Bone Morphogenetic Protein: The Molecularization of Skeletal System Development

Progress in science appears part and parcel with the introduction of new experimental methods. Three landmarks in the history of experimental embryology emerged from new methods. The first was von Baer's1 experiments showing how the chick embryo develops from a more general to a specific, detailed body design. The second was Spemann's2 microsurgical experiments on embryonic tissues showing the effects of one tissue upon another leading to differentiation of a third, and hypothetical chemical organizers of embryonic development. The third was in situ hybridization (Pardue et al., 1970)3 modified and improved by Harland4 for localization of bone morphogenetic protein (BMP) including BMP receptors.5-10 BMP is a hydrophobic low molecular weight polypeptide42 that evolved in species in the fossil record 500 million years ago. It is distinguished by heterotopic bone development in response to implants of nanogram quantities with a carrier in muscle. In this issue of JBMR, Nifuji et al.11 present circumstantial evidence of BMP-2– and -4–induced skeletal system development. The evidence is based on whole mounts of chick embryos and in situ hybridization, using digoxigenin-labeled mRNA probes to localize endogenous BMP-2 and BMP-4. Exogenous BMP (rhBMP-2) was implanted in pellets of glass in dorsal mesoderm. Transcripts for BMP-4 ligand and its receptors were expressed in dorsal ectoderm and mesoderm. The implants of exogenous BMP-4 and BMP-2 produced anomalies of vertebra, ribs, and scapulae. A basic assumption is that BMP generates form and controls the morphogenetic or predifferentiation stages of development. The cytodifferentiation phase delineates the time of differentiation of chondrocytes, adipocytes, osteocytes, and fibrocytes. Before the cytodifferentiation phase takes place, cells aggregate and proliferate in condensation centers. BMP concentration gradients arise in condensation centers.12-14 Nifuji et al.11 show that BMP-4 and BMP-2 expression occurs earlier than heretofore shown, and in reviewing the literature, they point out that previous research on skeletal cell condensation in the developing mammalian embryos appeared coincidental with the expression of BMP-5.7 The authors also point out that BMP-2 and BMP-4 are mammalian homologs of dpp and 60 A proteins associated with dorsoventral patterning in the fruit fly embryo, and of activan in the amphibian Xenopus.15-21 Similarly, in sea urchins,22 silkworms,23 and earthworms,24 to baboons25 and human beings,26 BMP or BMP antecedent molecules pervade the earliest stages of development of all animal life. BMPs have been observed in nearly all developing visceral and somatic organs, i.e., brain and sympathetic neurons,27-31 heart, liver, lung, skin, hair follicles,32, 33 craniofacial structures,34 branchial arches,35 placenta,36 and skeletal elements.37-41 Improved methods of in situ hybridization and immunocytochemistry made it possible to colocalize BMP-2 to BMP-4 and two number I BMP receptors in various extraskeletal, paraskeletal, and skeletal tissues. Hogan8 proposed that, depending on the concentration, at low levels BMP would stimulate cell proliferation, whereas at high levels BMP might promote differentiation. In embryogenesis, BMPs appeared to be omnipresent, as essential as components of the cytoskeleton, i.e., actin, tublin, filament networks, vitamin D, binding protein, depactin, actophorin, villin, β-actinin, etc. Activin, inhibin, and retinoic acid are also closely associated with BMP and related proteins. In postfetal mammalian species, BMP-2 to BMP-7 are expressed in bone generation, regeneration, modeling, and remodeling of cartilage and bone, including ligament or tendon connective tissue insertions. In general, depending on concentration gradients and locations, BMP inhibits cell proliferation and induces cytodifferentiation. The history of BMP evolved from observations of allogeneic bone matrix–induced cartilage and bone development in mammalian species.13, 42, 43 The name stems from the demonstration of a hydrophobic noncollagenous glycoprotein that induced mesenchymal-type cells to differentiate into a spherical ossicle with a medulla containing hematopoietic bone marrow.12-14 The isolation of BMP from bone matrix, with the aid of chaotropic sovents,13 and the identity of BMP were demonstrated by bioassays in adult mammals. Lyophilized protein fractions, either as purified native or as recombinant proteins, were implanted in the hind quarter muscles of mice. Soluble BMP was also adsorbed to deactivated demineralized bone matrix and implanted in muscle tissue in rats. Transforming growth factor-β (TGF-β) was classified as a cytokine found in demineralized bone matrix in larger quantities than BMPs.44 BMP was classified as a morphogen.45 The significance of BMP in developmental biology and evolution was appreciated when Drosophila sequences were amplified with PCR, and molecules were generated with primary structures resembling BMP-5, -6, and -7.6, 19, 46 Classified as a morphogen, BMP is a molecule diffusing in a concentration gradient detected by cells to activate or repress genes.45 Diffusing from a central point related to a source, BMP may control the development of framework and three-dimensional orientation in vertebrates, as well as invertebrates. The framework may be composed of many different kinds of exo- or endoskeletal molecules, collagen, chitin, fibroin, elastin, sclerites, and other substances to reach near perfection in cartilage and bone in the highest orders of vertebrate species.47 In a heterotropic site in muscle, BMP induces perivascular mesenchymal-type cells to switch from a fibroprogenitor to osteoprogenitor pathway of development. No other assay system is known to provide more conclusive evidence of BMP. Names other than BMP, i.e., DPP-Vg protein,27 osteogenic protein-1 (BMP-7), and osteogenin (BMP-3) are among others to appear in the literature. TGF-β, epidermal growth factor (EGF), insulin-like growth factor (ILGF),56 and other growth factors may synergize the response to BMP but fail to induce heterotopic bone formation. The name BMP relates to a role in morphogenesis, the central problem of bioscience. The name BMP also refers to the morphogenetic phase that precedes the cytodifferentiation phase of bone development.5 In the morphogenetic phase, cells disaggregate, migrate, and reaggregate in condensation centers. In the morphogenetic phase, the size, shape, and local constraints upon growth are outlined. In the first 72 h, bromodeoxyuridine (BrdU), a thymidine analog, inhibits bone morphogenesis by blocking chondro-osteogenetic DNA transcription; mesenchymal-type cells proliferate but do not differentiate under the influence of BrdU. If cells are exposed to BrdU for 72 h, after completion of the cytodifferentiation phase, BrdU does not inhibit chondro-osteogenetic progenitor cell differentiation, and bone development occurs as normal. The same phase and time-dependent relationships are noted with irradiation, and with nonsteroidal anti-inflammatory drug (NSAID) treatment in experimental animals and patients with heterotopic bone formation. Once past the morphogenetic phase, not before, the capacity to differentiate into cartilage and bone is heritable and uninhibited.42, 43, 47-49 In a series of epochal experiments, Wang et al.50 and Wozney et al.51-54 digested partially purified BMP with trypsin, and the amino acid sequences of the tryptic peptides were used to derive and express cDNA clones. Four isoforms of BMP were produced in a culture of hamster ovarian cells by recombinant gene technology. The recombinant proteins in the mixture were fractionated and purified; more than 15 different BMP molecules have been isolated to date.49, 53 For bioassay, the proteins were recombined with deactivated rat bone matrix and implanted in muscle in allogeneic rats. Neither the primary structure of native BMP nor the active domain of BMP have been characterized in physiochemical terms. Native BMP has about 10 times more activity measured in terms of quantity of induced bone development than any one of the presently isolated recombinant BMPs.52 BMP-1, neither a cytokine, mitogen, nor morphogen, was recently identified as procollagen C-proteinase (PCP) which cleaves COOH propeptides of procollagens I, II, and III to yield the major fibrous components of vertebrate bone matrix. Kessler et al.55 also suggested that PCP may link bone synthesis to genes involved in pattern formation. rhBMP and TGF-β may show as much as 25–35% identity in amino acid composition, but not necessarily in sequences. Native BMP has not yet been purified to homogeneity. The question of whether TGF-β may be a contaminant with synergistic action on induced bone formation remains to be answered. Whether the active domain of native BMP includes any peptide sequences characteristic of TGF-β would be revealed by further purification of native BMP. The primary target of BMP is not known. Wall and Hogan35 observed substantial evidence that in vertebrates BMP plays a key role in mesoderm formation and differentiation of the nervous system. At H-H stage 12 and later in chick embryo development, BMP-4 regulated signaling molecules in branchial arch development. BMP-2 and BMP-4 may model and control growth of skeletal elements with both molecules recruiting nonchondrogenic precursors to a chondrogenic fate.39 In chick embryo limb buds, beads of BMP-2, FGF-2, and TGF-β modified digital ray formation.36 Prior to implantation of a BMP-2 pellet (50–70 μm in length) expression patterns of BMP-2 and BMP-4, including type I receptors, were localized by in situ hybridization. Transcripts of both BMP-4 ligands and its receptors were localized in the dorsal ectoderm and mesoderm. Anomalies developed at the site of implantation in vertebrae, ribs, and scapulae. Employing the bead implant method previously designed for retinoic acid,15 Nifuji et al.11 implanted pellets of glass coated with exogenous BMP-2 and BMP-4. The BMP ligands and their receptors were colocalized in the dorsal ectoderm and mesoderm, but not in the ventral part of the somite. Extreme changes in BMP concentration gradients culminated in malformations of the axial skeleton. Thus, a possible link was observed between BMP expression patterns and axial skeleton development. The link was not observed for BMP-7 (OP-1), TGF-β1, or bFGF. The response was time dependent, occurring only on day 2 but not on day 3 of incubation, possibly a manifestation of competence. In systems in vitro, EGF and various other growth factors augmented cell proliferation but failed to initiate cartilage development.56 The quantity of BMP producing anomalies in the chick embryo axial skeleton was in the concentration range of 0.1–100 ng/ml.11 These quantities were not detectable by the heterotopic bone assay in adult mammals. The chick assay, however, was validated by negative effects of TGF-β1, FGF, and OP-1 (BMP-7) controls in comparable concentration ranges. The concentrations of the BMP receptors would be even lower than for the ligand. The appearance of BMP and two BMP-II receptors, so early in embryogenesis, fetal, and postfetal life in so many organ systems and in the cytoplasm of so many different cells of invertebrate as well as vertebrate species, invites investigation of BMP transmission through the cytoskeleton, the inconspicuous internal framework of all cells.57 Connections with the genome through the cytoskeleton could be important for investigation with transmission electron microscopy and radioisotope-labeled BMP, including blocking and activating agents. The above cited literature and editorial speculations are just the beginning of the molecularization of knowledge of skeletal system development.