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M. Jiang, R. Krishnamoorthy, T. Yorio; Downregulation of Glucocorticoid Receptor Beta Increases Glucocorticoid Sensitivity in Trabecular Meshwork Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):157.
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© ARVO (1962-2015); The Authors (2016-present)
Previously studies from our laboratory have shown that glaucomatous trabecular meshwork (TM) cells have lower expression of glucocorticoid receptor beta (GR-beta), compared to normal TM cells. Overexpression of glucocorticoid receptor beta was found to attenuate glucocorticoid sensitivity in glaucomatous TM cells. The purpose of this study was to determine if downregulating GR-beta expression could increase glucocorticoid sensitivity in cultured transformed normal trabecular meshwork cells (NTM5 cells).
siRNA duplexes specific for GR beta were designed and custom synthesized. NTM5 cells were transfected with either GR beta siRNA duplexes or a non-target siRNA duplex (control) and cotransfected with a GRE-luciferase reporter and a SV40 promoter-beta galactosidase construct to normalize for efficiency of transfection. Promoter-reporter assays were carried out to determine the effect of GR beta knockdown on glucocorticoid-mediated luciferase reporter activity.
NTM5 cells transfected with the non-targeting siRNA duplex showed a 7- to 8- fold increase in luciferase activity after dexamethasone treatment. After knock down of GRbeta expression using siRNA duplexes specific for GR beta, there was 18- to 23- fold increase in luciferase activity after dexamethasone treatment, compared to non-targeting siRNA controls .
Decreased expression of glucocorticoid receptor beta in trabecular meshwork could be a key mechanism accounting for glucocorticoid sensitivity that is observed in most primary open angle glaucoma patients.
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