Sp1 trans-activates and is required for maximal aldosterone induction of the αENaC gene in collecting duct cells.
Sp1 trans-activates and is required for maximal aldosterone induction of the αENaC gene in collecting duct cells.
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Sp1 反式激活，是集合管细胞中 αENaC 基因最大醛固酮诱导所必需的。
DOI：
10.1152/ajprenal.00177.2013
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发表时间：
2013
期刊：
影响因子：
--
通讯作者：
Kone,BruceC
中科院分区：
文献类型：
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作者：
Yu,Zhiyuan;Kong,Qun;Kone,BruceC
远端肾单位的上皮Na+通道（ENaC）构成肾钠重吸收的限速步骤。醛固酮在很大程度上通过增加集合管主细胞中的αENaC转录来增加肾小管钠吸收。我们之前报道过，Af9 与 αENaC 的 +78/+92 结合，并招募 Dot1a 来抑制小鼠内髓集合管 (mIMCD)3 细胞中基础和醛固酮敏感的 αENaC 转录。尽管存在这种表观遗传抑制，但 basalαENaC 转录仍然明显且具有生理必要性，表明正调节因子的基础操作。在本研究中，我们将 Sp1 确定为这样的调节器之一。使用 +208/+240 探针进行的凝胶位移和抗体竞争测定揭示了 mIMCD3 细胞中含有 DNA-Sp1 的复合物。 +222/+229 元件的突变消除了 Sp1 的体外结合以及在 mIMCD3 细胞中稳定表达的启动子-报告基因构建体中的结合。与野生型启动子相比，具有+222/+229突变的αENaC启动子-荧光素酶构建体在Sp1过表达实验中表现出低得多的活性和受损的反式激活。相反，Sp1 敲低抑制内源 αENaC mRNA 和野生型 αENaC 启动子的活性，但不抑制突变构建体的活性。醛固酮触发 Sp1 募集到 αENaC 启动子，这是最大限度诱导 αENaC 启动子活性所必需的，但被螺内酯阻断。 +78/+92 和 +222/+229αENaC 启动子突变体的连续染色质免疫沉淀测定和功能测试表明，虽然 Sp1、Dot1a 和 Af9 共同占据 αENaC 启动子，但 Sp1 的作用在功能上独立于 Dot1a 和 Af9。总之，Sp1 在 +222/+229 处与 acis 元件结合代表了第一个确定的 αENaC 转录的组成型驱动因素，它有助于最大程度地激活 αENaC 醛固酮反式激活。
The epithelial Na+channel (ENaC) in the distal nephron constitutes the rate-limiting step for renal sodium reabsorption. Aldosterone increases tubular sodium absorption in large part by increasingαENaCtranscription in collecting duct principal cells. We previously reported that Af9 binds to +78/+92 ofαENaCand recruits Dot1a to repress basal and aldosterone-sensitiveαENaCtranscription in mouse inner medullary collecting duct (mIMCD)3 cells. Despite this epigenetic repression, basalαENaCtranscription is still evident and physiologically necessary, indicating basal operation of positive regulators. In the present study, we identified Sp1 as one such regulator. Gel shift and antibody competition assays using a +208/+240 probe revealed DNA-Sp1-containing complexes in mIMCD3 cells. Mutation of the +222/+229 element abrogated Sp1 binding in vitro and in promoter-reporter constructs stably expressed in mIMCD3 cells. Compared with the wild-type promoter, anαENaCpromoter-luciferase construct with +222/+229 mutations exhibited much lower activity and impairedtrans-activation in Sp1 overexpression experiments. Conversely, Sp1 knockdown inhibited endogenous αENaC mRNA and the activity of the wild-typeαENaCpromoter but not the mutated construct. Aldosterone triggered Sp1 recruitment to theαENaCpromoter, which was required for maximal induction ofαENaCpromoter activity and was blocked by spironolactone. Sequential chromatin immunoprecipitation assays and functional tests of +78/+92 and +222/+229αENaCpromoter mutants indicated that while Sp1, Dot1a, and Af9 co-occupy theαENaCpromoter, the Sp1 effects are functionally independent from Dot1a and Af9. In summary, Sp1 binding to acis-element at +222/+229 represents the first identified constitutive driver ofαENaCtranscription, and it contributes to maximal aldosteronetrans-activation ofαENaC.