Hospital for Children and Adolescents, Research Laboratory, University of Helsinki, Biomedicum Helsinki, 00014 University of Helsinki, Finland
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ABSTRACT |
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Xanthine
oxidoreductase (XOR) may produce reactive oxygen species and play a
role in ischemia-reperfusion injury. Because tissue iron levels
increase after ischemia, and because XOR contains functionally
critical iron-sulfur clusters, we studied the effects of intracellular
iron on XOR expression. Ferric ammonium citrate and FeSO4
elevated intracellular iron levels and increased XOR activity up to
twofold in mouse fibroblast and human bronchial epithelial cells. Iron
increased XOR protein and mRNA levels, whereas protein and RNA
synthesis inhibitors abolished the induction of XOR activity. A human
XOR promoter construct (nucleotides +42 to 1937) was not induced by
iron in human embryonic kidney cells. Hydroxyl radical scavengers did
not block induction of XOR activity by iron. Iron chelation by
deferoxamine (DFO) decreased XOR activity but did not lower endogenous
XOR protein or mRNA levels. Furthermore, DFO reduced the activity of
overexpressed human XOR but not the amount of immunoreactive protein.
Our data show that XOR activity is transcriptionally induced by iron
but posttranslationally inactivated by iron chelation.
deferoxamine; gene regulation; iron-sulfur proteins; reactive oxygen species; ischemia-reperfusion injury
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INTRODUCTION |
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XANTHINE
OXIDOREDUCTASE (XOR) catalyzes the last two reactions of purine
catabolism by oxidizing hypoxanthine to xanthine and further to urate.
The enzyme occurs in two forms; the dehydrogenase form (XDH, EC
1.1.1.204), using NAD+, and the oxidase form (XO, EC
1.1.3.22), using molecular oxygen as the electron acceptor. In the
latter reaction, reactive oxygen species (ROS), i.e., superoxide anion
(O
Increased XOR activity after hypoxia is interesting in view of the putative role of XOR in postischemic tissue injury (19, 32). Cytokines also induce XOR activity (14, 16, 31), suggesting that XOR may contribute to ROS production in inflammatory conditions. Deferoxamine (DFO), an iron chelator, has been shown to reduce XOR activity in bovine pulmonary artery endothelial cells, which was proposed to account for the protective role of DFO in lipopolysaccharide-induced cytotoxicity (34).
In considerations of the pathophysiological role of XOR in vivo, its tissue expression is crucial. In humans, only the liver, mammary gland, and intestine express high levels of XOR (26, 35), whereas in rodents, the enzyme is strongly expressed in several organs (24). In the mammary gland, XOR protein level clearly increases during lactation (26), and the enzyme is present in human milk (38), where it has been proposed to have a microbicidal function (41).
Even though iron is a prerequisite for normal cellular functions, an increase in catalytically active iron after tissue ischemia can exacerbate ischemia-reperfusion injury by favoring the formation of intracellular free radicals and lipid peroxidation (5, 10, 40). A marked deposition of iron can be detected in rat brain after focal ischemia (6), and another study, using perfused rabbit lung as a model, showed that after prolonged ischemia iron can be released from its intracellular stores into the vascular space (21).
XOR is a homodimer with two equivalent active sites. Electrons are
transferred from purine substrate to molybdopterin cofactor and further
through two nonidentical [2Fe2S] centers of the ferredoxin type to
FAD cofactor (30). Studies on the relationship between intracellular iron levels and XOR activity have not been reported. However, because XOR contains iron-sulfur clusters, and because it may
play a pathophysiological role under conditions of varying tissue iron
levels, the effect of iron on XOR regulation is of interest. In this
study, we tested whether and by what mechanism intracellular iron
levels influence XOR expression and activity.
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MATERIALS AND METHODS |
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Cell culture. NIH/3T3 mouse fibroblasts from American Type Culture Collection (ATCC; Manassas, VA) and human embryonic kidney 293T cells (from Prof. K. Saksela, University of Tampere, Finland) were cultured as described previously (27). Transformed human bronchial epithelial BEAS2B cells (ATCC) were cultured in bronchial epithelial cell growth medium (Cytotech, Helleback, Denmark) supplied with 100 U/ml penicillin and 100 µg/ml streptomycin. For treatments with different compounds, the cells were grown to subconfluence. Ferric ammonium citrate (FAC) was from Riedel-de Haen (Seelze, Germany); FeSO4, CuSO4, and dimethyl sulfoxide (DMSO) were from Merck (Darmstadt, Germany); and actinomycin D, cycloheximide, DFO, 1,3-dimethyl-2-thiourea (DMTU), N-acetylcysteine (NAC), and 1,10-phenanthroline were from Sigma (St. Louis, MO).
Intracellular iron measurement. NIH/3T3 cells were treated with iron or iron chelators, washed three times with PBS, harvested in PBS, frozen and thawed twice, and centrifuged at 4°C at 15,800 g for 8 min in an Eppendorf centrifuge. The iron content of the supernatant was measured by using Rauta kit 141010 (Reagena, Kuopio, Finland), in which iron is first released in denaturing conditions (pH 4.8) and then reduced by ascorbic acid to ferrous iron, which forms a stable complex with ferene. This complex is then measured spectrophotometrically.
XOR activity measurement.
Total XOR and XO activities were determined by using
[14C]xanthine (NEN Life Science Products, Boston, MA) as
substrate in the presence or absence of NAD+, respectively,
and separating the product (uric acid) by HPLC as described previously
(36). For the activity measurements, cells were washed
twice with PBS and harvested in 50 mM potassium phosphate buffer, pH
7.8, containing 0.5 mM dithiothreitol, 1 mM EDTA, 0.5 µg/ml
leupeptin, and 0.2 mM phenylmethylsulfonyl fluoride or 50 µl/ml
protease inhibitor cocktail (Sigma). Subsequently, the cell suspension
was lysed as described above. The protein concentration of the
supernatant was measured with a Bio-Rad DC protein assay (Bio-Rad,
Hercules, CA), and the supernatant was stored at 70°C.
Expression of XOR protein. 293T cells were transiently transfected with pcDNA3-expression vector (Invitrogen, Croningen, The Netherlands) carrying the complete coding sequence of human XOR (36), named pcDNA3-XOR. When the cells in 10-cm-diameter dishes reached ~50% confluence, they were transfected with 2 µg of pcDNA3-XOR by using FuGene6 transfection reagent (Roche Molecular Biochemicals, Mannheim, Germany) as described by the manufacturer, and after 17 h, fresh medium with FAC or DFO was added. After 24-h incubation, the cells were harvested as for the activity measurements.
Western blot analysis.
Proteins from NIH/3T3 (10 µg) or 293T cells overexpressing XOR (2.5 µg), harvested as described above, were separated by 7.5% SDS-PAGE
and transferred to Immobilon-P membrane (Millipore, Bedford, MA). The
blotted membrane was blocked with 5% (wt/vol) skim milk in 0.1 M Tris,
1 M NaCl, and 0.1% (vol/vol) Tween 20 for 1 h. For detection of
XOR protein, polyclonal human anti-XOR antibodies (38) at
a dilution of 1:1,000 (for NIH/3T3 proteins) or 1:2,000 (for cells
overexpressing XOR) were used, followed by horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) at a dilution of 1:5,000.
To control for the protein loading, we applied monoclonal anti--tubulin antibody (Sigma) at a dilution of 1:40,000 on the same
Western blot. The protein bands were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech, Amersham, UK).
Northern blot analysis. Total RNA was extracted by using the RNeasy Mini kit (Qiagen, Hilden, Germany) and separated on a 1% agarose-formaldehyde gel (10 µg/lane) for Northern blot analysis. RNAs were transferred to Biodyne A nylon filters (PALL Gelman Sciences, Portsmouth, UK) and fixed by UV cross-linking. Membranes were hybridized by using standard procedures (37) with 32P-labeled complementary RNA probe corresponding to nucleotides 321 to 3021 of the rat XOR cDNA (kindly provided by Prof. T. Nishino, Nippon Medical School, Tokyo, Japan) (3). The membranes were exposed to autoradiography films (Curix Ortho HT-L PLUS; Agfa, Mortsel, Belgium). The same membranes were reprobed with random primed (Prime-a-Gene labeling system; Promega, Madison, WI) mouse 18S ribosomal gene DNA probe (Ambion, Austin, TX) to control for RNA loading. The X-ray films were scanned (Hewlett Packard ScanJet 6300C) and analyzed with the Scion Image beta 4.0.2 analysis software (Scion, Frederick, MD).
Transfection and reporter gene analysis.
Human XOR promoter luciferase reporter gene constructs (XOR1, from +42
relative to the translational start site to 1937, and XOR5, from +42
to
142) were generated, and 293T cells were transiently transfected
with XOR promoter constructs and
-galactosidase expression vector as
described previously (27). Medium containing FAC (1 or 2 mM) was changed 16 h after transfection, and the cells were
further incubated for 24 h. Luciferase activity was determined as
described previously (27).
Statistical analysis. Data are expressed as means ± SD, and means were compared by using a two-tailed t-test with unequal variations. P values <0.05 were considered significant.
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RESULTS |
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Intracellular iron.
The culture medium contained 2.3 µM iron, and the basal iron
concentration of NIH/3T3 cells was 0.7 nmol/mg protein. Compared with
cells grown in normal medium, the intracellular iron content in NIH/3T3
cells increased progressively up to 12-fold when FAC was present at
concentrations between 180 µM and 1.8 mM in the medium and up to
19-fold when 1 mM FeSO4 was added to the cell culture
(Table 1). The effect of iron chelators
on intracellular iron content could not be reliably determined because
the concentration of iron in the control cells was near the lower limit
of the assay.
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Intracellular XOR activity.
The basal XOR activity in NIH/3T3 cells was 849 ± 238 nmol · min1 · mg
protein
1. Total XOR activity in NIH/3T3 cells incubated
with FAC at concentrations between 180 µM and 1.8 mM increased
progressively up to twofold and somewhat less upon incubation with
FeSO4 (1 mM) (Fig.
1A). The
increase in enzyme activity was not detectable after 12 h of
incubation with iron but was clearly seen after 24 h (Fig. 1B). Neither iron compound altered the ratio of XDH to XO.
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Intracellular XOR protein.
The amount of XOR protein in NIH/3T3 cells was assessed by Western blot
analysis. The ~150-kDa band corresponding to XOR protein intensified
after 24 h of incubation with FAC, whereas DFO did not change the
level of immunoreactive XOR protein (Fig.
3A). The increase in XOR
protein was inhibited by cycloheximide and actinomycin D. This finding
is in accordance with the effect of cycloheximide on XOR activity,
indicating that new protein synthesis is needed for the iron-induced
increase in XOR activity.
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Effect of intracellular iron on XOR transcription. The effects of FAC and DFO on XOR mRNA levels in NIH/3T3 cells were determined by Northern blot analysis. FAC (180 µM) raised the level of XOR mRNA 1.5-fold (Fig. 3, B and C). Surprisingly, DFO (100 µM) did not decrease but consistently somewhat increased the amount of XOR message (Fig. 3, B and C), suggesting that the suppression of XOR enzyme activity by DFO does not take place at the transcriptional level. Induction of XOR mRNA by iron occurred in the presence of cycloheximide (Fig. 3B), indicating that the transcriptional induction of XOR by iron is independent of de novo protein synthesis. Actinomycin D decreased the amount of XOR mRNA after 24 h of incubation and inhibited the rise in XOR mRNA in iron-treated cells (Fig. 3B). FeSO4 also increased the level of XOR mRNA (Fig. 3C). On the basis of these findings, we conclude that iron-induced increase in XOR activity is associated with transcriptional activation of the XOR gene.
To explore whether iron has an influence on the activity of the human XOR promoter, we applied FAC onto 293T cells transiently transfected with XOR promoter constructs. Iron did not, however, increase the activity of either XOR1, corresponding to ~2 kb of the XOR 5'-flanking region, or XOR5, representing the proximal promoter and being the most active of our XOR promoter constructs, in 293T cells (27).Effect of DFO on XOR expressed in 293T cells.
To study the possible direct effect of DFO on the formation of
immunoreactive and active XOR protein, we expressed human XOR in 293T
cells that have no measurable endogenous XOR activity. The plasmid
pcDNA3-XOR produced enzymatically active XOR protein that could be
detected as one band with human XOR antibody in Western blot analysis
(Fig. 4), whereas nontransfected 293T
cells exhibited neither detectable amounts of XOR protein nor activity (data not shown). During 24 h of incubation, DFO did not decrease the amount of immunoreactive XOR protein produced by pcDNA3-XOR as
determined by Western blot analysis (Fig. 4). However, DFO reduced the enzyme activity of the expressed XOR protein to <20% of
the activity found in cells grown in standard culture medium (Fig. 4),
indicating posttranslational inhibition of XOR activity.
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Effects of hydroxyl radical scavengers and copper on XOR activity.
To evaluate the possible role of ROS, generated in iron-catalyzed
reactions, in the signaling pathway leading to increased XOR activity,
we treated NIH/3T3 cells with the hydroxyl radical scavengers NAC (1 mM), DMTU (10 mM), or DMSO (30 mM) with or without iron. None of the
hydroxyl radical scavengers inhibited the increase in XOR activity
caused by iron (Table 2). To assess
whether another transition metal, copper, could increase XOR activity,
we treated NIH/3T3 cells with CuSO4. Instead of increasing
XOR activity, CuSO4 decreased XOR activity (Fig.
1A).
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DISCUSSION |
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Using XOR activity measurements, Western and Northern blotting, and protein and RNA synthesis inhibitors, we have shown that increased intracellular iron induces XOR in mouse fibroblast NIH/3T3 cells at the transcriptional level. These cells were used because they have relatively high endogenous XOR activity. In human bronchial epithelial BEAS2B cells, both the basal activity and the iron induction were smaller than in NIH/3T3 cells, which may reflect species- or tissue-specific differences in regulation of XOR expression or in iron metabolism.
Several proteins involved in iron metabolism are regulated at the
posttranscriptional level by well-defined mechanisms involving sequence-specific mRNA-binding iron regulatory proteins (IRPs). In
iron-depleted cells, IRPs bind to iron-responsive elements (IREs) and
either promote mRNA stability (transferrin receptor) or inhibit protein
translation (ferritin, 5-aminolevulinate synthase) depending on the
location on the mRNA (15). The expression of proteins
carrying iron-sulfur clusters may also be regulated by IRPs binding to
IREs (17, 22). Furthermore, IRP1 itself is believed to be
converted into non-RNA-binding cytosolic aconitase by assembly of a
[4Fe4S] cluster in iron-replete cells (23). Even
though the rat XOR mRNA has been suggested to carry a 5'-IRE consensus
sequence element (CAGUGA) (7), which is conserved in the
human promoter, our data strongly support iron effects at the
transcriptional rather than the mRNA level. Furthermore, actinomycin D
prevented the iron-induced increase of both enzyme activity and mRNA,
indicating that de novo RNA synthesis is required.
Iron has been shown to directly modify the transcription of several
genes, e.g., protein kinase C, cyclin-dependent kinase inhibitor p21,
retinoblastoma susceptibility protein pRb, several stress proteins, and
inducible nitric oxide synthase (iNOS), but little is known of the
underlying mechanisms (1, 2, 12, 43). The transcription
factor NF-B has been implicated (25), and the human XOR
promoter has been suggested to carry a consensus NF-
B binding site
(42). Because our XOR promoter constructs failed to be
activated by iron, this potential mechanism could not be studied
further. NF-
B is also a major transcription factor mediating the
effects of reactive oxygen metabolites on gene regulation (39). Increased intracellular iron may catalyze the
formation of hydroxyl radicals (4, 18), which could
account for increased transcription of the XOR gene. However, hydroxyl
radical scavengers failed to prevent the induction of XOR by iron, thus
lending no support for the role of oxidants and, indirectly, of
NF-
B.
Iron chelation by the intracellular Fe(III) chelator DFO (33) substantially decreased XOR activity in both NIH/3T3 and BEAS2B cells. The rate of decrease in XOR activity caused by DFO was more rapid than the induction of XOR enzyme activity by iron, suggesting that the underlying mechanisms may be different. Paradoxically, even though DFO decreased enzyme activity, XOR mRNA levels were increased in NIH/3T3 cells, whereas XOR protein remained unchanged. These data suggest that DFO influences XOR activity at the posttranslational level. This conclusion is supported by our findings in cells overexpressing XOR, in which DFO strongly suppressed XOR activity without causing a decrease in XOR protein levels. The phenomenon may be analogous to the inactivation of ribonucleotide reductase by iron chelation, which is associated with an increase in the proportion of the apoprotein lacking iron (9).
The mechanism of the increase in XOR mRNA caused by DFO was not studied
further and remains unclear. In murine macrophages and NIH/3T3 cells,
iron chelation upregulates iNOS expression by a mechanism involving the
transcription factor NF-IL6 (C/EBP-) (12). This factor
has been shown to have a role in the transcriptional regulation of the
rat XOR promoter (8), but it fails to bind onto human XOR
promoter constructs (27).
We have explored the regulation of the XOR gene to better understand its potential pathophysiological role. In ischemic tissue, hypoxanthine, a substrate for XOR, accumulates, and during reperfusion XOR is converted into the ROS-producing oxidase form. Under the same conditions, tissue iron content increases. Our data, showing upregulation of XOR activity by iron, suggest another mechanism for exacerbation of tissue damage in ischemia-reperfusion injury and iron-overload states. Furthermore, because DFO rapidly and effectively suppresses XOR activity, the therapeutic potential of iron chelation in these clinical situations should be explored. Increased body iron stores are associated with an increased risk for cardiovascular events (11), and DFO has been shown to improve endothelial function in patients with coronary artery disease (13).
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ACKNOWLEDGEMENTS |
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We thank Dr. Aija Helin for the iron measurements and Sari Lindén and Ritva Löfman for skillful technical assistance. We thank Dr. Markku Heikinheimo and Dr. Anna-Liisa Levonen for valuable comments on the manuscript.
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FOOTNOTES |
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This study was supported by the Helsinki Biomedical Graduate School, University of Helsinki (E. Martelin) and the Research Fund of the Helsinki University Central Hospital.
Address for reprint requests and other correspondence: E. Martelin, Hospital for Children and Adolescents, Research Laboratory, Biomedicum Helsinki, Rm. B524b, PO Box 63, 00014 Univ. of Helsinki, Finland (E-mail: eeva.martelin{at}hus.fi).
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.
August 22, 2002;10.1152/ajpcell.00280.2002
Received 18 June 2002; accepted in final form 16 August 2002.
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