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 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. METHODS AND 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.

[1]  F. Verrecchia,et al.  Identification of Novel TGF-β/Smad Gene Targets in Dermal Fibroblasts using a Combined cDNA Microarray/Promoter Transactivation Approach* , 2001, The Journal of Biological Chemistry.

[2]  G. Owens,et al.  Transforming growth factor-beta-induced growth inhibition and cellular hypertrophy in cultured vascular smooth muscle cells , 1988, The Journal of cell biology.

[3]  A. Grace,et al.  The serum concentration of active transforming growth factor-β is severely depressed in advanced atherosclerosis , 1995, Nature Medicine.

[4]  Ester Piek,et al.  Role of Transforming Growth Factor-β Signaling in Cancer , 2000 .

[5]  M. Daemen,et al.  Transforming growth factor-beta mediates balance between inflammation and fibrosis during plaque progression. , 2002, Arteriosclerosis, thrombosis, and vascular biology.

[6]  J. Ioannidis,et al.  Meta-analysis methods for genome-wide association studies and beyond , 2013, Nature Reviews Genetics.

[7]  Y Okada,et al.  Collagens in human atherosclerosis. Immunohistochemical analysis using collagen type-specific antibodies. , 1992, Arteriosclerosis and thrombosis : a journal of vascular biology.

[8]  J. Grande,et al.  TGF-β1 is an Autocrine Mediator of Renal Tubular Epithelial Cell Growth and Collagen IV Production , 2002 .

[9]  L. Bruckner-Tuderman,et al.  Basement membranes and human disease , 2009, Cell and Tissue Research.

[10]  E. Pöschl,et al.  Cooperative and competitive interactions of regulatory elements are involved in the control of divergent transcription of human Col4A1 and Col4A2 genes , 1997, FEBS letters.

[11]  P. Kwok,et al.  Association of TGFBR2 polymorphism with risk of sudden cardiac arrest in patients with coronary artery disease. , 2009, Heart rhythm.

[12]  A. Tedgui,et al.  Inhibition of Transforming Growth Factor- Signaling Accelerates Atherosclerosis and Induces an Unstable Plaque Phenotype in Mice , 2001 .

[13]  H. Shimokawa,et al.  Prognostic significance of plasma concentrations of transforming growth factor-&bgr; in patients with coronary artery disease , 2002, Coronary artery disease.

[14]  Andrew G. Clark,et al.  Gene-Based Testing of Interactions in Association Studies of Quantitative Traits , 2013, PLoS genetics.

[15]  Cheuk-Man Yu,et al.  TGF-β/Smad3 signaling promotes renal fibrosis by inhibiting miR-29. , 2011, Journal of the American Society of Nephrology : JASN.

[16]  Peiyong Jiang,et al.  Inhibition of miR-29 by TGF-beta-Smad3 Signaling through Dual Mechanisms Promotes Transdifferentiation of Mouse Myoblasts into Myofibroblasts , 2012, PloS one.

[17]  C. Hill,et al.  Smad4 Dependency Defines Two Classes of Transforming Growth Factor β (TGF-β) Target Genes and Distinguishes TGF-β-Induced Epithelial-Mesenchymal Transition from Its Antiproliferative and Migratory Responses , 2005, Molecular and Cellular Biology.

[18]  J. Opitz,et al.  Identification of a novel sequence element in the common promoter region of human collagen type IV genes, involved in the regulation of divergent transcription. , 1993, The Biochemical journal.

[19]  H. Schnaper,et al.  The transforming growth factor-βbgr/SMAD signaling pathway is present and functional in human mesangial cells , 1999 .

[20]  Manuel A. R. Ferreira,et al.  PLINK: a tool set for whole-genome association and population-based linkage analyses. , 2007, American journal of human genetics.

[21]  G. Liau,et al.  Transforming growth factor β1 is a powerful modulator of platelet‐derived growth factor action in vascular smooth muscle cells , 1992 .

[22]  A. Lusis,et al.  Systems genetics approaches to understand complex traits , 2013, Nature Reviews Genetics.

[23]  Thomas W. Mühleisen,et al.  Large-scale association analysis identifies 13 new susceptibility loci for coronary artery disease , 2011, Nature Genetics.

[24]  R. Kalluri,et al.  The contribution of vascular basement membranes and extracellular matrix to the mechanics of tumor angiogenesis , 2004, APMIS : acta pathologica, microbiologica, et immunologica Scandinavica.

[25]  K. Lackner,et al.  Overexpression of TGF-ß1 in Macrophages Reduces and Stabilizes Atherosclerotic Plaques in ApoE-Deficient Mice , 2012, PloS one.

[26]  E. Genersch,et al.  Differential effects of DNA-binding proteins on bidirectional transcription from the common promoter region of human collagen type IV genes COL4A1 and COL4A2. , 1993, Biochimica et biophysica acta.

[27]  R. Kalluri.  Basement membranes: structure, assembly and role in tumour angiogenesis , 2003, Nature reviews. Cancer.

[28]  S. Katsuda,et al.  Collagen Synthesis by Cultured Arterial Smooth Muscle Cells during Spontaneous Phenotypic Modulation , 1990, Acta pathologica japonica.

[29]  A. Hata,et al.  Targeting the TGFβ signalling pathway in disease , 2012, Nature Reviews Drug Discovery.

[30]  J. Madri,et al.  Phenotypic modulation of endothelial cells by transforming growth factor-beta depends upon the composition and organization of the extracellular matrix , 1988, The Journal of cell biology.

[31]  E. Pöschl,et al.  The genes for the alpha 1(IV) and alpha 2(IV) chains of human basement membrane collagen type IV are arranged head‐to‐head and separated by a bidirectional promoter of unique structure. , 1988, The EMBO journal.

[32]  C. Heldin,et al.  Smad regulation in TGF-beta signal transduction. , 2001, Journal of cell science.

[33]  Allan Balmain,et al.  TGF-β signaling in tumor suppression and cancer progression , 2001, Nature Genetics.

[34]  Oliver Eickelberg,et al.  High Throughput Determination of TGFβ1/SMAD3 Targets in A549 Lung Epithelial Cells , 2011, PloS one.

[35]  F. Verrecchia,et al.  Transforming Growth Factor-β Signaling Through the Smad Pathway: Role in Extracellular Matrix Gene Expression and Regulation , 2002 .

[36]  A. Zinsmeister,et al.  Modulation of collagen gene expression by cytokines: stimulatory effect of transforming growth factor-beta1, with divergent effects of epidermal growth factor and tumor necrosis factor-alpha on collagen type I and collagen type IV. , 1997, The Journal of laboratory and clinical medicine.

[37]  A. Reith,et al.  SB-431542 is a potent and specific inhibitor of transforming growth factor-beta superfamily type I activin receptor-like kinase (ALK) receptors ALK4, ALK5, and ALK7. , 2002, Molecular pharmacology.

[38]  Ayellet V. Segrè,et al.  Analysis of Genome-Wide Association Study Reveals Novel Associations Between Key Biological Processes and Coronary Artery Disease , 2015 .

[39]  J. Qian,et al.  miR-29 is a major regulator of genes associated with pulmonary fibrosis. , 2011, American journal of respiratory cell and molecular biology.

[40]  Ying E. Zhang,et al.  Smad-dependent and Smad-independent pathways in TGF-β family signalling , 2003, Nature.

[41]  M. Bendeck,et al.  Collagens in the progression and complications of atherosclerosis , 2009, Vascular medicine.

[42]  P. Galéra,et al.  Modulation of transforming growth factor beta signalling pathway genes by transforming growth factor beta in human osteoarthritic chondrocytes: involvement of Sp1 in both early and late response cells to transforming growth factor beta , 2011, Arthritis Research & Therapy.

[43]  J. Danesh,et al.  Large-scale association analysis identifies new risk loci for coronary artery disease , 2013 .

[44]  J. Pérez-Ortín,et al.  Cytoplasmic 5′-3′ exonuclease Xrn1p is also a genome-wide transcription factor in yeast , 2013, Front. Genet..

[45]  E. Pöschl,et al.  Regulation of Divergent Transcription of the Genes Coding for Basement Membrane Type IV Collagen , 1990, Annals of the New York Academy of Sciences.

[46]  I. Jackson,et al.  Col4a1 mutation in mice causes defects in vascular function and low blood pressure associated with reduced red blood cell volume. , 2010, Human molecular genetics.

[47]  B. Uvelius,et al.  Mir-29 Repression in Bladder Outlet Obstruction Contributes to Matrix Remodeling and Altered Stiffness , 2013, PloS one.

[48]  Arif Yurdagul,et al.  Molecular Mechanisms of Collagen Isotype-Specific Modulation of Smooth Muscle Cell Phenotype , 2009, Arteriosclerosis, thrombosis, and vascular biology.

[49]  A. Bobik.  Transforming Growth Factor-&bgr;s and Vascular Disorders , 2006, Arteriosclerosis, thrombosis, and vascular biology.

[50]  K. Miyazono,et al.  Two major Smad pathways in TGF‐β superfamily signalling , 2002, Genes to cells : devoted to molecular & cellular mechanisms.

[51]  C. Gieger,et al.  Genomewide association analysis of coronary artery disease. , 2007, The New England journal of medicine.

[52]  A. Zinsmeister,et al.  Transforming growth factor-β1 induces collagen IV gene expression in NIH-3T3 cells , 1993 .

[53]  Y. Yamada,et al.  Genetic risk for myocardial infarction determined by polymorphisms of candidate genes in a Japanese population , 2007, Journal of Medical Genetics.

[54]  K Kühn,et al.  Basement membrane (type IV) collagen. , 1995, Matrix biology : journal of the International Society for Matrix Biology.

[55]  J. Bertram,et al.  Resveratrol inhibits renal fibrosis in the obstructed kidney: potential role in deacetylation of Smad3. , 2010, The American journal of pathology.

[56]  Toshiko Tanaka,et al.  COL4A1 Is Associated With Arterial Stiffness by Genome-Wide Association Scan , 2009, Circulation. Cardiovascular genetics.

[57]  D. Grainger,et al.  Transforming growth factor , 2003 .

[58]  Marylyn D. Ritchie,et al.  Analysis pipeline for the epistasis search – statistical versus biological filtering , 2014, Front. Genet..