ABSTRACT
OBJECTIVE: The tonicity-responsive transcription factor, nuclear factor of activated T cells 5 (NFAT5/tonicity enhancer binding protein [TonEBP]), has been well characterized in numerous cell types; however, NFAT5 function in vascular smooth muscle cells (SMCs) is unknown. Our main objective was to determine the role of NFAT5 regulation in SMCs. METHODS AND RESULTS: We showed that NFAT5 is regulated by hypertonicity in SMCs and is upregulated in atherosclerosis and neointimal hyperplasia. RNAi knockdown of NFAT5 inhibited basal expression of several SMC differentiation marker genes, including smooth muscle α actin (SMαA). Bioinformatic analysis of SMαA revealed 7 putative NFAT5 binding sites in the first intron, and chromatin immunoprecipitation analysis showed NFAT5 enrichment of intronic DNA. Overexpression of NFAT5 increased SMαA promoter-intron activity, which requires an NFAT5 cis element at +1012, whereas dominant-negative NFAT5 decreased SMαA promoter-intron activity. Because it is unlikely that SMCs experience extreme changes in tonicity, we investigated other stimuli and uncovered 2 novel NFAT5-inducing factors: angiotensin II, a contractile agonist, and platelet-derived growth factor-BB (PDGF-BB), a potent mitogen in vascular injury. Angiotensin II stimulated NFAT5 translocation and activity, and NFAT5 knockdown inhibited an angiotensin II-mediated upregulation of SMαA mRNA. PDGF-BB increased NFAT5 protein, and loss of NFAT5 inhibited PDGF-BB-induced SMC migration. CONCLUSIONS: We have identified NFAT5 as a novel regulator of SMC phenotypic modulation and have uncovered the role of NFAT5 in angiotensin II-induced SMαA expression and PDGF-BB-stimulated SMC migration.
Subject(s)
Atherosclerosis/metabolism , Carotid Artery Injuries/metabolism , Muscle, Smooth, Vascular/metabolism , Myocytes, Smooth Muscle/metabolism , NFATC Transcription Factors/metabolism , Tunica Intima/metabolism , Actins/genetics , Actins/metabolism , Angiotensin II/metabolism , Animals , Apolipoproteins E/deficiency , Apolipoproteins E/genetics , Atherosclerosis/genetics , Atherosclerosis/pathology , Becaplermin , Binding Sites , Carotid Artery Injuries/genetics , Carotid Artery Injuries/pathology , Cell Movement , Cell Proliferation , Cells, Cultured , Chromatin Immunoprecipitation , Coculture Techniques , Computational Biology , Disease Models, Animal , Gene Expression Regulation , Genes, Reporter , Humans , Hyperplasia , Introns , Mice , Mice, Knockout , Muscle, Smooth, Vascular/pathology , Myocytes, Smooth Muscle/pathology , NFATC Transcription Factors/genetics , Phenotype , Platelet-Derived Growth Factor/metabolism , Promoter Regions, Genetic , Protein Transport , Proto-Oncogene Proteins c-sis , RNA Interference , RNA, Messenger/metabolism , Rats , Transcription Factors/metabolism , Transfection , Tunica Intima/pathologyABSTRACT
OBJECTIVE: The goal of this study was to assess the activity of ß-catenin/T-cell-specific transcription factor (TCF) signaling in atherosclerosis development and its regulation of fibronectin in vascular endothelium. METHODS AND RESULTS: Histological staining identified preferential nuclear localization of ß-catenin in the endothelium of atheroprone aorta before and during lesion development. Transgenic reporter studies revealed that increased levels of TCF transcriptional activity in endothelium correlated anatomically with ß-catenin nuclear localization and fibronectin deposition. Exposure of endothelial cells to human-derived atheroprone shear stress induced nuclear localization of ß-catenin, transcriptional activation of TCF, and expression of fibronectin. Activation of fibronectin expression required ß-catenin, TCF, and the transcriptional coactivator CRBP-binding protein. Finally, we identified platelet endothelial cell adhesion molecule-1 as a critical regulator of constitutive ß-catenin and glycogen synthase kinase-3ß activities. CONCLUSIONS: These data reveal novel constitutive activation of the endothelial ß-catenin/TCF signaling pathway in atherosclerosis and regulation of fibronectin through hemodynamic shear stress.
Subject(s)
Atherosclerosis/metabolism , Endothelial Cells/metabolism , Fibronectins/metabolism , Hemodynamics , Inflammation/metabolism , Signal Transduction , TCF Transcription Factors/metabolism , beta Catenin/metabolism , Animals , Apolipoproteins E/deficiency , Apolipoproteins E/genetics , Atherosclerosis/genetics , Atherosclerosis/pathology , Atherosclerosis/physiopathology , Cell Nucleus/metabolism , Cells, Cultured , Disease Models, Animal , Endothelial Cells/pathology , Genes, Reporter , Glycogen Synthase Kinase 3/metabolism , Glycogen Synthase Kinase 3 beta , Humans , Inflammation/genetics , Inflammation/pathology , Inflammation/physiopathology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , NF-kappa B/metabolism , Platelet Endothelial Cell Adhesion Molecule-1/genetics , Platelet Endothelial Cell Adhesion Molecule-1/metabolism , RNA Interference , Stress, Mechanical , TCF Transcription Factors/genetics , Time Factors , Transcriptional Activation , Transfection , beta Catenin/geneticsABSTRACT
OBJECTIVE: Smooth muscle cell (SMC) phenotypic modulation, an important component of atherosclerosis progression, is critically regulated by the matrix, with normal components of the healthy SMC matrix limiting modulation and atherosclerosis-associated transitional matrix proteins promoting phenotypic modulation. We sought to determine how collagen IV (which comprises the healthy artery wall) and monomeric collagen I (which comprises atherosclerotic lesions) differentially affect SMC phenotype. METHODS AND RESULTS: Plating SMCs on collagen IV resulted in elevated expression of SMC contractility proteins compared to collagen I. Concurrent with enhanced contractile gene expression, collagen IV stimulates binding of SRF to CArG boxes in the promoters of smooth muscle actin and smooth muscle myosin heavy chain. Coll IV also stimulated the expression of myocardin, a critical SRF coactivator required to drive expression of SMC specific genes. In contrast to collagen IV, collagen I stimulated enhanced expression of the inflammatory protein vascular cell adhesion molecule (VCAM)-1. NF-kappaB and NFAT-binding sites in the VCAM-1 promoter are critical for collagen I-mediated expression of VCAM-1 promoter activity. However, only inhibitors of NFAT, not NF-kappaB, were able to reduce collagen I-associated VCAM expression, and collagen I but not collagen IV stimulated NFAT transcriptional activity. CONCLUSIONS: These results show for the first time that collagen IV and collagen I differentially affect smooth muscle phenotypic modulation through multiple pathways.
