Blood pressure–associated polymorphism controls ARHGAP42 expression via serum response factor DNA binding
Xue Bai, Kevin D. Mangum, Rachel A. Dee, George A. Stouffer, Craig R. Lee, Akinyemi Oni-Orisan, Cam Patterson, Jonathan C. Schisler, Anthony J. Viera, Joan M. Taylor, Christopher P. Mack
Xue Bai, Kevin D. Mangum, Rachel A. Dee, George A. Stouffer, Craig R. Lee, Akinyemi Oni-Orisan, Cam Patterson, Jonathan C. Schisler, Anthony J. Viera, Joan M. Taylor, Christopher P. Mack
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Research Article Genetics Vascular biology

Blood pressure–associated polymorphism controls ARHGAP42 expression via serum response factor DNA binding

Abstract

We recently demonstrated that selective expression of the Rho GTPase-activating protein ARHGAP42 in smooth muscle cells (SMCs) controls blood pressure by inhibiting RhoA-dependent contractility, providing a mechanism for the blood pressure–associated locus within the ARHGAP42 gene. The goals of the current study were to identify polymorphisms that affect ARHGAP42 expression and to better assess ARHGAP42’s role in the development of hypertension. Using DNase I hypersensitivity methods and ENCODE data, we have identified a regulatory element encompassing the ARHGAP42 SNP rs604723 that exhibits strong SMC-selective, allele-specific activity. Importantly, CRISPR/Cas9–mediated deletion of this element in cultured human SMCs markedly reduced endogenous ARHGAP42 expression. DNA binding and transcription assays demonstrated that the minor T allele variation at rs604723 increased the activity of this fragment by promoting serum response transcription factor binding to a cryptic cis-element. ARHGAP42 expression was increased by cell stretch and sphingosine 1-phosphate in a RhoA-dependent manner, and deletion of ARHGAP42 enhanced the progression of hypertension in mice treated with DOCA-salt. Our analysis of a well-characterized cohort of untreated borderline hypertensive patients suggested that ARHGAP42 genotype has important implications in regard to hypertension risk. Taken together, our data add insight into the genetic mechanisms that control blood pressure and provide a potential target for individualized antihypertensive therapies.

Authors

Xue Bai, Kevin D. Mangum, Rachel A. Dee, George A. Stouffer, Craig R. Lee, Akinyemi Oni-Orisan, Cam Patterson, Jonathan C. Schisler, Anthony J. Viera, Joan M. Taylor, Christopher P. Mack

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Figure 1

ARHGAP42 expression in SMCs is regulated by allele-specific mechanisms and controls BP.

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ARHGAP42 expression in SMCs is regulated by allele-specific mechanisms ...
(A) Total RNA isolated from HuAoSMCs heterozygous at the rs604723 SNP (C/T) was subjected to first-strand cDNA synthesis using reverse transcriptase. Reaction products were then subjected to a TaqMan-based PCR assay using allele-specific primers to the ARHGAP42 rs604723 variation. Data represent mean ± SEM of n = 4 experiments; *P < 0.01 vs. the major C allele (Student’s t test). (B) ARHGAP42 mRNA levels were measured by the Genotype-Tissue Expression (GTEx) Consortium. The minor T ARHGAP42 allele at the rs604723 polymorphism was significantly associated with increased ARHGAP42 expression in aortic and coronary artery samples. (C) Schematic of the Arhgap42 gene-trap and SM-MHCCreERT2 mice used for SMC-specific ARHGAP42 rescue experiments. (D) WT and Arhgap42gt/gt SM-MHCCreERT2 mice were injected i.p. with vehicle (corn oil) or tamoxifen (100 mg/kg) for 5 consecutive days as indicated. Two weeks after the last injection, BP was measured by tail cuff method, and Arhgap42 mRNA levels in the aorta were measured by semiquantitative RT-PCR analysis using primers to exons 1 and 4. Data are expressed as mean ± SEM; n = 6 for WT and Arhgap42gt/gt SM-MHCCreERT2 mice with vehicle treatment, n = 5 for Arhgap42gt/gt SM-MHCCreERT2 mice with tamoxifen treatment. *P < 0.05 vs. WT; **P < 0.05 vs. corn oil–treated (ANOVA). Note that tamoxifen treatment restored Arhgap42 expression and reduced BP to WT levels (representative of 3 separate experiments).