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A. Jain, R. J. Wordinger, T. Yorio, A. F. Clark; Sr Splicing Proteins: Key Players in Regulating Gr/grβ Ratio and Glucocorticoid Response in Trabecular Meshwork Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3216.
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© ARVO (1962-2015); The Authors (2016-present)
Glucocorticoids (GCs) are commonly used anti-inflammatory agents, but can be associated with increased intraocular pressure that can lead to iatrogenic glaucoma. However, there is heterogeneity in this GC-induced ocular hypertension, with a subset of the population being "steroid responders". The GC receptor (GR) exists as two alternatively spliced isoforms, GRα and GRβ. GRβ has a unique C-terminal 9β exon and acts as a dominant negative regulator of GC activity. Knocking down GRβ enhances GC responsiveness in trabecular meshwork (TM) cells. Alternative mRNA splicing is mediated by spliceosomes, which include SR proteins (SRps). The purpose of our study was to determine whether SR proteins in TM regulate the differential expression of these two alternatively spliced isoforms of GR (GRα and GRβ).
Putative binding sites for SRp20 and SRp40 in GR exon 9 that may regulate alternate splicing of GR were identified using Splicing Rainbow software. Quantitative RT-PCR and western immunoblotting were used to determine the differential expression of SRp20, SRp30c, and SRp40 in GTM3 and NTM5 cell lines. Over and under-expression of SRp20, SRp30c, and SRp40 via treatment with bombesin or by transfection with expression vectors or siRNAs were used to determine effects on GRα/GRβ ratios and dexamethasone (100 nM) response in TM cells. Dex responsiveness was determined using a GRE-luciferase reporter vector as well as western immunoblotting for fibronectin and myocilin.
Modulating the levels of SRp20, SRp30c, and SRp40 altered GRα/GRβ ratios in TM cells. Dex responsiveness correlated with the GRβ/GRα ratio. The bombesin peptide increased SRp30c expression, increased the GRβ/GRα ratio, and decreased Dex responsiveness in GTM-3 cells.
Relative levels of SRp20, SRp30c, and SRp40 in TM cells control differential expression of the two alternatively spliced isoforms of GR and thereby regulate GC responsiveness. Different levels and/or activities of these SRps may account for differential GC sensitivity among the population.
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