Glucocorticoids with different chemical structures but similar glucocorticoid receptor potency regulate subsets of common and unique genes in human trabecular meshwork cellsReport as inadecuate




Glucocorticoids with different chemical structures but similar glucocorticoid receptor potency regulate subsets of common and unique genes in human trabecular meshwork cells - Download this document for free, or read online. Document in PDF available to download.

BMC Medical Genomics

, 2:58

First Online: 10 September 2009Received: 18 February 2009Accepted: 10 September 2009

Abstract

BackgroundIn addition to their well-documented ocular therapeutic effects, glucocorticoids GCs can cause sight-threatening side-effects including ocular hypertension presumably via morphological and biochemical changes in trabecular meshwork TM cells. In the present study, we directly compared the glucocorticoid receptor GR potency for dexamethasone DEX, fluocinolone acetonide FA and triamcinolone acetonide TA, examined the expression of known GRα and GRβ isoforms, and used gene expression microarrays to compare the effects of DEX, FA, and TA on the complete transcriptome in two primary human TM cell lines.

MethodsGR binding affinity for DEX, FA, and TA was measured by a cell-free competitive radio-labeled GR binding assay. GR-mediated transcriptional activity was assessed using the GeneBLAzer beta-lactamase reporter gene assay. Levels of GRα and GRβ isoforms were assessed by Western blot. Total RNA was extracted from TM 86 and TM 93 cells treated with 1 μM DEX, FA, or TA for 24 hr and used for microarray gene expression analysis. The microarray experiments were repeated three times. Differentially expressed genes were identified by Rosetta Resolver Gene Expression Analysis System.

ResultsThe 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 TA, respectively. All four GRα translational isoforms A-D were expressed in TM 86 and TM 93 total cell lysates, however, the C and D isoforms were more highly expressed relative to A and B. All four GRβ isoforms A-D were also detected in TM cells, although GRβ-D isoform expression was lower compared to that of the A, B, or C isoforms. Microarray analysis revealed 1,968 and 1,150 genes commonly regulated by DEX, FA, and TA in TM 86 and TM 93, respectively. These genes included RGC32, OCA2, ANGPTL7, MYOC, FKBP5, SAA1 and ZBTB16. In addition, each GC specifically regulated a unique set of genes in both TM cell lines. Using Ingenuity Pathway Analysis IPA software, analysis of the data from TM 86 cells showed that DEX significantly regulated transcripts associated with RNA post-transcriptional modifications, whereas FA and TA modulated genes involved in lipid metabolism and cell morphology, respectively. In TM 93 cells, DEX significantly regulated genes implicated in histone methylation, whereas FA and TA altered genes associated with cell cycle and cell adhesion, respectively.

ConclusionHuman trabecular meshwork cells in culture express all known GRα and GRβ translational isoforms, and GCs with similar potency but subtly different chemical structure are capable of regulating common and unique gene subsets and presumably biologic responses in these cells. These GC structure-dependent effects appear to be TM cell-lineage dependent.

Electronic supplementary materialThe online version of this article doi:10.1186-1755-8794-2-58 contains supplementary material, which is available to authorized users.

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Author: Alissar Nehmé - Edward K Lobenhofer - W Daniel Stamer - Jeffrey L Edelman

Source: https://link.springer.com/



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