Functional polymorphism of the Anpep gene increases promoter activity in the Dahl salt-resistant rat
Functional polymorphism of the Anpep gene increases promoter activity in the Dahl salt-resistant rat
复制标题
DOI：
10.1161/01.hyp.0000256303.40359.38
复制
发表时间：
2007-03-01
期刊：
影响因子：
8.3
通讯作者：
Danziger, Robert S.
中科院分区：
文献类型：
--
作者：
Kotlo, Kumar;Hughes, Douglas E.;Danziger, Robert S.
我们已经报道，氨肽酶N/CD 13，代谢血管紧张素III血管紧张素IV，表现出更大的肾小管表达在达尔盐耐（SR/Jr）大鼠比盐敏感（SS/Jr）对应。在这项工作中，SS/Jr和SR/Jr菌株的氨肽酶N（Anpep）基因进行了比较。编码区只含有沉默的单核苷酸多态性菌株之间。5'侧翼区也含有多个单核苷酸多态性，使用肾上皮细胞（HK-2）核提取物和与含有单核苷酸多态性的区域对应的寡核苷酸通过电泳迁移率变动分析来分析。在SR/Jr Anpep基因5'侧翼区的CCAAT/增强子结合蛋白基序（核苷酸-2256至-2267）上游4个核苷酸处的独特单核苷酸多态性与DNA-蛋白质复合物的形成相关，而SS大鼠中的相应序列与此无关。来自Dahl SR/Jr大鼠的含有约4.4Kb的Anpep 5'侧翼的嵌合报告基因在HK-2细胞中的表达比来自SS株的相应构建体高2.5至3倍（P < 0.05）。用来自SR品系的CCAAT/增强子结合蛋白顺式作用元件替换来自SS大鼠的CCAAT/增强子结合蛋白顺式作用元件使报告基因表达增加了2.5倍（P < 0.05），并且消除了这种差异。CCAAT/增强子结合蛋白的关联通过染色质免疫沉淀证实，并与表达相关，表明Dahl SR/Jr大鼠中Anpep 5'侧翼的功能性CCAAT/增强子结合蛋白多态性的选择。这些结果突出了Anpep基因与Dahl大鼠高血压的可能关联，并提出了增加的Anpep可能在适应高盐中起机制作用的前景。
We have reported that aminopeptidase N/CD13, which metabolizes angiotensin III to angiotensin IV, exhibits greater renal tubular expression in the Dahl salt-resistant (SR/Jr) rat than its salt-sensitive (SS/Jr) counterpart. In this work, aminopeptidase N (Anpep) genes from SS/Jr and SR/Jr strains were compared. The coding regions contained only silent single nucleotide polymorphisms between strains. The 5' flanking regions also contained multiple single nucleotide polymorphisms, which were analyzed by electrophoretic mobility-shift assay using renal epithelial cell (HK-2) nuclear extracts and oligonucleotides corresponding with single nucleotide polymorphism-containing regions. A unique single nucleotide polymorphism 4 nucleotides upstream of a putative CCAAT/enhancer binding protein motif (nucleotides -2256 to -2267) in the 5' flanking region of the SR/Jr Anpep gene was associated with DNA- protein complex formation, whereas the corresponding sequences in SS rats were not. A chimeric reporter gene containing approximate to 4.4 Kb of Anpep 5' flank from the Dahl SR/Jr rat exhibited 2.5- to 3-fold greater expression in HK-2 cells than the corresponding construct derived from the SS strain (P < 0.05). Replacing the CCAAT/enhancer binding protein cis-acting element from the SS rat with that from the SR strain increased reporter gene expression by 2.5- fold (P < 0.05) and abolished this difference. CCAAT/enhancer binding protein association was confirmed by chromatin immunoprecipitation and correlated with expression, suggesting selection for a functional CCAAT/enhancer binding protein polymorphism in the 5' flank of Anpep in the Dahl SR/Jr rat. These results highlight a possible association of the Anpep gene with hypertension in Dahl rat and raise the prospect that increased Anpep may play a mechanistic role in adaptation to high salt.