Antagonistic Regulation of Tight Junction Dynamics by Glucocorticoids and Transforming Growth Factor-βin Mouse Mammary Epithelial Cells

Abstract

The synthetic glucocorticoid, dexamethasone, stimulated the transepithelial electrical resistance and suppressed the DNA synthesis of 31EG4 nontransformed mouse mammary epithelial cells. The addition of transforming growth factor-beta 1 (TGF-beta) to mammary cells simultaneously with or up to 24 h after dexamethasone treatment prevented the steroid induction of transepithelial electrical resistance and stimulated the incorporation of [3H]thymidine. However, the TGF-beta inhibition of tight junction formation did not require de novo DNA synthesis. Confocal microscopy revealed that the organized immunostaining pattern of the tight junction protein, ZO-1, and F-actin at the cell periphery was disrupted by TGF-beta, resulting in disorganized and diffuse staining patterns throughout the cell. Western blot analysis demonstrated that TGF-beta did not alter the protein levels of ZO-1. In contrast to cells not treated or pretreated with steroid for up to 24 h, TGF-beta had no effect on cells pretreated with dexamethasone for 48 h. Transfection of chimeric reporter genes containing promoters responsive to either glucocorticoid or TGF-beta demonstrated that the mutual antagonism of tight junction dynamics by dexamethasone and TGF-beta occurs in the presence of intact signaling pathways. Taken together, our results establish for the first time that glucocorticoids and TGF-beta can antagonistically regulate tight junction formation in a nontransformed mammary cell line.

Role for TGF-β in Cyclosporine-Induced Modulation of Renal Epithelial Barrier Function

“…TGF-␤1 not only upregulates colonic epithelial barrier function but also curtails the effects of barrier-reducing cytokines such as IFN-␥, IL-4, and IL-10 (12). In contrast, TGF-␤1 prevented glucocorticoid-stimulated tight junction formation and reduced TER in murine mammary epithelial cells (18). Therefore, it is probable that TGF-␤1 may produce converse effects on epithelial barrier function in various tissues.…”

Selective glucocorticoid control of Rho kinase isoforms regulate cell–cell interactions

“…This time point was chosen because approximately 3-4 days of steroid treatment is needed for complete apical junction organization and formation of highly sealed tight junctions [6][7][8]20,23]. As shown in Figure 1, dexamethasone strongly induced the levels of ROCK2 protein, but had no effect on ROCK1 protein levels.…”

“…These junctions control intercellular adhesion and permeability properties and have a highly dynamic structure [1,5]. Using rodent Con8 mammary epithelial tumor cells, we have uncovered a glucocorticoid hormone mediated signaling cascade that stimulates adherens junction organization and induces tight junction sealing as well as polarization of cell monolayers [6][7][8][9]. We further established that the glucocorticoid down-regulation of the small GTPase RhoA is required for formation of organized intercellular junctions [10].…”

“…We have previously shown that tight junctions sealing of monolayers of Con8 mammary epithelial tumor cells can be stimulated by either treatment with glucocorticoids [6][7][8][9][10]20,23] or by expression of the Rnd3/RhoE antagonist of RhoA [11]. To determine whether Rho kinase activity plays a role in the stimulation of cell-cell interactions by either glucooorticoids or Rnd3/RhoE, the effects Y-27632, a relatively specific pharmacological inhibitor of Rho kinase activity [25], was analyzed on tight junction sealing.…”

“…Cells were cultured at 37°C in DMEM/F-12 medium with 10% calf serum in the presence of penicillin/streptomycin, and the transepithelial electrical resistance (TER) measurements were recorded using an EVOM Epithelial Voltohmmeter (World Precision Instruments) as describerd previously (6)(7)(8)(9). Raw TER values were normalized by subtracting the background resistance values from an empty filter and multiplying the difference by the area of the monolayer of cells.…”