AVP-induced VIT32 gene expression in collecting duct cells occurs via trans-activation of a CRE in the 5'-flanking region of the VIT32 gene.
AVP-induced VIT32 gene expression in collecting duct cells occurs via trans-activation of a CRE in the 5'-flanking region of the VIT32 gene.
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AVP 诱导的集合管细胞中的 VIT32 基因表达是通过 VIT32 基因 5 侧翼区域中 CRE 的反式激活而发生的。
DOI：
10.1152/ajprenal.00107.2004
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发表时间：
2004
期刊：
影响因子：
--
通讯作者：
Liu,KangZ
中科院分区：
文献类型：
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作者：
Thomas,ChristieP;Loftus,RandyW;Liu,KangZ
VIT 32，一种加压素诱导的转录物，当与非洲爪蟾细胞中的上皮钠通道共表达时，抑制Na+转运（EMBO J21：5109-5117，2002）。为了了解VIT 32基因调控的机制，我们检测了DDAVP和cAMP刺激对M-1小鼠集合管细胞和H441人气道上皮细胞中VIT 32表达的影响。用forskolin和IBMX升高cAMP可增加VIT 32基因表达，2 h达高峰。H89和放线菌素D可抑制基因表达的增加，表明cAMP通过PKA介导的基因转录增加刺激VIT 32 mRNA的表达。克隆了VIT 32的5′侧翼区的一个约1.5kb的片段，当转染M-1和H441细胞时，该片段能够赋予cAMP刺激的报告基因活性。通过缺失分析和定点突变，在近端启动子区域内鉴定出cAMP反应元件（CRE），其足以解释用DDAVP观察到的INVITE 32基因表达增加和cAMP升高。此外，DDAVP刺激的VIT 32启动子-报告子活性被H89和显性负性CREB构建体抑制。最后，我们能够确定CREB作为一个核蛋白结合的VIT 32 CRE在凝胶迁移率变动分析。总之，DDAVP通过位于VIT 32基因近端启动子区域内的CRE刺激VIT 32的转录。
VIT32, a vasopressin-induced transcript, inhibits Na+transport when coexpressed with the epithelial sodium channel inXenopus laevisoocytes (EMBO J21: 5109–5117, 2002). To understand the mechanism ofVIT32gene regulation, we examined the effect of DDAVP and cAMP stimulation onVIT32expression in M-1 mouse collecting duct cells and in H441 human airway epithelial cells. Elevation of cAMP with forskolin and IBMX increasedVIT32gene expression with a peak effect at 2 h. The increase in gene expression was abolished by H89 and by actinomycin D, suggesting that cAMP stimulatesVIT32mRNA expression by a PKA-mediated increase in gene transcription. An ∼1.5-kb fragment of the 5′-flanking region ofVIT32was cloned and was able to confer cAMP-stimulated reporter gene activity when transfected into M-1 and H441 cells. By deletion analysis and site-directed mutagenesis, a cAMP response element (CRE) was identified within the proximal promoter region that was sufficient to account for the increase inVIT32gene expression seen with DDAVP and elevation of cAMP. Furthermore, DDAVP-stimulatedVIT32promoter-reporter activity was inhibited by H89 and by a dominant negative CREB construct. Finally, we were able to identify CREB as a nuclear protein that bound to theVIT32CRE in gel mobility shift assays. In summary, DDAVP stimulates transcription ofVIT32via a CRE within the proximal promoter region of theVIT32gene.