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

V. L. Sylvia, B. D. Boyan, David D Dean, Zvi Schwartz

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Abstract

Growth plate chondrocytes from both male and female rats have nuclear receptors for 17β-estradiol (E2); however, recent studies indicate that an alternative pathway involving a membrane receptor may also be involved in the female cell response. E2 directly affects the fluidity of chondrocyte membranes derived from female, but not male, rats. In addition, E2 activates PKC in a nongenomic manner in female cells, and chelerythrine, a specific inhibitor of PKC, inhibits E2-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 E2 on chondrocyte proliferation, differentiation, and matrix synthesis; and (2) to determine the pathway that mediates the membrane effect of E2 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 E2 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 E2 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 A2 [PLA2]), and melittin (an activator of PLA2). Alkaline phosphatase specific activity and proteoglycan sulfation were increased and [3H]thymidine incorporation was decreased by E2. The effects of E2 on all parameters were blocked by chelerythrine. Treatment of the cultures with E2 produced a significant dose-dependent increase in PKC. U73122 dose-dependently inhibited the activation of PKC in E2-stimulated female chondrocyte cultures. However, the classical receptor antagonist ICI 182780 was unable to block the stimulatory effect of E2 on PKC. Moreover, the classical receptor agonist diethylstilbestrol (DES) had no effect on PKC, nor did it alter the stimulatory effect of E2. Inhibition of tyrosine kinase and PLA2 had no effect on the activation of PKC by E2. The PLA2 activator also had no effect on PKC activation by E2. E2 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 E2 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.

Original languageEnglish (US)
Pages (from-to)211-224
Number of pages14
JournalJournal of Steroid Biochemistry and Molecular Biology
Volume73
Issue number5
DOIs
StatePublished - 2000

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Type C Phospholipases
Chondrocytes
GTP-Binding Proteins
Protein Kinase C
Estradiol
Chemical activation
Rats
Phospholipases A2
Membranes
Alkaline Phosphatase
Proteoglycans
Protein-Tyrosine Kinases
Thymidine
Steroid hormones
Melitten
Quinacrine
Signal transduction
Diethylstilbestrol
Fluidity
Genistein

Keywords

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

ASJC Scopus subject areas

  • Biochemistry
  • Endocrinology

Cite this

The membrane effects of 17β-estradiol on chondrocyte phenotypic expression are mediated by activation of protein kinase C through phospholipase C and G-proteins. / Sylvia, V. L.; Boyan, B. D.; Dean, David D; Schwartz, Zvi.

In: Journal of Steroid Biochemistry and Molecular Biology, Vol. 73, No. 5, 2000, p. 211-224.

Research output: Contribution to journalArticle

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AU - Sylvia, V. L.

AU - Boyan, B. D.

AU - Dean, David D

AU - Schwartz, Zvi

PY - 2000

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N2 - Growth plate chondrocytes from both male and female rats have nuclear receptors for 17β-estradiol (E2); however, recent studies indicate that an alternative pathway involving a membrane receptor may also be involved in the female cell response. E2 directly affects the fluidity of chondrocyte membranes derived from female, but not male, rats. In addition, E2 activates PKC in a nongenomic manner in female cells, and chelerythrine, a specific inhibitor of PKC, inhibits E2-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 E2 on chondrocyte proliferation, differentiation, and matrix synthesis; and (2) to determine the pathway that mediates the membrane effect of E2 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 E2 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 E2 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 A2 [PLA2]), and melittin (an activator of PLA2). Alkaline phosphatase specific activity and proteoglycan sulfation were increased and [3H]thymidine incorporation was decreased by E2. The effects of E2 on all parameters were blocked by chelerythrine. Treatment of the cultures with E2 produced a significant dose-dependent increase in PKC. U73122 dose-dependently inhibited the activation of PKC in E2-stimulated female chondrocyte cultures. However, the classical receptor antagonist ICI 182780 was unable to block the stimulatory effect of E2 on PKC. Moreover, the classical receptor agonist diethylstilbestrol (DES) had no effect on PKC, nor did it alter the stimulatory effect of E2. Inhibition of tyrosine kinase and PLA2 had no effect on the activation of PKC by E2. The PLA2 activator also had no effect on PKC activation by E2. E2 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 E2 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.

AB - Growth plate chondrocytes from both male and female rats have nuclear receptors for 17β-estradiol (E2); however, recent studies indicate that an alternative pathway involving a membrane receptor may also be involved in the female cell response. E2 directly affects the fluidity of chondrocyte membranes derived from female, but not male, rats. In addition, E2 activates PKC in a nongenomic manner in female cells, and chelerythrine, a specific inhibitor of PKC, inhibits E2-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 E2 on chondrocyte proliferation, differentiation, and matrix synthesis; and (2) to determine the pathway that mediates the membrane effect of E2 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 E2 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 E2 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 A2 [PLA2]), and melittin (an activator of PLA2). Alkaline phosphatase specific activity and proteoglycan sulfation were increased and [3H]thymidine incorporation was decreased by E2. The effects of E2 on all parameters were blocked by chelerythrine. Treatment of the cultures with E2 produced a significant dose-dependent increase in PKC. U73122 dose-dependently inhibited the activation of PKC in E2-stimulated female chondrocyte cultures. However, the classical receptor antagonist ICI 182780 was unable to block the stimulatory effect of E2 on PKC. Moreover, the classical receptor agonist diethylstilbestrol (DES) had no effect on PKC, nor did it alter the stimulatory effect of E2. Inhibition of tyrosine kinase and PLA2 had no effect on the activation of PKC by E2. The PLA2 activator also had no effect on PKC activation by E2. E2 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 E2 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.

KW - 17β-Estradiol

KW - Chondrocyte cultures

KW - G-proteins

KW - Phospholipase C

KW - Protein kinase C

KW - Signal transduction

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