17-BETA-ESTRADIOL REGULATION OF PROTEIN-KINASE-C ACTIVITY IN CHONDROCYTES IS SEX-DEPENDENT AND INVOLVES NONGENOMIC MECHANISMS

17 beta-Estradiol (E-2) regulates growth plate chondrocyte differentia tion in both a sex-and cell maturation-dependent manner, and the sex-s pecific effects of E-2 appear to be mediated in part by membrane event s. In this study, we examined whether E-2 regulates protein kinase C ( PKC) in a cell-maturation and sex-specific manner and whether E-2 uses a nongenomic mechanism in regulating this enzyme. In addition, we det ermined if PKC mediates the E-2-dependent stimulation of alkaline phos phatase activity seen in chondrocytes. Confluent, fourth passage resti ng zone (RC) and growth zone (GC) chondrocytes from male and female ra t costochondral cartilage were treated with 10(-10) to 10(-7) M E-2. E -2 caused a dose-dependent increase in PKC in RC and GC cells from fem ale rats. Peak stimulation was at 90 min. Increased PKC was evident by 3 min in both RC and CC and was still evident in RC cells at 720 min, but in CC 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 decr ease in E-2-stimulated PKC in RC treated with cycloheximide at 90 and 270 min and in GC treated for 90 min. E-2 increased cytosolic and memb rane PKC at 9 min and by 90 min promoted translocation of PKC activity from the cytosol to the membranous compartment of female RC cells. An tibodies specific for the alpha, beta, delta, epsilon, and zeta isofor ms of PKC revealed that PKC alpha in female CC and RC cells is activat ed by E-2. There was a small, but statistically significant, increase in PKC in male RC cells in response to E,, but it was not dose-depende nt, and no effect of E-2 was noted in male GC cells. 17 alpha-estradio l, an inactive isomer of E-2 I did not affect PKC specific activity in RC or GC cells from either female or male rats. Chelerythrine, a spec ific inhibitor of PKC, inhibited E-2 dependent alkaline phosphatase ac tivity, indicating that E-2 mediates its rapid effects on alkaline pho sphatase via PKC. (C) 1998 Wiley-Liss, Inc.