Delayed mechanism for induction of γ-glutamylcysteine synthetase heavy subunit mRNA stability by oxidative stress involving p38 mitogen-activated protein kinase signaling

Expression of the γ-glutamylcysteine synthetase heavy subunit (γ-GCSh), which encodes the rate-limiting enzymes for glutathione biosynthesis, is regulated by many cytotoxic agents. Moreover, γ-GCSh mRNA expression is elevated in colorectal cancer, but how γ-GCSh expression is regulated is not completely understood. By using actinomycin D, which inhibits new RNA synthesis, we showed that treatment of human colorectal cancer cells with the prooxidant sulindac increased the half-life of γ-GCSh mRNA. By using a tetracycline-regulated γ-GCSh mRNA assay system, we systematically dissected the cis-acting sequence and trans-acting factors that regulate the stability of γ-GCSh by cytotoxic prooxidants. We demonstrated that a HuR recognition sequence, AUUUA, in the 3′-untranslated region is responsible for the decay of γ-GCSh mRNA. Oxidative stress enhanced cytoplasmic content of HuR. Overexpression of HuR by transfection stabilized γ-GCSh mRNA, whereas overexpression of a dominant-negative HuR mutant suppressed the induced stability. Furthermore, prooxidant-induced γ-GCSh mRNA stabilization and HuR binding were blocked by p38 mitogen-activated protein kinase inhibitors. We provide the first evidence that reduction-oxidation regulation of γ-GCSh expression, itself a reduction-oxidation sensor and regulator, is mediated at least in part by the p38 mitogen-activated protein kinase signaling through the HuR RNA-binding protein.