Activation of Nrf2-Antioxidant Response Element Mediated Glutamate Cysteine Ligase Expression in Hepatoma Cell line by Homocysteine

Background: Homocysteine is a sulfur-containing amino acid which formed (mainly in the liver) during the metabolism of methionine. Prior studies indicated the important role of hyperhomocysteinemia in pathogenesis and progression of alcoholic liver disease, liver steatosis and cirrhosis. One of the most important mechanisms by which homocysteine promote the development of hepatic injury is oxidative stress. Transcription factor Nrf2-mediated antioxidant response, represents critical cellular defense mechanism that serves to maintain intracellular redox homeostasis and limit oxidative stress. Glutamate cysteine ligase catalytic (GCLc) is rate limiting enzyme in the synthesis of glutathione, an important endogenous antioxidant. Objectives: This study was conducted to investigate whether homocysteine induces the Nrf2 dependent expression of GCLc in hepatoma cell line (HepG2) and whether this induction is mediated by antioxidant response element (ARE) which present within its promoter. Materials and Methods: Glutathione (GSH) content was measured by flow cytometry. Using electro mobility shift assay (EMSA) and western blotting, ARE-binding activity of Nrf2 for GCLc was demonstrated. Real time RT-PCR and western blotting were performed to evaluate whether homocysteine was able to induce mRNA and protein expression of GCL. Results: Exposure of HepG2 cells to 50 mu MD/L homocysteine and western blotting of nuclear extracts revealed that Nrf2 is strongly stabilized and became detectable in nuclear extracts. EMSA demonstrated increased binding of Nrf2 to oligomers containing GCL promoter - specific ARE - binding site. A time-dependent increase in the gene and protein expression of GCL was observed. Additionally, GSH, which is prime scavenger of free radicals in cells, decreased initially. Elevation of GSH, following the initial decline, closely correlated with gene expression profile of GCLc, which is a rate-limiting enzyme in GSH synthesis. Conclusions: Altogether, we provide direct evidence that homocysteine activates Nrf2-mediated antioxidant response, which protects HepG2 cells from oxidative damage.