Bone morphogenetic protein 2 (BMP-2) and osteogenic protein 1 (OP-1, also termed BMP-7) are potent apoptotic signals for the undifferentiated limb mesoderm but not for the ectoderm or the differentiating chondrogenic cells. They promote intense radial growth of the differentiating cartilages and disturb the formation of joints accompanied by alterations in the pattern of Indian hedgehog expression. Interestingly, the effects of these two BMPs on joint formation are found to be different. While the predominant effect of BMP-2 is alteration in joint shape, OP-1 is a potent inhibitory factor for joint formation. In situ hybridizations to check whether this finding was indicative of specific roles for these BMPs in the formation of joints reveals a distinct and complementary pattern of expression for these genes during the formation of the skeleton of the digits. While OP-1 exhibits an intense expression in the perichondrium of the developing cartilages with characteristic interruptions in the zones of joint formation, BMP-2 expression is a positive marker for the articular interspaces. These data suggest that, in addition to the proposed role for BMP-2 and OP-1 in the establishment of the anteroposterior axis of the limb, they may also play direct roles in limb morphogenesis: (1) in regulating the amount and spatial distribution of the undifferentiated prechondrogenic mesenchyme and (2) in controlling the location of the joints and the diaphyses of the cartilaginous primordia of the long bones, once the chondrogenic aggregates are established (Macias, 1997).

BMP-7-deficient mice show among other mesodermal and skeletal patterning defects, polydactyly in the hindlimbs. A more detailed analysis of the limb phenotype in BMP-7-deficient mice is reported here using in situ hybridization to monitor expression of molecules implicated in patterning processes of the developing vertebrate limb. Sonic hedgehog (Shh) is expressed normally, but Hoxd-13 expression in limb mesenchyme is lower in BMP-7 mutant limbs. Hoxd-11 expression domains are also contracted and decreased in intensity in mutant limbs, suggesting that 5' genes of the Hoxd cluster are coordinately downregulated, while another BMP, BMP-2, which can be activated by Shh, is unaffected. The mutant limb buds are broader than normal buds, and fibroblast growth factor Fgf-8 is expressed throughout the extended ridge. However, expression of the homeobox gene Msx-1, which has been shown to be involved in epithelial-mesenchymal interactions during limb development, is decreased in the mesenchyme of BMP-7 mutant limbs. Taken together, these data suggest that BMP-7 is involved in regulating proliferation and/or epithelial-mesenchymal interactions in the developing limb (Hofmann, 1997).

Osteogenic protein-1 (OP-1, BMP-7), a bone morphogenetic protein in the transforming growth factor-beta superfamily, induces endochondral bone formation in vivo, but the mechanism of action of OP-1 in osteogenesis is not yet established. Three murine clonal cell lines in different stages of differentiation exhibit graded responses to recombinant human OP-1: the mouse embryonal carcinoma ATDC5 cell, with potential for chondroblastic differentiation; the osteoblast-like MC3T3-E1 cell derived from mouse calvaria, and the multipotent fibroblastic C3H10T1/2 cell derived from mouse embryo connective tissue. OP-1 acts on early stage mesenchymal progenitor cells (ATDC5, C3H10T1/2) to induce chondroblastic differentiation. OP-1 also strongly enhances the osteoblastic phenotype of committed osteoblasts (MC3T3-E1), possibly explaining its induction of the endochondral ossification cascade in vivo. Markers of osteoblastic, chondroblastic, and adipocytic differentiation are compared. OP-1 is strongly mitogenic for ATDC5, showing dose-dependent induction of Alcian blue staining, alkaline phosphatase activity, and mRNA expression for collagen types II and IX, and matrix Gla protein. MC3T3-E1 cells do not proliferate or stain with Alcian blue in response to OP-1, but express elevated levels of alkaline phosphatase and osteocalcin. While low-dose OP-1 treatment of C3H10T1/2 induces only adipocyte-like cells filled with lipid droplets, a high dose (500 ng/ml) causes the same cells to also exhibit chondrocytic properties. Thus, OP-1 can induce differentiation along elements of the endochondral ossification pathway according to the stage and potential of the target cell (Asahina, 1996).

Glucocorticoids (GCs) at physiological concentrations promote osteoblast differentiation from fetal calvarial cells, calvarial organ cultures, and bone marrow stromal cells; however, the cellular pathways involved are not known. Bone morphogenetic proteins (BMPs) are recognized as important mediators of osteoblast differentiation. Specific roles for individual BMPs during postembryonic membranous bone formation have yet to be determined. GCs potentiate the osteoblast differentiation effects of BMP-2 and BMP-4, but not of BMP-6, which, by itself, is the most potent of the three. Fetal rat secondary calvarial cultures were used to study the role of BMP-6 during early osteoblast differentiation. Treatment with the GC triamcinolone results in a 5- to 8-fold increase in BMP-6 steady-state messenger RNA levels, peaking at 12 h. In contrast, BMPs-2, -4, -5, -7, and transforming growth factor (TGF)-beta1 messenger RNA levels increase by less than 2-fold, after GC treatment. BMP-6 protein secretion increases 6- to 7-fold by 12 h and 12-fold by 24 h. Treatment of cells with oligodeoxynucleotides antisense to BMP-6 diminishes secretion of BMP-6 protein and significantly inhibits the GC-induced differentiation, as determined by a 10-fold decrease in the number of mineralized bone nodules, compared with controls that were treated with sense oligonucleotides or no oligonucleotides. The antisense oligonucleotide inhibition of differentiation is rescued by treatment with exogenous recombinant human BMP-6. It is concluded that GC-induced differentiation of osteoblasts from the pluripotent precursors is mediated, in part, by BMP-6. These results suggest that BMP-6 has an important and unique role during early osteoblast differentiation (Boden, 1997).

Bone morphogenetic proteins (BMPs) have the unique ability to convert mesenchymal cells into matrix-producing osteoblasts. To understand the mechanism(s) by which a BMP produces a multitude of effects on bone cells, the effects of recombinant human osteogenic protein (OP)-1 (referred to as BMP-7) were examined on the insulin-like growth factor (IGF) regulatory system, an important growth factor system in bone. After 48 h of treatment, OP-1 increases the level of IGF-II (3- and 2-fold, respectively) in the conditioned medium (CM) of SaOS-2 and TE85 human osteosarcoma cells with osteoblastic characteristics, whereas IGF-I levels are low to undetectable in the CM of either cell type. OP-1 treatment has no significant effect on the messenger RNA (mRNA) level for type 1 and type 2 IGF receptors. In TE85 and SaOS-2 cells, 100 ng/ml OP-1 increases the level of IGF binding protein (BP)-3 more than 10-fold, decreases the IGFBP-4 level by 50%, and increases the level of the 29-32.5 kDa IGFBP-5 3-fold in the CM. The effect of OP-1 on IGFBP production is time and dose dependent. The OP-1 induced changes in the levels of IGFBPs are associated with decreased IGFBP-3 and -5 protease activity (29% and 71%, respectively) and proportional changes in IGFBP mRNA levels. OP-1 increases the level of IGFBP-3 mRNA (2- and 10-fold, respectively, after 4 and 24 h of treatment) and of IGFBP-5 mRNA (more than 5-fold after 24 h of treatment) but decreases the level of IGFBP-4 mRNA. OP-1 treatment has no effect on IGFBP-4 protease activity. These results collectively demonstrate that OP-1 can act locally by modulating the IGF regulatory system, suggesting that the mitogenic/differentiative effect of OP-1 on human bone cells may in part be mediated via IGF-II by increasing its secretion, and by regulating the balance between the stimulatory (e.g. IGFBP-5) and inhibitory (e.g. IGFBP-4) classes of IGFBPs both at the level of production (mRNA) and at the level of degradation but not by up-regulating the IGF receptor (Knutsen, 1995).

The effect of recombinant human osteogenic protein-1 (OP-1, or bone morphogenetic protein-7) on growth and maturation was studied in day 11, 15 and 17 chick sternal chondrocytes in high density monolayers, suspension and agarose cultures for up to 5 weeks. OP-1 dose-dependently promotes chondrocyte maturation associated with enhanced alkaline phosphatase activity, and increases mRNA levels and protein synthesis of type X collagen in both the presence and absence of serum. In serum-free conditions, OP-1 promotes cell proliferation and chondrocyte maturation, without requiring either thyroid hormone or insulin, agents known to support chick chondrocyte differentiation in vitro. When grown in agarose under the same conditions, TGF-beta 1 and retinoic acid neither initiated nor promoted chondrocyte differentiation. The results demonstrate that OP-1, as the sole medium supplement, supports the maturation of embryonic chick sternal chondrocytes in vitro (Chen, 1995).

The definitive mammalian kidney forms as the result of reciprocal interactions between the ureteric bud epithelium and metanephric mesenchyme. Since osteogenic protein 1 (OP-1/bone morphogenetic protein 7) is expressed predominantly in the kidney, its involvement during metanephric induction and kidney differentiation was examined. OP-1 mRNA is expressed in the ureteric bud epithelium before mesenchymal condensation and is subsequently seen in the condensing mesenchyme and during glomerulogenesis. Mouse kidney metanephric rudiments cultured without ureteric bud epithelium fail to undergo mesenchymal condensation and further epithelialization, while exogenously added recombinant OP-1 is able to substitute for ureteric bud epithelium in restoring the induction of metanephric mesenchyme. This OP-1-induced nephrogenic mesenchyme differentiation follows a developmental pattern similar to that observed in the presence of the spinal cord, a metanephric inducer. Blocking OP-1 activity using either neutralizing antibodies or antisense oligonucleotides in mouse embryonic day 11.5 mesenchyme, cultured in the presence of metanephric inducers or in intact embryonic day 11.5 kidney rudiment, greatly reduces metanephric differentiation. These results demonstrate that OP-1 is required for metanephric mesenchyme differentiation and plays a functional role during kidney development (Vukicevic, 1996).