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 TGFb signaling, but the specific regulatory mechanisms, especially in humans, have not been defined. Our aim was to decipher TGFb 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 TGFb1 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 TGFb1 stimulation. In contrast, inhibition of the non-canonical TGFb signaling pathways was without effect. siRNA mediated knockdown of SMAD3 and SMAD4 abolished the stimulatory effects of TGFb1 on COL4A1/COL4A2 (p < 0.001) whereas SMAD2 knockdown had no effect. In luciferase reporter assays, neither SMAD3 overexpression nor TGFb1 treatment altered COL4A1 or COL4A2 promoter activity, supportive of more complex regulation of type IV collagen gene expression by the TGFb/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 TGFb-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. (C) 2015 Elsevier Ireland Ltd. All rights reserved.