17beta-estradiol regulation of protein kinase C activity in chondrocytes is sex-dependent and involves nongenomic mechanisms.

17Beta-estradiol (E2) regulates growth plate chondrocyte differentiation in both a sex- and cell maturation-dependent manner, and the sex-specific effects of E2 appear to be mediated in part by membrane events. In this study, we examined whether E2 regulates protein kinase C (PKC) in a cell-maturation and sex-specific manner and whether E2 uses a nongenomic mechanism in regulating this enzyme. In addition, we determined if PKC mediates the E2-dependent stimulation of alkaline phosphatase activity seen in chondrocytes. Confluent, fourth passage resting zone (RC) and growth zone (GC) chondrocytes from male and female rat costochondral cartilage were treated with 10(-10) to 10(-7) M E2. E2 caused a dose-dependent increase in PKC in RC and GC cells from female rats. Peak stimulation was at 90 min. Increased PKC was evident by 3 min in both RC and GC and was still evident in RC cells at 720 min, but in GC cells activity returned to baseline by 270 min. Actinomycin D had no effect at 9, 90, 270, or 720 min, but there was a small decrease in E2-stimulated PKC in RC treated with cycloheximide at 90 and 270 min and in GC treated for 90 min. E2 increased cytosolic and membrane PKC at 9 min and by 90 min promoted translocation of PKC activity from the cytosol to the membranous compartment of female RC cells. Antibodies specific for the alpha, beta, delta, epsilon, and zeta isoforms of PKC revealed that PKCalpha in female GC and RC cells is activated by E2. There was a small, but statistically significant, increase in PKC in male RC cells in response to E2, but it was not dose-dependent, and no effect of E2 was noted in male GC cells. 17Alpha-estradiol, an inactive isomer of E2, did not affect PKC specific activity in RC or GC cells from either female or male rats. Chelerythrine, a specific inhibitor of PKC, inhibited E2-dependent alkaline phosphatase activity, indicating that E2 mediates its rapid effects on alkaline phosphatase via PKC.