17β-estradiol-BSA conjugates and 17β-estradiol regulate growth plate chondrocytes by common membrane associated mechanisms involving PKC dependent and independent signal transduction

Nuclear receptors for 17β-estradiol (E₂) are present in growth plate chondrocytes from both male and female rats and regulation of chondrocytes through these receptors has been studied for many years; however, recent studies indicate that an alternative pathway involving a membrane receptor may also be involved in the cell response. E₂ was found to directly affect the fluidity of chondrocyte membranes derived from female, but not male, rats. In addition, E₂ activates protein kinase C (PKC) in a nongenomic manner in female cells, and chelerythrine, a specific inhibitor of PKC, inhibits E₂-dependent alkaline phosphatase activity and proteoglycan sulfation in these cells, indicating PKC is involved in the signal transduction mechanism. The aims of the present study were: (1) to examine the effect of a cell membrane-impermeable 17β-estradiol-bovine serum albumin conjugate (E₂-BSA) on chondrocyte proliferation, differentiation, and matrix synthesis; (2) to determine the pathway that mediates the membrane effect of E₂-BSA on PKC; and (3) to compare the action of E₂-BSA to that of E₂. Confluent, fourth passage resting zone (RC) and growth zone (GC) chondrocytes from female rat costochondral cartilage were treated with 10^-9 to 10^-7 M E₂ or E₂-BSA and changes in alkaline phosphatase specific activity, proteoglycan sulfation, and [³H]-thymidine incorporation measured. To examine the pathway of PKC activation, chondrocyte cultures were treated with E₂-BSA in the presence or absence of GDPβS (inhibitor of G-proteins), GTPγS (activator of G-proteins), U73122 or D609 (inhibitors of phospholipase C [PLC]), wortmannin (inhibitor of phospholipase D [PLD]) or LY294002 (inhibitor of phosphatidylinositol 3-kinase). E₂-BSA mimicked the effects of E₂ on alkaline phosphatase specific activity and proteoglycan sulfation, causing dose-dependent increases in both RC and GC cell cultures. Both forms of estradiol inhibited [³H]-thymidine incorporation, and the effect was dose-dependent. E₂-BSA caused time-dependent increases in PKC in RC and GC cells; effects were observed within three minutes in RC cells and within one minute in GC cells. Response to E₂ was more robust in RC cells, whereas in GC cells, E₂ and E₂-BSA caused a comparable increase in PKC. GDPβS inhibited the activation of PKC in E₂-BSA-stimulated RC and GC cells. GTPγS increased PKC in E₂-BSA-stimulated GC cells, but had no effect in E₂-BSA-stimulated RC cells. The phosphatidylinositol-specific PLC inhibitor U73122 blocked E₂-BSA-stimulated PKC activity in both RC and GC cells, whereas the phosphatidylcholine-specific PLC inhibitor D609 had no effect. Neither the PLD inhibitor wortmannin nor the phosphatidylinositol 3-kinase inhibitor LY294022 had any effect on E₂-BSA-stimulated PKC activity in either RC or GC cells. The classical estrogen receptor antagonist ICI 182780 was unable to block the stimulatory effect of E₂-BSA on PKC. Moreover, the classical receptor agonist diethylstilbestrol (DES) had no effect on PKC, nor did it alter the stimulatory effect of E₂-BSA. The specificity of the membrane response to E₂ was also demonstrated by showing that the membrane receptor for 1 α,25-(OH)₂D₃ was not involved. These data indicate that the rapid nongenomic effect of E₂-BSA on PKC activity in RC and GC cells is dependent on G-protein-coupled PLC and support the hypothesis that many of the effects of E₂ involve membrane-associated mechanisms independent of classical estrogen receptors.