RELATIONSHIP OF SERINE THREONINE PHOSPHORYLATION/DEPHOSPHORYLATION SIGNALING TO GLUCOCORTICOID REGULATION OF TIGHT JUNCTION PERMEABILITY AND ZO-1 DISTRIBUTION IN NONTRANSFORMED MAMMARY EPITHELIAL-CELLS/

The synthetic glucocorticoid dexamethasone regulates tight junction pe rmeability resulting in an increased transepithelial electrical resist ance (TER) of cultured 31EG4 mammary epithelial cells. Inhibition of c ellular type 1 and type 2A protein phosphatase activity by okadaic aci d reduced the TER of dexamethasone-treated monolayers of 31EG4 cells t o basal levels within 24 h. Coincident with the increase in tight junc tion permeability, immunofluorescence revealed that okadaic acid cause d a partial cellular redistribution of the ZO-1 tight junction-associa ted protein. The potent glucocorticoid antagonist RU486 had no effect on TER or ZO-1 distribution, indicating that the effects of okadaic ac id are not a result of disrupting glucocorticoid receptor function. Im munoprecipitation of P-32-labeled cells and V8 protease peptide mappin g demonstrated that dexamethasone did not alter ZO-1 phosphorylation. However, consistent with the changes in TER, dexamethasone induced a 2 .3-fold stimulation in ZO-1 protein levels which was reduced to 73% of basal levels by okadaic acid. No effects on ZO-1 transcript levels we re observed. Monolayers grown in the presence of glucocorticoids had o nly 28% less junction density and 16.5% more Linear junction/cell, whi ch cannot account for the observed increases of TER and ZO-1 protein l evels. Taken together, our results have shown that a disruption of pho sphorylation/dephosphorylation activity overrides the glucocorticoid r egulation of tight junction permeability in 31EG4 mammary cells.