ROCK1 expression is regulated by TGFβ3 and ALK2 during valvuloseptal endocardial cushion formation

During early heart development at the looped heart stage, endothelial cells in the outflow tract and atrioventricular (AV) regions transform into mesenchyme to generate endocardial cushion tissue. This endocardial epithelial-mesenchymal transition (EMT) is regulated by several regulatory pathways, including the transforming growth factor-beta (TGF beta), bone morphogenetic protein (BMP), and Rho-ROCK pathways. Here, we investigated the spatiotemporal expression pattern of ROCK1 mRNA during EMT in chick and examined whether TGF beta or BMP could induce the expression of ROCK1. At the onset of EMT, ROCK1 expression was up-regulated in endothelial/mesenchymal cells. A three-dimensional collagen gel assay was used to examine the mechanisms regulating the expression of ROCK1. In AV endocardium co-cultured with associated myocardium, ROCK1 expression was inhibited by either anti-TGF beta 3 antibody, anti-ALK2 antibody or noggin, but not SB431542 (ALK5 inhibitor). In cultured preactivated AV endocardium, TGF beta 3 protein induced the expression of ROCK1, but BMP did not. AV endothelial cells that were cultured in medium supplemented with TGF beta 3 plus anti-ALK2 antibody failed to express ROCK1. These results suggest that the expression of ROCK1 is up-regulated at the onset of EMT and that signaling mediated by TGF beta 3/ALK2 together with BMP is involved in the expression of ROCK1.