Rho-associated kinases play a role in endocardial cell differentiation and migration.

Development of the endocardial cushions in the heart involves cell migration and cell differentiation, which is known as epithelial-mesenchymal transformation (EMT). These processes are regulated by cell signaling systems. Yet, the roles of intracellular GTPases and their effectors on these cellular activities remain to be addressed. This study investigated the role Rho GTPase-associated kinases (ROCKs) in endocardial cushion development. Using reverse transcription (RT) and polymerase chain reaction (PCR), expression of the rock1 and rock2 genes was found in the endocardial cushions during development. To investigate the role of ROCKs in development, the ROCK inhibitor Y27632 and adenoviruses containing a dominant negative form of the rock gene were used to treat cultured endocardial cushions and cells. In monolayer cell culture and three-dimensional tissue culture, blockade of ROCK inhibited EMT development. Using three-dimensional collagen gel assays and confocal microscopy, we also observed inhibition of cell migration with ROCK inhibition. Examination of cell morphology and actin cytoskeleton revealed that inhibition of ROCK activity disturbed cytoskeletal organization and blocked the formation of lamellipodia and filopodia. Collectively, these data show that ROCKs play an essential role in endothelial cell differentiation and migration during endocardial cushion development.