Crosstalk between the mitogen-activated protein kinase signaling and protease pathways in a model of glutamate-induced oxidative toxicity

The molecular mechanisms of alternative forms of cell death are becoming increasingly common, particularly in the case of neurodegenerative diseases. Proteases pathways, such as calpains and caspases, and their interactions with the mitogen-activated protein kinase (MAPK) signaling pathways have not been well-explored, particularly in the context of a pro-toxic role for the extracellular-related kinase (ERK). HT22 cells lack ionotropic glutamate receptors, but are still sensitive to high concentration of extracellular glutamate, which depletes glutathione and causes oxidative toxicity in an ERK-dependent manner. Using a purified clone of HT22 cells which were consistently responsive to glutamate-induced toxicity in an ERK-dependent manner, we have found that caspase-1- and -2-like inhibitors were effective at blocking cell death. Delayed addition of these inhibitors remained protective when added up to 3 hours after glutamate exposure. Both inhibitors decreased ERK phosphorylation at 9 hours following glutamate treatment, a timepoint where the second rise in ERK activation has been found previously. Contrary to previous reports using pharmacological calpain inhibitors, we have found that molecular inhibition of calpains by overexpression of calpastatin, the endogenous calpain inhbitor, was effective at delaying cell toxicity in glutamate treated HT22 cells. Evidence for calpain activation was demonstrated using two separate assay systems. In an in vitro enzymatic assay, calpains were found to be activated in the glutamate-induced toxicity of HT22 cells, and that this activation appears to be biphasic in nature, with the early activation MEK-independent and the late activation MEK-dependent. Furthermore, the endogenous calpain target PARP was cleaved to a fragment consistent with calpain-mediated cleavage at late timepoints following glutamate exposure. The appearance of this fragment, consistent with the late MEK-dependent activation, was also dependent on MEK activity. These results indicate for the first time that calpains are indeed activated and involved in cell death execution, that they can be at least in part regulated by MEK activity. Taken together, we have found that glutamate-induced oxidative stress in HT22 cells represents a form of cell death where caspase-1- or -2-like proteases may function upstream of ERK activation, but that ERK activation is required at least in part for a further, downstream calpain activation