The membrane effects of 17beta-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 17beta-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 [3H]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 [3H]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. U73122 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.
Sylvia
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VL
Department of Orthopaedics, The University of Texas Health Science Center at San Antonio, 7703 Floyd Carl Drive, San Antonio, TX 78284-7774, USA.
Boyan
B D
BD
Dean
D D
DD
Schwartz
Z
Z
D000469:Alkaline Phosphatase / Q000378:metabolism*; D000470:Alkaloids; D000818:Animals; D053119:Benzophenanthridines; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D002772:Cholera Toxin / Q000494:pharmacology; D019902:Chondrocytes / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D004054:Diethylstilbestrol / Q000494:pharmacology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004958:Estradiol / Q000031:analogs & derivatives* / Q000494:pharmacology*; D005260:Female; D000077267:Fulvestrant; D008297:Male; D008560:Membrane Fluidity / Q000187:drug effects / Q000502:physiology; D010617:Phenanthridines / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011960:Receptors, Estrogen / Q000187:drug effects / Q000502:physiology*; D012727:Sex Characteristics; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D000470:Alkaloids; D053119:Benzophenanthridines; D004791:Enzyme Inhibitors; D010617:Phenanthridines; D011960:Receptors, Estrogen; D010566:Virulence Factors, Bordetella; D000077267:Fulvestrant; D004958:Estradiol; D004054:Diethylstilbestrol; D002772:Cholera Toxin; C016299:chelerythrine; D011493:Protein Kinase C; D000469:Alkaline Phosphatase