Glucocorticoid Down-regulation of RhoA Is Required for the Steroid-induced Organization of the Junctional Complex and Tight Junction Formation in Rat Mammary Epithelial Tumor Cells

Abstract

In Con8 mammary epithelial tumor cells, we have documented previously that the synthetic glucocorticoid dexamethasone induces the reorganization of the tight junction and adherens junction (apical junction) and stimulates the monolayer transepithelial electrical resistance (TER), which is a reliable in vitro measurement of tight junction sealing. Western blots demonstrated that dexamethasone treatment down-regulated the level of the RhoA small GTPase prior to the stimulation of the monolayer TER. To test the role of RhoA in the steroid regulation of apical junction dynamics functionally, RhoA levels were altered in Con8 cells by transfection of either constitutively active (RhoA.V14) or dominant negative (RhoA.DN19) forms of RhoA. Ectopic expression of constitutively active RhoA disrupted the dexamethasone-stimulated localization of zonula occludens-1 and ␤-catenin to sites of cell-cell contact, inhibited tight junction sealing, and prevented the complete formation of the F-actin ring structure at the apical side of the cell monolayer. In a complementary manner, dominant negative RhoA caused a precocious organization of the tight junction, adherens junction, and the F-actin rings in the absence of steroid, whereas the monolayer TER remained glucocorticoid-responsive. Taken together, our results demonstrate that the glucocorticoid down-regulation of RhoA is a required step in the steroid signaling pathway which controls the organization of the apical junctional complex and the actin cytoskeleton in mammary epithelial cells.

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

“…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]. Our recent evidence shows that the functional ratio of RhoA to Rnd3/RhoE, a recently discovered natural RhoA antagonist, controls both adherens junction formation and tight junction sealing [11], suggesting that key downstream effectors of RhoA and Rnd3/RhoE signaling may be regulated in a steroiddependent manner.…”

“…Con8 cells are rat mammary epithelial tumor cells that have been previously described [9,10,20,21]. 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).…”

“…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.…”

Impaired tight junction sealing and precocious involution in mammary glands of PKN1 transgenic mice

“…This is of particular interest, because Rho activation has previously been linked to a perturbation of tight junction function in various experimental systems (Hopkins et al, 2003;Jou et al, 1998;Wojciak-Stothard et al, 2001). In Con8 cells, expression of constitutively activated RhoA prevented tight junction formation upon glucocorticoid treatment (Rubenstein et al, 2003) and expression of constitutively activated RhoA increased tight junction permeability in MDCK cells (Jou et al, 1998). However, Rho inhibition has also been demonstrated to perturb tight junction function, suggesting that the activity of these molecules is carefully balanced and highly dependent on cellular context (Matter and Balda, 2003).…”

“…A potential signaling pathway, the Rho family of small GTPases is linked by some tantalizing evidence to the regulation of tight junctions in the mammary gland and other epithelial and endothelial cells (Matter and Balda, 2003). In con8 mammary epithelial tumor cells, it has been demonstrated that glucocorticoid downregulation of RhoA is required for the organization of the apical junctional complex and tight junction formation (Rubenstein et al, 2003). Furthermore, Jou et al studying MDCK cells, found that both constitutively active and dominant-negative RhoA and Rac1 impaired tight junction functionality in a dose-dependent and reversible manner (Jou et al, 1998).…”