During embryogenesis, the epicardium undergoes proliferation, migration, and differentiation into several cardiac cell types which contribute to the coronary vessels. This process requires epithelial to mesenchymal transition (EMT) and directed cellular invasion. The Type III Transforming Growth Factor-beta Receptor (TGFβR3) is required for epicardial cell invasion and coronary vessel development. Using primary epicardial cells derived from Tgfbr3+/+ and Tgfbr3−/− mouse embryos, high-molecular weight hyaluronan (HMWHA) stimulated cellular invasion and filamentous (f-actin) polymerization are detected in Tgfbr3+/+ cells, but not in Tgfbr3−/− cells. Furthermore, HMWHA-stimulated cellular invasion and f-actin polymerization in Tgfbr3+/+ epicardial cells are dependent on Src kinase. Src activation in HMWHA-stimulated Tgfbr3−/− epicardial cells is not detected in response to HMWHA. RhoA and Rac1 also fail to activate in response to HMWHA in Tgfbr3−/− cells. These events coincide with defective f-actin formation and deficient cellular invasion. Finally, a T841A activating substitution in TGFβR3 drives ligand-independent Src activation. Collectively, these data define a TGFβR3–Src–RhoA/Rac1 pathway that is essential for hyaluronan-directed cell invasion in epicardial cells.

During embryogenesis, the epicardium undergoes proliferation, migration, and differentiation into several cardiac cell types which contribute to the coronary vessels. The type III transforming growth factor-b receptor (TGFbR3) is required for epicardial cell invasion and development of coronary vasculature in vivo. Bone Morphogenic Protein-2 (BMP2) is a driver of epicardial cell migration. Utilizing a primary epicardial cell line derived from Tgfbr3 C/C and Tgfbr3 ¡/¡ mouse embryos, we show that Tgfbr3 ¡/¡ epicardial cells are deficient in BMP2 mRNA expression. Tgfbr3 ¡/¡ epicardial cells are deficient in 2-dimensional migration relative to Tgfbr3 C/C cells; BMP2 induces cellular migration to Tgfbr3 C/C levels without affecting proliferation. We further demonstrate that Src kinase activity is required for BMP2 driven Tgfbr3 ¡/¡ migration. BMP2 also requires Src for filamentous actin polymerization in Tgfbr3 ¡/¡ epicardial cells. Taken together, our data identifies a novel pathway in epicardial cell migration required for development of the coronary vessels.

The epicardium originates from the proepicardial organ (PEO). Both the PEO and epicardium require directed cell‐motility to cover the heart and instruct epicardially derived cells for coronary vasculature formation. The type III transforming growth factor‐beta receptor (Tgfbr3) is required for epicardial cell invasion and development of coronary vasculature in vivo. Utilizing a primary epicardial cell line derived from Tgfbr3(+/+) and Tgfbr3(‐/‐) e13.5 mouse embryos , we show that Tgfbr3(‐/‐) epicardial cells are 34.4% deficient in 2D migration relative to wild type. Furthermore, we show that high‐molecular weight Hyaluronan (HMWHA) drives 3D cellular invasion in wild type epicardial cells, but not Tgfbr3(‐/‐) epicardial cells. We show Src kinase is required for HMWHA stimulated filamentous actin (f‐actin) polymerization in wild‐epicardial cells. Furthermore, HMWHA stimulates Y416Src phosphorylation in wild type, but Src is not activated in Tgfbr3(‐/‐) epicardial cells. RhoA and Rac1 GTPase are downstream of Src kinase, and directly upstream of f‐actin polymerization; HMWHA robustly induces RhoA and Rac1 GTP binding and activity in wild type, but not Tgfbr3(‐/‐) epicardial cells. This deficit in RhoA and Rac1 activation results in reduced formation of f‐actin stress fibers in HMWHA stimulated Tgfbr3(‐/‐) epicardial cells. Taken together, we demonstrate that the type III TGFβ receptor is required for activation of basic cell motility pathways stimulated by Hyaluronan that are required for proper coronary vasculature formation. Grant Funding Source: NIH ES 04940; ES06694; SWEHSC P30ES006694