Tumor necrosis factor-induced modulation of glyoxalase I activities through phosphorylation by PKA results in cell death and is accompanied by the formation of a specific methylglyoxal-derived AGE

Abstract

Tumor necrosis factor (TNF) is a pleiotropic cytokine that plays a role in the host defense against microorganisms and bacterial pathogens and in the pathophysiology of various diseases (1, 2). Furthermore, TNF has potent antitumor and antimalignant cell effects. Depending on the cell type, cell death by TNF can occur by apoptosis or necrosis (3, 4). TNF-induced cell death in the murine fibrosarcoma cell line L929, the cell line used in this study, is characterized by a necrosis phenotype that is caspase-independent and does not involve DNA fragmentation, but instead requires the increased production of reactive oxygen species (ROS) in the mitochondria (5–7). Much effort has been directed at elucidating the molecular mechanisms of caspase-dependent cell death, but relatively little is yet known about the TNF-induced ROS-dependent cell death pathway.

To identify signaling molecules downstream of the receptor-proximal events that are involved in this cell death pathway, protein phosphorylation changes in L929 cells were examined by comparative two-dimensional (2D) gel electrophoresis after stimulation with TNF for 1.5 h (that is, just before cells start to die). Previous work has shown that TNF-induced increase in oncoprotein 18 (Op18, stathmin) phosphorylation is responsible for TNF-induced microtubule stabilization, which in turn promotes cell death (8). Now, we demonstrate that phosphorylation of glyoxalase I (lactoylglutathione lyase, EC 4.4.1.5) is a key step in TNF-induced cell death.

The glyoxalase system, consisting of the enzymes glyoxalase I (GLO1) and glyoxalase II (GLO2), is an integral component of cellular metabolism in mammalian systems (see review in ref. 9). Although the glyoxalase pathway was reported as early as 1913 and is ubiquitous in nature, its full biological function has never been elucidated. The work of Szent-Györgyi suggested that GLO1 and methylglyoxal (MG) were involved in the regulation of cellular growth, but a direct mechanistic link has yet to be identified (reviewed in ref. 10). A major function of the glyoxalase pathway is believed to be detoxification of α-ketoaldehydes, especially MG. MG is a cytotoxic metabolite produced primarily as a by-product of glycolysis through nonenzymatic phosphate elimination from the glycolytic pathway intermediates dihydroxyacetone phosphate and glyceraldehyde 3-phosphate (for a review on MG, see ref. 11). The glyoxalase system requires reduced glutathione (GSH) as a cofactor and catalyzes the conversion of MG to D-lactate. The substrate for GLO1 is the hemithioacetal formed through the nonenzymatic conjugation of MG with GSH. The product of the GLO1-catalyzed reaction is S-D-lactoylglutathione, which is then hydrolyzed by glyoxalase II to D-lactate; GSH is regenerated by the action of GLO2. D-Lactate is further metabolized to pyruvate in some mammalian tissues.

Increased expression of GLO1 occurs in diabetic patients and in some tumors, such as colon carcinoma (12), breast cancer (13), and prostate cancer (14). Recently, it has been shown that GLO1 is involved in resistance of human leukemia cells to antitumor agent-induced apoptosis (15). Under normal physiological conditions, most MG is bound to cellular proteins as adducts formed with Lys, Arg, and Cys residues (16, 17). Although the reaction with Cys is considered reversible, elevated concentrations of MG can lead to irreversible modifications of Lys and Arg residues through formation of advanced glycation end products (AGEs) (18). AGE formation is thought to contribute to several pathophysiological conditions, such as tissue damage after ischemia/reperfusion (19), to aging (20), and to the development of complications in diabetic patients such as blindness, neuropathy, and diabetic vascular diseases (21).

Acknowledgments

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