Browsing by Subject "TM cells"
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Item Identification of Actin Binding Proteins Associated with Cross-Linked Actin Networks(2006-12-01) Mills, Christy E.; Clark, Abbot F.; Yorio, Thomas; Wordinger, Robert J.Mills, Christy E., Identification of Actin Binding Proteins Associated with Cross-Lined Actin Networks. Master of Science (Pharmacology and Neuroscience), December 2006, 95 pp., 9 tables, 16 figures, references, 122 titles. Glucocorticoid therapy can leady to ocular hypertension and glaucoma. The purpose of this study is to examine mechanisms contributing to increased intraocular pressure using tissue culture models of steroid-induced ocular hypertension through identification of specific actin-binding proteins associated with cross-linked actin network (CLANs). Human trabecular meshwork ™ cells were cultured to confluence and treated with dexamethasone or vehicle for 14 days. Total RNA was extracted for gene expression analysis to confirm steroid-induced expression of actin binding proteins in human TM cells. Western blots confirmed expression of actin binding proteins and demonstrated the specificity of selected antibodies. Fluorescence microscopy of treated TM cells showed cytoskeleton rearrangements from linear actin stress fibers to cross-linked actin networks and the position of candidate proteins in relation to CLANs. Dexamethasone treatment of TM cells altered the expression of actin-associated proteins that may be important in the formation of CLANs and increased outflow resistance.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