9p21.3 Coronary Artery Disease Risk Variants Disrupt TEAD Transcription Factor-Dependent TGFβ Regulation of p16 Expression in Human Aortic Smooth Muscle Cells
Background—The mechanism whereby the 9p21.3 locus confers risk for coronary artery disease (CAD) 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 (SNPs) at the 9p21.3 locus disrupt (or create) transcription factor binding sites. Electrophoretic mobility shift (EMSA) 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 TEAD transcription factor binding sites. TEAD3 and TEAD4 over-expression induced p16 in HAoSMCs homozygous for the non-risk allele, but not for the risk allele. TGFβ, 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 TGFβ-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 TGFβ induction of p16 in HAoSMCs. This mechanism accounts, in part, for the 9p21.3 CAD risk.
- Received December 18, 2014.
- Revision received August 5, 2015.
- Accepted August 28, 2015.