RECOMBINANT BONE MORPHOGENETIC PROTEIN (BMP)-2 REGULATES COSTOCHONDRAL GROWTH-PLATE CHONDROCYTES AND INDUCES EXPRESSION OF BMP-2 AND BMP-4 IN A CELL MATURATION-DEPENDENT MANNER

This study examined the effect of recombinant human bone morphogenetic protein-2 on several parameters of growth, differentiation, and matri x synthesis and on the endogenous production of mRNA of bone morphogen etic proteins 2 and 4 by growth plate chondrocytes in culture. Chondro cytes from resting and growth zones were obtained from rat costochondr al cartilage and cultured for 24 or 48 hours in medium containing 0.05 -100 ng/ml recombinant human bone morphogenetic protein-2 and 10% feta l bovine serum. Incorporation of [H-3]thymidine, cell number, alkaline phosphatase specific activity, incorporation of [H-3]proline into col lagenase-digestible protein and noncollagenase-digestible protein, and incorporation of [S-35]sulfate were assayed as indicators of cell pro liferation, differentiation, and extracellular matrix synthesis. mRNA levels for bone morphogenetic proteins 2 and 4 were determined by Nort hern blot analysis. Recombinant human bone morphogenetic protein-2 inc reased the incorporation of [H-3]thymidine by quiescent resting-zone a nd growth-zone cells in a similar manner, whereas it had a differentia l effect on nonquiescent cultures. At 24 and 48 hours, 12.5-100 ng/ml recombinant human bone morphogenetic protein-2 caused a dose-dependent increase in cell number and DNA synthesis in resting-zone chondrocyte s. No effect was seen in growth-zone cells. Recombinant human bone mor phogenetic protein-2 stimulated alkaline phosphatase specific activity in resting-zone chondrocytes in a bimodal manner, causing significant increases between 0.2 and 0.8 ng/ml and again between 25 and 100 ng/m l. In contrast, alkaline phosphatase specific activity in growth-zone chondrocytes was significantly increased only between 12.5 and 100 ng/ ml. Recombinant human bone morphogenetic protein-2 increased the produ ction of both collagenase-digestible protein and noncollagenase-digest ible protein by resting-zone and growth-zone cells, but incorporation of [S-35]sulfate was unaffected. Administration of recombinant human b one morphogenetic protein-2 also increased incorporation of [H-3]uridi ne in both resting-zone and growth-zone chondrocytes; these cells prod uced mRNA for bone morphogenetic proteins 2 and 4. Bone morphogenetic protein-2 mRNA levels in both resting-zone and growth-zone chondrocyte s increased in the presence of recombinant human bone morphogenetic pr otein-2; however, bone morphogenetic protein-4 mRNA levels in growth-z one cells decreased under its influence, and those in resting-zone cel ls were upregulated only with a dose of 10 ng/ml. This indicates that recombinant human bone morphogenetic protein-2 regulates chondrocyte p roliferation, differentiation, and matrix production, and the effects are dependent on the stage of cell maturation. Resting-zone chondrocyt es were more sensitive? suggesting that they are targeted by bone morp hogenetic protein-2 and that this growth factor may have autocrine eff ects on these cells.