Tumor necrosis factor increases hepatocellular glutathione by transcriptional regulation of the heavy subunit chain of gamma-glutamylcysteine synthetase.
Tumor necrosis factor (TNF) is an inflammatory cytokine that causes cell injury by generation of oxidative stress. Since glutathione (GSH) is a key cellular antioxidant that detoxifies reactive oxygen species, the purpose of our work was to examine the regulation of cellular GSH, the expression of heavy subunit chain of gamma-glutamylcysteine synthetase (gamma-GCS-HS), and control of intracellular generation of reactive oxygen species in cultured rat hepatocytes treated with TNF. Exposure of cells to TNF (10,000 units/ml) resulted in depletion of cellular GSH levels (50-70%) and overproduction of hydrogen peroxide (2-3-fold) and lipid peroxidation. However, cells treated with lower doses of TNF (250-500 units/ml) exhibited increased levels of GSH (60-80% over control). TNF treatment increased (70-100%) the levels of gamma-GCS-HS mRNA, the catalytic subunit of the regulating enzyme in GSH biosynthesis. Furthermore, intact nuclei isolated from hepatocytes treated with TNF transcribed the gamma-GCS-HS gene to a greater extent than control cells, indicating that TNF regulates gamma-GCS-HS at the transcriptional level. The capacity to synthesize GSH de novo determined in cell-free extracts incubated with GSH precursors was greater (50-70%) in hepatocytes that were treated with TNF; however, the activity of GSH synthetase remained unaltered by TNF treatment indicating that TNF selectively increased the activity of gamma-GCS. Despite activation of nuclear factor-kappaB (NF-kappaB) by TNF, this transcription factor was not required for TNF-induced transcription of gamma-GCS-HS as revealed by deletion constructs of the gamma-GCS-HS promoter subcloned in a chloramphenicol acetyltransferase reporter vector and transfected into HepG2 cells. In contrast, a construct containing AP-1 like/metal response regulatory elements increased chloramphenicol acetyltransferase activity upon exposure to TNF. Thus, TNF increases hepatocellular GSH levels by transcriptional regulation of gamma-GCS-HS gene, probably through AP-1/metal response element-like binding site(s) in its promoter, which may constitute a protective mechanism in the control of oxidative stress induced by inflammatory cytokines.
Morales
A
A
Instituto de Investigaciones Biomédicas, Consejo Superior Investigaciones Científicas and Liver Unit and Servicio de Bioquímica, Department of Medicine, Hospital Clinic i Provincial, Universidad de Barcelona, Barcelona 08036, Spa.
García-Ruiz
C
C
Miranda
M
M
Marí
M
M
Colell
A
A
Ardite
E
E
Fernández-Checa
J C
JC
D000818:Animals; D002478:Cells, Cultured; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D005721:Glutamate-Cysteine Ligase / Q000235:genetics*; D005978:Glutathione / Q000378:metabolism*; D008099:Liver / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism; D010084:Oxidation-Reduction; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D012402:Rotenone / Q000494:pharmacology; D013804:Thenoyltrifluoroacetone / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D016328:NF-kappa B; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D014409:Tumor Necrosis Factor-alpha; D012402:Rotenone; D013804:Thenoyltrifluoroacetone; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione