Phosphorylation of Xanthine Dehydrogenase/Oxidase in Hypoxia*

Usamah S. Kayyali, Cameron Donaldson, Hailu Huang, Raja Abdelnour, and Paul M. HassounDagger

From the Department of Medicine, Pulmonary and Critical Care Division, Tupper Research Institute, New England Medical Center and Tufts University School of Medicine, Boston, Massachusetts 02111

Received for publication, November 6, 2000, and in revised form, January 17, 2001




    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (~50-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 ~4-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.




    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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, XDH1 utilizes NAD+ 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 reduce its affinity for NAD+.

The notion that XDH/XO is a phosphoprotein has been the subject of controversy. Initial reports of the presence of a phosphoserine in milk xanthine oxidase concluded that, in addition to two phosphates on the FAD and one phosphate in the molybdopterin cofactor, there was a phosphate covalently bound to a serine residue of the protein (4, 5). Later reports indicated that no covalently attached phosphate to milk XDH/XO could be detected by NMR or chemical analysis (6, 7). However, using biochemical analysis of immunoprecipitated XDH from chick embryo hepatocytes, Schieber and Edmondson (8) demonstrated the incorporation of labeled phosphate into XDH and estimated 3 mol of phosphate/mol of protein. Although the latter report described the covalent phosphorylation of serine residues in chicken xanthine dehydrogenase (8), the phosphorylation of mammalian XDH remains to be demonstrated conclusively.

In this report, evidence is provided that rat XDH/XO can be phosphorylated in pulmonary microvascular endothelial cells. Moreover, the phosphorylation of XDH is greatly increased in response to hypoxia, which is known to induce XDH/XO (9). The increase in XDH/XO phosphorylation in hypoxia is accompanied by an increase in XDH/XO activity. Hypoxia-induced XDH/XO phosphorylation is partially blocked by inhibitors of p38 kinase. Furthermore, our data indicate that p38 is significantly activated in hypoxia. Hence, p38 kinase is postulated to be involved in the phosphorylation of XDH/XO in hypoxia.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials and Reagents-- RPMI 1640, phosphate-free Dulbecco's modified Eagle's medium, fetal bovine serum, dialyzed fetal bovine serum, penicillin G potassium, streptomycin, fungizone, and glutamine were obtained from Life Technologies, Inc. Protein kinase and phosphatase inhibitors were obtained from Calbiochem. [32P] Orthophosphate was obtained from PerkinElmer Life Sciences. EDTA, Tris, and dithiothreitol were from Sigma. Rabbit anti-XO antibody was from LabVision, Fremont, CA, and anti p38 antibodies were from New England Biolabs, Beverly, MA. Horseradish peroxidase-conjugated anti-rabbit IgG and the chemiluminescent horseradish peroxidase substrate SuperSignal were from Pierce.

Cell Culture and Exposure to Hypoxia-- Rat pulmonary microvascular endothelial cells (RPMEC) were a gift from Dr. Una Ryan (Avant Immunotherapeutics, Needham, MA) and were cultured as previously described (10). For hypoxic exposure, cells were placed in humidified airtight incubation chambers (Billups-Rothenberg, Del Mar, CA) and gassed with 3% O2, 5% CO2, and balance N2. The hypoxic chambers were kept in a 37 °C incubator for the duration of the experiment. Normoxic cells were kept in a tissue culture incubator maintained at 5% CO2 and 37 °C.

Xanthine Oxidase Activity Measurements-- The activities of xanthine dehydrogenase and xanthine oxidase in response to different treatments were assayed using a slight modification of a fluorimetric assay that measures both xanthine oxidase and xanthine dehydrogenase activity (11). The principle of the assay involves the conversion of pterin into the fluorescent product isoxanthopterin. The rate of product formation with oxygen as the electron acceptor represents the activity of xanthine oxidase, whereas the combined activities of xanthine oxidase and xanthine dehydrogenase are measured with methylene blue as the electron acceptor. In brief, cells were washed once in phosphate-buffered saline and then scraped off the plate in 50 mM sodium phosphate (pH 7.4), 1.5 mg/ml dithiothreitol, and 1× protease inhibitor mixture 3 (Calbiochem). The cells were sonicated for 5 s and centrifuged at 10,000 × g for 5 min. The supernatant was collected and assayed immediately or stored at -80 °C overnight. For testing the effect of phosphorylation on XDH or XO activity, lysates were prepared in 50 mM Tris-HCl (pH 7.4), 1.5 mg/ml dithiothreitol, and 1× protease inhibitor mixture 3, and aliquots were treated with calf intestine alkaline phosphatase (New England Biolabs, Beverly, MA) before the assay.

SDS-PAGE, Western Blotting, Phosphoprotein Analysis, and Immunoprecipitation-- Aliquots from the cell lysates prepared as described above were assayed for protein using the Bradford protein assay (12) and then diluted with 2× Laemmli loading buffer for SDS-PAGE (13). Equal amounts of protein were then loaded in each well of 4-20% Tris/glycine gels. After electrophoresis for 90 min at 125 V constant voltage, the gel was blotted onto an Immobilon-P membrane by electrophoretic transfer at 25 V constant voltage overnight. The membrane was then washed, blocked with 5% milk, and probed with antibodies against xanthine oxidase (LabVision). The immunoreactive bands were visualized using a secondary antibody conjugated to horseradish peroxidase and a chemiluminescent substrate according to the manufacturer's instructions (SuperSignal, Pierce). The intensity of the bands was quantified using a Molecular Dynamics densitometer and ImageQuant software.

For phosphoprotein analysis RPMEC, which had been passaged the day before, were incubated with 32PO4 at 0.5 mCi/ml in phosphate-free medium. The cells were exposed to normoxia, hypoxia, and/or kinase inhibitors for 1-18 h. At the end of the exposure, the cells were washed and lysed (0.3 ml of radioimmunoprecipitation assay lysis buffer/3.5-cm dish). XDH/XO was then immunoprecipitated with rabbit anti-XDH/XO antibody as described (14) and run on SDS-PAGE. In brief, cell lysates were first precleared with protein A-Sepharose. Then they were incubated with anti-XDH/XO antibody at 4 °C overnight. Next, protein A-Sepharose was added to the samples, which were then rocked for 2 h at 4 °C. The complex was washed 10 times with 0.5 ml of radioimmunoprecipitation assay buffer, and the specifically bound XDH/XO was eluted from the complex by heating in 2× Laemmli sample buffer followed by centrifugation. The samples were then run on SDS-PAGE as described above. The gels were dried on filter paper or electrophoretically transferred onto Immobilon-P membranes. Phosphorylation of XDH/XO was detected by autoradiography and quantified by either phosphorimaging or by scanning and then quantifying the bands on film.

Statistical Analysis-- The values plotted in all figures are means, and the error bars reflect the standard deviation from the mean. Statistical analysis was carried out using SigmaStat (Jandel Scientific). Student's t test was used to determine whether differences between normoxic and hypoxic enzyme activity were significant in Fig. 1. When comparisons between multiple groups were carried out, one-way analysis of variance was employed. Statistical significance was considered at p >0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Hypoxia Stimulates XDH/XO Phosphorylation and Enzymatic Activity in RPMEC-- Previous reports have indicated that exposure of endothelial cells to hypoxia increased XDH/XO activity (9, 15, 16) and mRNA expression within a few hours of exposure (9, 16). An increase in protein expression has been reported in vivo after 24 h of exposure to hypobaric hypoxia (17) and in vitro after exposure to 48 h of hypoxia (16). The following experiments were designed to examine the effect of acute exposure (4 h) of cells to hypoxia on posttranslational modification of the XDH/XO protein. RPMEC were exposed to hypoxia for 4 h before XDH/XO activity was assayed. Because the changes in XDH and XO activities always occurred in parallel, total XDH/XO activity is reported for all experiments (nmol of isoxanthopterin formed/min/mg of protein). A significant increase in XDH/XO activity was observed by 4 h of exposure of RPMEC to hypoxia (Fig. 1), a finding that is consistent with an earlier report (9). Because the increase in the XDH/XO enzyme activity in hypoxia was not due to a change in the amount of XDH/XO protein in hypoxia as determined by Northern blotting (data not shown), posttranslational modification of the protein was investigated as a mechanism of enzyme activation. Upon examining the primary sequence of XDH/XO, potential phosphorylation sites for several protein kinases were identified. Therefore, the possibility of XDH/XO phosphorylation was examined in cultured cells. RPMEC were metabolically labeled with 32P, and XDH/XO was immunoprecipitated at various time points (see "Experimental Procedures"). Bands that corresponded to XDH/XO incorporated a significant amount of 32P, indicating that XDH/XO was covalently phosphorylated (Fig. 2, Normoxia).



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Fig. 1.   Exposing RPMEC to hypoxia for 4 h causes a 2.3-fold increase in total (XDH/XO) activity. * indicates p < 0.05 versus normoxia.



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Fig. 2.   XDH/XO is phosphorylated in RPMEC, and its phosphorylation is significantly increased in hypoxia. Cells were incubated with [32P]orthophosphate (1 mCi/ml) for 4 h. XDH/XO was immunoprecipitated with a rabbit anti-XDH/XO antibody followed by SDS-PAGE, Western blotting, and autoradiography. Left panel represents a 1-h film exposure showing significant phosphorylation of XDH/XO in hypoxia. A longer film exposure (24 h) was needed to demonstrate phosphorylation of XDH/XO from normoxic cells (right panel).

The possibility that the observed increase in XDH/XO activity after 4 h of exposure to hypoxia could be due to posttranslational modification of the protein, specifically phosphorylation, was examined. When RPMEC were incubated with 32P and exposed to hypoxia for 1, 2, 4, and 24 h, there was a significant increase in XDH/XO phosphorylation as compared with normoxia with a maximum 50-fold increase at 4 h (Fig. 2). This increase was consistently observed in repeated experiments. The increase in phosphorylation was not due to an increase in the protein level because probing the same membrane with an anti-XDH/XO antibody after decay of the 32P radioactivity revealed no significant differences in the amount of XDH/XO between hypoxic and normoxic samples (data not shown).

Upon demonstrating that hypoxia stimulated XDH/XO phosphorylation and enzyme activity, the effect of dephosphorylation on enzyme activity was investigated in normoxic and hypoxic samples. Samples were incubated with either alkaline phosphatase or inactivated alkaline phosphatase (control) and then assayed for XDH/XO enzyme activity. As shown in Fig. 3, alkaline phosphatase treatment reduced the activity of both normoxic and hypoxic samples. However, only the reduction in enzyme activity in hypoxic samples (64%) was statistically significant. These results are consistent with a role for phosphorylation in modulating XDH/XO activity.



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Fig. 3.   Treatment of normoxic and hypoxic RPMEC samples with alkaline phosphatase reduces XDH/XO enzyme activity. This effect was statistically significant for hypoxic samples. * indicates p < 0.05 versus normoxic control and versus alkaline phosphatase-treated hypoxic samples.

Casein Kinase II and p38 Kinase Inhibitors Block Hypoxia-induced Phosphorylation of XDH/XO As Well As Hypoxia-stimulated XDH/XO Enzyme Activity-- To identify the kinase(s) or phosphatase(s) involved in XDH/XO phosphorylation, RPMEC were preincubated with various inhibitors prior to labeling and exposing to hypoxia. As shown in Table I, inhibitors of protein kinase A and protein kinase G reduced base-line XDH/XO phosphorylation but had no effect on hypoxia-stimulated phosphorylation. Inhibitors of protein kinase C had no effect on XDH/XO phosphorylation in either normoxic or hypoxic cells. A casein kinase II (CK2) inhibitor partially blocked hypoxia-stimulated XDH/XO phosphorylation (Fig. 4). Inhibitors of p38 kinase, a stress-activated kinase reported to be activated in hypoxia (18, 19), also partially blocked hypoxia-stimulated phosphorylation of XDH/XO (Fig. 4). In conclusion, these results suggest that phosphorylation of XDH/XO in hypoxia involves p38 kinase and CK2.


                              
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Table I
Percent of inhibition of XDH/XO phospholabeling
Inhibitors of protein kinases A, G, and C reduce base-line phosphorylation of XDH/XO but do not affect hypoxia-stimulated phosphorylation. Protein kinase A (PKA) inhibitor (adenosine 3',5'-cyclic monophosphorothioate, 8-bromo-, Rp isomer) was used at 50 µM. Protein kinase G (PKG) inhibitor (guanosine 3',5'-cyclic monophosphorothioate, 8-(4-chlorophenylthio)-, Rp isomer, triethylammonium salt) was used at 5 µM. Protein kinase C (PKC) inhibitors calphostin and bisindolylamide were used at 1 µM. Inhibitors of protein kinases A, G, and C inhibited XDH/XO phosphorylation in normoxia but had little or no effect on hypoxia-stimulated phosphorylation.



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Fig. 4.   Inhibitors of CK2 and p38 kinase block hypoxia-stimulated XDH/XO phosphorylation. ICK2, inhibitor of CK2 (5,6-dichloro-1-beta -D-ribofuranosylbenzimidazole, 50 µM). Ip38, inhibitor of p38 MAP kinase (SB 203580, 1 µM).

Phosphatase inhibitors, cypermethrin and cyclosporin A, which inhibit protein phosphatase 2B or calcineurin, had no effect on XDH/XO phosphorylation in either normoxia or hypoxia (results not shown). Okadaic acid, which inhibits protein phosphatase 2A, was toxic to RPMEC at a concentration of 100 nM, and therefore, its effect could not be assessed.

After identifying inhibitors that block hypoxia-induced phosphorylation of XDH/XO, the effect of these inhibitors on XDH/XO enzyme activity was investigated. As shown in Fig. 5, both p38 kinase and CK2 inhibitors blocked hypoxia-stimulated XDH/XO enzyme activity. Taken together, these results are consistent with a role for phosphorylation by p38 kinase and CK2 in modulating XDH/XO enzymatic activity.



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Fig. 5.   Inhibitors of CK2 and p38 kinase block hypoxia-stimulated XDH/XO enzyme activity. CK2 inhibitor (5,6-dichloro-1-beta -D-ribofuranosylbenzimidazole, 50 µM), and p38 MAP kinase inhibitor (SB 203580, 1 µM) were used. * indicates p < 0.05 versus normoxic control, hypoxic + CK2 inhibitor, and hypoxic + p38 inhibitor samples.

Hypoxia Stimulates p38 Kinase in Rat Pulmonary Microvascular Endothelial Cells-- The MAP kinase p38 has been reported to be induced in hypoxia in PC12 and other cell types (18, 19). This enzyme is involved in stress-activated pathways responding to stressors such as hyperosmolarity, ultraviolet radiation, and inflammatory cytokines. Because inhibitors of p38 kinase partially prevented hypoxia-stimulated phosphorylation of XDH/XO, the effect of hypoxia on activation of p38 kinase in pulmonary endothelial cells was examined. Because p38 kinase becomes phosphorylated upon activation, antibodies that recognize phosphorylated (activated) p38 were used to probe Western blots from normoxic and hypoxic cells. To ensure that the changes in phosphorylated p38 immunolabeling were due to increased phosphorylation of the protein rather than total amount of p38, duplicate blots were probed with antibodies that recognize total p38 protein (both phosphorylated and unphosphorylated). For quantification, films were scanned with a densitometer, and the intensities of the phosphorylated p38 kinase bands were normalized to the intensities of the corresponding total p38 kinase bands. Hypoxia resulted in an increase in phosphorylated p38 kinase after 30 min of hypoxia with a peak (a 4-fold increase over normoxic cells) at 1 h (Fig. 6, A and B). By 24 h of hypoxia there was no difference in phosphorylated p38 kinase between normoxic and hypoxic cells. These results indicate that hypoxia causes the activation of p38 kinase in pulmonary endothelial cells, a phenomenon that chronologically precedes the peak of XDH/XO phosphorylation observed at 4 h of hypoxia.



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Fig. 6.   Hypoxia stimulates p38 kinase in RPMEC. A, hypoxia increases the amount of phosphorylated p38 kinase relative to total p38 kinase. B, maximum increase in the relative amount of phosphorylated p38 kinase normalized to total p38 kinase was observed at 1 h of exposure. The data (mean ± S.D.) are derived from three different experiments. * indicates p < 0.05 versus normoxic control mean.

Hyperosmolarity and Arsenite Activate p38 Kinase but Do Not Significantly Alter the Phosphorylation of XDH/XO-- Because inhibitors of p38 kinase reduce the phosphorylation of XDH/XO in hypoxia and because p38 becomes activated in RPMEC in hypoxia, experiments were performed to determine whether p38 kinase activators other than hypoxia stimulate XDH/XO phosphorylation. When RPMEC were exposed to hyperosmolar sorbitol (400 mM) or sodium arsenite (0.5 mM), both of which have been reported to activate p38 kinase in other cell types (20, 21), there was activation of p38 kinase by both agents within 15 min (Fig. 7). However, when RPMEC were prelabeled with 32P and then treated with sorbitol or arsenite for 30 min, no significant increase in XDH/XO phosphorylation over control cells was observed (Fig. 8). Taken together, these results indicate that activation of p38 kinase in hypoxia might be necessary but not sufficient to cause the phosphorylation of XDH/XO.



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Fig. 7.   Sorbitol (400 mM) and sodium arsenite (0.5 mM) cause an increase in phosphorylated p38 kinase without change in total p38 kinase.



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Fig. 8.   Sorbitol and arsenite do not increase XDH/XO phosphorylation. Cells were labeled with 32P as in Fig. 2 and exposed to sorbitol (400 mM), sodium arsenite (0.5 mM), or hypoxia for 1 and 4 h.



    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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This study demonstrates that XDH/XO is a phosphoprotein. To the best of our knowledge, this is the first evidence of phosphorylation of XDH/XO in mammalian cells. Furthermore, our results demonstrate that the phosphorylation of XDH/XO is greatly increased in hypoxia and is accompanied by an increase in XDH/XO enzyme activity. A causal relationship between increased phosphorylation and increased XDH/XO enzyme activity is supported by the finding that dephosphorylating XDH/XO reduces the hypoxia-stimulated enzyme activity and the finding that kinase inhibitors that block hypoxia-stimulated XDH/XO protein phosphorylation also block hypoxia-stimulated enzyme activity. These results also implicate p38 in the mechanism of XDH/XO phosphorylation by demonstrating that p38 kinase is induced in hypoxia and that an inhibitor of this enzyme partially blocks the phosphorylation of XDH/XO. The possible implications of these findings in regard to the regulation of XDH/XO by hypoxia are discussed below.

Up-regulation of XDH/XO has been reported in several diseases. For example, XDH/XO has been implicated in the acute respiratory distress syndrome (22, 23), the multisystem organ failure (24), and reexpansion pulmonary edema (25). The ability of XDH/XO to generate reactive oxygen species is believed to be the basis for the role of this enzyme in the pathogenesis of these diseases. The production of reactive oxygen species by XDH/XO is significantly increased upon the conversion of XDH to XO either through proteolysis or through posttranslational modification (26, 27). Both proteolysis and oxidation of the cysteines in XDH cause conformational changes in the molecule, which reduce its affinity for NAD+ leaving molecular oxygen as the electron acceptor instead (3). In animal models of lung injury, XDH/XO has been shown to be greatly increased (400-fold) in the bronchoalveolar lavage and serum of mice infected with the influenza virus (28). Using an in vivo model of acute lung injury, our laboratory has demonstrated an increase in lung XDH/XO activity and gene expression in response to hypoxia, lipopolysaccharide, and interleukin-1beta treatment (17). Pharmacological inhibition of XDH/XO prevents the development of pulmonary edema in this animal model further supporting a role for this enzyme in the pathogenesis of acute lung injury.

Hypoxia is known to trigger a series of events that has been likened to an inflammatory reaction. Some of these events involve the activation of transcription through the action of transcription factors such as HIF-1 (29-31). Other events, however, are considered too rapid to be due to transcriptional processes. One example of a nontranscriptional hypoxic response is the mobilization of P-selectin and its release from membranous organelles, which allows it to bind and activate neutrophils (32). The present data suggest another nontranscriptional endothelial response to hypoxia, namely the phosphorylation of XDH/XO and subsequent up-regulation of its enzymatic activity.

Two kinases, CK2 and p38 kinase, were shown to be involved in hypoxia-stimulated phosphorylation of XDH/XO. CK2, a ubiquitously expressed protein kinase, is believed to play an important role in regulating DNA replication and transcription as well as in regulating cell growth and metabolism. Upon examination of the primary sequence of XDH/XO using a phosphorylation site prediction program (33), several CK2 sites were identified. Although there is no information related to the effect of hypoxia on CK2, hypoxia is known to increase the levels of polyamines in the lung (34), which in turn are strong activators of CK2 (35, 36). Our finding that the CK2 inhibitor partially blocks hypoxia-stimulated XDH/XO phosphorylation and hypoxia-stimulated XDH/XO activity strongly suggests a role for this kinase in mediating the effects of hypoxia on XDH/XO.

p38 kinase was also found to be important in mediating the phosphorylation of XDH/XO in hypoxia. This enzyme, which belongs to the family of MAP kinases, can be activated by a variety of stresses including hyperosmolarity and UV radiation. Recently, p38 kinase was found to be activated by hypoxia as well (18, 19). The partial blocking of hypoxia-induced XDH/XO phosphorylation by p38 kinase inhibitor implicates this kinase in the XDH/XO phosphorylation pathway. The involvement of p38 kinase in hypoxia-stimulated XDH/XO phosphorylation is further supported by the finding that p38 is activated in hypoxic RPMEC. However, the inability of other p38 kinase stimulators, such as sorbitol and arsenite, to significantly increase XDH/XO phosphorylation suggests that p38 kinase activation alone is not sufficient to cause the XDH/XO phosphorylation and that other components are involved in the activation pathway. Although it is yet unclear how the two kinases, p38 kinase and CK2, interact in the XDH/XO phosphorylation pathway, a recent report demonstrated that stress-activated p38 kinase directly interacts with CK2 causing its activation in HeLa cells (37).

The mechanisms by which phosphorylation leads to increased enzyme activity need further investigation and are beyond the scope of the current study. However, one might speculate that the negative charges in the XDH/XO molecule introduced by phosphorylation might affect the affinity of the enzyme for its substrates (for example, see Ref. 38). Alternatively, these charges might alter protein-protein interactions either between different XDH/XO molecules affecting their tendency to dimerize or between XDH/XO and other unidentified proteins. An example of the latter mechanism of enzyme regulation involves the reactive oxygen species-producing neutrophil NADPH oxidase. This enzyme is believed to become activated during oxidative burst through the phosphorylation of one of its subunits, p47phox. Upon phosphorylation p47phox is translocated from the cytosol to the membrane where it associates with other subunits to activate the enzyme (39). It is noteworthy that p38 kinase has been shown to cause the phosphorylation of p47phox as well as the redistribution of other oxidase components, such as flavocytochrome b558, from vesicles to the plasma membrane through exocytosis resulting in activation of the enzyme (40). Another example of phosphorylation indirectly affecting enzyme activity involves spinach nitrate reductase, which belongs to the same family of molybdopterin-FAD oxidoreductases that includes XDH/XO. Phosphorylation of nitrate reductase causes it to bind the protein 14-3-3, resulting in the inhibition of the enzyme activity (41, 42). 14-3-3 is a family of proteins known to be involved in protein-protein interactions with various signal transduction components (for review, see Ref. 43). Alternatively, the phosphorylation of XDH/XO might alter XDH/XO subcellular localization by modulating its interaction with other proteins. Further studies are required to decipher the consequences of XDH/XO phosphorylation at the molecular level such as its potential role in the interaction of the enzyme with other proteins and in modulating its subcellular localization.

In conclusion, the findings that XDH/XO becomes phosphorylated and activated in hypoxia and that this phosphorylation is mediated by kinases known to be involved in signal transduction processes in inflammation and hypoxia implicate XDH/XO as an important component of the inflammatory reaction. Furthermore, the findings point to kinases as novel potential targets in regulating XDH/XO physiology. For instance, the p38 kinase inhibitor SB 203580 has been shown to reduce inflammation and lipopolysaccharide-induced mortality in animals (44). One possible action of this inhibitor might include modulating XDH/XO phosphorylation.


    ACKNOWLEDGEMENT

We thank Dr. Una Ryan for providing the RPMEC.


    FOOTNOTES

* This work was supported by grants from the American Lung Association, Massachusetts Tobacco Control Program, and Alzheimer's Association and National Institutes of Health Grant HL49441.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Pulmonary and Critical Care Div., New England Medical Center, 750 Washington St., P. O. Box 257, Boston, MA 02111. Tel.: 617-636-6386; Fax: 617-636-5953; E-mail: phassoun@lifespan.org.

Published, JBC Papers in Press, January 22, 2001, DOI 10.1074/jbc.M010100200


    ABBREVIATIONS

The abbreviations used are: XDH, xanthine dehydrogenase; XO, xanthine oxidase; RPMEC, rat pulmonary artery microvascular endothelial cells; PAGE, polyacrylamide gel electrophoresis; CK2, casein kinase II; MAP, mitogen-activated protein.


    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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