The Journal of Steroid Biochemistry and Molecular Biology
Suppression by estrogen receptor β of AP-1 mediated transactivation through estrogen receptor α
Introduction
The estrogen receptor (ER) is a conditional transcription factor, which belongs to the nuclear receptor superfamily and activates transcription of the specific genes only when a ligand is bound [1], [2], [3]. An estrogen responsive element (ERE), a DNA motif with palindromic structure, has been identified as a binding site for ER in the promoter region of several estrogen responsive genes such as these for vitellogenin and prolactin [4]. Once ER binds to EREs, it recruits a p160/p300 coactivator complex and enhances gene expression by interactions with the basic transcriptional machinery [5]. However, several lines of evidence have shown that estrogen can also enhance transcription from gene promoters containing the AP-1 motif which is a binding site for c-fos/c-jun protein and also known to be the TPA responsive element. It has been reported that AP-1 sites in the promoter region are involved in E2 dependent transcription of collagenase, insulin-like growth factor 1 (IGF-1) and ovalbumin genes [6], [7], [8], [9].
Recently a novel type of ER was cloned and designated as ERβ [10], [11]. Differences in structure and tissue distribution between ERα and ERβ suggest different biological roles of the two receptor subtypes. Transfection experiments with ERE containing reporters have indicated, however, that there is no difference in E2 dependent transactivation through the two types of receptor, even through some ligands such as genistein and coumestrol have a higher affinity for ERβ [12], [13]. When the E2 dependent response of the collagenase promoter which contains AP-1 motifs was examined, it was observed only with ERα but not with ERβ. Interestingly partial estrogen antagonists or AF-1 agonists such as tamoxifen, which have known to induce ERα-AP-1 responses, also did activate ERβ-AP-1 mediated transcription. Although these findings suggested a biological importance of AP-1 mediated E2 dependent transactivation especially for understanding functional differences between the two types of ER, very limited numbers of models have been examined and all of them involve rather complex promoters. In the present study, we therefore reconstructed and examined a model for E2 dependent AP-1 mediated transactivation through ERs using a reporter containing only consensus AP-1 motifs.
Section snippets
Hormones
17β-Estradiol (E2) and OH-tamoxifen (OH-TAM) were purchased from Sigma Chemicals, St. Louis and dissolved in ethanol to give stock solutions. ICI182,782 (ICI) was generously provided by Zeneca KK (Hyogo, Japan).
Cell culture
The NIH 3T3 cell line was obtained from the Japanese Collection of Research Bioresources (JCRB) and maintained in DME (Sigma Chemicals) containing penicillin and streptomycin with 10% calf serum (CS, Life Technologies, Rockville, MD, USA). For hormone treatments, the medium was changed
ER dependence of AP1-luc transactivation by E2
E2 responsive AP1-luc transcription was activated only when the ER expression plasmid, pSG5-hERα was co-transfected in NIH 3T3. The maximum response of 2.8-fold induction was found at 10−9 M of E2 (Fig. 1A). With the same cell line, induction of the (ERE)3-SV40-luc response by E2 was also low, approximately 3.7 times the control level (Fig. 1B). Fig. 1C shows that E2 dependent AP-1 responses increase according to the amount of pSG5-hERα co-transfected.
Effects of E2 on c-fos and c-jun protein levels in NIH 3T3 cells
Fig. 2 indicates time dependent changes in
Discussion
In the present study, for the first time, we reconstructed a model system for AP-1 mediated E2 dependent transcription with a simple AP-1 reporter containing only consensus AP-1 motifs. An E2 dependent response with the AP-1 reporter in NIH/3T3 cells was only found with ERα but not with ERβ, while tamoxifen enhanced AP-1 mediated transcription through both ERα and ERβ. The results are consistent with data for the collagenase promoter containing AP-1 motifs previously described [14]. There was a
Acknowledgements
We thank Ms K. Hashimoto for her expert technical assistance, Dr J. Kanno for providing ICI182,782, and Dr M.A. Moore for reading the manuscript and suggesting English clarification and JCRB for providing NIH 3T3 cells. This work was supported in part by a Grant-in-Aid from the Ministry of Health and Welfare, Japan.
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