title: Fluorescent protein-labeled glucocorticoid receptor alpha isoform trafficking in cultured human trabecular meshwork cells.
abstract: OBJECTIVE
  To characterize the roles of the cytoskeleton and heat shock protein 90 (HSP90) in steroid-induced glucocorticoid receptor alpha (GRα) translocation in cultured human trabecular meshwork cells.
  
  
  METHODS
  Stably transfected red fluorescent protein (RFP)-GRα NTM5 cell lines were developed. Nuclear localization 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, colchicine, and 17-demethoxygeldanamycin (17AAG, an HSP90 inhibitor), were tested for their abilities to affect GRα trafficking. Nuclear export of RFP-GRα was studied using confocal microscopy following DEX or RU486 removal.
  
  
  RESULTS
  NTM5 cells transfected with RFP-GRα showed a clear cytosolic localization of receptor that underwent nuclear localization after DEX treatment. RFP-GRα translocation was temperature sensitive, occurring at 37°C but not at room temperature. Neither cytochalasin D nor colchicine blocked DEX-induced or RU486-induced RFP-GRα nuclear translocation; however, 17AAG prevented DEX-induced RFP-GRα nuclear translocation. Both nuclear import and export of DEX-induced RFP-GRα were faster than RU-486-induced nuclear shuttling.
  
  
  CONCLUSIONS
  RFP-GRα receptor behaves similarly to the wild-type GRα with its cytosolic localization and shuttling to nucleus after DEX or RU486 treatment. HSP90 is required for nuclear translocation, but the disruption of cytoskeleton had no effect on nuclear translocation of RFP-GRα.
authors.affiliation: Department of Pharmacology and Neuroscience, University of North Texas Health Science Center at Fort Worth, 3500 Camp Bowie Boulevard, Fort Worth, TX 76107, USA. adnan.dibas@unthsc.edu
authors.forename: Adnan
authors.initials: A
authors.lastname: Dibas
authors.forename: Ming
authors.initials: M
authors.lastname: Jiang
authors.forename: Rafal
authors.initials: R
authors.lastname: Fudala
authors.forename: Ignacy
authors.initials: I
authors.lastname: Gryczynski
authors.forename: Zygmunt
authors.initials: Z
authors.lastname: Gryczynski
authors.forename: Abbot F
authors.initials: AF
authors.lastname: Clark
authors.forename: Thomas
authors.initials: T
authors.lastname: Yorio
mesh_terms: D016227:Benzoquinones / Q000494:pharmacology; D015153:Blotting, Western; D002478:Cells, Cultured; D003078:Colchicine / Q000494:pharmacology; D015638:Cytochalasin D / Q000494:pharmacology; D003907:Dexamethasone / Q000494:pharmacology; D000431:Ethanol / Q000494:pharmacology; D005938:Glucocorticoids / Q000494:pharmacology; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D006727:Hormone Antagonists / Q000494:pharmacology; D006801:Humans; D047029:Lactams, Macrocyclic / Q000494:pharmacology; D049408:Luminescent Agents; D008164:Luminescent Proteins; D018613:Microscopy, Confocal; D015735:Mifepristone / Q000494:pharmacology; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D020033:Protein Isoforms / Q000378:metabolism; D021381:Protein Transport; D011965:Receptors, Glucocorticoid / Q000187:drug effects / Q000378:metabolism*; D014129:Trabecular Meshwork / Q000166:cytology*; D014162:Transfection; D050257:Tubulin Modulators / Q000494:pharmacology; D000097573:Red Fluorescent Protein
chemical_list: D016227:Benzoquinones; D003078:Colchicine; D015638:Cytochalasin D; D003907:Dexamethasone; D000431:Ethanol; D005938:Glucocorticoids; D018841:HSP90 Heat-Shock Proteins; D006727:Hormone Antagonists; D047029:Lactams, Macrocyclic; D049408:Luminescent Agents; D008164:Luminescent Proteins; D015735:Mifepristone; D019384:Nucleic Acid Synthesis Inhibitors; D020033:Protein Isoforms; D011965:Receptors, Glucocorticoid; D050257:Tubulin Modulators; D000097573:Red Fluorescent Protein; C112765:tanespimycin