The synergistic effect of TGFβ and 24,25-(OH)₂D₃ on resting zone chondrocytes is metabolite-specific and mediated by PKC

Transforming growth factor-β (TGFβ) and 24,25-(OH)₂D₃ play a major role in chondrocyte maturation during endochondral bone formation. TGFβ and vitamin D metabolites when added separately to resting zone (RC) or growth zone (GC) chondrocyte cultures, activate protein kinase C (PKC). The present study determined whether there is an additive or synergistic effect of vitamin D₃ metabolites and TGFβ on alkaline phosphatase and PKC specific activities and whether this effect is cell maturationdependent. GC and RC chondrocytes were isolated from rat costochondral cartilage and cultured to fourth passage. The cells were incubated with vitamin D₃ metabolites and TGFβ 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)₂D₃ with TGFβ to RC cells caused a synergistic effect on alkaline phosphatase activity; this result was not found if the vitamin D₃, metabolite was replaced by 1,25-(OH)₂D₃. The addition of 1,25-(OH)₂D₃ or 24,25-(OH)₂D₃ with TGFβ on GC cells had no synergistic or additive effect. The addition of 24,25-(OH)₂D₃ and TGFβ for 12 hours caused a synergistic effect on PKC activity; this effect was also observed if TGFβ was added first for 12 hours and 24,25-(OH)₂D₃ for the last 90 min. However, the addition of 24,25-(OH)₂D₃ for 90 rain followed by the addition of TGFβ for an additional 10.5 hours had no synergistic effect. This study indicates that TGFβ and 24,25-(OH)₂D₃ have a synergistic effect on chondrocyte differentiation as well as on PKC activity, suggesting that the synergistic effect of 24,25-(OH)₂D₃ and TGFβ on chondrocyte differentiation may be mediated through activation of PKC. The synergistic effect of 24,25-(OH)₂D₃ and TGFβ was cell maturation dependent.