Endocardial cell epithelial-mesenchymal transformation requires Type III TGFβ receptor interaction with GIPC

Abstract

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 TGFβ receptor (TGFβR3) is required for TGFβ2- or BMP-2-stimulated EMT in atrioventricular endocardial cushion (AVC) explants in vitro but the mediators downstream of TGFβR3 are not well described. Using AVC and ventricular explants as an in vitro assay, we found an absolute requirement for specific TGFβR3 cytoplasmic residues, GAIP-interacting protein, C terminus (GIPC), and specific Activin Receptor-Like Kinases (ALK)s for TGFβR3-mediated EMT when stimulated by TGFβ2 or BMP-2. The introduction of TGFβR3 into nontransforming ventricular endocardial cells, followed by the addition of either TGFβ2 or BMP-2, results in EMT. TGFβR3 lacking the entire cytoplasmic domain, or only the 3 C-terminal amino acids that are required to bind GIPC, fails to support EMT in response to TGFβ2 or BMP-2. Overexpression of GIPC in AVC endocardial cells enhanced EMT while siRNA-mediated silencing of GIPC in ventricular cells overexpressing TGFβR3 significantly inhibited EMT. Targeting of specific ALK’s by siRNA revealed that TGFβR3-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 TGFβR3-mediated endothelial cell EMT.

Common pathways regulate Type III TGFβ receptor-dependent cell invasion in epicardial and endocardial cells

“…Although the cytoplasmic domain of TGFβR3 is not required for ligand presentation to TGFβR1 & TGFβR2, the regulation of migration and invasion of several cell types have been shown to require the cytoplasmic domain of TGFβR3. These include several cancer cell lines [18, 19] as well as both endocardial [20] and epicardial cells [11]. Therefore, efforts to understand TGFβR3 signaling have focused on the identification of proteins that interact with the cytoplasmic domain.…”

“…The interaction between TGFβR3 and GIPC has been reported to mediate the inhibition of breast cancer cell migration in vitro and cancer progression in vivo [22]. However, in both epicardial [11] and endocardial [20] cells, ligand-stimulated cell invasion has been found to be dependent on the cytoplasmic domain of TGFβR3, specifically the 3 C-terminal amino acids that interact with GIPC. In a second, distinct region of the cytoplasmic domain, phosphorylation of Thr841 by TGFβR2 is required for βarrestin2 (βArr2) binding which leads to TGFβR3 internalization [23].…”

Epithelial-mesenchymal transitions: insights from development

“…In chick embryos, TGF induces epithelial to mesenchymal transition (EMT) and cell invasion into the extracellular matrix via activation of TGFRII and TGFRIII, an essential co-receptor (Townsend et al, 2012). TGF signaling is linked to actin remodeling, possibly in part through zyxin, a focal adhesionassociated LIM protein implicated in EndMT under TGF-Twist1-zyxin regulation (Mori et al, 2009).…”

“…EndMT occurs in endothelial cells invading the cardiac jelly to form a cardiac cushion, which subsequently establishes the atrioventricular valves (Nakajima et al, 2000). EndMT is controlled by three distinct signaling pathways: TGF, Notch and Erbb3.In chick embryos, TGF induces epithelial to mesenchymal transition (EMT) and cell invasion into the extracellular matrix via activation of TGFRII and TGFRIII, an essential co-receptor (Townsend et al, 2012). TGF signaling is linked to actin remodeling, possibly in part through zyxin, a focal adhesionassociated LIM protein implicated in EndMT under TGF-Twist1-zyxin regulation (Mori et al, 2009).…”

Reprogramming during epithelial to mesenchymal transition under the control of TGFβ

“…51 These 2 cytokines, TGFb2 and BMP2, seem to contribute to EndoMT and invasiveness by binding to the type III co-receptor b-glycan, which coordinates signaling by both TGFb-specific type I receptors, activin receptor-like 5 (ALK5), and BMP-specific type I receptors, ALK2 and ALK3. 52 Upstream of the action of TGFb2 lies the MAP kinase kinase MEKK3, whose signaling in the developing heart of mice regulates the transcriptional induction of TGFb2. 53 Thus, heart valve morphogenesis is a good example of TGFb2-dependent EMT.…”