EMPs. In studies of porcine cementogenesis, little evidence was found to support a significant role of enamel matrix derivatives (in this case, amelogenin and ameloblastin) based on absence of significant quantities of these ameloblast products in the HERS and on the developing root surface

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(Bosshardt and Nanci, 2004). In mouse molars, immunocytochemistry and in situ hybridization failed to detect any trace of amelogenin in HERS cells, and amelogenin was also absent in porcine cementum extracts assayed by Western blot (Diekwisch, 2001). Janones et al. used microwave processing in conjunction with immunocytochemistry to demonstrate that in developing rat molars, amelogenin was present in early tooth formation, but gone before initiation of cementogenesis (Janones et al., 2005). These conflicting results, both old and new, should then be considered carefully for possibilities such as false positives and specificity or cross‐reactivity of antibodies. Ultimately, to confirm that EMPs are functionally important in cementogenesis, a consistent and regular expression of these proteins would be expected in association with developing cementum, and up to now, this standard remains to be met in a convincing way. Better probes, antibodies, etc., should assist in solving this puzzle. For example, an immortalized murine HERS cell line expressed ameloblastin (but not amelogenin or enamelin) in vitro. HERS conditioned media was found to induce BSP and OCN expression, as well as in vitro mineralization (Zeichner‐David, 2006). Further, these HERS cells were also observed to undergo an apparent phenotypic transformation to a morphologically distinct fibroblastic cell expressing cementum‐associated transcripts BSP, OCN, and OPN, supporting the potential not only for HERS to induce cementogenesis, but also secrete cementum matrix proteins directly (Zeichner‐David et al., 2003). Furthermore, if EMPs play an important role in tooth root formation, a cementum phenotype might be expected in animals deficient in these proteins. While a root phenotype has been suggested in amelogenin knockout mice, it is unclear whether this is a direct or indirect result (Hatakeyama et al., 2003, 2006), and this will be addressed in the next section under Question 3, as well as in Table II focusing on HERS‐ and ERM‐associated products. Though the role of epithelial proteins in root formation remains controversial, a treatment derived from porcine enamel organ known as EmdogainÒ is currently used clinically with the aim to promote periodontal regeneration. The applications of EmdogainÒ will be discussed in more detail under Question 3.

C. Differences Between Cementoblasts and Osteoblasts A common origin for cementum and alveolar bone has been proposed in the form of the dental follicle and perifollicular cells. Yet in the absence of a clear understanding of cementum origins, how can progress be made toward improving tissue engineering and promoting periodontal regeneration? Cementoblasts and osteoblasts and their respective tissues may be compared

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with respect to cells and regulators of their diVerentiation, and structural and functional properties of the cementum versus bone matrices. It is outside the scope of this review to exhaustively catalog points of comparison between cementoblasts and cementum versus osteoblasts and bone; for this the reader is recommended to excellent reviews on the topic (Bosshardt, 2005; Diekwisch, 2001; Nanci and Bosshardt, 2006; Saygin et al., 2000; Zeichner‐ David, 2006). Potential areas for progress in characterizing cementoblasts including identification of cementum marker proteins, performing comparisons to other cell types, and using in vitro models of cementoblasts and precursor cells in conjunction with in vivo observations. 1. Cementum‐Specific Markers Attempts have been made to identify unique cementum‐specific marker proteins that would distinguish cementum from bone. In the study of dental tissues, many ‘‘specific markers’’ have even been declared, later to be reported in other tissues as well. For example, DSPP and DMP‐1, formerly thought dentin‐specific, have subsequently been localized to bone and cementum and their respective cells, in vivo and in vitro (Baba et al., 2004a; Foster et al., 2006; Qin et al., 2002). Amelogenin, thought to be an ameloblast‐specific product, is expressed by pulp cells and odontoblasts during tooth development (Nagano et al., 2003; Oida et al., 2002; Papagerakis et al., 2003; Veis et al., 2000). There is a history of putative cementum‐specific factors as well. A cementum‐ derived growth factor (CGF) originally isolated from a human cementoblastoma and posited to be a novel growth factor and mitogen (Yonemura et al., 1992, 1993) was identified in human and bovine cementum, as well as in PDL cells and furthermore, on detailed analysis recognized as being very similar in composition to IGF1 (Narayanan et al., 1995). Cementum attachment protein (CAP) was identified from a human cementum tumor and proposed to be an extracellular matrix protein functioning in migration and attachment of cementoblast precursors to the root surface (Arzate et al., 1992; Bar‐Kana et al., 2000; Pitaru et al., 1995, 2002; Saito et al., 2001); CAP was later found to be expressed in PDL cells and alveolar bone cells, and to share homology with some collagen domains (BarKana et al., 1998; Wu et al., 1996). Another protein identified from cementum tumor was termed cementum‐protein 23 (CP‐23) (Alvarez‐Perez et al., 2006). Antibodies made to this protein cross‐reacted with a cartilage type collagen, type X collagen, and CP‐23 was identified within the PDL region, cementum and around blood vessels in the PDL. While CGF, CAP, and CP‐23 may play roles in periodontal development, they are not, strictly speaking, markers of cementoblasts or cementum. Importantly, these proteins were identified from a human cementoma and cementomas by definition are composed of a variety of cells, for example, fibroblasts, osteoblasts, and cementoblasts. Additional examples include lumican and fibromodulin,

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reported to be more highly expressed in cementum than bone (Bosshardt, 2005). Glucose transporter‐1 (GLUT‐1) was suggested to be a factor separating cementoblasts from osteoblasts (Koike et al., 2005), and though this protein is widely expressed, it is tenfold higher in human cementoblasts versus osteoblasts in vitro. While these proteins may not be unique cementum markers, they may still be useful in defining cementum matrix versus bone. These and other proteins are thought to be enriched or relatively highly expressed in cementum versus bone and have potential to be used to assemble a panel of markers characteristic or suggestive of cementum. As of yet, there is not any marker by itself that is unique or specific to this tissue.

2. Comparisons of Cementoblasts to Other Cell Types As no conclusive study demonstrating cementoblast origin has yet been reported and no cementum‐specific marker is likely, a very practical option may be direct cell‐to‐cell comparison, as between cementoblasts and osteoblasts. In vivo studies are limited by the need for specific probes and antibodies and the laborious nature of screening, while in vitro studies make many aspects of analysis easier, but results must be analyzed cautiously because of removal of cells from the natural milieu. Head‐to‐head comparisons of cells have yielded valuable insights when confirmed by other methods such as in situ hybridization and immunohistochemistry. Examples of such comparison technologies include laser capture, microarray analysis, proteomics, and subtractive hybridization. All, except laser capture, have been used to begin to define markers for dental cells (Hao et al., 2005; Koike et al., 2005; Lallier et al., 2005; Reichenberg et al., 2005; Shi et al., 2001). Care must be taken in the choice and preparation of cells to be compared in such experiments, as this sort of analysis may result in misleading conclusions if precautions are not used. For example, the cell populations being compared may be derived from diVerent developmental stages, which would strongly influence gene and protein profiles expressed. The logical comparison for cementoblasts would be osteoblasts lining the surrounding alveolar bone. Although alveolar bone is generally thought to be consistent with other bone tissues in cell and matrix components, it is a local, specialized bone tissue with unique features, including proximity to the tooth and the cellular/molecular influence of the tooth tissues, and a very high rate of remodeling relative to other bone tissues of the body (Sodek and McKee, 2000). There is some evidence that bone marrow stromal cells (BMSCs) within the same individuals diVer in a skeletal site‐specific fashion, and that orofacial stem cells may represent a unique population (Akintoye et al., 2006). If cementoblasts and alveolar osteoblasts share a direct precursor cell, it is

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possible that they share a more similar genetic profile than cementoblasts versus other osteoblast or osteoblast precursor populations. A cleverly designed experiment by Kaneda et al. used a strategy of consecutive enzymatic digestions of extracted mouse molars to explore diVerences between subpopulations of PDL cells, from cells obtained midway across the PDL space to those closest to the root surface, including cementum‐lining cells (Kaneda et al., 2006). As subpopulations were characterized closer to the root, their alkaline phosphatase activity and potential for promoting in vitro mineralization increased, as well as expression of BSP mRNA. Further studies employing a similar approach should yield insights into characteristics of subpopulations of cells located in the PDL region, and into the potential of these various subtypes to diVerentiate toward a cementoblast phenotype. 3. In Vitro Cell Models for Cementoblasts and Precursor Cells Establishment of in vitro cementoblast models in parallel with studying in vivo cementum development can be a powerful way to progress our understanding of the origins and characteristics of this tissue. To date, cementoblast cell lines for use in vitro have been prepared from mice (Berry et al., 2003; D’Errico et al., 2000; MacNeil et al., 1998), rats (Kitagawa et al., 2005), cows (Saito et al., 2005), human (Grzesik et al., 1998), and human cementoblastoma (Arzate et al., 1992). These cells express high levels of BSP, OCN, and OPN, and can produce mineralized nodules in vitro and ectopic ossification in an in vitro SCID mouse model. Additionally, putative cementoblast precursors, dental follicle cells, have been isolated and cultured from mice (Zhao et al., 2001), rats (Yao et al., 2004), and humans (Morsczeck et al., 2005), and these may provide clues as to potential mechanisms required cementoblast diVerentiation. An immortalized HERS‐derived cell line has been established from mice, and has been characterized as producing enamel‐related proteins prior to a phenotypic shift toward a mesenchymal cell type that produces a mineralized matrix resembling acellular cementum (Zeichner‐David et al., 2003). While species diVerences, phenotypic drift, and secondary eVects of immortalization must be considered, these approaches have already yielded considerable insight into the nature of ‘‘cementoblasts’’ and will continue to do so in future research.

D. Tooth Stem Cell Populations The nature and regenerative capacities of stem cell populations in tooth tissues have been one of the most exciting revelations in dental research in the last five years, with enormous potential for future application in

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designing regenerative therapies and tooth engineering in the future (Bartold et al., 2006a; Chai and Slavkin, 2003; Fong et al., 2005; Ohazama et al., 2004b; Risbud and Shapiro, 2005; ThesleV and Tummers, 2003). Embryonic stem cells are pluripotent, that is, they have the capability to diVerentiate into all cell types with appropriate conditions and stimulation. Stem cell research eVorts focus on the sizeable probability for such cells to be used in adult tissue regeneration and gene therapy. However, the current number of embryonic stem cell lines is limited and their use is controversial and subject to government regulation. As a result, there has been great interest in exploring stem cell populations in adults. Adult stem cells are undiVerentiated cells that remain in developed tissues of the adult organism and are multipotent, meaning they have the capability to diVerentiate into multiple cell types within a tissue, organ, or system. Adult stem cells have been identified in several locations including bone marrow, blood, neural and muscle tissue, and tooth environment (Fuchs and Segre, 2000). While the breadth of potential for diVerentiation, or potency, for most of these adult stem cell types remains to be fully explored, the therapeutic possibilities for an adult‐derived, unlimited population of multipotent stem cells are quite exciting (Robey, 2000). The identification and characterization of these adult stem cell populations in the tooth region has been one of the most exciting and promising discoveries of the last five years. 1. Dental Pulp Stem Cells The dental pulp holds promise for regeneration of dentin in response to trauma (Goldberg and Smith, 2004), and this has been recognized for many years. This knowledge, coupled with advances in technology, has enhanced our understanding of the underlying mechanisms involved in pulp cell maturation. A human adult stem cell population was identified and isolated from pulp chambers of impacted third molars. In cell culture, these dental pulp stem cells (DPSCs) were demonstrated to be clonogenic, rapidly proliferative, able to diVerentiate and form mineralized nodules in vitro, and produce a structure resembling a dentin/pulp complex in ex vivo transplantation experiments with SCID mice (Gronthos et al., 2000). In the same experiment, bone marrow stromal stem cells (BMSSCs) formed a more distinctly bone‐like tissue. In subsequent studies, the DPSC profile was further developed by identifying mesenchymal stem cell markers STRO‐1 and CD146, and transplant experiments were performed with DPSCs, with cells exhibiting odontoblast‐like gene and protein expression, and producing dentin‐like tissues (Batouli et al., 2003; Shi et al., 2001). The DPSC niche was hypothesized to be a perivascular location within the pulp. Subsequent work demonstrated the ability to harvest similar mesenchymal stem cells from human exfoliated deciduous teeth, cells that were termed SHED

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(Miura et al., 2003). Sorting and ex vivo expansion of pulp stem cells from exfoliated deciduous teeth allowed for the cells to be directed to adipocyte and myotube phenotypes, as well as osteoblast‐like cells that produced a mineralized tissue consistent with woven bone (Laino et al., 2006). 2. Periodontal Ligament Stem Cells The PDL demonstrates some limited potential for repair of periodontal tissues should they be damaged by trauma or disease, however, while there are currently several strategies aimed at regenerating periodontal tissues, sometimes successful, they are not predictable (Grzesik and Narayanan, 2002; Taba et al., 2005; Wang et al., 2005b; Zohar and Tenenbaum, 2005). This repair potential of the PDL is thought to result from the presence of a population of multipotent stem cells within the local region or recruited from the vasculature that are capable of regenerating cementum, bone, and PDL fibers (Bartold et al., 2000; Gould et al., 1980; McCulloch, 1985, 1995; Melcher, 1985). Although several groups have demonstrated the regenerative potential of a compartment of periodontal cells, recent studies have confirmed a stem cell population and characterized the nature of these cells. Human postnatal PDL stem cells (PDLSCs) were isolated, cultured, and characterized in vitro (Seo et al., 2004). PDLSCs were fibroblast‐like, clonogenic and rapidly proliferative, and were positive for mesenchymal stem cell markers STRO‐1 and CD146, similar to DPSCs and BMSSCs, indicating a possible common perivascular origin. Interestingly, expanded PDLSCs also expressed relatively high levels of a tendon‐associated transcription factor, scleraxis. In vitro studies showed that after incubation in diVerentiation media, PDLSCs expressed proteins characteristic of cementoblasts, including BSP, OCN, MEPE, ALP, and TGF R1, and had the ability to promote the formation of mineralized nodules. PDLSCs transplanted into SCID mice produced collagen fibers suggestive of the PDL and a mineralized tissue consistent with cellular cementum. It remains unclear what signals may be necessary to cue precursor cells to a cementum versus bone phenotype, and at present, no specific markers have been established for cementum versus bone (as described in detail above). Subsequent studies added to this work by showing that viable PDLSCs could be retrieved from frozen PDL tissues (Seo et al., 2005), increasing the practical potential for these stem cells to be used clinically (Bartold et al., 2006a; Shi et al., 2005). While the origins of cementoblasts remain in question, studies with periodontal ligament stem cells (PDLSCs) showing ability to produce cementum‐like tissues in SCID mouse transplant experiments lend some support to the mesenchymal origin of cementum, or at least cellular cementum. Indeed, expression of common transcription factors, cell surface markers, growth factors, and matrix proteins in postnatal

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stem cell populations suggests common regulatory pathways for cementum, dentin, and bone. 3. Epithelial Stem Cells of the Continuously Erupting Incisor The postnatal stem cells of the bone marrow, dental pulp, and PDL are multipotent mesenchymal stem cells with a capacity for limited generation of mesenchymally derived tissues. In the teeth of humans and the molars of rodents (teeth of limited eruption), the ameloblasts that form the tooth enamel are lost on tooth eruption, and there seems to be no epithelial self‐ renewing stem cell population remaining. However, in the continuously erupting incisor of rodents, new enamel (as well as dentin and cementum) are constantly generated apically to compensate for attrition on the incisal edge (Harada et al., 2002a). Therefore, new ameloblasts must be available from postnatal epithelial stem cell populations in order to synthesize enamel in the adult. A specialized apical bud structure proposed to be the epithelial stem cell niche was identified at the apical end of incisors of mice and guinea pigs (Ohshima et al., 2005). The apical bud was characterized by large amounts of stellate reticulum and basal epithelium, and a candidate molecule for maintenance of the apical bud niche was identified as fibroblast growth factor 10 (FGF‐10) by adjacent mesenchymal cells, with epithelial Notch signaling also implicated as playing a role (Harada et al., 2002b). Ameloblast diVerentiation and patterning in the rodent incisor was shown to be dependent on downregulation of follistatin in the epithelium on the labial edge, and subject to regulation by the antagonistic actions of BMP‐4 from odontoblasts and activin from dental follicle (Yamashiro et al., 2004). In a primary cell culture study, apical bud stem cells were shown to require mesenchymal cell interaction to be prompted to diVerentiate to an ameloblast‐like phenotype (Morotomi et al., 2005). 4. Crown and Root Developmental Fates The developmental diVerence between teeth of limited eruption versus continuous eruption may lie in the ‘‘choice’’ between maintenance of the epithelial stem cell niche as opposed to loss of this niche and development of a root. The developmental fate of the continuously erupting incisor is determined by maintenance of an apical bud epithelial stem cell population (Ohshima et al., 2005), while if crown development is arrested, a root fate is pursued (Tummers and ThesleV, 2003). The root fate is characterized by transformation and flattening of the stellate reticulum into the double‐ layered HERS, which lengthens to define the root shape outline, and fenestrates just prior to cementum formation. The fate of the HERS is a matter of debate, potentially becoming ERMs, secreting pro‐cementum factors, or contributing

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to acellular and/or cellular cementum formation. Crown and root fates were investigated in an elegant study of the molar of the sibling vole, which intriguingly develops root analog areas while remaining continuously erupting. In the root‐like regions of vole molars, FGF‐10, Notch 1 and 2, and BMP‐4 signals, thought to contribute to the epithelial stem niche, disappear coincident with development of root‐like tissues, similar to in mouse molars (Harada et al., 2002b; Ohshima et al., 2005; Tummers and ThesleV, 2003; Yokohama‐Tamaki et al., 2006). Therefore, mechanisms that downregulate signals specific for crown formation may be required for initiation of root formation. These findings are intriguing to consider in light of the failure of tissue recombination experiments to form tooth roots.

IV. Question 3. What Genes and Associated Proteins Are Important for Root/Periodontal Tissue Formation? In the last 5 years, newly discovered factors and new roles for already known factors regulating root/periodontal development have emerged and have changed how we view odontogenesis. Much of the recent progress in understanding factors that regulate root/periodontal tissue (R/PT) development has naturally arisen from studies of molecules that control mineralized tissue formation. The development of transgenic mice, designed to over‐ or underexpress specific genes, and the study of mice with well‐defined mutations has provided much insight into potential factors required for R/PT development. Tables II–V highlight factors that have been reported within (approximately) the last 5 years to play a role in R/PT development, including genes/ proteins that result specifically in a root phenotype when deficient or overexpressed. In addition, factors already established to play a role in root/ periodontal development will be briefly discussed, with an update of relevant references. It is well established that specific genes and associated proteins are required for patterning, proliferation, and diVerentiation of cells during crown development, that is ameloblasts for enamel and odontoblasts for dentin. Much attention has been given to factors involved in epithelial–mesenchymal interactions, including fibroblast growth factors (FGFs), sonic hedgehog (SHH), bone morphogenetic proteins (BMP)s, Wnts and associated receptors, as well as downstream transcription factors such as distal‐less homeobox (Dlx), Msx, AP‐1 factors, Pax‐9, and runt‐related transcription factor 2 (RUNX 2). Many of the knockout (KO) models developed to understand the specific roles for these genes during tooth development have resulted in severe phenotypes (and sometimes death in utero) because of the critical role these genes/proteins play during early development. This has in some cases

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prevented any detailed analysis of the teeth, because if the animals survived, tooth development was arrested at stages prior to root formation. Consequently, there is limited direct evidence for the role of such genes and their products in R/PT formation (Zhao, 2003). Several excellent reviews on the roles of specific genes and signaling molecules during crown development are available (Jernvall and ThesleV, 2000; ThesleV and Aberg, 1999; ThesleV and Mikkola, 2002; ThesleV et al., 1995, 2003; Tucker and Sharpe, 2004; Zhang et al., 2005). One major finding from research targeting crown development has been the recognition of niche areas, specifically the enamel knot, and cervical loop/apical bud regions, where a plethora of genes crucial for regulating crown development are expressed. While the aforementioned studies have focused primarily on molecules regulating crown development, they have also raised new questions related to investigation into R/PT development. Specifically, do ‘‘niche’’ regions for R/PT development exist, similar to the enamel knot region? And can we identify the signaling molecules that regulate cell diVerentiation toward a cementoblast, PDL cell, and osteoblast cell fate? Discussed below and described in Tables II–V are factors, both well‐established and putative, that have been implicated in regulating R/PT development, with an emphasis on cementum formation. Some molecules that have been reviewed previously (Bosshardt, 2005; Bosshardt and Nanci, 1997; Diekwisch, 2001; Popowics et al., 2005; Saygin et al., 2000) or implicated in R/PT formation with limited evidence to support their function in root development are mentioned below, but not included in the tables.

A. Factors Associated with the Putative Epithelial Niche (HERS and ERM) and Surrounding Mesenchyme After crown formation is completed, but prior to eruption, the outer and inner epithelia form a double‐layered sheath called Hertwig’s epithelial root sheath (HERS), and proliferate apically to outline the form the root will take, as detailed in the previous section. It is generally accepted that HERS cells of the inner enamel epithelium (IEE) regulate cells of the dental papilla to diVerentiate into odontoblasts and secrete matrix proteins required for forming root dentin, yet the events and molecular factors directly responsible for this sequence of events remain unknown (Thomas, 1995). With further studies, specific factors critical for directing root dentin formation may be identified (Table II). The role of HERS with regard to cementum formation is even less clear, and hypotheses are discussed in the previous section and summarized in Fig. 2. Hypotheses discussed above include the induction of cementogenesis by epithelial products from HERS, epithelial–mesenchymal transformation

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of HERS cells into a cementoblasts, and potential for diVerent contributions in the development of acellular versus cellular cementum. After development of the R/PT, remnant epithelial cells may reside in mature PDL as epithelial cell rests of Malassez (ERM). Several groups have begun to characterize HERS cells and their proteins to determine the contribution of these cells/factors during tooth root development. These investigations have demonstrated unique signaling molecules within the HERS region, with preliminary evidence of a unique niche region. A specialized structure termed the ‘‘apical bud’’ in the continuously erupting rodent incisor has been identified as an attractive candidate for consideration as an epithelial stem cell niche region for the crown (labial) side, with a less well‐defined region on lingual side, that still may serve as a niche region (Harada and Ohshima, 2004). In order to confirm that the HERS and labial epithelial regions are stem cell niche regions, specific gene signals and target cells need to be further characterized, and the possibility for epithelial‐ mesenchymal transformation in some select group of cells considered. Some of the investigations to date that support a role for HERS/epithelial products in R/PT formation are discussed below and also presented in Table II. Signaling between cervical dental tissues directs the behavior of cells and decision to act toward formation of crown versus root tissues. In the case of continuous crown formation, dental papilla cells surrounding the cervical loop express BMP‐4 and FGF‐10 (Table II). By regulating cell division, FGF‐10 promotes survival of epithelial stem cells within the cervical loop and allows continuous growth of rodent incisors. The data to date provide evidence that the absence of these signals from the cervical region of mouse molars, teeth of limited eruption, switches cellular activities from crown formation to root formation (Tummers and ThesleV, 2003; Yokohama‐ Tamaki et al., 2006). Signaling between HERS cells and adjacent mesenchyme occurs during root formation and appears to regulate the proliferation and diVerentiation of both epithelial and mesenchymal cells. Because the BMP–Msx pathway is known to elicit reciprocal interactions between epithelial and mesenchymal cells during early tooth development, Yamashiro and colleagues examined the expression of BMP–Msx signaling pathway molecules within the HERS region (Yamashiro et al., 2003). During tooth morphogenesis, expression of BMP‐4 in the apical mesenchyme precedes Msx2 expression in the root sheath. None of the BMPs (i.e., BMP‐2, 3, 4, or 7) were detected in the root sheath epithelium, nor transcripts for Msx1 or 2 in the mesenchyme. In contrast, Yamamoto and colleagues found BMP‐2 and 4 expression in HERS cells (Yamamoto et al., 2004a), suggesting that these signaling molecules may play a role in developing root shape. In relation to cell diVerentiation, BMP‐2 and 7 are transiently expressed in both preodontoblasts and diVerentiating odontoblasts, and may signal epithelial diVerentiation and/or have

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autocrine or paracrine eVects on diVerentiating odontoblasts (Yamashiro et al., 2003). Msx2 null mice have been reported to have irregularly shaped molar roots, although the extent of these alterations is not clear (Satokata et al., 2000). The expression of Msx2 in cells within the HERS region and of BMPs in the surrounding mesenchyme/follicle cells, coupled with the root phenotype in Msx2 KO mice, suggests that these molecules play a role in regulating root patterning and cell diVerentiation similar to their function during crown development. Additional signals between HERS cells and mesenchyme control root elongation. Sonic hedgehog (SHH) expression within HERS cells is thought to signal target genes Patched1 (Ptc1) and Gli1 and promote proliferation of dental mesenchyme (Nakatomi et al., 2006). PTC mutants show reduced cell division within dental mesenchyme, shortened tooth roots, and disturbances in tooth eruption. Autocrine or paracrine expression of insulin‐like growth factor‐1 (IGF‐1) by HERS cells has also been implicated in regulating root elongation. IGF‐1 receptors are present on HERS cells, and application of IGF‐1 to mouse HERS organ cultures, in vitro, resulted in elongation of the root sheath, possibly from increased cell proliferation from the OEE (Fujiwara et al., 2005). Some enamel proteins, including amelogenins and ameloblastin, in addition to their roles in crown formation (Section II and Table I), have been proposed as regulators of R/PT formation (Table II). Reports have indicated expression of these molecules in the HERS region and in the pulp region, although at low levels and with a great deal of variability between species (Bosshardt and Nanci, 1997, 2004; Fong and Hammarstrom, 2000; Janones et al., 2005; Nebgen et al., 1999; Oida et al., 2002; Papagerakis et al., 2003; Zeichner‐David et al., 1997). Amelogenin KO mice exhibit a tooth phenotype resembling amelogenesis imperfecta (AI) in humans, namely hypoplastic enamel characterized by poorly organized hydroxyapatite crystals, resulting in chalky‐white, fragile teeth (Gibson et al., 2001a). Additional studies of amelogenin KO mice have implicated a role for amelogenin in root development or maintenance. Increased osteoclastic root resorption (Hatakeyama et al., 2003) and decreased BSP expression (Viswanathan et al., 2003) by root surface cells have been reported in amelogenin KO mice compared to controls. It has been further proposed that amelogenin and the alternatively spliced product LRAP may be involved in regulating levels of receptor activator of NF‐B ligand (RANKL) within the local tooth root environment, thereby acting as a protector of against osteoclast‐mediated resorption of the root surface (Hatakeyama et al., 2006). In amelogenin KO mice, RANKL on the tooth root surface was increased, as determined by immunohistochemistry. Binding of RANKL (secreted from osteoblasts, PDL cells, cementoblasts, etc.) to RANK on osteoclast precursor cell surfaces results in maturation/activation to functioning osteoclasts. Further,

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it was reported that adding LRAP to cocultures of PDL/cementum cells and bone marrow cells resulted in decreased RANKL expression. Boabaid et al. additionally report that LRAP increased OPG and had a slight eVect (not statistically significant) on decreasing RANKL expression in immortalized cementoblasts, in vitro (Boabaid et al., 2004b). The epithelial cell rests of Mallasez (ERM) that correspond with the remnants of the root sheath within the periodontal ligament have been known to express transcription factors and signaling molecules including OPN, and BMP‐2 and 4 (Mouri et al., 2003; Rincon et al., 2005). These findings have led some researchers to suggest that ERMs aVect periodontal tissues within the local environment (Table II). Additional evidence that ERMs may have a role in repair of root tissues has been provided by Hasegawa et al., who reported that in early stages of cementum repair and adjacent to sites of root resorption, ERM cells express OPN, ameloblastin, and BMP‐2, molecules associated with regulation of mesenchymal cell behavior (Hasegawa et al., 2003). While the potential role of epithelial products in root development warrants further study, EmdogainÒ (Straumann Biologics, Waltham, MA, USA), an epithelial protein derivative aimed at regenerating periodontal tissues has been in use clinically for many years. EmdogainÒ, an extract of porcine tooth germs, promotes periodontal regeneration with varied reports of successful outcomes (Bartlett et al., 2006; Esposito et al., 2005; Giannobile and Somerman, 2003; Heden and Wennstrom, 2006; Venezia et al., 2004). In vitro studies have suggested that EmdogainÒ may preferentially promote proliferation, matrix production, and diVerentiation/mineralization in PDL fibroblasts (Gestrelius et al., 1997; Lyngstadaas et al., 2001), with increased proliferation but varying influence on gene expression in dental follicle cells (Hakki et al., 2001), osteoblasts, and putative cementoblasts (Tokiyasu et al., 2000). While the predominant protein in EmdogainÒ is amelogenin, several other factors have been reported, including alternatively spliced and proteolytically cleaved amelogenins, LRAP and TRAP respectively, as well as ameloblastin, TGF‐ , and BMPs (Kawase et al., 2001, 2002; Maycock et al., 2002; Suzuki et al., 2005; Takayama et al., 2005). Both in vitro and in vivo EmdogainÒ and the amelogenins have been confirmed to have bioactive signaling properties (Bartlett et al., 2006; Boabaid et al., 2004b; Esposito et al., 2005; Giannobile and Somerman, 2003; Swanson et al., 2006; Tompkins and Veis, 2002; Tompkins et al., 2005; Veis, 2003; Venezia et al., 2004; Viswanathan et al., 2003). Evidence for a signaling role for amelogenins has been bolstered by identification on myoblast cells of an LRAP‐interactive cell surface binding protein, lysosomal adhesion membrane protein‐1 (LAMP‐1) (Tompkins et al., 2006). Using a yeast two hybrid system, enamel matrix proteins were also found to interact with a large number of secreted and

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membrane proteins, notably LAMP‐3 (Wang et al., 2005a). Further studies of these enamel matrix proteins will help to clarify the significance of these putative receptors and interacting proteins, and subsequently elucidate their signaling role, if any, in root/periodontal tissue formation.

B. Bone Morphogenetic Proteins The importance of BMPs and BMP antagonists for contolling crown development is well established (Bei et al., 2000; Ferguson et al., 1998; Iwata et al., 2002; Kratochwil et al., 1996; Laurikkala et al., 2003; Maas and Bei, 1997; Zhang et al., 2000). More recent studies have provided insights into the interactions of various BMPs and their associated antagonists during bone and crown development, for example, negative regulators ectodin and follistatin. However, the significance of these interactions during root development warrants further investigation (Kassai et al., 2005), especially with the awareness that BMPs have very complex interactions in other tissues. For example, BMP‐2, 4, 7 can induce ectopic bone formation individually, but BMPs also form heterodimers, for example, BMP‐2/7, and 4/7, with greater mineral stimulating ability than their constituents (Franceschi, 2005) (Tables II and III). As one approach to defining the role of BMPs in tooth development, Plikus and colleagues evaluated the eVects of downregulating expression of BMP signaling in oral and dental tissues by creating keratin 14‐Noggin transgenic mice (Plikus et al., 2005). These mice developed a wide spectrum of tooth phenotypes that included abnormal histogenesis and diVerentiation of ameloblasts and odontoblasts, a decrease in the number of teeth developed, reduction and/or alteration in size and shape of teeth, as well as changes in the size and shape of roots. Root alterations included failure of molar teeth to form multiple roots and lack of definition in the cementum‐ enamel junction (CEJ) region. Using a similar strategy to better define the role of gremlin, a BMP antagonist, in osteoblastic diVerentiation and function, Gazzero and colleagues generated mice with conditional gremlin overexpression by employing an osteocalcin promoter driven—gremlin construct (Gazzerro et al., 2005). Mice overexpressing gremlin exhibited an osteopenic bone phenotype that included impaired bone formation, bone fractures, disorganized collagen bundles at the endosteal cortical surface, a marked decrease in osteoblast numbers, and reduced mineral apposition and bone formation rates versus WT littermate controls. In addition, although not detailed, incisor teeth were observed to be fragile in gremlin overexpressing mice compared to WT controls. BMP‐3 has emerged as a signaling factor that unlike other BMPs that promote osteoblast/cementoblast, odontoblast diVerentiation, antagonizes

Table III Factor Periostin

Factors Associated with Reported Root Phenotypes Cells/Tissues

Function/Putative Function

Models

References

Preferentially expressed in cells associated with bone, lung, kidney, heart valve, although found in many other tissues, including cancerous tissues. Also expressed at high levels in embryonic periosteum Teeth: PDL region (restricted to PDL after postnatal day 7 in mice) Epithelia Papillae cells Odontoblasts Follicle cells Alveolar bone region

Periostin is a secreted 90‐kDa protein with strong homology to the insect cone guidance protein fasciclin I family (includes ig‐h3). Periostin is thought to be involved in cell adhesion via v 3 and 5 integrins, although periostin does not contain an RGD motif. Exact function for this protein remains to be established Suggested general functions include: (1) Induction of angiogenesis, (2) Regulation of hard‐soft tissue interfaces, (3) Regulation of deposition and organization of other ECM molecules, (4) Protective function during stress/ mechanical load (e.g., in teeth: chewing/tooth movement/ tooth eruption), and (5) promotion of cell migration and adhesion. Periostin gene and protein are reported to be induced by several factors including BMP‐2, TGF‐ , PDGF, and angiotensin II In humans, five alternatively spliced forms have been identified, but functions not established

Periostin KO: exhibits a strong tooth/ periodontal phenotype: widening of PDL; root resorption; increased osteoclasts all suggestive of aggressive periodontal disease. Further, the incisors exhibit compression of enamel and dentin suggested to be related to a proposed role for periostin in controlling shear force/collagen degradation At birth, KO mice appear normal but many die before weaning and those that survive have growth retardation. Trabecular bone is decreased, although this phenotype is not as dramatic as that of the tooth. Partial correction of defects, most notably incisor enamel, is noted when the animals are given a soft diet Mice with periostin overexpression exhibit cardiac dilation and dysfunction BMP‐4 KO mice have decreased levels of periostin in mesenchymal tissues and MSX2 KO mice have increased evels of periostin

Gillan et al. (2002); Horiuchi et al. (1999); Kii et al. (2006); Kruzynska‐Frejtag et al. (2004); Li et al. (2005a); Rios et al. (2005); Suzuki et al. (2004); Wilde et al. (2003)

NF1‐C/CTF (nuclear factor I‐C) NF1 protein family of site‐specific DNA‐binding proteins (also known at CTF or CAAT box transcription factor)

General: found in many tissues during development and in mature tissues as well Tooth associated include: dental papilla region Ameloblasts Odontoblasts/ preodontoblasts (strong expression during root formation) Mesenchymal cells Stellate reticulum region PDL region Bone region HERS

Suggested function: in NF1‐C KO mice, HERS cells fail to proliferate and/or fail to induce odontoblast diVerentiation required for root formation. However, the specific transcription factors and cell‐ signaling pathways disrupted in cells from NF1‐C KO mice remain to be defined NF1 protein family: functions both in viral DNA replication and in the regulation of gene expression

NF‐1C KO: defective root development Incisors: Maxillary: KO mice fail to form roots but enamel and dentin appear normal Mandible: more severely aVected; histologically, disorganized tissues occur in place of incisors Molars: crowns form, but no root development is seen. Jaw bones seem normal although during preparation of heads for histology, the teeth fall out and sockets are shallow with an organized mesh of bony spicules vs. WT tissues with deep sockets and intact teeth. Also, mandibles appeared approx. 10% smaller vs. WT, but maxillary size diVerences were not reported Gene expression: Noted decreased expression (50%) of tooth‐associated genes dentin sialoprotein, ameloblastin, amelogenin in mandibles of KO vs. WT; but normal transcripts for 1 type I col and Nfia, b, x

Steele‐Perkins et al. (2003, 2005)

(Continued )

Table III

Continued

Factor DMP‐1 (dentin matrix protein‐1)

Cells/Tissues Odontoblasts, osteoblasts, osteocytes, hypertrophic chondrocytes, cementoblasts, cementocytes, cementum matrix, brain neurons, other tissues (salivary glands, muscle)

Function/Putative Function

Models

References

Role in mineral formation: dentin matrix assembly and crystal growth Multifunctional: attachment, diVerentiation, activation of MMP‐ 9, role in osteocyte response to mechanical stress

DMP‐1 KO: Bone and cartilage: decrease in mineral to matrix ratio, increase in crystal size in bones, osteomalacia, disordered canalicular network, cartilage defect, chondrodysplasia‐ like phenotype Newborn DMP‐1 KO mice have slightly expanded hypertrophic zones and modest increase in bone diameter vs. WT—DMP‐1 not essential for early bone and tooth development Tooth: reduced dentin, increase in predentin; reduced rate of dentin formation; abnormal dentin tubules; delay in/absent 3rd molars; cementum—cementocytes not healthy, but details on root defects have not been reported Overexpression of DMP‐1 in pluripotent and mesenchymal cells promotes an odontoblast phenotype

Almushayt et al. (2006); Baba et al. (2004b); Feng et al. (2003); Foster et al. (2006); Kalajzic et al. (2004); Ling et al. (2005); Ye et al. (2005)

BMP‐3 (bone morphogenetic protein 3)

Osteoblasts Follicle cells Cementoblasts PDL cells

BMP‐2/4 are expressed in all tissues involved in tooth formation, while BMP‐3 expression is limited to the follicle region and later in development to the PDL region. Existing data suggest that BMP‐3 acts as an antagonist to BMP‐2/4. Based on this data it has been suggested that BMP‐3 acts as a regulator of soft–hard tissue interfaces

BMP‐3 KO: increased bone mass, however no specific tooth phenotype has been reported BMP‐3 overexpression: a 2006 abstract from Gagari et al. reported decreased mass of dentin and cementum, enlarged pulp chambers, and widened PDL in BMP‐3 transgenic mice vs. WT littermate controls

Aberg et al. (1997); Chen et al. (2004); Daluiski et al. (2001); Gagari et al. (2006); Gamer et al. (2005); Zhao (2003)

BMPs/BMP antagonists

All cells associated with tooth development and maturation

Known to have major roles in controlling patterning and diVerentiation of odontoblasts and ameloblasts, BMPs have been shown to enhance regeneration of periodontal tissues (Nifusojeckii, other reviews), but their specific role during development of root/ periodontal tissues has not been reported

Keratin14‐Noggin transgenic mice: significant tooth phenotype with alterations in number, size, shape, and cell diVerentiation Root specific: no mandibular molars, maxillary molars—fail to form multiple roots, poor or no CEJ, small teeth with limited roots, HERS forms, but proliferation of cells in this region is limited Osteocalcin‐gremlin transgenic mice: General: impaired bone formation and osteopenia Tooth Phenotype: not explored in depth but reported tooth fragility

Aberg et al. (1997); Gazzerro et al. (2005); Nadiri et al. (2004); Nifuji and Noda (1999); Plikus et al. (2005); Ripamonti (2005); Yanagita (2005)

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these BMPs and so inhibits cementoblast and osteoblast function (Bahamonde and Lyons, 2001). Gagari et al. (to date in abstract form only) have reported that collagen type 1 promoter‐driven BMP‐3 transgenic mice exhibited a tooth phenotype that included decreased cementum and dentin mass, enlarged pulp chambers, and a widened PDL region (Gagari et al., 2006). In contrast, BMP‐3 null mice have been reported to have decreased bone density, but a tooth phenotype was not reported (Daluiski et al., 2001). Additionally, Gamer and colleagues found that BMP‐3 interferes with both activin and BMP signaling by binding to AcTRIIB, the common type II receptor for BMPs (Gamer et al., 2005). These data, coupled with previous studies demonstrating high expression for BMP‐3 in the follicle/PDL region, support a role for BMP‐3 in regulating hard–soft tissue interfaces in the periodontium (Aberg et al., 1997; Ripamonti and Reddi, 1997; Takahashi and Ikeda, 1996; Yamashiro et al., 2003). The existing evidence is strong that BMPs and associated antagonists are critical for R/PT development and future studies in this area may contribute to novel therapies for regenerating tooth structures.

C. Periostin and Nuclear Factor I‐C/CAAT Box Transcription Factor While many of the molecules described in this section play a role in mineralized tissue development, two molecules that may have a more specific critical role in root/periodontal tissue development have been identified from their respective KO mouse phenotypes, which were remarkable for the condition of their periodontia. These molecules include the extracellular matrix protein, periostin, and the nuclear factor I‐C/CAAT box transcription factor, NFI‐C/CTF. Periostin has been detected in many tissues (Nakamura et al., 2005), but mice null for periostin exhibit a very specific tooth phenotype (Rios et al., 2005) (Table III). Mice lacking the periostin gene appear relatively normal at birth but develop a condition resembling an aggressive form of periodontal disease by 3 months of age. The defect seems to be selective to the root/ periodontal region, with odontoblasts and dentin only mildly altered when compared with the periodontal apparatus. Some enamel defects are observed, but are limited to incisors and suggested to be associated with increased enamel stress due to a weakened PDL (Rios et al., 2005), or possibly due to disruption of the shear zone important for continuously erupting teeth (Kii et al., 2006). These data suggest the periostin may play a key role in protecting the root surface from root resorption, as well as for maintaining a functional periodontal ligament. The knowledge gained from continued studies directed at defining the factors regulating periostin expression during root formation, and the relationship between periostin and other root/PDL‐associated extracellular

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matrix molecules should prove valuable long‐term for designing regenerative therapies. The nuclear factor I‐C/CAAT box transcription factor (NFI‐C/CTF) KO mice have provided new insights into molecules that may be involved in directly regulating root development. There are four genes encoding nuclear factor I transcription‐replication proteins in mammals, NFI‐A, ‐B, ‐C, and ‐X. Members of the NFI protein family of site‐specific DNA‐binding proteins function both in viral DNA replication and in the regulation of gene expression. NFI‐C/CTF contains a prototypical proline‐rich transcription activation domain and a heptamer repeat that is homologous to the C‐terminal domain of RNA polymerase II (Gronostajski, 2000). To elucidate the physiological roles for this family of nuclear transcription factors, Gronostajaski’s laboratory has focused on disrupting their expression (Steele‐Perkins et al., 2003, 2005). Although NFI‐C is expressed in many organ systems, including developing teeth, disruption of the Nfic gene in mice resulted primarily in a unique tooth phenotype: molars lacking roots, thin and brittle mandibular incisors, and weakened and abnormal maxillary incisors. Molar crown development is normal and animals on a soft diet are fertile and live as long as their littermates (Steele‐Perkins et al., 2003).

D. Regulators of Phosphate and Pyrophosphate Metabolism 1. Progressive Ankylosis Protein, Plasma Cell Membrane Glycoprotein 1, and Tissue Nonspecific Alkaline Phosphatase Regulators of phosphate metabolism have received considerable attention within the last 5 years, with convincing evidence that inorganic phosphate (Pi), beyond its known role as an important component of hydroxyapatite mineralization, may also regulate cell behaviors and mineralization as a signaling molecule. Conversely, pyrophosphate (PPi) functions as a well‐ known and potent inhibitor of hydroxyapaptite formation. Table IV provides information on the role of Pi and PPi associated genes and their protein products in regulating mineralized tissues. These include mouse progressive ankylosis gene (ANK, as well as human homolog, ANKH), a putative transporter of PPi from the intracellular compartment to the extracellular space (Ho et al., 2000), the PPi‐generating nucleoside triphosphate pyrophosphohydrolase plasma cell membrane glycoprotein‐1 (PC‐1) (Goding et al., 1998), and tissue nonspecific alkaline phosphatase (TNAP), an enzyme proposed to cleave PPi substrate to its Pi constituents (Whyte et al., 1995). Further information regarding the functions of these proteins is provided in Table IV, including implications of their roles based on mouse models, for example, mutation or KO. Results from studies to date suggest that local

Table IV Regulators of Phosphate (Pi) and Pyrophosphate (PPi) Metabolism Factor

Cells

Function/Putative Function

Models

References

PC‐1: (NPP1—nucleotide pyrophosphatase phosphodiesterase‐1) gene symbol: Enpp1 ANK: proteins of mouse progressive ankylosis (ank) gene. (ANKH, human homologue) gene symbol: Ank

Expressed by a diverse group of cells including osteoblasts, PDL cells, odontoblasts, follicle cells, and cementoblasts, among others

PC‐1: increases intra/ extracellular and matrix vesicle PPi; inhibits apatite deposition ANK: transporter/ cotransporter of PPi from intracellular to extracellular matrix; inhibits apatite deposition

Fedde et al. (1999); Fong et al. (2005); Harmey et al. (2004); Ho et al. (2000); Johnson et al. (2003); Nociti et al. (2002); Nurnberg et al. (2001); Okawa et al. (1998); Reichenberger et al. (2001); Rutsch et al. (2000); Rutsch et al. (2001); Terkeltaub (2001)

TNAP: (TNSALP, ALKP, ALK) tissue nonspecific alkaline phosphatase gene symbol: Akp2

Expressed by a diverse group of cells including osteoblasts, PDL cells, odontoblasts, follicle cells, and cementoblasts, among others

A marker of cell (osteoblast/ cementoblast) diVerentiation; catalytic function in mineralization; may transport ions across membrane; hydrolyzes the mineralization inhibitor, PPi

PC‐1/ANK mutations: In animals/humans with mutations in these genes, ectopic calcification and decreased extracellular PPi occur. In murine models with mutations in either PC‐1 or Ank, there is a marked increase (10‐fold) in cementum formation (appears to be cellular), while dentin, enamel, PDL region, and surrounding alveolar bone appear normal (Fong et al., 2005; Nociti et al., 2002). There have been no reports of a tooth phenotype in humans TNAP mutations: In animals/humans there are a variety of forms of TNAP‐associated hypophosphatasia, which result in locally increased levels of PPi and osteopenia. In the area of the tooth root, cementum deficiency severely compromises anchoring of the PDL between bone and the tooth, with severe periodontal disease and eventually tooth loss TNAP KO: appears to aVect cementum formation selectively vs. dentin/enamel. There is no or minimal cementum formation and hence no PDL attachment and teeth are exfoliated

Chapple (1993); Beertsen et al. (1999); van den Bos et al. (2005); Whyte, (2002)

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control of PPi/Pi is critical for normal root/periodontal tissue development, and further that cementum may be a uniquely sensitive tissue to PPi and Pi in the local area. In cases of TNAP deficiency (TNAP mutation or KO, the condition hypophosphatasia in humans), bones are osteopenic and root cementum is disrupted, generally with a lack of acellular cementum and severely disrupted cellular cementum (Beertsen et al., 1999; van den Bos et al., 2005). Lack of cementum prevents insertion of PDL (Sharpey’s) fibers, leading to lack of attachment and exfoliation of teeth. In contrast, humans and animals with loss of function of PC‐1 or ANK exhibit low levels of PPi in the local extracellular environment, resulting in ectopic calcifications in joints, with mice exhibiting an arthritis‐like condition (Ho et al., 2000; Terkeltaub, 2001). Humans with mutations in these genes also present pathologies resulting from deficient PPi, including craniometaphyseal dysplasia (CMD) and idiopathic infantile arterial calcification (IIAC) (Nurnberg et al., 2001; Reichenberger et al., 2001; Rutsch and Terkeltaub, 2005; Rutsch et al., 2001). An unexpected and intriguing tooth phenotype has been reported in mice with mutations in either PC‐1 or ANK. Rather than observing ectopic calcification in the PDL, a marked increase in cementum formation was observed, while PDL, dentin, and alveolar bone appeared unaVected (Nociti et al., 2002). Ongoing studies are directed at examining the mechanical and structural properties of all of these mineralized tissues, under situations in mice, where PPi and/or Pi have been altered. The importance of maintaining the appropriate concentration of Pi in the extracellular environment for regulation of mineralization was highlighted by the elegant studies of Murshed and colleagues (Murshed et al., 2005). By studying a variety of KO mice, the critical nature of modulating extracellular Pi concentration both for regulating physiological mineralization and preventing pathological calcification was described. However, details on R/PT regulation and development were not included, and require further analysis using the animal models developed by this group. Studies focusing on teeth and surrounding regions may provide insight into mechanisms leading to altered cementum versus apparently normal enamel and dentin phenotypes in cases of altered Pi/PPi homeostasis. In addition to ANK, PC‐1, and TNAP, several other Pi‐regulating proteins have been demonstrated to be important in controlling mineralization, although tooth phenotypes in some cases have not yet been reported. These are described in the following sections. 2. Phosphate‐Regulating Gene with Homology to Endopeptidase on the X‐Chromosome X‐linked hypophosphatemic rickets (XLH/HYP), the most common form of rickets in humans, is caused by a mutation in the PHEX gene. The murine

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homologue (Hyp mice) of the human disease is marked by renal phosphate wasting, abnormal regulation of vitamin D metabolites, rickets, osteomalacia, growth retardation, resistance to vitamin D therapy, hypophosphatemia, and high levels of FGF‐23 and MEPE (Quarles, 2003; Rowe, 2004). Additionally, cartilage abnormalities were reported in Hyp mice, resulting potentially from participation of PHEX in regulation of growth plate cartilage (Miao et al., 2004). Dentin and bone hypomineralization in Hyp mice may result from not only low serum Pi, but also some intrinsic osteoblast/ odontoblast defect (Ogawa et al., 2006). A tooth root phenotype has not been reported. 3. Fibroblast Growth Factor 23 FGF‐23 regulates phosphate homeostasis, and FGF‐23 mutation in humans causes autosomal dominant hypophosphatemic rickets (ADHR) (Rowe, 2004; White et al., 2006; Yu and White, 2005). FGF‐23 null mice exhibit growth retardation, hyperphosphatemia, increased levels of 1, 25 vitamin D levels, increased total‐body Bone Mineral content but decreased Bone mineral density of limbs, and premature death by 13 weeks of age (Razzaque et al., 2006; Sitara et al., 2004). Although a general increase in mineralization resulted, there was also an accumulation of unmineralized osteoid associated with limb deformities and excessive mineralization of soft tissues such as heart and kidney. Crossing of FGF‐23 null with Hyp mice (PHEX mutation, equivalent to X‐linked hypophosphatemia) resulted in a mouse phenotype resembling the FGF‐23 null in skeletal phenotype and serum phosphate, suggesting that FGF‐23 is upstream of PHEX (Sitara et al., 2004). There has been some evidence for PHEX involvement in degradation of FGF‐23 (Bowe et al., 2001), and results from the Hyp/FGF‐23 null mice are consistent with the hypothesis that increased FGF‐23 in PHEX mutated mice and humans may be responsible for the observed Pi disorder (Sitara et al., 2004). 4. Matrix Extracellular Phosphoglycoprotein Matrix extracellular phosphoglycoprotein, a member of the SIBLING extracellular matrix protein family (Fisher and Fedarko, 2003) is expressed by several mineralized tissue‐associated cells, including osteoblasts and osteocytes, hypertrophic chondrocytes, dental pulp cells, and odontoblasts, as well as other tissues (Argiro et al., 2001; Liu et al., 2005b; Lu et al., 2004; MacDougall et al., 2002; Nampei et al., 2004; Rowe et al., 2000). Increased expression of MEPE protein has been noted in humans with XLH and their Hyp mouse counterparts (Argiro et al., 2001; Guo et al., 2002; Liu et al., 2005c; Rowe, 2004). MEPE is thought to control renal phosphate excretion and to modulate mineralization. MEPE contains an acidic serine aspartic

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rich motif, ASARM, that is cleaved by cathepsin B, and PHEX inhibits this activity (Rowe et al., 2004). One hypothesis is that the ASARM motif inhibits crystal growth, with MEPE KO mice therefore exhibiting accelerated mineralization and bone formation. Interestingly, it was reported that dentonin, a fragment of MEPE isolated from erupted human molars promoted the proliferation of dental pulp stem cells, in vitro, and it was speculated that such molecules may have potential to participate in repair of lost or damaged dentin (Liu et al., 2004).

E. Factors Known to Regulate Osteoprogenitor Cells and Osteoblasts 1. Wnt, Hedgehog, Osterix, and Nuclear Factor of Activated T Cells Pluripotent mesenchymal stem cells (MSCs) have the potential to diVerentiate into several diVerent cell types, and specific transcription factors that have been found to commit MSC diVerentiation to the osteoblast lineage require further study for their potential role in root and periodontal tissue development. Sequential expression of Indian hedgehog (Ihh) and canonical Wnt signals at progressive stages of osteoblast development has been found to coordinate the expression of transcription factors directing osteoblast diVerentiation (Hu et al., 2005). For example, osterix (Osx) regulates downstream genes that commit MSCs to an osteoblast lineage, while NFAT was found to cooperate with Osx to accelerate osteoblast diVerentiation and bone formation (Koga et al., 2005; Tai et al., 2004, 2005). Further, the expression of p53 had been found to repress Osx expression, inhibiting osteoblast diVerentiation and favoring osteoblast contributions to osteoclastogenesis (Wang et al., 2006) (Table V). 2. Runt‐Related Transcription Factor 2 and TaVazin Runt‐related transcription factor 2 (Runx2) expression is also necessary for osteoblast diVerentiation and function and a role in developing tooth crown has been identified (Aberg et al., 2004a,b; D’Souza et al., 1999). In osteoblasts, Runx2 directly stimulates transcription of osteoblast‐related genes such as OCN, type I collagen, OPN, and collagenase type III (Ducy et al., 1997; Franceschi et al., 2003; Kern et al., 2001). Canonical Wnt signaling upregulates Runx2 expression (Gaur et al., 2005), and Runx2 subsequently coordinates diverse signals involved in osteoblast diVerentiation and activity (Franceschi et al., 2003). For example, the transcription factor, taVazin (TAZ), is an endogenous co‐activator of Runx2 in cells, and therefore an endogenous regulator of osteoblast diVerentiation (Cui et al., 2003). Interestingly, TAZ simultaneously represses gene transcription associated with

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the adipocyte diVerentiation pathway (Hong et al., 2005). Stimuli that promote bone formation via regulation of transcription have been found to upregulate both TAZ and Runx2 (Hong et al., 2005). Runx2 expression during tooth development also has several tooth‐specific downstream targets (Gaikwad et al., 2001). The formation of successional teeth is inhibited by Runx2 activity (Wang et al., 2005c), and during tooth morphogenesis Runx2 mediates FGF signaling between epithelium and mesenchyme (Aberg et al., 2004b). Runx2 has also been identified in periodontal ligament cells; however, its function appears to be suppressed, preventing diVerentiation of PDL cells toward osteoblasts (Saito et al., 2002). 3. Activating Transcription Factor 4 Activating transcription factor 4 (ATF4) was identified as a factor for osteoblast diVerentiation, and it is in turn the substrate for p90 ribosomal S6 kinase 2 (RSK2), a growth factor‐regulated kinase (Yang et al., 2004). A mutation in RSK2 was mapped as the cause for CoYn‐Lowry Syndrome (CLS), which is associated with skeletal abnormalities in addition to mental retardation. Mice null for ATF4 displayed a phenotype indicative of osteoblast defects, namely delayed bone formation in early development and low bone mass. In addition to regulating type I collagen, ATF4 was found to act cooperatively with Runx2 in regulating the OCN promoter in osteoblasts (Xiao et al., 2005). No details on a tooth phenotype were provided. 4. Receptor Activator of NF‐kB Ligand and Osteoprotegerin Receptor activator of NF‐B ligand (RANKL) and osteoprotegerin (OPG) have emerged as the primary factors in the axis of regulation of osteoclasts and their precursors (Takahashi et al., 1999; Tsuda et al., 1997), and parallel roles in tooth development and eruption have been described (Ohazama et al., 2004a; Rani and MacDougall, 2000; Wise et al., 2002). Little is known of the role of cementoblasts in osteoclast‐mediated turnover. Cementoblasts, as well as cells of the nearby PDL and their precursors, express RANKL and OPG (Boabaid et al., 2004a; Liu et al., 2005a; Sakata et al., 1999) and so may be considered to take part in the regulation of osteoclastogenesis, though it is currently not clear how these tissues participate in conditions of health and disease. Cementum itself does not undergo significant physiological remodeling unlike the nearby alveolar bone that undergoes rapid turnover throughout life. Additionally, though cementum resorption is not unknown, it is relatively infrequent compared to osteoclast‐mediated resorption of bone. Even in advanced periodontal disease, alveolar bone may be severely eroded while cementum remains intact. This indirect evidence supports a protective role for cementum against biological resorption, a hypothesis supported

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by results from in vitro (Boabaid et al., 2004a; Hatakeyama et al., 2006; Nociti et al., 2004) and in vivo investigations (Hatakeyama et al., 2003). Further study of the involvement of cementoblasts and PDL in osteoclastogenesis should elucidate their role in osteoclastogenesis in the periodontium.

5. Dlx Transcription Factors Members of the Dlx family of transcription factors, a subfamily of divergent homeobox genes related to the Drosophila distal less (Dll) gene, have been implicated as key regulators of tissue development and cell diVerentiation (Stock et al., 1996). Currently, six Dlx genes have been identified, in both humans and mice, with convincing evidence that they play critical roles in diVerentiation of bone‐forming cells. Dlx KO mice have profound craniofacial defects and absence of molars (Thomas et al., 1997). In humans, tricho‐dento‐osseous (TDO) syndrome, characterized by enamel defects, enlarged pulp chambers, and distorted roots (taurodontism), has been linked to a mutation in Dlx3 (Price et al., 1998). Interestingly, in a very preliminary study, Morsczeck reported that Dlx3 increased in dental follicle cells during ‘‘osteogenic’’ diVerentiation, in vitro.

F. Emerging and Other Factors to Consider 1. Proteoglycans Another important group of molecules present in cementum and known to play critical roles in tooth development are the proteoglycans (PGs). Proteoglycans are macromolecules composed of core proteins and glycosaminoglycans (GAGs). Small leucine rich proteoglycans (SLRPs) including decorin, biglycan, lumican, osteoadherin/osteomodulin, and fibromodulin have been suggested to play important roles in collagen‐linked mineralization (Buchaille et al., 2000; Couble et al., 2004; Embery et al., 2001; Iozzo, 1998). The importance of PGs for appropriate crown development has been highlighted (Goldberg et al., 2005) and indicates that further studies are warranted to determine their respective roles during root formation. Several studies have implicated SLRPs as being important for controlling mineralization of dental tissues, and it was demonstrated that defective enamel and dentin formation resulted from loss of biglycan and decorin in KO mice (Goldberg et al., 2005). Although roles for PGs in root development have not been clarified, SLRPs are apparently present in all mineralized tissues, and it can therefore be safely asserted that there is some important, conserved function for this class of PGs in mineralized tissues. Immunohistochemical studies have shown that

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cementum in several species (rat, human, mouse) is immunoreactive to various PG species (Yamamoto et al., 2004b). PG presence and involvement in development was further supported in a rat molar model by intense immunoreactivity for chondroitin 4‐sulfate, chondroitin 6‐sulfate, and unsulfated chondroitin, during early phases of a cellular cementogenesis. GAGs and PGs have been detected in mature PDL, and taken together these data provide strong evidence that PGs are important for regulating collagen fibril formation during cementogenesis and in the mature periodontium (Buchaille et al., 2000; Hakkinen et al., 1993, 2000; Kaneko et al., 2001; Matias et al., 2003). 2. Small Integrin‐Binding Ligand N‐Linked Glycoprotein Family The SIBLING (Small Integrin‐Binding Ligand N‐Linked Glycoprotein) family is composed of genes located on human chromosome 4 (mouse chromosome 5) encoding noncollagenous extracellular matrix‐associated proteins associated with bones and teeth. While sequence homology among SIBLINGs is not high, their relatedness is suggested by common organizational features and similar, functionally important post‐translational modifications (Fisher and Fedarko, 2003; Huq et al., 2005; Qin et al., 2004). Genes in this family include bone sialoprotein (BSP), osteopontin (OPN), dentin matrix protein‐1 (DMP‐1), dentin sialophosphoprotein (DSPP), and matrix extracellular phosphoglycoprotein (MEPE). The DSPP transcript is processed and results in two proteins, namely dentin sialoprotein (DSP) and dentin phosphoprotein (DPP). SIBLINGs have common sequences including an arginine‐glycine‐aspartate (RGD) integrin‐binding domain that likely functions in signaling and cell attachment (Ganss et al., 1999; Sodek et al., 2000), and an ASARM or similar motif (in all except BSP) that may be a mineral inhibitory domain. SIBLINGs typically undergo extensive post‐ translational modification, including enzymatic cleavage (in DSPP and DMP‐1), phosphorylation, glycosylation, and likely more complex processing such as polymerization, in vivo (Gericke et al., 2005; He et al., 2005b; Kaartinen et al., 2005; Qin et al., 2004). Several lines of evidence support a role for SIBLINGs in root development (Bosshardt, 2005; Diekwisch, 2001; Saygin et al., 2000), including the timed and spatial expression of BSP and OPN during development and repair of root/periodontal tissue (Bosshardt et al., 1998; D’Errico et al., 1997; MacNeil et al., 1994; Shigeyama et al., 1996), coupled with their suggested roles in nucleation and regulation of crystal growth (Boskey et al., 2000; Gericke et al., 2005; He et al., 2005a; Tartaix et al., 2004). However, mice null for OPN, BSP, or MEPE have not been reported to exhibit a root/periodontal phenotype, suggestive of some redundancy with other molecules. DMP‐1 and DSPP, originally identified in dentin and thought to be specific for this tissue, have been shown to not only be critical for dentin development, but also present in other mineralized

Table V

Factors Known to Regulate Osteoprogenitor cells and Osteoblasts (Role in Cementogenesis Unknown)

Factor

Cells/Tissues

Function/Putative Function

Models

References

Wnt: wingless int Hh: hedgehog Ihh: Indian hedgehog

Mesenchymal stem cells (MSCs), many others

Osteoblast diVerentiation; Hh and Wnt signals control osteoblast development in a sequential manner Ihh is expressed in prehypertrophic and early hypertrophic chondrocytes and signals to immature chondrocytes and perichondrial cells. Canonical Wnt signaling is downstream of Ihh signaling During tooth development Hh and Wnt signals are emitted from the cap stage enamel knot and are considered to have a role in crown morphogenesis

Many participants in the Wnt pathway have been associated with mineralized tissues, in health and disease: Dickkopfs (Dkks), secreted frizzled‐ related proteins (sFRPs), Wnt inhibitory factor 1 (Wif1), LDL receptor protein 5 (LRP5), and Wnts 4, 10b, and others Ihh KO: Shows dysregulation of chondrocyte maturation and absence of expression of target genes for the Wnt canonical pathway in the perichondrium Lrp5 KO: Lrp5 is a transmembrane protein that forms part of the cell surface receptor complex binding Wnt within the Wnt canonical pathway. Lrp5 deficient mice exhibit osteopenia with fewer total osteoblasts per bone area, and a 50% reduction in bone formation

Canalis et al. (2005); Hu et al. (2005); Kato et al. (2002); Li et al. (2005b); Nusse (2005); St‐Jacques et al. (1999); ThesleV et al. (2001); Vaes et al. (2005); Westendorf et al. (2004)

Osx: osterix NFAT: nuclear factor of activated T cells

MSCs, osteoblasts

Osterix is a transcription factor required for osteoblast diVerentiation, operating downstream of Runx2; NFAT cooperates with Osx to accelerate osteoblast diVerentiation and bone formation

Osx null mice: Homozygotes for the mutation are not viable, and lack both endochondral and intramembranous bone formation; developing tooth germs appear unaVected NFAT KO: Mice are embryonically lethal

Koga et al. (2005); Nakashima et al. (2002); Tai et al. (2005)

(Continued )

Table V Continued Factor Runx2: runt‐related transcription factor 2

TAZ: TaVazin

ATF4 activating transcription factor 4

Cells/Tissues In bone, MSCs; in developing teeth: dental mesenchyme, including papilla, follicle cells, and periodontal ligament during pre‐ eruptive tooth development MSCs

Osteoblasts

Function/Putative Function

Models

References

Transcription factor necessary for osteoblast diVerentiation; focal point for integration of a variety of signals aVecting osteoblast activity Mediates epithelial‐mesenchymal interactions during tooth development

Runx2 KO: Functional osteoblasts, mineralized bone, and hypertrophic cartilage are absent Tooth morphogenesis arrested in the transition from the bud to cap stages

Aberg et al. (2004a,b); Bronckers et al. (2001); D’Souza et al. (1999); Franceschi and Xiao (2003); Franceschi et al. (2003); Komori et al. (1997); Otto et al. (1997)

Acts as a transcriptional modulator during osteoblast diVerentiation; endogenous coactivator of Runx2; promotes osteoblast formation, inhibits adipocyte formation

No mouse models reported. In humans, mutations in the TAZ gene are responsible for Barth’s syndrome (BTHS), X‐linked endocardial fibroelastosis (EFE), X‐linked fatal infantile dilated cardiomyopathy (CMD3A), and familial isolated noncompaction of left ventricular myocardium (INVM) ATF4 KO: Delayed osteoblast diVerentiation throughout the skeleton, and a reduction in the area of mineralized tissue visible in frontal and parietal bones, clavicles and long bones; no tooth phenotype reported

Brady et al. (2006); Hong and YaVe (2006); Hong et al. (2005)

ATF4 is a transcription factor that regulates osteoblast diVerentiation and function; cooperates with Runx2 in stimulating osteoblast‐ specific Ocn expression

Xiao et al. (2005); Yang et al. (2004)

RANKL: Receptor activator of NF‐B ligand

Osteoprotegerin

p53

RANKL: Osteoblasts, cementoblasts, PDL cells, dental follicle cells, and many others (e.g., cells of immune system) OPG: Osteoblasts, PDL cells, dental follicle cells, dental epithelial cells, dental papilla cells, and many others (e.g., cells of immune system)

In bone RANK/RANKL receptor/ ligand expression in osteoblasts promotes osteoclastogenesis; OPG operates as a decoy receptor for the RANK receptor, and inhibits osteoclastogenesis

MSCs

Osteoblast/osteoclast diVerentiation (negatively regulates osteoblast diVerentiation/function by repressing expression of Osx), p53 deficiency confers osteoblasts with an increased ability to promote osteoclastogenesis

Expression in dental tissues may coordinate bone and tooth development

RANKL KO: Severe osteopetrosis and lack of osteoclasts, absence of tooth eruption

OPG KO: Increased osteoclastic activity and bone remodeling, severe bone loss, destruction of growth plate cartilage and increased vascular calcification OPG overexpression: Osteopetrosis with normal tooth eruption p53 KO: Largely viable with a small proportion with defects in neural tube closure. Early onset of tumors, such as lymphomas and sarcomas. High bone mass, increased Osx expression, more rapid diVerentiation in osteoblasts, increased tendency for osteoblasts to promote osteoclastogenesis

Amizuka et al. (2003); Bucay et al. (1998); Katagiri and Takahashi (2002); Kong et al. (1999); Ohazama et al. (2004a); Yasuda et al. (1998); Yao et al. (2004)

Armstrong et al. (1995); Attardi and Donehower (2005); Sah et al. (1995); Wang et al. (2006)

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tissues and cells, including cementum (Baba et al., 2004a,b; Massa et al., 2005; Qin et al., 2002, 2003). Cementum/root phenotypes have been indicated in mice null for DMP‐1 and DSPP, but at present phenotypes have not been fully reported (Sreenath et al., 2003; Ye et al., 2004). See Table III for more details on the DMP‐1 deficient mouse model. SIBLING family members also share a tendency for an astonishing degree of multifunctionality, perhaps best embodied by the ubiquitous OPN. In addition to well‐established roles as mineral regulators, SIBLINGs have been identified as regulators of matrix metalloproteinase (MMP) function. The MMPs are an extensive family of secreted or cell surface enzymes that are critical in physiological development and remodeling of the extracellular matrix, as well as certain pathologies. Several MMPs have been specifically associated with tooth development and remodeling (Apajalahti et al., 2003; Bourd‐Boittin et al., 2005; Fanchon et al., 2004; Goldberg et al., 2003; Maruya et al., 2003; Randall and Hall, 2002; Takahashi et al., 2003; Tsubota et al., 2002), as well as periodontal and other oral diseases (Sorsa et al., 2004). SIBLING proteins, BSP, OPN, and DMP‐1 were shown to specifically activate MMP‐2, MMP‐3, and MMP‐9, respectively, even in the presence of tissue inhibitors of MMPs (TIMPs) (Fedarko et al., 2004). The significance of SIBLING–MMP interaction in bone and tooth development is not yet clear, but a model for localized interaction of matrix proteins and enzymes in tooth development and eruption will surely be an important subject for further study. An additional function for DMP‐1 as an intracellular transcriptional regulator involved in diVerentiation has been proposed, indicating additional mechanisms for SIBLING influence on tooth formation (Almushayt et al., 2006; Narayanan et al., 2003). 3. Cementum Protein‐23 Cementum protein 23 was identified as a potential cementum marker by screening a human cementum tumor cDNA library (Alvarez‐Perez et al., 2006). However, cementum tumors are reported to exhibit a mixed cell type so it is diYcult to determine the specificity of this protein/gene to cementum/ cementoblasts. Antibodies made to CP‐23 cross‐reacted with a cartilage type collagen, type X collagen. CP23 has been identified in cementum, subpopulations of cells in the PDL region, and specifically in PDL cells located around blood vessels. 4. Betaig‐h3 Betaig‐h3 ( ig‐h3) is a collagen‐associated protein containing an RGD motif that has been identified in several tissues and cells (Ohno et al., 2002). High concentrations are found in cartilage and in the PDL region. A function

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proposed is a negative regulator of osteogenesis, acting to maintain a structural balance between PDL and bone‐tooth interface. Further, there is evidence for mechanical induction of ig‐h3 and potential for this protein to regulate chondrocyte diVerentiation via the TGF‐ pathway (Doi et al., 2003; Ohno et al., 2005). 5. Brain‐Derived Neurotrophic Factor BDNF, a member of the neurotrophin family, is considered to play a role in survival and diVerentiation of central and peripheral neurons (Ebendal, 1992). In addition to being expressed in neural cells, BDNF is found in many non‐neural cells/tissues including tooth germ, mature PDL, bone, cartilage, heart, spleen, placenta, osteoblasts, immune cells, prostate, and kidney (Nakanishi et al., 1994; Nosrat et al., 1998; Yamashiro et al., 2001). Takeda et al., using a dog model of periodontal disease, reported that BDNF promoted periodontal regeneration, that is, new bone, connective tissue fibers, and new cementum (Takeda et al., 2005). 6. Bono 1 Bono 1 has been identified in bone cells, in secretory odontoblasts coexpressed with DSPP, but not in pre‐secretory ameloblasts (where one does see DSPP) and follicle cells (James et al., 2004). Bono 1 is associated with regions of mineralization in bone, dentin and cementum, leading James et al. to propose involvement in controlling mineral formation. 7. Connective Tissue Growth Factor Connective tissue growth factor (36–38 kDa) (Asano et al., 2005; Shimo et al., 2002; Yamaai et al., 2005) belongs to CCN family, that is, CTGF, CEF10, and Nov. CTGF is found in several cells, including PDL cells, fibroblasts, chondrocytes, dental mesenchyme cells, epithelial cells, vascular endothelial cells of the enamel knot, pre‐ameloblasts and dental lamina (Friedrichsen et al., 2003, 2005; Nakanishi et al., 2001). Studies to date indicated that expression of CTGF is regulated by TGF‐ 1/BMP‐2. Interestingly, CTGF promotes expression of gene/proteins associated with PDL homeostasis, for example, increased expression of type I collagen, periostin, and ALP, but with no eVect on OPN or OCN gene expression (Asano et al., 2005). Other studies indicate that CTGF is involved in a chemotactic and mitogenic eVect in fibroblast‐like cells in vitro, and further enhances cell proliferation and matrix synthesis in connective tissues linked to wound healing (Lin et al., 2003, 2005). KO animals have abnormal growth plates, while CTGF mutant mice have impaired endochondral ossification (Ivkovic et al., 2003), though no tooth

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phenotype has been reported. Other studies using tooth germ implants in vitro with CTGF antibodies report severe inhibition of proliferation of both epithelial and mesenchymal cells, and a delay in cytodiVerentiation of ameloblasts and odontoblasts (Shimo et al., 2002). Intriguingly, CTGF is not expressed in Cbfa1‐null mice embryos (Yamaai et al., 2005). Based on these studies, there is growing evidence that CTGF may have a significant role in the development of mineralized tissues and in promotion of endochondral ossification.

8. Ectodysplasin (Tabby/Downless) Ectodysplasin is associated with ectodermal tissues (Pispa and ThesleV, 2003; Sharpe, 2001). The tabby gene (Ta) encodes the soluble tumor necrosis factor (TNF) ligand ectodysplasin (Eda). Eda binds to the TNF receptor (EdaR), encoded by the downless gene (dl), and this interaction leads to NFB activation via the cytoplasmic death domain adapter, Edaradd, encoded by the crinkled locus gene (Cr) (Courtney et al., 2005). Mice with a mutation in Ta, dl, or Cr display an ectodermal dysplasia phenotype characterized by abnormal development of ectoderm derived structures, including teeth (Courtney et al., 2005; Drogemuller et al., 2001; Risnes et al., 2005; Tucker et al., 2000). Additionally, studies manipulating levels of signaling molecules in the Eda axis support a critical role for these signals in determining tooth shape and cusp number (Kangas et al., 2004; Pispa et al., 2004). Altered signals from the enamel knot have been demonstrated in mutant mice, and the most dramatic defects are seen in molars with reduced size and abnormal shape (including roots). Specific root/PDL targeted signals related to these genes (Ta, dl or Cr) have not been identified. Humans with mutations in EDA, EDAR or EDARADD genes have hypohidroitic ectodermal dysplasia, with more markedly aVected individuals exhibiting severe tooth deformities and tooth loss (Courtney et al., 2005).

9. Osteocrin Osteocrin was initially identified using a virus‐based signal‐trap proteomic approach, and further characterized as a bone‐selective molecule (MoVatt et al., 2002; Thomas et al., 2003). Data suggest expression of this molecule in developing mineralized tissues, osteoblasts, osteocytes, hypertropic chondrocytes, and additionally the PDL region (Bord et al., 2005). Currently, it is thought that osteocrin may play a role in regulating osteoblast maturation, and subsequently mineral formation. The role of osteocrin in tooth development, if any, is speculative and requires further investigation.

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10. Matrix Gla Protein Matrix gla protein (MGP), a mineral‐binding extracellular matrix protein, was originally identified from bone matrix (Price et al., 1983). MGP expression has subsequently been reported in several types of cells and tissues, including early in development in lung and limb buds and cells of chondrocytic lineage (Luo et al., 1995), and later in mineralized tooth‐associated areas including dentin and the PDL/cementum region (Camarda et al., 1987; Hale et al., 1988; Hashimoto et al., 2001). MGP was also found in tissues producing unmineralized matrices, cartilage and vascular smooth muscle. MGP has been proposed to be a negative regulator of mineralized tissues (Mori et al., 1998), and mice lacking MGP exhibit pathological calcification in arteries, aortic valves, and cartilage (Luo et al., 1997). MGP expression in the periodontium may regulate hard–soft tissue interactions during tooth root development, as well as in mature tissues, however, no tooth phenotype has been found in MGP null mice. It is possible that MGP has a role in root/PDL development, though its function may overlap that of other mineralization regulators, for example OPN. Mice deficient in both MGP and OPN had three times as much arterial calcification by age 4 weeks, and died earlier from vascular rupture, supporting a shared role of MGP and OPN as inhibitors of calcification in the vasculature (Speer et al., 2002). A tooth phenotype in MGP and OPN null mice has not been reported, and it remains to be seen whether they operate similarly in the periodontal region.

V. Conclusions and Future Directions In recent years, rapid advances in technologies and molecular biology have propelled all areas of biomedical research forward at an exciting pace. The exquisitely regulated, sequential, reciprocal, reiterative cell signaling that defines morphogenesis and diVerentiation during the development of a tooth has been well characterized and reported (Jernvall and ThesleV, 2000; ThesleV, 2003). Considered together, advances of the last 5–6 years, including increased understanding of molecular signaling in tooth development, characterization and employment of tooth stem cell populations, and recombination of cells and tissues of specific developmental ages to generate tooth structures in vitro, have truly shown the way to a bright future for tooth engineering. With this progress in mind, it is now time to delve into how the tooth root develops and explore the possibilities for regeneration of these tissues, for the success of bioengineering a ‘‘whole tooth’’ depends on it. Characterization of root and associated periodontal apparatus formation lags behind that of crown: what cells are involved, what signaling drives morphogenesis

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Figure 3 Root/PDL formation: key regulators and attractive candidates to consider. While mechanisms for tooth crown formation have been well established, cells and signals required for periodontal and tooth root formation are only now beginning to unfold. Naturally occurring mutations (mut) and knockout (KO) and gene overexpressing (O/E) animal models have driven the identification of some key regulators, while elucidation of mechanisms controlling root and periodontal formation is currently underway. During Late Bell stage, root formation is initiated, preceded by the conversion of the cervical loop to the bilayer Hertwig’s epithelial root sheath (HERS). A. With proper signaling, the healthy root with associated periodontal apparatus forms, composed of root dentin (D), cementum (C), periodontal ligament (P), and alveolar bone (B). B. Alterations in several genes/proteins are known to contribute to cementum phenotypes, including regulators of Pi/PPi homeostasis (ank, PC‐1, TNAP) and BMP signaling (BMP‐3). C. The development and maintenance of the periodontal ligament (PDL) is dramatically altered as a result of BMP‐3 overexpression (O/E) and loss of periostin. D. Alterations on the level of the whole root often lead to tooth loss, including phenotypes marked by absence of roots (NF1c KO) and distorted roots (Msx2 and DMP‐1 KO, noggin O/E). Text: Pi ¼ inorganic phosphate; PPi ¼ pyrophosphate; BMP ¼ bone morphogenetic protein; FGF ¼ fibroblast growth factor; HERS ¼ Hertwig’s epithelial root sheath; NF1c ¼ nuclear factor 1c; Shh ¼ sonic hedgehog; KO ¼ knockout; mut ¼ mutation; ank ¼ progressive ankylosis protein; PC‐1 ¼ plasma cell membrane glycoprotein 1; TNAP ¼ tissue nonspecific alkaline phosphatase; O/E ¼ overexpressing; PDL ¼ periodontal ligament; DMP‐1 ¼ dentin matrix protein 1. Insets: IEE ¼ inner enamel epithelium; OEE ¼ outer enamel epithelium; D ¼ dentin; C ¼ cementum; P ¼ periodontal ligament; B ¼ bone.

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and diVerentiation, how is the extracellular matrix synthesized, and how are cells dynamically aVected by the local environment? This chapter attempted to pose some of these questions and provide updates regarding current knowledge on the roles of cells/factors/genes in regulating root and periodontal tissue development, as well as directions for future research. While it is clear that there is yet much to accomplish, some clues have begun to emerge regarding tooth root formation. Animal models with periodontal phenotypes have provided a starting point by indicating transcription factors, signals and signaling pathways, and matrix proteins that are important in root formation. Attractive candidates required for controlling root/ periodontal tissue formation, homeostasis, and regeneration have been presented, and are summarized in Fig. 3. These last years have provided new insights into the possible triggers for cementum/periodontal tissue development and regeneration, and new directions for investigation that should result ultimately in improved clinical approaches for regeneration of lost periodontal tissues.

Acknowledgments Support for this research was provided from grants DE05932 and DE15109 (MJS), and T32 DE0 7023–29 (HKF), from the National Institute of Dental and Craniofacial Research, National Institutes of Health.

References Aberg, T., Wozney, J., and ThesleV, I. (1997). Expression patterns of bone morphogenetic proteins (Bmps) in the developing mouse tooth suggest roles in morphogenesis and cell diVerentiation. Dev. Dyn. 210(4), 383–396. Aberg, T., Cavender, A., Gaikwad, J. S., Bronckers, A. L., Wang, X., Waltimo‐Siren, J., ThesleV, I., and D’Souza, R. N. (2004a). Phenotypic changes in dentition of Runx2 homozygote‐null mutant mice. J. Histochem. Cytochem. 52(1), 131–139. Aberg, T., Wang, X. P., Kim, J. H., Yamashiro, T., Bei, M., Rice, R., Ryoo, H. M., and ThesleV, I. (2004b). Runx2 mediates FGF signaling from epithelium to mesenchyme during tooth morphogenesis. Dev. Biol. 270(1), 76–93. Abukawa, H., Papadaki, M., Abulikemu, M., Leaf, J., Vacanti, J. P., Kaban, L. B., and Troulis, M. J. (2006). The engineering of craniofacial tissues in the laboratory: A review of biomaterials for scaVolds and implant coatings. Dent. Clin. North Am. 50(2), 205–216, viii. Akintoye, S. O., Lam, T., Shi, S., Brahim, J., Collins, M. T., and Robey, P. G. (2006). Skeletal site‐specific characterization of orofacial and iliac crest human bone marrow stromal cells in same individuals. Bone [Epub ahead of print]. Alatli‐Kut, I., Hultenby, K., and Hammarstrom, L. (1994). Disturbances of cementum formation induced by single injection of 1‐hydroxyethylidene‐1,1‐bisphosphonate (HEBP) in rats: Light and scanning electron microscopic studies. Scand. J. Dent. Res. 102(5), 260–268. Almushayt, A., Narayanan, K., Zaki, A. E., and George, A. (2006). Dentin matrix protein 1 induces cytodiVerentiation of dental pulp stem cells into odontoblasts. Gene Ther. 13(7), 611–620.

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Anabolic Agents and the Bone Morphogenetic Protein Pathway I. R. Garrett OsteoScreen, San Antonio, Texas 78229

I. Introduction II. Bone Metabolism A. Bone Anabolic Growth Factors B. Platelet‐Derived Growth Factor C. Transforming Growth Factor
D. Fibroblast Growth Factors E. Vascular Endothelial Growth Factors F. Growth Hormone/Insulin‐Like Growth Factors G. Bone Morphogenetic Proteins III. The BMP Pathway and Bone Anabolic Therapies A. BMP/SMAD Signaling Pathway B. Agents That Act on the BMP Pathway C. Agents That Do Not Act on the BMP Pathway IV. Conclusions and Future Directions References

A major unmet need in the medical field today is the availability of suitable treatments for the ever‐increasing incidence of osteoporosis and the treatment of bone deficit conditions. Although therapies exist which prevent bone loss, the options are extremely limited for patients once a substantial loss of skeletal bone mass has occurred. Patients who have reduced bone mass are predisposed to fractures and further morbidity. The FDA recently approved PTH (1–34) (Teriparatidew) for the treatment of postmenopausal osteoporosis after both preclinical animal and clinical human studies indicated it induces bone formation. This is the only approved bone anabolic agent available but unfortunately it has limited use, it is relatively expensive and diYcult to administer. Consequently, the discovery of low cost orally available bone anabolic agents is critical for the future treatment of bone loss conditions. The intricate process of bone formation is co‐ordinated by the action of many diVerent bone growth factors, some stored in bone matrix and others released into the bone microenvironment from surrounding cells. Although all these factors play important roles, the bone morphogenetic proteins (BMPs) clearly play a central role in both bone cartilage formation and repair. Recent research Current Topics in Developmental Biology, Vol. 78 Copyright 2007, Elsevier Inc. All rights reserved.

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into the regulation of the BMP pathway has led to the discovery of a number of small molecular weight compounds as candidate bone anabolic agents. These agents may usher in a new wave of more innovative and versatile treatments for osteoporosis as well as orthopedic and dental indications. ß 2007, Elsevier Inc.

I. Introduction Skeletal architecture plays a crucial role in bone by providing both shape and structure to withstanding repetitive loading. The function of bone tissue is to aVord both a load‐bearing material and an available repository for mineral. One of the major medical issues today is the increasing incidence of osteoporosis with subsequent morbidity. This is a disabling disease characterized by compromised bone strength, predisposing patients to increased risks of fracture. It aVects at least one‐quarter of all postmenopausal white women in the United States and the proportion rises to 70% in women older than 80 years (Conference‐Report, 1993; Melton, 1997; WHO, 2003). One in three women older than 50 years will have an osteoporotic fracture that causes a considerable social and financial burden on society (Melton et al., 1992). One person in the European Union sustains an osteoporotic fracture every 30 s, and the annual first‐year direct cost of treating all osteoporotic fractures is estimated at 25 billion (Compston et al., 1998). The disease is not limited to women; older men can also be aVected (Akin et al., 2004). By 2050, the worldwide incidence of hip fracture is projected to increase by 310% and 240% in men and women, respectively (Gullberg et al., 1997). The combined lifetime risk for hip, forearm, and vertebral fractures presenting clinically is around 40%, equivalent to the risk for cardiovascular disease (Kanis, 2002). Osteoporotic fractures, therefore, cause substantial mortality, morbidity, and economic cost. Bone loss occurs in both adult women and men, probably due to a decline in the volume of bone formed as a natural part of aging. The rate of loss is slow in young adulthood because the remodeling rate is low; however, it accelerates in women at menopause due to increased bone turnover and results in trabecular thinning, disappearance and loss of connectivity, cortical thinning, and increased intracortical porosity. These changes compromise the material and structural properties of bone and lead to increased propensity for fracture. Most of the treatments in use today are antiresorptive medications including estrogens, selective estrogen receptor modulators (SERMs) such as raloxifene, bisphosphonates (alendronate, risedronate, and ibandronate), and calcitonins all aimed at inhibiting osteoclastic resorption reducing the progression of trabecular thinning, loss of connectivity, cortical thinning, and porosity.

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One of the main issues with these antiresorptive therapies is although they are successful at reducing loss of bone and its eventual devastating consequences, they lack the ability to replace lost bone. The ultimate eVectiveness of these agents is dependent on early diagnosis of the onset of this osteoporotic condition. If substantial bone has been lost at important sites in the skeleton, then these agents do little to augment skeletal structural integrity. Because of the mechanism of action of these antiresorptive agents, they are ineVective treatments for one of the most serious consequences of osteoporosis, which are fractures. In fact, they appear to delay remodeling of fracture callus woven bone into lamellar bone resulting in delayed healing of the fracture itself and possibly weaker bone (Komatsubara and Mori, 2005). Therefore, a critical need exists for inexpensive skeletal anabolic agents to treat bone deficit conditions such as osteoporosis as well as fractures and other orthopedic and dental indications. Recent clarification of the complex pathways involved in bone formation and repair indicates that growth factors play essential roles in the intricate cascade leading to mature bone formation. This has raised the possibility of using growth factors to help increase bone mass or repair bone tissues. However, while BMP‐2 and platelet‐derived growth factor (PDGF) are approved for local bone repair, in general, growth factors have obvious limitations in their ability to act or to be used systemically. In addition, together with their inherent high cost of manufacturing, they are restricted in their use for local indications only. For a successful anabolic agent, they must fit important therapeutic criteria such as ease of administration, strong therapeutic eYcacy, limited side eVects, and low toxicity. They should increase bone mass and reverse deficit conditions such as osteoporosis, while having the ability to enhance healing of fractures and replacement of bone tissue. To date, no acceptable drug exists according to these criteria. While most of the approved agents for bone act as antiresorptive agents, parathyroid hormone (PTH) is the only systemically active anabolic agent available. However, its use is extremely limited due to its diYculty to administer, side eVects, and cost. While BMP‐2 and PDGF are approved for local use, they are also limited in their use mainly due to their cost (Table I). Other factors and compounds, with anabolic activity, are being investigated as potential candidates as anabolic agents (Table II). However, in general, the larger the molecule, the higher the cost and the more diYcult it is to administer. With these important restrictions in mind, this chapter focuses attention on current anabolic therapies and possibility of small innovative and more versatile agents as future potential therapies. The use of clinically available small molecular weight systemic agents for local repair of fractures has met with a number of diYculties. For instance, although local application of bisphosphonates, in animal studies, increases

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Table I Clinical Use of Bone Anabolic Growth Factors Stage of Development* Drug

Oral Availability

Use

No No No No No No No No No

Local Local Systemic Local Local Local Local Local Systemic

BMP‐2 (Infusew) PDGF (GEM 215w) PTH (1–34) (Teriparatidew) PTH (Preos) FGF TGF‐
VEGF IGF‐1,2 Sclerostin antagonists

Pre

PI

PII

PIII

NDA

*Pre—Preclinical; PI—Phase clinical studies; PII—Phase II clinical studies; PIII—Phase III clinical studies; NDA—New drug application.

Table II

Clinical Use of Small Molecular Weight Bone Anabolic Agents Stage of Development*

Drug Strontium ranelate (PROTELOSw) Chrysalin Calcilytics Statins Proteasome inhibitors Prostaglandin agonists Flavonoids
‐Blockers AC100

Oral Availability

Use

Yes

Systemic

No Yes Yes Yes Yes Yes Yes No

Local Systemic Local/Systemic Local/Systemic Local/Systemic Local/Systemic Systemic Local

Pre

PI

PII

PIII

NDA

*Pre—Preclinical; PI—Phase clinical studies; PII—Phase II clinical studies; PIII—Phase III clinical studies; NDA—New drug application.

bone area next to dental implants (Miller and Marks, 1993a) and reduces bone resorption following surgical trauma (Binderman et al., 2000; Kaynak et al., 2000), it appears this approach leads to delayed healing and weaker bone (Komatsubara and Mori, 2005). These currently used clinical drugs for the treatment of osteoporosis (bisphosphonates, calcitonin, estrogen, and vitamin D analogs) inhibit bone resorption instead of primarily stimulating

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new bone formation and clearly would be of limited if any benefit for the treatment of bone conditions requiring the establishment of new bone growth. A key approach to finding new potential bone anabolic agents is to focus on those pathways critically important for bone formation. However, the action of these agents must be proximal enough, in these pathways, to activate the multitude of complex downstream signaling cascades responsible for initiating and sustaining the intricate process of bone formation.

II. Bone Metabolism Bone is a metabolically active organ in which the organizational pattern of the mineral and organic components determine the successful mechanical function of the skeleton. This is achieved by a combination of dense, compact, and cancellous (trabecular) bone, reinforced at points of stress (Glimcher, 1968, 1987). Defined agents and mechanisms regulate bone formation and bone resorption, the two major processes of bone remodeling. Bone formation in vivo is a complex phenomenon whereby recruitment and replication of mesenchymal precursors of osteoblasts, diVerentiation into preosteoblasts, osteoblasts, and mature osteoblasts ultimately result in the accumulation and mineralization of the extracellular matrix. Since the formation of new bone is primarily a function of the osteoblast, agents regulating bone formation act by either increasing or decreasing the replication of cells of the osteoblastic lineage or modifying the diVerentiated function of the osteoblast. Both systemic and local factors control bone formation. These local regulators of bone formation are growth factors that act directly on cells of the osteoblastic lineage. Growth factors are polypeptides with important eVects on cell function. Some are also present in the circulation and may function as systemic agents, but for the most part, work locally in specific tissues as regulators of cell metabolism. Production of new bone during embryogenesis occurs through a complex series of cellular interactions controlled by growth factors that communicate the information needed for correct pattern formation and the signals required for diVerentiation of cells into cartilage and bone. Most bones in the body start as cartilage models, then are ultimately replaced by bone through the process of endochondral bone formation. In contrast, bones of the craniofacial skeleton form directly by the conversion of mesenchymal progenitors into osteoblasts through intramembranous bone formation. The result of either developmental path, controlled by growth factors, is a bone surrounded by a periosteal layer rich in progenitor cells and containing a mature marrow cavity and vascular

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supply. It is the expression of these bone growth factors and the appropriate signaling of their pathways that plays a central role in the regulation of the anabolic response in bone.

A. Bone Anabolic Growth Factors Systemic factors do influence skeletal integrity including PTH, 1, 25 dihydroxy vitamin D3, and more recently the possible role of the sympathetic nervous system. A wide variety of locally derived growth factors positively impact bone formation including insulin‐like growth factors (IGFs), fibroblast growth factors (FGFs), hedgehogs (Shh and Ihh), transforming growth factor
(TGF‐
), PDGF, vascular endothelial growth factor (VEGF), Wnts, and BMPs probably being the most important of these local bone growth factors. Therefore, due to the importance of the BMPs in bone formation, factors that limit the eVect of BMP activity play very important roles in the regulation of both the temporal and spatial aspects of bone formation suggesting that they might be potential therapeutic targets. These include proteins such as noggin, dan, chordin, follistatin, and cerberus. The SOST gene product, sclerostin (a BMP antagonist) has been shown to exhibit a pattern of expression in trabecular bone only suggesting it as a potential therapeutic target (Kusu et al., 2003; Poole et al., 2005; van Bezooijen et al., 2005b). Although all these growth factors and hormones alone aVect bone formation, it is the interaction of all of these growth factors and their inhibitors together that results in the complex process of bone formation.

B. Platelet‐Derived Growth Factor hPDGF is the major growth factor of human blood serum. The hPDGF heterodimer and its two isoforms, the PDGF‐1(A) and PDGF‐2(B) homodimers, are potent mitogens and chemoattractants for target cells such as diploid fibroblasts, osteoblasts, arterial smooth muscle cells, and brain glial cells. PDGF, an osteoblast mitogen, accelerates fracture healing and periodontal bone repair when applied locally in vivo (Giannobile et al., 1994), while systemic administration of PDGF increases bone density and strength throughout the skeleton (Mitlak et al., 1996). PDGF was recently shown to improve periodontal regeneration in humans (Camelo et al., 2003; Nevins et al., 2003, 2005) and has been approved by the FDA for the treatment of bone loss associated with advanced periodontal disease when combined with the synthetic bone matrix,
‐tricalcium phosphate (
‐TCP).

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133

C. Transforming Growth Factor b The first local bone growth factor purified to homogeneity was TGF‐
. Early on, TGF‐
promised to be one of the key factors involved in coupling bone formation to previous bone resorption (Mundy, 1991). This potent osteotropic polypeptide is abundant in the bone matrix, and produced in response to factors that stimulate osteoclastic bone resorption. It is a very potent stimulator of osteoblastic bone formation, causing chemotaxis, proliferation, and diVerentiation in committed osteoblasts. TGF‐
has complex eVects on bone resorption, it inhibits osteoclast formation and osteoclast activity (Dieudonne et al., 1991). TGF‐
is released from bone in a biologically inert state due to the presence of at least two proteins that appear to regulate its activity. Release of active TGF‐
from these latent complexes occurs during bone resorption and is mediated by osteoclasts (Bonewald et al., 1991). Knowledge of the mechanisms responsible for these activation processes may be vital to understanding the role of TGF‐
in bone remodeling. A single application of human recombinant TGF‐
1 to skull defects induced a dose‐dependent increase in intramembranous bone formation in rabbits (Beck et al., 1991, 1993) and defects in sheep (Moxham et al., 1996). Alternatively, evidence suggested that high dose TGF‐
delayed or inhibited mineralization of newly formed osteoid (Broderick et al., 2005). TGF‐
is also known to regulate chondrocyte proliferation and hypertrophic diVerentiation and has marked eVects on cartilage growth (Tuli et al., 2002). It is clear TGF‐
is a pivotal growth factor during osteogenesis and systemic bone disease.

D. Fibroblast Growth Factors Fibroblast growth factors and FGF receptors (FGFRs) comprise a signaling system conserved throughout evolution. Twenty‐two FGFs and four FGFRs have been identified in humans and mice (Itoh and Ornitz, 2004). FGFs are key regulators of several developmental processes in which cell fate and diVerentiation to various tissue lineages are determined. The importance of the proper spatial and temporal regulation of FGF signals is evident from human and mouse genetic studies which show that mutations leading to the dysregulation of FGF signals cause a variety of developmental disorders including dominant skeletal diseases and cancer (Dailey et al., 2005). A number of years ago, the overexpression of FGF‐2 caused a variety of skeletal malformations including shortening and flattening of long bones and moderate macrocephaly (CoYn et al., 1995), while continuous slow administration of a small amount of FGF‐2 accelerates bone‐derived osteogenic cytokine‐induced new bone formation (Kimoto et al., 1998). It was

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also shown that both local and systemic FGF‐1 increases new bone formation and bone density, and can restore bone microarchitecture and prevent bone loss associated with estrogen‐withdrawal (Dunstan et al., 1999). rhFGF‐4 was shown to stimulate bone formation around titanium implants in bone (Franke Stenport et al., 2003). Other uses in bone have been the acceleration of surgical angiogenesis in necrotic bone with a single injection of FGF‐2 (Nakamae et al., 2004). As with these other growth factors, FGF is an extremely important growth factor in the bone metabolism. E. Vascular Endothelial Growth Factors The family of vascular endothelial growth factors (VEGFs) currently includes VEGF‐A, ‐B, ‐C, ‐D, ‐E, and placenta growth factor (PlGF). Several of these factors, notably VEGF‐A, exist as diVerent isoforms, which appear to have unique biological functions. The VEGF family proteins bind in a distinct pattern to three structurally related receptor tyrosine kinases, denoted as VEGF receptors‐1, ‐2, and ‐3. One of the main influences of VEGFs is to stimulate angiogenesis (Baumgartner and Isner, 1998) which appears to play an important role in cancer (Carmeliet, 2005). This has led to the discovery of VEGF inhibitors as potential therapies in cancer treatment (Cardones and Banez, 2006; Ferrara, 2005). VEGF has been shown to promote bone growth (Peng et al., 2002; Young et al., 2002) probably by its eVects on angiogenesis (FilvaroV, 2003; Kent Leach et al., 2006; Kleinheinz et al., 2005; Peng et al., 2005).

F. Growth Hormone/Insulin‐Like Growth Factors The GH/IGF axis is important for long bone development, homeostasis, and disease (Fisher et al., 2005; Kasukawa et al., 2004; McCarthy and Centrella, 2001), and IGF 1 and IGF 2 have both systemic and local eVects on bone growth and fracture repair (Aspenberg et al., 1989; Isgaard et al., 1986; Kawata et al., 2002; Linkhart et al., 1996; Marie, 1997; McCarthy et al., 1989; Miyakoshi et al., 2001; Stabnov et al., 2002; Wildemann et al., 2004) through a complicated series of growth factor‐binding proteins which regulate their eVects on bone (Yamaguchi et al., 2006). There seems to be a role for IGF in the downstream signaling of PTH on bone where in mouse osteoblast cultures PTH treatment increased IGF‐I mRNA and protein levels, and alkaline phosphatase activity, which were accompanied by phosphorylations of IGF‐I receptor, insulin receptor substrate 1 (IRS‐1), and IRS‐2, essential adaptor molecules for the IGF‐I signaling (Miyakoshi et al., 2001). Further results indicate that the PTH bone anabolic action is

4. Anabolic Agents and the BMP Pathway

135

mediated by the activation of IRS‐1 as a downstream signaling of IGF‐I that acts locally as an autocrine/paracrine factor (Yamaguchi et al., 2005).

G. Bone Morphogenetic Proteins The BMPs are probably the most important bone growth factors and account for the major proportion of the osteoinductive potential of bone extracts (HoVmann and Gross, 2001). Over 20 BMPs family members have been identified and characterized (Cao and Chen, 2005; Wan and Cao, 2005; Wozney, 1992; Wozney et al., 1988). All these BMPs are members of the TGF‐
superfamily. The BMPs are critically important for the regulation of bone formation, and it is these factors which is the focus of this chapter.

III. The BMP Pathway and Bone Anabolic Therapies Bone deficit conditions such as osteoporosis, osteopenia, nonunion factures, and bone loss from traumatic injury are extremely diYcult to treat with currently available agents that only prevent the loss of bone while lacking substantial bone anabolic activity. The stimulation of local bone formation by either local or systemic application of bone anabolic agents would vastly improve the clinical treatment and repair of bone fractures, help integrating and stabilizing orthopedic implants, and markedly improve the repair of isolated bony defects. Growth factors, including recombinant BMP‐2, FGF, and PDGF have the ability to stimulate bone formation and increase repair rates in animal models. Because of the lack of systemic availability, the use of these factors is restricted to their local application. Further, the use of these recombinant human growth factors for local application, to stimulate bone formation in humans, has been unfortunately variable and concerns have been raised about the expense and drug stability in such therapies (Schilephake, 2002). While understanding that there are many growth factors and hormones involved in bone formation, the discovery of small molecular weight compounds that can elicit bone anabolic activity will come from a closer understanding of the signaling pathway that are critical for bone formation. There are multiple signaling cascades and processes required to be either activated or inhibited to elicit bone formation. One of the main signaling cascade events that stimulates bone formation is the BMP/SMAD pathway. Although other pathways are involved in the anabolic response in bone, it is the BMP/SMAD pathway that has received the most attention to date and a number of compounds have been discovered which can aVect this pathway and lead to bone formation.

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A. BMP/SMAD Signaling Pathway Bone morphogenetic proteins are very powerful inducers of both bone and cartilage and are implicated in a variety of non‐osteogenic developmental processes. The BMPs are multifunctional growth factors that belong to the TGF‐
superfamily family. Type I and II BMP receptors and the downstream molecules, SMAD1, 5, and 8, mediate BMP signals. Phosphorylated SMAD1, 5, and 8 form complexes with SMAD4 and translocate to the nucleus where they interact with numerous other important transcription factors such as Runx2/cbfa1 to elicit bone formation. BMPs were first discovered by Marshall Urist when he found that the osteoinductive activity present in bone could not be accounted for by any known single growth factor or combination of growth factors, suggesting the existence of a novel bone‐inductive protein (Urist et al., 1977, 1979, 1982). To date, over 20 BMPs family members have been identified and characterized (Cao and Chen, 2005; Wan and Cao, 2005; Wozney, 1992; Wozney et al., 1988). Subsequent investigation led to the realization that the BMP pathway plays an integral role in bone formation not only in embryogenesis but also in the adult bone metabolism and plays a very important role in maintaining adult skeletal integrity. BMP‐2 and other BMPs heal bone defects when applied locally. However, there are limitations to its use. Its eYcacy appears limited to local use as no convincing evidence exists of its systemic activity. However, when used locally with an appropriate matrix such as collagen, BMPs show strong anabolic eVects. Preclinical models and clinical trials have shown the ability of BMP‐2 to stimulate bone formation. In these animal models, bone defects are large enough so they will not heal without a therapeutic intervention allowing the ability of BMP‐2 to induce bone and heal nonunion defects. Healing of long bone critical‐sized defects by BMP‐2 has been shown in a number of species including rats, rabbits, dogs, sheep, and non‐human primates (Murakami et al., 2002). Gene therapy studies indicate that bone defects can be healed by the local implantation of a bioresorbable polymer mixed with bone marrow mesenchymal stem cells to which adenovirus BMP‐2 is transferred (Chang et al., 2003). Studies show that rhBMP‐2 delivered in an injectable formula with a calcium phosphate carrier or with a liposome carrier accelerates bone healing in a rabbit ulna osteotomy model and a rat femoral bone defect model (Li et al., 2003; Matsuo et al., 2003). Clinical studies show rhBMP‐2 can be utilized as complete bone graft substitutes in spinal fusion surgery. In some circumstances, the eYcacy of BMP‐2 for inducing successful fusion is superior to that of autogenous bone graft, while BMIP‐2 has been shown to be eYcacious in several fusion applications, including intervertebral and lumbar posterolateral fusion (Boden et al., 2002; Sandhu, 2004). BMP‐2 has also been shown to induce new dentine formation and has a potential

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137

application as a substitute for root canal surgery and BMP‐2 is an eVective bone inducer around dental implants for periodontal reconstruction (Cochran and Wozney, 1999). One promising use of the BMPs is their application for local repair of bone or stimulation of new bone formation where loss has occurred (Asahina et al., 1997; Hong et al., 1998; Southwood et al., 2004; Springer et al., 2005). BMP‐2 is FDA approved for a spinal fusion indication, although its cost and lower than expected eYcacy have limited its usage in its current form (Boden, 2005; Khan and Lane, 2004). Unfortunately, BMP‐2 appears to be ineVective when administered systemically where it is unable to increase bone formation or prevent trabecular bone loss induced by unloading in rats (Zerath et al., 1998), probably because of its limited pharmacokinetics. One approach would be to stimulate the production of BMPs at local sites in bone by small compounds, resulting in bone formation where needed. This approach has led to the discovery of some small molecular weight anabolic agents that act by increasing BMP‐2 protein at local sites.

B. Agents That Act on the BMP Pathway There is growing interest in the discovery of new and more versatile small molecular weight anabolic agents. Although the time has not quite come for these agents to make their impact on the market, a lot of preclinical and developmental studies suggest the possible use of small molecular weight agents as anabolic agents. These include agents, which not only enhance the expression of anabolic growth factors such as BMPs in the bone microenvironment, but also agents that can directly enhance the signaling pathways critically involved in the process of bone formation. Interestingly, a great proportion of the agents that have been reported to have bone anabolic potential, have the ability to aVect the BMP pathway either directly or indirectly. Below is a table of the reported anabolic agents that aVect the BMP pathway (Table III). 1. Statins Initial identification of statins as small molecular weight anabolic agents was by the use of a cell‐based screening assay that enhances BMP‐2 transcription. This assay employed the 2T3 osteoblast cell line transfected with the murine BMP‐2 promoter operatively linked to the firefly luciferase reporter. Screening of a natural products collection led to the identification of an extract that specifically stimulated the BMP‐2 promoter in these cells. Purification of this extract identified lovastatin as the active constituent. Statins including lovastatin, simvastatin, pravastatin, atorvastatin, fluvastatin, rosuvastatin,

138 Table III

I. R. Garrett Anabolic Agents That AVect or Enhance the BMP Pathway BMP

Drug BMP‐2 Statins Proteasome inhibitors Flavonoids Sclerostin antagonists Prostaglandin agonists PTH (1–34) (Teriparatidew) PTH (Preos)

Expression

Signaling

Yes Yes Yes Yes ? Yes ? ?

Yes Yes Yes ? Yes ? Probably Probably

Table IV Increase of New Bone Formation In Vitro by Lovastatin Dose (mg/ml) 0 0.075 0.15 0.3 0.6 1.2

New Bone Formation (mm
103) 3.7 4.5 7.1 10.6 11.8 11.3

     

0.3 0.3 0.4* 0.5* 0.6* 0.4*

*Significantly greater than vehicle treated group. ANOVA p < 0.05.

pitavastatin, and cerivastatin are widely used agents for lowering cholesterol and reducing heart attacks. They provide an important and eVective approach to the treatment of hyperlipidemia and arteriosclerosis (Hunninghake, 1998; Spin and Vagelos, 2003) and all stimulated BMP‐2 promoter activity except pravastatin. Subsequently, statins have been shown to increase the expression of BMP‐2 in human and rodent bone cells (Mundy et al., 1999) confirmed by many others (Emmanuele et al., 2003; Maeda et al., 2001, 2004; Mundy et al., 1999; Sugiyama et al., 2000). In vitro models of bone formation indicate simvastatin elicits marked increases in osteoblast accumulation and new bone formation over 4–7 days of culture (Mundy et al., 1999) (Table IV), while pravastatin could not, consistent with its inability to stimulate the BMP‐2 expression (Maeda et al., 2001; Mundy et al., 1999; Sugiyama et al., 2000). Pravastatin cannot enter cells other than hepatocytes (Nakai et al., 2001; Yamazaki et al., 1996; Ziegler et al., 1994), resulting in its reduced pleiotropic eVects correlating with its inability to stimulate new bone formation.

4. Anabolic Agents and the BMP Pathway

139

It has been reported that compactin (mevastatin) at doses of 1–100 M suppresses osteoclastic bone resorption in vitro by inducing apoptosis of osteoclasts (Luckman et al., 1998). Studies confirm the mechanism is through inhibition of the fusion of preosteoclastic cells and the disruption of actin ring in osteoclasts (Woo et al., 2000). This eVect is because of the inhibition of prenylation of target proteins by prenyl protein transferases, similar to that seen with bisphosphonates. These findings suggest statins, while able to stimulate osteoblasts are also capable of inhibiting resorbing osteoclasts. However, these in vitro eVects occur at markedly diVerent doses where inhibition of osteoclastic activity is between 1–100 M, while the bone anabolic eVects of these agents occur at doses as low as 0.06 M. Given the hepatoselective nature of these statin drugs and the high doses required, it is unlikely they would be able to inhibit bone resorption in vivo as suggested previously (Luckman et al., 1998). Alternatively, it is clear statins, unlike bisphosphonates, can aVect osteoblastic activity at low doses, show anabolic eVects in vitro, and there is increasing evidence this translates to eVects in vivo. Statins stimulate local formation in mice resulting in a 30–50% increase in calvarial bone thickness after 21 days (Mundy et al., 1999). Lovastatin incorporated in biodegradable polymer discs, releasing lovastatin at a constant rate, resulted in a 50–70% increase in cranial bone thickness over a 21‐day period in mice (Whang et al., 2005). High dose of simvastatin formulated in a gel stimulates cranial bone apposition and the bone formed remained for up to 22 days after dosing (Thylin et al., 2002). These findings pose the possibility that statins can be utilized for local bone repair to enhance new bone formation in orthopedic indications such as device stabilization, in surgical repair of bone defects as well as in conditions such as periodontal bone disease. There are numerous advantages for the local use of statins to stimulate bone formation. First, it is an inexpensive drug to manufacture compared with recombinant proteins such as BMPs or FGFs, second, it has a long history of clinical systemic usage with very acceptable good toxicity profiles, and finally, it is relatively easy to incorporate into biodegradable matrices that regulate its release to enhance its eVectiveness. Systemically, when lovastatin is given to ovariectomized rats, there is a marked increase in bone density (Mundy et al., 1999). Long bones from rats treated with cerivastatin showed a 43% increase in tibial trabecular volumes and a 38% increase in tibial trabecular volumes in rats treated orally with simvastatin (Garrett et al., 2001b). Both cerivastatin and simvastatin increase trabecular bone in rats in a similar manner to that previously seen by acidic FGF (Dunstan et al., 1999). There was a 46% increase in both BFR and a 32% increase in MAR in the tibiae of rats treated with cerivastatin at 0.1 mg/ kg/day (Garrett et al., 2001b). Cerivastatin has also been shown to improve cortical bone strength in ovariectomized rats when used in doses as low as

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0.1 mg/kg/day, and in addition significantly increased bone mineral density (BMD), bone formation rate, osteocalcin mRNA levels, as well as resistance to fracture (Wilkie et al., 2000). Lovastatin, applied transdermally in a gel preparation to intact rats, increased bone volume up to 33% and the BFR was still increased by 166% after 30 days following the last dose (Gutierrez et al., 2000). Further studies show simvastatin given orally to rats significantly increases cancellous bone compressive strength in the vertebral bodies of these rats (Oxlund and Andreassen, 2004), while others show an increase in cortical bone in young male rats using a single local administration (Crawford et al., 2001). In mice, given a diet prepared with simvastatin, there was a marked improvement in fracture healing (Skoglund et al., 2002). Work has also shown that simvastatin, when administered locally to a fracture site, demonstrated a dramatic positive eVect on the strength of a healing fracture and indicated that the local delivery of statins could be used to promote fracture healing (Skoglund and Aspenberg, 2006). These findings indicate statins have an anabolic eVect by increasing the rate at which bone is forming and show statins have the potential to stimulate bone formation both in vitro and in vivo in rats. As statins appear to be bone anabolic agents in rats, with low relative toxicity in man, they could provide an important treatment when administered in the correct fashion for osteoporosis and fracture healing. a. Mechanism of Action. Statins inhibit the rate‐limiting step in the mevalonate pathway and the addition of downstream metabolites mevalonate, farnesyl pyrophosphate, or geranylgeranyl pyrophosphate inhibited statin‐stimulated bone formation. As geranylgeranyl pyrophosphate reverses these eVects, inhibition of prenylation appears to play a major role in the stimulation of bone formation by this drug. Prenylation is important for the activity of important intracellular molecules including GTPases, such as Rho, Rac, Rab, and Rap. These proteins play important roles in cellular proliferation and diVerentiation and therefore any perturbation of their activity influences cellular activity. Initial studies focused on nitric oxide synthase (eNOS) activity and NO which has been shown to be needed for bone formation in mice and in cultured mouse calvaria (Armour et al., 2001; Feron et al., 2001; Garrett et al., 2001a), and prenylation of Rho GTPase regulates eNOS (Ming et al., 2002). The inhibition of Rho GTPase prenylation upregulates eNOS activity providing the beneficial eVects in the endothelium (Laufs et al., 1997). Statin stimulation of bone resulted in upregulation of eNOS mRNA in murine osteoblasts and increased expression of mRNA for BMP‐2. Interestingly, the expression of both eNOS and BMP‐2 mRNAs peaked at 6 hours after exposure to the drug and disappeared by 24 hours. Furthermore, eNOS protein levels as well as NO production increased by 24 hours in human osteoblastic MG63 cells, which is at the same time

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141

BMP‐2 protein levels have been found to be increased in cells treated with statins (Garrett et al., 2001b). Another possible impact of prenylation inhibition is on the small GTPase Rab23, a negative regulator of hedgehog signaling. Downstream signaling of hedgehog is facilitated by the members of the Gli family of transcription factors (Cohen, 2003; Eggenschwiler et al., 2001, 2006; Evans et al., 2003, 2005). As regulation of the BMP‐2 promoter is known to be under the control of these Glis transcription factors (Garrett et al., 2003), reduction in prenylation by statins would cause a decrease in Rab23 prenylation and a subsequent increase in Gli signaling and BMP expression. These findings link the prenylated Rab23 and eNOS expression and activity with BMP‐2 mRNA in time, although the mechanism whereby the activation of eNOS activity and Rab23 would lead to BMP‐2 protein increases is still under investigation. b. Clinical Findings. Large databases were examined and indicated there a possible relationship between statin use, BMD, and subsequent fractures (Bauer, 2003). Since then, published studies indicate a significant increase of BMD associated with taking statins in postmenopausal women (Edwards et al., 2000), and a protective eVect against nonpathological fracture among older women (Chan et al., 2000). Another association between statin use by elderly patients and reduction in the risk of hip fracture was seen (Wang et al., 2000), while others suggested current exposure to statins is associated with a decreased risk of bone fractures in individuals aged 50 years and older (Meier et al., 2000a,b). Further studies indicated a 60% reduction in fracture risk in women is associated with statin use (Pasco et al., 2002) with similar findings in men (Funkhouser et al., 2002). In male patients with type 2 diabetes mellitus, it was shown HMG‐CoA reductase inhibitors increased BMD of the femur (Chung et al., 2000). A recent prospective 1‐year study found simvastatin treatment resulted in a significant increase in bone alkaline phosphatase with no significant decrease in the bone resorption marker C‐terminal fragment of type I collagen. Simvastatin increased BMD at 6 and 12 months in women, and the authors concluded simvastatin had a positive eVect on bone formation and BMD. Another study indicated simvastatin increased serum osteocalcin levels in patients (Chan et al., 2001). One prospective study of 91,052 patients indicated statin use was associated with a 36% (odds ratio, 0.64; 95% confidence interval, 0.58–0.72) reduction in fracture risk when compared with no lipid‐lowering therapy and a 32% (odds ratio, 0.67; 95% confidence interval, 0.50–0.91) reduction when compared with non‐statin lipid‐lowering therapy (Scranton et al., 2005). Other preliminary reports have indicated little or no effect of orally administered statins on bone (Cauley et al., 2000; LaCroix et al., 2003; Rejnmark et al., 2002, 2004; van Staa et al., 2001). The major drawback of all these studies is they are retrospective. The compliance of patients taking

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statins is unknown and statins ineVective on bone, such as pravastatin, are included, while the dose of statin used varies considerably. The in vitro findings suggest statins increase BMP‐2 expression and stimulate osteoblast diVerentiation leading to new bone formation in vivo. However, all of the statins currently on the market target the liver and decrease cholesterol biosynthesis. Consequently, the biodistribution of active statin or metabolites to bone and other peripheral tissues is small, making it uncertain if current statins administered orally for lipid lowering will have beneficial eVects on bone in humans. There are several possibilities for improving biodistribution to bone. The more recent potent statins such as cerivastatin or atorvastatin may get past the liver in suYcient amounts to cause beneficial eVects on bone, and animal studies suggest this may be the case. Alternative modes of administration of the statins such as transdermal application through a skin patch may also solve the problem of poor biodistribution to bone (Gutierrez et al., 2006). Another possibility would be to administer statins at local sites, bypassing the peripheral distribution problem, to stimulate bone formation (Skoglund and Aspenberg, 2006). This turns out to be a feasible mode of administration to stimulate new bone. A fourth possibility is that there may be other drugs of this class that were not selected for development as cholesterol‐lowering agents because of their relatively greater biodistribution to peripheral tissues. These may be ideal drugs for use as bone‐active agents. Perhaps the most important consequence of these findings is that not only would statins themselves be eVective drugs for bone loss conditions, but these findings focus attention on the mevalonate pathway and its relationship to BMP‐2 expression and bone formation. This could lead to the identification of other potential molecular targets for drug discovery as well as other therapeutic approaches to enhance bone formation and produce the ideal anabolic agent for osteoporosis. 2. Proteasome Inhibitors The proteasome is an abundant multicatalytic enzyme complex present in the cytoplasm and nucleus of all eukaryotic cells. The primary function of the proteasome is to degrade proteins. While it can act primarily as a cellular ‘‘garbage disposal’’ that removed damaged or misfolded proteins from cells, the proteasome also removes various short‐lived proteins that regulate the cell cycle, cell growth, and diVerentiation. By regulating the turnover of these proteins via timely degradation and recycling, the proteasome plays a critical role in the maintenance of cellular homeostasis. Substrates of the proteasome include cell‐cycle regulators, signaling molecules, tumor suppressors, transcription factors, and anti‐apoptotic proteins; over 80% of all cellular proteins are recycled through the proteasome.

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The ubiquitin/proteasome system plays an important role in the regulation of activity of bone cells. Osteoblastic function is suppressed by the cAMP pathway through proteolytic degradation of Cbfa1/Runx2 involving a ubiquitin/proteasome‐dependent mechanism (Tintut et al., 1999) and that the anti‐apoptotic eVect of PTH is prolonged by inhibition of proteasomal activity resulting in elevated levels of cbfa1/Runx2 (Bellido et al., 2003). Treatment of cells with the proteasome inhibitors also induced ATF4 accumulation, an important regulator of bone cell activity, resulted in activation of an osteocalcin promoter (Yang and Karsenty, 2004). Other evidence to supporting the role of the proteasome in bone cells shows that Smurf1, an E3 ligase responsible to targeting Cbfa1 and other important transcription factors to the proteasome, appears to be an important regulatory factor in osteoblast diVerentiation and a potential molecular target for identification of bone anabolic agents (Zhao et al., 2003) and confirmed where Smurf1 induces Runx2 degradation in a SMAD6‐dependent manner (Shen et al., 2006). Smurf1 also physically interacts with MEKK2, an important mediator of BMP signaling, and promotes the ubiquitination and turnover of MEKK2 which negatively regulates osteoblast activity and response to BMP through controlling MEKK2 degradation (Yamashita et al., 2005). Proteasomal activity in osteoblasts plays a pivotal role in regulating the intracellular levels of molecules important for many of the critical signaling pathways. These include the intricate hedgehog signaling mechanism of the Gli family of transcription factors where the activity of Gli2 and Gli3 are regulated by the proteasome (Cohen, 2003; Kalderon, 2002, 2005; van den Heuvel, 2003; Yu and Miller, 2004). Regulation of Gli transcription factors by proteasome inhibitors leads to the enhanced expression of BMP‐2 and results in bone formation (Garrett et al., 2003). Bone morphogenetic protein is a powerful stimulator of bone formation acting through its receptors BMPR‐I and ‐II and through the downstream eVector molecules known as SMADs. The cytoplasmic levels of these SMADs signaling molecules are also regulated by the proteasome (Heldin and ten Dijke, 1999; Johnsen et al., 2002; Nishimori et al., 2001; Shen et al., 2006; Zhang et al., 2001) indicating a probable enhancement of BMP signaling by proteasome inhibitors. Another pathway which plays an important role in the anabolic activity of osteoblasts in the Wnt/
‐catenin pathway (Holmen et al., 2005; Iwao et al., 1999; Mbalaviele et al., 2005; Urano, 2006). Upregulation of this pathway has been shown to play a role in bone metabolism and appears to play a cooperative role with the BMP pathway in stimulating bone formation (Mbalaviele et al., 2005). Although the Wnt/
‐catenin pathway is extremely complicated, it has been shown that endogenous regulators such as sFRP (soluble Frizzled‐related peptide) play a critical role in its eVect on bone (Bodine et al., 2005).
‐catenin levels inside the cell are maintained by action

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Table V Increase of New Bone Formation In Vitro by Proteasome Inhibitor 1 Dose (mg/ml) 0 0.005 0.0125 0.025 0.05 0.1

New Bone Formation (mm
103) 4.1 4.2 6.1 8.6 9.9 8.3

     

0.2 0.4 0.4* 0.6* 0.5* 0.5*

*Significantly greater than vehicle treated group. ANOVA p < 0.05.

of the proteasome (Aberle et al., 1997; Bonvini et al., 1999; Easwaran et al., 1999; Salomon et al., 1997). This indicates another potential pathway in which proteasomes and their inhibitors may influence bone metabolic activity. Therefore, it is not surprising that inhibition of proteasomal activity in osteoblasts by proteasome inhibitors activity would lead to pronounced eVects on bone tissues. In fact proteasome inhibitors are very potent anabolic agents in vitro (Table V) in the low nanomolar range as well as in vivo (Garrett et al., 2003). In the clinic Bortezomibw or Velcadew (PS‐341), the first proteasome inhibitor evaluated in human clinical trials, has been approved by the US Food and Drug Administration for use in patients with refractory or relapsed multiple myeloma with minimal side eVects and has been shown to significantly elevate alkaline phosphatase from baseline (Zangari et al., 2005). The rise in alkaline phosphatase together with a parallel increase in PTH after Bortezomibw suggested that response to Bortezomibw in myeloma is closely associated with osteoblastic activation (Shimazaki et al., 2005; Zangari et al., 2005) indicating a possible therapeutic activity not only treat myeloma itself but also reverse the bone loss common in the associated myeloma bone disease. Proteasome inhibitors, such as Bortezomibw, therefore have the potential to be potent anabolic agents, systemically to treat bone deficit conditions locally to eVectively enhance fracture healing, where bone regeneration is required.

3. Flavonoids Flavonoids are found ubiquitously in higher plants and constitute an important component of the majority of people’s daily diets. The biological activities of flavonoids cover a very broad spectrum, from anticancer and antibacterial activities to eVects on bone. Although flavonoids may not be developed as

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pharmaceutical agents for the treatment of bone deficit conditions, mostly because of the multitude of eVects ascribed to them, numerous papers have suggested these agents act on bone in many ways. The eVects of flavonoids on bone tissues were first noted sometime ago when the flavonoid Catergen (Zyma) showed an improvement in bone quality (Jones et al., 1984). Since then, many flavonoids have been shown to have eVects on bone. A synthetic flavonoid, Ipriflavone, has been shown to have eVects on bone cells in vitro (Benvenuti et al., 1991; Bonucci et al., 1992; Hagiwara et al., 1999; Kakai et al., 1992; Ribari and Sziklai, 1987). It also has eVects on bone metabolism in rats (Cecchini et al., 1997; Foldes et al., 1988; Ozawa et al., 1992; Shino et al., 1988; Yamazaki, 1987) and in patients (Agnusdei et al., 1989; de Aloysio et al., 1997; Gambacciani et al., 1993; Kovacs, 1994; Mazzuoli et al., 1992; Melis et al., 1992; Nakamura et al., 1992; Passeri et al., 1992; Reginster, 1993) and is now used in the clinic in many countries. However, there have been a number of papers showing no eVect of this agent either in vitro or in the clinic (Alexandersen et al., 2001; Deyhim et al., 2005; Ghezzo et al., 1996). ‐glucosylhesperidin significantly prevented this bone loss in rats (Chiba et al., 2003) while kaempferol positively aVected osteoblasts (Miyake et al., 2003). Quercetin has pronounced eVects on bone cells preventing bone loss as well as having marked bone‐building properties (Horcajada‐ Molteni et al., 2000; Prouillet et al., 2004; Rassi et al., 2005; Ross, 2005; Singh et al., 2001; Son et al., 2006; Sziklai and Ribari, 1995; Wattel et al., 2003, 2004; Woo et al., 2004; Wood, 2004; Yamaguchi and Jie, 2001; Zhang et al., 1996). The flavonoids eupalitin 3‐O‐
‐D‐galactopyranosyl‐(1‐‐ > 2)‐
‐D‐glucopyranoside, eupalitin3‐O‐
‐D‐galactopyranoside, and 6‐methoxykaempferol 3‐O‐
‐D‐(1‐‐ > 6)‐robinoside were shown to have eVects on bone (Li et al., 1996). Another flavonoid naringin, which can be found in citrus fruit, apparently can act to inhibit HMG‐Co reductase and can increase local new bone formation possibly being used as a bone graft material (Wong and Rabie, 2006). A great portion of these flavonoid molecules seem to have their eVects on bone through an estrogen‐like activity and have been therefore termed phytoestrogens (Vaya and Tamir, 2004). Although the majority of the eVects of flavonoids are on preventing resorption, recently it has been shown they are capable of stimulating new bone formation and therefore can be considered anabolic agents. An example of the eVects of these flavonoids on the BMP‐2 promoter activity is shown in Fig. 1 where Robustone from the Derris robusta plant (Dakora, 1995; Dakora and Ndakidemi, 2003) caused a dose‐ dependent increase in BMP‐2 promoter activity in osteoblastic cells. These cells were stably transfected with a BMP‐2‐promoter—Luciferase construct and exposed to this flavonoid for 24 hours. There was a marked increase

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200 180

Relative Luciferase Activity

160 140 120 100 80 60 40 20 0 Cont BMP2

Figure 1

0.1 0.2 0.4 0.8 1.6 3.2 6.25 12.5 25 Concentration of Flavonoid (mM)

50

Increase in BMP‐2 promoter stimulated by the flavonoid Robustone.

Table VI Increase of New Bone Formation In Vitro by Robustone Dose (mg/ml) 0 0.02 0.2 2 20

New Bone Formation (mm
103) 4.2 4.1 5.3 6.4 8.3

    

0.2 0.3 0.3* 0.2* 0.2*

*Significantly greater than vehicle treated group. ANOVA p < 0.05.

in BMP‐2 promoter activity around 1 M and a maximum stimulation at 5 M. This flavonoid also dose dependently stimulated new bone formation when assessed in an in vitro murine calvarial assay (Table VI, Fig. 2), where over 7 days, there is a significant increase in new bone formation in cultured neonatal murine calvaria. Many flavonoids aVect bone metabolism and most inhibit osteoclastic resorption and reduce bone loss. However, there is a subset of these compounds that aVects BMP expression and the BMP signaling pathway stimulating and

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Robustone (20 mg/ml)

New Bone Formation Figure 2

Histology of neonatal calvaria treated with Robustone.

enhancing bone formation. Therefore, some flavonoids do aVect the BMP pathway and are potential anabolic agents. Whether these agents can clinically increase bone mass and reduce fractures is still to be determined. They do have the potential to be clinically relevant bone anabolic agents. Although, it is unclear if these agents, mostly from dietary intake, would reach suYcient serum concentrations to elicit a systemic bone anabolic response this may not preclude their use for local administration for fracture and bone defect repair.

4. Antagonists of Sclerostin Unlike the other growth factors, sclerostin is an osteocyte‐expressed negative regulator of bone formation with amino acid sequence similarity with the DAN family of secreted glycoproteins that share the capacity to antagonize BMP activity. While it binds BMPs and antagonizes their bone forming capacity, it cannot antagonize all BMP responses. Sclerostin’s mechanism of action is, therefore, distinct from that described for classical BMP antagonists. Because of its unique expression pattern and role in reducing bone formation (Kusu et al., 2003; Ohyama et al., 2004; Sutherland et al., 2004; van Bezooijen et al., 2004; Winkler et al., 2003, 2004, 2005), it has been suggested that blocking the activity of this factor would lead to anabolic activity leading to enhanced bone formation (Ott, 2005; Poole et al., 2005; van Bezooijen et al., 2005a,b). It has been shown that antibodies developed to sclerostin cause marked increases in bone mass and bone formation (Paszty, 2006). This alternative approach to the treatment of bone deficit

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conditions is particularly interesting and provides us with another way to treat bone diseases. The eVectiveness of this treatment would be limited to sites where sclerostin is expressed and regulating bone formation. It is therefore unclear if this would be true at fracture sites or other sites of bone formation.

5. Prostaglandin Agonists Prostaglandins play a significant role in bone metabolism (Tashjian et al., 1972). Strong evidence indicates that prostaglandins play a role in stimulating bone resorption (Caniggia et al., 1978; Dietrich and Raisz, 1975; Dowsett et al., 1976; Harris et al., 1973a,b; Kato, 1980; Katz et al., 1981; Raisz and Koolemans‐Beynen, 1974; Raisz et al., 1974, 1979; Robinson et al., 1975; Sakamoto et al., 1979; Tashjian and Levine, 1978; Tashjian et al., 1977; Yamasaki et al., 1980; Yu et al., 1979). Early evidence indicated prostaglandin E2 could not only cause bone resorption but it could also stimulate bone formation (Chyun and Raisz, 1984). Subsequent work has confirmed this where prostaglandins either enhance bone formation or prevent bone loss (Akamine et al., 1992; Ito et al., 1993; Jee et al., 1990, 1992; Jorgensen et al., 1988; Ke et al., 1993; Li et al., 1993, 1995; Lorenzo and Sousa, 1988; Ma et al., 1994; Marks and Miller, 1988; McCarthy et al., 1991; Miller and Marks, 1993a,b; Mori et al., 1990, 1992; Raisz and Fall, 1990; Raisz et al., 1993; Schmid et al., 1992; Yang et al., 1993). This early work led to the discovery that PGE2 mediates its tissue‐specific pharmacological activity via four diVerent G‐protein‐coupled receptor subtypes, EP1–4 (Paralkar et al., 2003; Raisz and Woodiel, 2003). Bone eVects have been reported with many of the prostanoid receptors, with most interest focused on the anabolic eVects of EP2, EP4, and FP receptors. Current data suggests activity of the EP2 receptor stimulates formation and activity of the EP4 receptor stimulates resorption (and possibly formation), while activity of the FP receptor produces new trabeculae (Hartke and Lundy, 2001). These studies have led to the synthesis of a number of EP4 receptor selective prostaglandin E2 agonists which enhance bone formation (Hagino et al., 2005; Ito et al., 2006; Ke et al., 2006; Shamir et al., 2004) and can augment BMP‐induced bone formation (Toyoda et al., 2005). These agents have been further investigated in preclinical studies for their ability to enhance fracture healing (Paralkar et al., 2003; Tanaka et al., 2004). Although no evidence exists which suggests these agents utilizes the BMP signaling pathway to elicit its activity, these agents do appear to enhance the eVects of BMP on bone tissues (Arikawa et al., 2004; Toyoda et al., 2005).

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6. Parathyroid Hormone Full‐length human parathyroid hormone (PTH), like PTH‐peptide (Teriparatidew), is an eVective anabolic agent in preclinical studies and is being developed under the name of Preos for treatment in osteoporosis although unlike PTH‐peptide it has not yet been approved. Preos demonstrated significant fracture risk reductions in postmenopausal women with osteoporosis, but noted the higher incidence of hypercalcemia with Preos compared to placebo (NPS, 2006). PTH is expensive to manufacture and can only be given by injection which like the PTH‐peptide (Teriparatidew) limits its use for the treatment of osteoporosis and bone diseases. Of the agents available on the market today to treat bone deficit conditions, PTH‐peptide remains the only agent with significant bone anabolic activity. While PTH has been shown to interact with BMP to increase osteoblastogenesis and decrease adipogenesis (Chang et al., 2003), other studies show that PTHrP and PTH inhibited BMP induced osteogenesis (van der Horst et al., 2005). PTH has been shown to reduce sclerostin, a very potent inhibitor of the BMP signaling pathway, and it was suggested that this is one of the mechanisms of action of PTH (Keller and Kneissel, 2005).

C. Agents That Do Not Act on the BMP Pathway A few reported anabolic agents exist which elicit their bone anabolic activity apparently through BMP independent pathways. Unlike Strontium, AC100, and Chrysalin, the Calcilytics agents increase PTH production. And from studies PTH aVects the BMP pathway by interacting with BMP (van der Horst et al., 2005) and possibly by inhibiting sclerostin expression (Keller and Kneissel, 2005). So it is entirely possible that the calcilytics do aVect the BMP pathway although to date this has not been shown (Table VII).

Table VII Bone Agents That Do Not AVect or Enhance the BMP Pathway BMP Drug Strontium ranelate Chrysalin Calcilytics
‐blockers AC100

Protein Expression

Signaling

No No No No No

No No No No No

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1. Strontium Ranelate Strontium ranelate (PROTELOS) is a newer anti‐osteoporotic agent that appears to reduce bone resorption by decreasing osteoclast diVerentiation and activity (Ammann, 2005). The di‐strontium salt strontium ranelate, a novel orally active agent consisting of two atoms of stable strontium and the organic moiety ranelic acid, has been developed for the treatment of osteoporosis (Reginster, 2002; Reginster et al., 2003). It has been reported to enhance osteoblastic cell replication and increase collagen synthesis while decreasing pre‐osteoclast diVerentiation and bone‐resorbing activity of mature osteoclasts in vitro (Marie, 2005). The anti‐fracture eYcacy of strontium ranelate was assessed in two large, randomized, controlled trials conducted in postmenopausal women. Over the 3‐year treatment period significantly fewer patients had height loss and fewer patients reported new or worsening back pain in the treated group than in the control group (Delmas, 2005; Rizzoli, 2005). These results demonstrate strontium ranelate is a new therapeutic option in the prevention of osteoporotic vertebral fractures in postmenopausal women. Pharmacological studies in animals have shown that strontium ranelate decreases bone resorption and increases bone formation, resulting in increased bone mass. In ovariectomized rats, strontium ranelate prevented the reduction in bone mineral content and the decrease in trabecular bone volume induced by estrogen deficiency suggesting a possible anabolic activity. In this model, strontium ranelate decreased bone resorption, whereas bone formation was maintained at a high level as documented by plasma biochemical markers and histomorphometric indices of bone formation (Marie, 2005). The mechanism of action of strontium ranelate is unclear, however, no evidence exists indicating this agent elicits any of its eVects through modulation of the BMP pathway.

2. Chrysalin The ‐thrombin peptide, TP508 or Chrysalin, accelerates the healing of full‐ thickness wounds in both normal and ischemic skin. In wounds treated with TP508, a pattern of increased vascularization is consistently observed both grossly and microscopically when compared to wounds treated with saline (Norfleet et al., 2000). TP508, binds to high‐aYnity thrombin receptors and mimics cellular eVects of thrombin at sites of tissue injury (Stiernberg et al., 2000). This thrombin peptide was shown to be active in repairing segmental bone defects in rabbits (Sheller et al., 2004), and its mode of action was thought to be through thrombin‐induced angiogenesis (Tsopanoglou et al., 2004). This thrombin‐related peptide was then shown to enhance bone formation during distraction osteogenesis (Li et al., 2005). TP508 was shown

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to promote fracture repair through a mechanism that involves an increased induction of a number of growth factors, enhanced expression of inflammatory mediators, and angiogenesis‐related genes (Wang et al., 2005). This peptide has also been shown to exert maturation specific eVects on chondrocytes in the endochondral lineage, promoting cartilage extracellular matrix synthesis over endochondral diVerentiation in resting zone cells and proliferation over diVerentiation of growth zone cells (Schwartz et al., 2005). Phase 3 clinical trials with this synthetic peptide Chrysalin (R) (TP508) in unstable, displaced distal radius (wrist) fractures showed that the treatment with 10 g Chrysalin did not demonstrate a statistically significant benefit compared to placebo in the primary eYcacy endpoint of time to removal of immobilization. A secondary endpoint, radiographic evidence of time to radial cortical bridging, showed a statistically significant benefit for Chrysalin‐treated subjects (p ¼ 0.049) (Othologic, 2006). The mechanism of action of Chrysalin does not appear to be due to eVects on BMP expression or signaling and so the agent probably works through an alternative independent pathway. 3. Calcilytics A number of years ago, the receptor sensing calcium (CaR) was discovered on the surface of parathyroid cells. Although Ca2þ receptors are expressed throughout the body and in many tissues that are not intimately involved in systemic Ca2þ homeostasis, their physiological and/or pathological significance remains speculative and their value as therapeutic targets is unknown (Nemeth, 2004a,b). Stimulation of CaR induced by an increase of extracellular ionized calcium concentration resulted in an increase of intracellular calcium and subsequent decrease of PTH secretion from parathyroid cells (Hoppe and Rybczynska, 2000). Agents were then synthesized that block this receptor activity and act as antagonist at the Ca2þ receptor. When infused intravenously in normal rats, they caused a rapid and large increase in plasma levels of PTH (Nemeth et al., 2001). These are known as calcilytic agents and they increase endogenous levels of circulating PTH to an extent that stimulates new bone formation (Nemeth, 2004a,b). These agents could replace the use of exogenous PTH or its peptide fragments in treating osteoporosis (Nemeth, 2002a,b). There have been designs and new synthesis, of new calcium receptor antagonist which are novel 3H‐pyrimidin‐4‐ones (Shcherbakova et al., 2005a,b). These newer calcilytic agents are promising new therapeutic tools allowing for tight control of plasma PTH and restoration of circadian PTH rhythmicity (Schmitt et al., 2005). Other eVects are now being seen with these agents where calcilytic drugs prevent NO‐induced damage and death of human neurons (Dal Pra et al., 2005). Although calcilytics show no direct eVects on the BMP pathway, it is interesting to

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postulate that due to their capacity to increase PTH expression they may still aVect this pathway. 4. b‐Blockers A number of years ago,
‐adrenergic blocking agent propranolol was shown to increase bone formation in rats (Minkowitz et al., 1991), and the direct eVects of physiologically relevant propranolol concentrations on osteoblastic cells can be attributed principally to
‐adrenergic blockade (Majeska et al., 1992). It has been shown that
‐adrenergic stimulation enhances osteoclastogenesis and bone resorption (Arai et al., 2003; Takeuchi et al., 2001). Because of these findings,
‐adrenergic antagonists have been investigated in the clinic and have been shown to be associated with a reduction in fracture risk and higher BMD (Pasco et al., 2004; Schlienger et al., 2004). While studies in rats using
‐agonists confirm the deleterious eVect of
2 agonists on bone mass (Bonnet et al., 2005), more recent studies indicate that
‐blockers might suppress bone resorption with relative preservation of bone formation (Pasco et al., 2005). However, others report no evidence of
‐blocker drugs stimulating bone formation and if anything, propranolol reduces osteoblast activity (Reid et al., 2005). Although there does appear to be a relationship between BMP and responses in adrenergic cells (Varley and Maxwell, 1996; Varley et al., 1998; Xu et al., 2003; Zhang et al., 2004), there appears to be little if any evidence that
‐blockers elicit their bone activity by acting on BMP or enhancing its activity. 5. AC‐100 (MEPE Peptide) Matrix extracellular phosphoglycoprotein (MEPE) is a 56.6‐kDa protein and is expressed exclusively in osteoblasts, osteocytes, and odontoblasts with markedly elevated expression found in X‐linked hypophosphatemic rickets (Hyp) osteoblasts and in oncogenic hypophosphatemic osteomalacia (OHO) tumors (Rowe et al., 2004). AC‐100, a central 23‐amino acid fragment of MEPE, contains motifs that are important in regulating cellular activities in the bone microenvironment although it is unclear just how this happens. This fragment AC‐100 apparently requires inducible cyclooxygenase‐2 to exert potent anabolic eVects on normal human marrow osteoblast precursors (Nagel et al., 2004). Work has focused on the ability of the synthetic peptide fragment of human MEPE stimulates new bone formation in vitro and in vivo (Hayashibara et al., 2004). It is therefore possible that this fragment of MEPE, AC‐100, represents a potential treatment for bone repair in periodontal and orthopedic applications and a novel biological approach to dentistry. A phase I clinical study demonstrated equivalent

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safety seen in preclinical studies. Two phase II clinical studies of AC‐100 were initiated in 2005, one for periodontal regeneration, the other for dental pulp protection. Results for these studies will be available in early 2006. In addition, this product is being developed for an orthopedic application (Acologix, 2006). No evidence exists which suggests this peptide utilizes the BMP signaling pathway to elicit its activity.

IV. Conclusions and Future Directions There are numerous factors, signaling pathways, and transcription factors critically involved in the stimulation of bone growth. These include BMP/ SMAD, Wnt/
‐catenin, Hedgehog/Gli, IGF, and FGF pathways. Lately, there has been the suggestion that the sympathetic nervous system is involved in bone metabolism, and it is intriguing to suggest that it might be possible to modulate bone metabolism by manipulating the important molecules that influence this pathway. While many of these pathways play an essential role in growth responses during patterning an embryogenesis of bone, there is also evidence these pathways play important roles in adult bone metabolism. It would be naive to think that any single pathway can stimulate bone formation without a major influence on these other pathways. Mounting evidence indicates that the BMP, Wnt, and hedgehog pathways interact as regulators and/or modulators of each other. This would be especially true when considering the intricate control and regulation of the cascade of events responsible for bone formation (Burstyn‐Cohen et al., 2004; Fisher et al., 2005; Garrett et al., 2003; Grotewold and Ruther, 2002; Huang and Klein, 2004; Kawai and Sugiura, 2001; Litsiou et al., 2005; Mbalaviele et al., 2005; Raible and Ragland, 2005; Rawadi et al., 2003; Theil et al., 2002; Tian et al., 2005; Tzahor et al., 2003; Zhang and Stott, 2004). Although these diVerent pathways control anabolic activity, it is vital to understand that the negative regulation of bone growth by inhibitors of these pathway may play a more important role in controlling where and when bone growth occurs. It is clear, however, that the BMP pathway is one of the major pathways responsible for initiating the complex process of bone formation and small molecular weight agents that enhance this pathway by either upregulating the expression of BMPs, increasing downstream signaling or reducing the negative regulators of this pathway would be good potential candidates for bone anabolic agents. A major need exists for low cost eVective anabolic agents that could be used orally or delivered locally for the treatment of bone deficit conditions. In addition, agents that influence the BMP pathway such as the statins or agents that aVect the proteasome clearly show promise as anabolic agents for the treatment for bone deficit conditions. These new and powerful small molecular

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weight bone anabolic agents may oVer new innovative and versatile treatments for bone disease in the future.

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The Role of Mammalian Circadian Proteins in Normal Physiology and Genotoxic Stress Responses Roman V. Kondratov, Victoria Y. Gorbacheva, and Marina P. Antoch Department of Cancer Biology, Lerner Research Institute Cleveland Clinic Foundation, Cleveland, Ohio 44195

I. Introduction II. Circadian Rhythms and the Organization of the Mammalian Circadian System III. Molecular Organization of the Circadian Oscillator in Mammals A. The Molecular Circadian Oscillator Is Composed of Multiple Interacting Loops B. Posttranslational Regulation of the Components of the Molecular Circadian Oscillator C. Peripheral Clocks and Circadian Control of Gene Expression in DiVerent Tissues IV. Human Disorders Associated with Altered Function of the Circadian System A. Sleep Disorders B. Jet Lag and Shift Work Maladaptation Syndromes C. Seasonal AVective Disorder D. Mood Disorders V. Pathologies and Developmental Defects in Circadian Mutant Mice A. CLOCK B. BMAL1 C. NPAS2 D. PERIODs E. TIMELESS F. CRYPTOCHROMEs G. REV‐ERB H. Circadian Proteins, Organism‐Environment Interaction, and Pathologies: A Hypothesis VI. Circadian Control of the Organism’s Response to Genotoxic Stress A. Chronotherapy of Cancer B. The Sensitivity of Normal Cells to Genotoxic Drugs Depends on the Functional Status of the CLOCK/BMAL1 Transactivation Complex C. Molecular Determinants of Sensitivity to Anticancer Therapy D. Functional Interplay Between the Circadian and Stress Response Systems: A Model VII. Circadian Proteins as Targets for Therapeutic Intervention Acknowledgments References

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The last two decades have significantly advanced our understanding of the organization of the circadian system at all levels of regulation–molecular, cellular, tissue, and systemic. It has been recognized that the circadian system represents a complex temporal regulatory network, which plays an important role in synchronizing various biological processes within an organism and coordinating them with the environment. It is believed that deregulation of this synchronization may result in the development of various pathologies. However, recent studies using various circadian mutant mouse models have demonstrated that at least some of the components of the molecular oscillator are actively involved in physiological processes not directly related to their role in the circadian clock. The growing amount of evidence suggests that, in addition to their circadian function, circadian proteins are important in maintaining tissue homeostasis under normal and stress conditions. In this chapter, we will summarize recent data about the regulation of the mammalian molecular circadian oscillator and will focus on a new role of the circadian system and individual circadian proteins in the organism’s physiology and response to genotoxic stress in connection with diseases treatment and prevention. ß 2007, Elsevier Inc.

I. Introduction Many essential life processes are driven by an intrinsic timekeeping system, called the circadian system, which allows adaptation of the timing of an organism’s physiology to the cyclic changes in the environment. In the past decade, significant progress has been made in our understanding of the molecular and cellular mechanisms required for generation of circadian rhythms. In addition to deciphering the details of the molecular clock function, we have also started to recognize how multiple clocks interact and how clock‐generated periodicities translate into oscillations in various biological processes. Most of these advances were made possible by the analysis of mice with targeted disruption of or mutations in major circadian genes. The study of these animal models has also shed new light on the importance of maintaining proper temporal organization for normal tissue homeostasis. In addition, detailed study of mice deficient in diVerent components of the circadian system revealed a number of pathologies that may not be directly dependent on the activity of the circadian clock per se. In this chapter, we give a very brief overview of the organization and functional role of the mammalian circadian system. We will focus on recent data, summarizing the role of the circadian system and individual circadian proteins in maintaining normal tissue homeostasis and in modulating the

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organism’s response to genotoxic stress. We also discuss possible clinical applications of these novel findings.

II. Circadian Rhythms and the Organization of the Mammalian Circadian System Circadian rhythms are 24‐hour oscillations in an organism’s behavior, physiology, and metabolism. It is believed that these rhythms evolved in the process of an organism’s adaptation to 24‐hour periodicities caused by the Earth’s rotation. A remarkable feature of the daily rhythms observed in diVerent organisms is that they are not simply a response to changes in the physical environment that occur during a 24‐hour period, but arise from an internal timekeeping mechanism. The internal clock is capable of generating and sustaining 24‐hour periodicities under constant environmental conditions (i.e., in constant darkness). To synchronize its internal phase with the environment and to orchestrate proper timing, the internal clock responds to various environmental stimuli (light in particular) and transmits this information through the output pathways to regulate a number of biological processes. Circadian clocks have been characterized in a broad range of organisms, from cyanobacteria and fungi to birds and mammals (Bell‐Pedersen et al., 2005; Cahill, 2002; Dunlap and Loros, 2004; Hall, 2003; Iwasaki and Kondo, 2004; Lowrey and Takahashi, 2004; Mas, 2005). The mammalian circadian clock is organized as a hierarchical network of numerous oscillators, with the master clock residing in the neurons of the hypothalamic suprachiasmatic nucleus (SCN) of the anterior hypothalamus. The master clock in the SCN is entrained by the light:dark (LD) cycle and, in turn, synchronizes the phases of multiple peripheral clocks residing in diVerent peripheral tissues (Reppert and Weaver, 2002). Identification of the SCN functions as the master circadian regulator resulted from the SCN lesion and transplantation studies, demonstrating that animals with surgical ablation of the SCN loose behavioral rhythmicity and that transplantation of SCN tissue from normal donor animals to the SCN‐ lesioned recipients restored the circadian rhythms of the locomotor activity with a period characteristic of the donor (Ralph et al., 1990; Sujino et al., 2003). Importantly, the peripheral clocks can respond to other synchronizing stimuli. Thus, it has been demonstrated that in the liver, the rhythmicity in gene expression (Damiola et al., 2000; Hara et al., 2001; Stokkan et al., 2001) and energy metabolism (Satoh et al., 2006) can be entrained by the feeding schedule independent of the SCN. When the master synchronizing signal is missing (i.e., in SCN‐lesioned animals), the peripheral clocks still function, however, the phases of their oscillation are uncoordinated (Yoo et al., 2004).

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The ubiquitous nature of the clock mechanism and its ability to respond to a variety of external stimuli underscores the important role that the circadian system plays in controlling diVerent physiological processes. Indeed, we now understand that synchronization of various oscillating processes is an important component of an organism’s health and that deregulations of the circadian system can be detrimental.

III. Molecular Organization of the Circadian Oscillator in Mammals A. The Molecular Circadian Oscillator Is Composed of Multiple Interacting Loops In all types of mammalian cells, the molecular mechanism underlying circadian clock function is based on the interconnected transcription/translation feedback loops. Two basic helix‐loop‐helix (bHLH) PAS‐domain transcription factors, CLOCK (or its closest homolog expressed in the forebrain and vasculature, NPAS2) and BMAL1, form the positive elements of the central oscillatory loop. The CLOCK/BMAL1 heterodimer binds to E‐box elements in the promoters of target genes and drives rhythmic transcription of three Period (Per1, Per2, and Per3) and two Cryptochrome (Cry1 and Cry2) genes. When PER and CRY proteins are translated, they form PER/CRY complexes that are translocated to the nucleus where they inhibit CLOCK/ BMAL1‐mediated transcription, forming a negative autoregulatory loop (Gekakis et al., 1998; GriYn et al., 1999; Kume et al., 1999; Sato et al., 2006). As a consequence, transcription of the Per and Cry genes is reduced, which results in decrease in the abundance of the corresponding proteins. This leads to release of the repression of CLOCK/BMAL1 and to initiation of a new, 24‐hour transcriptional cycle. CLOCK/BMAL1 also controls transcription of Rev‐erb, which periodically represses expression of Bmal1, thus forming an additional feedback loop (Preitner et al., 2002). Positive and negative elements of these regulatory loops function to coordinate transcriptional events at appropriate times to provide 24‐hour periodicities (Fig. 1). In addition to core components of the circadian oscillatory machinery, such as Per, Cry, and Rev‐erb genes, the CLOCK/BMAL1 complex also drives rhythmic expression of numerous output genes harboring E‐boxes in their promoter regions (Lowrey and Takahashi, 2004). Altogether, oscillations in the expression of multiple genes account for the circadian rhythmicity of physiological processes (Panda and Hogenesch, 2004). Extensive genetic studies have demonstrated the critical role of clock proteins in maintaining circadian rhythmicity and identified specific functions of

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Figure 1 Molecular organization of the circadian oscillator in mammals. The bHLH‐PAS domain transcription factors CLOCK and BMAL1 in a form of a heterodimer translocate to the nucleus and interact with histone acetyltransferase (HAT) to activate transcription of Periods, Cryptochromes, Timeless, and Rev‐Erb genes. The protein products of Periods, Cryptochromes, and Timeless form various complexes, enter the nucleus and inhibit CLOCK/ BMAL1‐mediated transcription, including their own, thus, closing the negative feedback loop. An interlocking positive feedback loop involves REV ERB‐dependent repression of Bmal1 transcription. In addition, Bmal1 gene is reported to be activated by PER2. It is believed that the interaction of these multiple loops is necessary for generation and maintenance of the 24‐hour periodicities in the expression of clock‐controlled genes.

individual proteins. Targeted mutations have been generated in all of the components of the circadian feedback loops and the resulting mutant mice have been tested for their locomotor activity and circadian gene expression profiles. Analysis of Clock mutant and Bmal1 knockout mice demonstrated that both of these positive components of the major circadian transactivation complex are necessary for persistence of circadian rhythmicity in behavior and gene expression. An ENU‐induced Clock mutation lengthens the circadian period both in heterozygous and homozygous animals, and results in complete loss of rhythmicity in homozygous mice after prolonged exposure to constant darkness (DD) (Vitaterna et al., 1994). Since the mutant CLOCK protein lacks the segment of the putative transactivation domain, expression of CLOCK/ BMAL1 transcriptional targets is downregulated in tissues of Clock/Clock mice (Jin et al., 1999; Kume et al., 1999; Oishi et al., 2000). The targeted disruption of

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the functional partner of Clock, Bmal1, results in an immediate and complete loss of behavioral and molecular rhythms (Bunger et al., 2000). Clock mutant mice were first reported in 1994 (Vitaterna et al., 1994), and, until recently, represented the only model of functional deficiency of this transcriptional activator. Based on the analysis of Bmal1‐deficient animals and on the current model of CLOCK/BMAL1 function, it was assumed that Clock null mice would have phenotypic and molecular characteristics similar to those of Bmal1‐deficient animals. Thus, it came as a surprise that when CLOCK‐deficient animals were finally generated and tested in behavioral assays, they did not display disruption of rhythmicity (Debruyne et al., 2006). At the molecular level, CLOCK‐deficient mice demonstrate some changes in the expression levels of circadian genes mRNA and proteins; however, these changes did not aVect circadian function (Debruyne et al., 2006). These results suggest that the cells of Clock‐deficient mice retain intact clock function due to BMAL1 dimerization with another partner, which functionally substitutes for CLOCK. The most likely candidate for this substitute is Neuronal PAS‐domain protein 2 (NPAS2), which is known to dimerize with BMAL1 to form functional transactivation complex (Hogenesch et al., 1998; Kondratov et al., 2006b; Reick et al., 2001). This possibility is supported by the observation of increased levels of the NPAS2 in the liver of Clock‐deficient mice (Debruyne et al., 2006). However, NPAS2 protein was not detected in the SCN of the Clock‐null mice, suggesting the existence of another yet unknown transcriptional partner for BMAL1. CRY1 and CRY2 are key negative regulators of the mammalian circadian system; and deficiency in either CRY component aVects circadian behavior albeit in an opposite way. Mice deficient for Cry1 demonstrate the period of the locomotor activity about 1 hour shorter than the WT littermates, whereas deficiency in Cry2 results in
1 hour lengthening of the circadian period. Cry double knockout animals display complete loss of circadian rhythmicity under constant environmental conditions (Thresher et al., 1998; van der Horst et al., 1999; Vitaterna et al., 1999). In mammals, CRYs function as inhibitors of CLOCK/BMAL1‐mediated transactivation (GriYn et al., 1999; Kume et al., 1999). Consistent with this, expression of CLOCK/BMAL1 transcriptional targets (Per1 and Per2) is increased in the SCN and peripheral tissues of Cry double knockout mice (Okamura et al., 1999). Targeted mutations have been generated in all three mammalian Per genes. However, only Per2‐mutants, which have an in‐frame deletion in the PAS‐B domain, show an obvious circadian phenotype with a
1.5 hour shortening of the free‐running period followed by complete loss of rhythmicity in DD (Zheng et al., 1999). Per1/Per2 double‐mutants have a very strong circadian phenotype, exhibiting complete arhythmicity immediately after being placed in DD (Bae et al., 2001; Zheng et al., 2001). Based on a report that PER1 forms a complex with TIM (the mammalian homolog of the Drosophila

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timeless protein) that inhibits the transcriptional activity of CLOCK/BMAL1 in transient transfection experiments, PERs were considered as negative components of the mammalian transcription/translation feedback loop (Sangoram et al., 1998). However, in contrast to CRYs, deficiency in PERs does not result in the upregulation of CLOCK/BMAL1 transcriptional targets (Zheng et al., 2001). One proposed explanation for this assumes that PER2 acts as a component of a complex that activates transcription of Bmal1, thus generating an additional loop in molecular circadian oscillator (Fig. 1). Indeed, PER2 deficiency results in downregulation of Bmal1 (Shearman et al., 2000b). Hence, the precise role of mammalian PERs in transcriptional regulation is still unclear and will require further investigation. Rev‐erb‐deficient mice demonstrate significantly shorter circadian period under both constant dark (DD) and constant light (LL) conditions confirming that REV‐ERB protein plays an important role in the mammalian circadian clock. In addition, circadian expression of Bmal1 mRNA is severely blunted in the tissues of Rev‐erb knockout animals and consistent with the proposed role of REV‐ERB as a negative regulator of Bmal1 expression, CLOCK and BMAL1 levels are constituvely elevated in the liver of these animals (Preitner et al., 2002). Since circadian regulation is based on periodic CLOCK/BMAL1‐mediated transcriptional activation and PER/CRY‐dependent transcriptional repression, it has been suggested that, as in many other systems, chromatin remodeling constitutes an important regulatory step governing the circadian clock machinery. Consistent with this hypothesis, it has been demonstrated that CLOCK/BMAL1‐dependent transactivation is coupled to circadian changes in histone acetylation. Using chromatin immunoprecipitation, Etchegaray and colleagues showed that rhythmic transcription of circadian genes in vivo correlates with rhythms in histone H3 acetylation, suggesting that the CLOCK/ BMAL1 complex may recruit histone acetyl transferase (HAT) for modification of chromatin structure (Etchegaray et al., 2003) (Fig. 1). The importance of this mechanism was extended to a distinct peripheral clock in the vasculature, where it involves the CLOCK paralog NPAS2 (Curtis et al., 2004). It has also been proposed that CRY executes its repressor function through disruption of interaction between CLOCK/BMAL1 and HAT, thereby preventing recruitment of HAT to the promoters of target genes (Etchegaray et al., 2003). However, data from Doi et al. show that CLOCK possesses intrinsic HAT activity. Therefore, the CLOCK/BMAL1 complex can induce chromatin modification without recruitment of external HAT (Doi et al., 2006). In addition to rhythms in histone acetylation, other chromatin modifications may play a role in circadian gene regulation. For example, the promoter region of Dbp gene displays rhythmic methylation/demethylation patterns and CLOCK/BMAL1 binding resulting in daily variations in transition

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between euchromatin‐like and heterochromatin‐like states (Ripperger and Schibler, 2006). It has also been shown that the CLOCK/BMAL1 complex itself can function as transcriptional repressor and that the transition from transactivation to transrepression is induced by CRY‐dependent posttranslational modifications of the CLOCK and BMAL1 proteins (Kondratov et al., 2006b,c). Finally, it is quite possible that transcription from diVerent promoters is regulated by diVerent mechanisms. For example, the CLOCK/ BMAL1 complex does not display rhythmicity in its binding to the regulatory regions of the Per1 and Per2 promoters (Etchegaray et al., 2003; Lee et al., 2001), but does bind to E‐box motifs in the promoter of Dbp gene in a rhythmic manner (Ripperger and Schibler, 2006), although at this point we cannot exclude the possibility that observed diVerence could be explained by variations in the stringency of conditions used in these studies (Ripperger and Schibler, 2006). The role of PERs in CRY‐dependent transcriptional control remains unclear. While formation of a complex with PERs is important for the nuclear translocation of CRYs (Yagita et al., 2002), CRYs can nevertheless eYciently suppress CLOCK/BMAL1 transcriptional activity in Per1/ Per2/ cells (Shearman et al., 2000b). In addition, changes in the expression patterns of CLOCK/BMAL1 transcriptional targets are rather opposite in the tissues of Per‐ and Cry‐deficient mice (Zheng et al., 2001). One of the possible interpretations suggest that while CRYs play a major role in transcriptional inhibition, PERs regulate the timing of the inhibition through posttranslational mechanisms (Lee et al., 2001).

B. Posttranslational Regulation of the Components of the Molecular Circadian Oscillator Posttranslational modifications, intracellular distribution, and stabilization/ degradation of circadian proteins provide an additional level of regulation of the molecular circadian oscillator. It is generally believed that these modifications introduce temporal delays in transcriptional inhibition, ensuring circadian rhythms maintain periodicities close to 24 hours. Both positive and negative components of the mammalian transcriptional feedback loops are subject to posttranslational modifications that aVect their function in the molecular clock mechanism (schematically summarized in Fig. 2). The best studied posttranslational modification of clock proteins at this time is phosphorylation. PERs, CRYs, CLOCK, and BMAL1 all undergo time‐dependent phosphorylation that regulates their ability to form multimeric complexes and enter the nucleus (Lee et al., 2001). Several kinases have been reported to phosphorylate mammalian clock proteins, incliding casein kinase I epsilon [CKI, (Eide et al., 2002)], casein kinase I delta [CKI,

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Figure 2 Posttranslational modifications of circadian proteins provide additional level of regulation. Interaction of CLOCK and BMAL1 proteins initiates a cascade of posttranslational events: phosphorylation by yet unknown kinase(‐s), degradation, nuclear translocation and interaction with other circadian proteins. PERIODs get phosphorylated by casein kinase I epsilon (CKI). This phosphorylation destabilizes the protein and initiates proteasome‐dependent degradation. Interaction with CRYs blocks degradation of PERs and stimulates nuclear accumulation of PER/CRY complexes. Glycogen synthase kinase 3 (GSK‐3) phosphorylates REV‐ERB protein and targets it for proteasome‐dependent degradation.

(Akashi et al., 2002)], glycogen synthase kinase [GSK (Yin et al., 2006)], and mitogen‐activated protein kinase (MAPK) (Sanada et al., 2002). CKI phosphorylates all three PER proteins in a circadian manner (Lee et al., 2001, 2004). This phosphorylation destabilizes the protein and initiates proteasome‐dependent degradation (Akashi et al., 2002; Eide et al., 2005). In addition to regulating the proteins’ stability, phosphorylation of PER proteins aVects their intracellular distribution in a sequence‐specific way. Thus, CKI induces either nuclear translocation or cytoplasmic retention of PER1 by phosphorylating diVerent residues in the PER1 protein (Takano et al., 2004a; Vielhaber et al., 2000). The functional significance of PER phosphorylation is further underscored by its association with one of the disorders of the human circadian system, Familial Advanced Sleep Phase Syndrome (FASPS, see Section IV.A.1).

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Importantly, posttranslational regulation of PERs is aVected by their interaction with CRYs. When expressed individually, PERs and CRYs are shuttled between the nucleus and cytoplasm. Formation of the PER/CRY complex blocks nuclear export and induces nuclear accumulation of both proteins (Yagita et al., 2002). Similar to PERs, both CRY1 and CRY2 are phosphorylated in vivo in a time‐dependent manner (Harada et al., 2005; Lee et al., 2001). Two kinases have been associated with CRYs’ phosphorylation in vitro—MAPK (Sanada et al., 2004) and GSK‐3 (Harada et al., 2005). Functionally, phosphorylation of CRYs at specific Ser residues perturbs their activities to suppress CLOCK/BMAL1‐mediated transactivation (Sanada et al., 2004) and causes degradation of CRYs by a proteasome‐dependent pathway (Harada et al., 2005). Positive components of the circadian transcription/translational feedback loop are also subject to multiple posttranslational modifications. In the mouse liver, both CLOCK and BMAL1 display circadian oscillations in their phosphorylation status and intracellular distribution (Kondratov et al., 2003; Lee et al., 2001; Tamaru et al., 2003). Phosphorylation of CLOCK and BMAL1 proteins is triggered by their coexpression and interaction and is closely coupled with the nuclear/cytoplasm distribution, stability and transcriptional activity of the CLOCK/BMAL1 complex. Consequently, phosphorylation and nuclear localization of CLOCK is significantly impaired in Bmal1‐deficient cells (Kondratov et al., 2003). The sites of CLOCK and BMAL1 phosphorylation and the specific kinases involved have not been identified yet. Experiments with chemical kinase inhibitors and dominant‐ negative mutants have demonstrated that known clock‐related kinases, such as various isoforms of CKI, CKII, and GSK‐3 are not major regulators of the CLOCK/BMAL1 complex (Kondratov et al., 2003). MAP kinase and CKI can phosphorylate BMAL1 protein and negatively regulate its functional activity in vitro (Eide et al., 2002; Sanada et al., 2002); however, the role of these kinases in vivo remains unclear. CLOCK can be phosphorylated in vitro by active protein kinase G‐II (PKG‐II), and PKG‐II‐like immunoreactivity can be detected in CLOCK immunoprecipitates. However, any eVect of PKG‐II on the transcriptional activity, stability or localization of CLOCK protein has not been reported (Tischkau et al., 2004). REV‐ERB undergoes phosphorylation by another clock‐related kinase‐ GSK‐3, and this modification is important for the turnover of this transcriptional regulator. Mutation in phosphorylation site stabilizes REV‐ERB and interferes with circadian rhythms in gene expression in cell culture (Yin et al., 2006). Another type of posttranslational modification that has been reported for BMAL1 protein is sumoylation (Cardone et al., 2005). Similar to phosphorylation, sumoylation of BMAL1 depends on its interaction with CLOCK

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and aVects the stability of the protein. Mutation of the critical Lys residue that abrogates sumoylation of BMAL1, increases BMAL1 half‐life, but does not interfere with the transcriptional activity of the CLOCK/BMAL1 complex. Sumoylation and SUMO‐dependent degradation of BMAL1 are necessary for rhythmic expression of circadian genes at least in fibroblast cell culture (Cardone et al., 2005). It is important to emphasize that interactions between clock proteins may aVect their posttranslational modification and, as a result, contribute to functional regulation of the molecular clock. For example, CLOCK/BMAL1 dimerization initiates both proteins’ phosphorylation, nuclear translocation, and subsequent degradation (Kondratov et al., 2003). Interaction of ectopically expressed CRY with CLOCK/BMAL1 complex increases nuclear accumulation of unphosphorylated forms of both CLOCK and BMAL1 proteins, which correlates with the decrease in transcriptional activity monitored in cell culture by luciferase reporter assay (Kondratov et al., 2006b). Based on this correlation, Kondratov et al. suggested that the phosphorylated forms of CLOCK and BMAL1 are associated with CLOCK/BMAL1‐dependent transactivation, while the unphosphorylated—with CLOCK/BMAL1‐ dependent transcriptional repression (Kondratov et al., 2006b). Although the molecular details of the proposed mechanism require additional studies, the data clearly demonstrate the functional significance of posttranslational modifications of core clock components initiated by their interactions with one another. Various posttranslational modifications of clock proteins may also contribute to the observed significant phase shift in temporal expression patterns of CLOCK/BMAL1 transcriptional targets. Temporal mRNA profiling for Dbp and Rev‐Erb genes in several tissues has demonstrated that the peak of their expression is in anti‐phase with the expression of Per1 and Per2 mRNA. One possible explanation suggests that the expression of each phase group is regulated by diVerent combinations of circadian transcriptional factors CLOCK/BMAL1, DBP, REV‐ERB (Etchegaray et al., 2003; Ueda et al., 2005). Because each of these factors has its own phase of activity, the phase of combined activity can be diVerent on diVerent promoters. In addition to this, the binding aYnity of the CLOCK/BMAL1 complex to diVerent promoters may depend on its interaction with other circadian proteins or on posttranslational modifications induced by these interactions. Therefore, the phase of the target genes’ expression will depend on the phase in phosphorylation, sumolyation, or other putative modification that have not yet been characterized. Consistent with this hypothesis, CLOCK/BMAL1 complex display diVerent phases in binding to mCry1 and Dbp promoters (Etchegaray et al., 2003; Ripperger and Schibler, 2006) but show no circadian variations in binding to Per1 promoter (Etchegaray et al., 2003; Lee et al., 2001). Similar type of regulation, when diVerent posttranslational modifications of transcription factors

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diVerentially aVect their interaction with diVerent promoters, has been described for other than circadian systems. For example, diVerent types of stress induce diVerent modifications of p53 that results in diVerent spectra in the expression of p53 target genes and as a result, diVerent cellular outcome (i.e., cell cycle arrest, apoptosis, or senescence) (reviewed in Gudkov and Komarova, 2003).

C. Peripheral Clocks and Circadian Control of Gene Expression in Different Tissues Insight into how oscillations in the expression of clock genes translate into physiological periodicities was made possible by the development of high‐ density oligonucleotides microarray technology for gene expression profiling and its application to the circadian system. In mice, global temporal gene expression profiling has been reported for several diVerent tissues, including SCN, liver, heart, fibroblasts and adipose tissue (Akhtar et al., 2002; Ando et al., 2005; DuYeld, 2003; Grundschober et al., 2001; Panda et al., 2002; Stokkan et al., 2001; Ueda et al., 2002). These profiling studies were performed under constant environmental conditions (constant darkness) to ensure that observed oscillations in gene expression were truly circadian in nature. Taken together, the microarray studies demonstrated several important points. First, they showed that expression of a significant portion of the genome is under circadian control (in mammals, approximately 3–10% of all detectably expressed transcripts (DuYeld, 2003; Panda et al., 2002)). Second, comparison of circadian patterns of gene expression among diVerent tissues revealed remarkable tissue specificity of this regulation. Comparison of the sets of cycling transcripts in the SCN, the master regulator of mammalian clock, and the liver revealed that only 5–10% of clock‐controlled genes are shared between these tissues (Akhtar et al., 2002; Panda et al., 2002). Similar results were obtained from global temporal gene expression profiling of other mammalian tissues, including heart (Storch et al., 2002) and adipose tissue (Ando et al., 2005) as well as cultured fibroblasts after serum synchronization (Grundschober et al., 2001). In many cases, tissue‐specific clock‐controlled genes were recognized to be involved in rate‐limiting steps of processes critical for an organ function. For example, genes demonstrating circadian regulation in the SCN include some involved in protein/neuropeptide synthesis, processing and degradation as well as in regulation of redox state and energy utilization (Panda et al., 2002). In the liver, the major metabolic organ, coordinated circadian expression of genes encoding components of sugar, lipid, cholesterol, and xenobiotic metabolic pathways has been reported (Akhtar et al., 2002; Panda et al., 2002; Storch et al., 2002). In adipose tissue, numerous genes involved in adipocyte function showed

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24‐hour periodicities in their expression profiles (Ando et al., 2005). These findings suggest that profiling of additional tissues is likely to reveal more circadian‐regulated genes important for the function of diVerent organs. The gene expression profiling studies described above suggest that clock‐ controlled fluctuations in gene expression levels could potentially provide the mechanistic background for daily variations in physiology, metabolism, and various responses to external signals. However, there are two considerations that need to be taken into account before extrapolating these oscillations directly to downstream physiological eVects. First, most of the changes in mRNA levels detected in the microarray studies have relatively small amplitudes of oscillation, and second, it is not yet known if they are translated into corresponding oscillations in protein abundance and/or activity. Thus, additional mechanisms are likely to be involved in circadian regulation of various types physiological processes and responses.

IV. Human Disorders Associated with Altered Function of the Circadian System Given the important role that the circadian system plays in proper timing of various physiological processes with the daily environmental changes, it is likely that dysfunction of the internal clock or disruption of its synchronization with the environment could aVect an organism’s health. This appears to be particularly true for human sleep disorders since the timing of sleep and wakefulness is, in part, mediated by a clock‐dependent mechanism (Dijk and Czeisler, 1995). Recent studies have conclusively demonstrated the association of several types of sleep disorders with functional variations in circadian genes. A. Sleep Disorders 1. Advanced Sleep Phase Syndrome Advanced sleep phase syndrome (ASPS) is a disorder, in which the major sleep episode is advanced in relation to the desired clock time. This results in symptoms of compelling evening sleepiness, early sleep onset (bedtimes around 6–9 p.m.), and awakening that is earlier than desired (around 1–4 a.m.) (Wagner, 1996). A familial form of ASPS (FASPS) results from an autosomal dominant circadian rhythm variant. AVected individuals display a very short circadian period with a 4‐hour advance in the phase of the sleep‐wake cycle, plasma melatonin and body core temperature rhythms (Jones et al., 1999). Linkage analysis followed by single‐strand conformational polymorphism analyses of aVected and unaVected individuals attributed

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FASPS to a missence mutation in the hPer2 gene (Toh et al., 2001). This mutation results in a serine to glycine change within the CKI binding region of hPER2, causing hypophosphorylation of hPER2. Studies of other aVected families demonstrated that some are unlinked to the hPer2 locus, suggesting the existence of locus heterogeneity in FASPS. Indeed, a missense mutation (T44A) in another clock‐relevant kinase, CKI, also results in FASPS (Xu et al., 2005). Interestingly, FASPS is phenotypically similar the tau mutant hamster, which displays a short circadian period (Ralph and Menaker, 1988) and has a mutation in the gene encoding CKI (Lowrey et al., 2000). This mutation leads to PER hypophosphorylation due to deficiency in CKI enzymatic activity. Taken together, the human and hamster studies suggest that reduced levels of PER2 phosphorylation might induce faster accumulation of PER2, resulting in an acceleration of the feedback loop and a shorter circadian period (Lowrey et al., 2000; Toh et al., 2001). 2. Delayed Sleep Phase Syndrome In contrast to ASPS, patients with delayed sleep phase syndrome (DSPS), which is the most common circadian rhythm sleep disorder, demonstrate an inability to fall asleep and awaken spontaneously at the desired times due to a phase delay in the main sleep episode. These patients are extreme ‘‘night owls’’ with bedtimes occur around 3 a.m.–6 a.m. and wake‐up times around 10 a.m.–3 p.m. (Wagner, 1996). Similar to patients with other sleep disorders, DSPS patients cannot synchronize their sleep‐wake cycle to the desired time schedule and have diYculties in their social lives (Campbell et al., 1999; Wagner, 1996). Genetic studies suggest that the pathogenesis of DSPS may be related to structural polymorphisms in the hPer3 gene. Mutation screening of the hPer3 gene revealed five polymorphisms occurring in four haplotypes, one of which was more frequent in DSPS patients (Ebisawa et al., 2001). Another study reports a link between a length polymorphism in hPer3 and both extreme diurnal preference and DSPS (Archer et al., 2003). The length polymorphism occurs in a region of PER3 protein containing a cluster of potential CKI phosphorylation sites. Interestingly, the longer allele was associated with morningness, while the shorter one was associated with eveningness. The shorter allele was also associated with DSPS patients, 75% of whom were homozygous for this allele (Archer et al., 2003). Thus, the genetic studies suggest that diVerential PER3 phosphorylation may contribute to phenotypic diVerences. 3. Non‐24‐Hour Sleep‐Wake Syndrome The non‐24‐hour sleep‐wake syndrome (N‐24) is characterized by a chronic pattern of daily delays in sleep onset and waking times. Over time, the sleep period shifts out of phase with conventional hours, resulting in insomnia and

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an inability to stay alert during the daytime. Depending on concurrent synchrony of their internal sleep‐wake rhythm with the external 24‐hour period, patients with N‐24 often alternate between symptomatic and asymptomatic episodes (Wagner, 1996). The syndrome is extremely rare and is more frequently observed in blind patients or subjects living under artificial light‐dark conditions (Kennaway and Van Dorp, 1991). Recent studies identified an amino acid substitution in CKI, S408N. This substitution eliminates one of the putative autophosphorylation sites in the C‐terminal region of CKI and results in higher enzymatic activity (Takano et al., 2004b). The N408 allele was found to be more common in both DSPS and N‐24 patients than the S408 allele. These results indicate that the N408 allele of CKI plays a protective role in the development of both disorders through alteration of the enzyme’s activity (Takano et al., 2004b).

B. Jet Lag and Shift Work Maladaptation Syndromes Jet lag and shift work maladaptation syndromes develop as a result of large abrupt changes in light conditions. Jet lag is commonly experienced following rapid travel across several time zones and is manifested by daytime sleepiness, inability to sleep during local nighttime, fatigue and decreased alertness, and performance. It is often accompanied by dyspepsia and constipations, which are probably caused by shifts in meal schedule (Wagner, 1996). The severity and duration of this disorder vary depending on age, number of time zones crossed, direction of the travel, and times of takeoV and arrival. Shift work sleep disorder or shift maladaptation syndrome symptoms are similar to those of jet lag: sleep disturbances, fatigue and gastrointestinal symptoms often followed by depression (Healy and Waterhouse, 1995). Shift maladaptation syndrome is also characterized by higher rates of accidents or near‐misses, alcohol abuse, and personality changes. Night workers have poorer daytime sleep and poorer performance at night. Adjustment to night work is usually slower than adjustment to a time‐zone transition. The work schedule of night workers is out of phase with the intrinsic circadian sleep‐wake rhythm, and their internal clocks typically never fully entrain to their reverse light/dark regime even after years of steady night work (Rajaratnam and Arendt, 2001). Experimental confirmation for the association of these two disorders with desynchronization of the phase of the internal clock and the environment has been made possible due to generation of transgenic animals expressing a reporter luciferase gene under control of the circadian Per1 promoter (Yamazaki et al., 2000). Yamazaki and colleagues monitored clock function in various tissues of these animals following exposure to shifted light schedules and found that the rhythmicity in the SCN, muscle, and lung (but not in the liver) resume correct phase with respect to each other and the light

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cycle within 6 days. However, during these 6 days, phase relationships were disturbed and some individual rhythms were severely disrupted (Yamazaki et al., 2000). Notably, frequent exposure to shifted schedules may result in further medical complications not directly related to circadian system malfunctioning. Thus, frequent travelers may develop chronic sleep disturbances. An increased risk of cancer among long‐distance flight crews and night workers has also been reported (Hansen, 2001a,b). These findings have also been confirmed in the animal models demonstrating accelerated tumor growth in mice subject to experimental jet lag (Filipski et al., 2006). Although systematic research on a possible link between jet lag and psychiatric disorders is still lacking, clinical and pathophysiological data suggest that jet lag may trigger either the exacerbation of existing aVective disorders or the de novo appearance of mood disorders in predisposed persons (Katz et al., 2001).

C. Seasonal Affective Disorder Seasonal aVective disorder (SAD) is a major depressive disorder occurring with the seasonal pattern. AVected individuals experience regular recurring episodes of depression during either the fall‐winter months (winter form) or the spring‐summer months (summer form) with remission during the other months (Rosenthal et al., 1984). SAD is accompanied by such symptoms as increased or decreased appetite followed by weight gain or loss, changes in sleep habit, irritability, poor concentration, and reduced energy level. This disorder is relatively common with a prevalence rate ranging from 1.5% to 9% across the country (Modell et al., 2005). The cause of SAD is unknown; however, it has been proposed that a genetic factor may be associated with at least the winter form of this disorder (Madden et al., 1996). The involvement of the circadian system in the pathogenesis of SAD was revealed by studies demonstrating that patients with SAD have disturbances in their circadian cycle that can be explained by a weakened entrainment to the 24‐hour period. In these people, the circadian cycle appears to be more variable across days, deviating more from 24 hours and peaking at less regular times (Teicher et al., 1997). This disturbance may be explained by inconsistent resetting of the circadian pacemaker and caused by impaired transmission of serotonin or neuropeptide Y—two neurotransmitters that are thought to be essential for regulating mood and appetite, respectively (Partonen and Lonnqvist, 1998). Consistent with this, genetic analysis revealed a short‐allele polymorphism in the serotonin transporter that was more common in patients with SAD than in healthy controls (Rosenthal et al., 1998). In addition, more recent studies identified circadian

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clock‐related polymorphisms, NPAS2 471 Leu/Ser, as potentially implicated in SAD (Johansson et al., 2003).

D. Mood Disorders It has been recognized for quite some time that certain dysfunctions of the circadian system may be associated with various mood disorders. Several lines of evidence suggest that patients with bipolar disorder (BD) display marked alterations in their sleep/activity patterns as well as in daily variations in hormones secretion (Mansour et al., 2005b). Correction of these abnormalities usually accompanies clinical stabilization. Recent genetic studies confirmed that mutations in circadian genes leading to either deregulation of the internal clock or impairment of its ability to interact with the environment indeed may be associated with psychiatric disorders. For example, the alterations in the expression of PER1 were reported in postmortem temporal cortex from patients with schizophrenia (Aston et al., 2004). In addition, a strong association between CLOCK variants and sleep disturbances leading to a higher recurrence of manic or depressive episodes has been reported in patients with major depressive disorder (MDD) and BD (Serretti et al., 2003). A recent study including more that 1450 individuals revealed a modest association between single‐nucleotide polymorphism in the Bmal1 and Timeless genes and BD1. Associations with Tim and Per3 were also detected in patients with schizophrenia (Mansour et al., 2006). However, the most compelling molecular evidence of a functional link between the circadian system and mood disorders come from two independent studies. The first one identified a single nucleotide polymorphism within the GSK‐3 promotor that was associated with an earlier age of onset for BD (Benedetti et al., 2004a,b). The second study demonstrated that, in cultured cells, GSK‐3 phosphorylates and stabilizes REV‐ERB, a negative component of the circadian clock. Lithium, commonly used to treat BD, is known to modulate circadian rhythms by inhibiting the kinase activity of GSK‐3 (Abe et al., 2000; Healy and Waterhouse, 1995; Mansour et al., 2005a). Lithium treatment of cells leads to rapid proteasomal degradation of REV‐ERB protein and results in an increase in the expression level of Bmal1 (Yin et al., 2006). Importantly, REV‐ERB degradation was required for the generation and maintenance of rhythmic gene expression. In summary, elucidation of the molecular clock function identifies new directions for the search of potential links between human circadian pathologies and malfunction of particular circadian proteins. These studies are further supported by extensive work in animal model systems, in which the consequences of such malfunctions are easier to trace. Specifically, the availability of

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mice with targeted deletions or mutations in core clock genes have allowed to describe pathologies associated with deficiency in particular components of the circadian system. Results of the animal studies, which significantly advance our current view on the role of circadian clock proteins in normal physiology, are summarized below.

V. Pathologies and Developmental Defects in Circadian Mutant Mice Since the majority of human circadian disorders involve various sleep disturbances, in order to address the role of individual clock proteins in sleep regulation, circadian mutant mice were tested for their sleep patterns. Sleep is a complex physiological process governed by circadian and homeostatic components. The SCN regulates the timing and consolidation of the sleep‐ wake cycle, while a homeostatic mechanism controls the accumulation of sleep debt and sleep recovery (Borbely et al., 2001). Interestingly, several animal studies reported altered homeostatic regulation of sleep in mice lacking both Cry genes (Wisor et al., 2002), Bmal1 (Laposky et al., 2005) or Npas2 (Dudley et al., 2003; Franken et al., 2006), and in Clock mutant mice (Naylor et al., 2000) suggesting a noncircadian role of clock genes in sleep regulation (Shaw and Franken, 2003). Furthermore, the detailed analysis of mice deficient in various components of the molecular clock revealed a number of pathologies specific for each mutation. These studies have demonstrated that circadian proteins play important roles in normal tissue homeostasis and that, in some cases, these roles might be distinct from their role in circadian regulation.

A. CLOCK Clock was the first circadian gene identified in a mammalian system (Vitaterna et al., 1994). It belongs to the family of bHLH‐PAS domain containing transcription factors. In complex with its partner, BMAL1, CLOCK regulates transcription of multiple genes containing E‐box regulatory sequence in their promoters (Lowrey and Takahashi, 2004). CLOCK null mice have not been reported until very recently; therefore, all of the data regarding the role of CLOCK protein in circadian rhythms and normal physiology were obtained using Clock/Clock mutant mice. These mice carry a mutation in a splice donor site that results in production of a mutant protein with a 51‐amino acid deletion within the C‐terminal domain (King et al., 1997). The mutation aVects transactivation properties of CLOCK and results in disruption

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of rhythmic gene expression and downregulation of CLOCK/BMAL1 target genes in tissues of Clock mutant mice, but has no eVect on its repressor properties (Kondratov et al., 2006c). Although Clock/Clock mutant mice do not display any obvious anatomical abnormalities, they cannot be maintained through homozygous matings (at least on the C57Bl/6J background), suggesting that some reproductive system defects are present. These defects are female specific and are manifested both by disruption of the estrous cycle and by inability to maintain a full‐term pregnancy (Miller et al., 2004). Even though Clock/Clock females display irregular and lengthened estrous cycles, they show no gross morphological abnormalities in ovary tissue and no change in serum levels of estradiol and progesterone. Detailed analysis of hormonal levels demonstrated that the estrous cycle is disrupted through the failure of Clock/Clock females to produce a luteinizing hormone (LH) surge. Despite these defects in estrous cycle and initial low levels of LH, initiation and progression of pregnancy in Clock/Clock females are normal. However, about 40% of pregnant females have non‐productive labor or failed to enter labor at all, fully reabsorbing full‐ term fetuses. Together, these data suggest that Clock/Clock mutant females lack a proper circadian‐regulated signal that is required to coordinate hypothalamic hormone secretion (Miller et al., 2004). This lack of hormonal coordination also disrupts nursing behavior and growth and survival of pups when crossed to ICR strain (Hoshino et al., 2006). Another significant abnormality characteristic of Clock/Clock mutant mice is obesity caused by various metabolic deregulations including hyperleptinemia, hyperlipidemia, hepatic steatosis (fatty liver), hyperglycemia, and hypoinsulinemia (Turek et al., 2005). Homozygous Clock mutant mice demonstrate significant disruptions of diurnal feeding rhythms, which are accompanied by an increase in caloric intake and a decrease in energy expenditure. As a result, serum levels of triglycerides, cholesterol, glucose, and leptin are significantly elevated in Clock/Clock mice compared to their WT littermates. Changes in feeding rhythms are associated with significantly reduced levels of transcripts encoded selected orexigenic (orexin) and anorexigenic (ghrelin) neuropeptides expressed in the mediobasal hypothalamus. Since human clinical data has shown that disruption of circadian rhythmicity and sleep patterns are often associated with drug addiction, several murine circadian mutants were tested for behavioral responses for commonly used drugs of abuse. Clock/Clock mice display high levels of cocaine sensitization and are more sensitive to the rewarding eVects of cocaine (McClung et al., 2005). These phenotypes are associated with an elevated rate of excitability of dopamine neurons in the midbrain ventral tegmental area (VTA), a region of the brain important for drug reward behavior. Elevated dopaminergic transmission in the reward circuit of Clock/Clock mice was accompanied by increased levels of mRNA and protein for tyrosine hydroxylase (TH), the

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rate‐limiting enzyme in dopamine synthesis. Importantly, the additional TH protein was present in its active phosphorylated form. In addition, expression of several genes important for dopaminergic transmission was altered in the VTA of Clock/Clock mutant mice (McClung et al., 2005). Similar phenotypes have been described for mouse mutants with deficiency of PER1 and PER2 (see Section V.C).

B. BMAL1 BMAL1 is a functional partner of CLOCK, which belongs to the same family of bHLH‐PAS domain transcription factors. Upon heterodimerization with CLOCK, it regulates expression of target genes. Mice with targeted disruption of BMAL1 do not display circadian rhythms in behavior and gene expression (Bunger et al., 2000). The critical role of BMAL1 in an organism’s physiology and maintenance of normal tissue homeostasis came from detailed analysis of BMAL1 null mice. Both male and female Bmal1/ mice are sterile, however, the reasons for this are not yet known. Male infertility, at least in part, might be explained by the observed significant reduction in the size of seminal vesicles and coagulation glands, suggesting possible defects in sexual maturation (R. Kondratov, unpublished observations). Female mice do not display any visible abnormalities in their reproductive system; however, they are not able to produce progeny even when mated with wild‐type males (R. Kondratov, unpublished observations). Similar to Clock mutant mice, Bmal1/ mice have a metabolic defect, although it is somewhat diVerent. BMAL1 appears to play an important role in control of glucose homeostasis and recovery from insulin‐induced hypoglycemia (Rudic et al., 2004). Both deletion of Bmal1 and mutation of Clock abolish diurnal variations in glucose and triglyceride levels. In addition, gluconeogenesis as measured by conversion of exogenously administered pyruvate to glucose was seriously impaired by deletion of Bmal1, yet the counterregulatory responses of corticosterone and glucagon were retained. Interestingly, these defects were more pronounced in Bmal1‐deficient mice, while Bmal1 þ/ and Clock/Clock mutant mice demonstrated intermediate phenotypes (Rudic et al., 2004). It appeared that BMAL1 is also important for maintaining normal bone joint soft‐tissue architecture. According to Bunger and colleagues, by 35 weeks of age, Bmal1‐deficient mice develop severe joint ankylosis (Bunger et al., 2005). This pathology is characterized by ossification of joint soft tissues leading to replacement of normal tendons and ligaments by bones. Observed elevated levels of osteocalcin and reduced levels of calcitonin in the serum of Bmal1‐null animals are indicative of formation of new bones. Interestingly,

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such abnormal proliferation of osteoblasts occurs mainly at joints and has no eVects on the architecture of long bones. The mechanism underlying this mineralization remains unknown; no changes in serum calcium, phosphorus, and creatinine levels that might be associated with these processes have been detected (Bunger et al., 2005). In a series of in vitro experiment with mouse embryonic fibroblasts and 3T3‐L1 preadipocytes, Shimba and colleagues demonstrated that BMAL1 protein is important for diVerentiation of these cells into adipocytes (Shimba et al., 2005). BMAL1‐deficient MEFs and BMAL1 knock‐down 3T3‐L1 cells failed to accumulate lipid droplets. In contrast, overexpression of BMAL1 in adipocytes induced significant lipid accumulation through activation of several lipid metabolism‐related factors (such as SREBP‐1a and Rev‐erb). While the role of BMAL1 in adipocytes diVerentiation has been clearly demonstrated in cell culture, its role in vivo remains somewhat unclear, since young Bmal1‐deficient animals do not show reduction in adipose tissue and the progressive weight loss leading to a ‘‘lean’’ phenotype develops only with age (Kondratov et al., 2006a). Interestingly, while CLOCK and BMAL1 are components of the same transcriptional complex, Clock mutant mice develop obesity (Turek et al., 2005) and BMAL1 knockouts develop a ‘‘lean’’ phenotype (reduced amount of adipose tissue) with age (Bunger et al., 2005; Kondratov et al., 2006a). The reasons for these contrasting phenotypes remain unclear. On one hand, they may reflect diVerences in the molecular nature of these defect that, according the proposed model of a dual functional role of CLOCK/BMAL1, result in loss of both transactivation and transrepression in Bmal1‐deficient mice, but only loss of transactivation in Clock/Clock mutants (Kondratov et al., 2006c). On the other hand, the observed phenotypic diVerences of Bmal1/ and Clock/Clock mice may be specific to putative alternative partners of CLOCK and BMAL1 proteins that modify functional characteristics of the complex. A report of rhythmic circadian behavior in Clock‐null mice indirectly supports such a possibility (Debruyne et al., 2006). In any case, the data obtained in Bmal1‐ and Clock‐deficient mice clearly show that both CLOCK and BMAL1 play important roles in mammalian energy balance. Another major pathological defect caused by Bmal1‐deficiency was identified by observation of a large group of Bmal1/ and WT mice over the course of their lifespan. Although BMAL1‐null mice do not display any visible developmental defects and are undistinguishable from their WT littermates at birth, at 16–18 weeks of age they start to develop a progeria‐ like (premature aging) syndrome (Kondratov et al., 2006a). At this age, both males and females stop growing and, after a short period of time, start to lose weight. The decrease in body weight is primarily due to progressive loss of muscle and bone mass and intraabdominal and subcutaneous adipose tissue. Several major organs including the spleen and kidney also demonstrate

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age‐dependent shrinkage. Most of the mutant mice develop cataracts (another hallmark of aging in mammals) as early as 6 months of age. Chronic cornea inflammation was also detected in more than 50% of mutants. In addition, abnormally high levels of monocytes and neutrophiles are present in the blood of aged Bmal1/ animals. As a result, Bmal1‐null mice die prematurely with an average lifespan of less than 37 weeks (ranging from 26 to 52 weeks), which is more than twofold shorter than the lifespan of wild‐type mice. The molecular mechanisms underlying the early‐aging phenotype of Bmal1/ mice are still unclear. Most likely, such a complex phenotype results from the superposition of deficiencies in multiple pathways that are normally controlled by BMAL1. These could include defects in metabolism (Rudic et al., 2004) and stress responses (Gorbacheva et al., 2005) as well as BMAL1‐dependent regulation of the balance of reactive oxygen species (ROS) (Kondratov et al., 2006a). ROS play important roles in a number of physiological processes, however, their excess may result in oxidative stress that damages biological macromolecules and, in turn, accelerates or even initiates aging (Balaban et al., 2005). Since BMAL1‐deficiency results in an increase in the concentration of ROS in several tissues (Kondratov et al., 2006a), this may contribute to the development of early aging in the knockout mice.

C. NPAS2 Neuronal PAS domain protein 2 (NPAS2) is the closest homolog of CLOCK and, like CLOCK, functions as a transcriptional partner of BMAL1. Npas2/ mice display normal circadian behavior that is comparable to that of wild‐type animals. However, since expression of NPAS2 is restricted to tissues in which CLOCK is expressed at extremely low levels (forebrain cortices, basal ganglia and vasculature), is has been proposed that NPAS2 might be involved in clock mechanism in these tissues (McNamara et al., 2001; Reick et al., 2001). NPAS2‐deficient mice do not show any obvious abnormalities; they are reported to be active, fertile, and indistinguishable from their wild‐type and heterozygous littermates (Garcia et al., 2000). However, since the temporal and special expression of NPAS2 in wild‐type mice coincides with regions of the brain involved in learning and memory function, NPAS2‐deficient mice were subject to a series of behavioral tests. These experiments demonstrated that the null mice exhibit deficits in the long‐term memory arm of the cued and contextual fear task suggesting that NPAS2 may serve a dedicated regulatory role in the acquisition of specific types of memory (Garcia et al., 2000).

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D. PERIODs Three homologs of the Drosophila period gene have been identified in mammals, Per1, Per2, and Per3 (Bae et al., 2001). Per1 and Per2 are required for maintenance of normal circadian function while Per3 appears dispensable (Shearman et al., 2000a). Multiple complexes of PERIODs with other circadian proteins have been reported (Field et al., 2000; Kume et al., 1999; Lee et al., 2001; Shearman et al., 2000b). Interaction between PERIODs and CRYs is important for both proteins’ posttranslational regulation and intracellular distribution (see Section III.B). PERIODs are considered to be components of the negative limb of the circadian transcriptional‐translational feedback loop and inhibitors of CLOCK/BMAL1 transcriptional activity. However, while Drosophila per directly inhibits the activity of CLC/CYC (the analog of the mammalian CLOCK/BMAL1 transcriptional complex) by interfering with its binding to DNA, the molecular details of mammalian PER function are still unclear. Moreover, the pattern of circadian gene expression in the tissues of Period‐deficient mice argues against it functioning as a transcriptional inhibitor (Zheng et al., 2001). Several reports suggest that deficiency of Per1 and Per2 genes in mice aVects drug abuse‐related behaviors. Similar to what has been reported for CLOCK, both PER1 and PER2 mediate behavioral responses to cocaine in mice (Abarca et al., 2002). Thus, daily variations in sensitized responses to cocaine and cocaine reward correlates with the expression of Per1 in the SCN of wild‐ type mice. Furthermore, behavioral responses to repeated administration of cocaine are strongly attenuated in mice with a targeted disruption of the Per1 gene. In contrast, Per2 mutant mice (expressing a truncated PER2 protein) show hypersensitization to cocaine (Abarca et al., 2002). However, there are no diVerences between WT and Per mutants in their acute responses to cocaine administration. Together, these data suggest that PERs, at least partially, are involved in regulation of drug addiction (Abarca et al., 2002). The exact mechanism of PER‐mediated drug addiction is still unknown, since Per‐ deficient mice show no changes in the dopamine transporter, which is the key component of cocaine‐induced sensitization and reward. It has been suggested, however, that PERs may aVect the dopamine and/or N‐methyl‐D‐aspartate receptors. This view is supported by a recent report demonstrating that Per2 mutant mice display increased voluntary alcohol consumption resulting from hyper‐glutamatergic tonus. Expression of the glutamate transporter Eaat1 in these mice was reduced resulting in accumulation of glutamate in the brain (Spanagel et al., 2005). In addition, PER1 may play a role in morphine dependence; however, the mechanism is not known (Liu et al., 2005). The loss of Per2 results in deficiencies in DNA damage responses. Fu and colleagues have reported that Per2 mutant mice display higher sensitivity

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in vivo to the long‐term eVects of low dose ionizing radiation (Fu et al., 2002). This sensitivity is manifested by early hair graying and higher rates of tumor formation. Interestingly, however, the primary thymocytes from Per2 mutant animals are more resistant to high doses of radiation in the short‐ term experiments. The observed diVerences in response to radiation and tumor formation were associated with changes in the mRNA expression patterns of several oncogenes and tumor suppressor genes in the liver of Per2‐deficient mice (Fu et al., 2002). However, it remains unclear whether the observed phenotypes are due to direct regulation of genes aVecting cell death or proliferation within individual cells or if they reflect the long‐term eVects of disruption of circadian rhythmicity at the level of the organism. In the latter case, Per2 would qualify as a gene involved in tumor prevention rather than as a classical tumor suppressor gene (Fu et al., 2002). Involvement of the Per1 and Per2 genes in leptin‐mediated bone formation has been reported (Fu et al., 2005). Mice deficient in Per1 or Per2 or expressing a truncated mutant Per2m/m have normal bone mass and structure. In contrast, double knockouts (Per1/;Per2/) or Per1/;Per2m/m mice exhibit age‐progressive increases in bone mass that aVect both vertebrae and long bones. In contrast to bone formation, bone resorption is not aVected in these mice. The most striking feature reported in Per1/;Per2/ or Per1/;Per2m/m double knockout mice is their increased levels of osteoblasts. Based on these data, the authors suggest that clock proteins mediate leptin‐dependent sympathetic bone formation by preventing osteoblast proliferation. Thus, in the absence of both PERs, osteoblasts undergo uncontrolled proliferation. Indeed, osteoblasts isolated from Per‐deficient mice display altered growth characteristic in cell culture (Fu et al., 2005), which correlate with the in vivo data. At the molecular level, these diVerences are associated with upregulation of cyclin D1 expression. The molecular clock might contol cyclin D1 expression indirectly by inhibiting its critical regulator, c‐myc, and by interfering with the activity of the AP‐1 transcription factor, which controls both c‐myc and cyclin D1 expression in osteoblasts (Fu et al., 2005).

E. TIMELESS Whereas the role of timeless in the Drosophila circadian clock is well documented, the function of its mammalian orthologue is still under debate. In flies, TIM and PER heterodimerize are translocated to the nucleus and inhibit CLK/CYC‐mediated transcription (Hardin, 2005). Mammalian TIM interacts with PER in cell culture (Takumi et al., 1999) and with CRYs both in cell culture and in SCN extracts (Field et al., 2000; Kume et al., 1999) and can inhibit CLOCK/BMAL1‐mediated transcription (Jin et al., 1999; Sangoram

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et al., 1998). The disruption of neuronal activity rhythms on brain slices by antisense oligonucleotides for Tim strongly supports the requirement of mammalian Timeless for circadian rhythmicity in vivo (Barnes et al., 2003). Unfortunately, these observations cannot be supported by the analysis of a null mutant since targeted disruption of Tim results in embryonic lethality (Gotter et al., 2000). Thus, even if TIM is a core component of the molecular circadian oscillator, it must also be involved in other fundamental non‐circadian processes. It has been shown recently that TIM protein interacts with the cell cycle checkpoint proteins Chk1 and ATR and plays an important role in the DNA damage checkpoint response. Downregulation of TIM in human cells seriously compromises replication and intra‐S checkpoints. These findings suggest that TIM may function as a checkpoint protein that directly couples the cell cycle and circadian cycle (Unsal‐Kacmaz et al., 2005).

F. CRYPTOCHROMEs CRYPTOCHROMEs are members of the blue‐light photoreceptor/photolyases family that play a central role in the inhibitory limb of the mammalian circadian autoregulatory loop. Similar to Per1/Per2/ double knockout animals, mice with deficiency of both CRYPTOCHROMEs exhibit high bone mass and increased levels of osteoblasts (Fu et al., 2005). These phenotypes presumably reflect similarities in the functional roles of PER and CRY proteins in transcriptional regulation. On a pure C57BL/6J background, Cry1/Cry2/ animals are significantly smaller than their WT littermates (body weight of both males and females is reduced by 10–15%); however, they do not show any obvious pathologies and remain fertile such that they can be maintained through homozygous matings (Kondratov et al., unpublished observations).

G. REV‐ERBa REV‐ERB belongs to the nuclear receptor superfamily, which contains receptors for steroids, thyroid hormones, retinoic acid, and vitamin D, as well as ‘‘orphan’’ receptors. No ligand has been found for REV‐ERB to date, making it one of these orphan receptors. Similar to some other orphan receptors, REV‐ERB has been shown to bind DNA as a monomer on a specific sequence (REV‐ERB‐responsive element), but its transcriptional activity remains unclear. REV‐ERB acts as a negative regulator of transcription binding to the same response element than another orphan nuclear receptor, ROR. In addition to its role in circadian system, REV‐ERB is involved in adipocyte diVerentiation (Laitinen et al., 2005) and in control of

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inflammation in vascular smooth muscle cells by regulating NFB responsive genes, such as IL‐6 and COX‐2 (Migita et al., 2004). Rev‐erb‐deficient animals appear indistinguishable from wild‐type littermates (Guillaumond et al., 2005). They do not show any obvious phenotype in either adipose tissue or skeletal muscle, despite a previous report demonstrating a role for REV‐ERB protein in adipocyte diVerentiation and myotube diVerentiation in vitro (Laitinen et al., 2005). However, during the second week of life, the cerebellum of Rev‐erb/ mice presents several unexpected abnormalities, such as alterations in the development of Purkinje cells, delay in the proliferation and migration of granule cells from the external granule cell layer and increased apoptosis of neurons in the internal granule cell layer. In addition, mutant females are less fertile than wild type (Chomez et al., 2000). In summary, monitoring the mice with defects in various components of the circadian clock system revealed that in addition to shared phenotype of disrupted rhythmicity, each mutant line develops a unique set of phenotypic characteristics, which in some cases are associated with the development of pathological defects. These new findings extend our view on the role of the circadian proteins in normal physiology of an organism.

H. Circadian Proteins, Organism‐Environment Interaction, and Pathologies: A Hypothesis It has been generally assumed that the circadian system has evolved to coordinate various biological processes with the environment and that disruption of this coordination may lead to pathological defects. According to this view, deficiency in any circadian protein that aVects the function of the molecular circadian oscillator would lead to desynchronization of multiple physiological processes and disrupt normal tissue homeostasis. This, in turn, would lead to development of various pathologies (schematically illustrated in Fig. 3A). In this case all circadian mutant mice with defects in circadian clock function should have a similar spectrum of pathological changes. Few examples are consistent with this scenario. Per1/, Per2/, and Clock mutant mice all display alterations in cocaine sensitization and rewards (Abarca et al., 2002; McClung et al., 2005), and abnormal bone growth has been detected in both Cry1/Cry2/ and Per1/Per2/ mutant mice (Fu et al., 2005). However, there is growing evidence that mice with deficiencies in diVerent components of the molecular clock may display diVerent types of pathological changes. Thus, increased tumorigenesis has been reported specifically for Per2 mutants (Fu et al., 2002) and ossification is only observed in Bmal1‐null mice (Bunger et al., 2005). In addition, out of all of the circadian genes, only the Tim knockout has an embryonic lethal

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Figure 3 Role of the circadian system, circadian proteins, and organism/environment interactions in development of pathologies. (A) Deficiency in any circadian protein results in deregulation of the circadian system, desynchronization of multiple physiological processes, and development of pathologies. (B) Individual circadian proteins play unique roles in maintaining normal tissue homeostasis that may be relatively independent from their roles in the molecular circadian oscillator. Impaired activity of a particular circadian protein will result in a specific primary pathology (Pathology 2). At the same time, CLOCK deficiency will aVect circadian function leading to a variety of secondary pathologies. (C) Improper interaction of an organism with the environment may aVect the expression and/or activity of circadian proteins and induce specific pathologies without directly aVecting clock function.

phenotype (Gotter et al., 2000). The early aging phenotype described for Bmal1‐deficient mice is another striking example of a phenotype that is specific to deficiency of a particular clock component (Kondratov et al., 2006a). None of the other circadian mutant mice that were monitored for a similar period of time (Clock mutant or Cry double‐knockout mice) developed any signs of premature aging. These examples suggest that the traditional view of the functional role of circadian proteins in normal physiology needs to be reassessed. An alternative model suggests that individual circadian proteins play unique roles in maintaining normal tissue homeostasis that may be relatively independent from their roles in the molecular circadian oscillator. In this case, deficiency in a particular circadian protein would be predicted to result in a specific primary pathology. For example, defective transactivation function of CLOCK in tissues of Clock mutant mice results in metabolic syndrome and obesity.

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This syndrome may develop due to deregulation of the expression pattern of CLOCK/BMAL1 transcriptional targets, rather than general impairment of circadian function (schematically shown in Fig. 3B as Pathology 2). However, in addition to specific CLOCK‐related pathologies, Clock mutation also aVects circadian function by disrupting the expression of other circadian proteins. The impaired functions of these proteins, in turn, may lead to secondary pathologies (pathologies 1, 3, 4, and 5 shown in Fig. 3B). Since the secondary pathologies result from general circadian deregulation rather than complete deficiency of a particular circadian protein, they may have milder phenotypes and develop later in time compared to primary pathologies. This model suggests that diVerent circadian mutants may have their own spectra of diseases that may develop independent of LD conditions, although in some cases these spectra may overlap. The suggested model of clock‐independent function of the circadian proteins in normal physiology also provides an alternative interpretation for the increased risk of diseases such as cardiovascular disease, diabetes, and cancer in workers with abnormal schedules (frequent time zone travels, shift workers, etc.) (Knutsson, 2003). It is believed that this increased risk is a consequence of desynchronization between the internal clock and the environment, resulting in deregulation of multiple clock‐controlled biological processes. At the same time, various environmental stimuli (light, food, etc.) can aVect the activity of major circadian proteins and disrupt their functions important for normal tissue homeostasis. Improper interaction with the environment leading to impaired function of clock proteins important for normal physiology may induce specific pathologies without directly aVecting clock function (schematically shown in Fig. 3C). Thus, the development and progression of various diseases will occur through unbalanced internal/external interactions rather than through disruption of circadian rhythmicity per se. The proposed model of interconnection between the circadian system, circadian proteins, and an organism’s physiology provides new insight into molecular links connecting life style and disease development. It also underscores the essential role of the molecular components of the circadian clock in response to external signals including those that are important for clinical applications.

VI. Circadian Control of the Organism’s Response to Genotoxic Stress A. Chronotherapy of Cancer There are numerous indications that, in addition to regulating an organism’s physiology, circadian proteins play an important role in modulating responses

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to various stress conditions including those associated with DNA damage induced by anticancer agents such as chemotherapeutic drugs and radiation. While designed to selectively kill tumor cells, both treatments also cause non‐ specific damage to normal cells and tissues, resulting in undesirable side eVects. One of the approaches that were developed empirically to minimize these side eVects was based on the observation that in vivo responses to various therapies vary significantly depending on the time of treatment. It has been suggested that these variations may result from diVerential daily responses of both tumor and normal cells and that the final outcome (i.e., the therapeutic index associated with a specific treatment) reflects superposition of these eVects. The first study designed to test whether the timing of chemotherapy aVects therapeutic index demonstrated that the circadian pattern of arabinosyl cytosine administration determined both its toxicity and antitumor activity (Haus et al., 1972). Over 30 chemotherapeutic drugs have been tested since then in animal model systems and all demonstrated daily variations in toxicity and eYcacy (Levi, 1997). These initial observations were extended in a series of clinical trials in which the eYciency of conventional and chronomodulated delivery schedules were directly compared. In one study, performed for doxorubicin/cisplatin treatment of advanced ovarian cancer, a significant advantage of timed drug administration was reported. Patients that received both drugs at specific times associated with reduced toxicity in experimental systems demonstrated better performance and showed half as many complications with the same therapeutic eVect (Hrushesky, 1985). Improved response to chemotherapy on a chronomodulated schedule has been also shown in patients with metastatic colorectal cancer treated with 5‐fluorouracyle (5‐FU) in combination with leucovorin and oxaliplatin (Giacchetti, 2002) and in patients with bladder cancer treated with 5‐FU (Kobayashi et al., 2002). Other clinical studies demonstrated that when colorectal cancer patients with unresectable liver metastases were treated with a combination of oxaliplatin with 5‐FU and folinic acid on a chronomodulated schedule, the liver metastases were able to be surgically resected, resulting in 39–50% 5‐year survival (Eriguchi et al., 2003). Both experimental and clinical studies have conclusively demonstrated that in vivo responses to anticancer therapies show significant daily variations. These variations may depend on daily variations in any of a number of parameters including the pharmacokinetics of a particular therapeutic drug, the blood and tissue concentrations of various growth factors, cytokines and hormones, and the activity of the intracellular genotoxic stress response system. In any case, the chronotherapeutic approach takes advantage of natural daily variability in the physiological response of an organism, which along with many other parameters, is controlled by the circadian clock system. An improved understanding of the molecular mechanisms underlying

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clock‐controlled responses of normal and tumor tissues to a given treatment will likely lead to improved eYcacy of cancer therapies.

B. The Sensitivity of Normal Cells to Genotoxic Drugs Depends on the Functional Status of the CLOCK/BMAL1 Transactivation Complex While the data on daily variations in therapeutic response of tumors are still sporadic and sometimes inconsistent (reviewed in Antoch et al., 2005), daily variations in drug and radiation responses of normal tissues were studied in more detail. The identification of clock genes and generation of genetically altered mice with well‐characterized defects in clock function provided a new direction for studying the molecular links between the circadian system and drug‐induced stress response. Thus, the key role of major circadian proteins in genotoxic stress response was demonstrated by testing the sensitivity of Clock/Clock mutant, Bmal1/ knockout and Cry1/Cry2/knockout animals to toxicity induced by the chemotherapeutic drug cyclophosphamide (CY). Normally, mice demonstrate a robust daily rhythm in sensitivity to CY, with mortality and morbidity rates peaking when the drug is administered at the times of the day when the CLOCK/BMAL1 complex has the lowest activity as measured by Per1 and Per2 gene expression in peripheral tissues. In contrast, drug‐induced mortality and morbidity were lowest when CY was administered at the time of maximal transcriptional activity of the CLOCK/BMAL1 complex (Gorbacheva et al., 2005). This correlation suggests that the functional activity of CLOCK/BMAL1 plays a role in determining sensitivity to CY. Consistent with this, Clock/Clock mutant mice and Bmal1/ mice, which are both characterized by reduced CLOCK/BMAL1 transcriptional activity, demonstrated heightened drug sensitivity as compared to wild‐type mice regardless of the time of administration. Moreover, Cry1/Cry2/ double knockout mice, which have high CLOCK/ BMAL1 activity, demonstrated resistance to CY toxicity. Thus, comparison of wild‐type mice to three diVerent models of circadian mutant mice showed that both survival and morbidity following administration of sublethal doses of CY were directly correlated with CLOCK/BMAL1 functional activity (Gorbacheva et al., 2005). Interestingly, as for many of the pathologies described above that develop in circadian mutant mice, drug sensitivity does not depend on persistence of rhythmicity per se since phenotypically, all three types of mutants are characterized by disrupted circadian rhythmicity at the behavioral and gene expression levels (Bunger et al., 2000; van der Horst et al., 1999; Vitaterna et al., 1994) yet they have diVerent drug sensitivity phenotypes. Rather, the data demonstrate that drug sensitivity is aVected by functional activity of the CLOCK/BMAL1 complex and suggest that the

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CLOCK/BMAL1 complex may directly control the molecular determinants of drug sensitivity at the transcriptional level.

C. Molecular Determinants of Sensitivity to Anticancer Therapy Traditionally, daily variations in sensitivity to chemotherapy have been attributed to rhythms in the activity of multiple drug metabolizing enzymes (Levi, 1997). In line with this, global gene expression analysis of circadian transcriptional output demonstrated that many genes encoding drug‐ metabolizing enzymes display 24‐hour periodicities in their expression pattern in the liver and other tissues (Panda et al., 2002; Storch et al., 2002). However, the circadian patterns of sensitivity to diVerent genotoxic stresses in several tissues suggest that additional mechanisms contribute to circadian responses to anticancer treatment. Indeed, radiation‐induced apoptosis in mouse small intestine exhibits a clear circadian pattern (Ruifrok et al., 1998). Moreover, daily variations in sensitivity to CY cannot be explained completely by diVerences in pharmacokinetics (Gorbacheva et al., 2005). Cell sensitivity to genotoxic stress depends strongly on cell cycle stage. Therefore, the observed circadian rhythms in the proliferation of hematopoietic cells, vascular endothelial cells, and intestinal epithelial cells (Bjarnason and Jordan, 2002; Bjarnason et al., 2001; Smaaland et al., 2002) provide one possible explanation for the time‐of‐day variations in drug‐induced toxicity in these tissues. One example of the molecular connections between the circadian system and cell proliferation is circadian regulation of G2/M transition time in the process of liver regeneration, which depends on circadian‐controlled expression of the Wee1 kinase that is involved in phosphorylation of the cell cycle regulator CDC2 (Matsuo et al., 2003). It is possible that the CLOCK/BMAL1 complex may be directly involved in regulating expression of other genes involved in cell cycle regulation and apoptosis. Indeed, Gadd45, c‐Myc, and Cyclin D1 all demonstrate circadian variation in their transcript levels and, their expression patterns are disrupted in the tissues of circadian mutant mice (Fu et al., 2002). Low‐ amplitude circadian variations in the expression profiles of some pro‐ and anti‐apoptotic genes have been described in mouse bone marrow and tumors (Granda et al., 2005); however, it is still not clear how these fluctuations may be mechanistically translated into diVerences in cell survival rate. It is also possible that extracellular signaling molecules such as cytokines, growth factors and hormones may aVect cell survival following genotoxic stress. Some of these agents, such as fibroblast growth factor, epidermal growth factor, and transforming growth factor display daily variations in their plasma concentrations (Haus et al., 2001; Holzheimer et al., 2002; Liu, 2002). It is likely that circadian modulation of responses to genotoxic stress

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in vivo reflects the complex superposition of multiple levels of regulation involving a number of factors.

D. Functional Interplay Between the Circadian and Stress Response Systems: A Model It has been generally assumed that circadian control of various pathways occurs through CLOCK/BMAL1‐dependent transcriptional activation/repression of numerous target genes that is translated into circadian variations in the abundance and/or activity of the corresponding protein products. However, this type of regulation would only result in changes of significant amplitude for genes that are controlled primarily by circadian transcription factors and whose basal level of expression is low. In reality, the promoter regions of many clock‐ controlled genes contain multiple regulatory elements for other factors that may keep the promoter constitutively active. In these cases, basal promoter activity is often high, and additional circadian activation will not induce significant changes in expression. Consistent with this view, very few of the reported circadian‐regulated genes display high amplitude oscillations in mRNA abundance (Panda et al., 2002; Storch et al., 2002). For most of them, quantitative changes are modest and are therefore unlikely to wholly account for the dramatic diVerences observed in in vivo responses. These responses might result from amplification or cumulative eVects due to interplay between components of the circadian system, and other transcription regulators involved in stress response pathways. The proposed circadian repressor model provides a mechanistic background for the functional interaction of the CLOCK/BMAL1 complex with other transcriptional regulators (Kondratov et al., 2006c). Each cell in an organism is constantly balancing between life and death every time it enters a new round of cycle division, starts the process of diVerentiation or receives external signals from the environment. These external signals include changes in nutrients, temperature or light conditions, exposure to hypoxia, UV‐light, ionizing radiation, chemical mutagens, etc. Many of these agents are harmful for an organism as they directly aVect DNA. To prevent detrimental eVects on the genome, cells have evolved a response system that induces cell cycle arrest to allow suYcient time to repair the incurred damage, activates the appropriate DNA repair pathway, or, in the case of irreparable damage, induces apoptosis. Thus, response to genotoxic stress is a complex process that involves induction of multiple genes associated with cell cycle control, replication, DNA repair, apoptosis, signal transduction, etc. For example, ionizing radiation (or other DNA damaging stress) activates p53. This regulation occurs at the posttranslational level through multiple

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modifications of the p53 protein (Gudkov and Komarova, 2003). Various cytokines activate NFB by disrupting its interaction with the repressor, IkB (Bonizzi and Karin, 2004). The activated p53 and NFB transcription factors, in turn, upregulate expression of their target genes, the products of which determine the organism’s response to the original stressor(s) (schematically shown in Fig. 4A). While some of these stress‐induced genes may be circadian‐independent, others may be transcriptional targets of CLOCK/ BMAL1 and, therefore, their induction will vary according to the time of day that the stress signal is imposed. If the stress signal is received at the time when the CLOCK/BMAL1 complex acts as circadian activator and cooperates with other stress‐induced transcription factors, the overall response will be amplified (Fig. 4B). However, if the stress signal is imposed at the time when CLOCK/BMAL1 complex acts as circadian repressor and interferes with the activity of other transcription factors, induction of the same genes may be significantly attenuated or completely suppressed (Fig. 4C). Therefore, the pattern of stress‐induced gene expression will depend on whether the cells were exposed to stress at a time of the day of maximal activity of the circadian activator or of the circadian repressor. The resulting pattern of gene expression will, in turn, determine the in vivo response. Importantly, the stress response system activates both pro‐death and pro‐ survival components of cellular homeostasis. For example, genotoxic stress simultaneously induces the tumor suppressor gene p53, which is considered as an important mediator of DNA damage‐induced cell death, and NFB, which enhances cell survival pathways. In addition, in response to particular type of stress, the same transcription factor may simultaneously activate diverse spectra of genes, some of which may display alternative functions in determining cell fate. Thus, p53 activates transcription of pro‐apoptotic (Bax, Puma and p53AIP1) and anti‐apoptotic (Slug and Mdm2) genes, regulators of cell cycle progression (p21/WAF1), and regulators of ROS levels (sestrins) (Bonizzi and Karin, 2004; Budanov et al., 2004; Gudkov and Komarova, 2003; Sablina et al., 2005). Cell fate on stress depends on the balance of these pro‐ and anti‐apoptotic factors. For example, an excess of BCL2 or BCLX will suppress apoptosis while an excess of BAX or PUMA will promote the process. Stress responses are further complicated by the fact that the same pattern of gene expression may enhance protection in one type of tissue and decrease protection in another. For example, activation of p53 in response to high doses of radiation results in increased sensitivity in lymphoid tissues, but plays a protective role in small intestine (Komarova et al., 2004). Overall, cell fate following genotoxic stress will be determined by the superposition of multiple stress response pathways and will depend on cell type, cell status, the level and type of damage, and environmental conditions.

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A A B B C C D D E E FF Cytoplasm Cytoplasm

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Figure 4 Circadian control of genotoxic stress response. (A) Genotoxic stress results in activation of various transcription factors, which, in turn, activate expression of multiple stress response genes (genes A, B, C, D, E, and F). The balance of the protein products of these genes determines cell’s and organism’s responses to genotoxic stress. (B) and (C) Some of the stress response genes (A, B, and E) function under circadian transcriptional control being regulated either directly by the CLOCK/BMAL1 complex or indirectly through the downstream transcriptional regulators such as DBP or REV‐ERB. The activity of circadian transcriptional regulators may both cooperate and interfere with the activity of stress‐induced transcription factors. CLOCK/BMAL1 complex displays dual functional activity acting as both transcriptional activator and transcriptional repressor depending on its posttranslational modifications. Therefore, at time of the day when the CLOCK/BMAL1 complex acts as an activator (CA, circadian activator), it will cooperate with stress‐induced transcription factors resulting in increased expression of genes A, B, and E compared to genes C, D, and F. However, when the CLOCK/BMAL1 complex acts as transcriptional repressor (CR, circadian repressor), the induction of genes A, B, and E by genotoxic stress will be either attenuated or completely blocked. As a result, the pattern of stress‐induced genes will depend on whether the cells were exposed to stress at a time of the day of maximal activity of the circadian activator or of the circadian repressor. The resulting pattern of gene expression will, in turn, determine the in vivo response.

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VII. Circadian Proteins as Targets for Therapeutic Intervention Recent reports describing multiple developmental and/or pathological defects in various tissues of circadian mutant mice highlight the potential importance of components of the circadian system as targets for therapeutic intervention. Traditionally, circadian proteins have been considered for clinical applications predominantly in relation to sleep, mood, and other mental disorders. However, increasing amounts of data demonstrate that the circadian clock is involved in regulation of almost every aspect of mammalian physiology. Moreover, the non‐overlapping spectra of defects in diVerent circadian mutants suggest that they might arise as a consequence of deficiency in the activity of a particular circadian protein rather than as a result of disruption of circadian clock function. Thus, it is likely that at least some circadian proteins are actively involved in physiological processes not directly related to their role in the circadian clock. Further study of the status and function of diVerent circadian proteins in human pathologies will be necessary to reveal their full potential as targets for therapeutic manipulation. Our growing understanding of the molecular links between the circadian and stress response systems provides a rationale for potential use of these mechanisms in clinical practice. The direct correlation between CLOCK/ BMAL1 function and sensitivity to genotoxic stress suggests that existing therapeutic schedules may be rationally modulated to increase the therapeutic index of a given cancer treatment. In summary, recent discoveries connecting the circadian clock, normal physiology, and diseases make the circadian system and its molecular components potential targets for therapeutic intervention and provide a basis for development of new medications. Drug targeting the circadian system might be useful not only for treatment of circadian disorders (such as sleep and mood disorders of shift workers and frequent time zone travelers), but also for manipulating sensitivity genotoxic anticancer treatments to improve their therapeutic index.

Acknowledgments This work was supported by grant from the National Cancer Institute CA102522 (to M.P.A.). We thank Dr. P. Stankope‐Baker for the editorial assistance with manuscript preparation.

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Autophagy and Cell Death Devrim Gozuacik and Adi Kimchi Department of Molecular Genetics, Weizmann Institute of Science Rehovot 76100, Israel

I. Introduction II. Description of Programed Cell Death Morphologies III. Autophagic Cell Death A. Autophagy May Kill: Molecular Evidence B. How Does Autophagy Kill? IV. Autophagy and Autophagic Cell Death Regulatory Mechanisms A. Class III Phosphatidylinositol 3‐Kinase, Beclin‐1, and Autophagy B. Two Ubiquitin‐Like Pathways Involved in Autophagic Vesicle Formation C. The Tor Pathway D. Class I Phosphatidylinositol 3‐Kinase and Autophagy E. Protein Transcription/Translation‐Related Pathways F. G Proteins G. DAPk Family H. Sphingolipid Pathways I. Role of Mitochondria and the Bcl‐2 Family Proteins in Autophagy J. RIP Protein‐Related Pathways and Autophagic Cell Death V. Autophagy–Apoptosis Crosstalks VI. Survival Versus Death Aspects of Autophagy VII. Conclusions Acknowledgments References

Autophagy is a physiological and evolutionarily conserved phenomenon maintaining homeostatic functions like protein degradation and organelle turnover. It is rapidly upregulated under conditions leading to cellular stress, such as nutrient or growth factor deprivation, providing an alternative source of intracellular building blocks and substrates for energy generation to enable continuous cell survival. Yet accumulating data provide evidence that the autophagic machinery can be also recruited to kill cells under certain conditions generating a caspase‐independent form of programed cell death (PCD), named autophagic cell death. Due to increasing interest in nonapoptotic PCD forms and the development of mammalian genetic tools to study autophagy, autophagic cell death has achieved major prominence, and is recognized now as a legitimate alternative death pathway to apoptosis. This chapter aims at summarizing the recent data in the field of autophagy signaling and autophagic cell death. ß 2007, Elsevier Inc. Current Topics in Developmental Biology, Vol. 78 Copyright 2007, Elsevier Inc. All rights reserved.

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I. Introduction Programed cell death (PCD) is an evolutionarily conserved phenomenon observed especially in multicellular organisms, which is crucial for several vital functions, including developmental morphogenesis, tissue homeostasis, and defense against pathogens. In their seminal article published in 1972, Kerr, Wyllie, and Currie described two major types of cell death: apoptosis, the genetically controlled PCD, and necrosis, the nonprogramed and accidental type of cell death (Kerr et al., 1972). In the following three decades, the term ‘‘apoptosis’’ was used as a general term to describe PCD and an impressive amount of information has accumulated regarding the molecular mechanisms governing this phenomenon. Because the apoptosis versus necrosis concept of cell death was so dominant, early observations about the existence of alternative, nonapoptotic PCD types were ignored by the majority of the scientific community (Schweichel and Merker, 1973). In recent years, however, increasing interest in alternative PCD types emerged once diVerent tools to study these other genetically controlled systems at the molecular level became available. One type, autophagic cell death, recently received considerable momentum in light of the identification of the mammalian orthologues of the yeast autophagic genes. As a consequence, autophagic cell death is currently recognized as one of the major alternative or complementary cell death pathways to apoptosis in several experimental systems. In this chapter, we will describe the morphological and molecular basis of autophagy and autophagic cell death, document what is known so far about proteins and pathways regulating this phenomenon and finally discuss its crosstalk with apoptosis. A crucial issue in the field is how to reconcile the catabolic and survival‐related role of autophagy with its cell death‐inducing properties. We will also discuss this important issue in light of recent observations.

II. Description of Programed Cell Death Morphologies Revisiting the earlier work of Schweichel and Merker (Schweichel and Merker, 1973), in a review article from 1990, Clarke described three major cell death morphologies of cell death during embryonic development or after toxin treatment (Clarke, 1990). Clarke classified classical apoptosis as Type I cell death. This type of cell death is characterized morphologically by cell shrinkage, chromatin condensation, nucleosomal DNA degradation and finally, fragmentation of the cell into so‐called apoptotic bodies. Caspases are the key mediators of this type of cell death and most of the

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observed morphological changes can be attributed to the cleavage of key cellular target proteins by these cysteine proteases. The final destination of the apoptotic bodies is the lysosomes of phagocytes or neighboring cells after heterophagocytosis. Autophagic cell death was classified as the Type II cell death by Clarke. The most prominent morphological change observed in this type of cell death is the appearance of double‐ or multiple‐membrane enclosed vesicles in the cytoplasm that engulf portions of cytoplasm and/or organelles such as mitochondria and endoplasmic reticulum. These vesicles fuse with lysosomes and deliver their cargo for degradation by lysosomal enzymes of the same cell. This process is termed as autophagy (self‐eating in Greek), corresponds to the previous definition of macroautophagy, referred to as the term autophagy in this text. While the initial observation that certain dying cells display morphological hallmarks of autophagy is beyond dispute, the question of whether autophagy is causative for cell death became a major issue for intensive research. One working hypothesis suggested that under specific circumstances that depend on the character of the stimulus, its amplitude, and duration, extensive autophagy may cause cell death. According to this possibility, the cell cannibalizes itself from inside, and the ultimate cause(s) of cellular demise should be identified as will be detailed in this chapter. Of note, nuclear changes, such as chromatin condensation, appear later in autophagic cell death than they do in apoptosis, and there is no DNA fragmentation or formation of apoptotic bodies. Clearance of the remnants of the dead cells by phagocytosis may occur later and more sporadically than that seen in apoptosis. Type III cell death, defined by Clarke as nonlysosomal vesiculate degradation, which represent a less well‐studied type of cell death, is out of the scope of this chapter, therefore it will not be discussed further. Cells simultaneously harboring the morphological characteristics of more than one of the types of cell death described above have also been observed in certain tissues and under certain conditions. This suggests the existence of additional mixed types of PCD that proceed with concomitant activation of several death mechanisms in the same cell (Clarke, 1990).

III. Autophagic Cell Death A. Autophagy May Kill: Molecular Evidence Morphological analysis of cells and tissues revealed an increased autophagic activity during developmental cell death in several organisms. The list includes cell death during insect metamorphosis, limb bud morphogenesis in birds, and palatal closure in mammals (Bursch, 2001; Clarke, 1990;

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Schweichel and Merker, 1973). Certain toxins also caused cell death with ultrastructural characteristics of autophagic cell death (Schweichel and Merker, 1973). Although a causal relationship between the autophagic activity and cell death could not be measured and the observation stayed at the correlative level, these studies opened the way to subsequent studies dissecting the role of autophagy in cell death. The discovery that some chemical compounds like 3‐MA, wortmannin, and LY294002 inhibited autophagy, was a turning point in the analysis of autophagic cell death (Blommaart et al., 1997; Seglen and Gordon, 1982). Several independent groups using diVerent cell types and death stimuli reported the existence of a caspase‐independent cell death which proceeded with the accumulation of autophagic vesicles and increased lysosomal activity, and which was attenuated by inhibitors of autophagy. Antiestrogen‐induced death of the breast cancer line MCF‐7 (Bursch et al., 1996), TNF‐‐induced death of leukemia cells (Jia et al., 1997), oncogenic Ras‐induced death of gastric or glioma cells (Chi et al., 1999) or growth factor withdrawal induced death of neuronal cells (Xue et al., 1999) may be cited as examples of these studies. Yet the most commonly used autophagy inhibitor, 3‐MA, used at the dose range that optimally inhibits autophagy, also inhibited JNK and p38 activation (Xue et al., 1999), attenuated mitochondrial permeability transition pore opening (Xue et al., 2002) and increased lysosomal pH (Caro et al., 1988). Due to these multiple pleiotropic eVects, the results obtained from these studies were evaluated with caution by the scientific community and the role of autophagy observed in dying cells remained a debated issue (reviewed by Gozuacik and Kimchi, 2004; Levine and Yuan, 2005). More convincing indications for the relevance of this phenomenon to death regulation was provided by studies demonstrating autophagic cell death induction by well‐established death‐ promoting proteins like BNIP3 (Vande Velde et al., 2000), death‐associated protein kinase (DAPk) and DRP‐1/DAPk2 (Inbal et al., 2002), and from studies documenting the suppression of autophagy by death protective proteins like Bcl‐2 (Cardenas‐Aguayo Mdel et al., 2003; Saeki et al., 2000; Vande Velde et al., 2000; Xue et al., 2001; Yanagisawa et al., 2003). The discovery of genes which are part of the basic machinery of autophagy, initially made in yeast by several independent groups, opened a new era in autophagy studies and led to a better understanding of molecular events underlying this biological phenomenon (Kim and Klionsky, 2000; Ohsumi, 2001). Orthologues of the yeast genes were identified soon thereafter in several organisms including Dictyostelium, C. elegans, Drosophila, mouse, and human (Klionsky et al., 2003). Further studies confirmed that the autophagic function of these orthologues in higher organisms was conserved. These major advances opened the way to study the role of autophagy in cell death, using genetic approaches like knockout of autophagic genes

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and RNAi or antisense‐mediated knockdown strategies. It was shown that the RNAi‐mediated knockdown of autophagic proteins like Atg5, Atg7, and Beclin‐1 (see below) attenuated cell death developing under certain conditions. This included death of L929 mouse fibroblastic cells, U937 monocytic cells and macrophages in the presence of the pan‐caspase inhibitor zVAD (Xu et al., 2006; Yu et al., 2004), etoposide and staurosporine‐induced death of Bax/Bak knockout fibroblasts (Shimizu et al., 2004) and cell death‐ induced by overexpression of a short mitochondrial form of the p19ARF tumor suppressor (smARF) (Reef et al., 2006). The finding that the inhibition of autophagic activity by knocking down these autophagic genes could under certain conditions attenuate the death responses of cells supported very significantly the existence of a nonapoptotic, caspase‐independent cell death type proceeding with accumulation of autophagic vesicles, and depending on autophagy proteins. In light of these new molecular strategies to inhibit autophagy, the earlier studies that utilized less specific autophagic inhibitors such as 3‐MA and wortmannin, which nevertheless conferred a protection from cell death comparable to the RNAi knockdown studies, should be revisited.

B. How Does Autophagy Kill? In principle, autophagic activity above a certain threshold which destroys a major portion of the cytosol and organelles could lead to an irreversible type of cellular atrophy and cause a total collapse of cellular functions. Indeed, it was observed that during extensive autophagy, the total area of autophagic vacuoles and dense bodies may be roughly equal to, or greater than, that of cytosol and organelles outside the vacuoles (Clarke, 1990; Lum et al., 2005). It is therefore plausible to think that such a degree of cellular destruction could lead to cellular demise. In line with this hypothesis, it was shown that autophagy induction by growth factor deprivation in neurons, or by staurosporine treatment of HeLa or CHO cell lines exposed to caspase inhibitors, was accompanied by the destruction of the majority of the mitochondria and by irreversible commitment to death (Xue et al., 2001). In IL‐13‐dependent bone marrow cells derived from the Bax/Bak knockout mouse, cytokine deprivation led to progressive atrophy by autophagy and finally, cell death ensued (Lum et al., 2005). Nevertheless, in this cellular setting, damage to the cell was reversible up until a certain threshold, and the cell death observed in these studies was relatively slow, indicating that other mechanisms contributing to cellular demise may exist in autophagic cell death. A second possible mechanism of killing by autophagy may include the selective degradation of vital proteins in the cell. Although macroautophagy,

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the relevant type of autophagy discussed in this chapter, is generally considered a nonselective phenomenon, there is evidence indicating a selective destruction of certain vital cellular components which could contribute to death induction by autophagy. Selective autophagic elimination of depolarized mitochondria, endoplasmic reticulum, and peroxisomes was observed by several independent groups (Elmore et al., 2001; Hamasaki et al., 2005; Iwata et al., 2006; Kissova et al., 2004). Likewise, long‐lived proteins, mutant protein aggregates, and certain intracellular bacteria and viruses were specifically recognized and selectively eliminated by autophagy, indicating that selectivity may be a general property of autophagy under certain circumstances (Reggiori and Klionsky, 2005; Webb et al., 2003; Yu et al., 2005). Therefore, an alternative death‐induction mechanism by autophagy may consist of selective elimination of vital organelles and/or proteins involved in cell survival and homeostasis, although the mechanisms regulating this selectivity still remain obscure. Evidence in support of the contribution of selective protein elimination to autophagic cell death in mammalian cells comes from a recent study of zVAD‐induced autophagic cell death observed in certain cell types. Lenardo and coworkers elegantly showed that catalase, a key enzyme of the cellular antioxidant defense mechanism, was selectively eliminated during autophagic cell death (Yu et al., 2006). Oxidative stress induced by the degradation of catalase was directly responsible for cellular demise, since inhibitors of autophagy, RNAi‐mediated knockdown of autophagic genes, or various ROS inhibitors, prevented cell death. Thus, catalase may be an example of the selective protein target(s) of autophagy contributing to the irreversible cellular damage that leads to cell death. Discovery of additional selective autophagy targets and mechanisms controlling their degradation may further clarify how a catabolic and prosurvival mechanism can be used for cellular suicide.

IV. Autophagy and Autophagic Cell Death Regulatory Mechanisms Although initial morphology‐based studies were performed in mammalian cells and tissues, genes regulating autophagy were discovered by several independent groups in yeast (Harding et al., 1995; Thumm et al., 1994; Tsukada and Ohsumi, 1993). These studies resulted in the cloning of a partially overlapping set of APG, AUT, and CVT genes by independent groups. The confusing nomenclature has been simplified by consensus to a unified nomenclature of ATG genes (autophagy‐related genes) (Klionsky et al., 2003). The list to date includes 27 proteins that are involved in various stages of the autophagic

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process, including vesicle enucleation and their subsequent expansion, autophagic vesicle fusion to late endosome/lysosome, and cargo degradation. Although we are still far from having a complete picture, upstream pathways regulating autophagy started to be uncovered and some of the genes which mediate the formation of the autophagosomes were organized along molecular pathways. In this section, we will describe these pathways and discuss their relevance to autophagic cell death.

A. Class III Phosphatidylinositol 3‐Kinase, Beclin‐1, and Autophagy Phosphatidylinositol 3‐phosphate (PI3‐P) generated by the Class III PI3‐ kinase is the major lipid signal controlling autophagic vesicle formation (Petiot et al., 2000). Indeed, the widely used chemical inhibitors of autophagy, 3‐MA, wortmannin and LY294002, target the Class III PI3‐kinase through competition for ATP binding in the active site of its kinase domain, with variable eYciency and specificity (Petiot et al., 2000; Seglen and Gordon, 1982). Consequently, treating cells with the Class III product, PI3‐P, accelerates autophagy, while Class I PI3‐kinase products inhibit it (Petiot et al., 2000). Studies performed mainly in yeast and mammalian cells showed that a protein complex consisting of Atg6 (Vps30 or Beclin‐1 in mammalians) and myristylated serine kinase Vps15/p150 regulates the activity of the autophagy‐related Class III PI3‐kinase Vps34 (Stack et al., 1995). In line with this, expression of Vps15/p150 or Beclin‐1 stimulated autophagy in mammalian cell lines (Liang et al., 1999; Petiot et al., 2000). Furthermore, Class III PI3‐kinase inhibitors and knockdown of Beclin‐1 attenuated death in several diVerent cell types (Reef et al., 2006; Shimizu et al., 2004; Xu et al., 2006; Yu et al., 2004). Atg14 is also part of the complex in the yeast. Unlike the other members of the complex, the mammalian homologue of Atg14 has yet to be discovered. In the yeast, Atg14 localizes to yeast‐specific autophagy organizing structures called preautophagosomal structures (PAS) and serves as an adaptor for the Atg6/Vps34 complex (Kim et al., 2002). Mammalian Beclin‐1 localizes to the trans‐Golgi network and the endoplasmic reticulum, suggesting that multiple foci in these compartments may be the sites of PI3‐P formation and autophagic vesicle nucleation in mammals (Kihara et al., 2001; Liang et al., 1998; Pattingre et al., 2005). In general, PI3‐P mediates docking of FYVE (for conserved in Fab1, YOTB, Vac1, and EEA1) or PX (Phox homology) domain‐containing proteins to nucleation sites (Gillooly et al., 2001; Wishart et al., 2001). The identity of the PI3‐P‐binding proteins that regulate autophagy pathways have yet to be discovered. Beclin‐1, the mammalian orthologue of Atg6, was cloned as a Bcl‐2 interacting protein (Liang et al., 1998). In fact, Bcl‐2 blocked the Beclin‐1

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interaction with Vps34, decreased Class III PI3‐kinase activity in cells and downregulated starvation‐induced autophagy, both in cell culture and in mouse cardiac muscle (Pattingre et al., 2005). In line with this, immunoprecipitation of Beclin‐1 from starved cells brought down less Bcl‐2 compared to controls grown in nutrient rich conditions, indicating that the Bcl‐2/Beclin‐1 interaction may be regulated by autophagy‐inducing signals. Strikingly, Beclin‐1 mutants which can no longer bind Bcl‐2 caused excessive autophagy and cell death, highly suggesting that the protective eVect of Bcl‐2 on autophagic cell death may be related to its ability to sequester Beclin‐1 and thus inhibit Vps34 PI3‐kinase activity (Canu et al., 2005; Cardenas‐Aguayo Mdel et al., 2003; Saeki et al., 2000; Vande Velde et al., 2000).

B. Two Ubiquitin‐Like Pathways Involved in Autophagic Vesicle Formation Two ubiquitin‐like pathways play a central role in autophagic vesicle formation. The first system involves Atg12, which is covalently conjugated to Atg5 through the sequential action of the E1 ligase‐like protein Atg7 and E2‐ like protein Atg10 (Mizushima et al., 1998; Shintani et al., 1999; Tanida et al., 1999). Atg12 is conjugated to Atg5 just after its synthesis, and this event seems not to be regulated by autophagy‐inducing signals. The Atg12/ Atg5 complex then binds to Atg16 which, through its homo‐oligomerization capacity, leads to the formation and stabilization of larger protein complexes (350 kDa in yeast and 800 kDa in mammals) (Mizushima et al., 1999, 2003). Atg12 conjugation to Atg5 is necessary for the second ubiquitin‐like pathway to proceed and autophagic vesicles to form (Mizushima et al., 2001; Suzuki et al., 2001). The second ubiquitin‐like pathway involves the conjugation of Atg8 protein (mammalian MAP LC3 protein) to a lipid molecule, phosphatidylethanolamine (PE) (Ichimura et al., 2000) or, at least in vitro, phosphatidylserine (Sou et al., 2006). In this manner, several Atg8 proteins recruit lipids molecules to expand the autophagic membranes. As soon as Atg8 is translated, a protease called Atg4 cleaves oV a portion of its C‐terminus, exposing a critical C‐terminal glycine residue (Kirisako et al., 2000). Following the ubiquitination‐like reaction mediated by Atg7 and Atg3, the amino group of the lipid molecule is conjugated via an amide bond to this glycine residue (Ichimura et al., 2000). Lipid conjugation leads to the conversion of the soluble 18 kDa form of Atg8 (mammalian LC3‐I) to an autophagic vesicle‐ associated, faster migrating 16 kDa form on SDS‐PAGE gels (mammalian LC3‐II). Immunostaining of Atg8/LC3 indicates a change from a diVuse cytoplasmic or nuclear distribution to punctate dots which reflect autophagic

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vesicles (Kamada et al., 2000; Kirisako et al., 1999). Analysis of LC3 by SDS‐PAGE and immunolocalization is now, in addition to the more labor‐ intensive transmission electron microscopy, commonly used to detect and quantify autophagic activity in cells and whole animals (Mizushima, 2004).

C. The Tor Pathway Discovered as the target of the drug rapamycin, the Tor serine/threonine (Ser/Thr) kinase (also known as RAFT1, FRAP or SEPT) is the central player of a complex signaling network regulating several cellular events, including protein synthesis, cell growth and proliferation, as well as autophagy (Fingar and Blenis, 2004). The Tor pathway is modulated by growth factor, nutrient and energy availability, osmotic stress and DNA damage, and its activation suppresses autophagy. In yeast under nutrient rich conditions, Tor phosphorylates autophagy‐ related protein Atg13, leading to it dissociation from a protein complex containing Atg1 kinase. Atg13 dissociation attenuates Atg1 kinase activity, which correlates with autophagy induction (Kamada et al., 2000; Scott et al., 2000). Under nutrient limitation, Tor activity is blocked, Atg13 is rapidly dephosphorylated, and it tightly associates with Atg1 kinase. This promotes activation of Atg1, leading to autophagy induction (Abeliovich et al., 2003; Kamada et al., 2000). Additionally, the Tor complex regulates the function of the three yeast orthologues of the Ser/Thr phosphatase PP2Ac (PPH21/22 proteins), through phosphorylation of their regulatory subunit TAP42 (Di Como and Arndt, 1996). The relevance of these pathways to mammalian autophagy and autophagic cell death is yet to be discovered. The mammalian Tor orthologue, mTor, exists in the cell as part of two distinct multiprotein complexes (Fingar and Blenis, 2004): raptor/mTor and rictor/mTor complexes. The complex containing the raptor protein regulates cell survival and autophagy through p70S6K and 4E‐BP1 phosphorylation. p70S6K phosphorylates a wide spectrum of proteins involved in transcription, protein synthesis and RNA splicing, including the 40S ribosomal protein S6, eukaryotic elongation factor 2 kinase (eEF‐2 kinase), and transcription and splicing related proteins. mTor mediated activation of the 4E‐BP1 also contributes to the prosurvival role of Tor, since it leads to upregulation of cap‐dependent translation. Furthermore, p70S6K phosphorylates and inactivates the proapoptotic BH3‐only protein Bad (Harada et al., 2001). Another p70S6K target, eEF‐2 kinase, inhibits protein synthesis through phosphorylation and inactivation of elongation factor 2. Its phosphorylation by p70S6K relieves this inhibition by inactivation of the kinase (Wang et al., 2001).

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Binding of the small GTPase Rheb is a critical step in the activation of the mTor complex. Rheb is under strict control through regulation of its GTPase activating proteins TSC1 (hamartin) and TSC2 (tuberin) (Fingar and Blenis, 2004). Phosphorylation of TSC2 by an intracellular energy status sensor and an AMP‐activated kinase, AMPK, leads to the stabilization of the TSC1/2 complex, increases its GTPase activity toward Rheb, inactivating it and thereby inhibiting the raptor/mTor complex and activating autophagy (Inoki et al., 2003). On the other hand, survival‐related kinases Akt/PKB, ERK (Ma et al., 2005), and RSK1 (Roux et al., 2004) activate the mTor pathway through phosphorylation and inhibition of TSC2. Hypoxia also inhibits mTor activity through hypoxia inducible factor (HIF) transcriptional targets REDD1 (Brugarolas et al., 2004), REDD2 (Corradetti et al., 2005) and in a TSC1/2‐dependent manner. Thus, TSC1/2‐dependent control of Rheb is a convergence point of several metabolic, prosurvival, and stress‐ related signals regulating mTor activity. AMPK and TSC1/2 may also connect DNA damage‐induced stress to mTor inhibition and to autophagy activation, since this pathway was necessary for etoposide‐induced and p53‐dependent autophagy activation (Feng et al., 2005). In line with this, the DNA damaging agent etoposide was capable of inducing autophagic cell death in Bax/Bak knockout cells (Shimizu et al., 2004). The second Tor complex, consisting of rictor and mTor, is not inhibited by rapamycin, and therefore its relevance to autophagy pathways is not clear.

D. Class I Phosphatidylinositol 3‐Kinase and Autophagy Class I PI3‐kinase antagonizes apoptosis and autophagy through activation of the PDK1 and Akt/PKB pathway. Following growth factor binding to cell surface receptors, Class I PI3‐kinase is activated to generate PI3,4‐ diphosphate and PI3,4,5‐triphosphate. These lipid products recruit PDK1 and Akt/PKB to cell membranes through their Pleckstrin homology (PH) domains, where PDK1 activates Akt/PKB by phosphorylation. Akt/PKB promotes cell survival through phosphorylation of several substrates. Phoshorylation of the cell death‐related Bcl‐2 family member Bad leads to its inactivation through sequestration to the cytoplasm by 14‐3‐3 proteins. Transcriptional induction of death‐related genes is also blocked following the nuclear exclusion of forkhead transcription factors as a result of their phosphorylation by Akt/PKB. In another survival promoting mechanism, Mdm2 phosphorylation by Akt/PKB enhances p53 ubiquitination and degradation (Zhou et al., 2001). Akt/PKB has also been shown to activate the antiautophagic mTor pathway either by directly phosphorylating and inhibiting TSC2 (Inoki et al., 2002) or by decreasing AMP/ATP ratios and hence

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suppressing AMPK activity (Hahn‐Windgassen et al., 2005). The tumor suppressor PTEN, which inactivates the Akt/PKB pathway by dephosphorylation of Class I PI3,4,5‐triphosphate, upregulated autophagy in HT‐29 colon cancer cells (Arico et al., 2001). Indeed, PTEN overexpression was shown to counteract Akt/PKB‐mediated survival signals and caused cell death in various cell types (Li et al., 1998; Wang et al., 2000). Interestingly, the antiautophagic nature of Class I PI3‐kinase is not universal. Class I PI3‐kinases and growth hormones that activate this pathway can actually induce autophagic cell death in glucose‐deprived muscle derived H9c2 and C2C12 cell lines, but not in PC12 or HepG2 carcinoma cell lines. In other cell types such as HT‐29 colon cancer cell lines, treating cells with Class I PI3‐kinase products, expression of activated Akt/PKB kinase or IL‐13 treatment inhibited autophagy, while Akt/PKB inhibitors stimulated autophagy (Petiot et al., 2000; Takeuchi et al., 2005). Therefore, cell type and/or tissue‐specific signaling connections may modulate the outcome of the stimulation of this pathway (Aki et al., 2003).

E. Protein Transcription/Translation‐Related Pathways Phosphorylation of the eukaryotic initiation factor 2?(eIF2) on serine‐51 by a conserved family of protein kinases represents a central and evolutionarily conserved mechanism of stress‐induced translation regulation and autophagy. In yeast during amino acid starvation, eIF2 phophorylation by the yeast eIF2 kinase GCN2 inhibits global protein synthesis and favors translation of GCN4, a stress‐related transcription factor, by an alternative mechanism (Dever et al., 1992). In addition to the transcriptional activation of several stress‐related genes, including those involved in amino acid biosynthesis and transport, GCN4 also activates the transcription of autophagy‐regulating genes ATG1, ATG13, and ATG14 (Natarajan et al., 2001). Indeed, GCN2 and GCN4, and eIF2 phosphorylation were essential for starvation‐induced autophagy. In addition, GCN4 was also necessary for rapamycin‐induced autophagy (Talloczy et al., 2002). In mammalians, the eIF2 kinase family consists of four proteins that connect diVerent stress conditions to cellular responses, including autophagy. GCN2 is activated during amino acid starvation by uncharged tRNAs. PKR is activated during viral infection by double stranded RNA. PERK is activated during endoplasmic reticulum stress by unfolded proteins, and HRI is activated by low heme levels. In addition to its role in starvation‐induced autophagy, as in the yeast, mammalian eIF2 phosphorylation was also found to be necessary for virus‐induced autophagy in primary murine embryonic fibroblasts (Talloczy et al., 2002). The eIF2 kinase PKR was also shown

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to be essential for virus‐induced autophagy, and it was able to complement the yeast GCN2 to induce autophagy in response to starvation (Talloczy et al., 2002). Therefore, in mammalian cells, stress responses triggered by eIF2 phosphorylation regulate autophagy activation by viral infection or amino acid starvation. Whether this pathway plays a role in the autophagic cell death observed following additional cellular stresses still needs to be studied. Other molecular events controlling global protein synthesis include 4E‐BP1 phosphorylation and eEF‐2 kinase activation. 4E‐BP1 (also known as PHAS‐I) is a repressor of the mRNA cap‐binding factor eIF4E. Growth factor stimulation under nutrient rich conditions leads to 4E‐BP1 phosphorylation by mTor and other kinases, and induces its dissociation from eIF4E. eIF4E is then free to start a cascade of events leading to the correct positioning of the initiation complex and the 40S ribosomal subunit at the 50 end of mRNA, leading to cap‐dependent translation. mTor inhibition was shown to block 4E‐BP1 phopshorylation and thus cap‐dependent translation (Beretta et al., 1996). eEF‐2 kinase, another protein involved in translation regulation, is a target of the mTor downstream kinase p70S6K (Wang et al., 2001). eEF‐2 kinase was shown to block the elongation phase of translation by phosphorylation of eEF‐2. A study showed that eEF‐2 kinase was crucial for nutrient deprivation‐triggered autophagy (Wu et al., 2006). Like some other autophagy‐related kinases [e.g., DAPk or DRP‐1, see below], eEF‐2 kinase is also regulated by calcium/calmodulin (Nairn et al., 1985). Therefore, eEF‐2 kinase may also connect calcium‐dependent signals to autophagy induction through translation regulation. As observed in yeast, inhibition of mammalian cap‐dependent protein translation through the above‐mentioned mechanisms could lead to upregulation of certain proteins involved in the transcriptional regulation of autophagy‐ related genes. Upregulation of these autophagy‐related proteins could stimulate or support the progress of autophagy and even autophagic cell death. In fact, in several mammalian experimental systems, accumulation of autophagy regulating proteins was observed by several independent groups (Daido et al., 2004; Kanzawa et al., 2003; Scarlatti et al., 2004; Shimizu et al., 2004). Furthermore, overexpression of key autophagy‐regulating proteins like Beclin‐1 and Atg5, induced cell death with autophagic morphology, indicating the importance of transcriptional and/or translational regulation of autophagic cell death (Pattingre et al., 2005; Pyo et al., 2005). Supporting the concept of transcriptional control of autophagy, in Drosophila, a transcriptional program involving the steroid hormone ecdysone regulates autophagic cell death‐mediated destruction of the salivary gland (Baehrecke, 2003). On the other hand, the protein translation inhibitor cycloheximide did not block the initial stages of autophagy in mammalian liver cells (Lawrence and Brown, 1993). Therefore, further studies are necessary to fully appreciate

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the contribution of these transcriptional and/or translational mechanisms to autophagy and autophagic cell death.

F. G Proteins In HT‐29 colon cancer cells, amino acids and the Ras/Raf1/ERK1/2 pathway were shown to modulate autophagy through regulation of ER‐ and Golgi‐ localized heterotrimeric G proteins (Houri et al., 1993; Ogier‐Denis et al., 1995; Pattingre et al., 2003a). Amino acid starvation was shown to relieve Raf1 from an inhibitory dephosphorylation and enhance phosphorylation of ERK1/2 by Raf1. Activated ERK1/2 in turn caused direct activation of the GAIP protein (Ogier‐Denis et al., 2000; Pattingre et al., 2003a). GAIP is a GTPase activating protein for the Gi3 class of G proteins. Activation of GTP hydrolysis by Gi3 and stabilization of its GDP‐bound form by AGS3 protein resulted in the stimulation of autophagy (Ogier‐Denis et al., 1995, 1997; Pattingre et al., 2003b). To date, the mechanism of autophagy induction by the heterotrimeric G protein complex is unknown. Considering the Golgi/ER localization of the complex, hypothesized that Gi3 could contribute to the control the flux of membrane from these compartments to autophagy organizing centers (Meijer and Codogno, 2004). Akt/PKB can also modulate this G protein‐dependent pathway through direct phosphorylation and inactivation of Raf1. Consequently, simultaneous stimulation of the Akt/PKB and Ras/Raf1/ERK1/2 pathways by EGF treatment failed to induce autophagy (Pattingre et al., 2003a). On the other hand, PTEN, which is a negative regulator of Akt, stimulated autophagy in the same system (Arico et al., 2001). Thus, a complex network which consists of at least three modulators, namely amino acid availability, Ras/Raf1/ERK1/2 signaling, and the Akt/PKB pathway, regulate autophagy activation in this system. In line with this, overexpression of Ras was able to activate autophagy and autophagic cell death in several systems (Chi et al., 1999; Pattingre et al., 2003a).

G. DAPk Family A functional genetic screen for mediators of interferon‐ ‐induced cell death led to the isolation of several new death‐associated proteins (Deiss et al., 1995). One of the genes isolated by this approach, DAPk (DAPk or DAPk1), and its homologues DRP‐1/DAPk2 and ZIPk/DAPk3, were later shown to be involved in cell death‐induced by other stimuli as well, including activation of Fas receptors, TNF‐, TGF‐ , detachment from extracellular

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matrix and oncogenes (Cohen and Kimchi, 2001; Inbal et al., 2002; Jang et al., 2002; Kawai et al., 2003; Raveh et al., 2001; Wang et al., 2002). While the DAPk family of Ser/Thr kinases has been shown to modulate the apoptotic death in some experimental settings, their overexpression in HEK 293 or HeLa cells induced autophagic Type II cell death (Gozuacik and Kimchi, 2004; Inbal et al., 2002; Shani et al., 2004). Although physical interactions and a complex cascade‐like signaling connection exist among the members of the DAPk family (Inbal et al., 2000; Shani et al., 2004), accumulating data indicate that individual members may have specific functions in diVerent autophagy pathways. A dominant negative DRP‐1/DAPk2, but not DAPk was able to attenuate tamoxifen‐ induced autophagy and amino acid starvation‐induced in MCF‐7 breast carcinoma cells (Inbal et al., 2002). Knocking down DAPk on the other hand attenuated the interferon‐ ‐induced autophagic cell death of HeLa cells (Inbal et al., 2002). In contrast, DAPk knockout primary fibroblasts and hepatocytes showed similar level of autophagy activation in response to amino acid starvation and inhibition of mTor with rapamycin, indicating that DAPk is not involved in these phenomena (D. Gozuacik and A. Kimchi, unpublished observations). Moreover, while DAPk associates with the actin cytoskeleton (Cohen et al., 1997), overexpressed DRP‐1 was highly concentrated in the lumen of autophagic vesicles (Inbal et al., 2002), raising the possibility that DRP‐1 may have a direct role in autophagic vesicle formation, possibly through phosphorylation of the components of autophagic vesicle formation machinery. Thus, the DAPk family of proteins seems to be stress‐activated kinases linking diVerent cellular stresses like interferon‐ exposure, starvation, or growth factor deprivation to autophagy pathways and to autophagic Type II cell death.

H. Sphingolipid Pathways One of the classes of lipid found in eukaryotic membranes is sphingolipids. The biological function of these molecules goes far beyond the structural role that had been initially attributed to them. The sphingolipid metabolites ceramide, sphingosine, and sphingosine 1‐phosphate (S1P) are now recognized as lipid messengers that regulate cell growth, survival, and death. Recent work also provides evidence that ceramide and S1P are involved in autophagy regulation. Ceramide may be derived from the hydrolysis of sphingomyelin by sphingomyelinase enzymes (SMase) or it may be synthesized de novo through the action of serine palmitoyl transferase and ceramide synthase. Studies in cancer cell lines have implied that ceramide may act as an autophagic cell death signaling molecule, and provided evidence that classical autophagic

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cell death‐inducers like tamoxifen, used ceramide as a second messenger (Daido et al., 2004; Scarlatti et al., 2004). Ceramide can induce autophagy activation through several mechanisms: Ceramide treatment blocked the antiautophagic Akt/PKB and mTor pathways, upregulated expression of key autophagy regulators Beclin‐1, LC3, and the pro‐death Bcl‐2 family member BNIP3, and led to mitochondrial membrane potential loss. Of note, the autophagic cell death kinase DAPk was also upregulated and activated on ceramide treatment, and it was shown to be play a central role in ceramide‐induced cell death (Pelled et al., 2002; Shohat et al., 2001). S1P and one of the enzymes responsible for its production, sphingosine kinase 1 (SK1), also induced autophagy in MCF‐7 cells (Lavieu et al., 2006). S1P is generated on phosphorylation by sphingosine kinases of sphingosine (which itself is produced by deacylation of ceramide by ceramidases). Unlike ceramide, SK1 and S1P seem to be involved in survival‐related autophagy under nutrient limiting conditions, rather than autophagic cell death, since blockage of autophagy induced by SK1 and S1P killed the cells by apoptosis rather than rescuing them from death. In line with this, nutrient starvation in mammalian or yeast cells stimulated endogenous SK1 activity (Lanterman and Saba, 1998; Lavieu et al., 2006) and starvation‐induced autophagy was blocked by a SK1 inhibitor or a dominant negative SK1 expression (Lavieu et al., 2006). At the molecular level, there are diVerences between ceramide and S1P‐induced autophagy. In contrast with ceramide, SK1 did not lead to the inactivation of the Akt/PKB prosurvival pathway, but while blocked the mTor pathway by an independent mechanism. Also, upregulation of Beclin‐1 expression by SK1 occurred to a lesser extent compared to that observed after ceramide treatment (Lavieu et al., 2006). All these studies implicate the sphingolipids ceramide and S1P and their upstream modulators in autophagy signaling. The potential outcome of the opposing eVects of these sphingolipids will be discussed in detail in the following sections.

I. Role of Mitochondria and the Bcl‐2 Family Proteins in Autophagy Mitochondria are one of the most prominent targets of autophagy, and several studies in yeast and mammalian cells addressed the role of mitochondrial destruction by autophagy in cell survival and death. In yeast, Uth1p, an outer mitochondrial membrane protein, was necessary for mitochondrial autophagy (Kissova et al., 2004), and inactivation of its gene conferred resistance to Bax‐ or rapamycin‐induced cell death (Camougrand et al., 2003). Under nonstarvation conditions, yeast mutations causing mitochondrial dysfunction, loss of mitochondrial membrane potential, and defects in mitochondrial biogenesis were shown to trigger mitochondrial autophagy

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and cell death (Priault et al., 2005). Interestingly, despite the extensive elimination of mitochondria, the site of aerobic ATP production, intracellular ATP levels actually increased during autophagy in wild‐type cells, probably due to the shutdown of energy‐consuming processes such as protein translation. This observation may indicate that deficiencies of mitochondrial functions other than respiration and energy supply (e.g., lipid, heme, amino acid, and nucleotide synthesis) may be the critical contributors to cell death induction by mitochondrial dysfunction (Priault et al., 2005). Similar observations concerning the role of mitochondrial integrity as a signal for the activation of autophagy were also made in mammalian cells. Mutations in mitochondrial DNA or drug‐induced loss of mitochondrial membrane potential led to selective elimination of these organelles by autophagy (Elmore et al., 2001; Gu et al., 2004). According to Lemasters and colleagues, elimination of damaged mitochondria by autophagy could in fact block cell death by limiting the release of proapoptotic mitochondrial factors to the cytoplasm (Lemasters et al., 1998). Therefore, mitochondrial autophagy could act as a mechanism to protect the cells from cell death in this setting. Work by others, however, challenged this hypothesis. Amino acid starvation, which induces mitochondrial autophagy, caused death of primary hepatocytes, and the autophagy inhibitor, 3‐MA, prevented cell death (Schwarze and Seglen, 1985). Furthermore, preferential destruction of mitochondria by autophagy, accompanied by a decrease in cell size, was critical to commit cells to death in the presence of caspase inhibitors (Xue et al., 2001). The members of the Bcl‐2 family are critical regulators of the mitochondrial‐based intrinsic apoptotic pathway (Gross et al., 1999). Accumulating data indicate that Bcl‐2 family members may also be important for the regulation of autophagy and autophagic cell death. Section IV.A discussed the role that Bcl‐2 plays in regulating autophagy through direct interaction with key autophagy proteins, such as Beclin‐1 in the ER. Mitochondrial‐ localized Bcl‐2 family members also contribute to the regulation of autophagy. First, overexpression of the antideath members, Bcl‐2 or Bcl‐XL, protected cells from autophagic cell death (Cardenas‐Aguayo Mdel et al., 2003; Saeki et al., 2000; Vande Velde et al., 2000; Xue et al., 2001; Yanagisawa et al., 2003), while overexpression of death‐inducing Bcl‐2 family members containing BH3 domains, such as BNIP3 or Bax, caused autophagic cell death (Camougrand et al., 2003; Vande Velde et al., 2000). Autophagic cell death induced by BNIP3 overexpression was dependent on mitochondrial permeability transition pore opening and membrane potential loss, and inhibitors of pore opening blocked cell death (Vande Velde et al., 2000). Nevertheless, cytochrome c release and AIF nuclear translocation was not observed on BNIP3 overexpression, indicating that autophagic cell death does not depend on these factors. Additionally, upregulation of BNIP3 expression and BNIP3 activation

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by dimerization accompanied ceramide and arsenic trioxide induced autophagic cell death (Daido et al., 2004; Kanzawa et al., 2005). In another study, a dominant negative BNIP3 was able to block ischemia/reperfusion‐induced cell death of cardiac myocytes, but inhibition of BNIP3‐induced autophagy in this system accelerated cell death with apoptotic characteristics (Hamacher‐Brady et al., 2006). Overexpression of another pro‐death Bcl‐2 family member, Bax, in yeast, caused autophagic cell death (Camougrand et al., 2003). In this system, Bax led to the accumulation of reactive oxygen species, lipid oxidation and plasma membrane changes. Cell death was dependent on the Uht1 protein. Cytochrome c release was observed in this system but was not essential for cell death (Priault et al., 1999). Overexpression of HSpin1, a transmembrane protein containing a BH3 (Bcl‐2 homology)‐like domain that localizes to mitochondria, also caused a nonapoptotic cell death with characteristics reminiscent of autophagic cell death (Yanagisawa et al., 2003). All these data indicate that, as in the case of apoptosis, prosurvival Bcl‐2 members seem to inhibit autophagy and block autophagic cell death, while, prodeath Bcl‐2 members have the opposite eVects.

J. RIP Protein‐Related Pathways and Autophagic Cell Death Recent studies revealed the importance of death pathways involving the death‐receptor associated kinase, RIP protein, in autophagy signaling. In fibroblast L929 cells, U937 monocytes, RAW 264.7 macrophages, and primary mouse peritoneal macrophages, the pancaspase inhibitor zVAD‐ induced cell death with autophagic characteristics and this event was blocked by RNAi‐mediated knockdown of autophagy‐related proteins Beclin‐1 and Atg7 (Yu et al., 2004). zVAD’s eVects were attributed to the lack of proteolytic cleavage of RIP by caspase‐8, which triggered a novel autophagic cell death pathway, involving MKK7, JNK, and c‐Jun. Of note, de novo protein synthesis was required for autophagic cell death in this system, since the protein synthesis inhibitor cycloheximide prevented zVAD‐induced cell death. In a second study, the same group further expanded these results by showing that zVAD‐activated and RIP‐dependent cell death in L929 cells resulted in selective degradation of the antioxidant enzyme catalase through autophagy, which killed the cells by causing the accumulation of ROS (Yu et al., 2006). An independent group studied zVAD‐induced death in RAW 264.7 macrophages and primary mouse peritoneal macrophages, in the presence of lipopolysaccharides (LPS) (Xu et al., 2006). Under these conditions, the cells died by autophagic cell death which could be inhibited by the chemical inhibitors of autophagy or RNAi‐mediated knockdown of Beclin‐1. In line

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with the observations by Yue et al., Xue and colleagues observed that autophagic cell death depended on the accumulation of ROS. Interestingly, PARP was also involved in cell death and was activated downstream of ROS production. Autophagic cell death activated by the combination of zVAD and LPS was attenuated in Toll‐Like Receptor 4 adaptor (TRIF) knockout cells. Therefore, the authors proposed that the combination of LPS‐ stimulated Toll Receptor/TRIF and zVAD mediated stabilization of RIP contributed to the activation of a RIP‐dependent autophagic death pathway in this system.

V. Autophagy–Apoptosis Crosstalks Despite the fact that apoptosis and autophagy proceed through independent mechanisms, several lines of evidence point out the existence of crosstalk between the two pathways. This concept stems from several independent observations. One basic observation is that the cellular response to the same stimuli may manifest itself predominantly by autophagic or apoptotic characteristics depending on cellular context or experimental setting. Furthermore, in some cases, apoptotic and autophagic morphologies may coexist in the same cells, and the cell death may show mixed characteristics even at the molecular level. On the other hand, apoptosis and autophagy may depend on each other in some systems, so that blockage of one may aVect the progress of the other. In other scenarios, one mechanism may counteract the other, or they may manifest themselves in a mutually exclusive manner. Consequently, inhibition of one PCD type may lead to enhancement or inhibition of the other type. This section will summarize the emerging data regarding the molecular basis of the connection between apoptosis and autophagic cell death. In some systems, the very same signals or molecules that trigger apoptosis were shown to stimulate autophagy. Etoposide (Shimizu et al., 2004), staurosporine (Shimizu et al., 2004), interferon‐ (Inbal et al., 2002), TRAIL (Mills et al., 2004; Thorburn et al., 2005), FADD (Thorburn et al., 2005), ceramide (Scarlatti et al., 2004), NGF withdrawal (Xue et al., 1999), Ras (Kanzawa et al., 2003; Pattingre et al., 2003a), and DAPk (Inbal et al., 2002) were all shown to induce cell death with apoptotic characteristics in some systems, while in others they were capable of killing the cells by autophagic cell death. According to one possibility, the same upstream signaling pathways may impinge on both apoptotic and autophagic directions, and the cellular context, for example tissue specificity or diVerent experimental settings, may determine whether one pathway may predominate over the other during cell death induction. For example, ectopically

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expressed DAPk leads to apoptotic cell death in fibroblasts, and to autophagic cell death in 293T or HeLa cells (Inbal et al., 2002; Jang et al., 2002; Raveh et al., 2001). In another example it has been reported that while genotoxic stress activates in wild‐type fibroblasts the canonical p53 pathway which leads to caspase dependent apoptosis, in the Bax/Bak‐deficient fibroblasts autophagic cell death dominated (Shimizu et al., 2004). In line with this, blockage of apoptosis by the inhibition of caspases was able to switch cell death from apoptosis to autophagic cell death in several systems. On the other hand, knockdown of key autophagy genes in some systems accelerates nutrient deprivation‐induced apoptotic cell death (Boya et al., 2005). Apoptosis and autophagy may also be activated simultaneously. Gene expression profiling studies of steroid‐triggered development in Drosophila revealed that several apoptosis‐related genes are upregulated together with autophagy‐related genes (Gorski et al., 2003; Lee et al., 2003). The concept of mixed type of cell death is also a central issue to be studied from a clinical point of view, since several disorders such as neurodegenerative diseases or myocardial infarction seem to involve cell death with mixed morphologies, although the contribution of individual pathways to cell death is still a matter of controversy in this field. In our hands, certain cellular stresses activated caspases (typical sign of apoptosis) and autophagy in the same cells, and both phenomena contributed to cellular demise, indicating cooperation between the two pathways to ensure irreversible elimination of damaged cells (D. Gozuacik and A. Kimchi, unpublished results). Alternative scenarios also exist. In neurons, apoptosis may be preceded by autophagy, and full manifestation of apoptosis may depend on prior autophagy activation, since autophagy inhibitors like 3‐MA was able to delay cytochrome c release and caspase activation (Xue et al., 1999). Another example of autophagy‐dependent apoptosis is the TNF‐‐induced apoptosis of T‐lymphoblastic leukemia cell lines (Jia et al., 1997). In some systems, autophagy may also depend on apoptosis, since caspase activation was shown to be necessary for ecdysone‐induced autophagic cell death in the Drosophila salivary gland (Martin and Baehrecke, 2004). In other cellular settings, autophagy may antagonize apoptosis. For example, in sulindac sulfide (a nonsteroidal anti‐inflammatory drug) treated HT‐29 colon carcinoma cells, inhibition of autophagy increased the sensitivity of the cells to apoptotic signals, which manifested itself by faster cytochrome c release (Bauvy et al., 2001). Another example of this antagonism is nutrient or growth factor deprivation‐induced cell death. Treatment of deprived cells with autophagy inhibitors or blockage of autophagy by gene knockdown accelerated apoptotic cell death (Boya et al., 2005). All the scenarios presented above underline the complexity of the apoptosis/autophagy interconnection. Presumably, there must be molecular

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regulators that control the switch between the two pathways. Any of the proteins that have been shown to regulate both apoptosis and autophagy are possible candidates, such as the Bcl‐2 family members, DAPk, and FADD. In fact, Atg5 was shown to interact with the proapoptotic protein FADD and overexpression of Atg5‐induced apoptosis through FADD (Pyo et al., 2005). The list will probably continue to grow in the future.

VI. Survival Versus Death Aspects of Autophagy Intense scientific debate revolved around the question of whether the autophagic activity observed in dying cells plays a causal role in cellular demise. The initial skepticism stemmed from the well‐established catabolic role of autophagy. Even under normal growth conditions, cells use autophagy as the major pathway for the degradation and recycling of long‐lived proteins, some ubiquitinated proteins and organelles like mitochondria (Komatsu et al., 2005; Kuma et al., 2004). Stress condition imposed on cultured cells or in the context of the entire organism, such as nutrient and/or growth factor deprivation, strongly upregulate autophagy to allow sustained viability under these unfavorable circumstances (Levine and Yuan, 2005). Examples of the prosurvival role of autophagy include yeast (Tsukada and Ohsumi, 1993) and plant (Doelling et al., 2002; Hanaoka et al., 2002; Liu et al., 2005) autophagy under nutrient deprivation, C. elegans autophagy during dauer diapause (Melendez et al., 2003), autophagy observed in human carcinoma cells during nutrient starvation (Boya et al., 2005), autophagy in Bax/Bak knockout hematopoietic cells under growth factor deprivation (Lum et al., 2005), and autophagy observed in postnatal mice during the starvation period that exists between the interruption of placental blood supply and the beginning of nursing (Kuma et al., 2004). Deletion or knockdown of key autophagic genes during nutrient and growth factor deprivation led to increased cell death by apoptosis, rather than protecting from cell death, indicating that, under these circumstances, autophagy functions as a protective mechanism. In light of this substantial body of data, how can it be that the same cellular mechanism is involved in both survival and death of cells? Although survival‐related autophagy and autophagic cell death may share the same morphological features and the same basic molecular machinery of autophagic vesicle formation, several lines of evidence indicate diVerences at molecular level. For example, zVAD‐induced autophagic cell death in L929 mouse fibroblastic cells involved the selective autophagic degradation of antioxidant protein catalase, but such elimination of catalase was not observed in starvation‐induced autophagy in these same cells (Yu et al., 2006). Furthermore, although both ceramide and nutrient starvation

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activate autophagy via sphingolipid signaling and subsequent activation of mTor, only the former, which leads to cell death, involved inhibition of the prosurvival PKB/Akt pathway, and upregulation of Beclin‐1 and BNIP3 (Daido et al., 2004). The critical factor determining the final cellular outcome of death or survival may be the relative levels between the two sphingolipids SK1 and S1P, with accumulation of S1P favoring survival. This hypothesis has been termed the ‘‘S1P rheostat’’ (Cuvillier et al., 1996). In line with these observations, Levine and coworkers proposed a model of cell survival versus death, regulated by the balance between Beclin‐1 and Bcl‐2 proteins (Pattingre et al., 2005). According to this model, when cellular Bcl‐2 levels are high, Bcl‐2 binds to and inactivates Beclin‐1, thereby inhibiting autophagy. Imbalances in the Beclin‐1/Bcl‐2 ratios due to modest increases in Beclin‐1 may lead to controlled catabolic and prosurvival autophagy activation. When Beclin‐1 levels are induced above a certain threshold and no longer bound by Bcl‐2, however, extensive autophagy and autophagic cell death will result. In support of this hypothesis, overexpression of Beclin‐1 mutants unable to bind Bcl‐2 caused autophagic cell death (Pattingre et al., 2005). All data presented above underline the importance of the cellular context created by the combinatory activation/inactivation of prosurvival or death pathways, intracellular levels of key regulatory molecules, and the regulation of selective degradation in converting autophagy from a protective mechanism to a killing machine. Therefore, although the morphological characteristics of autophagy may seem similar in both cases, survival‐related autophagy and autophagic cell death mechanisms diVer at the molecular level.

VII. Conclusions The field of autophagy has greatly benefited from increasing scientific interest in the last decade, and this fact is documented by the dramatic increase in autophagy‐related publications in recent years. Orthologues of the yeast autophagy genes begin to be characterized, and several model organisms lacking key autophagic genes or carrying autophagy markers are now available and under analysis. Signaling pathways regulating autophagy and autophagic cell death are beginning to be characterized. Advances in this field will not only broaden our knowledge about a fundamental process regulating cell survival and death, but it may also help to better understand certain human diseases and to characterize new drug targets, since autophagy was shown to play a role in several pathological conditions in various organisms from plants to human beings.

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Acknowledgments We thank Shani Bialik for reading this chapter. This work was supported by the European Union (LSHB‐CT‐2004–511983) and the Center of Excellence grant from Flight Attendant Medical Research Institute (FAMRI). A.K. is the incumbent of Helena Rubinstein Chair of Cancer Research.

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Index A AC100, 149, 152–153 Acellular extrinsic fiber cementum (AEFC), 65 Activating transcription factor 4 (ATF4), 92 Adenomatous polyposis coli (APC) gene, 8 ADHR. See Autosomal dominant hypophosphatemic rickets Adult stem cells, 73 Advanced sleep phase syndrome (ASPS), 185–186 AEFC. See Acellular extrinsic fiber cementum AFC. See Alveolar fluid clearance AFM. See Atomic force microscopy Agrin, 3 Airway surface liquid (ASL), 39 Akt/PKB kinase, 226–227, 229 Aldosterone eVects, 37 Aldosterone-sensitive distal nephron (ASDN), 34 Alendronate, 128 Alveolar fluid clearance (AFC), 31–32 Ameloblastin, 53, 57 Amelogenin, 52–53 Amelotin, 53, 57 Amino acid starvation, 229 AMP-activated kinase (AMPK), 226–227 AMPK. See AMP-activated kinase Antiestrogen-induced cell death, 220 APC. See Adenomatous polyposis coli AP-1 gene, 76 APG genes, 222 Apoptosis. See also Programmed cell death morphologies crosstalk and autophagy, 234–236 Aprotinin, 32 ASARM motif, 91 ASDN. See Aldosterone-sensitive distal nephron ASGP2/ErbB2 complex, 11 ASGP1 protein, 10–11 ASGP2 protein, 10–11

ASL. See Airway surface liquid ASPS. See Advanced sleep phase syndrome ATF4. See Activating transcription factor 4 ATG genes, 222 Atg kinases, 221, 223–224, 228 Atg14 mammalian homologue, 223 Atg6/Vps34 complex, 223 Atomic force microscopy (AFM), 52 AUT genes, 222 Autophagic cell death, 219–222 regulatory mechanism of beclin-1, in autophagic vesicle formation, 223–224 class III phosphatidylinositol 3-kinase, in autophagic vesicle formation, 223–224 class I phosphatidylinositol 3-kinase, in autophagy, 226–227 DAPk family in, 229–230 G proteins in, 229 protein transcription/translation-related pathways in, 227–229 RIP protein-related pathways in, 233–234 sphingolipid pathways in, 230–234 tor pathway in, 225–226 ubiquitin-like pathways, in autophagic vesicle formation, 224–225 Autophagy, 217. See also Autophagic cell death apoptosis crosstalks, 234–236 inhibitors of, 220 LY294002 inhibition in, 220 survival vs death aspects of, 236–237 Autosomal dominant hypophosphatemic rickets (ADHR), 90 B Bax/Bak knockout cells, 226 Bax/Bak knockout fibroblasts, 221 Bax/Bak knockout hematopoietic cells, 236

247

248 BAY 39–9437, 25 Bcl-2, 220 Bcl-2/Beclin-1 interaction, 224 Bcl-2 family, 232 BD. See Bipolar disorder BDNF. See Brain derived neurotrophic factor Beclin-1 protein, 221, 223–224, 228, 231–233, 237 Betaig-h3, 98–99 Biglycan, 53, 57 Bipolar disorder (BD), 189 Bisphosphonates, 128 -blockers, 152 BMAL1 gene, 192–194 BMAL1 protein, 176–177 BMP. See Bone morphogenetic proteins BMP–Msx signaling pathway molecules, 78 BMP/SMAD signaling pathway, 136–137 BMSC. See Bone marrow stromal cells BNIP3 genes, 220, 232–233 Bone deficit conditions BMP pathway and bone anabolic therapies agents directly aVecting, 137–149 agents indirectly aVecting, 149–153 BMP/SMAD signaling pathway, 136–137 bone metabolism anabolic growth factors, 132 bone morphogenetic proteins, 135 fibroblast growth factors, 133–134 growth hormone/insulin-like growth factors, 134–135 platelet-derived growth factors, 132 transforming growth factor , 133 vascular endothelial growth factors, 134 Bone marrow stromal cells (BMSC), 71, 73 Bone metabolism. See Bone deficit conditions Bone morphogenetic proteins (BMP), 48, 76, 135 Bono 1, 99 Bortezomibw, 144 Brain derived neurotrophic factor (BDNF), 99 C C. elegans, 220, 236 Calcilytics, 149, 151–152 Calcitonins, 128 CAP. See Channel-activating peptidase

Index CAP1. See Channel-activating peptidase 1 CAP3/MT-SP1 matriptase, 34, 38–39 CAP1/prostasin, 24, 32 CAP2/TMPRSS4 matriptase, 24, 30 Caspases, 218–220 -Catenin, 7–10 CD146, 73 Cdc42 GTP-binding protein, 15 CEJ. See Cementum enamel junction Cellular intrinsic fiber cementum (CIFC), 65 Cementoblasts, 69 Cementum enamel junction (CEJ) region, 81 Cementum-protein 23 (CP-23), 70, 98 Ceramide synthase, 230 CF. See Cystic fibrosis CFTR. See Cystic fibrosis transmembrane conductance regulator Channel-activating peptidase (CAP), 24 Channel-activating peptidase 1 (CAP1), 25 CHO cell lines, 221 Chronotherapy, of cancer, 200–202 Chrysalin, 149–151 CIFC. See Cellular intrinsic fiber cementum c-Jun protein, 233 ‘‘Classical’’ mesenchymal hypothesis, 66–67 Class I PI3,4,5-triphosphate, 227 Clock gene, 190–192, 199 CLOCK protein, 176–177 CLS. See CoYn-Lowry Syndrome c-myc gene, 8 CNC. See Cranial neural crest CoYn-Lowry Syndrome (CLS), 92 Connective tissue growth factor (36–38 kDa), 99–100 CP-23. See Cementum-protein 23 Cranial neural crest (CNC) cells, 64, 66 Cryptochrome genes, 176 CRYPTOCHROMEs, 197 CTCK. See C-terminal cysteine-knot C-terminal cysteine-knot (CTCK) domain, 3 C-terminal glycine residue, 224 C-terminal peptide anchor, 30 CVT genes, 222 cyclin-D1 gene, 8 Cycloheximide, 228 Cysteine proteases, 219 Cystic fibrosis (CF), 24 Cystic fibrosis transmembrane conductance regulator (CFTR), 40 Cytosol, 221

249

Index D DAPk. See Death-associated protein kinase DAPk family, 229–230, 234, 236 Death-associated protein kinase (DAPk), 220 Decorin, 53 DEJ. See Dentin-enamel junction Delayed sleep phase syndrome (DSPS), 186 Dental pulp stem cells, 73–76 Dentin-enamel junction (DEJ), 51–52 Dentin sialoprotein (DSP), 65 2-Deoxyglucose, 5 Developmental cell death, 219 Dictyostelium, 220 1, 25 dihydroxy vitamin D3, 132 Distal-less homeobox (Dlx), 86 Dlx. See Distal-less homeobox Dlx-2 expression, 67 Dlx genes, 93 Doxorubicin, 5 Drosophila, 220, 228, 235 DRP-1/DAPk2 homologue, 220, 229 DSP. See Dentin sialoprotein DSPS. See Delayed sleep phase syndrome E 4E-BP1 kinase, 228 phosphorylation of, 225 E-cadherin, 8 Ectodysplasin, 100 eEF-2 kinases, 225 EGFR. See ErbB1/epidermal growth factor receptor eIF2 phosphorylation, 227 Emdogainw, 69 EMP. See Enamel matrix proteins Enamel matrix proteins (EMP), 53–57, 68–69 Enamel structure, of tooth. See Tooth crown, morphogenesis and development of Endoplasmic reticulum, 219 Enterokinase, 3 Epithelial cells rests, of Malassez (ERM), 64, 78, 80 Epithelial-mesenchymal (E-M) signaling, in crown formation, 49 Epithelial Naþ channel, regulation by peptidases in vitro evidences inhibition of Naþ transportation, 25–26

role of candidate physiological inhibitors, 30 role of CAPs in mammalian epithelia, 28–30 role of peptidases, 26–28 in vivo evidences CAPs as drug targets, 39–41 potential role of prostasin in regulating aldosterone production and hypertension, 37 regulation of colonic prostasin expression by aldosterone and dietary Naþ, 33–34 regulation of prostasin by aldosterone in kidney, 34–36 role of CAPs in maintaining the epidermal permeability barrier, 37–39 role of serine peptides, 31–33 Epithelial stem cells, 75 ER. See Estrogen receptor ErbB1/epidermal growth factor receptor (EGFR), 6 ErbB2/ErbB3 complex functions, 7 ErbB2/ErbB3 receptor tyrosine phosphorylation, 12 ErbB2 gene, 5 ErbB2/Her2/Neu receptor, 6 ErbB3 receptor, 6 ErbB4 receptor, 6 ErbB2 tyrosine phosphorylation, 11 ERK kinase, 226 ERM. See Epithelial cells rests of Malassez Estrogen receptor (ER), 9 Etoposide, 234

F FADD, 234, 236 Familial advanced sleep phase syndrome (FASPS), 181 FASPS. See Familial advanced sleep phase syndrome FGF–10. See Fibroblast growth factor 10 FGF–23. See Fibroblast growth factor 23 Fibroblast growth factor 10 (FGF-10), 75–76, 78 Fibroblast growth factor 23 (FGF-23), 90 Fibroblast growth factors (FGF), 76, 132–134 Flavonoids, 144–147 FYVE protein, 223

250

Index

G

J

GAG. See Glycosaminoglycans GCN2 kinase, 227 Glioma cells, 220 -Glucosylhesperidin, 145 Glycosaminoglycans (GAG), 93 G-protein–coupled receptors, 13 G proteins, 229 Growth hormone/insulin-like growth factors, 134–135 GTPase Rheb, 226

JME/CF15 airway cells, 25 JNK protein, 233

H HAP. See Hydroxyapatite HEK 293 expression, 230 HeLa cell lines, 221, 230, 235 Hepatocyte growth factor activator (HGFA) inhibitor-1B, 30 Hepatocyte growth factor (HGF), 14 Herceptin, 5 Hertwig’s epithelial root sheath (HERS) cells, 64–65, 68, 77 HGF. See Hepatocyte growth factor HGFA. See Hepatocyte growth factor activator HIF. See Hypoxia inducible factor Homeostatic functions, 217 HT-29 colon cancer cells, 227, 229, 235 Human neutrophil elastase (hNE), 41 Human prostasin, 28, 30, 35, 37 Hydroxyapatite (HAP), 51 Hyperaldosteronism, 33 Hypoxia inducible factor (HIF), 226

I Ibandronate, 128 Idiopathic infantile arterial calcification (IIAC), 89 IEE. See Inner enamel epithelium IGF-1. See Insulin-like growth factor-1 IIAC. See Idiopathic infantile arterial calcification IL-13-dependent bone marrow cells, 221 Inner enamel epithelium (IEE), 64, 77 Insect metamorphosis, 219 Insulin-like growth factor-1 (IGF-1), 79 Interferon- , 234

K Kallikrein-4 (KLK-4), 57–58 Keratinocytes, 38 KLK-4. See Kallikrein-4 L L929 cells, 233 LD. See Light:dark Liddle’s syndrome, 25, 28 Light:dark (LD) cycle, 175 Limb bud morphogenesis, 219 Lipopolysaccharides (LPS), 233 L929 mouse fibroblastic cells, 221 LPS. See Lipopolysaccharides LY294002, 220, 223 Lysosomal enzymes, 219 M 3-MA, 220, 223, 232, 235 Macroautophagy, 219–220 Major depressive disorder (MDD), 189 Mammalian circadian system circadian control on genotoxic stress chronotherapy of cancer, 200–202 circadian patterns of sensitivity, 203–204 circadian regressor model, 204–206 role of CLOCK/BMAL1 transactivation complex, 202–203 circadian proteins, as therapeutic agents, 207 circadian rhythms, 175–176 human disorders related to jet lag and shift work maladaptation syndromes, 187–188 mood disorders, 189–190 seasonal aVective disorder, 188–189 sleep disorders, 185–187 molecular organization of circadian oscillator multiple interacting loops, 176–180 peripheral clocks and circadian control of gene expression in tissues, 184–185

251

Index posttranslational regulation of components, 180–184 organization of, 175–176 pathologies and developmental defects, in mutant mice BMAL1, 192–194 circadian protein, deficiency of, 198–199 clock gene, 190–192 CRYPTOCHROMEs, 197 neuronal PAS domain protein2 (NPAS2), 194 organism-environmental interaction, 198–199 PERIODs, 195–196 REV-ERB, 197–198 role of timeless gene, 196–197 Mammalian MAP LC3 protein, 224 Mammalian Tor orthologue (mTor), 225 Matrix extracellular phosphoglycoprotein (MEPE), 90–91, 152–153 Matrix gla protein (MGP), 101 Matrix metalloproteinase-20 (MMP-20), 57–58 MCF–7 breast carcinoma cells, 220, 230–231 MDD. See Major depressive disorder Mdm2 phosphorylation, 226 MEPE. See Matrix extracellular phosphoglycoprotein Mesenchymal stem cells (MSCs), 91 Metastasis, 14 MGP. See Matrix gla protein Mitochondria, 219, 222, 231, 236 Mitogen-activated protein kinase (MAPK), 181 MKK7 protein, 233 MMP-20. See Matrix metalloproteinase-20 Mood disorders, 189–190 Msx gene, 76 mTor. See mammalian Tor orthologue MUC1, role in tumor cell growth signaling by ErbB RTKs, 6–7 growth regulatory mechanisms of, 9 interaction with -catenin, 7–9 interaction with EGFR, 7 mouse models, 9–10 MUC4, role in tumor cell growth signaling ErbB2 localizations, 11–13 and metastasis, 13–14 MUC5AC protein, 3 MUC5B protein, 3 MUC4/ErbB2 complex, 11

MUC20 gene, 14–15 Mucins contributions to tumor cell growth signaling, See MUC1, role in tumor cell growth signaling; MUC4, role in tumor cell growth signaling inhibition of signaling by, 14–15 membrane of, and tumor progression, 4–5 perspectives, 15–16 structure of, 2–3 MUC6 protein, 3 MUC19 protein, 3 N N408 allele, 187 Necrosis, 218 Nedd4 kinase, 28 Neuregulin-1 (NRG1), 9 Neuronal PAS domain protein 2 (NPAS2), 194 NGF, 234 N-linked glycans, 2 N-linked sugars, 26 Non-24-hour sleep-wake syndrome (N-24), 186–187 NPAS2. See Neuronal PAS domain protein2 NPAS2 protein, 178 NRG1. See Neuregulin-1 O Odontogenesis, 63 OEE. See Outer enamel epithelium O-glycosylation, 2 OHO. See Oncogenic hypophosphatemic osteomalacia Oncogenic hypophosphatemic osteomalacia (OHO), 152–153 OPG. See Osteoprotegerin Organelle turnover, 217 Orthologues, of yeast genes, 220 Osteoblasts, 69–72 factors of, 73–75 Osteocrin, 100 Osteoprogenitors, 73–75 Osteoprotegerin (OPG), 92–93 Osterix (Osx), 91 Osx. See Osterix Outer enamel epithelium (OEE), 64, 67, 79

252 Overexpressing (O/E) ameloblastin, 57 Oxidative stress, 222 P Palatal closure, in mammals, 219 Parathyroid hormone (PTH), 132, 149 p19ARF tumor suppressor (smARF), 221 PAS. See Preautophagosomal structures Pax-9 gene, 76 PCL. See Periciliary liquid layer PDGF. See Platelet-derived growth factor PDL. See Periodontal ligament PE. See Phosphatidylethanolamine Periciliary liquid layer (PCL), 39 Period gene, 176, 195–196 Periodontal development and regeneration. See Tooth crown, morphogenesis and development of Periodontal ligament (PDL), 49, 63 stem cells of, 74–75 Periostin, 86–87 PERK protein, 227 PER1 protein, 181 PG. See Proteoglycans PH. See Pleckstrin homology PHEX mutation, 90 Phosphate metabolism, factors of, 72, 87–91 Phosphatidylethanolamine (PE), 224 Phosphatidylinositol 30 -kinase (PI3K), 6 Phosphatidylinositol 3-phosphate (PI3-P), 223 PI3K. See Phosphatidylinositol 30 -kinase PI3-P. See Phosphatidylinositol 3-phosphate PI3-P-binding proteins, 223 PI8 serpin, 30 PKR kinase, 227 Platelet-derived growth factor (PDGF), 129, 132 Pleckstrin homology (PH) domains, 226 Polyoma virus middle T antigen (PyMT), 9 Postnatal PDL stem cells (PDLSC), 74 PPH21/22 proteins, 225 Preautophagosomal structures (PAS), 223 Programmed cell death morphologies, 218–219. See also Autophagic cell death Prostaglandins, 148 Proteases, 57–58 Proteasome inhibitors, 142–144 Protein degradation, 217 Protein kinase G-II (PKG-II), 182

Index Proteoglycans (PG), 93 p70S6K phosphorylation, 225, 228 PTEN, 229 PTEN genes, 227 PTH. See Parathyroid hormone p53 ubiquitination, 226 PX (Phox homology), 223 PyMT-induced mammary tumorigenesis, 9 Pyrophosphate metabolism, factors of, 72, 87–91 R Raloxifene, 128 RANKL. See Receptor activator of NF-B ligand Rapamycin, 225 Raptor/mTor complex, 225 Ras-induced death, of gastric/glioma cells, 220 Ras/Raf1/ERK1/2 pathways, 229 Rat MUC4, 10 RAW 264.7 macrophages, 233 Receptor activator of NF-B ligand (RANKL), 79–80, 92–93 Receptor tyrosine kinases (RTK), 5 REDD1, 226 REDD2, 226 REV-ERB  genes, 197–198 REV-ERB protein, 189 Rhodamine 123, 5 Rictor/mTor complex, 225 Risedronate, 128 RNAi-mediated knockdown, 221 Root/periodontal tissue (R/PT), development of. See Tooth crown, morphogenesis and development of Root phenotypes, factors of, 68–70 ROS inhibitors, 222 RSK1 kinase, 226 RTK. See Receptor tyrosine kinases Runt-related transcription factor 2 (Runx2), 91–92 RUNX 2 gene, 76 S Saccharomyces cerevisiae, 15 SAD. See Seasonal active disorder SCID mouse model, 72 Sclerostin, 132, 147–148

253

Index SCN. See Suprachiasmatic nucleus SCNN1B gene, 40 SCNN1G gene, 40 SDS-PAGE gels, 224–225 SEA. See Sea urchin sperm protein Seasonal active disorder (SAD), 188–189 Sea urchin sperm protein (SEA), 3 Selective estrogen receptor modulators (SERM), 128 Serine palmitoyl transferase, 230 Serine peptidases, role of, 31, 33 SERM. See Selective estrogen receptor modulators Ser/Thr kinases, 7–8, 10, 15 SHH. See Sonic hedgehog SIBLING extracellular matrix protein family, 90, 94–98 SLRP. See Small leucine rich proteoglycans Small integrin-binding ligand N-linked glycoprotein family. See SIBLING extracellular matrix protein family Small leucine rich proteoglycans (SLRP), 93 SMase. See Sphingomyelinase enzymes Sonic hedgehog (SHH), 76, 79 Soybean trypsin, 33 S1P. See Sphingosine 1-phosphate Sphingolipid metabolites ceramide, 230 Sphingolipids, 230–233 Sphingomyelinase enzymes (SMase), 230 Sphingosine, 230 Sphingosine 1-phosphate (S1P), 230, 237 SPINT1, 30 S1P rheostat, 237 S6 protein, 225 Statins, 137–140 clinical findings, 141–142 mechanism of actions, 140–141 Staurosporine, 234 STRO-1; 81 Strontium ranelate (PROTELOS), 150 Suprachiasmatic nucleus (SCN), 175 T TaVazin (TAZ), 91–92 TAP42, 225 Taxol, 5 TAZ. See TaVazin T84 colon cancer cells, 34 TDO. See Tricho-dento-osseous Teriparatidew, 149

TGF. See Transforming growth factor Timeless gene, 196–197 Tissue nonspecific alkaline phosphatase (TNAP), 87 TMPRSS3 mutations, 30 TNAP. See Tissue nonspecific alkaline phosphatase TNF--induced death, of leukemia cells, 220 Toll-like receptor 4 adaptor (TRIF) knockout cells, 234 Tooth crown, morphogenesis and development of cells in periodontal development and regeneration cementoblasts vs osteoblasts, 69–72 developmental cells, 63–65 role of cementoblast, 65–69 tooth stem cell populations, 72–76 genes in periodontal tissue formation bone morphogenetic proteins, 81–86 factors responsible for regulation of osteoprogenitor cells and osteoblasts, 91–93 HERS and ERM, 77–81 other factors, 93–101 role of nuclear factor I-C/CAAT box transcription factor, 86–87 role of periostin, 86–87 role of phosphate metabolism, 87–91 role of pyrophosphate metabolism, 87–91 replication of enamel enamel biomineralization, 52–58 enamel regeneration, 58–63 enamel structure, 51–52 TRAIL, 234 Transforming growth factor (TGF)- 1, 41, 133 Tricho-dento-osseous (TDO) syndrome, 93 TSC1/2 complex, 226 TSC1 protein, 226 Tuftelin, 57 Tumor necrosis factor ligand ectodysplasin, 100 Type I cell death, 218 Type II cell death, 219, 230 Type III cell death, 219 U U937 monocytes, 221, 233 US Food and Drug Administration, 144 Uth1p protein, 231–232

254

Index

V

X

Variable number tandem repetition (VNTR) polymorphisms, 2 Vascular endothelial growth factor (VEGF), 132, 134 VEGF. See Vascular endothelial growth factor Velcadew, 144 Vinblastine, 5 VNTR. See Variable number tandem repetition von Willebrand factor C (VWC), 3 Vps34, 224 Vps15/p150 kinase, 223

Xenopus CAP1, 30 Xenopus kidney cells, 24–25 X-linked hypophosphatemic rickets (XLH/HYP), 89–90

W Wnt1-Cre, R26R, 66 Wnt signaling pathway, 7 Wortmannin, 221, 223

Y Yeast autophagic genes, 218 Z ZIPk/DAPk3 homologue, 229 zVAD, 221 induced cell death, 222, 233, 236

Contents of Previous Volumes Volume 47 1 Early Events of Somitogenesis in Higher Vertebrates: Allocation of Precursor Cells during Gastrulation and the Organization of a Moristic Pattern in the Paraxial Mesoderm Patrick P. L. Tam, Devorah Goldman, Anne Camus, and Gary C. Shoenwolf

2 Retrospective Tracing of the Developmental Lineage of the Mouse Myotome Sophie Eloy-Trinquet, Luc Mathis, and Jean-Franc¸ois Nicolas

3 Segmentation of the Paraxial Mesoderm and Vertebrate Somitogenesis Olivier Pourqule´

4 Segmentation: A View from the Border Claudio D. Stern and Daniel Vasiliauskas

5 Genetic Regulation of Somite Formation Alan Rawls, Jeanne Wilson-Rawls, and Eric N. Olsen

6 Hox Genes and the Global Patterning of the Somitic Mesoderm Ann Campbell Burke

7 The Origin and Morphogenesis of Amphibian Somites Ray Keller

8 Somitogenesis in Zebrafish Scott A. Halley and Christiana Nu¨sslain-Volhard

9 Rostrocaudal Differences within the Somites Confer Segmental Pattern to Trunk Neural Crest Migration Marianne Bronner-Fraser

255

256

Contents of Previous Volumes

Volume 48 1 Evolution and Development of Distinct Cell Lineages Derived from Somites Beate Brand-Saberi and Bodo Christ

2 Duality of Molecular Signaling Involved in Vertebral Chondrogenesis Anne-He´le`ne Monsoro-Burq and Nicole Le Douarin

3 Sclerotome Induction and Differentiation Jennifer L. Docker

4 Genetics of Muscle Determination and Development Hans-Henning Arnold and Thomas Braun

5 Multiple Tissue Interactions and Signal Transduction Pathways Control Somite Myogenesis Anne-Gae¨lle Borycki and Charles P. Emerson, Jr.

6 The Birth of Muscle Progenitor Cells in the Mouse: Spatiotemporal Considerations Shahragim Tajbakhsh and Margaret Buckingham

7 Mouse–Chick Chimera: An Experimental System for Study of Somite Development Josiane Fontaine-Pe´rus

8 Transcriptional Regulation during Somitogenesis Dennis Summerbell and Peter W. J. Rigby

9 Determination and Morphogenesis in Myogenic Progenitor Cells: An Experimental Embryological Approach Charles P. Ordahl, Brian A. Williams, and Wilfred Denetclaw

Volume 49 1 The Centrosome and Parthenogenesis Thomas Ku¨ntziger and Michel Bornens

2 g-Tubulin Berl R. Oakley

Contents of Previous Volumes

257

3 g-Tubulin Complexes and Their Role in Microtubule Nucleation Ruwanthi N. Gunawardane, Sofia B. Lizarraga, Christiane Wiese, Andrew Wilde, and Yixian Zheng

4 g-Tubulin of Budding Yeast Jackie Vogel and Michael Snyder

5 The Spindle Pole Body of Saccharomyces cerevisiae: Architecture and Assembly of the Core Components Susan E. Francis and Trisha N. Davis

6 The Microtubule Organizing Centers of Schizosaccharomyces pombe Iain M. Hagan and Janni Petersen

7 Comparative Structural, Molecular, and Functional Aspects of the Dictyostelium discoideum Centrosome Ralph Gra¨f, Nicole Brusis, Christine Daunderer, Ursula Euteneuer, Andrea Hestermann, Manfred Schliwa, and Masahiro Ueda

8 Are There Nucleic Acids in the Centrosome? Wallace F. Marshall and Joel L. Rosenbaum

9 Basal Bodies and Centrioles: Their Function and Structure Andrea M. Preble, Thomas M. Giddings, Jr., and Susan K. Dutcher

10 Centriole Duplication and Maturation in Animal Cells B. M. H. Lange, A. J. Faragher, P. March, and K. Gull

11 Centrosome Replication in Somatic Cells: The Significance of the G1 Phase Ron Balczon

12 The Coordination of Centrosome Reproduction with Nuclear Events during the Cell Cycle Greenfield Sluder and Edward H. Hinchcliffe

13 Regulating Centrosomes by Protein Phosphorylation Andrew M. Fry, Thibault Mayor, and Erich A. Nigg

14 The Role of the Centrosome in the Development of Malignant Tumors Wilma L. Lingle and Jeffrey L. Salisbury

15 The Centrosome-Associated Aurora/IpI-like Kinase Family T. M. Goepfert and B. R. Brinkley

258

Contents of Previous Volumes

16 Centrosome Reduction during Mammalian Spermiogenesis G. Manandhar, C. Simerly, and G. Schatten

17 The Centrosome of the Early C. elegans Embryo: Inheritance, Assembly, Replication, and Developmental Roles Kevin F. O’Connell

18 The Centrosome in Drosophila Oocyte Development Timothy L. Megraw and Thomas C. Kaufman

19 The Centrosome in Early Drosophila Embryogenesis W. F. Rothwell and W. Sullivan

20 Centrosome Maturation Robert E. Palazzo, Jacalyn M. Vogel, Bradley J. Schnackenberg, Dawn R. Hull, and Xingyong Wu

Volume 50 1 Patterning the Early Sea Urchin Embryo Charles A. Ettensohn and Hyla C. Sweet

2 Turning Mesoderm into Blood: The Formation of Hematopoietic Stem Cells during Embryogenesis Alan J. Davidson and Leonard I. Zon

3 Mechanisms of Plant Embryo Development Shunong Bai, Lingjing Chen, Mary Alice Yund, and Zinmay Rence Sung

4 Sperm-Mediated Gene Transfer Anthony W. S. Chan, C. Marc Luetjens, and Gerald P. Schatten

5 Gonocyte–Sertoli Cell Interactions during Development of the Neonatal Rodent Testis Joanne M. Orth, William F. Jester, Ling-Hong Li, and Andrew L. Laslett

6 Attributes and Dynamics of the Endoplasmic Reticulum in Mammalian Eggs Douglas Kline

7 Germ Plasm and Molecular Determinants of Germ Cell Fate Douglas W. Houston and Mary Lou King

Contents of Previous Volumes

259

Volume 51 1 Patterning and Lineage Specification in the Amphibian Embryo Agnes P. Chan and Laurence D. Etkin

2 Transcriptional Programs Regulating Vascular Smooth Muscle Cell Development and Differentiation Michael S. Parmacek

3 Myofibroblasts: Molecular Crossdressers Gennyne A. Walker, Ivan A. Guerrero, and Leslie A. Leinwand

4 Checkpoint and DNA-Repair Proteins Are Associated with the Cores of Mammalian Meiotic Chromosomes Madalena Tarsounas and Peter B. Moens

5 Cytoskeletal and Ca2+ Regulation of Hyphal Tip Growth and Initiation Sara Torralba and I. Brent Heath

6 Pattern Formation during C. elegans Vulval Induction Minqin Wang and Paul W. Sternberg

7 A Molecular Clock Involved in Somite Segmentation Miguel Maroto and Olivier Pourquie´

Volume 52 1 Mechanism and Control of Meiotic Recombination Initiation Scott Keeney

2 Osmoregulation and Cell Volume Regulation in the Preimplantation Embryo Jay M. Baltz

3 Cell–Cell Interactions in Vascular Development Diane C. Darland and Patricia A. D’Amore

4 Genetic Regulation of Preimplantation Embryo Survival Carol M. Warner and Carol A. Brenner

260

Contents of Previous Volumes

Volume 53 1 Developmental Roles and Clinical Significance of Hedgehog Signaling Andrew P. McMahon, Philip W. Ingham, and Clifford J. Tabin

2 Genomic Imprinting: Could the Chromatin Structure Be the Driving Force? Andras Paldi

3 Ontogeny of Hematopoiesis: Examining the Emergence of Hematopoietic Cells in the Vertebrate Embryo Jenna L. Galloway and Leonard I. Zon

4 Patterning the Sea Urchin Embryo: Gene Regulatory Networks, Signaling Pathways, and Cellular Interactions Lynne M. Angerer and Robert C. Angerer

Volume 54 1 Membrane Type-Matrix Metalloproteinases (MT-MMP) Stanley Zucker, Duanqing Pei, Jian Cao, and Carlos Lopez-Otin

2 Surface Association of Secreted Matrix Metalloproteinases Rafael Fridman

3 Biochemical Properties and Functions of Membrane-Anchored Metalloprotease-Disintegrin Proteins (ADAMs) J. David Becherer and Carl P. Blobel

4 Shedding of Plasma Membrane Proteins Joaquı´n Arribas and Anna Merlos-Sua´rez

5 Expression of Meprins in Health and Disease Lourdes P. Norman, Gail L. Matters, Jacqueline M. Crisman, and Judith S. Bond

6 Type II Transmembrane Serine Proteases Qingyu Wu

7 DPPIV, Seprase, and Related Serine Peptidases in Multiple Cellular Functions Wen-Tien Chen, Thomas Kelly, and Giulio Ghersi

Contents of Previous Volumes

261

8 The Secretases of Alzheimer’s Disease Michael S. Wolfe

9 Plasminogen Activation at the Cell Surface Vincent Ellis

10 Cell-Surface Cathepsin B: Understanding Its Functional Significance Dora Cavallo-Medved and Bonnie F. Sloane

11 Protease-Activated Receptors Wadie F. Bahou

12 Emmprin (CD147), a Cell Surface Regulator of Matrix Metalloproteinase Production and Function Bryan P. Toole

13 The Evolving Roles of Cell Surface Proteases in Health and Disease: Implications for Developmental, Adaptive, Inflammatory, and Neoplastic Processes Joseph A. Madri

14 Shed Membrane Vesicles and Clustering of Membrane-Bound Proteolytic Enzymes M. Letizia Vittorelli

Volume 55 1 The Dynamics of Chromosome Replication in Yeast Isabelle A. Lucas and M. K. Raghuraman

2 Micromechanical Studies of Mitotic Chromosomes M. G. Poirier and John F. Marko

3 Patterning of the Zebrafish Embryo by Nodal Signals Jennifer O. Liang and Amy L. Rubinstein

4 Folding Chromosomes in Bacteria: Examining the Role of Csp Proteins and Other Small Nucleic Acid-Binding Proteins Nancy Trun and Danielle Johnston

262

Contents of Previous Volumes

Volume 56 1 Selfishness in Moderation: Evolutionary Success of the Yeast Plasmid Soundarapandian Velmurugan, Shwetal Mehta, and Makkuni Jayaram

2 Nongenomic Actions of Androgen in Sertoli Cells William H. Walker

3 Regulation of Chromatin Structure and Gene Activity by Poly(ADP-Ribose) Polymerases Alexei Tulin, Yurli Chinenov, and Allan Spradling

4 Centrosomes and Kinetochores, Who needs ‘Em? The Role of Noncentromeric Chromatin in Spindle Assembly Priya Prakash Budde and Rebecca Heald

5 Modeling Cardiogenesis: The Challenges and Promises of 3D Reconstruction Jeffrey O. Penetcost, Claudio Silva, Maurice Pesticelli, Jr., and Kent L. Thornburg

6 Plasmid and Chromosome Traffic Control: How ParA and ParB Drive Partition Jennifer A. Surtees and Barbara E. Funnell

Volume 57 1 Molecular Conservation and Novelties in Vertebrate Ear Development B. Fritzsch and K. W. Beisel

2 Use of Mouse Genetics for Studying Inner Ear Development Elizabeth Quint and Karen P. Steel

3 Formation of the Outer and Middle Ear, Molecular Mechanisms Moise´s Mallo

4 Molecular Basis of Inner Ear Induction Stephen T. Brown, Kareen Martin, and Andrew K. Groves

5 Molecular Basis of Otic Commitment and Morphogenesis: A Role for Homeodomain-Containing Transcription Factors and Signaling Molecules Eva Bober, Silke Rinkwitz, and Heike Herbrand

Contents of Previous Volumes

263

6 Growth Factors and Early Development of Otic Neurons: Interactions between Intrinsic and Extrinsic Signals Berta Alsina, Fernando Giraldez, and Isabel Varela-Nieto

7 Neurotrophic Factors during Inner Ear Development Ulla Pirvola and Jukka Ylikoski

8 FGF Signaling in Ear Development and Innervation Tracy J. Wright and Suzanne L. Mansour

9 The Roles of Retinoic Acid during Inner Ear Development Raymond Romand

10 Hair Cell Development in Higher Vertebrates Wei-Qiang Gao

11 Cell Adhesion Molecules during Inner Ear and Hair Cell Development, Including Notch and Its Ligands Matthew W. Kelley

12 Genes Controlling the Development of the Zebrafish Inner Ear and Hair Cells Bruce B. Riley

13 Functional Development of Hair Cells Ruth Anne Eatock and Karen M. Hurley

14 The Cell Cycle and the Development and Regeneration of Hair Cells Allen F. Ryan

Volume 58 1 A Role for Endogenous Electric Fields in Wound Healing Richard Nuccitelli

2 The Role of Mitotic Checkpoint in Maintaining Genomic Stability Song-Tao Liu, Jan M. van Deursen, and Tim J. Yen

3 The Regulation of Oocyte Maturation Ekaterina Voronina and Gary M. Wessel

4 Stem Cells: A Promising Source of Pancreatic Islets for Transplantation in Type 1 Diabetes Cale N. Street, Ray V. Rajotte, and Gregory S. Korbutt

264

Contents of Previous Volumes

5 Differentiation Potential of Adipose Derived Adult Stem (ADAS) Cells Jeffrey M. Gimble and Farshid Guilak

Volume 59 1 The Balbiani Body and Germ Cell Determinants: 150 Years Later Malgorzata Kloc, Szczepan Bilinski, and Laurence D. Etkin

2 Fetal–Maternal Interactions: Prenatal Psychobiological Precursors to Adaptive Infant Development Matthew F. S. X. Novak

3 Paradoxical Role of Methyl-CpG-Binding Protein 2 in Rett Syndrome Janine M. LaSalle

4 Genetic Approaches to Analyzing Mitochondrial Outer Membrane Permeability Brett H. Graham and William J. Craigen

5 Mitochondrial Dynamics in Mammals Hsiuchen Chen and David C. Chan

6 Histone Modification in Corepressor Functions Judith K. Davie and Sharon Y. R. Dent

7 Death by Abl: A Matter of Location Jiangyu Zhu and Jean Y. J. Wang

Volume 60 1 Therapeutic Cloning and Tissue Engineering Chester J. Koh and Anthony Atala

2 a-Synuclein: Normal Function and Role in Neurodegenerative Diseases Erin H. Norris, Benoit I. Giasson, and Virginia M.-Y. Lee

3 Structure and Function of Eukaryotic DNA Methyltransferases Taiping Chen and En Li

4 Mechanical Signals as Regulators of Stem Cell Fate Bradley T. Estes, Jeffrey M. Gimble, and Farshid Guilak

Contents of Previous Volumes

265

5 Origins of Mammalian Hematopoiesis: In Vivo Paradigms and In Vitro Models M. William Lensch and George Q. Daley

6 Regulation of Gene Activity and Repression: A Consideration of Unifying Themes Anne C. Ferguson-Smith, Shau-Ping Lin, and Neil Youngson

7 Molecular Basis for the Chloride Channel Activity of Cystic Fibrosis Transmembrane Conductance Regulator and the Consequences of Disease-Causing Mutations Jackie F. Kidd, Ilana Kogan, and Christine E. Bear

Volume 61 1 Hepatic Oval Cells: Helping Redefine a Paradigm in Stem Cell Biology P. N. Newsome, M. A. Hussain, and N. D. Theise

2 Meiotic DNA Replication Randy Strich

3 Pollen Tube Guidance: The Role of Adhesion and Chemotropic Molecules Sunran Kim, Juan Dong, and Elizabeth M. Lord

4 The Biology and Diagnostic Applications of Fetal DNA and RNA in Maternal Plasma Rossa W. K. Chiu and Y. M. Dennis Lo

5 Advances in Tissue Engineering Shulamit Levenberg and Robert Langer

6 Directions in Cell Migration Along the Rostral Migratory Stream: The Pathway for Migration in the Brain Shin-ichi Murase and Alan F. Horwitz

7 Retinoids in Lung Development and Regeneration Malcolm Maden

8 Structural Organization and Functions of the Nucleus in Development, Aging, and Disease Leslie Mounkes and Colin L. Stewart

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Contents of Previous Volumes

Volume 62 1 Blood Vessel Signals During Development and Beyond Ondine Cleaver

2 HIFs, Hypoxia, and Vascular Development Kelly L. Covello and M. Celeste Simon

3 Blood Vessel Patterning at the Embryonic Midline Kelly A. Hogan and Victoria L. Bautch

4 Wiring the Vascular Circuitry: From Growth Factors to Guidance Cues Lisa D. Urness and Dean Y. Li

5 Vascular Endothelial Growth Factor and Its Receptors in Embryonic Zebrafish Blood Vessel Development Katsutoshi Goishi and Michael Klagsbrun

6 Vascular Extracellular Matrix and Aortic Development Cassandra M. Kelleher, Sean E. McLean, and Robert P. Mecham

7 Genetics in Zebrafish, Mice, and Humans to Dissect Congenital Heart Disease: Insights in the Role of VEGF Diether Lambrechts and Peter Carmeliet

8 Development of Coronary Vessels Mark W. Majesky

9 Identifying Early Vascular Genes Through Gene Trapping in Mouse Embryonic Stem Cells Frank Kuhnert and Heidi Stuhlmann

Volume 63 1 Early Events in the DNA Damage Response Irene Ward and Junjie Chen

2 Afrotherian Origins and Interrelationships: New Views and Future Prospects Terence J. Robinson and Erik R. Seiffert

3 The Role of Antisense Transcription in the Regulation of X-Inactivation Claire Rougeulle and Philip Avner

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4 The Genetics of Hiding the Corpse: Engulfment and Degradation of Apoptotic Cells in C. elegans and D. melanogaster Zheng Zhou, Paolo M. Mangahas, and Xiaomeng Yu

5 Beginning and Ending an Actin Filament: Control at the Barbed End Sally H. Zigmond

6 Life Extension in the Dwarf Mouse Andrzej Bartke and Holly Brown-Borg

Volume 64 1 Stem/Progenitor Cells in Lung Morphogenesis, Repair, and Regeneration David Warburton, Mary Anne Berberich, and Barbara Driscoll

2 Lessons from a Canine Model of Compensatory Lung Growth Connie C. W. Hsia

3 Airway Glandular Development and Stem Cells Xiaoming Liu, Ryan R. Driskell, and John F. Engelhardt

4 Gene Expression Studies in Lung Development and Lung Stem Cell Biology Thomas J. Mariani and Naftali Kaminski

5 Mechanisms and Regulation of Lung Vascular Development Michelle Haynes Pauling and Thiennu H. Vu

6 The Engineering of Tissues Using Progenitor Cells Nancy L. Parenteau, Lawrence Rosenberg, and Janet Hardin-Young

7 Adult Bone Marrow-Derived Hemangioblasts, Endothelial Cell Progenitors, and EPCs Gina C. Schatteman

8 Synthetic Extracellular Matrices for Tissue Engineering and Regeneration Eduardo A. Silva and David J. Mooney

9 Integrins and Angiogenesis D. G. Stupack and D. A. Cheresh

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Volume 65 1 Tales of Cannibalism, Suicide, and Murder: Programmed Cell Death in C. elegans Jason M. Kinchen and Michael O. Hengartner

2 From Guts to Brains: Using Zebrafish Genetics to Understand the Innards of Organogenesis Carsten Stuckenholz, Paul E. Ulanch, and Nathan Bahary

3 Synaptic Vesicle Docking: A Putative Role for the Munc18/Sec1 Protein Family Robby M. Weimer and Janet E. Richmond

4 ATP-Dependent Chromatin Remodeling Corey L. Smith and Craig L. Peterson

5 Self-Destruct Programs in the Processes of Developing Neurons David Shepherd and V. Hugh Perry

6 Multiple Roles of Vascular Endothelial Growth Factor (VEGF) in Skeletal Development, Growth, and Repair Elazar Zelzer and Bjorn R. Olsen

7 G-Protein Coupled Receptors and Calcium Signaling in Development Geoffrey E. Woodard and Juan A. Rosado

8 Differential Functions of 14-3-3 Isoforms in Vertebrate Development Anthony J. Muslin and Jeffrey M. C. Lau

9 Zebrafish Notochordal Basement Membrane: Signaling and Structure Annabelle Scott and Derek L. Stemple

10 Sonic Hedgehog Signaling and the Developing Tooth Martyn T. Cobourne and Paul T. Sharpe

Volume 66 1 Stepwise Commitment from Embryonic Stem to Hematopoietic and Endothelial Cells Changwon Park, Jesse J. Lugus, and Kyunghee Choi

Contents of Previous Volumes

269

2 Fibroblast Growth Factor Signaling and the Function and Assembly of Basement Membranes Peter Lonai

3 TGF- Superfamily and Mouse Craniofacial Development: Interplay of Morphogenetic Proteins and Receptor Signaling Controls Normal Formation of the Face Marek Dudas and Vesa Kaartinen

4 The Colors of Autumn Leaves as Symptoms of Cellular Recycling and Defenses Against Environmental Stresses Helen J. Ougham, Phillip Morris, and Howard Thomas

5 Extracellular Proteases: Biological and Behavioral Roles in the Mammalian Central Nervous System Yan Zhang, Kostas Pothakos, and Styliana-Anna (Stella) Tsirka

6 The Genetic Architecture of House Fly Mating Behavior Lisa M. Meffert and Kara L. Hagenbuch

7 Phototropins, Other Photoreceptors, and Associated Signaling: The Lead and Supporting Cast in the Control of Plant Movement Responses Bethany B. Stone, C. Alex Esmon, and Emmanuel Liscum

8 Evolving Concepts in Bone Tissue Engineering Catherine M. Cowan, Chia Soo, Kang Ting, and Benjamin Wu

9 Cranial Suture Biology Kelly A Lenton, Randall P. Nacamuli, Derrick C. Wan, Jill A. Helms, and Michael T. Longaker

Volume 67 1 Deer Antlers as a Model of Mammalian Regeneration Joanna Price, Corrine Faucheux, and Steve Allen

2 The Molecular and Genetic Control of Leaf Senescence and Longevity in Arabidopsis Pyung Ok Lim and Hong Gil Nam

3 Cripto-1: An Oncofetal Gene with Many Faces Caterina Bianco, Luigi Strizzi, Nicola Normanno, Nadia Khan, and David S. Salomon

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4 Programmed Cell Death in Plant Embryogenesis Peter V. Bozhkov, Lada H. Filonova, and Maria F. Suarez

5 Physiological Roles of Aquaporins in the Choroid Plexus Daniela Boassa and Andrea J. Yool

6 Control of Food Intake Through Regulation of cAMP Allan Z. Zhao

7 Factors Affecting Male Song Evolution in Drosophila montana Anneli Hoikkala, Kirsten Klappert, and Dominique Mazzi

8 Prostanoids and Phosphodiesterase Inhibitors in Experimental Pulmonary Hypertension Ralph Theo Schermuly, Hossein Ardeschir Ghofrani, and Norbert Weissmann

9 14-3-3 Protein Signaling in Development and Growth Factor Responses Daniel Thomas, Mark Guthridge, Jo Woodcock, and Angel Lopez

10 Skeletal Stem Cells in Regenerative Medicine Wataru Sonoyama, Carolyn Coppe, Stan Gronthos, and Songtao Shi

Volume 68 1 Prolactin and Growth Hormone Signaling Beverly Chilton and Aveline Hewetson

2 Alterations in cAMP-Mediated Signaling and Their Role in the Pathophysiology of Dilated Cardiomyopathy Matthew A. Movsesian and Michael R. Bristow

3 Corpus Luteum Development: Lessons from Genetic Models in Mice Anne Bachelot and Nadine Binart

4 Comparative Developmental Biology of the Mammalian Uterus Thomas E. Spencer, Kanako Hayashi, Jianbo Hu, and Karen D. Carpenter

5 Sarcopenia of Aging and Its Metabolic Impact Helen Karakelides and K. Sreekumaran Nair

6 Chemokine Receptor CXCR3: An Unexpected Enigma Liping Liu, Melissa K. Callahan, DeRen Huang, and Richard M. Ransohoff

Contents of Previous Volumes

271

7 Assembly and Signaling of Adhesion Complexes Jorge L. Sepulveda, Vasiliki Gkretsi, and Chuanyue Wu

8 Signaling Mechanisms of Higher Plant Photoreceptors: A Structure-Function Perspective Haiyang Wang

9 Initial Failure in Myoblast Transplantation Therapy Has Led the Way Toward the Isolation of Muscle Stem Cells: Potential for Tissue Regeneration Kenneth Urish, Yasunari Kanda, and Johnny Huard

10 Role of 14-3-3 Proteins in Eukaryotic Signaling and Development Dawn L. Darling, Jessica Yingling, and Anthony Wynshaw-Boris

Volume 69 1 Flipping Coins in the Fly Retina Tamara Mikeladze-Dvali, Claude Desplan, and Daniela Pistillo

2 Unraveling the Molecular Pathways That Regulate Early Telencephalon Development Jean M. He´bert

3 Glia–Neuron Interactions in Nervous System Function and Development Shai Shaham

4 The Novel Roles of Glial Cells Revisited: The Contribution of Radial Glia and Astrocytes to Neurogenesis Tetsuji Mori, Annalisa Buffo, and Magdalena Go¨tz

5 Classical Embryological Studies and Modern Genetic Analysis of Midbrain and Cerebellum Development Mark Zervas, Sandra Blaess, and Alexandra L. Joyner

6 Brain Development and Susceptibility to Damage; Ion Levels and Movements Maria Erecinska, Shobha Cherian, and Ian A. Silver

7 Thinking about Visual Behavior; Learning about Photoreceptor Function Kwang-Min Choe and Thomas R. Clandinin

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8 Critical Period Mechanisms in Developing Visual Cortex Takao K. Hensch

9 Brawn for Brains: The Role of MEF2 Proteins in the Developing Nervous System Aryaman K. Shalizi and Azad Bonni

10 Mechanisms of Axon Guidance in the Developing Nervous System Ce´line Plachez and Linda J. Richards

Volume 70 1 Magnetic Resonance Imaging: Utility as a Molecular Imaging Modality James P. Basilion, Susan Yeon, and Rene´ Botnar

2 Magnetic Resonance Imaging Contrast Agents in the Study of Development Angelique Louie

3 1H/19F Magnetic Resonance Molecular Imaging with Perfluorocarbon Nanoparticles Gregory M. Lanza, Patrick M. Winter, Anne M. Neubauer, Shelton D. Caruthers, Franklin D. Hockett, and Samuel A. Wickline

4 Loss of Cell Ion Homeostasis and Cell Viability in the Brain: What Sodium MRI Can Tell Us Fernando E. Boada, George LaVerde, Charles Jungreis, Edwin Nemoto, Costin Tanase, and Ileana Hancu

5 Quantum Dot Surfaces for Use In Vivo and In Vitro Byron Ballou

6 In Vivo Cell Biology of Cancer Cells Visualized with Fluorescent Proteins Robert M. Hoffman

7 Modulation of Tracer Accumulation in Malignant Tumors: Gene Expression, Gene Transfer, and Phage Display Uwe Haberkorn

8 Amyloid Imaging: From Benchtop to Bedside Chungying Wu, Victor W. Pike, and Yanming Wang

9 In Vivo Imaging of Autoimmune Disease in Model Systems Eric T. Ahrens and Penelope A. Morel

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273

Volume 71 1 The Choroid Plexus-Cerebrospinal Fluid System: From Development to Aging Zoran B. Redzic, Jane E. Preston, John A. Duncan, Adam Chodobski, and Joanna Szmydynger-Chodobska

2 Zebrafish Genetics and Formation of Embryonic Vasculature Tao P. Zhong

3 Leaf Senescence: Signals, Execution, and Regulation Yongfeng Guo and Susheng Gan

4 Muscle Stem Cells and Regenerative Myogenesis Iain W. McKinnell, Gianni Parise, and Michael A. Rudnicki

5 Gene Regulation in Spermatogenesis James A. MacLean II and Miles F. Wilkinson

6 Modeling Age-Related Diseases in Drosophila: Can this Fly? Kinga Michno, Diana van de Hoef, Hong Wu, and Gabrielle L. Boulianne

7 Cell Death and Organ Development in Plants Hilary J. Rogers

8 The Blood-Testis Barrier: Its Biology, Regulation, and Physiological Role in Spermatogenesis Ching-Hang Wong and C. Yan Cheng

9 Angiogenic Factors in the Pathogenesis of Preeclampsia Hai-Tao Yuan, David Haig, and S. Ananth Karumanchi

Volume 72 1 Defending the Zygote: Search for the Ancestral Animal Block to Polyspermy Julian L. Wong and Gary M. Wessel

2 Dishevelled: A Mobile Scaffold Catalyzing Development Craig C. Malbon and Hsien-yu Wang

3 Sensory Organs: Making and Breaking the Pre-Placodal Region Andrew P. Bailey and Andrea Streit

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4 Regulation of Hepatocyte Cell Cycle Progression and Differentiation by Type I Collagen Structure Linda K. Hansen, Joshua Wilhelm, and John T. Fassett

5 Engineering Stem Cells into Organs: Topobiological Transformations Demonstrated by Beak, Feather, and Other Ectodermal Organ Morphogenesis Cheng-Ming Chuong, Ping Wu, Maksim Plikus, Ting-Xin Jiang, and Randall Bruce Widelitz

6 Fur Seal Adaptations to Lactation: Insights into Mammary Gland Function Julie A. Sharp, Kylie N. Cane, Christophe Lefevre, John P. Y. Arnould, and Kevin R. Nicholas

Volume 73 1 The Molecular Origins of Species-Specific Facial Pattern Samantha A. Brugmann, Minal D. Tapadia, and Jill A. Helms

2 Molecular Bases of the Regulation of Bone Remodeling by the Canonical Wnt Signaling Pathway Donald A. Glass II and Gerard Karsenty

3 Calcium Sensing Receptors and Calcium Oscillations: Calcium as a First Messenger Gerda E. Breitwieser

4 Signal Relay During the Life Cycle of Dictyostelium Dana C. Mahadeo and Carole A. Parent

5 Biological Principles for Ex Vivo Adult Stem Cell Expansion Jean-Franc¸ois Pare´ and James L. Sherley

6 Histone Deacetylation as a Target for Radiosensitization David Cerna, Kevin Camphausen, and Philip J. Tofilon

7 Chaperone-Mediated Autophagy in Aging and Disease Ashish C. Massey, Cong Zhang, and Ana Maria Cuervo

8 Extracellular Matrix Macroassembly Dynamics in Early Vertebrate Embryos Andras Czirok, Evan A. Zamir, Michael B. Filla, Charles D. Little, and Brenda J. Rongish

Contents of Previous Volumes

275

Volume 74 1 Membrane Origin for Autophagy Fulvio Reggiori

2 Chromatin Assembly with H3 Histones: Full Throttle Down Multiple Pathways Brian E. Schwartz and Kami Ahmad

3 Protein–Protein Interactions of the Developing Enamel Matrix John D. Bartlett, Bernhard Ganss, Michel Goldberg, Janet Moradian-Oldak, Michael L. Paine, Malcolm L. Snead, Xin Wen, Shane N. White, and Yan L. Zhou

4 Stem and Progenitor Cells in the Formation of the Pulmonary Vasculature Kimberly A. Fisher and Ross S. Summer

5 Mechanisms of Disordered Granulopoiesis in Congenital Neutropenia David S. Grenda and Daniel C. Link

6 Social Dominance and Serotonin Receptor Genes in Crayfish Donald H. Edwards and Nadja Spitzer

7 Transplantation of Undifferentiated, Bone Marrow-Derived Stem Cells Karen Ann Pauwelyn and Catherine M. Verfaillie

8 The Development and Evolution of Division of Labor and Foraging Specialization in a Social Insect (Apis mellifera L.) Robert E. Page Jr., Ricarda Scheiner, Joachim Erber, and Gro V. Amdam

Volume 75 1 Dynamics of Assembly and Reorganization of Extracellular Matrix Proteins Sarah L. Dallas, Qian Chen, and Pitchumani Sivakumar

2 Selective Neuronal Degeneration in Huntington’s Disease Catherine M. Cowan and Lynn A. Raymond

3 RNAi Therapy for Neurodegenerative Diseases Ryan L. Boudreau and Beverly L. Davidson

4 Fibrillins: From Biogenesis of Microfibrils to Signaling Functions Dirk Hubmacher, Kerstin Tiedemann, and Dieter P. Reinhardt

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5 Proteasomes from Structure to Function: Perspectives from Archaea Julie A. Maupin-Furlow, Matthew A. Humbard, P. Aaron Kirkland, Wei Li, Christopher J. Reuter, Amy J. Wright, and G. Zhou

6 The Cytomatrix as a Cooperative System of Macromolecular and Water Networks V. A. Shepherd

7 Intracellular Targeting of Phosphodiesterase-4 Underpins Compartmentalized cAMP Signaling Martin J. Lynch, Elaine V. Hill, and Miles D. Houslay

Volume 76 1 BMP Signaling in the Cartilage Growth Plate Robert Pogue and Karen Lyons

2 The CLIP-170 Orthologue Bik1p and Positioning the Mitotic Spindle in Yeast Rita K. Miller, Sonia D’Silva, Jeffrey K. Moore, and Holly V. Goodson

3 Aggregate-Prone Proteins Are Cleared from the Cytosol by Autophagy: Therapeutic Implications Andrea Williams, Luca Jahreiss, Sovan Sarkar, Shinji Saiki, Fiona M. Menzies, Brinda Ravikumar, and David C. Rubinsztein

4 Wnt Signaling: A Key Regulator of Bone Mass Roland Baron, Georges Rawadi, and Sergio Roman-Roman

5 Eukaryotic DNA Replication in a Chromatin Context Angel P. Tabancay, Jr. and Susan L. Forsburg

6 The Regulatory Network Controlling the Proliferation–Meiotic Entry Decision in the Caenorhabditis elegans Germ Line Dave Hansen and Tim Schedl

7 Regulation of Angiogenesis by Hypoxia and Hypoxia-Inducible Factors Michele M. Hickey and M. Celeste Simon

Volume 77 1 The Role of the Mitochondrion in Sperm Function: Is There a Place for Oxidative Phosphorylation or Is this a Purely Glycolytic Process? Eduardo Ruiz-Pesini, Carmen Dı´ez-Sa´nchez, Manuel Jose´ Lo´pez-Pe´rez, and Jose´ Antonio Enrı´quez

Contents of Previous Volumes

277

2 The Role of Mitochondrial Function in the Oocyte and Embryo Re´mi Dumollard, Michael Duchen, and John Carroll

3 Mitochondrial DNA in the Oocyte and the Developing Embryo Pascale May-Panloup, Marie-Franc¸oise Chretien, Yves Malthiery, and Pascal Reynier

4 Mitochondrial DNA and the Mammalian Oocyte Eric A. Shoubridge and Timothy Wai

5 Mitochondrial Disease—Its Impact, Etiology, and Pathology R. McFarland, R. W. Taylor, and D. M. Turnbull

6 Cybrid Models of mtDNA Disease and Transmission, from Cells to Mice Ian A. Trounce and Carl A. Pinkert

7 The Use of Micromanipulation Methods as a Tool to Prevention of Transmission of Mutated Mitochondrial DNA Helena Fulka and Josef Fulka, Jr.

8 Difficulties and Possible Solutions in the Genetic Management of mtDNA Disease in the Preimplantation Embryo J. Poulton, P. Oakeshott, and S. Kennedy

9 Impact of Assisted Reproductive Techniques: A Mitochondrial Perspective from the Cytoplasmic Transplantation A. J. Harvey, T. C. Gibson, T. M. Quebedeaux, and C. A. Brenner

10 Nuclear Transfer: Preservation of a Nuclear Genome at the Expense of Its Associated mtDNA Genome(s) Emma J. Bowles, Keith H. S. Campbell, and Justin C. St. John