Abstract
Endothelial to Mesenchymal Transition (EnMT) is an important process during cardiac valve formation. It is believed the general mechanism of EnMT is similar to that of epithelial to mesenchymal transition (EMT). Previous studies indicate that EnMT can be induced by transforming growth factor β (TGF-β). TGF-β first triggers the downstream signal transduction, such as SMAD pathway, and then activates transcription factor like Snail. The activated Snail represses the expression of endothelial markers while promotes the expression of mesenchymal markers, to drive the EnMT process. Nevertheless, the EnMT has a complex molecular mechanism with many details still remains unclear. PRSS23 is a novel endothelial serine protease identified in our previous studies. In previous in vivo assays, we have used morpholino to knockdown PRSS23 expression in zebrafish embryo, and found the loss of PRSS23 could cause cardiac valve malformation in developing heart. Immunohistochemcal data further showed the cardiac endothelial cells fail to initiate EnMT during valve formation. To characterize the role of PRSS23 in EnMT, human aortic endothelial cells (HAECs) and shRNA knockdown were used as key experimental systems in this study. The results indicated that knockdown of PRSS23 inhibited TGF-β2-induced EnMT in HAECs. Further studies revealed knockdown of PRSS23 did not affect the phosphorylation of SMAD, but repressed the transcription of Snail in TGF-β2-treated HAECs. By co-immunoprecipitation (co-IP), we also validated the interaction of PRSS23 and TCF12. TCF12 is a transcription factor involved in heart development, and is known to bind E-box (CANNTG) in promoter. Interestingly, we found E-box is conserved in the Snail promoter of various vertebrates. Therefore, PRSS23/TCF12 complex may bind the promoter of Snail to regulate EnMT. Our results suggest that PRSS23 is essential for Snail-dependent EnMT, and provide clue to reveal the possible mechanism of PRSS23 in EnMT.