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J. L. Edelman, A. Nehme, E. Lobenhofer, D. W. Stamer; Dexamethasone (DEX), Fluocinolone Acetonide (FA), and Triamcinolone Acetonide (TA) Generate Unique Patterns of Gene Expression in Human Trabecular Meshwork Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):5716.
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. In addition to their well-documented ocular therapeutic effects, glucocorticoids (GCs) can cause sight-threatening side-effects including ocular hypertension and cataract. Although the molecular mechanisms of steroid-induced ocular hypertension are not well-defined, GCs appear to increase aqueous humor outflow resistance and IOP via morphological and biochemical changes in trabecular meshwork (TM) cells. In the present study, we used Agilent microarray analysis to compare the effects of DEX, FA, and TA on the complete transcriptome in two primary human TM cell lines.
. Binding to the glucocorticoid receptor (GR) was assessed by a cell-free competitive radio-labeled binding assay (Cerep), and transactivation of GR was determined in HeLa cells by the GeneBLAzer beta-lactamase reporter gene assay (Invitrogen). Expression of GR translational isoforms was assessed by Western Blot. For Agilent whole human genome microarray studies, TM 86 and TM 93 cells were treated with 1 µM DEX, FA, or TA for 24 hr. Differentially expressed genes were identified using the Rosette Resolver analysis package, and identification of specific signaling pathways and biologic functions was performed using Ingenuity pathway Analysis (IPA) software.
. The GR binding affinity (IC50) for DEX, FA, and TA was 5.4, 2.0, and 1.5 nM, respectively. These values are similar to the GR transactivation EC50 of 3.0, 0.7, and 1.5 nM for DEX, FA, and TAA, respectively. All four GR translational isoforms (A-D) were detected in TM 86 and TM 93 total cell lysates, however the C and D isoforms were more highly expressed relative to A and B. Microarray analysis revealed 1,968 and 1,150 genes commonly regulated by DEX, FA, and TAA in TM 86 and TM 93, respectively. In addition, each GC modulated a unique set of genes in both TM cell lines. IPA analysis showed that in TM 86, DEX significantly regulated TGF-β signaling pathways, whereas FA and TAA significantly modulated FGF and Wnt/β-catenin signaling, respectively. In TM 93, Dex significantly affected PPAR/ RXR activation signaling, whereas FA and TAA regulated hypoxia and 14-3-3-mediated signaling, respectively.
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