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首页> 外文期刊>American Journal of Physiology >Molecular cloning and functional analysis of the human Na(+)/H(+) exchanger NHE3 promoter.
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Molecular cloning and functional analysis of the human Na(+)/H(+) exchanger NHE3 promoter.

机译：人Na（+）/ H（+）交换子NHE3启动子的分子克隆和功能分析。

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摘要

Na(+)/H(+) exchanger (NHE) isoforms NHE2 and NHE3, colocalized to the brush border membrane of the epithelial cells, exhibit differences in their pattern of tissue expression and regulation by various molecular signals. To investigate the mechanisms involved in regulation of NHE3 gene expression, the human NHE3 promoter region was cloned and characterized. Primer extension experiments located the transcription start site to a position 116 nucleotides upstream from the translation start codon. The 5'-flanking region lacked a CCAAT box but contained a TATA-like sequence. Nucleotide sequencing of the 5'-flanking region revealed the presence of a number of cis elements including Sp1, AP-2, MZF-1, CdxA, Cdx-2, steroid and nonsteroid hormone receptor half sites, and a phorbol 12-myristate 13-acetate-response element. Transient transfection experiments using C2/bbe cell line defined a maximal promoter activity in -95/+5 region. The regulatory response elements clustered within this region include a potential transcription factor IID (TF IID), a CACCC, two Sp1, and two AP-2 motifs. Deletion of a fragment containing the AP-2 and Sp1 motifs resulted in a drastic decrease in promoter activity. In gel mobility shift assays, an oligonucleotide spanning from -78 to -56 bp bound a recombinant AP-2, and the corresponding binding activity in nuclear extracts was supershifted with anti-AP2alpha antibody. Our studies suggest that the NHE3 expression is regulated by a combination of cis elements and their cognate transcription factors that include the AP-2 and Sp1 family members.
机译：Na（+）/ H（+）交换子（NHE）亚型NHE2和NHE3，共定位于上皮细胞的刷状缘膜，在组织表达和通过各种分子信号调控的模式中表现出差异。为了研究调节NHE3基因表达的机制，克隆并表征了人NHE3启动子区。引物延伸实验将转录起始位点定位在翻译起始密码子上游116个核苷酸的位置。 5'侧翼区域缺少CCAAT盒，但包含TATA样序列。 5'侧翼区域的核苷酸测序表明存在许多顺式元件，包括Sp1，AP-2，MZF-1，CdxA，Cdx-2，类固醇和非类固醇激素受体半位点以及佛波醇12-肉豆蔻酸酯13 -乙酸盐响应元素。使用C2 / bbe细胞系的瞬时转染实验在-95 / + 5区定义了最大的启动子活性。聚集在该区域内的调节应答元件包括潜在的转录因子IID（TF IID），CACCC，两个Sp1和两个AP-2基序。删除包含AP-2和Sp1基序的片段会导致启动子活性急剧下降。在凝胶迁移率迁移分析中，跨度为-78到-56 bp的寡核苷酸与重组AP-2结合，而核提取物中的相应结合活性被抗AP2α抗体超迁移。我们的研究表明，NHE3的表达受顺式元件及其相关转录因子（包括AP-2和Sp1家族成员）的组合调控。

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