Glutathione Peroxidase-1 Regulates Mitochondrial Function to Modulate Redox-dependent Cellular Responses

Glutathione peroxidase-1 (GPx-1) is a selenocysteine-containing enzyme that plays a major role in the reductive detoxification of peroxides in cells. In permanently transfected cells with approximate 2-fold overexpression of GPx-1, we found that intracellular accumulation of oxidants in response to exogenous hydrogen peroxide was diminished, as was epidermal growth factor receptor (EGFR)-mediated Akt activation in response to hydrogen peroxide or EGF stimulation. Knockdown of GPx-1 augmented EGFR-mediated Akt activation, whereas overexpression of catalase decreased Akt activation, suggesting that EGFR signaling is regulated by redox mechanisms. To determine whether mitochondrial oxidants played a role in these processes, cells were pretreated with a mitochondrial uncoupler prior to EGF stimulation. Inhibition of mitochondrial function attenuated EGF-mediated activation of Akt in control cells but had no additional effect in GPx-1-overexpressing cells, suggesting that GPx-1 overexpression decreased EGFR signaling by decreasing mitochondrial oxidants. Consistent with this finding, GPx-1 overexpression decreased global protein disulfide bond formation, which is dependent on mitochondrially produced oxidants. GPx-1 overexpression, in permanently transfected or adenovirus-treated cells, also caused overall mitochondrial dysfunction with a decrease in mitochondrial potential and a decrease in ATP production. GPx-1 overexpression also decreased EGF- and serum-mediated [³H]thymidine incorporation, indicating that alterations in GPx-1 can attenuate cell proliferation. Taken together, these data suggest that GPx-1 can modulate redox-dependent cellular responses by regulating mitochondrial function.Accumulation of reactive oxygen species (ROS),² such as superoxide anion and hydrogen peroxide, is thought to contribute to cellular damage, apoptosis, and cell death (1–3); however, ROS production is part of normal cellular metabolism, and evidence is accumulating that hydrogen peroxide, in particular, may function as a signaling molecule necessary for cell growth and survival (4–8). Superoxide is generated as a byproduct of mitochondrial respiration and by cellular redox enzymes, such as NADPH oxidase, that are stimulated through receptor-mediated mechanisms (9). Hydrogen peroxide is formed from the dismutation of superoxide, which occurs spontaneously or can be catalyzed by superoxide dismutase (10) or, alternatively, is produced by the two-electron enzymatic reduction of molecular oxygen by various oxidases, such as xanthine oxidase (11). Recent studies also suggest that hydrogen peroxide may be directly generated by receptor-ligand interactions (12). One mechanism by which hydrogen peroxide may modulate signal transduction is through the reversible oxidation of proteins at redox-active cysteines, including, for example, thiols in tyrosine kinase phosphatases. Oxidation and inactivation of phosphatases, such as PTEN, have been shown to promote the activity of the pro-growth and -survival kinase, Akt (13).Antioxidant enzymes, such as glutathione peroxidase, catalase, and peroxiredoxins, serve to eliminate hydrogen peroxide, thereby regulating cellular responses to this endogenous oxidant. GPx-1 is a selenoprotein and one of a family of peroxidases that reductively inactivate peroxides using glutathione as a source of reducing equivalents (14, 15). GPx-1, in particular, is a major intracellular antioxidant enzyme that is found in the cytoplasm and mitochondria of all cell types. In cell culture models as well as in genetic mouse models, GPx-1 overexpression is associated with enhanced protection against oxidative stress (16–19); however, GPx-1-overexpressing mice can become obese and insulin-resistant, and have attenuated insulin-mediated activation of Akt (20). Thus, to study how GPx-1 modulates the effects of cellular oxidants on cell signaling and cell growth, we analyzed cellular responses to hydrogen peroxide and EGF in permanently transfected cells overexpressing GPx-1.