The membrane effects of 17β-estradiol on chondrocyte phenotypic expression are mediated by activation of protein kinase C through phospholipase C and G-proteins

Growth plate chondrocytes from both male and female rats have nuclear receptors for 17 beta -estradiol (E-2); however, recent studies indicate that an alternative pathway involving a membrane receptor may also be involved in the female cell response. E-2 directly affects the fluidity of chondrocyte membranes derived from female, but not male, rats. In addition, E-2 activates PKC in a nongenomic manner in female cells, and chelerythrine, a specific inhibitor of PKC, inhibits E-2-dependent alkaline phosphatase activity in these cells, indicating PKC is involved in the signal transduction mechanism. The aims of this study were: (1) to examine if PKC mediates the effect of E-2 on chondrocyte proliferation, differentiation, and matrix synthesis; and (2) to determine the pathway that mediates the membrane effect of E-2 on PKC. Confluent, fourth passage resting zone (RC) and growth zone (GC) chondrocytes from female rat costochondral cartilage were treated with 10(-10) to 10(-7) M E-2 in the presence or absence of the PKC inhibitor chelerythrine, and changes in alkaline phosphatase specific activity, proteoglycan sulfation, and [H-3]thymidine incorporation were measured. To examine the pathway of PKC activation, chondrocyte cultures were treated with E-2 in the presence or absence of genistein (an inhibitor of tyrosine kinases), U73122 or D609 (inhibitors of phospholipase C [PLC]), quinacrine (an inhibitor of phospholipase A(2) [PLA(2)]), and melittin (an activator of PLA(2)). Alkaline phosphatase specific activity and proteoglycan sulfation were increased and [H-3]thymidine incorporation was decreased by E-2. The effects of E-2 on all parameters were blocked by chelerythrine. Treatment of the cultures with E-2 produced a significant dose-dependent increase in PKC. U73132 dose-dependently inhibited the activation of PKC in E-2-stimulated female chondrocyte cultures. However, the classical receptor antagonist ICI 182780 was unable to block the stimulatory effect of E-2 on PKC. Moreover, the classical receptor agonist diethylstilbestrol (DES) had no effect on PKC, nor did it alter the stimulatory effect of E-2. Inhibition of tyrosine kinase and PLA(2) had no effect on the activation of PKC by E-2. The PLA(2) activator also had no effect on PKC activation by E-2. E-2 stimulated PKC activity in membranes isolated from the chondrocytes, demonstrating a direct membrane effect for this steroid hormone. These data indicate that the rapid nongenomic effect of E-2 on PKC activity in chondrocytes from female rats is sex-specific and dependent upon a G-protein-coupled phospholipase C. (C) 2000 Elsevier Science Ltd. All rights reserved.