Regulation of gamma-glutamylcysteine synthetase subunit gene expression in retinal Müller cells by oxidative stress.
OBJECTIVE
To study regulation of gamma-glutamylcysteine synthetase (GCS) heavy and light subunit gene expression in Müller cells under conditions of oxidative stress.


METHODS
Experiments were carried out with an SV40 transformed cell line (rMC-1) that exhibits the phenotype of rat retinal Müller cells. Endogenous glutathione levels were modified by treating cells with diethyl maleate (DEM), D,L-buthionine sulfoximine (BSO), or tert-butylhydroquinone (TBH). In other experiments, cells were grown in either high (28 mM) or normal (5.5 mM) glucose medium for 1 week to examine the effects of hyperglycemia. Cells were processed for reduced glutathione (GSH) measurement, RNA extraction, cell count, and, in some cases, lactate dehydrogenase activity. The steady state mRNA levels of GCS heavy and light subunits were measured by northern blot analysis using specific cDNA probes. Changes in mRNA levels were normalized to beta-actin or 18S rRNA.


RESULTS
Treatment with DEM for 30 minutes depleted cell GSH to 20% to 30% of the normal value. GSH content recovered completely 6 hours after returning to normal medium. BSO treatment for 12 hours followed by a medium change for 6 hours resulted in a cell GSH level that was 26% that of untreated cells. If cells were left in BSO for 18 hours, however, GSH levels were reduced to < 1%. Treatment with TBH for 12 hours led to a 77% increase in cellular GSH level. Treatment with DEM, TBH, or BSO for 18 hours led to a significant induction of the mRNA level of the GCS subunits, regardless of glucose concentration in the medium. Shorter BSO treatment exerted no effect. Prolonged hyperglycemia resulted in 30% lower GSH level, 55% lower GCS heavy subunit, and 30% lower GCS light subunit mRNA levels.


CONCLUSIONS
Oxidative stress induced the gene expression of GCS heavy and light subunits in Müller cells. The effect of BSO on mRNA levels correlated with the degree of GSH depletion. Prolonged hyperglycemia lowered GCS subunit mRNA and GSH levels.
Lu
S C
SC
Department of Medicine, USC Liver Disease Research Center, University of Southern California School of Medicine, Los Angeles, USA.
Bao
Y
Y
Huang
Z Z
ZZ
Sarthy
V P
VP
Kannan
R
R
D000818:Animals; D015152:Blotting, Northern; D019328:Buthionine Sulfoximine / Q000494:pharmacology; D002461:Cell Line, Transformed; D015971:Gene Expression Regulation, Enzymologic; D005721:Glutamate-Cysteine Ligase / Q000235:genetics*; D005978:Glutathione / Q000378:metabolism; D006873:Hydroquinones / Q000494:pharmacology; D006943:Hyperglycemia / Q000201:enzymology; D008298:Maleates / Q000494:pharmacology; D009457:Neuroglia / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D018384:Oxidative Stress; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D012160:Retina / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D013539:Simian virus 40
D006873:Hydroquinones; D008298:Maleates; D012333:RNA, Messenger; D019328:Buthionine Sulfoximine; C018855:2-tert-butylhydroquinone; D005721:Glutamate-Cysteine Ligase; C014476:diethyl maleate; D005978:Glutathione