Maturation-dependent regulation of protein kinase C activity by vitamin D3 metabolites in chondrocyte cultures.

Vitamin D3 metabolites regulate the differentiation of chondrocytes isolated from the growth zone or resting zone of rat costochondral cartilage. Since some of the direct membrane effects of vitamin D metabolites are nongenomic, we hypothesized that protein kinase C (PKC) plays a role in signal transduction for these chondrocyte differentiation factors and that the regulation of PKC by the vitamin D metabolites is cell maturation dependent. Confluent, fourth passage cultures of growth zone and resting zone chondrocytes were treated with vitamin D3 metabolites for up to 24 h, lysed, and cell extracts assayed for kinase activity using a specific PKC substrate peptide. The addition of 1,25-(OH)2D3 to growth zone cell cultures resulted in a rapid dose-dependent stimulation of PKC, significant at 10(-9)-10(-7) M, beginning at 3 min and sustained until 90 min; 1,25-(OH)2D3 had no effect on PKC activity in resting zone chondrocyte cultures. The addition of 24,25-(OH)2D3 to resting zone cultures showed a slower PKC activation, with significant stimulation seen at 90-360 min for 10(-8)-10(-7) M 24,25-(OH)2D3. However, 24,25-(OH)2D3 had no effect on PKC activity in growth zone cell cultures at all times and concentrations examined. The specificity of PKC stimulation by the vitamin D3 metabolites was verified using a specific pseudosubstrate region peptide inhibitor, which reduced PKC activity when included in the reaction mixture. Pretreatment of the cultures with U73, 122, a phospholipase C inhibitor, decreased 1,25-(OH)2D3-stimulated PKC activity but had no effect upon 24,25-(OH)2D3-induced activity. The tyrosine kinase inhibitor, genistein, did not inhibit the PKC response in either vitamin D3 metabolites-treated culture. Neither actinomycin D nor cycloheximide affected 1,25-(OH)2D3-induced PKC activity in growth zone chondrocyte cultures, while both compounds inhibited 24,25-(OH)2D3-induced activity in resting zone chondrocyte cultures. The results of this study indicate that vitamin D metabolites stimulate PKC activity in a metabolite- and cell-maturation-specific manner. Effects of 1,25-(OH)2D3 appear to be nongenomic, whereas the effects of 24,25-(OH)2D3 probably involve a genomic mechanism.