Division of Molecular Medicine, Department of Anesthesiology, UCLA, Los Angeles, California 90095-1778, USA.
J Biol Chem. 2011 Sep 9;286(36):31064-71. doi: 10.1074/jbc.M111.235457. Epub 2011 Jul 11.
Estrogen receptor α (ERα) regulates gene transcription via "genomic" (binding directly or indirectly, typically via Sp1 or AP-1 sites, to target genes) and/or "nongenomic" (signaling) mechanisms. ERα activation by estrogen up-regulates the murine Ca(2+)-activated K(+) channel α subunit gene (mSlo1) via genomic mechanisms. Here, we investigated whether ERα also drives transcription of the human (hSlo1) gene. Consistent with this view, estrogen increased hSlo1 transcript levels in primary human smooth muscle cells. Promoter studies revealed that estrogen/hERα-mediated hSlo1 transcription was nearly 6-fold more efficient than for mSlo1 (EC(50), 0.07 versus 0.4 nM). Unlike the genomic transcriptional mechanism employed by mSlo1, hSlo1 exhibits a nongenomic hERα-mediated regulatory mechanism. This is supported by the following: 1) efficient hSlo1 transcription after disruption of the DNA-binding domain of hERα or knockdown of Sp1, and 2) lack of AP-1 sites in the hSlo1 promoter. Three nongenomic signaling pathways were explored: Src, Rho, and PI3K. Inhibition of Src with 10 μM PP2, and reported downstream ERK with 25 μM PD98059 did not prevent estrogen action but caused an increase in hSlo1 basal transcription; conversely, constitutively active c-Src (Y527F) decreased hSlo1 basal transcription even preventing its estrogen/hERα-mediated transcriptional activation. Rho inhibition by coexpressed Clostridium botulinum C3 transferase did not alter estrogen action. In contrast, inhibition of PI3K activity with 10 μM LY294002 decreased estrogen-stimulated hSlo1 transcription by ∼40%. These results indicate that the nongenomic PI3K signaling pathway plays a role in estrogen/hERα-stimulated hSlo1 gene expression; whereas c-Src activity leads to hSlo1 gene tonic repression independently of estrogen, likely through ERK activation.
雌激素受体 α（ERα）通过“基因组”（直接或间接结合，通常通过 Sp1 或 AP-1 位点与靶基因结合）和/或“非基因组”（信号转导）机制调节基因转录。雌激素激活 ERα可通过基因组机制上调鼠钙激活钾通道 α亚基基因（mSlo1）。在这里，我们研究了 ERα 是否也驱动人（hSlo1）基因的转录。与这一观点一致，雌激素增加了原代人平滑肌细胞中 hSlo1 的转录水平。启动子研究表明，雌激素/hERα 介导的 hSlo1 转录比 mSlo1 高近 6 倍（EC50，0.07 对 0.4 nM）。与 mSlo1 采用的基因组转录机制不同，hSlo1 表现出非基因组 hERα 介导的调节机制。这得到了以下几点的支持：1）破坏 hERα 的 DNA 结合域或敲低 Sp1 后，hSlo1 转录效率高，2）hSlo1 启动子中缺乏 AP-1 位点。探讨了三种非基因组信号通路：Src、Rho 和 PI3K。用 10 μM PP2 抑制 Src，用 25 μM PD98059 抑制报道的下游 ERK，不能阻止雌激素的作用，但会增加 hSlo1 的基础转录；相反，组成型激活的 c-Src（Y527F）降低了 hSlo1 的基础转录，甚至阻止了其雌激素/hERα 介导的转录激活。用表达的梭菌肉毒杆菌 C3 转移酶抑制 Rho 不改变雌激素的作用。相反，用 10 μM LY294002 抑制 PI3K 活性可使雌激素刺激的 hSlo1 转录减少约 40%。这些结果表明，非基因组 PI3K 信号通路在雌激素/hERα 刺激的 hSlo1 基因表达中发挥作用；而 c-Src 活性通过 ERK 激活导致 hSlo1 基因持续抑制，而与雌激素无关。
