Protein-DNA Interactions at the Opossum Npt2a Promoter are Dependent upon NHERF-1.
Protein-DNA Interactions at the Opossum Npt2a Promoter are Dependent upon NHERF-1.
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Opossum Npt2a 启动子处的蛋白质-DNA 相互作用依赖于 NHERF-1。
DOI：
10.1159/000445601
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发表时间：
2016
期刊：
影响因子：
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通讯作者：
Lederer,EleanorD
中科院分区：
文献类型：
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作者：
Clark,BarbaraJ;Murray,RebeccaD;Salyer,SarahA;Tyagi,SamuelC;Arumugam,Cibi;Khundmiri,SyedJ;Lederer,EleanorD
背景/目的磷酸盐体内平衡是由位于近端小管刷状缘膜的IIa型磷酸钠协同转运蛋白Npt 2a对Pi的肾重吸收控制的。Npt 2a表达的调节是维持磷酸盐稳态的关键控制点，大多数研究集中在调节刷状缘膜中的蛋白水平。然而，控制Npt 2a mRNA的分子机制仍有待确定。我们已经报道了Npt 2a mRNA和蛋白水平与使用负鼠肾（OK）细胞和NHERF-1缺陷的OK-H细胞的Na+/H+交换调节因子1（NHERF-1）的表达直接相关。本研究的目的是确定NHERF-1是否有助于转录和/或转录后机制控制Npt 2a mRNA levels.MethodsNpt2a mRNA半衰期之间的OK和NHERF-1缺陷的OK-H细胞系进行了比较。结果在OK和OK-H细胞中，oNpt 2a mRNA的半衰期基本相同。NHERF-1反应区位于近端启动子内，该区域含有高度保守的CAATT盒和富含G的元件。特定的蛋白质-DNA复合物的形成与CAATT元素的情况下，NHERF-1（OK v OK-H EMSA）的改变，虽然NHERF-1不直接有助于复杂formation.ConclusionNHERF-1有助于维持稳态Npt 2a mRNA水平在OK细胞通过间接的机制，有助于促进蛋白质-DNA相互作用在Npt 2a近端启动子。
Background/AimsPhosphate homeostasis is controlled by the renal reabsorption of Pi by the type IIa sodium phosphate cotransporter, Npt2a, which is localized in the proximal tubule brush border membrane. Regulation of Npt2a expression is a key control point to maintain phosphate homeostasis with most studies focused on regulating protein levels in the brush border membrane. Molecular mechanisms that control Npt2a mRNA, however, remain to be defined. We have reported that Npt2a mRNA and protein levels correlate directly with the expression of the Na+/H+ exchanger regulatory factor 1 (NHERF-1) using opossum kidney (OK) cells and the NHERF-1-deficient OK-H cells. The goal of this study was to determine whether NHERF-1 contributes to transcriptional and/or post-transcriptional mechanisms controlling Npt2a mRNA levels.MethodsNpt2a mRNA half-life was compared between OK and NHERF-1 deficient OK-H cell lines. oNpt2a promoter-reporter gene assays and electrophoretic mobility shift assays (EMSA) were used identify a NHERF-1 responsive region within the oNpt2a proximal promoter.ResultsNpt2a mRNA half-life is the same in OK and OK-H cells. The NHERF-1 responsive region lies within the proximal promoter in a region that contains a highly conserved CAATT box and G-rich element. Specific protein-DNA complex formation with the CAATT element is altered by the absence of NHERF-1 (OK v OK-H EMSA) although NHERF-1 does not directly contribute to complex formation.ConclusionNHERF-1 helps maintain steady-state Npt2a mRNA levels in OK cells through indirect mechanisms that help promote protein-DNA interactions at the Npt2a proximal promoter.