title: Functional interaction between COL4A1/COL4A2 and SMAD3 risk loci for coronary artery disease.
abstract: 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 TGFβ signaling, but the specific regulatory mechanisms, especially in humans, have not been defined. Our aim was to decipher TGFβ 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.
  
  
  RESULTS
  Experiments were performed in primary human aortic smooth muscle cells and HT1080 fibroblasts. Pharmacological inhibition of the TGFβ1 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 TGFβ1 stimulation. In contrast, inhibition of the non-canonical TGFβ signaling pathways was without effect. siRNA mediated knockdown of SMAD3 and SMAD4 abolished the stimulatory effects of TGFβ1 on COL4A1/COL4A2 (p < 0.001) whereas SMAD2 knockdown had no effect. In luciferase reporter assays, neither SMAD3 overexpression nor TGFβ1 treatment altered COL4A1 or COL4A2 promoter activity, supportive of more complex regulation of type IV collagen gene expression by the TGFβ/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 TGFβ-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.
authors: Atherogenomics Laboratory, University of Ottawa Heart Institute, Ottawa, K1Y 4W7, Canada.
authors: Adam W
authors: AW
authors: Turner
authors: Ruddy Canadian Cardiovascular Genetics Centre, University of Ottawa Heart Institute, Ottawa, K1Y 4W7, Canada.
authors: Majid
authors: M
authors: Nikpay
authors: Atherogenomics Laboratory, University of Ottawa Heart Institute, Ottawa, K1Y 4W7, Canada.
authors: Anada
authors: A
authors: Silva
authors: Atherogenomics Laboratory, University of Ottawa Heart Institute, Ottawa, K1Y 4W7, Canada.
authors: Paulina
authors: P
authors: Lau
authors: Atherogenomics Laboratory, University of Ottawa Heart Institute, Ottawa, K1Y 4W7, Canada.
authors: Amy
authors: A
authors: Martinuk
authors: Atherogenomics Laboratory, University of Ottawa Heart Institute, Ottawa, K1Y 4W7, Canada.
authors: Tara A
authors: TA
authors: Linseman
authors: Atherogenomics Laboratory, University of Ottawa Heart Institute, Ottawa, K1Y 4W7, Canada.
authors: Sebastien
authors: S
authors: Soubeyrand
authors: Atherogenomics Laboratory, University of Ottawa Heart Institute, Ottawa, K1Y 4W7, Canada; Ruddy Canadian Cardiovascular Genetics Centre, University of Ottawa Heart Institute, Ottawa, K1Y 4W7, Canada. Electronic address: rmcpherson@ottawaheart.ca.
authors: Ruth
authors: R
authors: McPherson
mesh_terms: D001011:Aorta / Q000473:pathology; D002460:Cell Line; D015331:Cohort Studies; D024141:Collagen Type IV / Q000378:metabolism*; D003324:Coronary Artery Disease / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000737:chemistry; D004843:Epistasis, Genetic; D005347:Fibroblasts / Q000378:metabolism; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006801:Humans; D009131:Muscle, Smooth, Vascular / Q000166:cytology; D032389:Myocytes, Smooth Muscle / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012307:Risk Factors; D015398:Signal Transduction; D051900:Smad3 Protein / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000378:metabolism; D015854:Up-Regulation
chemical_list: C547475:COL4A1 protein, human; C502625:COL4A2 protein, human; D024141:Collagen Type IV; D004791:Enzyme Inhibitors; D034741:RNA, Small Interfering; C497790:SMAD3 protein, human; D051900:Smad3 Protein; D016212:Transforming Growth Factor beta
keywords: Atherosclerosis; COL4A1; COL4A2; Coronary artery disease; Epistasis; SMAD3; TGFβ; Type IV collagen