cAMP motifs regulating transcription in the aquaporin 2 gene
cAMP motifs regulating transcription in the aquaporin 2 gene
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DOI：
10.1152/ajpcell.1996.270.6.c1695
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发表时间：
1996-06-01
影响因子：
5.5
通讯作者：
Ausiello, DA
中科院分区：
文献类型：
--
作者：
Hozawa, S;Holtzman, EJ;Ausiello, DA
分离包括AQP2基因5‘侧翼区的基因组克隆，瞬时将启动子-荧光素酶报告融合基因导入肾上皮细胞，检测启动子区域。在AQP2基因5‘侧翼区的近3kb处发现了一个定位特异性启动子。缺失分析发现，AQP2基因的启动子活性在转录起始点上游198个碱基范围内最低。对转录活性区域进行测序发现，在其近端1.2-kb区域有一个典型的TATA盒、3‘，5’-环单磷酸(CAMP)反应元件(CRE)和三个可能的CCAAT盒。值得注意的是，在该区域还发现了GATA基序、AP1、AP2和SP1转录因子共识位点。CAMP增强剂(1 NM加压素或20 mM Forsklin和250 mM 3-异丁基-1-甲基黄嘌呤)可平行增加荧光素酶活性，提示加压素诱导的AQP2基因转录是通过至少一种肾细胞类型LLC-PK1细胞内cAMP增加而介导的。利用一系列肾上皮细胞和其他细胞类型的缺失突变体，进一步研究了AQP2基因转录的cAMP反应机制。在LLC-PK1细胞中，cAMP调控基序存在于-340至-290之间的50bp序列(含Cre)和-150至ATG起始点之间的65bp序列(含AP2位点)中。在大鼠内髓集合管(IMCD)细胞中，cAMP调控基序也存在于-340~-290(含Cre)的50bp序列和-160~-150(含SP1位点)的10bp序列中。这些独立的区域可以协同作用，以细胞特异性的方式赋予AQP2基因完全的cAMP诱导性。
Genomic clones including the 5' flanking regions of the AQP2 (aquaporin 2) gene were isolated, and the promoter region was examined by transiently transfecting a promoter-luciferase reporter fusion gene into renal cultured epithelial cells. An orientation specific promoter for the AQP2 gene was found within the proximal 3 kb of 5'-flanking region. Minimal basal promoter activity of the AQP2 gene was found within 198 bp upstream from the transcription start site by deletion analysis. Sequencing the transcriptionally active region revealed a typical TATA box, adenosine 3',5'-cyclic monophosphate (cAMP) responsive element (CRE) and three putative CCAAT boxes in the proximal 1.2-kb region. Significantly, a GATA motif, AP1, AP2, and SP1 transcriptional factor consensus sites were also found in this region. Exposure to cAMP-enhancing agents (1 nM vasopressin or 20 mM forskolin and 250 mM 3-isobutyl-1-methylxanthine) showed that these agents increased luciferase activity in a parallel fashion, suggesting that vasopressin-induced AQP2 gene transcription is mediated through increases in intracellular cAMP in at least one renal cell type, the LLC-PK1 cells. The mechanism of cAMP responsiveness of AQP2 gene transcription was further studied using a series of deletion mutants in renal epithelial cells and other cell types. The cAMP regulatory motifs were shown to exist in a 50-bp sequence between -340 and -290 (containing CRE) and a 65-bp sequence (containing an AP2 site) between -150 and the ATG start site in LLC-PK1 cells. In rat inner medullary collecting duct (IMCD) cells, the cAMP regulatory motifs also exist in a 50-bp sequence between -340 and -290 (containing CRE) and in a 10-bp sequence between -160 and -150 (containing an SP1 site). These separate regions may cooperate to confer full cAMP inducibility to the AQP2 gene in a cell-specific manner.