Tumour necrosis factor alpha induces co-ordinated activation of rat GSH synthetic enzymes via nuclear factor kB and activator protein-1

摘要

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 TNF alpha (tumour necrosis factor a) 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. TNFa induces the expression and recombinant promoter activities of GCLC, GCLM and GSS in H4IIE cells. TNFa induces NF-kappa B (nuclear factor kappa B) and AP-1 (activator protein 1) nuclear-binding activities. Blocking AP-1 with dominant negative c-Jun or NF-kappa B with I kappa BSR (I kappa B super-repressor, where I kappa B stands for inhibitory kappa B) lowered basal expression and inhibited the TNF alpha-mediated increase in mRNA levels of all three genes. While all three genes have multiple AP-1-binding sites, only GCLC has a NF-kappa B-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-kappa B also lowered basal c-Jun expression and blunted the TNFa-mediated increase in c-Jun mRNA levels. TNFa 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, TNFa no longer induced the GCLM promoter activity. In conclusion, both c-Jun and NF-kappa B are required for basal and TNFa-mediated induction of GSH synthetic enzymes in H4IIE cells. While NF-kappa B may exert a direct effect on the GCLC promoter, it induces the GCLM and GSS promoters indirectly via c-jun.