Nrf2-dependent ARE activation in neural cells.

摘要

The antioxidant responsive element (ARE) is a cis -acting regulatory element of genes encoding phase II detoxification enzymes and antioxidant proteins, such as NAD(P)H:quinone oxidoreductase 1 (NQO1), glutathione S-transferases, and glutamate-cysteine ligase. Numerous studies demonstrated that NF-E2-related factor 2 (Nrf2) mediates the ARE-driven gene expression in hepatoma cell lines. This study was performed to investigate the molecular mechanism of ARE activation in neural cells.; Initial work on IMR-32 human neuroblastoma cells showed that tert-butylhydroquinone (tBHQ)-induced ARE activation is dependent on Nrf2. Overexpression of wild-type Nrf2 activated the ARE, and the tBHQ-induced ARE activation was inhibited by dominant negative Nrf2. In addition, a dramatic nuclear translocation of Nrf2 by tBHQ supports a role for Nrf2 in ARE activation in this cell line.; We also observed that tBHQ increased NQO1 protein without activation of Erk1/2, and a selective MEK inhibitor PD98059 did not block tBHQ-induced ARE activation. A selective phosphatidylinositol 3-kinase (PI3-kinase) inhibitor LY294002, however, inhibited the ARE activation. In support of this, we demonstrated; (1) constitutively active PI3-kinase selectively activated the ARE that was inhibited by LY294002, (2) LY294002 or wortmannin decreased tBHQ-induced Nrf2 nuclear translocation, and (3) ARE activation by constitutively active PI3-kinase was completely blocked by dominant negative Nrf2. These data suggest that ARE activation by tBHQ is dependent on PI3-kinase, not Erk1/2.; Moving into primary cultures, we found that Nrf2−/− astrocytes and neurons have decreased levels of both basal and inducible NQO1 gene expression compared to Nrf2+/+ counterparts. We also observed that Nrf2 protected primary astrocytes and neurons from oxidative stress. Supporting these observations, oligonucleotide microarray analysis revealed that Nrf2 coordinately upregulates the expression of many protective genes. The gene list includes detoxification enzymes, glutathione-related proteins, antioxidant proteins, NADPH producing enzymes, anti-inflammatory, growth factors, and calcium homeostasis proteins. Genes within these functional categories are critical to the maintenance and responsiveness of a cells defense system. Taken together, these observations imply that PI3-kinase-Nrf2 pathway regulates the ARE-driven gene expression in neural cells, and an orchestrated regulation of gene expression via the Nrf2-ARE pathway yields a synergistic protective effect against oxidative stress.