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Estrogen Receptor-Dependent Activation of AP-1 via Non-Genomic Signalling

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Estrogen Receptor-Dependent Activation of AP-1 via Non-Genomic Signalling

Linda Björnström et al. Nucl Recept.

Abstract

BACKGROUND: Ligand-bound estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta) modulate AP-1-dependent transcription via protein-protein interactions on DNA, in a manner that depends on the type of cells and the subtype of ER. We present here evidence for an additional mechanism by which ERs modulate the transcriptional activity of AP-1. RESULTS: We show that ERs located in the cytoplasm efficiently activate transcription at AP-1 sites in response to 17beta-estradiol, while ERs present in the nucleus repress transcription under the same conditions. 17beta-estradiol-induced activation of the coll-73-luc reporter correlated with cytoplasmic localization of various ERalpha and ERbeta mutant receptors, and was inhibited in the presence of the full estrogen antagonist ICI 182,780 and the MAP-kinase inhibitor UO126. We also show that the selective estrogen receptor modulator (SERM) tamoxifen is as potent as 17beta-estradiol in inducing activation of AP-1 when ERalpha is present in the cytoplasm. CONCLUSIONS: These results suggest that non-genomic signalling is involved in the mechanism by which ERalpha and ERbeta influence AP-1-dependent transcription. We have previously shown that Stat3 and Stat5 are targeted by non-genomic actions of ERs, and the results presented here allow us to conclude that ERs bound to 17beta-estradiol mediate the transcriptional activation of promoters regulated by AP-1 and by Stat proteins via different combinations of signal transduction pathways. Our observations thereby provide new insights into the mechanisms by which ERs act at alternate response elements, and suggest a mechanism by which tamoxifen exerts its action as a tissue-selective agonist.

Figures

Figure 1
Figure 1
Cytoplasmic variants of ERα and ERβ display reversed activity at an AP-1 response element. (A) HC11 cells were transfected with expression vectors for ERα wt, NLSA, deleted in the hinge domain (Δ245-307), ERβ wt, ERβ Flag 148-530 or ERβ 148-530 Flag. The cellular localization of the receptor proteins was analysed by indirect immunofluorescence as described in Experimental Procedures. HC11 cells were transiently co-transfected with (B) 500 ng coll-73-luc reporter gene or (C) 1 μg β-casein reporter gene and 200 ng of ERα wt, NLSA, ERβ wt, ERβ Flag 148-530 or ERβ 148-530 Flag. Cells were treated with either no hormone (NH) or 10-8 M 17β-estradiol (E2), and the reporter activity was analysed 24 hours after treatment. Luciferase activity was normalised using β-gal as an internal control. Data are representative of at least three independent experiments performed in duplicate. Mean and ± SD are shown.
Figure 2
Figure 2
ERβ structurally disrupted in the DBD re-localizes to the cytoplasm and induces AP-1 activity in the presence of 17β-estradiol. (A) HC11 cells were transiently co-transfected with 500 ng coll-73-luc reporter gene or 1 μg β-casein reporter gene and 200 ng of ERβ wt or C201A/C204A. Cells were treated with either no hormone (NH) or 10-8 M 17β-estradiol (E2), as indicated, and the reporter activity was analysed 24 hours after treatment. Luciferase activity was normalised using β-gal as an internal control. Data are representative of at least three independent experiments performed in duplicate. Mean and ± SD are shown. (B) HC11 cells were transfected with expression vectors for ERβ wt or C201A/C204A. Cells were treated with either no hormone (NH) or 10-8 M 17β-estradiol (E2), as indicated, and the cellular localization of the receptor proteins was analysed 24 hours after treatment by indirect immunofluorescence as described in Experimental Procedures.
Figure 3
Figure 3
Ligand-dependent ER actions at an AP-1 response element. HC11 cells were transiently co-transfected with 500 ng coll-73-luc reporter gene and (A) 200 ng of NLSA or (B) ERβ 148-530 Flag. Cells were treated with either no hormone (NH), 10-8 M 17β-estradiol (E2), 10-7 M DES, 10-7 M PPT, 10-7 M estren, 10-7 M tamoxifen (OHT), 10-7 M raloxifene (Ral), 10-7 M idoxifene (Idox), 10-7 M nafoxidene (Nafox) or 10-7 M ICI 182,780 (ICI), as indicated, and the reporter activity was analysed 24 hours after treatment. Luciferase activity was normalised using β-gal as an internal control. Data are representative of at least three independent experiments performed in duplicate. Mean and ± SD are shown. (C) HC11 cells were transiently co-transfected with 500 ng TRE-tk-luc reporter gene and 200 ng of NLSA or ERβ 148-530 Flag. Cells were treated with either no hormone (NH), 10-8 M 17β-estradiol (E2), 10-7 M tamoxifen (OHT) or 10-7 M ICI 182,780 (ICI), as indicated, and further assayed as described in A and B. (D) HC11 cells were transiently co-transfected with 500 ng coll-73-luc reporter and 200 ng of ERα wt or ERβ wt. Cells were treated and analysed as described in A and B.
Figure 4
Figure 4
Intact MAP-kinase activity is important for non-genomic actions of ERs at AP-1 sites. (A) HC11 cells were transiently co-transfected with 500 ng coll-73-luc reporter gene and 200 ng of NLSA, ERβ 148-530 Flag or C201A/C204A. Cells were pretreated with 10 μM UO126 or 10 μM LY294002 for one hour before the addition of 10-8 M 17β-estradiol (E2), and the reporter activity was analysed 6 hours after treatment. (B) SYF cells were transiently co-transfected with 500 ng coll-73-luc reporter gene and 200 ng of NLSA, ERβ 148-530 Flag or C201A/C204A. Cells were treated with either no hormone (NH), 10-8 M 17β-estradiol (E2) or 10-7 M tamoxifen (OHT), and the reporter activity was analysed 24 hours after treatment. Luciferase activity was normalised using β-gal as an internal control. Data are representative of at least three independent experiments performed in duplicate. Mean and ± SD are shown.

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