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 existenceofthedemineralizedmatrixhasbeenknown for over a century, but a critical discovery was made in the 1960s when it was found that the formation of Morphogenesis is the developmental cascade of new bone could be induced by demineralized, pattern formation and body plan establishment, lyophilized segments of bone (Urist, 1965). The including the symmetry of most bilateral structures induction of new bone by demineralized extracellular and the asymmetry of some unilateral anatomical matrix occurs as a result of a sequential develop- sites, culminating in the adult form. Morphogenetic mentalcascade(ReddiandHuggins,1972;Reddiand proteins initiate morphogenesis. The first morpho- Anderson, 1976; Reddi, 1981), the key steps of which gens in mammalswere identifiedin adult bonetissue. arethechemotaxisofprogenitorstemcells,mitosisof Bone has considerable potential for regeneration and progenitor cells, and then differentiation first into repair. The biochemical basis for this is the family of cartilageandthenreplacementofcartilagebybone,a cytokines/morphogens called bone morphogenetic process commonly known as endochondral ossifica- proteins (BMPs). BMPs initiate, promote, and main- tion.Implantationofthedemineralizedmatrixresults tain cartilage and bone morphogenesis, differentia- in interaction and binding of plasma fibronectin to tion, and regeneration. This prototype paradigm implantedcollagenousmatrix(WeissandReddi,1980). demonstrates that a single cytokine signal can Peptides of fibronectin are chemotactic for mesen- function in prenatal development, postnatal growth, chymal cells. and regeneration of bone in the adult. BMPs have It was found that the migratory mesenchymal cells actionsbeyondboneineye,heart,kidney,skin,tooth, attached to the collagenous matrix and then andothertissuedevelopment.Thepleiotropicactions proliferated and the daughter cells emerged as chon- are mediated by discrete thresholds of concentration droblasts on day 5. With continued progression, of BMPs. The accrued knowledge may be used in chondrocytes were abundant on day 7. The chon- clinicalapplicationsofBMPsindentistry,orthopedic drocytes hypertrophied on days 8–9 and the surgery, and plastic and reconstructive surgery. 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 BACKGROUND alkaline phosphatase initially, and later, osteocalcin (also known as bone gla protein). With continued Discovery remodeling, hematopoietic marrow differentiation was evident in the ossicle. This entire sequence is Unlike most tissues in the body, bone has consider- a recapitulation of the stages of the embryonic limb able potential for repair and regeneration. In fact, bud development and differentiation (Reddi and bone grafts with associated hematopoietic cells are Anderson, 1976). routinelyusedtostimulate bonerepairin non-unions Thebioassayforboneinductionisbasedoninvivo of long bone fractures, although the molecular basis implantation. The demineralized bone matrix is in of these bone grafts is still not fully understood. The the solid state. Dissociative extractants, such as 4M 748 A. Hari Reddi and Dominik Haudenschild guanidine hydrochloride or 8M urea containing reductionindithiothreitolormercaptoethanolresults 1% NaCl and 1% sodium dodecyl were used to in loss of biological activity (Luyten et al., 1989). solubilize proteins. The soluble proteins were recon- stituted with insoluble collagenous matrix and bioassayed. This key advance (Sampath and Reddi, 1981,1983)wasakeystepintheeventualpurification GENE AND GENE REGULATION of bone morphogenetic protein (BMP). The BMPs are dimeric and the disulfide bonds are critical The incisive investigation of Wozney and colleagues for their activity (Wozney et al., 1988; Luyten et al., (1988) cloned BMP-2, BMP-2B (now called BMP-4), 1989). and BMP-3 (also called osteogenin). There are over 30membersintheBMPfamily,althoughinmammals there appear to be only about 15 BMPs (Table 1). Alternative names Ozkaynak and colleagues (1990) cloned osteogenic protein 1 (also known as BMP-7). The genes for CertainBMPsarealsoknownasosteogenin(BMP-3) BMPs revealed that they are induced by BMP (Luyten et al., 1989), osteogenic protein-1 (OP-1) treatment indicating an auto-stimulable. Two novel (Ozkaynak et al., 1991). genes related to BMPs were isolated from a cDNA library obtained from calf articular cartilage, and calledcartilage-derivedmorphogeneticproteins1and Structure 2 (Chang et al., 1994). These were isolated indepen- dentlybyLeeandcolleagues(Stormetal.,1994),and TheBMPsaredimericmoleculeswithasinglecritical called growth/differentiation factor 5 (GDF-5) and interchain disulfide bond. Disruption of this bond by GDF-6.BMP-4 stimulatedthe expression ofTGF(cid:12)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 BMP-3 Osteogenin BMP-3 4 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 – Others Growth/differentiation factor 2 (GDF-2) BMP-9 BMP-10 BMP-10 Growth/differentiation factor 11 (GDF-11) BMP-11 CDMP Cartilage-derived morphogenetic protein 1 (CDMP-1), BMP-14 20 growth/differentiation factor 5 (GDF-5) or Cartilage-derived morphogenetic protein 2 (CDMP-2), BMP-13 – growth/differentiation factor 6 (GDF-6) or Cartilage-derived morphogenetic protein 3 (CDMP-3), BMP-12 – growth/differentiation factor 7 (GDF-7) or BMP-15 BMP-15 Note:BMP-1isnotaBMPfamilymemberwithsevencanonicalcysteines.Itisaprocollagen-CproteinaserelatedtoDrosophila tolloidandmayplayaroleinmodulatingBMPactionsbyproteolysisofBMPantagonists/bindingproteins,suchasnoggin, chordin,andgremlin. BMP Family 749 indicating that members of the BMP family cross- Description of protein regulate each other (Cunningham et al., 1992). In early experiments the fact that demineralized bone Chromosome location matrix devoid of any cells induced de novo cartilage and bone differentiation pointed to the extracellular collagenous matrix and associated proteins as the See Table 1. inducer of bone. Since the extracellular matrix is insoluble, various dissociative extractants were used, including 4M spore guanidine hydrochloride, 8M PROTEIN urea containing1Msodium chlorideand1%sodium dodecyl sulfate buffered to pH 7.4 (Sampath and Sequence Reddi, 1981). Approximately 2–3% of the proteins were solubilized and bioassays in vivo revealed that EachBMPmonomercontainssevencysteineresidues, extract alone or matrix alone was devoid of the bone sixofwhichareinvolvedinintrachaindisulfidebonds induction activity. However, when the extract was andonedemonstratedinaninterchaindisulfidebond combined with the inactive matrix, the osteoinduc- (Figure1).TheBMPfamilymembersareinitiallysyn- tivity was restored. This critical reconstitution thesized as a precursor of 400 amino acids, then pro- experiment permitted further purification and char- cessedtoamaturemoleculeofabout110aminoacids. acterization of the bone morphogenetic proteins The alignment of amino acids among the 15 mem- (Sampath and Reddi, 1983; Luyten et al., 1989; bers of the BMP family, BMP-2 through BMP-15, Wozney et al., 1988). The reconstitution experiments reveals the characteristic motifs (Figure 2 and also demonstrated that the soluble extract and in- Figure 3). The determination of protein sequence led solubleextracellularmatrixsubstratumcollaborateto totheeventualmolecularcloningoftheBMPs.BMP- initiate new cartilage and bone formation. 1isametalloprotease-relatedDrosophilatolloid(Tld). Initial rough estimates revealed that about 1mg of Purified procollagen C-proteinase is involved in the purified osteogenic protein was present in 1kg of cleavage of the C-terminal precursor from procolla- bone (Luyten et al., 1989). In view of this, bovine gen, a biosynthetic precursor of collagens identical to bone was used for further work. However, deminer- BMP-1. Tolloid in Xenopus has been demonstrated alized bovine or human matrix is not osteoinductive to play a role in the proteolytic cleavage of a BMP antagonist called clordin. Thus, tolloid may play a criticalrolein theactivationofinactive BMPsduring morphogenesisofdiversetissueduringembryogenesis Figure 2 A phylogram of the mamma- and pattern formation and during regeneration of lian BMPs. The distinct subfamilies are tissues in adults. grouped BMP-3, BMP-2/4, BMP-7, and CDMPs. 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 Pro domain Active domain peptide Mature BMP-2 Intrachain disulfide bonds Interchain disulfide bond 25 aa 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 species- with active fractions, the induction of bone was specificity of bone morphogenetic proteins or to blocked.Also,ifsolublefractionsexceeding100kDa, the immunogenicity of the matrix. To investigate this obtained by chromatography, were added to the question, demineralized bovine and human matrices bioactive fractions, bone formation was inhibited. were extracted in 4.0M guanidine and the extracts Thus, these experiments demonstrated that bone fractionated by molecular sieve chromatography. It morphogeneticsignalsarenotspecies-specific.Never- is noteworthy that fractions containing proteins less theless, both the xenogeneic matrix and soluble than 50kDa were osteoinductive in rats when combi- components over 100kDa were, perhaps, immuno- nedwithinactivematrixpreparedfromrats;however, genic and/or contained inhibitors or binding proteins wheninactivehumanorbovinematrixwasimplanted to BMP (Sampath and Reddi, 1983). BMP Family 751 Further work with over a ton of bovine bone Important homologies allowed the complete purification of the bone mor- phogenetic protein called osteogenin (Luyten et al., The BMP family is related to TGF(cid:12), and related 1989). Amino acid sequences of tryptic peptides per- activins and inhibins implicated in mesoderm induc- mitted the eventual cloning and expression of bone tion and follicle-stimulating hormone release and morphogeneticproteins(BMPs).Theincisiveworkof Mullerian duct inhibitory substance (MIS) involved Wozney (1989) and colleagues revealed the presence in the degeneration of Mullerian duct male embryos. 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 CELLULAR SOURCES AND by others. TISSUE EXPRESSION Cellular sources that produce Discussion of crystal structure Initially, BMPs were identified, purified and cloned See Figure 4. frombone.WiththeavailabilityofrecombinantBMPs 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 Figure 4 Deduced structural models of BMP-7 and its is necessary for kidney morphogenesis. BMPs have similarity to TGF(cid:12)2 based on X-ray crystallography. The beenlocalizedbyimmunechannelmethodsinsmooth dimeric conformation is critical for biological actions. 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 alkalinephosphatase activity inthe mouse osteoblastcelllineMC3T3-E1(Vukicevicetal.,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 develo- pment and in regeneration (Yamaguchi et al., 1991; Vainioetal.,1993;Nakashimaetal.,1994;Vukicevic et al., 1994). 752 A. Hari Reddi and Dominik Haudenschild IN VIVO BIOLOGICAL these patients there is inexplicable formation of new endochondral bone in muscle (Shafritz et al., 1996). ACTIVITIES OF LIGANDS IN It is also possible that heterotopic bone formation, ANIMAL MODELS following hip surgery, is also triggered by BMPs. A variety of ectopic ossifications in aorta, kidney, and Normal physiological roles placenta potentially implicate BMPs in diverse tis- sues.This is notsurprising given that members ofthe BMP family play a critical role in cardiac, renal, and The fact that BMPs were identified, purified, and eye morphogenesis. 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 IN THERAPY identifiedon the basis ofin vitroassays. Inthe in vivo bone induction assay, the three critical steps are Preclinical – How does it affect chemotaxis of stem cells, mitosis of progenitor stem disease models in animals? cells, and differentiation of cartilage and bone. Thus the keyin vitrobioassaysare chemotaxis,mitogenesis and chondrogenesis. The recombinant BMP-2 and BMPs induce de novo bone formation in heterotopic BMP-7havebeenextensivelyinvestigatedastheseare and orthotopic sites in rats, rabbits, dogs, and the two key molecules under development for human primates, including baboons (Reddi, 1998b; Reidel therapeutic applications in orthopedic surgery, and Valentin-Opran, 1999; Cook, 1999). Recom- dentistry, and oral surgery. binant BMP-2 is effective in the rabbit ulnar non- union model and in sheep (Gerhart et al. 1993; Bostrom et al., 1996). BMPs are local cytokines Species differences functioning at the site of implantation, although they may also function in a systemic endocrine manner. BMPs are highly conserved in nature in organisms as Although bone grafts can stimulate new bone diverse as nematodes, such as Caenorhabditis elegans, formation at the sites of non-union, their use in insects, and humans. therapy is handicapped due to the limited number of available autograft harvest sites, such as iliac bone andfurthercomplicationsduetodonorsitemorbidity PATHOPHYSIOLOGICAL ROLES (Younger and Chapman, 1989). Although fractures IN NORMAL HUMANS AND are treated by both physical and chemical appro- aches, including electrical fields and ultrasound, the DISEASE STATES AND long-term solutions must mimic the biological DIAGNOSTIC UTILITY and mechanical microenvironment of the fracture (McKibbin,1978; Lanyon et al., 1982; Goodship and Normal levels and effects Kenwright, 1985; Bolander, 1992; Yasko et al., 1992; Einhorn, 1995; Carter et al., 1998; Marsh, 1998). The pathophysiology of BMPs is linked to their known physiological functions. It is now well docu- mented that BMPs play a critical role in embryonic Clinical results pattern formation and limb morphogenesis (Reddi, 1998a). In general, during fracture healing of long Human recombinant BMP-2 is being developed as a bones, BMPs are re-expressed in the callus (Nakase therapeutic product by the Genetics Institute. In et al., 1994; Bostrom et al., 1996). conjunctionwithanabsorbablecollagenspongeitwas found to be efficacious in augmentation of maxillary Role in experiments of nature and sinus floor and extensive clinical trials in orthopedic trauma are underway (Reidel and Valentin-Opron, disease states 1999). Human osteogenic protein 1 (OP-1), also known as BMP-7, in conjunction with bovine type I BMPs have been implicated in certain human insoluble collagen particles prepared from bone diseases, such as fibrodysplasia ossificans progressiva (Sampath and Reddi, 1983) is currently in clinical (also known as myositis ossificans progressiva). In trials in tibial non-unions in both Europe and the BMP Family 753 United States (Cook, 1999) sponsored by Stryker- arechemotacticforhumanmonocytesandstimulatetransform- Biotech. The imminent completion of the clinical ing growth factor-B1 mRNA expression. Proc. Natl Acad. Sci. USA89,11740–11744. trials may aid the healing of recalcitrant non-unions. Einhorn, T. A. (1995). Current concepts review enhancement of However, the progress in this area is limited by the fracture-healing.JBoneJointSurg.77,940–952. availability of optimal carriers. Collagen appears to Gerhart,T.N.,Kirker-Head,C.,andKriz,M.J.(1993).Healing beanidealcarrier,althoughresearchshouldcontinue segmental femoral defects in sheep using recombinent human on synthetic substrates with defined release kinetics. bonemorphogeneticprotein.Clin.Orthop.Rel.Res.293,317–326. Goodship, A. E., and Kenwright, J. (1985). The influence of (Ma et al., 1990; Hollinger et al., 1996). It is note- induced microenvironment upon the healing of experimental worthy that the geometry of the delivery system is tibialfractures.J.BoneJointSurg.67B,650–655. criticalforboneinductionbyBMPs(Ripamontietal., Hollinger, J., Mayer, M., Buck, D., Zegzula, H., Ron, E., 1992). Smith, J., and Wozney, J. M. (1996). Poly (a-hydroxy acid) The accelerating pace of advances in the role of carrierfordeliveringrecombinanthumanbonemorphogenetic protein-2forboneregeneration.J.ControlledRelease39,287– morphogens in the newly emerging science of tissue 304. engineering augers well for clinical applications of Khouri,R.K.,Koudsi,B.,andReddi,A.H.(1991).Tissuetrans- BMPs. Tissue engineering is the science of manufac- formationintoboneinvivo.JAMA266,1953–1955. ture of spare parts for human body based on Lanyon, L. E., Goodship, A. E., Pye, C. J., and MacFie, J. H. morphogenetic cytokines, responding stem cells and (1982). Mechanically adaptive bone remodeling. J. 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