TGFβ and BMP-2 regulate epicardial cell invasion via TGFβR3 activation of the Par6/Smurf1/RhoA pathway

Coronary vessel development requires transfer of mesothelial cells to the heart surface to form the epicardiurn where some cells subsequently undergo epithelial-mesenchymal transformation (EMT) and invade the subepicardial matrix. Tgfbr3(-/-) mice die due to failed coronary vessel formation associated with decreased epicardial cell invasion but the mediators downstream of TGF beta R3 are not well described. TGF beta R3-dependent endocardial EMT stimulated by either TGF beta 2 or BMP-2 requires activation of the Par6/Smurf1/RhoA 1pathway where Activin Receptor Like Kinase (ALK5) signals Par6 to act downstream of TGF beta to recruit Smurf1 to target RhoA for degradation to regulate apical-basal polarity and tight junction dissolution. Here we asked if this pathway was operant in epicardial cells and if TGF beta R3 was required to access this pathway. Targeting of ALK5 in Tgfbr3(+/+) cells inhibited loss of epithelial character and invasion. Overexpression of wild-type (wt) Par6, but not dominant negative (dn) Par6, induced EMT and invasion while targeting Par6 by siRNA inhibited EMT and invasion. Overexpression of Smurf1 and dnRhoA induced loss of epithelial character and invasion. Targeting of Smurf1 by siRNA or overexpression of constitutively active (ca) RhoA inhibited EMT and invasion. In Tgfbr3(-/-) epicardial cells which have a decreased ability to invade collagen gels in response to TGF beta 2, overexpression of wtPar6, Smurf1, or dnRhoA had a diminished ability to induce invasion. Overexpression of TGF beta R3 in Tgfbr3(-/-) cells, followed by siRNA targeting of Par6 or Smurf1, diminished the ability of TGF beta R3 to rescue invasion demonstrating that the Par6/Smurf1/RhoA pathway is activated downstream of TGF beta R3 in epicardial cells. (C) 2011 Elsevier Inc. All rights reserved.