Nitric oxide decreases expression of osmoprotective genes via direct inhibition of TonEBP transcriptional activity
Nitric oxide decreases expression of osmoprotective genes via direct inhibition of TonEBP transcriptional activity
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DOI：
10.1007/s00424-008-0540-3
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发表时间：
2009-02-01
影响因子：
4.5
通讯作者：
Beck, Franz-X.
中科院分区：
文献类型：
--
作者：
Neuhofer, Wolfgang;Fraek, Maria-Luisa;Beck, Franz-X.
在抗利尿期间，肾髓质细胞通过增加抗利尿剂保护基因的表达来适应高渗间质环境，这是由常见的转录激活因子、张力响应增强子结合蛋白（TonEBP）驱动的。由于一氧化氮（NO）在肾髓质中大量产生，本研究探讨了NO对MDCK细胞中肾脏保护基因表达和TonEBP激活的影响。几个结构无关的NO供体钝化张力诱导的上调TonEBP靶基因参与细胞内积累的有机渗透压。这些影响介导的TonEBP的转录活性降低，评估张力响应元件和醛糖还原酶启动子驱动的报告构建体。无论是总TonEBP丰度，也不TonEBP核转位的NO的影响。此外，8-溴-cGMP和过氧亚硝酸盐未能再现NO的抑制作用，表明NO直接作用于TonEBP，而不是通过经典的NO信号通路。在支持这一观点，电泳迁移率变动分析表明，减少TonEBP结合到其靶序列在核提取物制备的MDCK细胞处理NO在体内和核提取物暴露于NO在体外。此外，S-亚硝基化蛋白的免疫沉淀和生物素开关方法将TonEBP鉴定为S-亚硝基化的靶点，其与降低的DNA结合和转录活性相关。这些观察结果揭示了一种新的直接抑制作用的NO对TonEBP，一种现象，可能是相关的调节肾脏髓质中的保护性基因。
During antidiuresis, renal medullary cells adapt to the hyperosmotic interstitial environment by increased expression of osmoprotective genes, which is driven by a common transcriptional activator, tonicity-responsive enhancer binding protein (TonEBP). Because nitric oxide (NO) is abundantly produced in the renal medulla, the present studies addressed the effect of NO on expression of osmoprotective genes and TonEBP activation in MDCK cells. Several structurally unrelated NO donors blunted tonicity-induced up-regulation of TonEBP target genes involved in intracellular accumulation of organic osmolytes. These effects were mediated by reduced transcriptional activity of TonEBP, as assessed by tonicity-responsive elements- and aldose reductase promoter-driven reporter constructs. Neither total TonEBP abundance nor nuclear translocation of TonEBP was affected by NO. Furthermore, 8-bromo-cGMP and peroxynitrite failed to reproduce the inhibitory effect of NO, indicating that NO acts directly on TonEBP rather than through classical NO signaling pathways. In support of this notion, electrophoretic mobility shift assays showed reduced binding of TonEBP to its target sequence in nuclear extracts prepared from MDCK cells treated with NO in vivo and in nuclear extracts exposed to NO in vitro. Furthermore, immunoprecipitation of S-nitrosylated proteins and the biotin-switch method identified TonEBP as a target for S-nitrosylation, which correlates with reduced DNA binding and transcriptional activity. These observations disclose a novel direct inhibitory effect of NO on TonEBP, a phenomenon that may be relevant for regulation of osmoprotective genes in the renal medulla.