AIMTodeterminetheroleofcorticotropinreleasingfactorreceptor(CRF2)inepithelialpermeabilityandenterocytecelldifferentiation.METHODSForthispurpose,weusedratSpragueDawleyandvariouscoloncarcinomacelllines(SW620,HCT8R,HT-29andCaco-2celllines).ExpressionofCRF2proteinwasanalyzedbyfluorescentimmunolabelinginnormalratcolonandthenbywesternblotindissociatedcolonicepithelialcellsandinthelysatesofcoloncarcinomacelllinesorduringtheearlydifferentiationofHT-29cells(tenfirstdays).ToassesstheimpactofCRF2signalingoncoloniccelldifferentiation,HT-29andCaco-2cellswereexposedtoUrocortin3recombinantproteins(Ucn3,100nmol/L).Insomeexperiments,cellswerepre-exposedtotheastressin2b(A2b)aCRF2antagonistinordertoinhibittheactionofUcn3.Intestinalcelldifferentiationwasfirstanalyzedbyfunctionalassays:thetrans-cellularpermeabilityandthepara-cellularpermeabilityweredeterminedbyDextran-FITCintakeandmeasureofthetransepithelialelectricalresistancerespectively.Morphologicalmodificationsassociatedtoepithelialdysfunctionwereanalyzedbyconfocalmicroscopyafterfluorescentlabelingofactin(phaloidin-TRITC)andintercellularadhesionproteinssuchasE-cadherin,p120ctn,occludinandZO-1.Theestablishmentofmatureadherensjunctions(AJ)wasmonitoredbyfollowingthedistributionofAJproteinsinlipidraftfractions,afterseparationofcelllysatesonsucrosegradients.Finally,themRNAandtheproteinexpressionlevelsofcharacteristicmarkersofintestinalepithelialcell(IEC)differentiationsuchasthetranscriptionalfactorkrüppel-likefactor4(KLF4)orthedipeptidylpeptidaseIV(DPPIV)wereperformedbyRT-PCRandwesternblotrespectively.ThespecificactivitiesofDPPIVandalkalinephosphatase(AP)enzymesweredeterminedbyacolorimetricmethod.RESULTSCRF2proteinispreferentiallyexpressedinundifferentiatedepithelialcellsfromthecryptsofcolonandinhumancoloncarcinoma
