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

Abstract

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—indiuced 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.