Functional Validation of a Common Nonsynonymous Coding Variant in ZC3HC1 Associated With Protection From Coronary Artery Disease.

BACKGROUND: Although virtually all coronary artery disease associated single-nucleotide polymorphisms identified by genome-wide association studies (GWAS) are in noncoding regions of the genome, a common polymorphism in ZC3HC1 (rs11556924), resulting in an arginine (Arg) to histidine (His) substitution in its encoded protein, NIPA (Nuclear Interacting Partner of Anaplastic Lyphoma Kinase) is linked to a protection from coronary artery disease. NIPA plays a role in cell cycle progression, but the functional consequences of this polymorphism have not been established. METHODS AND RESULTS: Here we demonstrate that total ZC3HC1 expression in whole blood is similar across genotypes, despite expression being slightly biased toward the risk allele in heterozygotes. At the protein level, the protective His363 NIPA variant exhibits increased phosphorylation of a critical serine residue (Ser354) and higher protein expression as compared with the Arg363 variant. Binding experiments indicate that neither SKP1 (S-phase kinase-associated protein 1) nor CCNB1 binding were affected by the polymorphism. Despite similar nuclear distribution, NIPA His363 exhibits greater nuclear mobility. NIPA suppression results in a modest reduction of proliferation in vascular smooth muscle cells, but given low proliferative capacity, a significant effect of the variant was not noted. By contrast, we demonstrate that the protective variant reduces cell proliferation in HeLa cells. CONCLUSIONS: These findings extend the genetic association between rs11556924 and coronary artery disease risk by characterizing its effects on the encoded protein, NIPA. The resulting amino acid change Arg363His is associated with increased expression and nuclear mobility, as well as lower rates of cell growth in HeLa cells, further supporting a role for cell proliferation in atherosclerosis and its clinical consequences.

Functional interaction between COL4A1/COL4A2 and SMAD3 risk loci for coronary artery disease.

OBJECTIVE: The COL4A1/COL4A2 region on chromosome 13q34 is a highly replicated locus for coronary artery disease (CAD). In the normal arterial wall, type IV collagen acts to inhibit smooth muscle cell proliferation. Its production is in part a function of TGFß signaling, but the specific regulatory mechanisms, especially in humans, have not been defined. Our aim was to decipher TGFß signaling components important in the regulation of COL4A1 and COL4A2 and determine whether these components showed genetic interaction with the COL4A1/COL4A2 locus for CAD association. METHODS AND RESULTS: Experiments were performed in primary human aortic smooth muscle cells and HT1080 fibroblasts. Pharmacological inhibition of the TGFß1 receptor and subsequent SMAD protein phosphorylation by treatment with an ALK5 inhibitor prevented the increase in COL4A1/COL4A2 mRNA (p < 0.001) and protein expression in response to TGFß1 stimulation. In contrast, inhibition of the non-canonical TGFß signaling pathways was without effect. siRNA mediated knockdown of SMAD3 and SMAD4 abolished the stimulatory effects of TGFß1 on COL4A1/COL4A2 (p < 0.001) whereas SMAD2 knockdown had no effect. In luciferase reporter assays, neither SMAD3 overexpression nor TGFß1 treatment altered COL4A1 or COL4A2 promoter activity, supportive of more complex regulation of type IV collagen gene expression by the TGFß/SMAD3 signaling pathway. Epistasis analysis in 5 CAD case/control cohorts revealed that SMAD3 and COL4A1/COL4A2 display statistical interaction for CAD association. CONCLUSIONS: These findings demonstrate that SMAD3 is a necessary factor for TGFß-mediated stimulation of mRNA and protein expression of type IV collagen genes in human vascular smooth muscle cells. Epistasis analyses further supports the hypothesis that the SMAD3-dependent regulation of COL4A1/COL4A2 may be of functional significance for CAD pathogenesis.