Abstract
Purpose:
We previously showed the requirement of microfilaments and microtubules in nuclear export of GR-alpha and in the current study, new mechanisms involved in steroid-induced GRα receptor nuclear export in human trabecular meshwork cells are characterized.
Methods:
Stably-transfected RFP-GRα cell lines were developed. Nuclear export of RFP-GRα in NTM5 cells treated with vehicle (ethanol), dexamethasone (DEX) was studied using confocal microscopy and fluorometry in isolated nuclear and cytosolic fractions following DEX removal. Leptomycin B (an inhibitor of exportin-1), MG132 and lactacystin (proteasomal inhibitors), and rotenone (ATP depletor), were tested for their abilities to affect GRα-trafficking.
Results:
NTM5 cells transfected with RFP-GRα showed a clear cytosolic localization of receptor that underwent translocation to the nucleus after DEX treatment. Surprisingly, while both proteasomal inhibitors (MG132 and lactacystin) had no effect of nuclear import, both suppressed nuclear export of RFP-GRα. Leptomycin while not affecting nuclear import of RFP-GRα, also attenuated nuclear export of RFP-GRα following DEX removal. Finally, both nuclear import and export of RFP-GRα were abolished by rotenone.
Conclusions:
While RFP-GRα nuclear import is cytoskeleton-independent, nuclear export appears to involve cytoskeletal network rearrangement. Exportin-1 appears to mediate nuclear export of RFP-GRα receptor following DEX removal. Interestingly, proteasomal inhibitors blockade of export suggest a novel role for ubiquitination in the export of GRα. Finally, nuclear shuttling of GRα to and from the nucleus is absolutely ATP-dependent.
Keywords: 735 trabecular meshwork •
450 chaperones •
487 corticosteroids