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M. Jiang, R. Krishnamoorthy, T. Yorio; Downregulation of Glucocorticoid Receptor Beta (GR Beta) Expression Promotes Glucocorticoid Responsiveness in Transformed Normal Trabecular Meshwork Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):674.
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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 (GRß) compared to normal TM cells. Overexpression of glucocorticoid receptor ß was found to attenuate glucocorticoid sensitivity in glaucomatous TM cells. The pupose of this study was to determine if downregulating GRß expression could increase glucocorticoid responsiveness in cultured transformed normal trabecular meshwork cells (NTM5 cells).
siRNA oligonucleotide sequences specific for GRß were designed, cloned into expression vectors and sequenced. NTM5 cells were transfected with different siRNA constructs for GRß, while a scrambled sequence served as a negative control. Immunoblot analyses were carried out in the transfected NTM5 cells to determine knockdown of GRß expression. Another set of NTM5 cells were transfected with either a GRß siRNA contruct or an empty vector (control) and cotransfected with a GRE-luciferase reporter and a SV40 promoter-ß galactosidase construct to normalize for efficiency of transfection. Promoter-reporter assays were carried out to determine the effect of GRß knockdown on glucocorticoid-mediated luciferase reporter activity.
An appreciable 2-fold decrease in GRß protein expression was observed 72 hours post-transfection in GRß siRNA transfected NTM5 cells, compared to the scrambled sequence transfected cells. NTM5 cells transfected with the empty vector (control) showed a 3- to 4-fold increase in luciferase activity after dexamethasone treatment, which was further increased by knocking down GRß expression using siRNA construct specific for GRß.
Decreased expression of glucocorticoid receptor ß in trabecular meshwork could be a key mechanism responsible for increased glucocorticoid responsiveness that is seen in most primary open angle glaucoma patients.
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