Effects And Potential Mechanisms Of Glucocorticoid On Cytoskeleton Reorganization In Human Ovarian Cancer Cell Line HO-8910

Glucocorticoids(GCs) play an important role in regulating cell growth,differentiation,apoptosis and adhesion in many cell types through genomic and nongenomic mechanism. The cytoskeleton in eukaryotic cells controls cell morphology. Reorganization of the actin cytoskeleton in response to several extracellular signaling, such as chemotherapy factor,hormone,growth factor and so on, controls several cell events including cell adhesion,motility,growth,migration, differentiation,apoptosis and gene expression,which are fundamental to embryogenesis, carcinogenesis, and wound healing.We reported in our previous study that glucocorticoids (GCs)-dexamethasone (Dex) led to pronounced morphologic alteration in a human ovarian cancer cell line HO-8910. In this study, we explored the effects of Dex on cytoskeleton reorganization in HO-8910 cells and tried to clarify its potential mechanisms.Rhodamine-phalloidin stain assay was used to label F-actin filaments;cell morphology were captured by using Olympus camera and actin filaments were visualized using confocal microscopy;the expression of ROCK II protein and tyrosine phosphorylated FAK protein were detected by Western blots;the nuclear translocation of NF-kB was observed using confocal microscopy, and the transcriptional activity of NF-kB was detected by reporter gene assay.In our study, we found that vehicle-treated cells show a smooth peripheral actin ring and homogeneous distribution of actin in the cytoplasm. 10 nM Dex treatment resulted in the rapid polymerization of actin to form stress fiber at 2 h, with the maximal effect at 8 h, and then actin depolymerized and shifted to cell cortical to form actin ring again at 24 h.To understand the mechanisms responsible for the effect of Dex on the actin reorganization, the role of Dex on the tyrosine phosphorylation of a cytoskeleton-associated protein: focal adhesion kinase (FAK) was first assessed. Theresult showed that Dex increased levels of tyrosine phosphorylation of FAK within 20 minutes without a change in protein expression. It indicates that the rapid nongenomic effect of DEX on actin assembly may due to the tyrosine phosphorylation of FAK.We have reported that Dex induced the expression of small GTPase RhoB in both mRNA and protein levels. In this study, we presented the evidence that over-expression of RhoB could induce actin depolymerization similar to that of Dex treatment for long time (12h). while inhibition RhoB expression by RNA interference reversed the Dex-induced actin reorganization, indicating that RhoB signaling is involved in the Dex-induced actin reorganization.As the downstream effector of Rho proteins, ROCK II protein was thought as an important regulator of actin cytoskeleton. So, we detected the effect of Dex on the expression of ROCK II both in mRNA and protein levels. The result showed that Dex can induce the expression of ROCK II in a time and dose dependent manner. It indicated that ROCK II may play a role in the actin reorganization induced by Dex. The following experiment that inhibiting ROCK II activity by Y-27632 could reverse Dex-induced actin reorganization approved our assumption.We also observed that physiological dose of Dex treatment for 20 h could elevate but not inhibit the activity of the transcription factor NF-kB by reporter gene assay, and over-expression or activation of RhoB signaling also enhanced the transcriptional activity of NF-kB by accelerating nuclear transportation of NF-kB. Furthermore, inhibiting NF-kB activity by PDTC, an inhibitor of NF-kB, could reverse Dex-induced actin reorganization, indicating that Dex may elevate the activity of NF-kB through RhoB to regulate actin reorganization.In summary, our observations suggest that the rapid nongenomic effect of Dex on actin assembly may due to tyrosine phosphorylation of the cytoskeleton-associated proteins FAK. Moreover, the enhanced RhoB levels and it's downstream protein ROCK II and NF-kB activity following longtime treatment with Dex imply a mechanism for long-term effects of glucocorticoids on actin cytoskeleton.