Proteomic Analysis of Dexamethasone-Induced Changes in Human Trabecular Meshwork Cells

Abstract

Abstract: : Purpose: Chronic administration of steroids can produce dramatic rises in IOP resulting from reductions in outflow facility. To understand the cellular changes responsible for this increase in IOP, studies have examined how steroids alter the cellular signaling events that regulate transcription and gene expression in trabecular meshwork. However, one cannot assume a direct correlation between changes in mRNA and protein levels. The purpose of these studies was to evaluate how chronic exposure of human trabecular meshwork (TM) cells to dexamethasone (DEX) alters protein expression. Methods: Cultured human TM cells were maintained in the presence or absence of DEX (10-6 mol/L) for 6 days. Total protein from control and DEX-treated cells were labeled with light and heavy isotope-coded affinity tag (ICAT) reagents, respectively. Following trypsin digestion, proteomic analysis was then carried out by means of liquid chromatography/tandem mass spectrometry. A comparison of labeled peptide intensities was used to identify up- and downregulated proteins. Tandem mass spectrometry data was used for protein identification. Results: Proteomic analysis of DEX-treated cells identified twelve proteins upregulated over control levels, with the increase in protein expression ranging from 15 to 100%. Proteomic analysis of DEX-treated cells identified four proteins that were downregulated, with the decrease in protein expression ranging from 40 to 52%. Initial sequence analysis identified two of the upregulated proteins as RNA binding protein regulatory subunit and triose phosphate isomerase, and one of the downregulated proteins as tubulin beta-4 chain. Conclusions: These studies demonstrate that utilizing liquid chromatography/tandem mass spectrometry it is possible to analyze the entire proteome from trabecular meshwork cells for DEX-induced changes in protein expression. The protein identified from these studies should provide new insights into the cellular events responsible for steroid-induced elevation in IOP.

Keywords: trabecular meshwork • proteins encoded by disease genes • protein structure/function