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 permeability resulting in an increased transepithelial electrical resistance (TER) of cultured 31EG4 mammary epithelial cells. Inhibition of cellular type 1 and type 2A protein phosphatase activity by okadaic acid reduced the TER of dexamethasone-treated monolayers of 31EG4 cells to basal levels within 24 h. Coincident with the increase in tight junction permeability, immunofluorescence revealed that okadaic acid caused a partial cellular redistribution of the ZO-1 tight junction-associated protein. The potent glucocorticoid antagonist RU486 had no effect on TER or ZO-1 distribution, indicating that the effects of okadaic acid are not a result of disrupting glucocorticoid receptor function. Immunoprecipitation of 32P-labeled cells and V8 protease peptide mapping 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 were observed. Monolayers grown in the presence of glucocorticoids had only 28% less junction density and 16.5% more linear junction/cell, which cannot account for the observed increases of TER and ZO-1 protein levels. Taken together, our results have shown that a disruption of phosphorylation/dephosphorylation activity overrides the glucocorticoid regulation of tight junction permeability in 31EG4 mammary cells.
Singer
K L
KL
Department of Molecular and Cell Biology, University of California at Berkeley 94720.
Stevenson
B R
BR
Woo
P L
PL
Firestone
G L
GL
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000818:Animals; D015820:Cadherins / Q000235:genetics / Q000378:metabolism; D002451:Cell Compartmentation; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000502:physiology*; D003907:Dexamethasone / Q000494:pharmacology*; D017097:Electric Impedance; D004847:Epithelial Cells; D004848:Epithelium / Q000187:drug effects / Q000502:physiology; D004988:Ethers, Cyclic / Q000494:pharmacology; D007365:Intercellular Junctions / Q000502:physiology*; D007546:Isoquinolines / Q000494:pharmacology; D008321:Mammary Glands, Animal / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D015735:Mifepristone / Q000494:pharmacology; D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D010879:Piperazines / Q000494:pharmacology; D047428:Protein Kinase Inhibitors; D012333:RNA, Messenger / Q000032:analysis; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D062826:Zonula Occludens-1 Protein
D015820:Cadherins; D004988:Ethers, Cyclic; D007546:Isoquinolines; D008565:Membrane Proteins; D010750:Phosphoproteins; D010879:Piperazines; D047428:Protein Kinase Inhibitors; D012333:RNA, Messenger; C569043:Tjp1 protein, mouse; D062826:Zonula Occludens-1 Protein; D019319:Okadaic Acid; D015735:Mifepristone; D003907:Dexamethasone; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D010749:Phosphoprotein Phosphatases