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Adnan Dibas, Abbot F Clark, Thomas Yorio; Importin-2 and RAN GTP control nuclear import of glucocorticoid alpha (GRα) receptor following DEX treatment in human trabecular meshwork cell-line (NTM5). Invest. Ophthalmol. Vis. Sci. 2014;55(13):2904.
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Importins are proteins that carry out nuclear import of many proteins. However, their role in steroid-induced nuclear import of GRα has yet to be characterized in human trabecular meshwork. In the current study, we investigated the role of importins-2, 7, 13 and RAN-GTP in mediating dexamethasone-induced RFP-GRα nuclear trafficking.
Stably-transfected RFP-GRα cell lines were developed. siRNAs against importin-2, importin-7, importin-13, and RAN-GTP were transfected in cells and cells were allowed to reach confluency. Nuclear import of RFP-GRα in NTM5 cells treated with vehicle (ethanol) or dexamethasone (DEX) was studied using confocal microscopy and western blot in isolated nuclear and cytosolic fractions. Leptomycin B (an inhibitor of exportin-1), was tested for its ability to block RFP-GRα-nuclear export following DEX removal.
NTM5 cells transfected with siRNAs against importin-2 and RAN-GTP significantly inhibited DEX-induced nuclear import of RFP-GRα receptor that accumulated in the cytosol after DEX treatment. In contrast, both siRNAs against importin-7 and 13 failed to block DEX-induced nuclear import of RFP-GRα. Leptomycin while not affecting nuclear import of RFP-GRα, attenuated nuclear export of RFP-GRα following DEX removal.
While DEX-induced RFP-GRα nuclear import is importin-2 and RAN-GTP-dependent, neither importin-7 nor importin-13 appear involved in such pathway. However, exportin-1 appears to mediate nuclear export of RFP-GRα receptor following DEX removal.
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