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Recruitment of Specificity Protein 1 by CpG hypomethylation upregulates Na+-K+-2Cl cotransporter 1 in hypertensive rats

Cho, Hyun-Mina; Lee, Hae-Ahma,b; Kim, Hye Younga; Lee, Dong-Youba; Kim, In Kyeoma,b,c

doi: 10.1097/HJH.0b013e3283610fed
ORIGINAL PAPERS: Genetic aspects

Objectives: 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.

Methods: 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.

Results: 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.

Conclusion: Our results indicate that recruitment of Sp1 by CpG hypomethylation leads to upregulation of Na+-K+-2Cl cotransporter 1 in hypertensive rats.

aDepartment of Pharmacology

bCardiovascular Research Institute

cCell and Matrix Research Institute, Kyungpook National University School of Medicine, Daegu, Republic of Korea

Correspondence to In Kyeom Kim, MD, PhD, Department of Pharmacology, Kyungpook National University School of Medicine, 101 Dongin-2-Ga, Daegu, 700-422, Republic of Korea. Tel: +82 53 420 4833; fax: +82 53 426 7345; e-mail: inkim@knu.ac.kr

Abbreviations: Ang II, angiotensin II; Cfp1, CXXC finger protein 1; DNMT, DNA methyltransferase; EMSA, electrophoretic mobility shift assay; MeCP2, methyl cytosine binding protein 2; NKCC1, Na+-K+-2Cl cotransporter 1; Pol II, RNA polymerase II; SHR, spontaneously hypertensive rat; Sp1, Specificity Protein 1; WKY, Wistar–Kyoto

Received 9 October, 2012

Revised 11 January, 2013

Accepted 8 March, 2013

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