Departments of Physiology and Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226
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ABSTRACT |
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The present study hypothesized that
superoxide (O expression at
posttranscriptional levels in renal medullary interstitial cells
(RMICs) of rats. By Western blot analysis, it was found that incubation
of RMICs with O
levels and completely
blocked the increase in HIF-1
levels induced by ubiquitin-proteasome inhibition with CBZ-LLL in the nuclear extracts from these cells. Under
normoxic conditions, a cell-permeable O
levels in RMICs. Two
mechanistically different inhibitors of NAD(P)H oxidase, diphenyleneiodonium and apocynin, were also found to increase HIF-1
levels in these renal cells. Moreover, introduction of an anti-sense
oligodeoxynucleotide specific to NAD(P)H oxidase subunit,
p22phox, into RMICs markedly increased HIF-1
levels. In contrast, the OH· scavenger tetramethylthiourea had no
effect on the accumulation of HIF-1
in these renal cells. By
Northern blot analysis, scavenging or dismutation of
O
-targeted gene, heme oxygenase-1. These
results indicate that increased intracellular O
degradation independently of
H2O2 and OH· radicals in RMICs. NAD(P)H oxidase activity may importantly contribute to this posttranscriptional regulation of HIF-1
in these cells under physiological conditions.
reactive oxygen species; gene transcription; anoxia; renal interstitium; renal medulla
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INTRODUCTION |
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HYPOXIA-INDUCIBLE
factor-1 (HIF-1
) mediates the transcriptional activation of many
oxygen-sensitive genes such as erythropoietin, heme oxygenase-1 (HO-1),
inducible nitric oxide synthase, vascular endothelial growth factor,
transferrin, and several glycolytic enzymes (18, 31-35,
37). This nuclear factor forms a heterodimer complex with its
partner HIF-1
to activate gene transcription. It has been
demonstrated that HIF-1
can be induced by low tissue or cell
O2 concentrations and rapidly degraded via an
ubiquitin-proteasome pathway when O2 concentrations return
to normoxic conditions (13, 16, 25). HIF-1
constitutively appeared in cells and tissues under normoxic conditions.
The HIF-1 heterodimer complex recognizes a DNA consensus sequence
5'-CGTG-3' in enhancer or promoter regions of many hypoxia-responsive
genes, interacts with these binding sites in the major groove, and
activates the transcription of these genes (32). Although
much has been learned about the role of HIF-1 in activating the
transcription of hypoxia-responsive genes, the mechanism by which HIF-1
levels within cells are regulated under physiological conditions is
still poorly understood.
Reactive oxygen species (ROS) have been reported to be involved in the
oxygen-sensing mechanism and play a critical role in the regulation of
expression of oxygen-sensitive genes. Superoxide anions
(O levels in different cell lines (2, 3, 12,
25), which may serve as an important mechanism activating oxygen-sensitive genes. However, it remains to be elucidated how ROS
change HIF-1
levels and which species of ROS is importantly involved
in the regulation of HIF-1
levels. More recently, we demonstrated
that renal medullary cells more abundantly expressed HIF-1
compared
with cortical cells and that HIF-1
participated in the
transcriptional activation of oxygen-sensitive genes such as HO-1
(37, 46). The HIF-1
expression and related
transcriptional activation of target genes in these cells may play an
important role in the normal regulation of renal medullary oxygenation, renal medullary blood flow, and renal functions such as sodium excretion and osmolality adaptation (37, 43-46).
However, the mechanism regulating HIF-1
levels in renal medullary
cells is poorly understood. Given that ROS levels are higher in the
renal medulla than cortex (44), it is possible that
HIF-1
levels are importantly regulated by redox status in this
kidney region. The present study used renal medullary interstitial
cells (RMICs) as prototype cells to test whether HIF-1
levels in
renal medullary cells are regulated by ROS and whether ROS may alter
the transcriptional activation of some hypoxia-sensitive genes through
a HIF-1
-mediated mechanism. Moreover, the present study examined
which species of ROS contributes to the regulation of HIF-1
levels
and whether NAD(P)H oxidase-derived O
expression in these kidney cells.
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MATERIALS AND METHODS |
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Isolation and culture of RMICs. RMICs were isolated, cultured, and identified as we described previously (37). Briefly, inbred male Wistar rats weighing 300-350 g (Harlan Sprague Dawley, Madison, WI) were anesthetized with pentobarbital sodium (50 mg/kg body wt ip). Then, the left kidney was removed, and the renal papilla was dissected and finely minced. The minced tissue was resuspended in 3 ml of basic medium Eagle's (BME; Sigma) and injected subcutaneously in two to four vertical tracks on the abdominal wall of a recipient rat (from the same litter). Four days after injection, many firm and yellow nodules located at the site of injections were dissected carefully. These nodules were minced, trypsinized in 0.05% Trypsin-EDTA solution at 37°C for 20-30 min, and then washed and centrifuged to obtain a cell pellet. The cell suspension was transferred to plastic tissue culture flasks and then incubated with BME containing fetal bovine serum (10% vol/vol), amino acid mixtures (10% vol/vol), lactalbumin hydrolysate (0.25% wt/vol), yeast extracts (0.05% wt/vol), and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin) using a 37°C incubator with a 95% air-5% CO2 environment. The culture medium was first replaced with fresh medium in 5 days and then changed every 3 days. These cells formed a confluent monolayer in 18-21 days and then were trypsinized and subsequently replanted in flasks. The cells from passages 7 and 8 were used for all experiments. The identity of these cells was confirmed by a standard staining method and light and electron microscopy as we described previously (37).
Preparation of nuclear extracts.
Nuclear extracts from RMICs were prepared by a modification of the
protocol described by Semenza and Wang (28). The cell pellet was washed with 4 packed-cell volumes (PCV) of buffer
A [10 mM Tris · HCl (pH 7.8), 1.5 mM
MgCl2, 10 mM KCl] containing 0.5 mM DTT, 0.4 mM PMSF, 2 µg/ml leupeptin, 2 µg/ml pepstatin, 2 µg/ml aprotinin, and 1 mM
sodium vanadate (all obtained from Sigma), resuspended in buffer
A, and incubated on ice for 10 min. Then, the cell suspension was
homogenized, and the nuclei were pelleted by centrifugation at 3,000 rpm for 5 min, resuspended in 3 PCV of buffer B [20 mM
Tris · HCl (pH 7.8), 1.5 mM MgCl2, 0.42 M KCl, 20% glycerol] containing 0.5 mM DTT, 0.4 mM PMSF, 2 µg/ml leupeptin, 2 µg/ml pepstatin, 2 µg/ml aprotinin, and 1 mM
sodium vanadate, and mixed on a rotator at 4°C for 30 min. Finally,
nuclear extracts were collected by centrifugation of nuclei incubation
mixtures in buffer B for 30 min at 13,500 rpm. Aliquots were
frozen in liquid N2 and stored at 80°C. Protein concentrations were determined using a Bio-Rad protein assay kit with
bovine serum albumin standards. In our previous studies (37, 46), the nuclear extracts prepared according to this protocol were confirmed rich in HIF-1
.
Western blot analysis.
Western blotting was performed as we described previously
(37). Briefly, 40 µg of the nuclear extracts were
subjected to 8% SDS-PAGE and transferred onto nitrocellulose membrane.
Then, the membrane was washed and probed with 1:1,000 specific
polyclonal anti-HIF-1 antibody and subsequently with 1:4,000
horseradish peroxidase-labeled goat anti-rabbit IgG. This polyclonal
antibody against a 13-residue peptide from rat HIF-1
was prepared
and validated in our previous studies (37, 46). To detect
an immunoblotting signal, 10 ml of enhanced chemiluminescence detection
solution (Amersham Pharmacia) were added, and the membrane was wrapped and exposed to Kodak Omat film. HIF-1
was used as an internal control because HIF-1
is constitutively expressed and not inducible during hypoxia, CoCl2, and other stimuli (13, 16,
25).
cDNA probes for Northern blot analysis of HIF-1 and HO-1.
The HIF-1
and HO-1 cDNA from the rat kidney were cloned by RT-PCR
with primer pairs designed and synthesized based on the sequences of
rat HIF-1
and HO-1 cDNA in GenBank [accession number AF057308 for
HIF-1
and M12129 for HO-1 (42, 46)]. A First-Strand
cDNA Synthesis Kit (Amersham Pharmacia) was used to generate
single-strand cDNA by RT, which was then used as a template for PCR
with the primers for HIF-1
: 5'-CGGCGAAGCAAAGAGTCT-3' (sense) and
5'-TGAGGTTGGTTACTGTTG-3' (anti-sense); and for HO-1: 5'-GTCTATGCCCCGC
TCTACTTC-3' (sense) and 5'-GTCTTAGCCTCTTCTGACACC-3' (anti-sense). The
PCR products were fractionated on a 1.5% agarose gel, excised, and
extracted with the use of a QIAGEN Gel Extraction Kit. The resulting
cDNAs (542 bp for HIF-1
and 396 bp for HO-1) were cloned into
pCR2.1-TOPO vector as described by the manufacturer (Invitrogen) and
sequenced to confirm the identity of cDNA with an autosequencer by
McConnell. The inserts for these genes in plasmid DNA were dissected by
PCR or by enzyme digestion and used as probes for Northern blot
analysis. The probes were purified and stored at
80°C until used.
RNA extraction and Northern blot analysis.
Total RNA was extracted using TRIzol solution (Life Technologies)
according to the manufacturer's protocol. Northern blot analyses of
HIF-1 and HO-1 mRNAs were performed as described previously
(37, 43, 46). In brief, total RNA (10-20 µg) was
fractionated on a 1.0% formaldehyde-agarose gel, stained with ethidium
bromide (0.5 µg/ml), washed, photographed, transferred onto nylon
membrane (Pirece), and cross-linked to the membrane by UV irradiation.
The nylon membranes were first prehybridized with Rapid Hyb buffer
(Amersham Pharmacia) and then probed with 32P-labeled rat
HIF-1
or HO-1 cDNA, respectively, at 65°C for 2.5 h. After
being washed once at RT and then twice at 65°C, the membranes were
autoradiographed at
80°C for 24 or 36 h. The autoradiographed films were scanned with a laser densitometer (Hewlett Packard ScanJet
ADF) and then digitized by a UN-SCAN-IT software package (Silk
Scientific). The densitometric values of those specific bands for
corresponding gene expression were normalized to 28S rRNA.
Treatment of cells with various compounds.
Xanthine and xanthine oxidase (X/XO; 100 µM · 50 mU1 · ml
1) was used
to produce O
degradation. All
these reagents were directly added into the culture medium and
incubated for a time period indicated in RESULTS. The doses
or concentrations of these compounds for changing redox status in RMICs
were chosen based on previous studies in our laboratory or by others,
demonstrating that they effectively decreased or increased ROS levels
in the cells or tissues (4, 11, 19, 21, 36, 37, 40, 44).
NAD(P)H oxidase subunit, p22phox anti-sense
oligodeoxynucleotide transfection.
p22phox Anti-sense oligodeoxynucleotide (P22-AS)
was synthesized and introduced into RMICs as we previously described
(37, 38). Briefly, a phosphorothioation-modified
P22-AS was synthesized based on a cDNA sequence of
p22phox (AJ295951) and it contained
5'-GCCCACTCGATCTGCCCCAT-3' (antisense, OPERON). The modification of
five nucleotides on each side of P22-AS by phosphorothioation increased
the stability and prevented this oligonucleotide from being degraded by
intracellular nucleotide enzymes. The fluorescein attachment at the
5'-end was used as an indicator for transfection into the RMICs. The
P22-AS was wrapped by cationic liposome (Avanti Polar Lipids,
Alabaster, AL) and transfected into RMICs as described by the
manufacturer. The transfection efficiency was evaluated by using a
fluorescence microscope (Olympus, Tokyo, Japan) 3 h after
incubation of RMICs with liposome-P22-AS mixtures. Positively
transfected cells (70-80% cells) indicated by a remarkable
intracellular fluorescence were used to determine the effects of
p22phox blockade on HIF-1 levels.
Cell hypoxia. RMICs were plated in 100-mm2 tissue culture dishes 24 h before experiments and cultured to form a subconfluence. To decrease PO2 in the culture medium, these dishes were transferred to a sealed, humidified modular chamber and flushed for 2 h with 5% CO2-95% N2. PO2 in the culture medium was measured by a polarigraphic measurement described in our previous studies (45, 46), which is less than 10 mmHg after 2-h hypoxia.
Statistical analysis. Data are presented as means ± SE. The significance of difference in mean values within and between multiple groups was examined with an ANOVA for repeated measures followed by a Duncan's post hoc test. Student's t-test was used to evaluate the significance of differences between two groups of experiments (SigmaStat, SPSS). A value of P < 0.05 was considered statistically significant.
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RESULTS |
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Effects of X/XO and menadione on hypoxia- or
CoCl2-induced HIF-1 protein expression in RMICs.
Both X/XO and menadione have been commonly used to produce
extracellular or intracellular O
1 · ml
1) or
menadione (100 µM) was added to cell culture medium and incubated for
22 h. Then, cells were subjected to hypoxia for another 2 h.
It was found that HIF-1
protein levels significantly increased in
control RMICs exposed to hypoxia for 2 h. However, the
hypoxia-induced increase in HIF-1
protein levels was attenuated in
RMICs pretreated with X/XO or menadione as shown in Fig.
1A. Similarly, HIF-1
protein levels were found to increase in RMICs treated by 150 µM
CoCl2 for 4 h. In the presence of X/XO or menadione,
the HIF-1
increase induced by CoCl2 was inhibited (Fig.
1B). All these experimental interventions had no effects on
HIF-1
levels. Summarized data from these experiments by
densitometric analysis are presented in Fig. 1C. Increases
in the intensity of the HIF-1
-immunoreactive band during hypoxia
(n = 6) or treatment of CoCl2
(n = 6) were significantly attenuated by X/XO and
menadione.
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Effects of X/XO and menadione on CBZ-LLL-induced increase in
HIF-1 protein levels in RMICs.
To further determine the effects of altered cell redox status on
HIF-1
levels, we examined CBZ-LLL-induced alterations of HIF-1
levels in X/XO- or menadione-treated cells. CBZ-LLL is an inhibitor of
ubiquitin-proteasome, which inhibits degradation of HIF-1
in the
cells. In Fig. 2A are
representative gel documents showing that treatment of RMICs with 10 µM CBZ-LLL for 4 h markedly increased HIF-1
levels. However,
in the presence of X/XO or menadione, the CBZ-LLL-induced increase in
HIF-1
protein levels was attenuated. In these experiments, HIF-1
was not altered by any treatments. The data of these experiments are
summarized in Fig. 2B. CBZ-LLL produced a 3.5-fold increase
in HIF-1
protein levels in RMICs. In the presence of X/XO and
menadione, the CBZ-LLL-induced increase in HIF-1
levels was
completely blocked (n = 4).
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Effects of X/XO on HIF-1 mRNA expression in RMICs in response to
hypoxia, CoCl2, and CBZ-LLL.
To determine whether O
mRNA
expression, we detected changes in HIF-1
mRNA levels in RMICs under
control conditions or subjected to different stimuli. It was found that
HIF-1
mRNA levels increased in RMICs exposed to hypoxia for 2 h
or to 150 µM CoCl2 for 4 h. Pretreatment of the
cells with X/XO had no effect on hypoxia- and
CoCl2-increased HIF-1
mRNA expression (Fig.
3A). Although CBZ-LLL
significantly increased HIF-1
protein levels as shown above,
it did not increase, even decreased and, HIF-1
mRNA expression by an
unknown mechanism. However, in the presence of X/XO, this
CBZ-LLL-decreased HIF-1
mRNA level was not further altered (Fig.
3B).
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Effects of TEMPOL, PEG-SOD, and DPI on HIF-1 levels in
RMICs.
To confirm the role of endogenously produced
O
levels, the
effects of O
levels
in RMICs. As shown in Fig. 4A, incubation of RMICs with TEMPOL (0.1 or 0.4 mM) or PEG-SOD (50 or 100 U/ml) for 6 h markedly increased HIF-1
protein levels in RMICs.
Similarly, inhibition of O
protein
levels in these cells. Figure 4B summarizes the results from
these experiments. Both O
levels in
RMICs (n = 6).
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Effects of apocynin and P22-AS on HIF-1 levels in RMICs.
Although DPI has been reported to inhibit NAD(P)H oxidase and our
recent study demonstrated that this compound at concentrations less
than 50 µM had no effect on other O
levels in RMICs.
In one group of experiments, apocynin, another specific inhibitor that
blocks aggregation of NAD(P)H oxidase subunits and thereby inhibits its
activity, was used (10, 29). As shown in the
representative gel documents of Western blot analysis (Fig.
5A), apocynin (100 µM)
markedly increased HIF-1
levels, but it was without effect on
HIF-1
expression. Figure 5B summarized the results from
these experiments (n = 11), showing that HIF-1
, but
not HIF-1
levels were significantly increased in RMICs treated with
apocynin.
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Effects of TMTU on HIF-1 protein accumulation in RMICs in
response to TEMPOL.
Because H2O2 can be converted to form OH·,
which was reported to increase the degradation of HIF-1
(12,
17, 25), we examined the effect of OH· production on HIF-1
expression associated with an H2O2 increase by
TEMPOL. A specific scavenger of OH·, TMTU was used to examine the
effects of OH· on HIF-1
protein levels in RMICs. Treatment of
RMICs with 1 mM TMTU for 6 h had no effect on TEMPOL-induced
increase in HIF-1
protein levels (n = 6; Fig.
7).
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Effects of TEMPOL and PEG-SOD on HO-1 mRNA expression in RMICs.
Because the HO-1 gene is regulated transcriptionally by HIF-1, we
were wondering whether an endogenous O
levels influences the transcription of this gene.
Therefore, one protocol was designed to examine the effects of TEMPOL
and PEG-SOD on HO-1 mRNA expression. Consistent with an increase in
HIF-1
protein levels, HO-1 mRNA expression in RMICs was
significantly increased by TEMPOL and PEG-SOD (Fig. 8A). These results are
summarized in Fig. 8B. Dismutation of
O
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DISCUSSION |
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It has been reported that the activation of many oxygen-sensitive
genes during hypoxia is mediated by the binding of HIF-1, a HIF-1
and HIF-1
complex, to a hypoxia-responsive element containing 5'-CGTG-3' in the promoter or enhancers of these genes (27, 28,
34, 35, 39). Because HIF-1
can respond to changes in
PO2 at physiological range, this transcription
factor has been considered as one of the most important transcription
factors that are involved in the regulation of oxygen-sensitive genes (27, 34, 35). In previous studies, we demonstrated that HIF-1
mRNA and protein levels were enriched in the renal medulla, a
kidney region exposed to low PO2 (less than 10 mmHg) under physiological conditions (46). RMICs isolated
from this kidney region expressed HIF-1
even under normoxic
conditions and in these cells HIF-1
could be increased in response
to low PO2 or induced by different inducers
such as desferrioxamine and CoCl2 (37).
Therefore, RMICs represent an appropriate cell model to study the
regulation of HIF-1
expression and to explore the functional
significance of its regulatory mechanisms in the renal medulla.
In the first series of experiments, the exposure of these cells to
hypoxia or HIF-1 inducer CoCl2 was found to
significantly increase the levels of HIF-1
protein. In the presence
of a continuous O
protein during hypoxia or CoCl2
incubation was substantially blocked. This suggests that an increase in
the production of ROS in RMICs downregulates HIF-1
levels, which may
impair the adaptive response of many oxygen-sensitive genes to cell
hypoxia. This inhibition of ROS on the response of HIF-1
to hypoxia
in RMICs may be an important mechanism mediating the detrimental
effects of ROS in this kidney region. To further determine whether
intracellularly produced O
levels in RMICs, we examined the effects of an
intracellular stimulator of O
protein levels and its response to hypoxia. It
was found that menadione also significantly reduced hypoxia- or
CoCl2-induced accumulation of HIF-1
protein in RMICs. These results demonstrate that intracellular O
increase in response to hypoxia,
indicating that regardless of the resource of O
levels.
By Northern blot analysis, we found that X/XO did not have any
significant effect on hypoxia- and CoCl2-induced
upregulation of HIF-1 mRNA in RMICs. This suggests that the effect
of ROS on HIF-1
levels may primarily occur at posttranscriptional
levels, which is consistent with the previous studies indicating that ROS directly destabilize HIF-1
, resulting in its degradation through
ubiquitin-proteasome (13, 16, 25). Indeed, treatment of
RMICs with a selective ubiquitin-proteasome inhibitor, CBZ-LLL, significantly increased HIF-1
protein levels. In the presence of
X/XO or menadione, however, the increase in HIF-1
protein levels
induced by CBZ-LLL was substantially abolished. This antagonistic effect of ROS-generating systems on CBZ-LLL-induced inhibition of
ubiquitin-proteasome indicates that ROS may enhance HIF-1
degradation associated with this proteasome system. This view is
supported by a recent study showing that ROS directly activate a 26S
ubiquitin-proteasome enzyme activity in K562 cells (24).
However, recent studies challenged this view regarding ROS-induced
destabilization of HIF-1. Especially, in nonhypoxic activation of
this transcription factor, ROS seem to play a mediating role. For
example, different cytokines or inflammatory factors such as TNF-
,
IL-1
, and thrombin have been reported to increase the mRNA or
protein levels of HIF-1
or enhance its binding activity in several
cell types, and inhibition of ROS production or increased ROS
scavenging substantially blocked their effects on HIF-1
levels or
activity (6, 8, 9). The reason for this discrepancy is
still unclear. It is possible that there exist different regulatory mechanisms responsible for hypoxic and nonhypoxic activation of HIF-1
. Recent studies indicated that the different effects of ROS on
HIF-1
levels or activity may be associated with the extent of
oxidative stress. It has been proposed that moderate oxidative stress induces HIF-1
degradation by proteasomal system, whereas enhanced stress may inhibit the 26S proteasome, increasing HIF-1
levels or activity (23, 24, 26). On the basis of this
view, moderate ROS production under normoxic conditions may decrease HIF-1
levels due to its degradation, but exaggerated production of
ROS during inflammation or stimulation with inflammatory factors would
increase HIF-1
levels or activity due to inhibition of proteasome.
However, the present finding that incubation of the cells with X/XO and
menadione largely decreased HIF-1
levels does not support this view,
because it is obvious that exogenously induced oxidative stress by X/XO
or menadione should not be a moderate oxidative stress in these cells.
Considering a wide spectrum of the action of ROS on different signaling
pathways, the exaggerated ROS production induced by X/XO or menadione
may also alter HIF-1
levels or activity through other related
regulatory pathways such as phosphorylation, cAMP signaling, and other
mechanisms, which have been reported to regulate the activation or
degradation of HIF-1
(8, 9, 26).
It is the diversity of exogenous ROS in stimulating or blocking
HIF-1 production or degradation that prompted us to question the
role of endogenously generated ROS in the regulation of HIF-1
levels
in RMICs under normoxic conditions. The present study found that
incubation of RMICs with either TEMPOL, a cell-permeable SOD mimetic,
or PEG-SOD significantly increased HIF-1
protein concentrations.
These results support the view that endogenously produced
O
levels in these renal cells, which maintains HIF-1
at
appropriate intracellular levels. The results also suggest that
endogenous H2O2 may not decrease HIF-1
levels in RMICs, because H2O2 produced by
dismutation of O
levels. It is
O
levels in RMICs. This
conclusion is strengthened by the results obtained from the experiments
using an OH· scavenger, which showed that scavenging OH· by TMTU
had no effect on HIF-1
levels in RMICs irrespective of the absence
or presence of SOD mimetic TEMPOL. However, these results are not
consistent with those reported in previous studies in some cell lines
such as Hela cells (17). In those studies, the generation
of OH· from H2O2 via the iron-dependent
Fenton reaction was proposed to stimulate the degradation of HIF-1
,
thereby decreasing its levels in the cells. However, because those
studies mainly examined the effects of exogenous
H2O2 on HIF-1
protein levels, rather than
that of endogenously produced H2O2, one should
be cautious to conclude that H2O2 serves as an
intracellular messenger molecule to mediate the redox response of
HIF-1
as discussed above. Furthermore, recent studies demonstrated
that H2O2 at high concentrations can produce
O
levels observed in
those studies may simply be due to the production of
O
compared with cultured normal RMICs. Taken
together, our results suggest that the effect of endogenous
O
levels in RMICs is
independent of H2O2 or OH·.
Next, we addressed whether NAD(P)H oxidase contributes to endogenous
production of O
levels in RMICs. NAD(P)H oxidase was chosen because this enzyme has
been found to be a major enzyme to produce O
protein levels increased in RMICs incubated with DPI, a NAD(P)H oxidase inhibitor that was used to characterize the activity of this enzyme pharmacologically (7), suggesting that NAD(P)H oxidase may be involved in the regulation of HIF-1
. As a flavoprotein
oxidoreductase inhibitor, however, the specificity of DPI to inhibit
NAD(P)H oxidase activity has been often challenged, despite that it was demonstrated to have no effect on other
O
, DPI was even found
to block the stabilization of HIF-1
under certain circumstances
(3, 5, 36). As discussed above, this opposite effect of
DPI on HIF-1
levels may be associated with its use under conditions
with a different extent of oxidative stress. However, the nonspecific
effect may not be ruled out. To address this concern, we performed two
additional series of experiments to confirm the role of NAD(P)H oxidase
in the regulation of HIF-1
levels using a mechanistically different
inhibitor, apocynin, and P22-AS. Consistently, both apocynin and P22-AS
significantly increased HIF-1
levels in RMICs, but they had no
effect on HIF-1
levels. On the basis of these results, we believe
that NAD(P)H oxidase as an endogenous resource of ROS may be
importantly involved in the posttranscriptional regulation of HIF-1
in these renal medullary cells. In fact, recent studies also indicated
that a nonphagocytic NAD(P)H oxidase [namely cytochrome
b-type NAD(P)H oxidoreductase] may serve as an oxygen
sensor to enhance degradation of HIF-1
through production of ROS
(41, 42). It is possible that this NAD(P)H oxidase senses
oxygen and produces O
and ultimate
degradation (41). Recently, this
prolyl-4-hydroxylase-mediated hydroxylation of HIF-1
proline residue
has been demonstrated to be necessary and sufficient for the binding of
this transcription factor to von Hippel-Lindau protein, thereby
resulting in its ubiquitination and degradation by the proteasome
(14, 15, 20). However, the role of NAD(P)H oxidase in
proline hydroxylation of HIF-1
remains to be determined.
The next question we tried to answer was whether
O levels
influences the transcriptional activation of those genes regulated by
this transcription factor. In this regard, previous studies reported that ROS may increase HIF-1
degradation and thereby reduce the transcription of downstream genes (12, 17, 25). However, most of those studies were performed by exogenous addition of oxidants
such as H2O2 and then detection of the changes
in the expression of HIF-1
-targeted genes (12, 17, 22,
30). Little is known regarding the role of endogenously produced
oxidants on the expression of those HIF-1
-targeted genes. With the
use of HO-1 as a prototype gene, which was confirmed as a typical HIF-1
-activated gene (18, 37), the present study
examined the effects of TEMPOL and PEG-SOD on HO-1 mRNA expression.
Because TEMPOL and PEG-SOD have been found to increase HIF-1
levels
as discussed above, it is expected that HO-1 mRNA levels should be increased. Indeed, we demonstrated that HO-1 mRNA expression was upregulated by TEMPOL and PEG-SOD. This suggests that endogenously produced O
in the cells.
In summary, the present studies provide evidence that HIF-1 levels
in RMICs are regulated by cellular redox status in RMICs even under
physiological conditions. NAD(P)H oxidase-derived
O
levels. This redox regulation of HIF-1
may
be one of the essential mechanisms maintaining normal levels of
HIF-1
in renal medullary cells or resulting in tissue or cell injury
during exaggerated oxidative stress in the kidney.
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ACKNOWLEDGEMENTS |
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This study was supported by National Institutes of Health Grants HL-70726 and DK-54927 and Grant 96007310 from the American Heart Association.
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FOOTNOTES |
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Address for reprint requests and other correspondence: A-P Zou, Dept. of Physiology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226 (E-mail: azou{at}mcw.edu).
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.
First published February 20, 2003;10.1152/ajprenal.00017.2002
Received 14 January 2002; accepted in final form 11 February 2003.
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REFERENCES |
---|
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---|
1.
Carter, WO,
Narayanan PK,
and
Robinson JP.
Intracellular hydrogen peroxide and superoxide anion detection in endothelial cells.
J Leukoc Biol
55:
253-258,
1994[Abstract].
2.
Chandel, NS,
Maltepe E,
Goldwasser E,
Mathieu CE,
Simon MC,
and
Schumacker PT.
Mitochondrial reactive oxygen species trigger hypoxia-induced transcription.
Proc Natl Acad Sci USA
95:
11715-11720,
1998
3.
Chandel, NS,
McClintock DS,
Feliciano CE,
Wood TM,
Melendez JA,
Rodriguez AM,
and
Schumacker PT.
Reactive oxygen species generated at mitochondrial complex I.I.I. stabilize hypoxia-inducible factor-1 during hypoxia: a mechanism of O2 sensing.
J Biol Chem
275:
25130-25138,
2000
4.
Chen, YF,
Li PL,
and
Zou AP.
Oxidative stress enhances the production and actions of adenosine in the kidney.
Am J Physiol Regul Integr Comp Physiol
281:
R1808-R1816,
2001
5.
Gleadle, JM,
Ebert BL,
and
Ratcliffe PJ.
Diphenylene iodonium inhibits the induction of erythropoietin and other mammalian genes by hypoxia. Implications for the mechanism of oxygen sensing.
Eur J Biochem
234:
92-99,
1995[Abstract].
6.
Gorlach, A,
Diebold I,
Schini-Kerth VB,
Berchner-Pfannschmidt U,
Roth U,
Brandes RP,
Kietzmann T,
and
Busse R.
Thrombin activates the hypoxia-inducible factor-1 signaling pathway in vascular smooth muscle cells: role of the p22phox-containing NADPH oxidase.
Circ Res
89:
47-54,
2001
7.
Griendling, KK,
Sorescu D,
and
Ushio-Fukai M.
NAD(P)H cxidase: role in cardiovascular biology and disease.
Circ Res
86:
494-501,
2000
8.
Haddad, JJ.
Recombinant human interleukin (IL)-1-mediated regulation of hypoxia-inducible factor-1
(HIF-1
) stabilization, nuclear translocation and activation requires an antioxidant/reactive oxygen species (ROS)-sensitive mechanism.
Eur Cytokine Netw
13:
250-260,
2002[ISI][Medline].
9.
Haddad, JJ,
and
Land SC.
A nonhypoxic, ROS-sensitive pathway mediates TNF--dependent regulation of HIF-1
.
FEBS Lett
505:
269-274,
2001[ISI][Medline].
10.
Hamilton, CA,
Brosnan MJ,
Al-Benna S,
Berg G,
and
Dominiczak AF.
NAD(P)H oxidase inhibition improves endothelial function in rat and human blood vessels.
Hypertension
40:
755-762,
2002
11.
Heberlein, W,
Wodopia R,
Bartsch P,
and
Mairbaurl H.
Possible role of ROS as mediators of hypoxia-induced ion transport inhibition of alveolar epithelial cells.
Am J Physiol Lung Cell Mol Physiol
278:
L640-L648,
2000
12.
Huang, LE,
Arany Z,
Livingston DM,
and
Bunn HF.
Activation of hypoxia-inducible transcription factor depends primarily upon redox-sensitive stabilization of its alpha subunit.
J Biol Chem
271:
32253-32259,
1996
13.
Huang, LE,
Gu J,
Schau M,
and
Bunn HF.
Regulation of hypoxia-inducible factor 1 is mediated by an O2-dependent degradation domain via the ubiquitin-proteasome pathway.
Proc Natl Acad Sci USA
95:
7987-7992,
1998
14.
Ivan, M,
Kondo K,
Yang H,
Kim W,
Valiando J,
Ohh M,
Salic A,
Asara JM,
Lane WS,
and
Kaelin WG, Jr.
HIF- targeted for VHL-mediated destruction by proline hydroxylation: implications for O2 sensing.
Science
292:
464-468,
2001
15.
Jaakkola, P,
Mole DR,
Tian YM,
Wilson MI,
Gielbert J,
Gaskell SJ,
Kriegsheim AV,
Hebestreit HF,
Mukherji M,
Schofield CJ,
Maxwell PH,
Pugh CW,
and
Ratcliffe PJ.
Targeting of HIF- to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation.
Science
292:
468-472,
2001
16.
Kallio, PJ,
Wilson WJ,
O'Brien S,
Makino Y,
and
Poellinger L.
Regulation of the hypoxia-inducible transcription factor 1 by the ubiquitin-proteasome pathway.
J Biol Chem
274:
6519-6525,
1999
17.
Kietzmann, T,
Fandrey J,
and
Acker H.
Oxygen radicals as messengers in oxygen-dependent gene expression.
News Physiol Sci
15:
202-208,
2000
18.
Lee, PJ,
Jiang BH,
Chin BY,
Iyer NV,
Alam J,
Semenza GL,
and
Choi AMK
Hypoxia-inducible factor-1 mediates transcriptional activation of the heme oxygenase-1 gene in response to hypoxia.
J Biol Chem
272:
5375-5381,
1997
19.
Luo, X,
Christie NA,
McLaughlin MA,
Belcastro R,
Sedlackova L,
Cabacungan J,
Freeman BA,
and
Tanswell AK.
H2O2 mediates O2 toxicity in cultured fetal rat distal lung epithelial cells.
Free Radic Biol Med
26:
1357-1368,
1999[ISI][Medline].
20.
Min, JH,
Yang H,
Ivan M,
Gertler F,
Kaelin WG, Jr,
and
Pavletich NP.
Structure of an HIF-1-pVHL complex: hydroxyproline recognition in signaling.
Science
296:
1886-1889,
2002
21.
Okada, H,
Woodcock-Mitchell J,
Mitchell J,
Sakamoto T,
Marutsuka K,
Sobel BE,
and
Fujii S.
Induction of plasminogen activator inhibitor type 1 and type 1 collagen expression in rat cardiac microvascular endothelial cells by interleukin-1 and its dependence on oxygen-centered free radicals.
Circulation
97:
2175-2182,
1998
22.
Panchenko, MV,
Farber HW,
and
Korn JH.
Induction of heme oxygenase-1 by hypoxia and free radicals in human dermal fibroblasts.
Am J Physiol Cell Physiol
278:
C92-C101,
2000
23.
Reinheckel, T,
Sitte N,
Ullrich O,
Kuckelkorn U,
Davies KJ,
and
Grune T.
Comparative resistance of the 20S and 26S proteasome to oxidative stress.
Biochem J
335:
637-642,
1998[ISI][Medline].
24.
Reinheckel, T,
Ullrich O,
Sitte N,
and
Grune T.
Differential impairment of 20S and 26S proteasome activities in human hematopoietic K562 cells during oxidative stress.
Arch Biochem Biophys
377:
65-68,
2000[ISI][Medline].
25.
Salceda, S,
and
Caro J.
Hypoxia-inducible factor 1 (HIF-1
) protein is rapidly degraded by the ubiquitin-proteasome system under normoxic conditions. Its stabilization by hypoxia depends on redox-induced changes.
J Biol Chem
272:
22642-22647,
1997
26.
Sandau, KB,
Fandrey J,
and
Brune B.
Accumulation of HIF-1 under the influence of nitric oxide.
Blood
97:
1009-1015,
2001
27.
Semenza, GL.
HIF-1: mediator of physiological and pathophysiological responses to hypoxia.
J Appl Physiol
88:
1474-1480,
2000
28.
Semenza, GL,
and
Wang GL.
A nuclear factor induced by hypoxia via de novo protein synthesis binds to the human erythropoietin gene enhancer at a site required for transcriptional activation.
Mol Cell Biol
12:
5447-5454,
1992[Abstract].
29.
Stolk, J,
Hiltermann TJ,
Dijkman JH,
and
Verhoeven AJ.
Characteristics of the inhibition of NADPH oxidase activation in neutrophils by apocynin, a methoxy-substituted catechol.
Am J Respir Cell Mol Biol
11:
95-102,
1994[Abstract].
30.
Tyrrell, RM,
and
Basu-Modak S.
Transient enhancement of heme oxygenase 1 mRNA accumulation: a marker of oxidative stress to eukaryotic cells.
Methods Enzymol
234:
224-235,
1994[ISI][Medline].
31.
Wang, GL,
Jiang BH,
Rue EA,
and
Semenza GL.
Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension.
Proc Natl Acad Sci USA
92:
5510-5514,
1995[Abstract].
32.
Wang, GL,
and
Semenza GL.
Characterization of hypoxia-inducible factor 1 and regulation of DNA binding activity by hypoxia.
J Biol Chem
268:
21513-21518,
1993
33.
Wang, GL,
and
Semenza GL.
Purification and characterization of hypoxia-inducible factor 1.
J Biol Chem
270:
1230-1237,
1995
34.
Wenger, RH,
and
Gassmann M.
Oxygen(es) and the hypoxia-inducible factor-1.
Biol Chem
378:
609-616,
1997[ISI][Medline].
35.
Wenger, RH,
Rolfs A,
Marti HH,
Guenet JL,
and
Gassmann M.
Nucleotide sequence, chromosomal assignment and mRNA expression of mouse hypoxia-inducible factor-1.
Biochem Biophys Res Commun
223:
54-59,
1996[ISI][Medline].
36.
Wiesener, MS,
Turley H,
Allen WE,
Willam C,
Eckardt KU,
Talks KL,
Wood SM,
Gatter KC,
Harris AL,
Pugh CW,
Ratcliffe PJ,
and
Maxwell PH.
Induction of endothelial PAS domain protein-1 by hypoxia: characterization and comparison with hypoxia-inducible factor-1.
Blood
92:
2260-2268,
1998
37.
Yang, ZZ,
and
Zou AP.
Transcriptional regulation of heme oxygenases by hypoxia-inducible factor-1 in renal medullary interstitial cells.
Am J Physiol Renal Physiol
281:
F900-F908,
2001
38.
Yang, ZZ,
and
Zou AP.
Homocysteine increases TIMP-1 expression and cell proliferation associated with NADH oxidase in rat mesangial cells.
Kidney Int
63:
1012-1020,
2003[ISI][Medline].
39.
Yu, AY,
Frid MG,
Shimoda LA,
Wiener CM,
Stenmark K,
and
Semenza GL.
Temporal, spatial, and oxygen-regulated expression of hypoxia-inducible factor-1 in the lung.
Am J Physiol Lung Cell Mol Physiol
275:
L818-L826,
1998
40.
Zhang, DX,
Zou AP,
and
Li PL.
Ceramide-induced activation of NADPH oxidase and endothelial dysfunction in small coronary arteries.
Am J Physiol Heart Circ Physiol
284:
H605-H612,
2003
41.
Zhu, H,
Jackson T,
and
Bunn HF.
Detecting and responding to hypoxia.
Nephrol Dial Transplant
17, Suppl 1:
3-7,
2002
42.
Zhu, H,
Qiu H,
Yoon HW,
Huang S,
and
Bunn HF.
Identification of a cytochrome b-type NAD(P)H oxidoreductase ubiquitously expressed in human cells.
Proc Natl Acad Sci USA
96:
14742-14747,
1999
43.
Zou, AP,
Billington H,
Su N,
and
Cowley AW, Jr.
Expression and actions of heme oxygenase in the renal medulla of rats.
Hypertension
35:
342-347,
2000
44.
Zou, AP,
Li N,
and
Cowley AW, Jr.
Production and actions of superoxide in the renal medulla.
Hypertension
37:
547-553,
2001
45.
Zou, AP,
Wu F,
and
Cowley AW, Jr.
Protective effect of angiotensin II-induced increase in nitric oxide in the renal medullary circulation.
Hypertension
31:
271-276,
1998
46.
Zou, AP,
Yang ZZ,
Li PL,
and
Cowley AW, Jr.
Oxygen-dependent expression of hypoxia-inducible factor-1 in renal medullary cells of rats.
Physiol Genomics
6:
159-168,
2001