Glucocorticoids stimulate human sgk1 gene expression by activation of a GRE in its 5′-flanking region
Glucocorticoids stimulate human sgk1 gene expression by activation of a GRE in its 5′-flanking region
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DOI：
10.1152/ajpendo.00021.2002
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发表时间：
2002-11-01
影响因子：
5.1
通讯作者：
Thomas, CP
中科院分区：
文献类型：
--
作者：
Itani, OA;Liu, KZ;Thomas, CP
在肺和集合管上皮细胞中，糖皮质激素 (GC) 刺激的 Na+ 转运先于蛋白激酶 sgk1 的增加，从而调节上皮 Na+ 通道 (ENaC) 的活性。我们研究了肺和肾上皮细胞中 GC 调节的人 sgk1 表达的机制。 GC 在这些上皮细胞中增加了 sgk1 mRNA，并且这种增加被放线菌素 D 抑制并被放线菌酮超级诱导，这与不需要蛋白质合成的转录效应一致。为了了解转录调控的基础，绘制了转录起始位点并通过 PCR 克隆了 5' 侧翼区域。将上游区域的 3 kb 片段与荧光素酶偶联并转染至 A549 细胞中。通过缺失分析，确定了一个不完美的 GC 响应元件 (GRE)，它对于 GC 响应是必要且充分的。当使用细胞提取物进行测试时，抗 GC 受体 (GR) 抗体识别的特定蛋白质在凝胶迁移率变化测定中与 GRE 结合。我们得出的结论是，GC 通过激活 sgk1 5' 侧翼区域的 GRE 来刺激人上皮细胞中 sgk1 的表达。
In lung and collecting duct epithelia, glucocorticoid (GC)-stimulated Na+ transport is preceded by an increase in the protein kinase sgk1, which in turn regulates the activity of the epithelial Na+ channel (ENaC). We investigated the mechanism for GC-regulated human sgk1 expression in lung and renal epithelia. sgk1 mRNA was increased in these epithelia by GCs, and this was inhibited by actinomycin D and superinduced by cycloheximide, consistent with a transcriptional effect that did not require protein synthesis. To understand the basis for transcriptional regulation, the transcription initiation site was mapped and the 5'-flanking region cloned by PCR. A 3-kb fragment of the upstream region was coupled to luciferase and transfected into A549 cells. By deletion analysis, an imperfect GC response element (GRE) was identified that was necessary and sufficient for GC responsiveness. When tested with cell extracts, a specific protein recognized by an anti-GC receptor (GR) antibody bound the GRE in gel mobility shift assays. We conclude that GCs stimulate sgk1 expression in human epithelial cells via activation of a GRE in the 5'-flanking region of sgk1.