Osteopontin

Osteopontin Gerard J. Nau* Infectious Diseases Unit, Massachusetts General Hospital, Boston, MA 02114, USA Whitehead Institute, Cambridge, MA 02142, USA * corresponding author tel: 617-726-3812, fax: 617-726-7416, e-mail: [email protected] DOI: 10.1006/rwcy.2000.07003.

SUMMARY Osteopontin (OPN) is a protein that is widely expressed and will likely have multiple biological functions based on structural modifications of the protein and the local environments in which it is expressed. Evidence is accumulating that OPN plays a role in cells derived from monocytes, including macrophages and osteoclasts. Presently there is strong evidence that OPN can activate the migration and adhesion of many cells in vitro and, therefore, may contribute to the accumulation of cells at sites of inflammation in vivo. The possibility exists that OPN does more than simply mediate the accumulation of cells; studies are needed that evaluate activating or inhibitory signals induced by OPN. In addition, more studies are required to understand OPN's function in normal and disease states.

BACKGROUND

Discovery Osteopontin (OPN) is a protein that is widely expressed in vivo (Denhardt and Guo, 1993) and it has been associated with immune/inflammatory responses (Uede et al., 1997). The protein may have multiple functions because it is expressed in disparate tissues such as kidney (Kohri et al., 1993), bone (Oldberg et al., 1986), breast milk (Senger et al., 1989), and epithelial surfaces (Brown et al., 1992). Within the inflammatory/immune system, OPN protein or its transcript has been identified in T cells (Patarca et al., 1989), macrophages (Miyazaki et al., 1990), and NK cells (Pollack et al., 1994). Because of findings in OPN null mice and in pathologic

specimens, experiments have only recently begun that study OPN's role as a cytokine.

Alternative names OPN has had several names, reflecting the fact that it has been identified in multiple systems. The protein was first described in the culture supernatants of transformed cell lines and was termed secreted phosphoprotein 1 (spp1) (Senger et al., 1979). The name bone sialoprotein II was applied after the protein was isolated from rat bone (Franzen and Heinegard, 1985). After cloning the cDNA for this protein, the same group identified an integrin-binding RGD sequence (Oldberg et al., 1986). They found that osteosarcoma cells bound to surfaces coated with OPN and reported that the protein adhered tightly to hydroxyapatite (Oldberg et al., 1986). The investigators proposed the name OPN because this cell-derived component of the osteoid matrix could form a bridge between cells and bone (Oldberg et al., 1986). Finally, the protein was found to be expressed in T lymphocytes early after activation, leading to the name early T lymphocyte activation 1 (Eta-1) (Patarca et al., 1989). Ultimately, it became clear that Eta-1 and OPN were identical proteins (Patarca et al., 1993).

Structure OPN is structurally complex. It is a secreted phosphoprotein composed of approximately 300 amino acids, depending on the species, with multiple posttranslational modifications including glycosylation and phosphorylation. In addition, a thrombin-cleavage site that is present within the protein is thought to be important in activating the biological properties of

690 Gerard J. Nau OPN (Senger et al., 1994). Although it is a secreted, soluble protein, it is possible for OPN to be immobilized by crosslinking to the extracellular matrix (Beninati et al., 1994). To date, the protein has been shown to be a ligand for integrin receptors and for CD44. Whether these receptors are responsible for the inflammatory properties of OPN or whether additional receptors are involved remains unclear.

Main activities and pathophysiological roles The most well-defined biologic activities of OPN are adhesion and migration. Multiple cell types have been shown to adhere to OPN (Liaw et al., 1994; Senger et al., 1994; Nasu et al., 1995). The RGD integrinbinding motif appears to be important for this process. Relevant for the immune/inflammatory systems, OPN can activate the migration of macrophages (Giachelli et al., 1998; Singh et al., 1990) as well as smooth muscle cells (Liaw et al., 1994). OPN has been identified in many pathologic states. OPN can bind calcium (Chen et al., 1992) and is associated with calcified atherosclerotic and neoplastic lesions (Hirota et al., 1993, 1995). The protein has been linked to various other inflammatory processes including the granulomas of tuberculosis (Nau et al., 1997) and sarcoidosis (Carlson et al., 1997). OPN is expressed in macrophages after myocardial injury (Murry et al., 1994) and in macrophages during skin wound healing (Liaw et al., 1998). Finally, the protein is not only associated with transformed cells (Craig et al., 1989; Senger et al., 1979), but expression of antisense OPN reduces the tumorigenicity and the metastatic potential of transformed cells (Behrend et al., 1994).

GENE AND GENE REGULATION

Accession numbers Rat cDNA: M14656 Mouse cDNA: J04806, X14882 Human cDNA: X13694, J04765, M83248

Sequence The genomic structure of OPN has been thoroughly investigated. There appears to be only one copy of the OPN gene in the mouse (Craig et al., 1989) and human (Young et al., 1990) genomes. Genomic clones for mouse and human OPN were published soon after

the cDNA sequence was determined (Miyazaki et al., 1990; Hijiya et al., 1994). The murine OPN gene was cloned from a murine liver library in which six exons and five introns were described (Miyazaki et al., 1990). The untranslated sequence of exon 1 corresponded to the 50 untranslated sequence of the Eta-1 cDNA (Patarca et al., 1989). However, the first intron identified by Miyazaki et al. conflicted with the structure of the human gene recovered by the same research group from a human liver library (Hijiya et al., 1994). The human gene contained an additional exon located 1083 bp 50 upstream of what was identified as exon 1 on the mouse (Hijiya et al., 1994). This apparent discrepancy was resolved by Craig and Denhardt when they isolated another OPN genomic clone from a murine embryo genomic library (Craig and Denhardt, 1991). This isolate contained an exon 1 homologous to that which had been identified in the human gene (Craig and Denhardt, 1991). A study of promoter site(s) in the murine OPN gene was conducted by primer extension analysis (Zhang et al., 1992) and confirmed the existence of one promoter site at exon 1 described by Craig and Denhardt (1991). OPN mRNA could not be identified that utilized the exon 1 proposed by Miyazaki et al. (Zhang et al., 1992; Behrend et al., 1993). Behrend and colleagues have suggested that the cDNA isolated by Miyazaki et al. and by Patarca et al. represents an incompletely processed nuclear transcript (Behrend et al., 1993).

Chromosome location Chromosomal localization has been performed for the murine and the human OPN genes. The mouse OPN gene was localized to chromosome 5 by a linkage analysis of inbred strains of mice, an estimated 0.00 cM from the Ric locus (see Pathophysiological roles in normal humans and disease states and diagnostic utility) (Fet et al., 1989). Young and colleagues performed chromosomal mapping of the human OPN gene using human/rodent hybrid cell DNAs demonstrating a single copy gene localized to chromosome 4 (Young et al., 1990). They carried their analysis further by performing in situ hybridization on human metaphase chromosome spreads and localized the OPN gene to 4q13 (Young et al., 1990). This chromosomal analysis is interesting because mouse chromosome 5 is homologous to the entire short arm and the proximal end of the long arm of human chromosome 4 (Lalley et al., 1988). This localization is also interesting because it places OPN in the same chromosomal location characteristically associated with human chemokines such as IL-8 (Modi et al., 1990; Sherry and Cerami, 1991).

Osteopontin 691

Regulatory sites and corresponding transcription factors Hijiya and others analyzed the promoter region 50 to the human OPN gene (Hijiya et al., 1994). A similar analysis was performed for the murine gene (Miyazaki et al., 1990), but is excluded here because of the confusion surrounding the identity of exon 1. Table 1 summarizes the sequence analysis performed by Hijiya et al. in which a number of potential regulatory motifs were identified (Hijiya et al., 1994). Of note, a number of NF-IL6 sites and one IRF-1 site were identified, suggesting the regulation of OPN expression by immune and inflammatory responses. In addition to transcription control regions upstream of the OPN gene, regulatory elements have been described in the first intron (Botquin et al., 1998). OPN is expressed in the inner cell mass and the Table 1 Regulatory elements in the 50 region upstream of human OPN Site

Number of elementsa

AP-1

1

AP-2

1

AP-3

1

CF-1

3

CTCF

4

E2A

2

E2BP

1

E4TF1

1

EtS-1

1

GATA-1

4

IRF-1

1

myb

2

NF-IL6

4

Oct-1

1

PEA-3

2

PPAR

1

SIF

1

SP-1

2

TCF-1 VDR-like a

Summarized from Hijiya et al. (1994).

11 2

hypoblast of the developing murine embryo. The transcription factors Oct-4 and Sox-2 bind specific sequences in the first intron of the OPN gene. A novel palindromic Oct recognition element contributes to the transcriptional activation of OPN and is antagonized by Sox-2 binding (Botquin et al., 1998).

Cells and tissues that express the gene After OPN was first identified in the culture supernatants of transformed cell lines, protein was subsequently purified from a number of different systems and the gene was cloned (Table 2). The murine cDNA, known as 2ar, was cloned from an epidermal cell line after treatment with phorbol esters (Smith and Denhardt, 1987). The gene is also induced in normal mouse epidermis after phorbol ester treatment and was found to be identical to secreted phosphoprotein 1 (spp1) and rat OPN (Craig et al., 1989). OPN transcription is developmentally regulated and can be found in calvaria, placenta, decidua, and kidney (Nomura et al., 1988). Additional studies of OPN expression in adult mice have shown that the transcript is detectable in kidney, ovary, lung, brain, and skin (Craig and Denhardt, 1991). Pregnant or lactating female mice have detectable OPN transcript in their deciduum and placenta and increased expression in the uterus, skin, and fatty tissue (Craig and Denhardt, 1991). In bone, the OPN gene is expressed in osteoclasts (Dodds et al., 1995; Tezuka et al., 1992) which are believed to differentiate from blood monocytes (Reinholt et al., 1990). Others have demonstrated that osteoblasts can produce OPN in vitro (Salih et al., 1997). Human OPN appears to exist as two different species in bone and decidua cells (Young et al., 1990). This is most likely related to differential splicing, though the functional consequences of the two species are unknown (Young et al., 1990). Cloned murine T lymphocytes, helper and cytolytic, and a cloned NK cell line produce the Eta-1 transcript after activation by Con A (Patarca et al., 1989). Cell lines with monocytic lineages express OPN mRNA (Miyazaki et al., 1990). The expression of OPN in tumors has been studied where splice variants also have been described (Saitoh et al., 1995). The function of these alternative forms of OPN is unclear but may be related to tumorigenicity (see Pathophysiological roles in normal humans and disease states and diagnostic utility).

692 Gerard J. Nau Table 2 OPN cDNA cloning Species

Tissue

Name

Accession numbers

Reference

Rat

Osteosarcoma

Bone sialoprotein

M14656

Oldberg et al., 1986

Mouse

Epidermal cell line

2ar

J04806/X14882

Craig et al., 1989

Spp-1 Human

Osteosarcoma

Op-30

X13694

Kiefer et al., 1989

Human

Primary bone cell culture

OPN

J04765

Young et al., 1990

Human

Kidney

Uropontin

M83248

Shiraga et al., 1992

Nephropontin

PROTEIN

Table 3 OPN protein sequences

Accession numbers

Species

Accession numbers

Amino acids

Reference

The accession numbers of murine and human OPN are summarized in Table 3. Although the contents of the table are not exhaustive of the multiple OPN entries available, the first two entries in the table summarize multiple different submissions to the database grouped under a single accession number. The final entry highlights the predicted proteins from splice variants that have been identified (Saitoh et al., 1995). The two smaller forms, OPN-b and c, have deletions in amino acid numbers 58±71 and 32±58, respectively.

Mouse

129261

294

Miyazaki et al., 1989, 1990; Craig et al., 1989; Patarca et al., 1989; Worcester et al., 1992

Human

129260

314

Kiefer et al., 1989; Young et al., 1990; Shiraga et al., 1992; Kohri et al., 1993; Hijiya et al., 1994

OPN-a

2119710

314

Saitoh et al., 1995

OPN-b

2119708

300

Saitoh et al., 1995

OPN-c

2119709

287

Saitoh et al., 1995

Description of protein OPN is a secreted soluble protein that is highly acidic with numerous posttranslational modifications. The protein is 294 amino acids in length in the mouse and 314 in humans (see Table 3). There is a 16 amino acid segment of hydrophobic residues that corresponds to a secretion signal sequence. The mature protein has a mass of 44,000 Da measured by sedimentation equilibrium centrifugation (Prince et al., 1987). However, the estimated molecular weight on SDS-PAGE is variable and can be up to 75,000 Da (Prince et al., 1987; Senger et al., 1989). This difference has been attributed to gel characteristics (Prince et al., 1987; Senger et al., 1989). Biochemical analyses indicate that OPN is a highly acidic protein (Patarca et al., 1989). Approximately 100 amino acids from the N-terminus there are stretches rich in aspartic acid residues: 11 of 12 amino acids in mouse (Craig et al., 1989; Miyazaki et al., 1990), 10 of 11 in rat (Oldberg et al., 1986), and 8 of 10 or 7 of 10 in human (Kiefer et al., 1989; Young et al., 1990). Overall, OPN has a pI of approximately 4.5 (Patarca et al., 1989).

Human (alternative splice)

Analyses of the amino acid sequence deduced from cDNA sequence have identified potential functional domains within OPN. First, there is an integrinbinding motif composed of GRGDS (Oldberg et al., 1986). This motif is highly conserved among the species studied thus far and is believed to be OPN's major domain for integrin binding. The GRGDS sequence is thought to be on a turn within a sheet and on the surface of the molecule (Craig et al., 1989). The domain rich in aspartic acid residues is thought to be important for binding hydroxyapatite (Oldberg et al., 1986) or calcium (Patarca et al., 1989). A seven amino acid stretch with homology to thrombospondin may also bind calcium (Patarca et al., 1989). Finally, there are two potential heparin-binding sites within OPN (Prince, 1989).

Osteopontin 693

Discussion of crystal structure The crystal structure of OPN has not yet been elucidated. The extensive posttranslational modifications may make accurate determination of the crystal structure difficult. Ribbon diagrams have been used to illustrate spatial relationships between domains of the protein (Denhardt and Guo, 1993; Uede et al., 1997). The predicted secondary structure includes eight  helix domains and six sheet domains (Denhardt and Guo, 1993; Uede et al., 1997). Of some interest, the GRGDS motif is located just 50 of a thrombin cleavage site (Denhardt and Guo, 1993). Thrombin treatment of OPN enhances integrin binding (see in vitro activities) (Senger et al., 1989). This enhancement may be the result of exposure of the GRGDS sequence.

are three potential N-linked sites based on sequence analysis (Sorensen et al., 1995). Although OPN is a secreted soluble protein, paradoxically it is thought to be a component of the extracellular matrix in some situations (Reinholt et al., 1990). The discovery that OPN is a substrate for transglutaminase provided one possible explanation for this paradox (Beninati et al., 1994). Transglutaminase was shown to catalyze the oligomerization of OPN and the crosslinking of OPN to fibronectin (Beninati et al., 1994). There are two glutamines in bovine OPN that are reactive with transglutaminase and these two residues are conserved in multiple species (Sorensen et al., 1994).

CELLULAR SOURCES AND TISSUE EXPRESSION

Important homologies

Cellular sources that produce

There are several structural homologies of note in OPN. As detailed above, an RGD integrin-binding motif is within OPN like the extracellular matrix proteins fibronectin and vitronectin (Oldberg et al., 1986). There are also domains with homology to thrombospondin. There does not, however, appear to be significant homology with other immunologically active proteins (Patarca et al., 1989).

Limited analyses have been performed on the in vitro production of OPN protein from various cell sources. In addition to transformed cells (Senger et al., 1979), OPN protein has been identified in the culture supernatants of activated T cells (Singh et al., 1990). Extensive immunohistochemical studies of normal tissue specimens have been performed. Using a rabbit antiserum raised against human breast milk OPN, the protein was found closely associated with many tissues such as lactating and nonlactating breast epithelium and luminal contents as well as the cement lines and trabecula in bone. Consistent with the results observed by immunohistochemistry, OPN has been biochemically purified from human (Senger et al., 1989) and bovine (Bayless et al., 1997) breast milk. Multiple epithelial surfaces are also positive on immunohistochemistry: (1) the gastrointestinal system (salivary gland, stomach, small and large intestine, bile duct, gallbladder, pancreas duct); (2) the urogenital system (urinary bladder epithelium, distal tubules of the kidney, testis, endocervix and endometrium, ovary, and placenta) (Brown et al., 1992). The fact that granulated metrial gland cells of the placenta/endometrium are derived from bone marrow and have an NK cell phenotype (Linnemeyer and Pollack, 1991) offers some explanation for the increased OPN gene expression in the pregnant uterus and deciduum (Craig and Denhardt, 1991). Immunohistochemical studies using a monoclonal antibody that recognizes OPN have demonstrated more extensive tissue expression (Nau et al., 1997), though this may be in part due to the nature of the tissues being examined (see Pathophysiological roles

Posttranslational modifications OPN is known to be heavily modified after translation. Isolation and analysis of purified rat bone OPN identified one phosphothreonine and 12 phosphoserine residues (Prince et al., 1987). Mass spectroscopy analysis of bovine milk OPN demonstrated 27 phosphoserine and one phosphothreonine residues (Sorensen et al., 1995). These residues were associated with recognition sequences for mammary gland casein kinase and casein kinase II (Sorensen et al., 1995). Glycosylation is a prominent posttranslational modification of OPN. Sequence analysis of OPN cDNAs predicted several O-linked and N-linked glycosylation sites (Oldberg et al., 1986; Patarca et al., 1989). Rat bone OPN contains 16.6% carbohydrate distributed between one N-linked and 5±6 O-linked oligosaccharides (Prince et al., 1987). Among these oligosaccharides, one-third of the monosaccharide residues are sialic acid (Prince et al., 1987). In contrast, bovine milk OPN contains three O-linked and no N-linked oligosaccharides, even though there

694 Gerard J. Nau in normal humans and disease states and diagnostic utility).

Eliciting and inhibitory stimuli, including exogenous and endogenous modulators Hormones and other small molecules can alter OPN expression. Consistent with the presence of steroid response elements in the promoter, topical administration of 17 -estradiol and progesterone, alone or in combination, leads to increased OPN mRNA in skin and subcutaneous adipose tissue (Craig and Denhardt, 1991). The treatment of osteosarcoma cells, osteoblasts, or fibroblasts with 1,25-dihydroxyvitamin D3 increases OPN protein production (Prince and Butler, 1987; Jin et al., 1990). Phorbol esters induce expression of OPN RNA in epidermal cells (Smith and Denhardt, 1987). Osteoblasts also upregulate OPN gene expression in response to mechanical stress (Toma et al., 1997). The effect of cytokines on OPN gene expression and protein production has been examined in several systems. Osteoblasts and fibroblasts increase OPN mRNA after exposure to TNF, and IL-1 (Jin et al., 1990). TGF induces the expression of OPN mRNA in osteosarcoma cells (Noda et al., 1988) and in vascular smooth muscle cells (Giachelli et al., 1993). Cardiac myocytes increase OPN mRNA after exposure to IL-1 and IFN (Singh et al., 1995). NK cells increase OPN gene expression after activation with IL-2 (Pollack et al., 1994). NF-IL6 and IRF-1 elements are present in the 50 region of the promoter (Hijiya et al., 1994). The expression of OPN mRNA in macrophages increases after infection by mycobacteria (Nau et al., 1997). This is of some interest because NF-IL6 regulatory elements are involved in IL-1 and TNF expression when monocytes are exposed to lipoarabinomannan from Mycobacterium tuberculosis (Zhang and Rom, 1993). LPS can alter OPN gene expression in the kidney (Madsen et al., 1997). Whereas OPN transcription is constitutive in the descending thin limb and the papillary surface, LPS induced expression in the distal tubules (Madsen et al., 1997). LPS also increases OPN mRNA in a mouse osteoblastic cell line and primary osteoblast-like cells (Jin et al., 1990). Growth factors also modulate the expression of OPN. Basic fibroblast growth factor enhances OPN message in vascular smooth muscle cells (Giachelli et al., 1993). PDGF and EGF, alone or in combination, induce OPN expression in quiescent Swiss 3T3 cells (Smith and Denhardt, 1987).

RECEPTOR UTILIZATION Studies into OPN receptor utilization have focused on integrins and CD44.

IN VITRO ACTIVITIES

In vitro findings The identification of a GRGDS integrin-binding motif within OPN (Oldberg et al., 1986) generated much interest in OPN as an activator of cell adhesion. A summary of several studies is presented in Table 4. In addition to characterizing the integrin receptors involved, several groups have tested the functional importance of the RGD motif. Altering the motif from RGD to RGE in recombinant rabbit OPN eliminates the protein's ability to promote adhesion or haptotaxis of P388D1 macrophages (Nasu et al., 1995). A deletion of the RGD motif or an RGD to RGE mutation abolishes the ability of recombinant murine OPN to promote adhesion and migration of fibroblast cells (Xuan et al., 1995). In addition to adhesion, several investigators have studied OPN's effect on cell migration. Several studies are listed in Table 4. OPN can trigger cell migration in a modified Boyden chamber assay for chemotaxis. Nasu et al. varied this slightly by examining haptotaxis (Nasu et al., 1995). In these experiments, OPN was dried on to the filters of a Boyden chamber apparatus before cells were added. OPN caused migration of the macrophage-like cell line P388D1 only when it was precoated on the filters, not when it was in fluid phase (Nasu et al., 1995). Several reports have shown an inhibitory effect of OPN on the induction of nitric oxide synthase type 2 (NOS2) in vitro. OPN appears to reduce nitric oxide synthesis and NOS2 mRNA in several cell types treated with LPS and IFN (Hwang et al., 1994; Rollo and Denhardt, 1996; Rollo et al., 1996; Singh et al., 1995). These intriguing findings appear at odds with many of the proinflammatory effects of OPN (see In vivo biological activities of ligands in animal models). One possible explanation for the conflicting results is the source of the OPN used in the experiments. OPN is heavily modified after translation and alterations of these modifications can have a significant impact on receptor binding (Shanmugam et al., 1997). Ultimately, more experiments in vitro and in vivo are required to fully understand OPN's pro- and anti-inflammatory properties. Finally, OPN's ability to bind Ca2‡ has been studied (Chen et al., 1992). OPN purified from rat

Osteopontin 695 Table 4 Summary of OPN in vitro activities Activity

Cell type

Adhesion

Carcinoma cells

Reference

ras-3T3, mammary

Xuan et al., 1994

Fibrosarcoma, bladder

Senger et al., 1994

Vascular smooth muscle and endothelial cells

Liaw et al., 1994

Embryonic kidney

Hu et al., 1995b

Fetal lung fibroblasts

Senger et al., 1994

Macrophage-like cells (P388D1)

Nasu et al., 1995

Platelets

Bennett et al., 1997

B lymphocytes Migration

Endothelial cells (chemotaxis)

Senger et al., 1996

Vascular smooth muscle cells (chemotaxis)

Liaw et al., 1994 a

Antagonize NOS2

Calcium binding a b

Macrophage-like cells (P388D1) (haptotaxis )

Nasu et al., 1995

Macrophage-like (RAW264.7)

Rollo et al., 1996

Peritoneal macrophages

Rollo and Denhardt, 1996

Cardiac myocytes

Singh et al., 1995

Kidney epithelial cells

Hwang et al., 1994

NAb

Chen et al., 1992

Haptotaxis defined as `the ability of cells to detect and move up gradients of substratum adhesiveness' (Nasu et al., 1995). Not applicable.

calvaria was shown to bind 45 Ca2‡ in an in vitro dot blot assay. Under conditions with excess 45 Ca2‡ , OPN was found to bind 50 atoms of Ca2‡ per molecule of protein (Chen et al., 1992). This value is significantly higher than what might be expected from the two Ca2‡ -binding sites predicted by the protein sequence (Patarca et al., 1989). Chen and colleagues attributed the high Ca2‡ -binding capacity to an electrostatic mode of binding rather than specific binding sequences (Chen et al., 1992).

Regulatory molecules: Inhibitors and enhancers OPN's ability to activate adhesion can be modulated by proteolytic cleavage. Analysis of OPN derived from milk revealed a specific cleavage site for thrombin (Senger et al., 1989). Treatment of OPN with thrombin greatly enhances the attachment and spreading of tissue culture cells (Senger et al., 1994). This activity was blocked almost entirely by antibodies recognizing the v 3 integrin (Senger

et al., 1994). The thrombin cleavage site of OPN is near the GRGDS integrin-binding site in multiple species (Bautista et al., 1994). Activation of OPN by thrombin cleavage has also been demonstrated in vivo where it participates in endothelial cell migration induced by VEGF (Senger et al., 1996). Divalent cations, Ca2‡ , Mg2‡ , or Mn2‡ , enhance cell adhesion to OPN (Hu et al., 1995a; Bennett et al., 1997; Bayless et al., 1998). However, adhesion via the v 3 integrin can be suppressed with calcium levels greater than 1±2 mM (Hu et al., 1995a; Bennett et al., 1997). Adhesion of platelets to OPN via v 3 integrin requires activation by treatment with ADP (Bennett et al., 1997). Adhesion of B cell lines requires pretreatment of the cells with phorbol esters (Bennett et al., 1997).

Bioassays used The bioactivity of OPN preparations are generally tested by adhesion mediated by the protein or by cell migration (see Table 4 for references).

696 Gerard J. Nau

IN VIVO BIOLOGICAL ACTIVITIES OF LIGANDS IN ANIMAL MODELS

Normal physiological roles Multiple models of OPN's in vivo function have been developed. Using immunogold staining techniques, Reinholt and colleagues localized high concentrations of OPN in bone at the clear zone between osteoclasts and the surface of bone (Reinholt et al., 1990). The v 3 vitronectin receptor also localized to the same region. The concentration of OPN was low elsewhere in the trabecular bone and the staining of osteoblasts did not appear significantly different than controls (Reinholt et al., 1990). These findings suggested a model where OPN anchors osteoclasts to the surface of bone. This model can be expanded with the knowledge that increased Ca2‡ concentration decreases adhesion to OPN (Hu et al., 1995a). The osteoclast would release from the bone surface after sufficient Ca2‡ was liberated in the local microenvironment; thus, a feedback mechanism would modulate bone resorption. Consistent with this model of osteoclast adhesion, bone resorption in vivo can be inhibited by a peptidomimetic antagonist of the v 3 integrin (Engleman et al., 1997). Also, osteoclast adherence to and resorption of bone particles can be inhibited by soluble OPN, RGD-containing peptides, or antibodies that bind OPN or the v 3 integrin (Ross et al., 1993). OPN has been purified from urine and is believed to be important in renal stone formation. Shiraga et al. purified a protein they named uropontin from human urine (Shiraga et al., 1992). N-terminal sequencing showed identity to human bone OPN though amino acid composition analysis showed some differences between the two proteins. Uropontin inhibited calcium oxalate crystal formation in vitro (Shiraga et al., 1992). The cDNA for uropontin was cloned and was found to be identical to OPN (Kohri et al., 1993). The protein was also purified from calcium oxalate stones (Kohri et al., 1993). In contrast to the in vitro results, expression of OPN message and protein in distal tubule cells increased when stone formation was induced by glyoxylic acid (Kohri et al., 1993). However, studies in mice appear to corroborate the results of Shiraga and colleagues in that OPN appears to inhibit crystal growth (Worcester et al., 1992). It seems likely that the normal role for OPN is to maintain the solubility of the calcium oxalate. Under conditions when stone formation is imminent and perhaps as a response to injury, OPN expression is

increased. Eventually, OPN is incorporated into the stone when the Ca2‡ and the oxalate concentrations reach a critical level. Kidney stones were not described in the two OPN knockout mice that have been generated thus far (see Knockout mouse phenotypes); this suggests that other proteins in urine also act to inhibit stone formation (Shiraga et al., 1992). OPN may have a protective role for humans at the interface between epithelial surfaces and the external environment (Brown et al., 1992). OPN was identified on the luminal surfaces of multiple epithelia and in sweat glands and ducts (Brown et al., 1992). There is also a significant amount of OPN in breast milk (Senger et al., 1989). Because certain bacteria use integrins for colonization, Brown and colleagues postulated that OPN may prevent colonization by blocking these receptors (Brown et al., 1992). OPN may also exert a bacteriostatic effect by limiting the amount of Ca2‡ available to pathogens.

Species differences To date there have been no functional comparisons between OPN protein from different species. In independent studies, OPN derived from different species was observed to have different effects (see Pharmacologic effects).

Knockout mouse phenotypes Liaw et al. generated an OPN-null mouse line to assess OPN's role in vivo (Liaw et al., 1998). Although OPN is expressed during embryonic development (Nomura et al., 1988), mice lacking OPN grow to adulthood and are fertile. The OPN-null animals demonstrate altered wound healing with delayed debridement and abnormal collagen fibril remodeling (Liaw et al., 1998). The delayed debridement was attributed to altered macrophage activation. Another series of experiments using the same OPN-null line have implicated abnormal macrophage activity in increased susceptibility to a mycobacterial infection (Nau, G. J. et al., unpublished results). Rittling et al. have generated another line of mice with a targeted disruption of the OPN gene (Rittling et al., 1998). In this line a high molecular weight, low abundance transcript that hybridized with OPN probes was seen on northern blot analysis. However, there was no mRNA species reminiscent of native OPN and there was no detectable OPN protein. Once again, the mutant mice are fertile and show normal development. A detailed analysis of osseous tissue revealed no abnormalities; in particular, an ultrastructural

Osteopontin 697 analysis using immunogold techniques showed normal cement lines though OPN is typically abundant at this site. The authors did identify an abnormal phenotype when osteoclastogenesis was tested in vitro. Splenocytes or bone marrow cells from null mice yield several fold more osteoclasts than cells from wild-type animals (Rittling et al., 1998).

Transgenic overexpression To date, there are no published reports on the effects of OPN overexpression in a transgenic mouse model.

Pharmacological effects Several groups have studied the pharmacologic effects of OPN administered in vivo. Subcutaneous injection of OPN derived from murine T cells into mouse skin led to an inflammatory cell infiltrate within 24 hours (Singh et al., 1990). The cellular composition of these infiltrates had a greater than expected proportion of macrophages (Singh et al., 1990). Similar results were obtained with E. coli-derived, recombinant murine OPN injected intradermally in rats (Giachelli et al., 1998). In contrast, Nasu and colleagues observed an inflammatory infiltrate composed mostly of polymorphonuclear leukocytes after injecting E. coliderived, recombinant rabbit OPN intradermally in rabbits (Nasu et al., 1995). It is unclear if the differences between these studies are related to species differences or to the source of the protein. Consistent with the pharmacologic effects detailed above, administration of an anti-OPN antiserum appears to block inflammation. N-Formyl-Met-LeuPhe (fMLP) injected intradermally in rats elicits a macrophage infiltrate in which the macrophages express OPN (Giachelli et al., 1998). The accumulation of macrophages at the site of fMLP was blocked by a neutralizing antiserum against OPN (Giachelli et al., 1998). The same neutralizing antiserum reduced inflammation in limited studies of a rat model for crescentic glomerulonephritis (Yu et al., 1998). Rats treated with the anti-OPN antiserum also showed diminished cutaneous delayed-type hypersensitivity reactions (Yu et al., 1998).

Interactions with cytokine network As discussed earlier (see Cellular sources and tissue expression), cytokines have been shown to modulate the expression of OPN. IL-1 and TNF increase the expression of OPN message in mouse osteoblast-like

cells (Jin et al., 1990). TGF increases both OPN transcription and protein synthesis in rat osteosarcoma cells (Noda et al., 1988).

PATHOPHYSIOLOGICAL ROLES IN NORMAL HUMANS AND DISEASE STATES AND DIAGNOSTIC UTILITY

Normal levels and effects The normal physiologic effects of OPN in humans are incompletely understood. Most understanding of its normal role is inferred from the observations that have been made using immunohistochemical analyses (Brown et al., 1992) and from studies in animals. OPN appears throughout the gastrointestinal system and secretory epithelium, including skin (Brown et al., 1992). Because OPN is a ligand for integrins, it may limit binding of pathogens that require surface integrins for colonization (Brown et al., 1992). Another possibility is that OPN may bind calcium, thereby reducing the availability of this cation to bacteria. An OPN-like protein is normally found in human urine and likely influences crystal formation (Shiraga et al., 1992).

Role in experiments of nature and disease states Inflammation Analyses of several pathologic conditions have provided more insight into OPN's role as a cytokine. In general, OPN is closely associated with tissue injury and the ensuing inflammation. Cryoinjury of rat myocardium and ischemic injury of human myocardium leads to increased OPN mRNA (Murry et al., 1994). In rats, the OPN message and protein are found in macrophages infiltrating the injured site (Murry et al., 1994). OPN expression is also associated with coronary atherosclerosis (Hirota et al., 1993; Panda et al., 1997). OPN is present in the plasma of patients with coronary artery atherosclerosis, but not in normal controls (Panda et al., 1997). Coronary atherectomy of atherosclerotic vessels leads to a sustained increase in plasma levels of OPN (Panda et al., 1997). OPN expression is increased in rat brain after a focal stroke and probably influences microglial, i.e. macrophage, infiltration (Wang et al., 1998). Serum levels of OPN also increase in patients with sepsis (Senger et al., 1988).

698 Gerard J. Nau OPN is also associated with other, more chronic, inflammatory conditions in humans. Granulomas of tuberculosis and silicosis demonstrate high levels of tissue OPN on immunohistochemical analysis (Nau et al., 1997). Other granulomatous conditions, such as sarcoidosis, histoplasmosis, temporal arteritis, and foreign-body reactions also express OPN message and protein (Carlson et al., 1997). OPN is also increased in two murine models of idiopathic pulmonary fibrosis (Nakama et al., 1998). Finally, OPN is elevated in mice predisposed to autoimmune disease (Patarca et al., 1990). While OPN can increase antibody production from B cells (Lampe et al., 1991), it is unclear if the elevated serum OPN causes the autoimmune disease in the mice (Patarca et al., 1990). Because OPN can activate the migration of cells, including macrophages, the protein may function as a soluble chemoattractant cytokine in the inflammatory conditions listed above. Alternatively, because OPN may be covalently linked to the extracellular matrix (Beninati et al., 1994), chronic OPN production and tissue accumulation may anchor macrophages that might have otherwise migrated through normal tissue. Infection From the standpoint of host resistance to infection, OPN has an intriguing history. Two alleles at the murine Ric locus determine the genetic resistance to a lethal rickettsial infection (Groves et al., 1980). OPN was subsequently mapped to the Ric locus (Fet et al., 1989). A comparison between a single resistant and a single susceptible allele revealed several amino acid changes, two were located in functional domains of the protein: a calcium-binding site and a heparinbinding site (Ono et al., 1995). Mice bearing the susceptible allele of Ric die from small inocula of the obligate intracellular pathogen Orientia (Rickettsia) tsutsugamushi, the causative agent of human scrub typhus (Groves et al., 1980; Patarca et al., 1993). Similar findings were made with group B arboviruses, though the genetic elements responsible have not been mapped as well (Goodman and Koprowski, 1962). In both cases, the defect was attributed to impaired macrophage activity (Goodman and Koprowski, 1962; Kokorin et al., 1976). Consistent with the increased susceptibility to a rickettsial infection, OPN-null mice show increased susceptibility to a mycobacterial infection (Nau, G.J. et al., unpublished results). Analysis of the human OPN gene has also suggested that a limited number of alleles exist (Young et al., 1990). However, these alleles have not yet been associated with particular disease states. In addition, there have been no reports of patients deficient in OPN.

Cancer OPN has a long association with cancer and tumorigenesis. As described earlier, the protein was first identified in the supernatants of tumor cells grown in vitro (Senger et al., 1979). Phorbol esters induce the expression of OPN mRNA (Craig et al., 1989). A thorough study of human carcinomas revealed limited expression of OPN by tumor cells (Brown et al., 1994). OPN mRNA is expressed by human renal and endometrial tumor cells (Brown et al., 1994). Multiple other carcinomas showed increased expression of OPN message, but this localized to macrophages within the tumor stroma (Brown et al., 1994). OPN protein was identified by immunohistochemistry in both macrophages and tumor cells, especially at the `tumor/host interface'. Gastrointestinal and breast tumor cells were negative for OPN mRNA on in situ hybridization analysis, suggesting that the OPN detected by immunohistochemistry was derived from the tumor stroma (Brown et al., 1994). Thus, OPN expression in human carcinomas generally is limited to the inflammatory macrophages within the stroma. Some carcinomas, in this case endometrial and renal, appear to make OPN independently. The fact that some tumor cells but not others express OPN was attributed to developmental origins, i.e. mesenchymal versus epithelial, of the cells (Brown et al., 1994). In other instances, not only is OPN produced by tumor cells but it also correlates with the severity of cancer. Saitoh and colleagues found OPN expression increased with more malignant grades of human glioblastoma cell lines (Saitoh et al., 1995). Studies in mice have shown that reducing OPN expression in transformed fibroblasts reduces tumorigenicity and malignant potential (Behrend et al., 1994; Gardner et al., 1994).

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