MATURATION-DEPENDENT REGULATION OF PROTEIN-KINASE-C ACTIVITY BY VITAMIN-D(3) METABOLITES IN CHONDROCYTE CULTURES

Vitamin D3 metabolites regulate the differentiation of chondrocytes is olated from the growth zone or resting zone of rat costochondral carti lage. Since some of the direct membrane effects of vitamin D metabolit es are nongenomic, we hypothesized that protein kinase C (PKC) plays a role in signal transduction for these chondrocyte differentiation fac tors and that the regulation of PKC by the vitamin D metabolites is ce ll maturation dependent. Confluent, fourth passage cultures of growth zone and resting zone chondrocytes were treated with vitamin D, metabo lites for up to 24 h, lysed, and cell extracts assayed for kinase acti vity using a specific PKC substrate peptide. The addition of 1,25-(OH) 2D3 to growth zone cell cultures resulted in a rapid dose-dependent st imulation of PKC, significant at 10(-9)-10(-7) M, beginning at 3 min a nd 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 significan t 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 ce ll cultures at all times and concentrations examined. The specificity of PKC stimulation by the vitamin D, metabolites was verified using a specific pseudosubstrate region peptide inhibitor, which reduced PKC a ctivity when included in the reaction mixture. Pretreatment of the cul tures with U73,122, a phospholipase C inhibitor, decreased 1,25-(OH)2D 3-stimulated PKC activity but had no effect upon 24,25-(OH)2D3-induced activity. The tyrosine kinase inhibitor, genistein, did not inhibit t he PKC response in either vitamin D3 metabolites-treated culture. Neit her actinomycin D nor cycloheximide affected 1,25-(OH)2D3-induced PKC activity in growth zone chondrocyte cultures, while both compounds inh ibited 24,25-(OH)2D3-induced activity in resting zone chondrocyte cult ures. The results of this study indicate that vitamin D metabolites st imulate PKC activity in a metabolite- and cell-maturation-specific man ner. Effects of 1,25-(OH)2D3 appear to be nongenomic, whereas the effe cts of 24,25-(OH)2D3 probably involve a genomic mechanism. (C) 1993 Wi ley-Liss, Inc.