Dissecting the molecular mechanism of glutathione-dependent regulation of cell proliferation and cell death.

Acute or chronic oxidative stress, accompanied by GSH depletion, has been frequently linked with many degenerative human diseases, such as atherosclerosis, myocardial infarction, stroke, and Parkinson’s disease. While the accumulation of reactive oxygen species is known being detrimental to cells and tissues, it has remained enigmatic if this is just a pleiotropic effect or whether a distinct signalling pathway is involved in oxidative stress-mediated cell death. Phospholipid hydroperoxide glutathione peroxidase (PHGPx) has emerged as an essential antioxidant enzyme in cells, efficiently detoxifying phospholipid hydroperoxides within biological membranes at the expense of GSH. To study PHGPx functions on life and death decisions in a cellular context, an inducible knockout model for PHGPx was established in mouse embryonic fibroblasts (MEFs), by using the 4- hydroxytamoxifen-inducible MerCreMer/loxP system. Targeted PHGPx abolition enabled to pinpoint a specific cell death inducing pathway, including activation of 15- lipoxygenase (15-LOX), lipid peroxidation, translocation of apoptosis inducing factor (AIF), and eventually cell death. The onset and execution of cell death could be efficiently prevented at each single step, either by the administration of the antioxidant α-tocopherol, by the highly specific 15-LOX inhibitor PD146176, or by siRNA-mediated knockdown of AIF. Since PHGPx is considered as the major GSHdependent enzyme, this cell death-inducing pathway may be a common signature in the etiology of many degenerative disorders, involving a detrimental accumulation of reactive oxygen species (ROS). To gain further insights into the role of GSH for cellular life and death decisions, a GSH-deficient in vitro model of γ-glutamylcysteine synthetase (γ-GCS) knockout cells was used. The glutamate/cystine (Cys)2 antiporter, system xc-, consists of two protein components, xCT light chain and 4F2 heavy chain, whereas xCT mediates transport specificity. The overexpression of xCT in γ-GCS knockout cells increased intra- and extracellular cysteine (Cys)-levels, rendering cell survival and proliferation independent from GSH. The elevated Cys levels are generated by the increased uptake of (Cys)2, intracellular reduction to Cys, and subsequent secretion to the extracellular space. These findings also imply that in the absence of GSH PHGPx accepts cysteine (Cys) as an alternative reducing equivalent when present in sufficient amounts. Since the maintenance of the cellular redox balance rather than GSH itself appeared to be essential for the maintenance of cell integrity, the Cys/(Cys)2-cycle has to be regarded as a distinct redox cycle of major importance. In conclusion, we propose that acute or chronic oxidative stress in cells and tissues triggers the herein described cell death pathway, including GSH depletion, impaired PHGPx enzyme activity, 15-LOX activation, lipid peroxidation, and AIF release. Whether up-regulation of xCT expression may represent a back-up system for low GSH levels, as observed under various pathophysiological conditions, certainly deserves further investigations