Integrative genomics identifies DSCR1 (RCAN1) as a novel NFAT-dependent mediator of phenotypic modulation in vascular smooth muscle cells

Monica Y. Lee 0 1 2 Sean M. Garvey 2 Alex S. Baras 6 Julia A. Lemmon 2 5 Maria F. Gomez 4 Pamela D. Schoppee Bortz 2 Guenter Daum 3 Renee C. LeBoeuf 7 Brian R. Wamhoff 0 1 2 0 Robert M. Berne Cardiovascular Research Center 1 Department of Biomedical Engineering 2 Cardiovascular Division, Department of Medicine 3 Department of Surgery 4 Department of Clinical Sciences, Lund University , Malmo , Sweden 5 Department of Pharmacology, University of Virginia , VA , USA 6 Department of Pathology 7 Division of Metabolism , Endocrinology and Nutrition , University of Washington , Seattle, WA , USA Vascular smooth muscle cells (SMCs) display remarkable phenotypic plasticity in response to environmental cues. The nuclear factor of activated T-cells (NFAT) family of transcription factors plays a critical role in vascular pathology. However, known functional NFAT gene targets in vascular SMCs are currently limited. Publicly available whole-genome expression array data sets were analyzed to identify differentially expressed genes in human, mouse and rat SMCs. Comparison between vehicle and phenotypic modulatory stimuli identified 63 species-conserved, upregulated genes. Integration of the 63 upregulated genes with an in silico NFAT-ome (a species-conserved list of gene promoters containing at least one NFAT binding site) identified 18 putative NFAT-dependent genes. Further intersection of these 18 potential NFAT target genes with a mouse in vivo vascular injury microarray identified four putative NFAT-dependent, injury-responsive genes. In vitro validations substantiated the NFAT-dependent role of Cyclooxygenase 2 (COX2/PTGS2) in SMC phenotypic modulation and uncovered Down Syndrome Candidate Region 1 (DSCR1/RCAN1) as a novel NFAT target gene in SMCs. We show that induction of DSCR1 inhibits calcineurin/NFAT signaling through a negative feedback mechanism; DSCR1 overexpression attenuates NFAT transcriptional activity and COX2 protein expression, whereas knockdown of endogenous DSCR1 enhances NFAT transcriptional activity. Our integrative genomics approach illustrates how the combination of publicly available gene expression arrays, computational databases and empirical research methods can answer specific questions in any cell type for a transcriptional network of interest. Herein, we report DSCR1 as a novel NFAT-dependent, injury-inducible, early gene that may serve to negatively regulate SMC phenotypic switching. - INTRODUCTION The biochemical, morphological and physiological phenotypes of a vascular smooth muscle cell (SMC) contribute to sustained vascular integrity and homeostasis. Unlike other terminally differentiated cell types, vascular SMCs display remarkable phenotypic plasticity. The adult, differentiated state is traditionally defined by expression of wellcharacterized SMC contractile genes including smooth muscle a-actin (SM-aA), smooth muscle myosin heavy chain (SMMHC) and SM22a (1). Extracellular cues, however, can induce contractile SMCs to remodel toward a synthetic state characterized by a spectrum of proliferative, migratory and inflammatory phenotypes (2). This synthetic phenotype is associated with downregulation of SMC contractile marker genes and upregulation of adhesion-, inflammation- and survival-related genes. SMC plasticity can be both beneficial and detrimental in response to acute vessel injury. A clinical example of adverse SMC phenotypic modulation involves late vascular inward remodeling in response to balloon angioplasty and intracoronary stent deployment (3). Previous studies also demonstrate a significant, linear correlation between degree of vascular injury and extent of restenosis (4). A thorough, molecular understanding of SMC phenotypic modulation is currently limited by the complexity of the transcriptional networks involved. The nuclear factor of activated T-cells (NFATc1-c4) family of transcription factors was originally identified in lymphocytes for its role in cytokine gene expression. Beyond the immune system, NFAT proteins are expressed in many cell types including cardiac, skeletal, and vascular smooth muscle. NFAT proteins are downstream effectors in the calcineurin (Cn) signaling pathwaya critical pathway in the transduction of many extracellular, adaptive stimuli. Calcineurin is a calcium-dependent, serine/threonine protein phosphatase that dephosphorylates NFAT to enable nuclear translocation and target gene transcription. Cn/NFAT activity has been shown to induce vascular SMC proliferation and migration in response to receptor tyrosine kinase (RTK) and G-protein-coupled receptor (GPCR) agonists, respectively (5,6). Interestingly, blocking Cn/NFAT signaling in vivo suppresses experimental balloon injury-induced neointimal hyperplasia, suggesting Cn/NFAT activity is involved in SMC phenotypic modulation (7). Tacrolimus (FK506), also a Cn/ NFAT inhibitor, has been used in clinical trials to counteract in-stent restenosis (8). While NFAT-dependent gene regulation has been widely studied in lymphocytes, cardiac and skeletal muscle, very few NFAT-dependent genes have been identified in SMCs (9). We have developed an unbiased, top-down integrative genomics approach to identify Cn/NFAT-dependent vascular SMC genes. Publicly available and experimentally obtained gene expression array data sets were integrated with a list of gene promoters containing putative NFAT binding sites in an effort to determine downstream targets of Cn/NFAT signaling. Our methodology illustrates how the combination of publicly available whole-genome expression arrays, computational databases and empirical in vivo and in vitro research methods can answer focused questions in a specific cell type for a transcriptional network of interest. Here we identified Down Syndrome Candidate Region 1 (DSCR1/RCAN1/MCIP1) as a novel Cn/NFATdependent, injury-responsive gene in vascular SMCs. Integrative genomics overview Empirical wet-bench research and in silico dry-bench bioinformatics can be combined to enhance the study of complex cell environment interactions. Recent studies from various fields demonstrate the utility of integrative genomics: the identification of the FOX family of transcription factors in human heart failure pathogenesis (10) and the identification of microRNA-126 in endothelial cell VCAM1 expression (11). This study addressed a fundamental question using integrative genomics (Fig. 1): What are the downstream gene targets of NFAT activation in vascular SMC phenotypic modulation? Genes associated with SMC phenotypic modulation were identified through analysis of multiple whole-genome expression array data sets: (i) serum-stimulated human coronary SMCs (12), (ii) PDGF-BB-stimulated mouse aortic SMCs (13) and (iii) PDGF-BB-stimulated rat aortic SMCs (Fig. 1C). Differentially upregulated genes were screened against an in silico orthologously conserved human NFAT-ome (Fig. 1A and B) to identify potential NFATdependent SMC genes (Fig. 1D). Putative NFAT-d (...truncated)