Tyrosine phosphorylation of focal adhesion kinase and paxillin regulates the signaling mechanism of the rapid nongenomic action of dexamethasone on actin cytoskeleton

We have previously shown that dexamethasone (DEX) stimulates rapid polymeri zation of actin and stabilization of microfilaments in human endometrial ad enocarcinoma cells. As the content of total cellular actin and the concentr ation of the actin transcript did not change, we concluded that polymerizat ion of actin by glucocorticoids involves nongenomic mechanisms. However, th e signaling events by which the latter is achieved remain unknown. In the p resent study we evaluated whether tyrosine phosphorylation is required for the rapid, nongenomic DEX effect on actin assembly. In cells preincubated w ith the tyrosine kinase inhibitors, genistein or erbstatin analogue (EA), b efore adding DEX the G-/total actin ratio remained unchanged, whereas DEX i n the absence of both inhibitors reduced the ratio by 25%. In addition, whe n cells were preincubated with the protein tyrosine phosphatase inhibitor p ervanadate and subsequently incubated with DEX, the G-/total actin ratio wa s dramatically reduced by 65%. Furthermore, DEX increased transiently the l evels of tyrosine phosphorylation of focal adhesion kinase (FAK) and paxill in within 2 to 15 min, without a change in their expression levels. Pervana date mimicked this effect of DEX and enhanced tyrosine phosphorylation of b oth proteins. Ln addition, when cells were exposed to the anticytoskeletal agent cytochalasin B, the basal levels of tyrosine phosphorylation of both proteins were reduced. This effect was reversed by DEX, indicating that act in cytoskeleton integrity is required for the effect of DEX on tyrosine pho sphorylation of FAK and paxillin. Finally, we documented enhanced expressio n of the Ras-related GTP-binding protein Rho-B after long-term (12- and 24- hr) treatment with DEX whereas Rho-B levels remained unchanged after short- term (3- and 6-hr) treatment. Our observations demonstrate a novel mechanis m through which the rapid nongenomic effect of DEX on actin assembly requir es tyrosine phosphorylation of the cytoskeleton-associated proteins FAK and paxillin. We also propose that the DEX-induced actin polymerization may co nstitute a mechanism for transduction of signals resulting in tyrosine phos phorylation of FAK and paxillin. Moreover, the enhanced Rho-B levels observ ed after long-term treatment with DEX imply a mechanism for the well-descri bed, long-term effects of glucocorticoids on actin cytoskeleton.