Tumour necrosis factor alpha induces co-ordinated activation of rat GSH synthetic enzymes via nuclear factor kappaB and activator protein-1.
GSH synthesis occurs via two enzymatic steps catalysed by GCL [glutamate-cysteine ligase, made up of GCLC (GCL catalytic subunit), and GCLM (GCL modifier subunit)] and GSS (GSH synthetase). Co-ordinated up-regulation of GCL and GSS further enhances GSH synthetic capacity. The present study examined whether TNFalpha (tumour necrosis factor alpha) influences the expression of rat GSH synthetic enzymes. To facilitate transcriptional studies of the rat GCLM, we cloned its 1.8 kb 5'-flanking region. TNFalpha induces the expression and recombinant promoter activities of GCLC, GCLM and GSS in H4IIE cells. TNFalpha induces NF-kappaB (nuclear factor kappaB) and AP-1 (activator protein 1) nuclear-binding activities. Blocking AP-1 with dominant negative c-Jun or NF-kappaB with IkappaBSR (IkappaB super-repressor, where IkappaB stands for inhibitory kappaB) lowered basal expression and inhibited the TNFalpha-mediated increase in mRNA levels of all three genes. While all three genes have multiple AP-1-binding sites, only GCLC has a NF-kappaB-binding site. Overexpression with p50 or p65 increased c-Jun mRNA levels, c-Jun-dependent promoter activity and the promoter activity of GCLM and GSS. Blocking NF-kappaB also lowered basal c-Jun expression and blunted the TNFalpha-mediated increase in c-Jun mRNA levels. TNFalpha treatment resulted in increased c-Jun and Nrf2 (nuclear factor erythroid 2-related factor 2) nuclear binding to the antioxidant response element of the rat GCLM and if this was prevented, TNFalpha no longer induced the GCLM promoter activity. In conclusion, both c-Jun and NF-kappaB are required for basal and TNFalpha-mediated induction of GSH synthetic enzymes in H4IIE cells. While NF-kappaB may exert a direct effect on the GCLC promoter, it induces the GCLM and GSS promoters indirectly via c-Jun.
Yang
Heping
H
Division of Gastroenterology and Liver Diseases, University of Southern California Research Center for Liver Diseases, Keck School of Medicine USC, Los Angeles, CA 90033, USA.
Magilnick
Nathaniel
N
Ou
Xiaopeng
X
Lu
Shelly C
SC
D024506:5' Flanking Region / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D045744:Cell Line, Tumor; D017353:Gene Deletion; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D005721:Glutamate-Cysteine Ligase / Q000235:genetics; D005978:Glutathione / Q000096:biosynthesis*; D005981:Glutathione Synthase / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D051267:NF-E2-Related Factor 2 / Q000378:metabolism; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D016755:Proto-Oncogene Proteins c-jun / Q000235:genetics / Q000378:metabolism; D051381:Rats; D020218:Response Elements / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D018808:Transcription Factor AP-1 / Q000378:metabolism*; D024363:Transcription Initiation Site; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D051267:NF-E2-Related Factor 2; D016328:NF-kappa B; D016755:Proto-Oncogene Proteins c-jun; D018808:Transcription Factor AP-1; D014409:Tumor Necrosis Factor-alpha; D005721:Glutamate-Cysteine Ligase; D005981:Glutathione Synthase; D005978:Glutathione