CRF Family Regulate Cell Migration And Proliferation Via PLA2 Family And The Underlying Mechanisms

Corticotropin-Releasing Factor(CRF),which was first isolated in 1981,acts through two G-protein-coupled transmenmbrane receptors of CRFR1 and CRFR2 to affect various pathophysiologic processes.Afterwards,three mammalian CRF-like paralogs,including UCN,UCN2 and UCN3,have been identified.These four peptides and their receptors constitute CRF family peptides&receptors system Among CRF family peptides,CRF binds to CRFR1 with higher affinity than CRFR2 while UCN2 binds to CRFR2 with higher affinity than CRFR1.UCN can bind to two types of CRF receptor with similar affinities.In this study,the effect of the CRFR1 and CRFR2 was investigated integrally and separately.It was found that there was difference between the roles of CRFR1 and CRFR2 in whatever tumor cell or vascular smooth muscle cell(VSMC).However,there existed a link between both receptors and phospholipase A2(PLA2)family in a variety of pathophysiologic processes,such as cellular signaling transduction,cell migration and proliferationRecent researches show that CRF family is a very promising target for tumor biotherapy.Our previous work demonstrated that UCN suppressed tumor growth via CRFR2 and similar outcomes were obtained in different tumor cells by other groups Moreover,some studies indicated that activation of CRFR1 also can inhibit the proliferation of multiple tumor cells such as Ishikawa(IK)human endometrial carcinoma cell,mouse melanoma cells and human breast cancer cells.Although most studies support that activation of CRFR1 or CRFR2 inhibit tumor growth,many reports indicate CRFR activation promote angiogenesis,tumor cell proliferation and migration.So far,it is not clear yet where the difference lies between the two CRF receptor’s roles in tumor development and progression.Therefore,the first part showed that the activation of the two CRF receptors affected hepatoma carcinoma cell migration oppositely via different regulation of two isoforms of PLA2,cPLA2 and iPLA2.PLA2 family is a wide class of enzyme that catalyze deacylation of the sn-2 position of glycerophospholipids producing fatty acid derivatives and lysophospholipids.Based on their cellular localization,substrate specificity,and calcium dependence,PLA2 have been classified into six categories:secretory PLA2(sPLA2),cytosolic PLA2(cPLA2),Ca2+-independent PLA2(iPLA2),platelet activating factor acetylhydrolases(PAFAH),lysosomes PLA2(LPLA2)and adipose specific PLA2(AdPLA2).Arachidonic acid(AA),the downstream of PLA2,has been reported to participate in migration of various cells,such as tumor cell,macrophage and VSMC.As known,inflammation and tumor have a close relationship.Many inflammatory factors promote tumor invasion and proliferation.UCN has been proven to promote inflammation in peripheral tissues and some inflammatory factors can induce the expression of UCN in vascular endothelial cell.In vivo and in vitro experiments in our lab have showed the important role of UCN in vascular inflammation.Furthermore,PLA2 family and its downstream bioactive products have also been reported to participate in development of vascular inflammation.Abnormal VSMC condition is an important appearance in vascular inflammation as atherosclerosis,including VSMC migration and proliferation.Thus,whether CRF family and PLA2 family could regulate the migration and proliferation of VSMC was studied in this thesis.Taken together,both CRF family and PLA2 family play a role in tumor progression and vascular inflammation.However,the complicated molecular mechanisms involving cross-talk among multiple signaling pathways are not well defined.The purpose of the study is to investigate whether there exist different roles of CRFR1 and CRFR2 in tumor development and vascular inflammation and whether these roles due to the regulation of PLA2.Part ⅠUCN affects migration of hepatoma carcinoma cell via regulating PLA2 familyGrowing evidence shows that UCN and PLA2 take part in development and progression of inflammation and tumor.The primary aim of this part was to identify a novel signaling pathway of CRF receptor activation leading to migration of two kinds of hepatoma carcinoma cell lines,HepG2 and SMMC-7721,linking the stimulation of PLA2 expression by UCN to UCN-induced tumor cell migration.Pharmacological inhibitors and genetic approaches(such as stable transfection and siRNAs)were used in this study.HepG2 cells express both of the CRF receptors while SMMC-7721 express neither of the CRF receptors.Thus stable transfection of CRFR1 or CRFR2 into SMMC-7721 cells was performed to study the effect of UCN.UCN was found to regulate two types of PLA2 enzymes,cPLA2 and iPLA2.The data showed that UCN raised cPLA2 expression but lowered iPLA2 expression.In addition,UCN was found to reduce iPLA2 transcription through acting on the certain region of its promoter.The results indicated that UCN promoted tumor cell migration by up-regulating cPLA2 expression via CRFR1 whereas it suppressed tumor cell migration by down-regulating iPLA2 expression via CRFR2.Taken together,our data suggest the dual roles for UCN in the hepatoma carcinoma cell migration,involving the regulation of both cPLA2 and iPLA2.Part ⅡCRF affects VSMC migration via regulating cPLA2Atherosclerosis is a kind of vascular inflammatory disease caused by a series of pathological processes,such as VSMC migration and proliferation.It is reported that PLA2 expression can contribute to VSMC migration and CRF receptor can regulate PLA2 expression.In this part,the role of CRF receptors in VSMC motility was studied and Sphingosine-1-phosphate(S1P)signaling pathway was found to take part in the regulation of cPLA2 induced by CRF.SIP is synthesized by sphingosine kinase 1 and 2(Sphkl and Sphk2)and binds to five G protein-coupled receptors designated S1PR1-5 to exert physiological functions.Similar to the results of part 1,activation of CRFR1 was found to promote cell migration and CRFR2 activation suppressed cell migration.Differently,CRFR1 activation was found to increase cPLA2 expression but decrease iPLA2 expression whereas CRFR2 activation was found to decrease cPLA2 expression but increase iPLA2 expression.In line with the regulation of cPLA2 and hence cell migration after the activation of CRFR1 or CRFR2,activation of CRFR1 regulated Sphkl expression oppositely to CRFR2.SIP release was also enhanced by CRFR1 activation,however,it was not influenced by activation by CRFR2.Furthermore,both the inhibitor of Sphk(dimethylsphingosine,DMS)and the selective inhibitor of S1PR3(CAY10444)abolished CRF-induced cPLA2 expression and hence cell migration.And CRF no longer promoted cell migration after the interference of S1PR3.These results indicated the pro-migratory role of CRFRl-Sphkl-S1P-S1PR3-cPLA2 signaling pathway in VSMC.Part Ⅲ UCN enhances TGF-β-mediated mitoinhibition of VSMC via counteracting TGF-β-induced cPLA2 expression and activationVSMC migration and proliferation has been demonstrated to participate in various cardiovascular diseases.It was showed above that CRF family and PLA2 family have an effect on VSMC migration.Some researchers have reported these two families’ role in VSMC proliferation separately.However,it is still not clear whether there is a connection between CRF family and PLA2 family in VSMC proliferation.Moreover,transforming growth factor-β(TGF-β)has been reported to show controversial effects on cell growth and growing evidence shows TGF-βcross-talk with multiple signaling pathway.Therefore,we investigated the regulation of VSMCs proliferation by UCN/TGF-β and whether cPLA2 was a link between their signaling pathways in this part.Our data showed that both UCN and TGF-βinhibited VSMC proliferation and an additive effect was observed when the cells were treated with UCN plus TGF-β.TGF-β increased the percentage of cells in G1-phase while UCN increased the cell percentage in G2-phase to inhibit VSMC proliferation.There existed a growth stimulative activity of TGF-β through activating cPLA2 and this signaling pathway could be blocked by UCN.UCN exerted its mitoinhibition role via decreasing cPLA2 expression and activation.These results indicated that UCN counteracted TGF-β-mediated cPLA2 expression and activation,thereby contributing to TGF-P-mediated mitoinhibition of VSMC.