ABSTRACT
Coronary vessel development depends on a subpopulation of epicardial cells that undergo epithelial to mesenchymal transformation (EMT) and invade the subepicardial space and myocardium. These cells form the smooth muscle of the vessels and fibroblasts, but the mechanisms that regulate these processes are poorly understood. Mice lacking the Type III Transforming Growth Factor ß Receptor (TGFßR3) die by E14.5 due to failed coronary vessel development accompanied by reduced epicardial cell invasion. BMP2 signals via TGFßR3 emphasizing the importance of determining the relative contributions of the canonical BMP signaling pathway and TGFßR3-dependent signaling to BMP2 responsiveness. Here we examined the role of TGFßR3 in BMP2 signaling in epicardial cells. Whereas TGFß induced loss of epithelial character and smooth muscle differentiation, BMP2 induced an ALK3-dependent loss of epithelial character and modestly inhibited TGFß-stimulated differentiation. Tgfbr3(-/-) cells respond to BMP2 indicating that TGFßR3 is not required. However, Tgfbr3(-/-) cells show decreased invasion in response to BMP2 and overexpression of TGFßR3 in Tgfbr3(-/-) cells rescued invasion. Invasion was dependent on ALK5, ALK2, ALK3, and Smad4. Expression of TGFßR3 lacking the 3 C-terminal amino acids required to interact with the scaffolding protein GIPC (GAIP-interacting protein, C terminus) did not rescue. Knockdown of GIPC in Tgfbr3(+/+) or Tgfbr3(-/-) cells rescued with TGFßR3 decreased BMP2-stimulated invasion confirming a requirement for TGFßR3/GIPC interaction. Our results reveal the relative roles of TGFßR3-dependent and TGFßR3-independent signaling in the actions of BMP2 on epicardial cell behavior and demonstrate the critical role of TGFßR3 in mediating BMP2-stimulated invasion.
Subject(s)
Bone Morphogenetic Protein 2/physiology , Epithelial-Mesenchymal Transition , Pericardium/cytology , Proteoglycans/metabolism , Receptors, Transforming Growth Factor beta/metabolism , Adaptor Proteins, Signal Transducing , Animals , Carrier Proteins/metabolism , Cell Differentiation , Cell Movement , Cells, Cultured , Coronary Vessels/cytology , Coronary Vessels/growth & development , Epithelial Cells/metabolism , Epithelial Cells/physiology , Mice , Mice, Knockout , Myocytes, Smooth Muscle/metabolism , Myocytes, Smooth Muscle/physiology , Neuropeptides/metabolism , Proteoglycans/genetics , Receptors, Transforming Growth Factor beta/genetics , Smad Proteins/metabolism , Transforming Growth Factors/physiologyABSTRACT
The epicardium is a major contributor of the cells that are required for the formation of coronary vessels. Mice lacking both copies of the gene encoding the Type III Transforming Growth Factor ß Receptor (TGFßR3) fail to form the coronary vasculature, but the molecular mechanism by which TGFßR3 signals coronary vessel formation is unknown. We used intact embryos and epicardial cells from E11.5 mouse embryos to reveal the mechanisms by which TGFßR3 signals and regulates epicardial cell behavior. Analysis of E13.5 embryos reveals a lower rate of epicardial cell proliferation and decreased epicardially derived cell invasion in Tgfbr3(-/-) hearts. Tgfbr3(-/-) epicardial cells in vitro show decreased proliferation and decreased invasion in response to TGFß1 and TGFß2. Unexpectedly, loss of TGFßR3 also decreases responsiveness to two other important regulators of epicardial cell behavior, FGF2 and HMW-HA. Restoring full length TGFßR3 in Tgfbr3(-/-) cells rescued deficits in invasion in vitro in response TGFß1 and TGFß2 as well as FGF2 and HMW-HA. Expression of TGFßR3 missing the 3 C-terminal amino acids that are required to interact with the scaffolding protein GIPC1 did not rescue any of the deficits. Overexpression of GIPC1 alone in Tgfbr3(-/-) cells did not rescue invasion whereas knockdown of GIPC1 in Tgfbr3(+/+) cells decreased invasion in response to TGFß2, FGF2, and HMW-HA. We conclude that TGFßR3 interaction with GIPC1 is critical for regulating invasion and growth factor responsiveness in epicardial cells and that dysregulation of epicardial cell proliferation and invasion contributes to failed coronary vessel development in Tgfbr3(-/-) mice.
Subject(s)
Carrier Proteins/chemistry , Carrier Proteins/metabolism , Neuropeptides/chemistry , Neuropeptides/metabolism , Pericardium/cytology , Pericardium/metabolism , Proteoglycans/chemistry , Proteoglycans/metabolism , Receptors, Transforming Growth Factor beta/chemistry , Receptors, Transforming Growth Factor beta/metabolism , Adaptor Proteins, Signal Transducing , Animals , Base Sequence , Carrier Proteins/antagonists & inhibitors , Carrier Proteins/genetics , Cell Differentiation/drug effects , Cell Movement/drug effects , Cell Movement/physiology , Cell Proliferation/drug effects , Coronary Vessel Anomalies/embryology , Coronary Vessel Anomalies/genetics , Coronary Vessel Anomalies/metabolism , DNA Primers/genetics , Epithelial-Mesenchymal Transition/drug effects , Female , Gene Expression Regulation, Developmental , Gene Knockdown Techniques , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Knockout , Models, Cardiovascular , Myocytes, Smooth Muscle/cytology , Myocytes, Smooth Muscle/drug effects , Myocytes, Smooth Muscle/metabolism , Neuropeptides/antagonists & inhibitors , Neuropeptides/genetics , Pericardium/embryology , Pregnancy , Protein Interaction Domains and Motifs , Proteoglycans/deficiency , Proteoglycans/genetics , Receptors, Transforming Growth Factor beta/deficiency , Receptors, Transforming Growth Factor beta/genetics , Signal Transduction , Time-Lapse Imaging , Transforming Growth Factor beta1/pharmacology , Transforming Growth Factor beta2/pharmacologyABSTRACT
In the developing heart, the epicardium is a major source of progenitor cells that contribute to the formation of the coronary vessel system. These epicardial progenitors give rise to the different cellular components of the coronary vasculature by undergoing a number of morphological and physiological changes collectively known as epithelial to mesenchymal transformation (EMT). However, the specific signaling mechanisms that regulate epicardial EMT are yet to be delineated. In this study we investigated the role of TGFß2 and hyaluronan (HA) during epicardial EMT and how signals from these two molecules are integrated during this important process. Here we show that TGFß2 induces MEKK3 activation, which in turn promotes ERK1/2 and ERK5 phosphorylation. TGFß2 also increases Has2 expression and subsequent HA production. Nevertheless, inhibition of MEKK3 kinase activity, silencing of ERK5 or pharmacological disruption of ERK1/2 activation significantly abrogates this response. Thus, TGFß2 promotes Has2 expression and HA production through a MEKK3/ERK1/2/5-dependent cascade. Furthermore, TGFß2 is able to induce epicardial cell invasion and differentiation but not proliferation. However, inhibition of MEKK3-dependent pathways, degradation of HA by hyaluronidases or blockade of CD44, significantly impairs the biological response to TGFß2. Taken together, these findings demonstrate that TGFß2 activation of MEKK3/ERK1/2/5 signaling modulates Has2 expression and HA production leading to the induction of EMT events. This is an important and novel mechanism showing how TGFß2 and HA signals are integrated to regulate changes in epicardial cell behavior.
Subject(s)
Cell Movement/physiology , Epithelial-Mesenchymal Transition/physiology , Hyaluronic Acid/metabolism , Pericardium/cytology , Stem Cells/cytology , Transforming Growth Factor beta2/pharmacology , Animals , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/pharmacology , Cell Line , Cell Movement/drug effects , Epithelial-Mesenchymal Transition/drug effects , Gene Expression/drug effects , Gene Expression/genetics , Glucuronosyltransferase/genetics , Glucuronosyltransferase/metabolism , Humans , Hyaluronan Receptors/immunology , Hyaluronan Synthases , Hyaluronoglucosaminidase/pharmacology , MAP Kinase Kinase Kinase 3/genetics , MAP Kinase Kinase Kinase 3/metabolism , Mice , Mice, Transgenic , Mitogen-Activated Protein Kinase 1/antagonists & inhibitors , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/antagonists & inhibitors , Mitogen-Activated Protein Kinase 3/metabolism , Mitogen-Activated Protein Kinase 7/genetics , Mitogen-Activated Protein Kinase 7/metabolism , Phosphorylation/drug effects , RNA, Small Interfering/genetics , Signal Transduction/drug effects , Signal Transduction/physiology , Stem Cells/drug effects , Stem Cells/metabolism , Vimentin/metabolismABSTRACT
During embryonic development, cells comprising the outermost layer of the heart or epicardium play a critical role in the formation of the coronary vasculature. Thus, uncovering the molecular mechanisms that govern epicardial cell behavior is imperative to better understand the etiology of cardiovascular diseases. In this study, we investigated the function of hyaluronan (HA), a major component of the extracellular matrix, in the modulation of epicardial signaling. We show that stimulation of epicardial cells with high molecular weight HA (HMW-HA) promotes the association of MEKK1 with the HA receptor CD44 and induces MEKK1 phosphorylation. This leads to the activation of two distinct pathways, one ERK-dependent and another NFkappaB-dependent. Furthermore, HMW-HA stimulates epicardial cells to differentiate and invade, as suggested by increased vimentin expression and enhanced invasion through a collagen matrix. Blockade of CD44, transfection with a kinase-inactive MEKK1 construct or the use of ERK1/2 and NFkappaB inhibitors significantly abrogates the invasive response to HMW-HA. Together, these findings suggest an important role for HA in the regulation of epicardial cell fate via activation of MEKK1 signaling cascades.
Subject(s)
Hyaluronic Acid/pharmacology , MAP Kinase Kinase Kinase 1/metabolism , MAP Kinase Signaling System , Pericardium/enzymology , Animals , Cell Differentiation , Cell Line , Cell Movement , Extracellular Signal-Regulated MAP Kinases/metabolism , Hyaluronan Receptors/metabolism , Mice , Mice, Transgenic , NF-kappa B/metabolism , Pericardium/cytology , Pericardium/drug effects , Serum Response Element , Snail Family Transcription Factors , Transcription Factors/metabolismABSTRACT
Cells derived from the epicardium are required for coronary vessel development. Transforming growth factor beta (TGFbeta) induces loss of epithelial character and smooth muscle differentiation in chick epicardial cells. Here, we show that epicardial explants from embryonic day (E) 11.5 mouse embryos incubated with TGFbeta1 or TGFbeta2 lose epithelial character and undergo smooth muscle differentiation. To further study TGFbeta Signaling, we generated immortalized mouse epicardial cells. Cells from E10.5, 11.5, and 13.5 formed tightly packed epithelium and expressed the epicardial marker Wilm's tumor 1 (WT1). TGFbeta induced the loss of zonula occludens-1 (ZO-1) and the appearance of SM22alpha and calponin consistent with smooth muscle differentiation. Inhibition of activin receptor-like kinase (ALK) 5 or p160 rho kinase activity prevented the effects of TGFbeta while inhibition of p38 mitogen activated protein (MAP) kinase did not. These data demonstrate that TGFbeta induces epicardial cell differentiation and that immortalized epicardial cells provide a suitable model for differentiation.
Subject(s)
Cell Differentiation/drug effects , Coronary Vessels/embryology , Muscle, Smooth/physiology , Pericardium/cytology , Transforming Growth Factor beta/pharmacology , Animals , Calcium-Binding Proteins/metabolism , Cell Culture Techniques , Cell Differentiation/physiology , Immunohistochemistry , Membrane Proteins/metabolism , Mice , Microfilament Proteins/metabolism , Muscle Proteins/metabolism , Pericardium/physiology , Phosphoproteins/metabolism , WT1 Proteins/metabolism , Zonula Occludens-1 Protein , CalponinsABSTRACT
The proepicardium (PE) migrates over the heart and forms the epicardium. A subset of these PE-derived cells undergoes epithelial-mesenchymal transformation (EMT) and gives rise to cardiac fibroblasts and components of the coronary vasculature. We report that transforming growth factor-beta (TGFbeta) 1 and TGFbeta2 increase EMT in PE explants as measured by invasion into a collagen gel, loss of cytokeratin expression, and redistribution of ZO1. The type I TGFbeta receptors ALK2 and ALK5 are both expressed in the PE. However, only constitutively active (ca) ALK2 stimulates PE-derived epithelial cell activation, the first step in transformation, whereas caALK5 stimulates neither activation nor transformation in PE explants. Overexpression of Smad6, an inhibitor of ALK2 signaling, inhibits epithelial cell activation, whereas BMP7, a known ligand for ALK2, has no effect. These data demonstrate that TGFbeta stimulates transformation in the PE and suggest that ALK2 partially mediates this effect.
