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 dexamethasone stimulates the remodeling of the apical junction (tight and adherens 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 regulatory subunit of phosphatidylinositol (PI) 3-kinase to regions of cell-cell contact, concurrently with the stimulation of TER. Expression of dominant-negative RasN17 abolished the dexamethasone stimulation in TER, whereas, dexamethasone induced the reorganization of tight junction and adherens junction proteins, ZO-1 and beta-catenin, as well as F-actin, to precise regions of cell-cell contact in a Ras-independent manner. Confocal microscopy revealed 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, respectively. Treatment with either of the PI 3-kinase inhibitors, wortmannin or LY294002, or the MEK inhibitor PD 098059, which prevents MAPK signaling, attenuated the dexamethasone stimulation of TER without affecting apical junction remodeling. Similar to dominant-negative RasN17, disruption of both Ras effector pathways using a combination of inhibitors abolished the glucocorticoid stimulation of TER. Thus, the glucocorticoiddependent remodeling of the apical junction and tight junction sealing can be uncoupled by their dependence on Ras and/or PI 3-kinase-dependent pathways, implicating a new role for Ras and PI 3-kinase cell signaling events in the steroid control of cell-cell interactions.
Woo
P L
PL
Department of Molecular and Cell Biology, The Cancer Research Laboratory, University of California, Berkeley, California 94720-3200, USA.
Ching
D
D
Guan
Y
Y
Firestone
G L
GL
D000199:Actins / Q000378:metabolism; D000818:Animals; D002867:Chromones / Q000494:pharmacology; D003907:Dexamethasone / Q000494:pharmacology; D017097:Electric Impedance; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D005938:Glucocorticoids / Q000494:pharmacology*; D007365:Intercellular Junctions / Q000187:drug effects / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D009025:Morpholines / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010750:Phosphoproteins / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D015398:Signal Transduction; D014162:Transfection; D014407:Tumor Cells, Cultured; D062826:Zonula Occludens-1 Protein; D018631:ras Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*
D000199:Actins; D002867:Chromones; D003907:Dexamethasone; D004791:Enzyme Inhibitors; D005938:Glucocorticoids; D008565:Membrane Proteins; D020928:Mitogen-Activated Protein Kinases; D009025:Morpholines; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010750:Phosphoproteins; D062826:Zonula Occludens-1 Protein; D018631:ras Proteins; C569044:Tjp1 protein, rat; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one