Kinase-mediated regulation of common transcription factors accounts for the bone-protective effects of sex steroids
Stavroula Kousteni, … , Teresita Bellido, Stavros C. Manolagas
Stavroula Kousteni, … , Teresita Bellido, Stavros C. Manolagas
Published June 1, 2003
Citation Information: J Clin Invest. 2003;111(11):1651-1664. https://doi.org/10.1172/JCI17261.
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Article Endocrinology

Kinase-mediated regulation of common transcription factors accounts for the bone-protective effects of sex steroids

Abstract

It has been found that 4-estren-3α,17β-diol, a synthetic ligand for the estrogen receptor (ER) or androgen receptor (AR), which does not affect classical transcription, reverses bone loss in ovariectomized females or orchidectomized males without affecting the uterus or seminal vesicles, demonstrating that the classical genotropic actions of sex steroid receptors are dispensable for their bone-protective effects, but indispensable for their effects on reproductive organs. We have now investigated the mechanism of action of this compound. We report that, identically to 17β-estradiol or dihydrotestosterone, but differently from raloxifene, estren alters the activity of Elk-1, CCAAT enhancer binding protein–β (C/EBPβ), and cyclic adenosine monophosphate–response element binding protein (CREB), or c-Jun/c-Fos by an extranuclear action of the ER or AR, resulting in activation of the Src/Shc/ERK pathway or downregulation of JNK, respectively. All of these effects are non–sex specific, require only the ligand-binding domain of the receptor, and are indispensable for the antiapoptotic action of these ligands on osteoblastic and HeLa cells. Moreover, administration of 17β-estradiol or 4-estren-3α,17β-diol to ovariectomized mice induces phosphorylation of ERKs, Elk-1, and C/EBPβ, downregulates c-Jun, and upregulates the expression of egr-1, an ERK/SRE target gene. Kinase-initiated regulation of commonly used transcription factors offers a molecular explanation for the profound skeletal effects of sex steroid receptor ligands, including synthetic ones that are devoid of classical transcriptional activity.

Authors

Stavroula Kousteni, Li Han, Jin-Ran Chen, Maria Almeida, Lilian I. Plotkin, Teresita Bellido, Stavros C. Manolagas

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Raloxifene does not mimic the nongenotropic effects of estren. Calvaria ...
Raloxifene does not mimic the nongenotropic effects of estren. Calvaria cells were pretreated with estren or raloxifene (Ral) and then exposed to etoposide as described in Figure 4b. Apoptosis was assayed by measuring caspase-3 activity in cell lysates (a). HeLa cells were transiently transfected with ERα or AR and the pCaspase3-Sensor vector (b). Osteoclasts were treated with the indicated concentrations of the two compounds for 24 hours, and apoptosis was assayed as described in a (c). HeLa cells were cotransfected with ERα and either SRE-SEAP (d) or IL-6–luciferase (IL-6–luc) (e) reporters, and SRE-SEAP activity was assessed as in Figure 1. PMA-induced IL-6 activity was determined as described in Methods. SRE-SEAP or IL-6–luciferase activity in the presence of vehicle is designated as 100%. MLO-Y4 cells were incubated for 5 minutes with estren or Ral, and ERK phosphorylation was assayed by Western blot analysis (f). GFP-ERK2 nuclear accumulation was assessed as in Figure 1b (g). Ten-week-old Swiss Webster mice were ovx, left untreated for 5 days, and then implanted with 21-day slow-release pellets containing estren (2.6 mg or 26 mg) or Ral (0.027 mg or 0.27 mg); 1×, 10×, 3×, or 30× refers to the correspondence of the given dose to the Kd of the respective compound for ERα. Forty-eight hours after pellet implantation, the animals were sacrificed and ERK phosphorylation was determined in protein extracts from L5 vertebrae; each lane represents one mouse (h). Bars indicate means ± SD of triplicate determinations; *P < 0.05 versus vehicle or ovx by ANOVA.