Epithelial-mesenchymal transformation in the embryonic heart is mediated through distinct pertussis toxin-sensitive and TGFβ signal transduction mechanisms

During early development, progenitors of the heart valves and septa are formed by epithelial-mesenchymal transformation (EMT) of endothelial cells in the atrioventricular (AV) canal. Previously, we showed that pertussis toxin, a specific inhibitor of a subset of G proteins, inhibited EMT in chick AV canal cultures. This study examines in detail the effects of pertussis toxin on the process of EMT. One of the major mediators of EMT is Transforming Growth Factor beta 3 (TGFβ3) which acts through the TGFβ Type II receptor. To determine whether pertussis toxin affects EMT via the TGFβ Type II receptor pathway, we compared AV cultures treated with pertussis toxin and TGFβ Type II receptor blocking antibody. Pertussis toxin inhibited several elements of EMT. At all stages tested, pertussis toxin blocked endothelial cell-cell separation, cell hypertrophy, and the cellular polarization associated with endothelial activation. These activities were unaffected by TGFβ Type II receptor antibodies. Pertussis toxin also reduced transformed mesenchymal cell migration by 61%. The expression patterns of several proteins (as markers of EMT) were analyzed in untreated, pertussis toxin- treated, and TGFβ Type II receptor blocking antibody-treated cultures. These markers were α-smooth muscle actin, Mox-1, fibrillin 2, tenascin, cell surface β 1,4 galactosyltransferase (GalTase), and integrin α6. Clear differences in marker expression were found between the two inhibitors. For example, in all cells, pertussis toxin inhibited expression of α-smooth muscle actin and GalTase while TGFβ Type II receptor antibody treatment increased expression of these two proteins. These data suggest that G protein-mediated signaling is required for several elements of EMT. Furthermore, distinct G protein and TGFβ signal transduction pathways mediate discrete components of EMT.