Protein expression in human trabecular meshwork: downregulation of RhoGDI by dexamethasone in vitro

[摘要] Purpose: The characterization of thehuman trabecular meshwork (TM) proteome is a valuable step towardunderstanding its role under normal and glaucomatous conditions. Thisstudy uses proteomic techniques to investigate the set of proteinsexpressed in normal human TM and to identify those differentiallyexpressed in response to dexamethasone (DEX) treatment of TM cells(TMCs) in vitro. Methods: TM tissue (TMT) was isolatedfrom human donor eyes and pooled. Immortalized human TMCs were culturedwith or without DEX. Protein extracts from each were separated bytwo-dimensional electrophoresis (2-DE). Protein spots in TMT gel wereexcised, destained, and subjected to in-gel tryptic digestion andidentification with matrix-assisted laser desorption/ionizationtime-of-flight mass spectrometry (MALDI-TOF-MS). To determine thoseproteins whose expression patterns were affected by glucocorticoids,TMCs were treated with DEX and assayed by3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) dyeand 2-DE. A differentially expressed protein, RhoGDI, was validated byboth western blotting and immunocytological staining. Results: The comprehensive protein setincluded more than 850 protein spots from both the TMT and TMCs, asvisualized on 2-DE gel. Two-hundred-and-thirty-five spots weresuccessfully identified in the TMT gel. The functional categories ofthe identified proteins were mainly comprised of metabolic process,cell adhesion, anti-apoptosis, cell motility, carbohydrate metabolicprocess, signal transduction, and regulation of transcription. Duringthree days of DEX treatment, TMCs’ proliferation was inhibited in atime- and dose-dependent manner, as evidenced by MTT assay. In the 48 hcultured cell group, RhoGDI expression was reduced, as detected by2-DE, western blotting, and immunocytological staining. In contrast,the expression of RhoA, a target of RhoGDI, increased in response toDEX treatment. Conclusions: Using the classic proteomicworkflow, the main protein complement of normal human TMT was detected,identified, and categorized. The DEX inhibition of RhoGDI expression inTMCs was evidenced.

[学科分类] 生物化学/生物物理