Src kinase regulates TGFbeta and hyaluronan induced epicardial cell invasion, differentiation and migration

Coronary vessel formation relies on growth factor as well as extracellular matrix (ECM) influences on cells of the epicardium that regulate proliferation, motility, invasion and differentiation. The Transforming Growth Factor beta (TGFbeta) family of receptors have been described to regulate cardiovascular development. The Type III TGFbeta receptor (TGFbetaR3) has been shown to be required for development of the coronary vessels. Mouse embryos lacking Hyaluronan Synthase 2 (Has2) are lethal at E 9.5 as a result of severely blocked cardiogenesis due to insufficient endocardial EMT. Src kinase is a non-receptor tyrosine kinase that functions in growth factor as well as ECM signal transduction, but its role in epicardial cell biology is unclear. We hypothesize Src kinase is a critical regulator of TGFbeta and Hyaluronan induced epicardial cell invasion, differentiation and migration during coronary vessel development. We show Src is required for TGFbeta2-induced vascular smooth muscle differentiation as well as TGFbeta2-induced EMT, cell invasion, and filamentous actin polymerization. Src activity is sufficient to drive epicardial activation of EMT, but not vascular smooth muscle differentiation. Next we demonstrate that TGFbetaR3 and Src are required for HMWHA-induced cell invasion and filamentous actin polymerization in epicardial cells. HMWHA induces activation of Src kinase in Tgfbr3+/+ epicardial cells, but not Tgfbr3-/- epicardial cells. Tgfbr3 -/- epicardial cells fail to activate Rac1 or RhoA GTPases in the presence of HMWHA. Finally, we demonstrate stimulus independent activation of TGFbetaR3 is sufficient to activate Src. These constitute novel findings establishing TGFbetaR3 as an HMWHA responsive receptor that is upstream of Src signal transduction. Migration of the epicardium to cover heart tube is an early step required for development of the coronary vessels. We demonstrate that Tgfbr3-/- epicardial cells are delayed in cell migration relative to Tgfbr3+/+ cells in a wound healing model of cell migration. BMP2 drives Tgfbr3 -/- (but not Tgfbr3+/+) cell migration to levels comparable to unstimulated Tgfbr3+/+ epicardial cells, without enhancing cell proliferation. We demonstrate that Src is required for this BMP2 induced cell migration and filamentous actin polymerization in Tgfbr3-/- cells. These pathways constitute important future targets for adult cardiovascular regeneration and cardioprotection in adult heart disease.