Abstract 18325: Functional Relationship of the COL4A1/COL4A2 Locus on Chromosome 13q34 to Coronary Artery Disease (CAD)

Abstract

Background: The COL4A1/COL4A2 locus has been linked to CAD in recent large GWAS meta-analyses. The COL4A1 and COL4A2 proteins constitute the major structural component of basement membranes, have important functional roles in angiogenesis, and mutations have been associated with diverse vascular abnormalities. We performed epistasis analysis for CAD-associated SNPs at the COL4A1/COL4A2 locus; this revealed genetic interaction with members of the TGFβ/Smad signalling pathway. Furthermore, pathway analysis of GWAS has implicated TGFβ and Smad signaling as well as the extracellular matrix as enriched CAD pathways. Bioinformatic analysis also predicted binding of Smad3 and Smad4 to the promoter region of COL4A1/COL4A2 . We sought to address the hypothesis that the TGFβ-mediated regulation of COL4A1 and COL4A2 is via Smad3/Smad4 and that this pathway is relevant to the pathogenesis of CAD. Methods and Results: Under basal conditions, HT-1080 fibroblast cells treated with TGFβ1 for 16h produced 5 fold and 3 fold increases in COL4A1 and COL4A2 mRNA respectively as measured by qRT-PCR. In contrast, 16h treatment with TGFβ1 in the presence of the specific Smad pathway inhibitor SB431542 (10nM) abolished COL4A1/COL4A2 upregulation (p<0.001). To rule out the possibility that TGFβ1 regulates COL4A1 and COL4A2 expression indirectly via a non-Smad pathway, we showed that inhibition of the JNK, p38 MAPK, and ERK pathways had no effect on COL4A1 and COL4A2 levels basally or in response to TGFβ1. siRNA knockdown (>85%) of SMAD3 in HT-1080 cells diminished the TGFβ1 upregulation of both COL4A1 and COL4A2 mRNA levels in comparison to nontarget siRNA (p<0.001). The effect of TGFβ1 on COL4A1 and COL4A2 mRNA expression was completely abolished when SMAD3 and SMAD4 were knocked-down in concert. These effects on COL4A1 and COL4A2 mRNA expression were recapitulated at the protein level by Western blotting. Conclusions: These data suggest that Smad3 and Smad4 are the downstream mediators by which TGFβ1 upregulates COL4A1 and COL4A2 expression in HT-1080 cells. This work further highlights the importance of the extracellular matrix and TGFβ/Smad signalling in the pathogenesis of CAD.