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.
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 beta-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.
Rubenstein
Nicola M
NM
Department of Molecular and Cell Biology and the Cancer Research Laboratory, University of California at Berkeley, 94720-3200, USA.
Guan
Yi
Y
Woo
Paul L
PL
Firestone
Gary L
GL
D000199:Actins / Q000378:metabolism; D022005:Adherens Junctions / Q000187:drug effects* / Q000502:physiology; D000818:Animals; D003907:Dexamethasone / Q000494:pharmacology; D015536:Down-Regulation; D017097:Electric Impedance; D004847:Epithelial Cells / Q000502:physiology; D005260:Female; D005938:Glucocorticoids / Q000494:pharmacology*; D008325:Mammary Neoplasms, Experimental / Q000378:metabolism* / Q000473:pathology; D051381:Rats; D019108:Tight Junctions / Q000187:drug effects* / Q000502:physiology; D014407:Tumor Cells, Cultured; D020742:rhoA GTP-Binding Protein / Q000032:analysis / Q000502:physiology*
D000199:Actins; D005938:Glucocorticoids; D003907:Dexamethasone; D020742:rhoA GTP-Binding Protein