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

Nuclear receptors for 17 beta -estradiol (E-2) 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 st udies indicate that an alternative pathway involving a membrane receptor ma y also be involved in the cell response. E-2 was found to directly affect t he fluidity of chondrocyte membranes derived from female, but not male, rat s. In addition, E-2 activates protein kinase C (PKC) in a nongenomic manner in female cells, and chelerythrine, a specific inhibitor of PKC, inhibits E-2-dependent alkaline phosphatase activity and proteoglycan sulfation in t hese 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 m embrane-impermeable 17 beta -estradiol-bovine serum albumin conjugate (E-2- BSA) on chondrocyte proliferation, differentiation, and matrix synthesis; ( 2) to determine the pathway that mediates the membrane effect of E-2-BSA on PKC; and (3) to compare the action of E-2-BSA to that of E-2. Confluent, f ourth passage resting zone (RC) and growth zone (GC) chondrocytes from fema le rat costochondral cartilage were treated with 10(-9) to 10(-7) M E-2 or E-2-BSA and changes in alkaline phosphatase specific activity, proteoglycan sulfation, and [H-3]-thymidine incorporation measured. To examine the path way of PKC activation, chondrocyte cultures were treated with E-2-BSA in th e presence or absence of GDP betaS (inhibitor of G-proteins), GTP gammaS (a ctivator of G-proteins), U73122 or D609 (inhibitors of phospholipase C [PLC ]), wortmannin (inhibitor of phospholipase D [PLD]) or LY294002 (inhibitor of phosphatidylinositol 3-kinase). E-2-BSA mimicked the effects of E-2 on a lkaline phosphatase specific activity and proteoglycan sulfation, causing d ose-dependent increases in both RC and GC cell cultures. Both forms of estr adiol inhibited [H-3]-thymidine incorporation, and the effect was dose-depe ndent. E-2-BSA caused time-dependent increases in PKC in RC and GC cells; e ffects were observed within three minutes in RC cells and within one minute in GC cells. Response to E-2 was more robust in RC cells, whereas in GC ce lls, E-2 and E-2-BSA caused a comparable increase in PKC. GDP betaS inhibit ed the activation of PKC in E-2-BSA-stimulated RC and GC cells. GTP gammaS increased PKC in E-2-BSA-stimulated GC cells, but had no effect in E-2-BSA- stimulated RC cells. The phosphatidylinositol-specific PLC inhibitor U73122 blocked E-2-BSA-stimulated PKC activity in both RC and GC cells, whereas t he phosphatidylcholine-specific PLC inhibitor D609 had no effect. Neither t he PLD inhibitor wortmannin nor the phosphatidylinositol 3-kinase inhibitor LY294022 had any effect on E-2-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 E-2-BSA on PKC. Moreover, the classical receptor agonist diethylstilbestrol (DES) had no effect oil PKC, nor did i t alter the stimulatory effect of E-2-BSA. The specificity of the membrane response to E-2 was also demonstrated by showing that the membrane receptor for 1 alpha ,25-(OH)(2)D-3 was not involved. These data indicate that the rapid nongenomic effect of E-2-BSA on PKC acti vity in RC and CC cells is dependent on G-protein-coupled PLC and support t he hypothesis that many of the effects of E-2 involve membrane-associated m echanisms independent of classical estrogen receptors. J. Cell. Biochem. 81 :413-429, 2001. (C) 2001 Wiley-Liss, Inc.