Protein extracts derived from bone can initiate the process that begins with cartilage formation and ends in de novo bone formation. The critical components of this extract, termed bone morphogenetic protein (BMP), that direct cartilage and bone formation as well as the constitutive elements supplied by the animal during this process have long remained unclear. Amino acid sequence has been derived from a highly purified preparation of BMP from bovine bone. Now, human complementary DNA clones corresponding to three polypeptides present in this BMP preparation have been isolated, and expression of the recombinant human proteins have been obtained. Each of the three (BMP-1, BMP-2A, and BMP-3) appears to be independently capable of inducing the formation of cartilage in vivo. Two of the encoded proteins (BMP-2A and BMP-3) are new members of the TGF-beta supergene family, while the third, BMP-1, appears to be a novel regulatory molecule.

Characterization of the polypeptides present in bone-inductive protein extracts from bovine bone has led to the cloning of seven regulatory molecules, six of which are distantly related to transforming growth factor .8. The three human bone morphogenetic proteins (BMPs) we describe herein, BMP-5, BMP-6, and BMP-7, show extensive sequence similarity to BMP-2, a molecule that by itself is sufficient to induce de novo bone formation in vivo. The additive or synergistic contribution of these BMP-2-related molecules to the osteogenic activity associated with demineralized bone is strongly implicated by the presence of these growth factors in the most active fractions of highly purified bone extract.Protein extracts of demineralized bone contain an activity known as bone morphogenetic protein (BMP) (1, 2). We have extensively purified this bone-inductive activity from bovine bone (3) utilizing an in vivo assay system for ectopic cartilage and bone formation (4, 5). Initial peptide sequence information derived from this highly purified extract enabled the cloning of four polypeptides, BMP-1, BMP-2, BMP-3, and BMP-4. Due to the increasing size of the BMP family of molecules described in this paper, we refer to the molecule previously called BMP-2A simply as BMP-2 and rename BMP-2B as BMP-4. Preliminary studies with these recombinant (r) human (h) molecules indicated that they all had some cartilage-forming activity in the in vivo assay system (6). Subsequent analysis has demonstrated that rhBMP-2 by itself is sufficient to initiate the developmental cascade resulting in chondrogenesis and osteogenesis in vivo (7). Further biochemical characterization of the proteins present in active BMP preparations indicated that additional molecules related to BMP-2 were present. As part of an effort to clearly define the roles of all these molecules in cartilage and bone induction, we have attempted to identify cDNA clones corresponding to each protein in the bovine (b) BMP mixture.In the present study we report the isolation ofcDNA clones encoding human BMP-5, BMP-6, and BMP-7.* These proteins represent three members of the transforming growth factor p (TGF-f3) family of growth and differentiation factors and are most closely related to the bone-inductive molecule rhBMP-2. MATERIALS AND METHODSProtein Purification and Peptide Sequence Analysis. Bovine bone extract, which was purified as in ref. dialyzed, concentrated, and subjected to SDS/PAGE under nonreducing conditions. The 28-to 30-kDa region of the gel was excised; protein was electroeluted, reduced, alkylated, and submitted to SDS/PAGE. The resulting 14-to 20-kDa region was excised; protein was electroeluted, digested with trypsin, and sequenced as described (3).RNA Preparation. RNA was isolated from the human osteosarcoma cell line U-2 OS by a modification of the Nonidet P-40 lysis method (8). Bovine bone RNA was prepared from primary cultures of fetal long bone (9). Enrichment of polyadenylylated RNA was obtained by ohgo(dT)-cellulose chromatography.cDNA Library Con...

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Clones of complementary DNA encoding the human lymphokine known as granulocyte-macrophage colony-stimulating factor (GM-CSF) were isolated by means of a mammalian cell (monkey COS cell) expression screening system. One of these clones was used to produce recombinant GM-CSF in mammalian cells. The recombinant hematopoietin was similar to the natural product that was purified to apparent homogeneity from medium conditioned by a human T-cell line. The human T-cell GM-CSF was found to be 60 percent homologous with the GM-CSF recently cloned from murine lung messenger RNA.

We purified a factor that induces bone formation >300,000-fold from guanidinium chloride extracts of demineralized bone. Fifty nanograms of highly purified protein was active in an in vivo cartilage and bone-formation assay. The activity resided in a single gel band, corresponding to a molecular mass of %30 kDa, which yielded proteins of 30, 18, and 16 kDa on reduction. The partial amino acid sequence obtained from these proteins confirmed our identification of specific factors that induce new bone formation in vivo.Bone is a complex tissue that undergoes constant remodeling in response to changing physical demands. The signals that control resorption and formation, whether from humoral or localized growth and differentiation factors, extracellular matrix, or other presently unknown controls, require much further study. One approach to studying bone development is use of in vivo ectopic bone formation-the best characterized model of which is induction by demineralized bone implanted intramuscularly or subcutaneously. During this sequence of events (i) mesenchymal cells are seen to migrate into the implant, proliferate after several days, and condense in regions. (ii) Chondroblasts, believed to be derived from the early-appearing mesenchymal cells, form a cartilaginous template in the area of presumptive bone. (iii) At 10-14 days, the cartilage hypertrophies, and the cartilage extracellular matrix is vascularized by hematopoietic and endothelial cells. (iv) The cartilage is gradually removed and replaced by bone, and at the end of 21 days an ossicle of bone, complete with marrow, has been formed. This response is localized to the implant itself. The morphological but not temporal developmental sequence is the same as seen in embryonic endochondral bone formation and adult fracture repair (1-3).This induction of the natural sequence of bone formation immediately suggested potential application for human therapeutics and for developmental studies. Thus began the search for a factor, or factors, named bone morphogenetic protein (BMP) by Urist (1), that could induce bone formation. BMP was characterized as an activity tightly bound to the matrix of demineralized bone and extractable by denaturing solvents (4). Implantation of protein itself was sufficient to induce bone, but reconstitution of the factor with a collagenous matrix (5, 6) or synthetic matrices (7, 8) enhanced sensitivity of the assay. Although purification and characterization have been hampered by the cumbersome in vivo assay, numerous reports have described osteoinductive factors (8-10). Additionally, many other growth factors, namely fibroblastic growth factor, platelet-derived growth factor, transforming growth factors 81 and /82, insulin-like growth factors I and II, and bone-derived growth factor, have been implicated in bone development by their presence in bone and their effect on cartilage and bone cells in vitro, although no direct osteoinductive role has yet been identified in vivo (for reviews, see refs. 11 and 12). We used the rat ...