Requirement for Ras and phosphatidylinositol 3-kinase signaling uncouples the glucocorticoid-induced junctional organization and transepithelial electrical resistance in mammary tumor cells

In Con8 rat mammary epithelial tumor cells, the synthetic glucocorticoid de xamethasone stimulates the remodeling of the apical junction (tight and adh erens junctions) and the transepithelial electrical resistance (TER), which reflects tight junction sealing. Indirect immunofluorescence revealed that dexamethasone induced the recruitment of endogenous Ras and the p85 regula tory subunit of phosphatidylinositol (PI) I-kinase to regions of cell-cell contact, concurrently with the stimulation of TER. Expression of dominant-n egative RasN17 abolished the dexamethasone stimulation in TER, whereas, dex amethasone induced the reorganization of tight junction and adherens juncti on proteins, ZO-1 and beta-catenin, as well as F-actin, to precise regions of cell-cell contact in a Res-independent manner. Confocal microscopy revea led that RasN17 and the p85 regulatory subunit of PI 3-kinase co-localized with ZO-1 and F-actin at the tight junction and adherens junction, respecti vely. Treatment with either of the PI S-kinase inhibitors, wortmannin or LY 294002, or the MEK inhibitor PD 098059, which prevents MAPK signaling, atte nuated the dexamethasone stimulation of TER without affecting apical juncti on remodeling. Similar to dominant-negative RasN17, disruption of both Ras effector pathways using a combination of inhibitors abolished the glucocort icoid stimulation of TER. Thus, the glucocorticoiddependent remodeling of t he apical junction and tight junction sealing can be uncoupled by their dep endence on Ras and/or PI S-kinase-dependent pathways, implicating a new rol e for Ras and PI 3-kinase cell signaling events in the steroid control of c ell-cell interactions.