PI3Kγ contributes to MEK1/2 activation in oxidative glutamate toxicity via PDK1

The role of phosphoinositide 3-kinase (PI3K) in oxidative glutamate toxicity is not clear. Here, we investigate its role in HT22 mouse hippocampal cells and primary cortical neuronal cultures, showing that inhibitors of PI3K, LY294002, and wortmannin suppress extracellular hydrogen peroxide (H2O2) generation and increase cell survival during glutamate toxicity in HT22 cells. The mitogen-activated protein kinase kinase (MEK) inhibitor U0126 also reduced glutamate-induced H2O2 generation and inhibited phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. LY294002 was seen to abolish phosphorylation of both ERK1/2 and Akt. A small interfering RNA (siRNA) study showed that PI3K and PI3K, rather than PI3K and PI3K, contribute to glutamate-induced H2O2 generation and cell death. PI3K knockdown also inhibited glutamate-induced ERK1/2 phosphorylation, whereas transfection with the constitutively active form of human PI3K (PI3K-CAAX) triggered MEK1/2 and ERK1/2 phosphorylation and H2O2 generation without glutamate exposure. This H2O2 generation was reduced by inhibition of MEK. Transfection with kinase-dead 3-phosphoinositide-dependent protein kinase 1 (PDK1-KD) reduced glutamate-induced ERK1/2 phosphorylation and H2O2 generation. Accordingly, cotransfection of cells with PDK1-KD and PI3K-CAAX suppressed PI3K-CAAX-triggered ERK1/2 phosphorylation and H2O2 generation. These results suggest that activation of PI3K induces ERK1/2 phosphorylation, leading to extracellular H2O2 generation via PDK1 in oxidative glutamate toxicity.