FIGURE 6.

CRAG activates antioxidative pathway via AP-1-mediated Srxn-1 activation. A, CRAG activates Srxn-1. Neuro2A cells were transfected with both Srxn-1-Luc and pRL-CMV together with either empty vector or indicated vector. Luciferase activities were assessed 48 h after the transfection. Error bars indicate ±S.D. (n = 3). *, p < 0.05 (Student's t test). B, both AP-1 sites in the Srxn-1 promoter are required for CRAG-dependent Srxn-1 activation. There are two AP-1 sites in the Srxn-1 promoter, and three different mutants were generated as indicated in the figure. The effect of CRAG expression on luciferase activity of Srxn-1 promoter mutants was assessed 48 h after the transfection. Error bars indicate ±S.D. (n = 4). *, p < 0.05; **, p < 0.01 (Student's t test). C, induction of Srxn-1 mRNA by CRAG. Neruro2A cells were transfected with either control vector or CRAG. At 48 h after transfection, quantitative RT-PCR was performed to quantify Srxn-1 mRNA in control and CRAG-transfected cells. D, CRAG reduces hydrogen peroxide-induced Prx-SO_2/3H. Neruro2A cells were transfected with either control vector or CRAG. At 48 h after transfection, cells were treated with 200 μm hydrogen peroxide for 1 h, following incubation in the fresh medium. Cells were harvested at the indicated time. Lysates of Neuro2A cells were immunoblotted with anti-Prx-SO_2/3H antibody. E, CRAG knockdown enhanced hydrogen peroxide-induced Prx-SO_2/3H accumulation. Neruro2A cells were transfected with either control or CRAG-specific siRNA. At 48 h after transfection, cells were treated with 100 μm hydrogen peroxide for 1 h, following incubation in the fresh medium. Cells were harvested at the indicated time. Lysates of Neuro2A cells were immunoblotted with anti-Prx-SO_2/3H antibody. F, a schematic model for the CRAG-mediated AP-1 signaling pathway.