Requirement for Ras and Phosphatidylinositol 3-Kinase Signaling Uncouples the Glucocorticoid-induced Junctional Organization and Transepithelial Electrical Resistance in Mammary Tumor Cells

Abstract

In Con8 rat mammary epithelial tumor cells, the synthetic glucocorticoid dexamethasone stimulates the remodeling of the apical junction (tight and adherens junctions) and the transepithelial electrical resistance (TER), which reflects tight junction sealing. Indirect immunofluorescence revealed that dexamethasone induced the recruitment of endogenous Ras and the p85 regulatory subunit of phosphatidylinositol (PI) 3-kinase to regions of cell-cell contact, concurrently with the stimulation of TER. Expression of dominant-negative RasN17 abolished the dexamethasone stimulation in TER, whereas, dexamethasone induced the reorganization of tight junction and adherens junction proteins, ZO-1 and ␤-catenin, as well as F-actin, to precise regions of cell-cell contact in a Ras-independent manner. Confocal microscopy revealed that RasN17 and the p85 regulatory subunit of PI 3-kinase co-localized with ZO-1 and F-actin at the tight junction and adherens junction, respectively. Treatment with either of the PI 3-kinase inhibitors, wortmannin or LY294002, or the MEK inhibitor PD 098059, which prevents MAPK signaling, attenuated the dexamethasone stimulation of TER without affecting apical junction remodeling. Similar to dominantnegative RasN17, disruption of both Ras effector pathways using a combination of inhibitors abolished the glucocorticoid stimulation of TER. Thus, the glucocorticoiddependent remodeling of the apical junction and tight junction sealing can be uncoupled by their dependence on Ras and/or PI 3-kinase-dependent pathways, implicating a new role for Ras and PI 3-kinase cell signaling events in the steroid control of cell-cell interactions.

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

“…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].…”

“…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.…”

“…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.…”

Pemphigus Vulgaris IgG and Methylprednisolone Exhibit Reciprocal Effects on Keratinocytes

“…Recently, it has been elucidated that PV IgG causes formation of Dsg 3-depleted desmosomes (51) and that this process is associated with increased phosphorylation of this desmosomal cadherin (52). On the other hand, the use of the synthetic corticosteroid agent dexamethasone, which can exhibit differential regulation of cadherin expression and reorganize adherens junction proteins in various cell types (53)(54)(55), has been shown to reverse the proinflammatory cytokine-induced abnormalities in the expression of E-cadherin (E-Cad), ␤-cat, and ␥-Cat in human bronchial epithelial cells (56). Therefore, we hypothesized that PV IgG and CH exhibit acantholytic and anti-acantholytic effects, respectively, by producing reciprocal effects on the expression/function of keratinocyte adhesion molecules that mediate intercellular cohesion in the epidermis.…”