FIGURE 4.

The cAMP target, Epac, suppresses EMT and cell migration in epicardial cell outgrowth from heart explants. A, Western blot showing that Epac1, p190RhoGAP, and Rap1 proteins were expressed in EMCs and the PE. B, the Epac activator, 007 (50 μm), increased active Rap1·GTP in EMCs. C, immunolabeling showed Rap1 localized to junctional membranes (arrows) and in the cytosol in 007 treated EMCs (panel a) stained with TRITC-labeled phalloidin (panel b) and co-localized with cortical actin (panel c). D, activation of Epac1 with 007 treatment suppressed EMT in epicardial cells grown out from E10.5 heart explants for 48 h in the presence of 50 ng of bFGF showing compact rounder cells outlined by cortical actin (panels c and d). bFGF treatment alone resulted in migratory cells with many processes (panel a) and actin stress fibers (panel b). E, migration of mesenchymal cells grown out from E10.5 heart explants into collagen gels in the presence of 50 ng of bFGF with/without 50 μm 007 for 6 days was retarded in the presence of 007. Counting of cells at different distances from the edge of the heart (distance = 0) is detailed under “Experimental Procedures.” F, Rap1 activity shown as a fraction of total Rap1 at 72 h decreased by 4-fold with TGF-β1-induced EMT in EECs consistent with a fall in Epac activity. Data are the means ± S.E., n = 3. G, epicardial cells grown out from WT and Epac1 null E10.5 heart explants for 48 h in the presence of 50 ng of bFGF, showing that the Epac1 null cells exhibit an increased migration indicated by the greater separation of the nuclei and the increased presence of lamellipodia; data for cell separation were from 6 WT and 9 Epac1 null hearts, number of cells counted were WT = 167 and Epac1 null = 73. The western blot shows absence of Epac1 protein in the Epac1 null cells.