9p21.3 Coronary Artery Disease Risk Variants Disrupt TEAD Transcription Factor–Dependent Transforming Growth Factor β Regulation of p16 Expression in Human Aortic Smooth Muscle Cells

Abstract

Background— The mechanism whereby the 9p21.3 locus confers risk for coronary artery disease remains incompletely understood. Risk alleles are associated with reduced expression of the cell cycle suppressor genes CDKN2A (p16 and p14) and CDKN2B (p15) and increased vascular smooth muscle cell proliferation. We asked whether risk alleles disrupt transcription factor binding to account for this effect. Methods and Results— A bioinformatic screen was used to predict which of 59 single nucleotide polymorphisms at the 9p21.3 locus disrupt (or create) transcription factor binding sites. Electrophoretic mobility shift and luciferase reporter assays examined the binding and functionality of the predicted regulatory sequences. Primary human aortic smooth muscle cells (HAoSMCs) were genotyped for 9p21.3, and HAoSMCs homozygous for the risk allele showed reduced p15 and p16 levels and increased proliferation. rs10811656 and rs4977757 disrupted functional TEF-1 TEC1 AbaA domain (TEAD) transcription factor binding sites. TEAD3 and TEAD4 overexpression induced p16 in HAoSMCs homozygous for the nonrisk allele, but not for the risk allele. Transforming growth factor β, known to activate p16 and also to interact with TEAD factors, failed to induce p16 or to inhibit proliferation of HAoSMCs homozygous for the risk allele. Knockdown of TEAD3 blocked transforming growth factor β–induced p16 mRNA and protein expression, and dual knockdown of TEAD3 and TEAD4 markedly reduced p16 expression in heterozygous HAoSMCs. Conclusions— Here, we identify a novel mechanism whereby sequences at the 9p21.3 risk locus disrupt TEAD factor binding and TEAD3-dependent transforming growth factor β induction of p16 in HAoSMCs. This mechanism accounts, in part, for the 9p21.3 coronary artery disease risk.

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

“…Our data add to a growing body of evidence that common noncoding variants alter cardiovascular risk by altering transcription factor binding and gene expression (34,35), and support previous studies implicating RhoA signaling in the regulation of BP homeostasis in mice (14,36,37). Our human genetic data from well-characterized untreated patients confirmed that the minor ARHGAP42 allele was associated with decreased BP.…”

Heritability and Genome-Wide Association Analyses of Intracranial Carotid Artery Calcification

“…Of particular note is a recent publication on how 9p21.3 risk variants disrupt specific transcription factor-dependent transforming growth factor-β regulation of p16 expression in human aortic smooth muscle cells. 27 An interesting hypothesis would be that this same mechanism is responsible for vascular disease in other vessel beds by affecting smooth muscle cells locally, such as in the carotid arteries, providing a potential mechanism on how the risk variants influence vascular disease.…”

Unveiling the Role of the Most Impactful Cardiovascular Risk Locus through Haplotype Editing

“…Given this heterogeneity, performing studies in a strongly implicated cell type such as human VSMCs would likely be most informative. However, such studies have been limited by difficulties in obtaining patient coronary arteries, by the tendency of VSMCs to senesce in vitro and by the known phenotypic heterogeneity of VSMCs across individuals, tissues, and disease states (Almontashiri et al, 2015). …”