The identification of TGF-beta signaling pathways that regulate endocardial cell epithelial-to-mesenchymal transformation

Valvular heart disease due to congenital abnormalities or pathology is a major cause of mortality and morbidity. Understanding the cellular processes and molecules that regulate valve formation and remodeling is required to develop effective therapies. In the developing heart, epithelial-to-mesenchymal transformation (EMT) in a subpopulation of endocardial cells in the atrioventricular cushion (AVC) is an important step in valve formation. Transforming Growth Factor beta (TGFbeta) has been shown to be an important regulator of AVC endocardial cell EMT in vitro and mesenchymal cell differentiation in vivo. TGFbeta signals through three high affinity cell surface receptors, the TGFbeta Type I (TGFbetaR1, also known as ALKs), Type II (TGFbetaR2), and Type III (TGFbetaR3) receptors. We have used targeted overexpression and knockdown experiments in the ventricle and AVC to score for gain- and loss-of-function respectively to determine the molecules that regulate EMT in vitro. First, we have demonstrated that a Par6/Smurf1/RhoA pathway is operative downstream of ALK5 to mediate endocardial cell EMT. Second, we identified BMP-2 as a ligand for TGFbetaR3 and demonstrated that BMP-2 can mediate EMT. Third, although dispensable for ligand presentation, we demonstrate that the cytoplasmic domain of TGFbetaR3 is absolutely required for TGFbeta2-stimulated EMT in vitro. Within this cytoplasmic domain, the three C-terminal amino acids comprise a Class I PDZ binding motif that binds to the PDZ domain containing protein GIPC to stabilize TGFbetaR3 surface expression. Deletion of the Class I PDZ binding motif is sufficient to abolish TGFbeta2-stimulated ventricular EMT in vitro, suggesting a role for GIPC in TGFbetaR3-mediated EMT. Consistent with GIPC interaction with TGFbetaR3 mediating EMT, overexpression of GIPC leads to an enhancement of AVC transformation, whereas targeting GIPC via siRNA inhibited TGFbetaR3-stimulated EMT. We establish that the Class I PDZ domain of TGFbetaR3 and interaction with GIPC are required for endocardial cell EMT in vitro. These data identify a novel ligand for TGFbetaR3 and reveal an essential role for TGFbetaR3 in mediating non-canonical signaling downstream of TGFbeta.