Transcriptional Profiling of Cultured, Embryonic Epicardial Cells Identifies Novel Genes and Signaling Pathways Regulated by TGFβR3 In Vitro

The epicardium plays an important role in coronary vessel formation and Tgfbr3-/- mice exhibit failed coronary vessel development associated with decreased epicardial cell invasion. Immortalized Tgfbr3-/- epicardial cells display the same defects. Tgfbr3+/+ and Tgfbr3-/- cells incubated for 72 hours with VEH or ligands known to promote invasion via TGFβR3 (TGFβ1, TGFβ2, BMP2), for 72 hours were harvested for RNA-seq analysis. We selected for genes >2-fold differentially expressed between Tgfbr3+/+ and Tgfbr3-/- cells when incubated with VEH (604), TGFβ1 (515), TGFβ2 (553), or BMP2 (632). Gene Ontology (GO) analysis of these genes identified dysregulated biological processes consistent with the defects observed in Tgfbr3-/- cells, including those associated with extracellular matrix interaction. GO and Gene Regulatory Network (GRN) analysis identified distinct expression profiles between TGFβ1-TGFβ2 and VEH-BMP2 incubated cells, consistent with the differential response of epicardial cells to these ligands in vitro. Despite the differences observed between Tgfbr3+/+ and Tgfbr3-/- cells after TGFβ and BMP ligand addition, GRNs constructed from these gene lists identified NF-ĸB as a key nodal point for all ligands examined. Tgfbr3-/- cells exhibited decreased expression of genes known to be activated by NF-ĸB signaling. NF-ĸB activity was stimulated in Tgfbr3+/+ epicardial cells after TGFβ2 or BMP2 incubation, while Tgfbr3-/- cells failed to activate NF-ĸB in response to these ligands. Tgfbr3+/+ epicardial cells incubated with an inhibitor of NF-ĸB signaling no longer invaded into a collagen gel in response to TGFβ2 or BMP2. These data suggest that NF-ĸB signaling is dysregulated in Tgfbr3-/- epicardial cells and that NF-ĸB signaling is required for epicardial cell invasion in vitro. Our approach successfully identified a signaling pathway important in epicardial cell behavior downstream of TGFβR3. Overall, the genes and signaling pathways identified through our analysis yield the first comprehensive list of candidate genes whose expression is dependent on TGFβR3 signaling.

[1]  C. Hong,et al.  Common pathways regulate Type III TGFβ receptor-dependent cell invasion in epicardial and endocardial cells. , 2016, Cellular signalling.

[2]  T. Camenisch,et al.  Type III TGFβ receptor and Src direct hyaluronan-mediated invasive cell motility. , 2015, Cellular signalling.

[3]  W. Pu,et al.  Cellular origin and developmental program of coronary angiogenesis. , 2015, Circulation research.

[4]  L. Birnbaumer,et al.  TLR4 activation of TRPC6-dependent calcium signaling mediates endotoxin-induced lung vascular permeability and inflammation , 2012, The Journal of experimental medicine.

[5]  M. Goumans,et al.  The arterial and cardiac epicardium in development, disease and repair. , 2012, Differentiation; research in biological diversity.

[6]  W. Pu,et al.  Endocardial and Epicardial Epithelial to Mesenchymal Transitions in Heart Development and Disease , 2012, Circulation research.

[7]  C. Hong,et al.  BMP2 signals loss of epithelial character in epicardial cells but requires the Type III TGFβ receptor to promote invasion. , 2012, Cellular signalling.

[8]  C. Tabin,et al.  Distinct compartments of the proepicardial organ give rise to coronary vascular endothelial cells. , 2012, Developmental cell.

[9]  S. Park,et al.  TRAF6 Mediates IL-1β/LPS-Induced Suppression of TGF-β Signaling through Its Interaction with the Type III TGF-β Receptor , 2012, PloS one.

[10]  Nora S. Sanchez,et al.  TGFβ and BMP-2 regulate epicardial cell invasion via TGFβR3 activation of the Par6/Smurf1/RhoA pathway. , 2012, Cellular signalling.

[11]  L. Santy,et al.  CNK3 and IPCEF1 produce a single protein that is required for HGF dependent Arf6 activation and migration. , 2012, Experimental cell research.

[12]  A. Czirók,et al.  The cytoplasmic domain of TGFβR3 through its interaction with the scaffolding protein, GIPC, directs epicardial cell behavior. , 2011, Developmental biology.

[13]  D. Welsh,et al.  Role of myosin light chain kinase and myosin light chain phosphatase in the resistance arterial myogenic response to intravascular pressure. , 2011, Archives of biochemistry and biophysics.

[14]  S. Baek,et al.  Epicardial-Derived Cell Epithelial-to-Mesenchymal Transition and Fate Specification Require PDGF Receptor Signaling , 2011, Circulation research.

[15]  Leah B. Honor,et al.  Adult mouse epicardium modulates myocardial injury by secreting paracrine factors. , 2011, The Journal of clinical investigation.

[16]  K. Brown,et al.  Differential Use of Chondroitin Sulfate to Regulate Hyaluronan Binding by Receptor CD44 in Inflammatory and Interleukin 4-activated Macrophages* , 2011, The Journal of Biological Chemistry.

[17]  A. Kispert,et al.  Notch Signaling Regulates Smooth Muscle Differentiation of Epicardium-Derived Cells , 2011, Circulation research.

[18]  J. Seidman,et al.  Construction of normalized RNA-seq libraries for next-generation sequencing using the crab duplex-specific nuclease. , 2011, Current protocols in molecular biology.

[19]  J. L. de la Pompa,et al.  Differential Notch Signaling in the Epicardium Is Required for Cardiac Inflow Development and Coronary Vessel Morphogenesis , 2011, Circulation research.

[20]  A. Moorman,et al.  Comprehensive Gene-Expression Survey Identifies Wif1 as a Modulator of Cardiomyocyte Differentiation , 2010, PloS one.

[21]  R. Vaillancourt,et al.  TGFβ2-mediated production of hyaluronan is important for the induction of epicardial cell differentiation and invasion. , 2010, Experimental cell research.

[22]  T. Mikawa,et al.  BMP signals promote proepicardial protrusion necessary for recruitment of coronary vessel and epicardial progenitors to the heart. , 2010, Developmental cell.

[23]  N. Rosenthal,et al.  Revealing New Mouse Epicardial Cell Markers through Transcriptomics , 2010, PloS one.

[24]  T. Camenisch,et al.  Involvement of the MEKK1 signaling pathway in the regulation of epicardial cell behavior by hyaluronan. , 2010, Cellular signalling.

[25]  E. Svensson,et al.  Epicardial-myocardial signaling directing coronary vasculogenesis. , 2010, Circulation research.

[26]  Teruhiko Yoshida,et al.  CADM1 Interacts with Tiam1 and Promotes Invasive Phenotype of Human T-cell Leukemia Virus Type I-transformed Cells and Adult T-cell Leukemia Cells* , 2010, The Journal of Biological Chemistry.

[27]  H. You,et al.  The type III transforming growth factor-beta receptor negatively regulates nuclear factor kappa B signaling through its interaction with beta-arrestin2. , 2009, Carcinogenesis.

[28]  T. Camenisch,et al.  Size-dependent regulation of Snail2 by hyaluronan: its role in cellular invasion. , 2009, Glycobiology.

[29]  J. Borg,et al.  Angiomotin-Like Protein 1 Controls Endothelial Polarity and Junction Stability During Sprouting Angiogenesis , 2009, Circulation research.

[30]  C. L. La Porta,et al.  AQP1 Is Not Only a Water Channel: It Contributes to Cell Migration through Lin7/Beta-Catenin , 2009, PloS one.

[31]  Yunfu Sun,et al.  Tbx18 and the fate of epicardial progenitors , 2009, Nature.

[32]  D. Horsfall,et al.  The biological role and regulation of versican levels in cancer , 2009, Cancer and Metastasis Reviews.

[33]  R. Schwartz,et al.  Signaling via the Tgf-beta type I receptor Alk5 in heart development. , 2008, Developmental biology.

[34]  C. Arteaga,et al.  Knockdown of the transforming growth factor-beta type III receptor impairs motility and invasion of metastatic cancer cells. , 2008, Cancer research.

[35]  R. Markwald,et al.  Periostin expression by epicardium-derived cells is involved in the development of the atrioventricular valves and fibrous heart skeleton. , 2008, Differentiation; research in biological diversity.

[36]  J. Tavernier,et al.  TLR-4, IL-1R and TNF-R signaling to NF-κB: variations on a common theme , 2008, Cellular and Molecular Life Sciences.

[37]  Osamu Takeuchi,et al.  Sequential control of Toll-like receptor–dependent responses by IRAK1 and IRAK2 , 2008, Nature Immunology.

[38]  G. Blobe,et al.  Bone Morphogenetic Proteins Signal through the Transforming Growth Factor-β Type III Receptor* , 2008, Journal of Biological Chemistry.

[39]  J. Barnett,et al.  Primary and immortalized mouse epicardial cells undergo differentiation in response to TGFβ , 2008, Developmental dynamics : an official publication of the American Association of Anatomists.

[40]  C. Arteaga,et al.  Modulation of NFκB Activity and E-cadherin by the Type III Transforming Growth Factor β Receptor Regulates Cell Growth and Motility* , 2007, Journal of Biological Chemistry.

[41]  J. Barnett,et al.  Coronary Vessel Development Is Dependent on the Type III Transforming Growth Factor &bgr; Receptor , 2007, Circulation research.

[42]  J. Haines,et al.  Peakwide mapping on chromosome 3q13 identifies the kalirin gene as a novel candidate gene for coronary artery disease. , 2007, American journal of human genetics.

[43]  M. Yost,et al.  A 3-D model of coronary vessel development , 2007, In Vitro Cellular & Developmental Biology - Animal.

[44]  M. Iruela-Arispe,et al.  Proteolytic cleavage of versican during cardiac cushion morphogenesis , 2006, Developmental dynamics : an official publication of the American Association of Anatomists.

[45]  T. Brand,et al.  BMP is an important regulator of proepicardial identity in the chick embryo. , 2006, Developmental biology.

[46]  K. Lewis,et al.  Identification of distinct inhibin and transforming growth factor beta-binding sites on betaglycan: functional separation of betaglycan co-receptor actions. , 2006, The Journal of biological chemistry.

[47]  J. Pérez-Pomares,et al.  In vivo and in vitro analysis of the vasculogenic potential of avian proepicardial and epicardial cells † , 2006, Developmental dynamics : an official publication of the American Association of Anatomists.

[48]  D. Mckinnon,et al.  Regional variation in mRNA transcript abundance within the ventricular wall. , 2006, Journal of molecular and cellular cardiology.

[49]  B. Ruffell,et al.  Chondroitin sulfate addition to CD44H negatively regulates hyaluronan binding. , 2005, Biochemical and biophysical research communications.

[50]  R. Kannagi,et al.  Expression of N-acetylglucosamine 6-O-sulfotransferases (GlcNAc6STs)-1 and -4 in human monocytes: GlcNAc6ST-1 is implicated in the generation of the 6-sulfo N-acetyllactosamine/Lewis x epitope on CD44 and is induced by TNF-alpha. , 2005, Glycobiology.

[51]  J. Barnett,et al.  Coronary vessel development: the epicardium delivers. , 2004, Trends in cardiovascular medicine.

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

[53]  Brad T. Sherman,et al.  DAVID: Database for Annotation, Visualization, and Integrated Discovery , 2003, Genome Biology.

[54]  T. Imamura,et al.  Differential roles of two N-acetylgalactosaminyltransferases, CSGalNAcT-1, and a novel enzyme, CSGalNAcT-2. Initiation and elongation in synthesis of chondroitin sulfate. , 2003, The Journal of biological chemistry.

[55]  D. Golenbock,et al.  Dysregulation of LPS-Induced Toll-Like Receptor 4-MyD88 Complex Formation and IL-1 Receptor-Associated Kinase 1 Activation in Endotoxin-Tolerant Cells1 , 2002, The Journal of Immunology.

[56]  Silvia Massari,et al.  Invasive behaviour of glioblastoma cell lines is associated with altered organisation of the cadherin-catenin adhesion system. , 2002, Journal of cell science.

[57]  H. Lodish,et al.  A novel mechanism for regulating transforming growth factor beta (TGF-beta) signaling. Functional modulation of type III TGF-beta receptor expression through interaction with the PDZ domain protein, GIPC. , 2001, The Journal of biological chemistry.

[58]  Harvey F. Lodish,et al.  Functional Roles for the Cytoplasmic Domain of the Type III Transforming Growth Factor β Receptor in Regulating Transforming Growth Factor β Signaling* , 2001, The Journal of Biological Chemistry.

[59]  Wei Zheng,et al.  Vascular Endothelial Growth Factor and Basic Fibroblast Growth Factor Differentially Modulate Early Postnatal Coronary Angiogenesis , 2001, Circulation research.

[60]  Christopher J. Morabito,et al.  Positive and negative regulation of epicardial-mesenchymal transformation during avian heart development. , 2001, Developmental biology.

[61]  J. Lin,et al.  Comparative studies on the expression patterns of three troponin T genes during mouse development , 2001, The Anatomical record.

[62]  L. Holmgren,et al.  Angiomotin: an angiostatin binding protein that regulates endothelial cell migration and tube formation. , 2001, The Journal of cell biology.

[63]  J. Stull,et al.  Dedicated Myosin Light Chain Kinases with Diverse Cellular Functions* , 2001, The Journal of Biological Chemistry.

[64]  A. Kispert,et al.  Cloning and expression analysis of the mouse T-box gene Tbx20 , 2001, Mechanisms of Development.

[65]  S. Orkin,et al.  FOG-2, a Cofactor for GATA Transcription Factors, Is Essential for Heart Morphogenesis and Development of Coronary Vessels from Epicardium , 2000, Cell.

[66]  A. Schedl,et al.  YAC complementation shows a requirement for Wt1 in the development of epicardium, adrenal gland and throughout nephrogenesis. , 1999, Development.

[67]  Raymond B. Runyan,et al.  Requirement of type III TGF-beta receptor for endocardial cell transformation in the heart. , 1999, Science.

[68]  J. Hecksher-Sørensen,et al.  YAC transgenic analysis reveals Wilms' Tumour 1 gene activity in the proliferating coelomic epithelium, developing diaphragm and limb , 1998, Mechanisms of Development.

[69]  R. Markwald,et al.  The Cspg2 gene, disrupted in the hdf mutant, is required for right cardiac chamber and endocardial cushion formation. , 1998, Developmental biology.

[70]  A. G. Gittenberger-de Groot,et al.  Epicardium-derived cells contribute a novel population to the myocardial wall and the atrioventricular cushions. , 1998, Circulation research.

[71]  J. Claverie,et al.  The significance of digital gene expression profiles. , 1997, Genome research.

[72]  G. Boivin,et al.  TGFbeta2 knockout mice have multiple developmental defects that are non-overlapping with other TGFbeta knockout phenotypes. , 1997, Development.

[73]  S. Virágh,et al.  Cell surface glycoconjugates and the extracellular matrix of the developing mouse embryo epicardium , 1995, Anatomy and Embryology.

[74]  R. Hynes,et al.  Cell adhesion events mediated by alpha 4 integrins are essential in placental and cardiac development. , 1995, Development.

[75]  A. G. Gittenberger-de Groot,et al.  Development of the cardiac coronary vascular endothelium, studied with antiendothelial antibodies, in chicken-quail chimeras. , 1993, Circulation research.

[76]  J. Massagué,et al.  Betaglycan presents ligand to the TGFβ signaling receptor , 1993, Cell.

[77]  J. Männer Experimental study on the formation of the epicardium in chick embryos , 1993, Anatomy and Embryology.

[78]  G. Proetzel,et al.  Targeted disruption of the mouse transforming growth factor-β1 gene results in multifocal inflammatory disease , 1992, Nature.

[79]  J. Tidball Identification and distribution of a novel, collagen-binding protein in the developing subepicardium and endomysium. , 1992, Journal of Biological Chemistry.

[80]  M. Lampugnani,et al.  A novel endothelial-specific membrane protein is a marker of cell-cell contacts , 1992, The Journal of cell biology.

[81]  J. Partanen,et al.  A novel endothelial cell surface receptor tyrosine kinase with extracellular epidermal growth factor homology domains , 1992, Molecular and cellular biology.

[82]  J. Massagué,et al.  Structure and expression of the membrane proteoglycan betaglycan, a component of the TGF-β receptor system , 1991, Cell.

[83]  J. Gorski,et al.  PECAM-1 (CD31) cloning and relation to adhesion molecules of the immunoglobulin gene superfamily. , 1990, Science.

[84]  S. Schiaffino,et al.  Troponin I switching in the developing heart. , 1989, The Journal of biological chemistry.

[85]  S. Schiaffino,et al.  Troponin T switching in the developing rat heart. , 1988, The Journal of biological chemistry.

[86]  C. E. Challice,et al.  The origin of the epicardium and the embryonic myocardial circulation in the mouse , 1981, The Anatomical record.

[87]  G. Blobe,et al.  Endocardial cell epithelial-mesenchymal transformation requires Type III TGFβ receptor interaction with GIPC. , 2012, Cellular signalling.

[88]  P. Lécine,et al.  The Amot/Patj/Syx signaling complex spatially controls RhoA GTPase activity in migrating endothelial cells. , 2009, Blood.

[89]  P. Doevendans,et al.  Regulation and characteristics of vascular smooth muscle cell phenotypic diversity , 2007, Netherlands heart journal : monthly journal of the Netherlands Society of Cardiology and the Netherlands Heart Foundation.

[90]  C. Arteaga,et al.  Modulation of NFkappaB activity and E-cadherin by the type III transforming growth factor beta receptor regulates cell growth and motility. , 2007, The Journal of biological chemistry.

[91]  R. Derynck,et al.  Smad-dependent and Smad-independent pathways in TGF-beta family signalling. , 2003, Nature.

[92]  H. Lodish,et al.  Functional roles for the cytoplasmic domain of the type III transforming growth factor beta receptor in regulating transforming growth factor beta signaling. , 2001, The Journal of biological chemistry.

[93]  S. Hoffman,et al.  Distinct spatial and temporal distributions of aggrecan and versican in the embryonic chick heart. , 1999, The Anatomical record.

[94]  J. Massagué,et al.  Betaglycan presents ligand to the TGF beta signaling receptor. , 1993, Cell.

[95]  J. Massagué,et al.  Structure and expression of the membrane proteoglycan betaglycan, a component of the TGF-beta receptor system. , 1991, Cell.