Department of Pharmacology, Kyungpook National University School of Medicine, Daegu, Republic of Korea.
J Hypertens. 2013 Jul;31(7):1406-13; discussion 1413. doi: 10.1097/HJH.0b013e3283610fed.
Promoter hypomethylation leads to upregulation of Na⁺-K⁺-2Cl⁻ cotransporter 1 (NKCC1) in the spontaneously hypertensive rat (SHR). We hypothesized that recruitment of Specificity Protein 1 (Sp1) by CpG hypomethylation would result in upregulation of Na⁺-K⁺-2Cl⁻ cotransporter 1 in hypertensive rats.
Sham-operated Wistar-Kyoto (WKY) rats (sham) and angiotensin II (Ang II)-infused WKY rats, as well as SHRs, were used in this study. We performed real-time PCR and western blot for determination of the expression levels of Nkcc1 mRNA and protein, respectively, and bisulphite sequencing for determination of the methylation status of the proximal promoter; an assay kit was used for assessment of the activity of DNA methyltransferase (DNMT), and the electrophoretic mobility shift assay (EMSA) was used for assessment of binding of Sp1 to cis-element, and promoter function was assessed using the luciferase assay.
Both Ang II-infused WKY rats and SHRs showed higher expression of Nkcc1 mRNA and protein and less DNA methylation, compared with sham. CpG methylation at Sp1 response elements interfered with binding of Sp1, resulting in disabled promoter activity. Both types of hypertensive rats showed hypomethylation of CpG dinucleotides in Sp1 response elements in accordance with the decrease of DNMT activity. DNMT3b and MeCP2 were highly recruited to the more methylated promoter of normotensive rats, whereas the CXXC finger protein 1 (Cfp1), Sp1 and RNA polymerase II were highly recruited to the less methylated promoter of hypertensive rats.
Our results indicate that recruitment of Sp1 by CpG hypomethylation leads to upregulation of Na⁺-K⁺-2Cl⁻ cotransporter 1 in hypertensive rats.
启动子低甲基化导致自发性高血压大鼠（SHR）中钠钾 2 氯共转运蛋白 1（NKCC1）的上调。我们假设 CpG 低甲基化通过特异性蛋白 1（Sp1）的募集导致高血压大鼠中钠钾 2 氯共转运蛋白 1 的上调。
本研究使用了假手术 Wistar-Kyoto（WKY）大鼠（假手术）和血管紧张素 II（Ang II）输注 WKY 大鼠以及 SHR。我们进行了实时 PCR 和 Western blot 分别用于测定 Nkcc1 mRNA 和蛋白的表达水平，并进行了亚硫酸氢盐测序以测定近端启动子的甲基化状态；使用试剂盒评估 DNA 甲基转移酶（DNMT）的活性，使用电泳迁移率变动分析（EMSA）评估 Sp1 与顺式元件的结合，使用荧光素酶测定评估启动子功能。
与假手术相比，Ang II 输注的 WKY 大鼠和 SHR 均表现出更高的 Nkcc1 mRNA 和蛋白表达水平以及更低的 DNA 甲基化。Sp1 反应元件的 CpG 甲基化干扰了 Sp1 的结合，导致启动子活性丧失。两种高血压大鼠均表现出 DNMT 活性降低导致的 Sp1 反应元件中 CpG 二核苷酸的低甲基化。DNMT3b 和 MeCP2 高度募集到正常血压大鼠中甲基化程度更高的启动子，而 CXXC 指蛋白 1（Cfp1）、Sp1 和 RNA 聚合酶 II 高度募集到高血压大鼠中甲基化程度较低的启动子。
我们的结果表明，CpG 低甲基化导致 Sp1 的募集导致高血压大鼠中钠钾 2 氯共转运蛋白 1 的上调。
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