Fluorescent protein-labeled glucocorticoid receptor alpha isoform trafficking in cultured human trabecular meshwork cells.

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Abstract

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. 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. 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. 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.

Original languageEnglish
Pages (from-to)2938-2950
Number of pages13
JournalInvestigative ophthalmology & visual science
Volume53
Issue number6
DOIs
StatePublished - 1 Jan 2012

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Trabecular Meshwork
Protein Isoforms
Dexamethasone
Proteins
tanespimycin
HSP90 Heat-Shock Proteins
Cell Nucleus Active Transport
Confocal Microscopy
Cytochalasin D
Colchicine
Cytoskeleton
glucocorticoid receptor alpha
red fluorescent protein
Mifepristone
Temperature
Cytoplasmic and Nuclear Receptors

Cite this

@article{2493bafcb3f244fda0bf6a279859c894,
title = "Fluorescent protein-labeled glucocorticoid receptor alpha isoform trafficking in cultured human trabecular meshwork cells.",
abstract = "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. 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. 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. 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.",
author = "Adnan Dibas and Ming Jiang and Rafal Fudala and Ignacy Gryczynski and Zygmunt Gryczynski and Abbot Clark and Thomas Yorio",
year = "2012",
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language = "English",
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TY - JOUR

T1 - Fluorescent protein-labeled glucocorticoid receptor alpha isoform trafficking in cultured human trabecular meshwork cells.

AU - Dibas, Adnan

AU - Jiang, Ming

AU - Fudala, Rafal

AU - Gryczynski, Ignacy

AU - Gryczynski, Zygmunt

AU - Clark, Abbot

AU - Yorio, Thomas

PY - 2012/1/1

Y1 - 2012/1/1

N2 - 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. 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. 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. 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.

AB - 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. 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. 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. 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.

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U2 - 10.1167/iovs.11-8331

DO - 10.1167/iovs.11-8331

M3 - Article

VL - 53

SP - 2938

EP - 2950

JO - Investigative Ophthalmology and Visual Science

JF - Investigative Ophthalmology and Visual Science

SN - 0146-0404

IS - 6

ER -