Stimulation of the expression of osteogenic and chondrogenic phenotypes in vitro by osteogenin.

Abstract

Osteogenin was recently purified and the amino acid sequences of tryptic peptides were determined. Osteogenin in conjunction with insoluble coliagenous bone matrix induces cartilage and bone formation in vivo. To understand the mechanism of action of osteogenin, we examined its influence on periosteal cells, osteoblasts, fibroblasts, chondrocytes, and bone marrow stromal cells in vitro. Osteogenin stimulated alkaline phosphatase activity and collagen synthesis in periosteal cells. The cAMP response to parathyroid hormone in periosteal cells was increased by osteogenin. In primary cultures of calvarial osteoblasts, osteogenin stimulated alkaline phosphatase activity, the cAMP response to parathyroid hormone, and the synthesis of collagenous and noncollagenous proteins; however, cell proliferation was not affected. Osteogenin increased the production of sulfated proteoglycans in fetal rat chondroblasts and in rabbit articular chondrocytes. The present experiments demonstrate the significant influence of osteogenin in the stimulation of osteogenic and chondrogenic phenotypes in vitro.Bone has a considerable potential for regeneration. However, the cellular and molecular mechanisms of bone regeneration are not clear. The presence of growth and differentiation factors in bone has been demonstrated by implantation of demineralized diaphyseal bone matrix in intramuscular and subcutaneous sites and results in local cartilage and bone formation (1, 2). The developmental cascade involves the following sequential steps: chemotaxis and attachment of mesenchymal stem cells, proliferation of progenitor cells, and differentiation of cartilage, bone, and hematopoietic marrow (3-5). Osteogenin, a bone-inductive protein, was isolated by heparin affinity chromatography (6). It was further purified and the amino acid sequences of tryptic peptides were determined and found to be unique (7). The amino acid sequence of bovine osteogenin (7) was similar to the amino acid sequence deduced from the cDNA clones of one of the human bone morphogenetic proteins, bone morphogenetic protein 3 (8). Whereas recombinant human bone morphogenetic proteins induced only cartilage (8), osteogenin purified from bovine bone matrix induces cartilage and bone formation in vivo (7, 9). To understand the mechanism of action of osteogenin, we have examined its influence on periosteal cells, osteoblasts, fibroblasts, chondrocytes, and bone marrow stromal cells in vitro. We report that osteogenin stimulates the expression of the osteogenic phenotype in periosteal cells, osteoblasts, and bone marrow stromal cells and of the chondrogenic phenotype in chondroblasts. MATERIALS AND METHODSPreparation of Purified Osteogenin. Osteogenin was purified as described (7). The method utilized extraction of demineralized bovine bone matrix with 6 M urea and purification by hydroxyapatite, heparin-Sepharose affinity chromatography, and molecular sieve chromatography on Sephacryl S-200. The most active fractions were further purified by preparative SDS/PAGE under nonr...

Identification and Functional Characterization of Distinct Critically Important Bone Morphogenetic Protein-specific Response Elements in the Id1 Promoter

“…B, An EMSA of E. coli-expressed GST-Smad4 or GST-Smad5⌬MH2 using Id1-(Ϫ1070/Ϫ1025) as a 32 P-labeled probe. Unlabeled (SBE) 4 was used as competitor in A and B. C, an EMSA was performed with nuclear extracts from C2C12 cells from non-treated or treated with BMP6 (100 ng/ml) and Id1-(Ϫ1133/Ϫ1071) containing GCЈ1-4 repeats flanked by CAGC motifs as a 32 P-labeled probe.…”

BMP-6 exerts its osteoinductive effect through activation of IGF-I and EGF pathways

“…Alkaline phosphatase (AP) activity was measured over a 28 day period at specific time points (6,10,14,19,23 and 28 days) as previously described [22] and expressed as product produced/min/μg protein. Adherent cell cultures from day 19 through day 28 were stained for the presence of minerals using the method of Von Kossa as previously described [4].…”

“…BMPs were shown to be important in osteoblast differentiation [23,24] and bone formation in vivo, where they, in concert with other cytokines and matrix components, induce sequential cascade of events for chondro-osteogenesis, consisting of chemotaxis, mesenchymal proliferation and differentiation, angiogenesis, and controlled synthesis of extracellular matrix [11,18,24].…”

Recombinant human bone morphogenetic protein-2 stimulates osteoblastic maturation and inhibits myogenic differentiation in vitro.

“…Furthermore, active recombinant human bone morphogenetic protein-2 (rhBMP-2) has been produced, which formed bone tissue in vivo when it was implanted (40). Although several bone-inducing factors were purified from bone matrix, the direct action of the isolated proteins on the osteoblastic cell lineage has not been fully investigated, except for osteogenln (37,38). Furthermore, the biological action of the factors on nonskeletal tissues has not been extensively explored; since their biological activity has been evaluated only by their in vivo implantation at ectopic sites (1,13,30,39,40,41).…”