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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Nongenotropic regulation of cytoplasmic kinases by sex steroids. HeLa ce...
Nongenotropic regulation of cytoplasmic kinases by sex steroids. HeLa cells were transfected with reporter constructs in which SRE or AP-1 drive the expression of SEAP. Aliquots of these cells were then cotransfected with either the full-length ERα, or its ligand-binding domain (E), or the E domain fused to a membrane localization sequence (E-Mem) or nuclear localization sequence (E-Nuc) and treated with vehicle (Veh) or E2 (10–8 M) for 15 minutes. The steroid-containing media were removed, the cells were washed twice, and the cultures were continued in fresh medium without E2. Supernatants were collected 6 hours later, and SEAP activity was assayed (a). MLO-Y4 cells transfected with GFP-ERK2 and nRFP were treated for 5 minutes with 10–7 M of the indicated compounds, and nuclear accumulation of GFP-ERK2 (left upper panels of b) was quantified as described in Methods and shown in left graph. The expression of nRFP in the nuclei is shown in the left lower panels of b. Regulation of IL-6 activity by 10–8 M E2, estren, or pyrazole is shown in the right graph (b). RLU, relative luciferase units. HeLa cells transfected with the ERα (c) or the AR (d) were exposed to vehicle or 10–8 M of the indicated steroids for 15 minutes as described in a, and SEAP activity was assessed 6 hours later. One hundred percent indicates activity in vehicle-treated cells. Bars indicate means ± SD of triplicate determinations; *P < 0.05 versus vehicle by ANOVA.