Effects Of CRH Family Peptide On EMT And Expression Of ICAM - 1 In Endothelial Cells And Its Mechanism

Part1CRH suppressed TGFβ1-induced Epithelial-Mesenchymal Transition via inducyion of E-cadherin expression in breast cancer cellsCorticotropin-Releasing Hormone (CRH) family is composed of CRH, Ucnl, Ucn2and Ucn3, and bind to their two known receptors:CRH receptor type l(CRHRl) and CRH receptor type2(CRHR2). A large number of studies have shown that peripheral CRH related peptides play an important role in tumor cell apoptosis and proliferation, but their role in metastasis and the relevant mechanism is not yet clear. Epithelial mesenchymal transition (EMT) is the process of the polarity epithelial cells converting into active mesenchymal cells to obtain invasion and migration ability. So far, transforming growth factor beta1(TGFβ1) is recognized as an EMT-inducing factor mainly through induction of transcriptional regulation factor Snaill and Twistl and hence inhibition of calcium epithelial mucin (E-cadherin) expression. Down-regulation of E-cadherin is considered as a hallmark of EMT. To study the mechanism of TGFβ1-induced EMT can help us better understand EMT and find new ways for interfering tumor invasion and metastasis.In this part, two breast cancer cell lines, MCF-7and MDA-MB-231were used to investigate the effect of CRH on TGFβ1-induced EMT by transwell chamber, western blot and real-time PCR. The experimental results showed that CRH inhibited breast cancer cell migration through down-regulation of Snaill and Twistl and hence increased the expression of E-cadherin. Further experiment results showed that CRH inhibited TGFβ1-mediated EMT in MCF-7via both CRHR1and CRHR2while this inhibition in MDA-MB-231was mainly via CRHR2. In order to confirm the exact role of each receptor, HEK293cells were transfected with CRHR1or CRHR2, respectively. Compared with the untransfected cells, higher levels of E-cadherin expression in two kinds of transfected cells were observed. Furthermore, CRH could repress expression of mesenchymal marker N-cadherin and induce Occludin expression.Taken together, our results suggest that CRH can suppress TGFβ1-mediated EMT mainly through CRHR1and CRHR2, contributing to uncovering the effects of CRH in breast tumorigenesis, invasion and metastasis. Part2Mechanism of Urocortin regulation ICAM-1expression on vascular endothelial cellsUrocortin (Ucnl), a member of corticotrophin-releasing hormone (CRH) family, has been reported to participate in inflammation. The increased expression of intercellular adhesion molecule1(ICAM-1) plays important roles in inflammation and immune responses. Our previous data demonstrated that Ucnl significantly increased the expression of ICAM-1. However, the underlying mechanisms are still unknown. Here, we used human umbilical vein endothelial cells to investigate the detailed mechanisms of Ucnl-induced ICAM-1expression.Our results revealed that Ucnl increased the expression and phosphorylation of cytosolic phospholipases A2(cPLA2) through CRHR1and CRHR2. By use of cyclooxygenase2(COX-2) and CRHR inhibitors, Ucnl was found to increase ICAM-1expression through COX-2, which was influenced by the CRHR2antagonist but not CRHR1antagonist. Besides, Ucnl increased the expression of prostaglandin E2(PGE2) which further activated protein kinase A (PKA)-CREB pathways dependening on cPLA2activation. Moreover, ablation of cPLA2by the inhibitor pyrrophenone or siRNA attenuated the ICAM-1upregulation induced by Ucnl. Therefore, cPLA2activation contributed to the activation of COX-2induced by Ucnl. Furthermore, inhibition of cPLA2abolished PKA-CREB pathways induced by Ucnl. Interestingly, cPLA2was activated via both CRHRs, but Ucnl increased cPLA2-dependent ICAM-1expression just via CRHR2in this study. The effects of cPLA2activated by Ucnl via CRHR1were not clear.In conclusion, these data suggest that Ucnl increased the ICAM-1expression via cPLA2-COX-2-PGE2-PKA-CREB pathways by means of CRHR2.