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Adnan Dibas, Yong Park, Abbot F. Clark, Thomas Yorio; Characterization Of Nuclear Import And Export Of GRα Following Dex Treatment In Human Trabecular Meshwork Cell-line. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1973.
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To characterize the role of the cytoskeleton and importins in steroid-induced glucocorticoid alpha (GRα) receptor nuclear import and export in human trabecular meshwork cells.
Stably-transfected RFP-GRα cell lines were developed. Nuclear accumulation of RFP-GRα in NTM5 cells treated with vehicle (ethanol), dexamethasone (DEX) or RU486 was measured in cytosolic and nuclear fractions by western blotting and laser confocal microscopy. Cytochalasin D, and colchicine were tested for their abilities to affect GRα-trafficking. Nuclear export of RFP-GRα was studied using confocal microscopy following DEX or RU486 removal. Importin and exportin proteins redistribution following DEX treatment was assessed by western blot.
NTM5 cells transfected with RFP-GRα showed a clear cytosolic localization of receptor that underwent translocation to the nucleus after DEX treatment. While neither cytochalasin D nor colchicine blocked DEX- or RU486-induced RFP-GRα nuclear translocation, both drugs blocked nuclear export of RFP-GRα following DEX and RU486 removal. Interestingly, both nuclear import and export of DEX-induced RFP-GRα was much faster than RU-486-induced nuclear shuttling. Importin-13 protein appears to translocate from nucleus to cytosol following DEX treatment.
RFP-GRα receptor behaves similarly to the wild type GRα with its cytosolic localization and shuttling to nucleus after DEX or RU486 treatment. DEX-induced nuclear import and export of RFP-GRα occurred at a much faster rate that RU-486-induced shuttling of RFP-GRα. It appears that the cytoskeleton network is needed for nuclear export but not import of RFP-GRα as only the disruption of the cytoskeleton blocked the nuclear export of RFP-GRα following DEX removal.
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