Rousseau Caroline, Pettersson Filippa, Couture Marie Claude, Paquin Andre, Galipeau Jacques, Mader Sylvie, Miller Wilson H
Departments of Oncology and Medicine, Lady Davis Institute for Medical Research, SMBD-Jewish General Hospital (McGill University), 3755 Cote Ste Catherine Road, Que., H3T 1E2, Montreal, Canada.
J Steroid Biochem Mol Biol. 2003 Jul;86(1):1-14. doi: 10.1016/s0960-0760(03)00255-3.
Transcriptional cross-talk exists between the estrogen receptor (ERalpha) and retinoic acid receptor (RAR) pathways in human breast cancer cells. We have previously shown that re-expression of ERalpha in ER-negative cells stimulates the transcriptional and growth inhibitory effects of all-trans-retinoic acid (tRA) by a mechanism that is independent of the ER ligands estradiol and tamoxifen. In this study, we generated cell lines stably expressing ERalpha-deletion mutants to elucidate the mechanism whereby ERalpha modulates RAR transcriptional activity. Using RT-PCR and RNAse protection assays, we observed that expression of ERalpha suppresses basal expression of the RA-responsive gene RARbeta2, while allowing it to be strongly induced by tRA. Repression of basal RARbeta2 transcription was confirmed by transient expression of the reporter plasmid betaRE-tk-CAT, containing the RARbeta2 promoter. In the ERalpha-negative cells, on the other hand, transcription was only weakly induced by RA. We further determined that this effect of ERalpha on RARbeta induction required the N-terminal AF-1-containing region, including the DNA-binding domain, but was independent of the C-terminal ligand-binding domain. Consistent with these results, the ER agonist estradiol and the AF-2 antagonist 4-hydroxytamoxifen had no significant effect on betaRARE activity. Conversely, the full ER antagonist ICI 182,780, which blocks ERalpha AF-1 activity, was able to completely relieve repression of basal betaRARE activity. The effect of ERalpha is specific for RAR-mediated transcription and does not occur on promoters containing typical response elements for the Vitamin D or thyroid hormone receptors. Moreover, the cross-talk between ERalpha and RAR does not seem to be mediated by sequestration of a number of common co-regulators, suggesting a novel mechanism whereby the N-terminal region of ERalpha modulates the transcriptional activity of RAR.
在人乳腺癌细胞中，雌激素受体（ERα）和视黄酸受体（RAR）信号通路之间存在转录相互作用。我们之前已经表明，在ER阴性细胞中重新表达ERα可通过一种独立于ER配体雌二醇和他莫昔芬的机制刺激全反式维甲酸（tRA）的转录和生长抑制作用。在本研究中，我们构建了稳定表达ERα缺失突变体的细胞系，以阐明ERα调节RAR转录活性的机制。通过逆转录聚合酶链反应（RT-PCR）和核糖核酸酶保护分析，我们观察到ERα的表达抑制了RA反应基因RARβ2的基础表达，同时使其能够被tRA强烈诱导。通过含有RARβ2启动子的报告质粒βRE-tk-CAT的瞬时表达，证实了基础RARβ2转录的抑制。另一方面，在ERα阴性细胞中，转录仅被RA微弱诱导。我们进一步确定，ERα对RAR诱导的这种作用需要包含DNA结合结构域的N端含AF-1区域，但独立于C端配体结合结构域。与这些结果一致，ER激动剂雌二醇和AF-2拮抗剂4-羟基他莫昔芬对βRARE活性没有显著影响。相反，阻断ERα AF-1活性的全ER拮抗剂ICI 182,780能够完全解除对基础βRARE活性的抑制。ERα的作用对RAR介导的转录具有特异性，在含有维生素D或甲状腺激素受体典型反应元件的启动子上不发生。此外，ERα和RAR之间的相互作用似乎不是由一些常见的共调节因子的隔离介导的，这表明ERα的N端区域调节RAR转录活性的一种新机制。
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