RESUMO
An early event in heart valve formation is the epithelial-mesenchymal transformation (EMT) of a subpopulation of endothelial cells in specific regions of the heart tube, the endocardial cushions. The Type III TGFß receptor (TGFßR3) is required for TGFß2- or BMP-2-stimulated EMT in atrioventricular endocardial cushion (AVC) explants in vitro but the mediators downstream of TGFßR3 are not well described. Using AVC and ventricular explants as an in vitro assay, we found an absolute requirement for specific TGFßR3 cytoplasmic residues, GAIP-interacting protein, C terminus (GIPC), and specific Activin Receptor-Like Kinases (ALK)s for TGFßR3-mediated EMT when stimulated by TGFß2 or BMP-2. The introduction of TGFßR3 into nontransforming ventricular endocardial cells, followed by the addition of either TGFß2 or BMP-2, results in EMT. TGFßR3 lacking the entire cytoplasmic domain, or only the 3C-terminal amino acids that are required to bind GIPC, fails to support EMT in response to TGFß2 or BMP-2. Overexpression of GIPC in AVC endocardial cells enhanced EMT while siRNA-mediated silencing of GIPC in ventricular cells overexpressing TGFßR3 significantly inhibited EMT. Targeting of specific ALKs by siRNA revealed that TGFßR3-mediated EMT requires ALK2 and ALK3, in addition to ALK5, but not ALK4 or ALK6. Taken together, these data identify GIPC, ALK2, ALK3, and ALK5 as signaling components required for TGFßR3-mediated endothelial cell EMT.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Coxins Endocárdicos/fisiologia , Transição Epitelial-Mesenquimal , Proteoglicanas/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Receptores de Ativinas/metabolismo , Sequência de Aminoácidos , Animais , Proteína Morfogenética Óssea 2/farmacologia , Proteína Morfogenética Óssea 2/fisiologia , Embrião de Galinha , Coxins Endocárdicos/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/fisiologia , Proteínas de Fluorescência Verde/biossíntese , Dados de Sequência Molecular , Domínios e Motivos de Interação entre Proteínas , Proteoglicanas/química , Receptores de Fatores de Crescimento Transformadores beta/química , Proteínas Recombinantes/biossíntese , Técnicas de Cultura de Tecidos , Fator de Crescimento Transformador beta2/farmacologia , Fator de Crescimento Transformador beta2/fisiologiaRESUMO
Blood circulation is dependent on heart valves to direct blood flow through the heart and great vessels. Valve development relies on epithelial to mesenchymal transition (EMT), a central feature of embryonic development and metastatic cancer. Abnormal EMT and remodeling contribute to the etiology of several congenital heart defects. Leptin and its receptor were detected in the mouse embryonic heart. Using an ex vivo model of cardiac EMT, the inhibition of leptin results in a signal transducer and activator of transcription 3 and Snail/vascular endothelial cadherin-independent decrease in EMT and migration. Our data suggest that an Akt signaling pathway underlies the observed phenotype. Furthermore, loss of leptin phenocopied the functional inhibition of alphavbeta3 integrin receptor and resulted in decreased alphavbeta3 integrin and matrix metalloprotease 2, suggesting that the leptin signaling pathway is involved in adhesion and migration processes. This study adds leptin to the repertoire of factors that mediate EMT and, for the first time, demonstrates a role for the interleukin 6 family in embryonic EMT.
