Protein expression in human trabecular meshwork: downregulation of RhoGDI by dexamethasone in vitro.
OBJECTIVE
The characterization of the human trabecular meshwork (TM) proteome is a valuable step toward understanding its role under normal and glaucomatous conditions. This study uses proteomic techniques to investigate the set of proteins expressed in normal human TM and to identify those differentially expressed in response to dexamethasone (DEX) treatment of TM cells (TMCs) in vitro.


METHODS
TM tissue (TMT) was isolated from human donor eyes and pooled. Immortalized human TMCs were cultured with or without DEX. Protein extracts from each were separated by two-dimensional electrophoresis (2-DE). Protein spots in TMT gel were excised, destained, and subjected to in-gel tryptic digestion and identification with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). To determine those proteins whose expression patterns were affected by glucocorticoids, TMCs were treated with DEX and assayed by 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) dye and 2-DE. A differentially expressed protein, RhoGDI, was validated by both western blotting and immunocytological staining.


RESULTS
The comprehensive protein set included more than 850 protein spots from both the TMT and TMCs, as visualized on 2-DE gel. Two-hundred-and-thirty-five spots were successfully identified in the TMT gel. The functional categories of the identified proteins were mainly comprised of metabolic process, cell adhesion, anti-apoptosis, cell motility, carbohydrate metabolic process, signal transduction, and regulation of transcription. During three days of DEX treatment, TMCs' proliferation was inhibited in a time- and dose-dependent manner, as evidenced by MTT assay. In the 48 h cultured cell group, RhoGDI expression was reduced, as detected by 2-DE, western blotting, and immunocytological staining. In contrast, the expression of RhoA, a target of RhoGDI, increased in response to DEX treatment.


CONCLUSIONS
Using the classic proteomic workflow, the main protein complement of normal human TMT was detected, identified, and categorized. The DEX inhibition of RhoGDI expression in TMCs was evidenced.
Yu
Minbin
M
Zhongshan Ophthalmic Center, State Key Laboratory of Ophthalmology, Sun Yat-sen University, Guangzhou, P.R. China.
Sun
Jing
J
Peng
Wei
W
Chen
Ziyan
Z
Lin
Xianchai
X
Liu
Xuyang
X
Li
Mingtao
M
Wu
Kaili
K
D000328:Adult; D003907:Dexamethasone / Q000494:pharmacology*; D015536:Down-Regulation / Q000187:drug effects*; D015180:Electrophoresis, Gel, Two-Dimensional; D005136:Eye Proteins / Q000378:metabolism*; D020730:Guanine Nucleotide Dissociation Inhibitors / Q000378:metabolism*; D006801:Humans; D013997:Time Factors; D014129:Trabecular Meshwork / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D055815:Young Adult; D062948:rho Guanine Nucleotide Dissociation Inhibitor alpha; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
C569638:ARHGDIA protein, human; D005136:Eye Proteins; D020730:Guanine Nucleotide Dissociation Inhibitors; D062948:rho Guanine Nucleotide Dissociation Inhibitor alpha; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors; D003907:Dexamethasone; D020742:rhoA GTP-Binding Protein