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

Abstract

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 (H 2O2) 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 PI3Kβ and PI3Kγ, rather than PI3Kα and PI3Kδ, contribute to glutamate-induced H2O2 generation and cell death. PI3Kγ knockdown also inhibited glutamate-induced ERK1/2 phosphorylation, whereas transfection with the constitutively active form of human PI3Kγ (PI3Kγ-CAAX) triggered MEK1/2 and ERK1/2 phosphorylation and H 2O2 generation without glutamate exposure. This H 2O2 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 H 2O2 generation. Accordingly, cotransfection of cells with PDK1-KD and PI3Kγ-CAAX suppressed PI3Kγ-CAAX-triggered ERK1/2 phosphorylation and H2O2 generation. These results suggest that activation of PI3Kγ induces ERK1/2 phosphorylation, leading to extracellular H2O2 generation via PDK1 in oxidative glutamate toxicity. Glutamate induces extracellular H2O2 generation by NADPH oxidase 4 (Nox4), leading to cell death in neurons. Our experiments, using siRNAs and chemical inhibitors, showed the PI3Kγ-PDK1-MEK axis but not Akt1, mTOR, or S6K to be involved in the glutamate-induced H2O2 generation and the subsequent toxicity in neurons. Glutamate induces extracellular H2O2 generation by NADPH oxidase 4 (Nox4), leading to cell death in neurons. Our experiments, using siRNAs and chemical inhibitors, showed the PI3Kγ-PDK1-MEK axis but not Akt1, mTOR, or S6K to be involved in the glutamate-induced H2O2 generation and the subsequent toxicity in neurons.