Phosphorylation of Xanthine Dehydrogenase/Oxidase in Hypoxia

Abstract

The enzyme xanthine oxidase (XO) has been implicated in the pathogenesis of several disease processes, such as ischemia-reperfusion injury, because of its ability to generate reactive oxygen species. The expression of XO and its precursor xanthine dehydrogenase (XDH) is regulated at pre-and posttranslational levels by agents such as lipopolysaccharide and hypoxia. Posttranslational modification of the protein, for example through thiol oxidation or proteolysis, has been shown to be important in converting XDH to XO. The possibility of posttranslational modification of XDH/XO through phosphorylation has not been adequately investigated in mammalian cells, and studies have reported conflicting results. The present report demonstrates that XDH/XO is phosphorylated in rat pulmonary microvascular endothelial cells (RPMEC) and that phosphorylation is greatly increased (ϳ50-fold) in response to acute hypoxia (4 h). XDH/XO phosphorylation appears to be mediated, at least in part, by casein kinase II and p38 kinase as inhibitors of these kinases partially prevent XDH/XO phosphorylation. In addition, the results indicate that p38 kinase, a stress-activated kinase, becomes activated in response to hypoxia (an ϳ4-fold increase after 1 h of exposure of RPMEC to hypoxia) further supporting a role for this kinase in hypoxia-stimulated XDH/XO phosphorylation. Finally, hypoxia-induced XDH/XO phosphorylation is accompanied by a 2-fold increase in XDH/XO activity, which is prevented by inhibitors of phosphorylation. In summary, this study shows that XDH/XO is phosphorylated in hypoxic RPMEC through a mechanism involving p38 kinase and casein kinase II and that phosphorylation is necessary for hypoxia-induced enzymatic activation.Xanthine dehydrogenase is the rate-limiting step in the catabolism of purines, where it catalyzes the conversion of hypoxanthine to xanthine and xanthine to uric acid. In this reaction, XDH 1 utilizes NAD ϩ preferentially as the electron acceptor. However, when XDH is converted to XO, the preferred electron acceptor becomes molecular oxygen resulting in the formation of superoxide and hydrogen peroxide. This generation of reactive oxygen species is thought to be the basis of XDH/XO involvement in various pathological conditions such as ischemia-reperfusion injury. Reversible conversion of XDH to XO can occur after the oxidation of eight cysteine residues in the molecule into four cystines by agents such as pyrimidines or oxidized glutathione (1, 2). This conversion may be reversed upon the addition of reducing agents such as dithiothreitol. XDH can also be converted into XO irreversibly through proteolysis (3). Experimental proteolysis by trypsin has allowed the identification of three different parts of the molecule: a 20-kDa N-terminal fragment, a 40-kDa flavin-binding fragment, and an 80-kDa molybdopterin-binding fragment, all of which remain attached after proteolysis (3). It is believed that both reversible and irreversible conversion of XDH to XO are due to conformational changes in the molecule that...

Sequential activation of JAKs, STATs and xanthine dehydrogenase/oxidase by hypoxia in lung microvascular endothelial cells

“…We found that hypoxia-mediated XDH/XO activation is dependent on the JAK/ STAT pathway in primary cultures of LMVEC. We demonstrated that the XDH/XO activity significantly increased by hypoxia, consistent with the increased phosphorylation or activation of XDH/XO in response to hypoxia in other cells (Terada et al, 1992;Dupont et al, 1992;Kayyali et al, 2001;Mervaala et al, 2001;Terada et al, 1997;Poss et al, 1996;Sohn et al, 2003;Kang et al, 2006). Interestingly, our data demonstrated that XDH/XO activation induced by could be inhibited by either depletion of IL6 using IL6 antibodies, pretreatment with the JAK2 inhibitor AG490 or transient expression of the JAK-STAT pathway blocker SOCS3 and their inhibitory effect on the XDH/XO activation was at a similar magnitude.…”

“…These data suggest that, in addition to the IL6-JAK-STAT pathway, other signaling pathways may also be involved in the activation of XDH/XO by hypoxia in LMVEC. Consistent with this possibility, it has been demonstrated that XDH/XO phosphorylation is partially mediated by p38 kinase and casein kinase II (Kayyali et al, 2001). Therefore, similar to STAT activation, XDH/XO activation by hypoxia in LMVEC is activated by multiple signal transduction pathways.…”

“…It has been demonstrated that hypoxia activates XDH/XO, an important source of reactive oxygen species (ROS) (Terada et al, 1992;Dupont et al, 1992;Kayyali et al, 2001;Mervaala et al, 2001;Terada et al, 1997;Poss et al, 1996;Sohn et al, 2003;Kang et al, 2006). However, the molecular mechanism underlying the hypoxia-mediated activation of XDH/XO remains largely unknown.…”

“…It has also been demonstrated that hypoxia activates the MAPK and PI3K pathways (Eguchi et al, 2007;Jin et al, 2007;Kayyali et al, 2001). To further dissect the pathway mediating STAT activation by hypoxia, we determined whether MAPK and PI3K were involved.…”

“…The XDH/XO activity was measured using a fluorimetric assay as described (Kayyali et al, 2001(Kayyali et al, , 2003Beckman et al, 1989). Cells were collected and sonicated in 50 mM sodium phosphate (pH 7.4), 1.5 mg/ml DTT and 1 × protease inhibitor mixture 3 (Calbiochem).…”