BMP Family

BMP Family A. Hari Reddi* and Dominik Haudenschild Department of Orthopedic Surgery, University of California, Davis, 4635 Second Avenue, Sacramento, CA 95817, USA * corresponding author tel: 916-734-5749, fax: 916-734-5750, e-mail: [email protected] DOI: 10.1006/rwcy.2000.08002.

SUMMARY Morphogenesis is the developmental cascade of pattern formation and body plan establishment, including the symmetry of most bilateral structures and the asymmetry of some unilateral anatomical sites, culminating in the adult form. Morphogenetic proteins initiate morphogenesis. The first morphogens in mammals were identified in adult bone tissue. Bone has considerable potential for regeneration and repair. The biochemical basis for this is the family of cytokines/morphogens called bone morphogenetic proteins (BMPs). BMPs initiate, promote, and maintain cartilage and bone morphogenesis, differentiation, and regeneration. This prototype paradigm demonstrates that a single cytokine signal can function in prenatal development, postnatal growth, and regeneration of bone in the adult. BMPs have actions beyond bone in eye, heart, kidney, skin, tooth, and other tissue development. The pleiotropic actions are mediated by discrete thresholds of concentration of BMPs. The accrued knowledge may be used in clinical applications of BMPs in dentistry, orthopedic surgery, and plastic and reconstructive surgery.

BACKGROUND

Discovery Unlike most tissues in the body, bone has considerable potential for repair and regeneration. In fact, bone grafts with associated hematopoietic cells are routinely used to stimulate bone repair in non-unions of long bone fractures, although the molecular basis of these bone grafts is still not fully understood. The

existence of the demineralized matrix has been known for over a century, but a critical discovery was made in the 1960s when it was found that the formation of new bone could be induced by demineralized, lyophilized segments of bone (Urist, 1965). The induction of new bone by demineralized extracellular matrix occurs as a result of a sequential developmental cascade (Reddi and Huggins, 1972; Reddi and Anderson, 1976; Reddi, 1981), the key steps of which are the chemotaxis of progenitor stem cells, mitosis of progenitor cells, and then differentiation first into cartilage and then replacement of cartilage by bone, a process commonly known as endochondral ossification. Implantation of the demineralized matrix results in interaction and binding of plasma fibronectin to implanted collagenous matrix (Weiss and Reddi, 1980). Peptides of fibronectin are chemotactic for mesenchymal cells. It was found that the migratory mesenchymal cells attached to the collagenous matrix and then proliferated and the daughter cells emerged as chondroblasts on day 5. With continued progression, chondrocytes were abundant on day 7. The chondrocytes hypertrophied on days 8±9 and the hypotrophic cartilage matrix mineralized on day 9. Concomitant angiogenesis and vascular invasion resulted in new bone formation on days 10±11. The appearance of osteoblasts was marked by increased alkaline phosphatase initially, and later, osteocalcin (also known as bone gla protein). With continued remodeling, hematopoietic marrow differentiation was evident in the ossicle. This entire sequence is a recapitulation of the stages of the embryonic limb bud development and differentiation (Reddi and Anderson, 1976). The bioassay for bone induction is based on in vivo implantation. The demineralized bone matrix is in the solid state. Dissociative extractants, such as 4 M

748 A. Hari Reddi and Dominik Haudenschild guanidine hydrochloride or 8 M urea containing 1% NaCl and 1% sodium dodecyl were used to solubilize proteins. The soluble proteins were reconstituted with insoluble collagenous matrix and bioassayed. This key advance (Sampath and Reddi, 1981, 1983) was a key step in the eventual purification of bone morphogenetic protein (BMP). The BMPs are dimeric and the disulfide bonds are critical for their activity (Wozney et al., 1988; Luyten et al., 1989).

Alternative names Certain BMPs are also known as osteogenin (BMP-3) (Luyten et al., 1989), osteogenic protein-1 (OP-1) (Ozkaynak et al., 1991).

Structure The BMPs are dimeric molecules with a single critical interchain disulfide bond. Disruption of this bond by

reduction in dithiothreitol or mercaptoethanol results in loss of biological activity (Luyten et al., 1989).

GENE AND GENE REGULATION The incisive investigation of Wozney and colleagues (1988) cloned BMP-2, BMP-2B (now called BMP-4), and BMP-3 (also called osteogenin). There are over 30 members in the BMP family, although in mammals there appear to be only about 15 BMPs (Table 1). Ozkaynak and colleagues (1990) cloned osteogenic protein 1 (also known as BMP-7). The genes for BMPs revealed that they are induced by BMP treatment indicating an auto-stimulable. Two novel genes related to BMPs were isolated from a cDNA library obtained from calf articular cartilage, and called cartilage-derived morphogenetic proteins 1 and 2 (Chang et al., 1994). These were isolated independently by Lee and colleagues (Storm et al., 1994), and called growth/differentiation factor 5 (GDF-5) and GDF-6. BMP-4 stimulated the expression of TGF 1,

Table 1 The BMP family in humans and chromosome location BMP subfamily

Generic name

BMP designation

Chromosome location

BMP-2/4

BMP-2A

BMP-2

20

BMP-2B

BMP-4

14

Osteogenin

BMP-3

4

BMP-3

Growth/differentiation factor 10 (GDF-10)

BMP-3B

10

OP-1

BMP-5

BMP-5

6

BMP-7

Vegetal related 1 (Vgr-1)

BMP-6

6

Osteogenic protein 1 (OP-1)

BMP-7

20

Osteogenic protein 2 (OP-2)

BMP-8

±

Osteogenic protein 3 (OP-3)

BMP-8B

±

Growth/differentiation factor 2 (GDF-2)

BMP-9

BMP-10

BMP-10

Growth/differentiation factor 11 (GDF-11)

BMP-11

Cartilage-derived morphogenetic protein 1 (CDMP-1), growth/differentiation factor 5 (GDF-5) or

BMP-14

20

Cartilage-derived morphogenetic protein 2 (CDMP-2), growth/differentiation factor 6 (GDF-6) or

BMP-13

±

Cartilage-derived morphogenetic protein 3 (CDMP-3), growth/differentiation factor 7 (GDF-7) or

BMP-12

±

BMP-15

BMP-15

Others

CDMP

Note: BMP-1 is not a BMP family member with seven canonical cysteines. It is a procollagen-C proteinase related to Drosophila tolloid and may play a role in modulating BMP actions by proteolysis of BMP antagonists/binding proteins, such as noggin, chordin, and gremlin.

BMP Family indicating that members of the BMP family crossregulate each other (Cunningham et al., 1992).

Chromosome location See Table 1.

PROTEIN

Sequence Each BMP monomer contains seven cysteine residues, six of which are involved in intrachain disulfide bonds and one demonstrated in an interchain disulfide bond (Figure 1). The BMP family members are initially synthesized as a precursor of 400 amino acids, then processed to a mature molecule of about 110 amino acids. The alignment of amino acids among the 15 members of the BMP family, BMP-2 through BMP-15, reveals the characteristic motifs (Figure 2 and Figure 3). The determination of protein sequence led to the eventual molecular cloning of the BMPs. BMP1 is a metalloprotease-related Drosophila tolloid (Tld). Purified procollagen C-proteinase is involved in the cleavage of the C-terminal precursor from procollagen, a biosynthetic precursor of collagens identical to BMP-1. Tolloid in Xenopus has been demonstrated to play a role in the proteolytic cleavage of a BMP antagonist called clordin. Thus, tolloid may play a critical role in the activation of inactive BMPs during morphogenesis of diverse tissue during embryogenesis and pattern formation and during regeneration of tissues in adults. Figure 1 A schematic diagram of the dimeric secreted BMP molecule showing the signal peptide, pro domain, and the active domain. The mature secreted BMP-2 dimer is held together by a single interchain disulfide bond. Signal peptide

Pro domain

Mature BMP-2

25 aa

Interchain disulfide bond

Active domain

Intrachain disulfide bonds

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Description of protein In early experiments the fact that demineralized bone matrix devoid of any cells induced de novo cartilage and bone differentiation pointed to the extracellular collagenous matrix and associated proteins as the inducer of bone. Since the extracellular matrix is insoluble, various dissociative extractants were used, including 4 M spore guanidine hydrochloride, 8 M urea containing 1 M sodium chloride and 1% sodium dodecyl sulfate buffered to pH 7.4 (Sampath and Reddi, 1981). Approximately 2±3% of the proteins were solubilized and bioassays in vivo revealed that extract alone or matrix alone was devoid of the bone induction activity. However, when the extract was combined with the inactive matrix, the osteoinductivity was restored. This critical reconstitution experiment permitted further purification and characterization of the bone morphogenetic proteins (Sampath and Reddi, 1983; Luyten et al., 1989; Wozney et al., 1988). The reconstitution experiments also demonstrated that the soluble extract and insoluble extracellular matrix substratum collaborate to initiate new cartilage and bone formation. Initial rough estimates revealed that about 1 mg of purified osteogenic protein was present in 1 kg of bone (Luyten et al., 1989). In view of this, bovine bone was used for further work. However, demineralized bovine or human matrix is not osteoinductive

Figure 2 A phylogram of the mammalian BMPs. The distinct subfamilies are grouped BMP-3, BMP-2/4, BMP-7, and CDMPs.

750 A. Hari Reddi and Dominik Haudenschild Figure 3 Alignment of amino acid sequences in the characteristics seven-cysteine domain of the mature functional BMPs. The remarkable conservation of the amino acid sequence is illustrated.

in rats. This is probably due either to the speciesspecificity of bone morphogenetic proteins or to the immunogenicity of the matrix. To investigate this question, demineralized bovine and human matrices were extracted in 4.0 M guanidine and the extracts fractionated by molecular sieve chromatography. It is noteworthy that fractions containing proteins less than 50 kDa were osteoinductive in rats when combined with inactive matrix prepared from rats; however, when inactive human or bovine matrix was implanted

with active fractions, the induction of bone was blocked. Also, if soluble fractions exceeding 100 kDa, obtained by chromatography, were added to the bioactive fractions, bone formation was inhibited. Thus, these experiments demonstrated that bone morphogenetic signals are not species-specific. Nevertheless, both the xenogeneic matrix and soluble components over 100 kDa were, perhaps, immunogenic and/or contained inhibitors or binding proteins to BMP (Sampath and Reddi, 1983).

BMP Family Further work with over a ton of bovine bone allowed the complete purification of the bone morphogenetic protein called osteogenin (Luyten et al., 1989). Amino acid sequences of tryptic peptides permitted the eventual cloning and expression of bone morphogenetic proteins (BMPs). The incisive work of Wozney (1989) and colleagues revealed the presence of BMP-2, BMP-2B (now called BMP-4) and BMP-3 (osteogenin). Osteogenic protein 1 (OP-1), cloned by Ozkaynak et al. (1990, 1992), is also called BMP-7 by others.

Discussion of crystal structure See Figure 4.

Figure 4 Deduced structural models of BMP-7 and its similarity to TGF 2 based on X-ray crystallography. The dimeric conformation is critical for biological actions.

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Important homologies The BMP family is related to TGF , and related activins and inhibins implicated in mesoderm induction and follicle-stimulating hormone release and Mullerian duct inhibitory substance (MIS) involved in the degeneration of Mullerian duct male embryos.

CELLULAR SOURCES AND TISSUE EXPRESSION

Cellular sources that produce Initially, BMPs were identified, purified and cloned from bone. With the availability of recombinant BMPs and cognate monospecific antibodies, considerable advances have accrued. BMP-2 is critical for cardiac morphogenesis (Reddi, 1998b). BMP-4 is involved in mesoderm formation in developing embryos. BMP-7 is necessary for kidney morphogenesis. BMPs have been localized by immune channel methods in smooth muscle cells, adrenal cortex, and kidney (Vukicevic et al., 1994).

RECEPTOR UTILIZATION See the chapter on BMP Receptors.

IN VITRO ACTIVITIES

In vitro findings BMPs are pleiotropic morphogens. A single BMP, such as recombinant human BMP-4 is chemotactic for human monocytes at femtomolar levels (Cunningham et al., 1992), mitogenic to cells and is bone inductive in vivo (Reddi, 1998b). BMPs are stimulatory to alkaline phosphatase activity and collagen synthesis in osteoblasts (Vukicevic et al., 1989; T.L. Chen et al., 1991). BMP-2 and BMP-3 stimulated alkaline phosphatase activity in the mouse osteoblast cell line MC3T3-E1 (Vukicevic et al., 1990; Takuwa et al., 1991; Yamaguchi et al., 1991). BMPs stimulate a chondrogenic phenotype in vivo (Chen et al., 1991). BMPs have been implicated in a wide variety of epithelial±mesenchymal interactions during development and in regeneration (Yamaguchi et al., 1991; Vainio et al., 1993; Nakashima et al., 1994; Vukicevic et al., 1994).

752 A. Hari Reddi and Dominik Haudenschild

IN VIVO BIOLOGICAL ACTIVITIES OF LIGANDS IN ANIMAL MODELS

Normal physiological roles The fact that BMPs were identified, purified, and amino acid sequence determined from extracts of bone, based on an in vivo bone induction bioassay (Wozney et al., 1988; Luyten et al., 1989; Reddi, 1998b), sets them apart from numerous cytokines identified on the basis of in vitro assays. In the in vivo bone induction assay, the three critical steps are chemotaxis of stem cells, mitosis of progenitor stem cells, and differentiation of cartilage and bone. Thus the key in vitro bioassays are chemotaxis, mitogenesis and chondrogenesis. The recombinant BMP-2 and BMP-7 have been extensively investigated as these are the two key molecules under development for human therapeutic applications in orthopedic surgery, dentistry, and oral surgery.

Species differences BMPs are highly conserved in nature in organisms as diverse as nematodes, such as Caenorhabditis elegans, insects, and humans.

PATHOPHYSIOLOGICAL ROLES IN NORMAL HUMANS AND DISEASE STATES AND DIAGNOSTIC UTILITY

Normal levels and effects The pathophysiology of BMPs is linked to their known physiological functions. It is now well documented that BMPs play a critical role in embryonic pattern formation and limb morphogenesis (Reddi, 1998a). In general, during fracture healing of long bones, BMPs are re-expressed in the callus (Nakase et al., 1994; Bostrom et al., 1996).

Role in experiments of nature and disease states BMPs have been implicated in certain human diseases, such as fibrodysplasia ossificans progressiva (also known as myositis ossificans progressiva). In

these patients there is inexplicable formation of new endochondral bone in muscle (Shafritz et al., 1996). It is also possible that heterotopic bone formation, following hip surgery, is also triggered by BMPs. A variety of ectopic ossifications in aorta, kidney, and placenta potentially implicate BMPs in diverse tissues. This is not surprising given that members of the BMP family play a critical role in cardiac, renal, and eye morphogenesis.

IN THERAPY

Preclinical ± How does it affect disease models in animals? BMPs induce de novo bone formation in heterotopic and orthotopic sites in rats, rabbits, dogs, and primates, including baboons (Reddi, 1998b; Reidel and Valentin-Opran, 1999; Cook, 1999). Recombinant BMP-2 is effective in the rabbit ulnar nonunion model and in sheep (Gerhart et al. 1993; Bostrom et al., 1996). BMPs are local cytokines functioning at the site of implantation, although they may also function in a systemic endocrine manner. Although bone grafts can stimulate new bone formation at the sites of non-union, their use in therapy is handicapped due to the limited number of available autograft harvest sites, such as iliac bone and further complications due to donor site morbidity (Younger and Chapman, 1989). Although fractures are treated by both physical and chemical approaches, including electrical fields and ultrasound, the long-term solutions must mimic the biological and mechanical microenvironment of the fracture (McKibbin, 1978; Lanyon et al., 1982; Goodship and Kenwright, 1985; Bolander, 1992; Yasko et al., 1992; Einhorn, 1995; Carter et al., 1998; Marsh, 1998).

Clinical results Human recombinant BMP-2 is being developed as a therapeutic product by the Genetics Institute. In conjunction with an absorbable collagen sponge it was found to be efficacious in augmentation of maxillary sinus floor and extensive clinical trials in orthopedic trauma are underway (Reidel and Valentin-Opron, 1999). Human osteogenic protein 1 (OP-1), also known as BMP-7, in conjunction with bovine type I insoluble collagen particles prepared from bone (Sampath and Reddi, 1983) is currently in clinical trials in tibial non-unions in both Europe and the

BMP Family United States (Cook, 1999) sponsored by StrykerBiotech. The imminent completion of the clinical trials may aid the healing of recalcitrant non-unions. However, the progress in this area is limited by the availability of optimal carriers. Collagen appears to be an ideal carrier, although research should continue on synthetic substrates with defined release kinetics. (Ma et al., 1990; Hollinger et al., 1996). It is noteworthy that the geometry of the delivery system is critical for bone induction by BMPs (Ripamonti et al., 1992). The accelerating pace of advances in the role of morphogens in the newly emerging science of tissue engineering augers well for clinical applications of BMPs. Tissue engineering is the science of manufacture of spare parts for human body based on morphogenetic cytokines, responding stem cells and extracellular matrix scaffolds (Reddi, 1998a). In fact, a prototype model has established the proof of the concept of tissue transformation in vivo with applications in surgery (Khouri et al., 1991). The evolution of tissue engineering strategies comes at a most opportune time when there is chronic shortages of donor tissues for transplantation. The advances in BMPs have provided the prototype bone tissue fabricated on the basis of cytokines. In conclusion, cytokines are at the center of the new biology of tissue engineering of other tissues, including heart and kidney.

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