Spliceosome protein (SRp) regulation of glucocorticoid receptor isoforms and glucocorticoid response in human trabecular meshwork cells.
OBJECTIVE
Glaucoma is a leading cause of visual impairment and blindness, with elevated intraocular pressure (IOP) as a major causative risk factor. Glucocorticoid (GC) therapy causes morphologic and biochemical changes in the trabecular meshwork (TM), an ocular tissue involved in regulating IOP, which can lead to the development of glaucoma in susceptible individuals (steroid responders). Steroid responders comprise 40% of the general population and are at higher risk of developing glaucoma. In addition, a majority of glaucoma patients are steroid responders. Differential distribution of various isoforms of GC receptor (GR) may be responsible for this heterogeneity in the steroid response. The alternatively spliced GRβ isoform acts as dominant negative regulator of classical GRα transcriptional activity. mRNA splicing is mediated by spliceosomes, which include serine-arginine rich proteins (SRps). The purpose of this study was to determine whether specific SRps regulate levels of these isoforms and thereby GC response in TM cells.


METHODS
Quantitative RT-PCR, Western blot analysis, and immunocytochemistry were used to determine the differential expression of different SRps (SRp20, 30c, and 40) in human normal and glaucomatous TM cell strains. Bioinformatics was used to find putative binding sites for SRp20 and SRp40 on exon 9 of the GR gene. A peptide modulator of splicing (bombesin) and SRp expression vectors were used to modulate SRp levels and determine their effects on GRα/GRβ ratios as well as dexamethasone (DEX) responsiveness via GRE- luciferase reporter activity, fibronectin, and myocilin induction in TM cells.


RESULTS
SRp20, SRp30c, and SRp40 regulate GR splicing and the GC response in TM cells. Modulation of SRp levels altered the GRβ/α ratio that correlated with DEX responsiveness. Bombesin decreased SRp20; increased SRp30c, SRp40 levels, and GRβ/α ratio, and suppressed DEX response in TM cells.


CONCLUSIONS
Relative levels of SRp20, SRp30c, and SRp40 in TM cells control differential expression of the two alternatively spliced isoforms of the GR and thereby regulate GC responsiveness. Different levels and/or activities of these SRps may account for differential GC sensitivity among the normal and glaucoma populations.
Department of Cell Biology and Anatomy, University of North Texas Health Science Center at Fort Worth, Fort Worth, Texas 76107, USA. anjain@live.unthsc.edu
Ankur
A
Jain
Robert J
RJ
Wordinger
Thomas
T
Yorio
Abbot F
AF
Clark
D015153:Blotting, Western; D002478:Cells, Cultured; D004587:Electrophoresis, Agar Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005786:Gene Expression Regulation; D005787:Gene Frequency; D005901:Glaucoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D005938:Glucocorticoids / Q000494:pharmacology*; D006801:Humans; D007150:Immunohistochemistry; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics*; D016601:RNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D011965:Receptors, Glucocorticoid / Q000096:biosynthesis / Q000235:genetics*; D000068103:Serine-Arginine Splicing Factors; D017381:Spliceosomes / Q000235:genetics* / Q000378:metabolism; D014129:Trabecular Meshwork / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*
D005938:Glucocorticoids; D009687:Nuclear Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011965:Receptors, Glucocorticoid; C491388:SRSF3 protein, human; C411470:glucocorticoid receptor alpha; C121943:glucocorticoid receptor beta; D000068103:Serine-Arginine Splicing Factors