Browsing by Subject "Glucocorticoid receptor β"
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Item Mechanisms of Glucocorticoid-induced Ocular Hypertension and Glaucoma(2017-12-01) Patel, Gaurang C.; Clark, Abbot F.; Pang, Iok-Hou; Mao, WeimingGlucocorticoid (GC)-induced ocular hypertension (OHT) is a serious side-effect of prolonged GC therapy that can lead to iatrogenic glaucoma and permanent vision loss. Patients with GCOHT have open iridocorneal angles and increased outflow resistance in the trabecular meshwork (TM) outflow pathway, similar to that seen in POAG. However, the molecular mechanisms responsible for GC-induced OHT are not entirely clear. GCs acts through glucocorticoid receptor (GR), and GR can regulate transcription both through transactivation and transrepression mechanisms. However, there is no evidence showing which of the two mechanisms play role in GC-induced OHT. In addition, the alternatively spliced isoform of glucocorticoid receptor GRβ acts as dominant negative regulator of GC activity, and it has been shown that overexpressing GRβ in TM cells inhibits GC-induced glaucomatous damage in TM cells. Therefore, we hypothesized that GR transactivation through the GRα isoform elevates IOP and that overexpression of GRβ decreases IOP upon GC treatment. We developed a mouse model that mimics many aspects of GC-OHT in humans to help us understand precise molecular mechanisms and etiology of GC-OHT, and we further demonstrated that myocilin does not play a major role in DEX-induced OHT in mice. We also provide the first evidence of the in vivo physiological role of GRβ in regulating GC-OHT and GC-mediated gene expression in the TM. Furthermore, we use GRdim transgenic mice (which have active transrepression and impaired transactivation) to determine the GR functions that regulate GC-OHT and provide first evidence of the role of GR transactivation in regulating GC-mediated gene expression in the TM and GC-OHT in mice. These studies all combine will significantly advance our knowledge in designing useful therapeutic approaches for GC-induced OHT and glaucoma.Item Role of Glucocorticoid Receptor β in Glucocorticoid-Induced Ocular Hypertension(2008-01-01) Jiang, Ming; Yorio, Thomas; Krishnamoorthy, Raghu R.; Dibas, AdnanJiang, Ming, Role of Glucocorticoid Receptor β in Glucocorticoid-Induced Ocular Hypertension, Master of Science. (Biomedical Sciences). Purpose: Previous studies from our laboratory have shown that glaucomatous trabecular meshwork ™ cells have a lower expression of glucocorticoid beta (GRβ) compared to normal TM cells. Overexpression of glucocorticoid receptor β was found to attenuate glucocorticoid responsiveness in glaucomatous TM cells. The purpose of this study was to determine if downregulating GRβ expression could increase glucocorticoid responsiveness in cultured transformed normal trabecular meshwork cells (NTM5 cells) and primary trabecular meshwork cell lines derived from normal individuals. Methods: siRNA oligonucleotide sequences specific for GRβ were designed, cloned into expression vectors and sequenced. TM cells were transfected with different siRNA constructs for GRβ, while a scrambled sequence served as a negative control. Immunoblot analyses were carried out in the transfected TM cells to determine knockdown of GRβ expression. In another set of experiments TM cells were transfected with either a GRβ siRNA construct or an empty vector (control) and co-transfected with a GRE-luciferase reporter and a SV40 promoter-β galactosidase construct to normalize for efficiency of transfection. Promoter-reporter assays were carried out to determine the effect of GRβ knockdown on glucocorticoid-mediated luciferase reporter activity. Results: Using MetafecteneTM rpo as the transfection reagent for siRNA, an appreciable 50% to 90% decrease in GRβ protein expression was observed 72 hours post-transfection in three GRβ siRNA constructs transfected in NTM5 cells, compared to the scrambled sequence transfected cells. NTM5 cells transfected with the empty vector (control) showed a 3.5 fold increase in luciferase activity after dexamethasone treatment, which was further increased (4 to 5 fold) by knocking down GRβ expression using a siRNA construct specific for GRβ. A similar trend was found using GRβ-siRNA#3 to transient knockdown GRβ in four primary TM cell lines – NTM210-05, NTM486-04, NTM174-04, and NTM153-00, respectively. To investigate to glucocorticoid responsiveness in cells permanently transfected for knockdown of GRβ, we made 6 clones from NTM5 cells. There were 10 to 20 fold induction in luciferase activity in GRβ siRNA stably transfected NTM5 cells, following glucocorticoid administration, respectively. Conclusion: In this study, using RNAi(s) which is specific for GRβ, we decreased GRβ expression in vitro in several different cell lines. Using luciferase assays it was found that the glucocorticoid responsiveness after knockdown of GRβ in vitro in trabecular meshwork cells was significantly enhanced. Decreased GRβ expression significantly increased glucocorticoid responsiveness not only in normal transformed human trabecular meshwork cells (NTM5 cells), but also in four primary trabecular meshwork ™ cell lines derived from normal individuals. These results further support the notion that GRβ negatively regulates responsiveness in TM cells. Keywords: glaucoma, glucocorticoid, glucocorticoid receptor beta, trabecular meshwork