Effects And Relevant Mechanisms Of CRH Related Peptides On Vascular Permeability And Transmigration, Invasion And Apoptosis Of Cancer Cells

Corticotropin-Releasing Hormone (CRH) is a major regulator of thehypothalamic-pituitary adrenal axis and principal coordinator of the stress response.CRH is originally found in central nervous system and subsequently severalstructure-related peptides are discovered in central and peripheral tissues, such ascardiovascular system, sites of inflammation and many types of cancers. TheCRH-related peptides are composed of CRH, Urocortin (Ucn1), Urocortin2(Ucn2)and Urocortin3(Ucn3), which bind with two CRH receptors，CRHR1and CRHR2with different affinity. Althouth long noted participation of the CRH-related peptidesin pathophysiology of inflammation and cancer, the detailed mechanisms of CRH onvascular permeability and carcinogenesis and metastasis of cancer are not wellunderstood.In the first part, we investigated the effect of Ucn1on the lipopolysaccharides(LPS)-induced hyperpermeability in Human Umbilical Vein Endothelial Cells(HUVEC). Pretreatment of HUVEC with Ucn1augmentated LPS-induced endothelialhyperpermeability, which was accompanied by significant downregulation ofVE-cadherin. Overexpression of VE-cadherin in HUVEC prevented thehyperpermeability caused by Ucn1+LPS. Moreover, Ucn1increased phosphorylationof protein kinase D (PKD) and heat shock protein27(HSP27) in a time-andCRHR2-dependent manner. Pharmacological inhibition or siRNA knockdown ofPKD and HSP27drastically inhibited Ucn1-induced downregulation of VE-cadherin.We also found that Ucn1+LPS induced greater β-catenin phosphorylation at Ser552,disruption of the cadherin-catenin complex, and increased expression of β-catenin, which translocated into the nucleus and enhanced the expression of vascularendothelial growth factor (VEGF). The above effects of Ucn1were blocked byantagonism of CRHR2and siRNA knockdown of PKD and HSP27. Taken together,we demonstrate that Ucn1may enhance the vascular permeability throughPKD-HSP27signaling pathway, which may increase the phosphorylation of β-cateninand then disruption of the VE-cadherin–β-catenin complex.In the second part, we investigated the effects and mechanisms of CRH onmigration and invasion with two breast cancer cell lines, MCF-7and MDA-MB-231.We firstly explored the pattern of CRH receptors expressed by these cells, and theresult showed both CRH receptors were present in MCF-7cell while MDA-MB-231expressed only CRHR2. We next treated cells with CRH to observe the effect onmigration and invasion using Transwell. CRH significantly, through activation ofCRHR2, inhibited the migration and invasion in both cell lines, while antagonism ofCRHR1was without effect. Activation of CRHR2also led to phosphoralation ofprotein kinase D (PKD) and increased expression of E-cadherin. In addition toE-cadherin, we also found abundance of several other epithelial-mesenchymaltransition (EMT) related proteins was changed by CRH via CRHR2. The epithelialmarker, Occludin, was increased while mesenchymal markers such as N-cadherin andVimentin were decreased. The above results indicate that CRH may prevent theinduction of EMT and inhibit cellular metastasis and invasion via CRHR2.In the third part, mice prostate cancer cell line RM-1was used to explore theeffect of CRH related peptides on apoptosis and the relevant mechanism. We firstlyconfirmed the expression of both CRFR1and CRFR2in RM-1cells using Westernblot. Treatment of RM-1cell with CRH induced apoptosis via CRHR1, which wasaccompanied by reduced anti-apoptotic Bcl-2expression and increased pro-apoptoticBax expression, and activation of caspases-9. On the contrary, activation of CRHR2with Ucn2suppressed apoptosis by increasing Bcl-2/Bax ratio. Ucn2rapidlyphosphorylated Akt and CREB，and inhibition of PI3K with LY294002prevented theactivation of Akt and CREB，and most importantly, the increment of Bcl-2expression.We also found activation of CRHR1increased the expression of cPLA2while activation of CRHR2downregulated it. These results suggest that CRH relatedpeptides may exert pro-or anti-apoptotic effects depending on the activation ofdifferent CRH receptors. Activation of Akt-CREB pathway and change of cPLA2expression and Bcl-2/Bax ratio may be involed in the anti-apoptotic mechanisms ofCRHR2.