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Adnan Dibas, Ming Jiang, Abbot F. Clark, Thomas Yorio; Generation Of Human Trabecular Meshwork Cell-lines With Fluorescently-labeled Glucocorticoid Alpha And Beta Receptors. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1506.
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© ARVO (1962-2015); The Authors (2016-present)
There are two human glucocorticoid receptor isoforms termed GRα and GRβ. While GRα undergoes hormone-dependent translocation from the cytoplasm to the nucleus, GRβ is primarily nuclear. GRβ acts as a dominant negative regulator of GC activity, a mechanism proposed to occur by dimerization. However, the proposed model of dimerization is not proven yet. Currently we report the production of the first human trabecular cell-line expressing both RFP-GRα and EGFP-GRβ that will allow characterization of trafficking as well as protein-protein interaction of both steroid receptors. Also, cell-lines with selective knock-down of GRβ receptor have been produced.
Transfection of RFP-GRα and EGFP-GRß was used to increase the expression of RFP-GRα and EGFP-GRβ in NTM5, a human trabecular meshwork cell-line. Cytosol and nuclear fractions were isolated followed by immunoprecipitation using specific GRα and GRβ antibodies. Western blotting and confocal microscopoy was used to measure the overexpression and nuclear accumulation of RFP-GRα and EGFP-GRβ in NTM5 cells treated with ethanol, dexamethasone (DEX), or RU486.
NTM5 cells transfected with RFP-GRα showed a clear cytosolic localization of receptor that underwent nuclear localization after dexamethasone treatment as judged by both western blot and confocal microscopy. However, while endogenous GRβ was primarily nuclear-bound, NTM5 cells transfected with EGFP-GRβ showed a predominant cytosolic localization of receptor that underwent nuclear localization after RU486 treatment as judged by both western blot and confocal microscopy. In cells expressing both RFP-GRα and EGFP-GRβ receptors, a clear co-localization of both receptors was observed in nucleus following DEX treatment.
We have, for the first time, produced human trabecular meshwork cell-lines expressing RFP-GRα receptor, EGFP- GRβ and both fluorescent receptors. RFP-GRα receptor behaves typically like the wild type GRα in terms of its cytosolic localization and shuttling to nucleus after DEX treatment. However, unlike the wild type GRß receptor that is nuclear-bound, the EGFP-GRβ receptor is primarily cytosolic but appears to translocate to nucleus following RU-486 treatment. Although it has been reported that GRβ exerts a negative effect on GRα 's action in the nuclear region, this is the first direct evidence for co-localization of RFP-GRα and EGFP-GRβ in nucleus of TM after DEX treatment. These human cell-lines are valuable tools for studying trafficking of GRα and GRβ and drug screening.
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