Section of Digestive Diseases and Nutrition, Department of Medicine, University of Illinois at Chicago, Chicago, Illinois, USA.
Am J Physiol Gastrointest Liver Physiol. 2012 Feb 1;302(3):G317-25. doi: 10.1152/ajpgi.00363.2011. Epub 2011 Nov 3.
The apical Na+/H+ exchanger (NHE) isoform NHE2 is involved in transepithelial Na+ absorption in the intestine. Our earlier studies have shown that mitogenic agent phorbol 12-myristate 13-acetate (PMA) induces the expression of NHE2 through activation of transcription factor early growth response-1 (Egr-1) and its interactions with the NHE2 promoter. However, the signaling pathways involved in transcriptional stimulation of NHE2 in response to PMA in the intestinal epithelial cells are not known. Chemical inhibitors and genetic approaches were used to investigate the signaling pathways responsible for the stimulation of NHE2 expression by PMA via Egr-1 induction. We show that, in response to PMA, PKCδ, a member of novel PKC isozymes, and MEK-ERK1/2 pathway of mitogen-activated protein kinases stimulate the NHE2 expression in C2BBe1 intestinal epithelial cells. PMA rapidly and transiently induced activation of PKCδ. Small inhibitory RNA-mediated knockdown of PKCδ blocked the stimulatory effect of PMA on the NHE2 promoter activity. In addition, blockade of PKCδ by rottlerin, a PKCδ-specific inhibitor, and ERK1/2 by U0126, a MEK-ERK inhibitor, abrogated PMA-induced Egr-1 expression. Immunofluorescence studies revealed that inhibition of ERK1/2 activation prevents translocation of PMA-induced Egr-1 into the nucleus. Consistent with these data, PMA-induced Egr-1 interaction with the NHE2 promoter region was prevented in nuclear extracts from U0126-pretreated cells. In conclusion, our data provide the first evidence that the stimulatory effect of PMA on NHE2 expression is mediated through the initial activation of PKCδ, subsequent PKCδ-dependent activation of MEK-ERK1/2 signaling pathway, and stimulation of Egr-1 expression. Furthermore, we show that transcription factor Egr-1 acts as an intermediate effector molecule that links the upstream signaling cues to the long-term stimulation of NHE2 expression by PMA in C2BBe1 cells.
顶端钠/氢交换器（NHE）同工型 NHE2 参与肠道中的跨上皮钠离子吸收。我们之前的研究表明，有丝分裂原佛波醇 12-肉豆蔻酸 13-乙酸酯（PMA）通过激活转录因子早期生长反应-1（Egr-1）及其与 NHE2 启动子的相互作用，诱导 NHE2 的表达。然而，在肠道上皮细胞中，PMA 诱导 NHE2 转录的信号通路尚不清楚。我们使用化学抑制剂和基因敲除方法研究了 PMA 通过诱导 Egr-1 诱导 NHE2 表达的信号通路。我们发现，在 PMA 刺激下，新型蛋白激酶 C 同工型 PKCδ和丝裂原活化蛋白激酶 ERK1/2 途径刺激 C2BBe1 肠道上皮细胞中 NHE2 的表达。PMA 可快速短暂地诱导 PKCδ 激活。小干扰 RNA 介导的 PKCδ 敲低阻断了 PMA 对 NHE2 启动子活性的刺激作用。此外，PKCδ 特异性抑制剂罗特林和 MEK-ERK 抑制剂 U0126 阻断了 PMA 诱导的 Egr-1 表达。免疫荧光研究表明，抑制 ERK1/2 激活可阻止 PMA 诱导的 Egr-1 向核内易位。与这些数据一致，在 U0126 预处理细胞的核提取物中，抑制 PMA 诱导的 Egr-1 与 NHE2 启动子区域的相互作用。总之，我们的数据首次表明，PMA 对 NHE2 表达的刺激作用是通过 PKCδ 的初始激活、随后 PKCδ 依赖性激活 MEK-ERK1/2 信号通路以及刺激 Egr-1 表达来介导的。此外，我们还表明，转录因子 Egr-1 作为一种中间效应分子，将上游信号线索与 PMA 在 C2BBe1 细胞中对 NHE2 表达的长期刺激联系起来。
