THE SYNERGISTIC EFFECT OF TGF-BETA AND 24,25-(OH)(2)D-3 ON RESTING ZONE CHONDROCYTES IS METABOLITE-SPECIFIC AND MEDIATED BY PKC

Transforming growth factor-beta (TGF beta) and 24,25-(OH)(2)D-3 play a major role in chondrocyte maturation during endochondral bone formati on. TGF beta and vitamin D metabolites when added separately to restin g zone (RC) or growth zone (GC) chondrocyte cultures, activate protein kinase C (PKC). The present study determined whether there is an addi tive or synergistic effect of vitamin D-3 metabolites and TGF beta on alkaline phosphatase and PBC specific activities and whether this effe ct is cell maturation-dependent. GC and RC chondrocytes were isolated from rat costochondral cartilage and cultured to fourth passage. The c ells were incubated with vitamin D-3 metabolites and TGF beta alone or in combination, and the specific activity of alkaline phosphatase, as well as the specific activity of PKC, were measured. The addition of 24,25-(OH)(2)D-3 with TGF beta to RC cells caused a synergistic effect on alkaline phosphatase activity; this result was not found if the vi tamin D-3 metabolite was replaced by 1,25-(OH)(2)D-3. The addition of 1,25-(OH)(2)D-3 or 24,25-(OH)(2)D-3 with TGF beta on GC cells had no s ynergistic or additive effect. The addition of 24,25-(OH)(2)D-3 and TG F beta for 12 hours caused a synergistic effect on PKC activity; this effect was also observed if TGF beta was added first for 12 hours and 24,25-(OH)(2)D-3 for the last 90 min. However, the addition of 24,25-( OH)(2)D-3 for 90 min followed by the addition of TGF beta for an addit ional 10.5 hours had no synergistic effect. This study indicates that TGF beta and 24,25-(OH)(2)D-3 have a synergistic effect on chondrocyte differentiation as well as on PKC activity, suggesting that the syner gistic effect of 24,25-(OH)(2)D-3 and TGF beta on chondrocyte differen tiation may be mediated through activation of PKC. The synergistic eff ect of 24,25-(OH)(2)D-3 and TGF beta was cell maturation dependent.