Figure 4.

Corticotropin releasing factor receptor 2 signaling increases para-cellular permeability and alteration of adherens junctions and tight junctions in spontaneously differentiated Caco-2 cells. A: The trans-cellular flux of dextran-FITC (4 kDa) was measured by spectrofluorimetry. Caco-2 cells were cultured 21 d after they reached confluence. After 5 h treatment or not (untreated cells) with 100 nmol/L Ucn3, dextran-FITC was added at the apical compartment. Then the green fluorescence incorporated in the cell monolayer was measured after 5, 10, 20, 30 and 40 min. The release of fluorescence in the basal compartment is associated to trans-cellular permeability. Ucn3 treatment seems to increase transcellular transport in well-differentiated Caco-2 cells. Two-way ANOVA, P < 0.001. B: Twenty-one days differentiated Caco-2 cells were pre-incubated with or without A2b (1 μmol/L) overnight before addition or not of Ucn3 (100 nmol/L). Intercellular junction integrity was evaluated by measuring transepithelial electrical resistance (TEER). Values are means of 5 different experiments ± SEM. ^aP < 0.05 vs the three other groups, ^fP < 0.01 vs the three other groups and ^b,c,d,eP < 0.001 vs the three other groups; C: Twenty-one days differentiated Caco-2 cells were treated or not with 100 nmol/L Ucn3 before immunostaining for E-cadherin (upper panels), occludin (middle panels) and ZO-1 (lower panels). Bar is 20 μm. Images were acquired by confocal microscopy on a LEICA TCS/SPE (objective × 100). Ucn3 treatment induces a time-dependent alteration of AJ and TJ protein localization.