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

V. L. Sylvia, J. Walton, D. Lopez, D. D. Dean, B. D. Boyan, Z. Schwartz

Research output: Contribution to journalArticle

72 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)413-429
Number of pages17
JournalJournal of Cellular Biochemistry
Volume81
Issue number3
DOIs
StatePublished - 2001

Keywords

  • 17β-estradiol-BSA
  • Chondrocyte cultures
  • G-proteins
  • Phospholipase C
  • Protein kinase C
  • Signal transduction

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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