Singer K L, Stevenson B R, Woo P L, Firestone G L
Department of Molecular and Cell Biology, University of California at Berkeley 94720.
J Biol Chem. 1994 Jun 10;269(23):16108-15.
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.
合成糖皮质激素地塞米松可调节紧密连接通透性，导致培养的31EG4乳腺上皮细胞的跨上皮电阻（TER）增加。冈田酸抑制细胞1型和2A型蛋白磷酸酶活性，可在24小时内将地塞米松处理的31EG4细胞单层的TER降低至基础水平。与紧密连接通透性增加同时发生的是，免疫荧光显示冈田酸导致紧密连接相关蛋白ZO-1发生部分细胞重新分布。强效糖皮质激素拮抗剂RU486对TER或ZO-1分布没有影响，表明冈田酸的作用不是破坏糖皮质激素受体功能的结果。对32P标记细胞进行免疫沉淀和V8蛋白酶肽图谱分析表明，地塞米松不会改变ZO-1的磷酸化。然而，与TER的变化一致，地塞米松诱导ZO-1蛋白水平增加2.3倍，而冈田酸将其降低至基础水平的73%。未观察到对ZO-1转录水平的影响。在糖皮质激素存在下生长的单层细胞的连接密度仅低28%，每个细胞的线性连接多16.5%，这无法解释观察到的TER和ZO-1蛋白水平的增加。综上所述，我们的结果表明，磷酸化/去磷酸化活性的破坏优先于糖皮质激素对31EG4乳腺细胞紧密连接通透性的调节。
