CREB trans-activates the murine H(+)-K(+)-ATPase alpha(2)-subunit gene.
CREB trans-activates the murine H(+)-K(+)-ATPase alpha(2)-subunit gene.
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CREB 反式激活鼠 H( )-K( )-ATPase α(2) 亚基基因。
DOI：
10.1152/ajpcell.00065.2004
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发表时间：
2004
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：
Xu,Xiangyang;Zhang,Wenzheng;Kone,BruceC
尽管H+-K+-ATP酶α2-亚基（HKα2）基因在钾稳态中起关键作用，但其在集合管中的转录控制仍不清楚。cAMP可增加集合管H+-K+-ATP酶活性，但其在激活HKα 2转录中的作用尚不清楚。以前，我们证明HKα 2启动子近端177 bp赋予基底集合管选择性表达。该区域包含几个潜在的cAMP/Ca 2 +-响应元件（CRE）。因此，我们研究了CREB在小鼠内髓集合管（mIMCD）-3细胞HKα 2转录调控中的作用。Forskolin和加压素可诱导HKα 2 mRNA的表达，CREB的过表达可刺激HKα 2 mRNA启动子-荧光素酶结构的活性。序列缺失分析显示，CREB诱导子保留在含有HKα 2启动子近端100 bp的构建体中。相反，显性负性抑制剂（A-CREB）的表达导致HKα 2启动子-荧光素酶活性降低60%，表明组成型CREB参与了基础HKα 2转录活性。CREB突变体（CREB-VP 16）能强烈诱导HKα 2启动子-荧光素酶活性，而过表达缺乏CREB DNA结合域的CREBdLZ-VP 16则能消除这种激活。用重组谷胱甘肽S-转移酶（GST）-CREB-1和mIMCD-3细胞核提取物对近端启动子进行体外DNA酶I足迹和凝胶位移/超位移分析，结果显示在−86/−60处存在序列特异性DNA-CREB-1复合物。在该区域内的三个CRE样序列的突变消除了CREB-1的DNA结合活性，并消除了CREB-VP 16对HKα 2启动子的反式激活。相反，邻近的-104/-94 κβ元件的突变并不改变CREB-VP 16对HKα 2启动子的反式激活。因此，CREB-1与−86/−60区域的一个或多个CREB样元件结合，反式激活HKα 2基因，并可能代表cAMP对HKα 2活性的快速和延迟效应之间的重要联系。
Despite its key role in potassium homeostasis, transcriptional control of the H+-K+-ATPase α2-subunit (HKα2) gene in the collecting duct remains poorly characterized. cAMP increases H+-K+-ATPase activity in the collecting duct, but its role in activating HKα2transcription has not been explored. Previously, we demonstrated that the proximal 177 bp of the HKα2promoter confers basal collecting duct-selective expression. This region contains several potential cAMP/Ca2+-responsive elements (CRE). Accordingly, we examined the participation of CRE-binding protein (CREB) in HKα2transcriptional control in murine inner medullary collecting duct (mIMCD)-3 cells. Forskolin and vasopressin induced HKα2mRNA levels, and CREB overexpression stimulated the activity of HKα2promoter-luciferase constructs. Serial deletion analysis revealed that CREB inducibility was retained in a construct containing the proximal 100 bp of the HKα2promoter. In contrast, expression of a dominant negative inhibitor (A-CREB) resulted in 60% lower HKα2promoter-luciferase activity, suggesting that constitutive CREB participates in basal HKα2transcriptional activity. A constitutively active CREB mutant (CREB-VP16) strongly induced HKα2promoter-luciferase activity, whereas overexpression of CREBdLZ-VP16, which lacks the CREB DNA-binding domain, abolished this activation. In vitro DNase I footprinting and gel shift/supershift analysis of the proximal promoter with recombinant glutathioneS-transferase (GST)-CREB-1 and mIMCD-3 cell nuclear extracts revealed sequence-specific DNA-CREB-1 complexes at −86/−60. Mutation at three CRE-like sequences within this region abolished CREB-1 DNA-binding activity and abrogated CREB-VP16trans-activation of the HKα2promoter. In contrast, mutation of the neighboring −104/−94 κβ element did not alter CREB-VP16trans-activation of the HKα2promoter. Thus CREB-1, binding to one or more CRE-like elements in the −86/−60 region,trans-activates the HKα2gene and may represent an important link between rapid and delayed effects of cAMP on HKα2activity.