Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226
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ABSTRACT |
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The present
study was designed to test the hypothesis that hypoxia-inducible
factor-1 (HIF-1
)-mediated transcriptional activation contributes
to increased expression of heme oxygenase (HO) genes in renal medullary
interstitial cells (RMICs). By Northern blot analysis, HO-1 mRNA
expression was found to significantly increase in response to reduction
of PO2 in culture medium. However, HO-2 mRNA
was not altered by hypoxia. This hypoxia-induced upregulation of HO-1
mRNA was significantly blocked by HIF-1
inhibition with ferrous
ammonium sulfate. To further determine the role of HIF-1
in the
activation of HO-1, the inducers of HIF-1
were used to address
whether induction of HIF-1
stimulates HO-1 mRNA expression. Both
desferrioxamine and CoCl2 markedly increased HIF-1
mRNA and protein levels and resulted in the upregulation of HO-1 mRNA but
not HO-2. Furthermore, inhibition of HIF-1
degradation by CBZ-LLL,
an inhibitor of ubiquitin-proteasome, significantly increased HIF-1
protein and HO-1 mRNA but not HO-2 in these cells. Using cis-element oligodeoxynucleotide transfection to
specifically decoy HIF-1
and block HIF-1
binding, increased mRNA
expression of HO-1 in response to hypoxia and CoCl2 was
attenuated. In vitro nuclear run-on assays further confirmed that
hypoxia and alterations of HIF-1
mRNA or protein levels
significantly affected the formation of HO-1 mRNA. Taken together, our
results indicate that HO-1, but not HO-2, is transcriptionally
activated by hypoxia through HIF-1
-mediated mechanism in RMICs. This
hypoxia-induced transcriptional activation may be one of the important
mechanisms mediating increased expression of HO-1 in the renal medulla.
hypoxia-inducible factor-1; gene transcription; anoxia
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INTRODUCTION |
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RECENT STUDIES HAVE INDICATED that heme oxygenases (HOs) are involved in the cellular degradation of heme and the production of endogenous CO, biliverdin, and iron in a variety of mammalian tissues (3, 15). CO is one of the important autocrines or paracrines similar to nitric oxide, which may participate in the control of cardiovascular and renal functions and neural activity (11, 12, 14). Biliverdin can be converted to bilirubin, which is of importance in the antioxidant mechanism. It has been demonstrated that there are two catalytically active isozymes of the oxygenase, HO-1 and HO-2, which are encoded by two different genes. Recently, the isoform HO-3 has been found to have 90% homology with HO-2 at the amino acid sequence level, but it displays very little heme-degrading activity (16). HO-1 is an inducible gene present at low levels in the tissues, and its expression can be rapidly and robustly accelerated in response to a variety of stimuli, including a number of physiological and pathological changes such as heat shock, ischemia, radiation, hypoxia, and cellular transformation. HO-2 has been considered as a constitutively expressed gene and relatively uninducible by stimuli (4, 6, 14, 15, 21, 22).
We have demonstrated that HO-1 and HO-2 were expressed more abundantly in the renal medulla compared with the renal cortex (33). The mechanism by which HO expression increased in this kidney region is poorly understood. Numerous studies have indicated that the renal medulla is characterized by a low PO2 level due to countercurrent exchange of O2 through vasa recta and a high metabolic rate in medullary thick ascending limb (5). Given the importance of hypoxia-induced adaptation in the gene expression, low PO2 in the renal medulla may importantly activate the expression of HOs. It has been reported that HO-1 expression, which is primarily regulated at the transcriptional level (3, 24), increased during ischemia or hypoxia in different tissues or organs. However, the mechanisms by which HO-1 is transcriptionally activated in the renal medullary cells are poorly understood.
More recently, a helix-loop-helix transcriptional factor,
hypoxia-inducible factor-1 (HIF-1), which consists of HIF-1 and HIF-1
, has been cloned and characterized as a transcriptional activator of many oxygen-sensitive genes, such as erythropoietin, vascular endothelial growth factor, transferrin, and several
glycolytic enzymes (25, 26, 28, 30). It has been
indicated that HIF-1
is an inducible protein by a decrease in tissue
or cellular O2. HIF-1
is not inducible, but it can be
bound to HIF-1
to form a dimer to activate the transcription of many
genes containing cis hypoxia-response element (HRE) in their
promoter or enhancer regions. We have demonstrated that HIF-1
was
more abundantly expressed in the renal medulla compared with the renal
cortex (34). This raised a question of whether HIF-1
contributes to the increase in the expression of HO genes in this
kidney region. Using the constructs of chloramphenicol
acetyltransferase reporter gene with the HRE of mouse HO-1 genes, Lee
et al. (13) have demonstrated that HIF-1 mediates
transcriptional activation of the mouse HO-1 gene in response to
hypoxia. The role of HIF-1 in the activation of HO-2 has not yet been
studied. The present study was designed to test the hypothesis that
HIF-1 mediates the transcription activation of HO in the renal medulla
of rats. Using renal medullary interstitial cells (RMICs) isolated from the renal inner medulla of Wistar rats, we determined the effect of
hypoxia and changes in HIF-1
levels by pharmacological and molecular
interventions on expression of both HOs. These experiments provided
evidence that HO-1, but not HO-2, is transcriptionally activated by
HIF-1-mediated mechanism in RMICs.
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MATERIALS AND METHODS |
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Isolation and culture of RMICs. RMICs were isolated and cultured as described previously (19, 20). Briefly, inbred male Wistar rats, weighing 300-350 g (Hanlan Sprague Dawley), 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 basal 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, yellow nodules located at the site of injections were carefully dissected. These nodules were minced, trypsinized in a 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) by 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 was changed every 3 days. The cells formed a confluent monolayer in 18-21 days and were then trypsinized and subsequently replanted in flasks. The cells of passages 7 and 8 were used for hypoxia experiments.
A standard staining method as described previously (19, 20) was used to confirm the identity of these cells. The cells grown on coverslips were stained with oil-red O and Sudan black B, and lipid droplets within the cells were stained as a specific marker for these cells. Under a light microscope, we observed that >98% of cells contained rich lipid granules (dark black spots) (Fig. 1). These granules represent a unique and specific feature of RMICs, because other renal cells exhibit very little, if any, lipid staining in their cytoplasm. In addition, the cultured cells had long processes and round or irregularly shaped vacuoles. Nuclei were small and irregular in shape, with deep invaginations. Under an electron microscope, the cells were found to have lipid droplets and multiple vesicular bodies. They had elongated mitochondria, abundant rough endoplasmic reticula, and bundles of actin filaments under the plasma membrane. These light and electron microscopic features were different from those observed in renal tubular cells, lymphocytes, granulocytes, and endothelial cells (19, 20).
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Western blot analysis.
Nuclear extracts were prepared by a modification of the protocol
described by Semenza and Wang (23). The cell pellet was washed with four packed-cell volumes of buffer A (10 mM
Tris · HCl, pH 7.8, 1.5 mM MgCl2, 10 mM KCl)
containing 0.5 mM dithiothreitol (DTT), 0.4 mM phenylmethylsulfonyl
fluoride (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 three
packed-cell volumes 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 by a Bio-Rad
assay with bovine serum albumin standards.
RNA extraction and Northern blot analysis.
The cDNA probes for HIF-1, HO-1, and HO-2 were generated by RT-PCR
with primer pairs designed and synthesized based on the sequences of
rat HIF-1
, HO-1, and HO-2 cDNA in GenBank (accession nos. AF057308
for HIF-1
, M12129 for HO-1, and J05405 for HO-2) (33).
The primer sequence is the following: for HIF-1
: 5'-CGGCGAAGCAAAGAGTCT-3' (sense) and 5'-TGAGGTTGGTTACTGTTG-3' (antisense); for HO-1: 5'-GTCTATGCCCCGCTCTACTTC-3' (sense) and 5'-GTCTTAGCCTCTTCTGACACC-3' (antisense); and for HO-2:
5'-GAATTCGGGACCAAGGAAGCACAT-3' (sense) and
5'-TCTAGACTATGTAGTACCAGGCCAAGA-3' (antisense). Total RNA was extracted
using TRIzol solution (Life Technologies), according to the
manufacturer's protocol. Northern blot analyses of HIF-1
, HO-1, and
HO-2 mRNAs were performed as we described previously (33).
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 membranes (Pirece), and
cross-linked to the membrane by ultraviolet irradiation. The nylon
membranes were first prehybridized with Rapid Hyb buffer (Amersham
Pharmacia) and then probed with 32P-labeled rat HIF-1
,
HO-1, or HO-2 cDNA, respectively, at 65°C for 2.5 h. After being
washed once at room temperature 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.
Cell hypoxia. RMICs were plated in 100-mm2 tissue culture dishes in 5 ml of media 24 h before the experiments to form a subconfluence. To decrease PO2 in the culture medium, the dishes were transferred to a sealed, humidified modular chamber and flushed for 2 or 4 h with 5% CO2-95% N2. PO2 in the culture medium was measured as we described previously (35). PO2 in the culture medium, as measured by an electrode and oxygenmeter, was ~10 Torr after hypoxia for 2 h. After hypoxia, the culture medium was rapidly replaced by TRIzol solution, and then total RNA was extracted.
Decoy of HIF-1.
It has been demonstrated that HIF-1
activates gene expression by
binding to a promoter or an enhancer site, HRE. This cis element contains a -CGTG-consensus sequence. A standard fluorescein-attached HRE containing oligodeoxynucleotides (ODN) was
synthesized with sequences of 5'-GCCCTACGTGCTGTCTCA-3' (sense) and 5'-TGAGACAGCACGTAGGGC-3' (antisense) and
scrambled ODN with sequences of 5'-GCCCTTACAAC TGTCTCA-3' (sense) and
5'-GAGACAGTTGTAAGGGC-3' (antisense) (26, 28). The
fluorescein attachment at the 5'-end was used as an indicator for
transfection into the cells. To make double-strand ODN (dsODN), both
sense and antisense ODNs (100 µM in TE, pH 8.0) were heated at 95°C
for 5 min and then cooled slowly down to room temperature
(17). These dsODNs were wrapped by using cationic
liposomes (GenePORTER Transfection Reagent; GTS) and transfected into
RMICs as described by the manufacturer. dsDNA (10 µg) was first mixed
with 50 µl of liposome and then added to 5 ml of serum-free
incubation medium. The transfection efficiency was evaluated by a
fluorescence microscope (Olympus, Tokyo, Japan) 24 h after
incubation of RMICs with the liposome-dsODN mixtures. Positively
transfected cells (60-80% cells), indicated by a remarkable
intracellular fluorescence, were used to determine the effects of the
HIF-1
decoy on the gene expression of HO-1 and HO-2 induced by
hypoxia or HIF-1
inducers and to prepare the nuclei for in vitro
transcription and nuclear protein extraction.
Nuclear run-on assay.
Nuclei were isolated from RMICs essentially according to a method
previously described (1). Briefly, RMICs (2 × 107 cells/assay) were washed twice with ice-cold PBS,
scraped, and collected in a 15-ml centrifuge tube by centrifugation at
500 g for 5 min at 4°C. Subsequent steps were performed at
4°C. The cells were resuspended in 4 ml of lysis buffer [10 mM
Tris · HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, 0.5%
Nonidet P-40], allowed to stand on ice for 5 min, and were
then centrifuged at 500 g at 4°C for 5 min. Nuclei were
resuspended in 200 µl of glycerol storage buffer [10 mM
Tris · HCl, pH 8.3, 40% (vol/vol) glycerol, 5 mM
MgCl2, 0.1 mM EDTA] and frozen in liquid N2
until used. Frozen nuclei (200 µl) were thawed and mixed with 200 µl of 2× reaction buffer [10 mM Tris · HCl (pH 8.0), 5 mM
MgCl2, 300 mM KCl], 1 mM each of ATP, CTP, and GTP and 100 µCi of [-32P]UTP (3,000 Ci/mmol, Amersham). Samples
were incubated at 30°C for 30 min with shaking and for 5 min in the
presence of 20 units of DNase I (RNase free, GIBCO). Extracted RNA was
resuspended in diethylpyrocarbonate-H2O at 5 × 106 cpm/ml, where cpm is counts per minute. Target DNA
membranes were prepared by denaturation, neutralization, and
immobilization of 10 µg of linearized plasmid DNA containing a 396-bp
fragment of rat HO-1, and a 356-bp fragment of rat
-actin served as
a loading control. Prehybridization and hybridization were performed as
described above in RNA extraction and Northern blot
analysis.
Statistical analysis. Data are presented as means ± SE. The significance of the difference in mean values within and among 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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Effect of hypoxia on HO and HIF-1 mRNA expression in
RMICs.
Figure 2 presents the results of
Northern blot analysis of HIF-1
, HO-1, and HO-2 mRNA. Figure
2A shows a typical autoradiographic document of the membrane
carrying RNAs extracted from RMICs under normoxic and hypoxic
conditions, which were probed with HIF-1
, HO-1, and HO-2 cDNA
probes, respectively. Exposure of RMICs to hypoxia produced a
remarkable increase in HIF-1
and HO-1 mRNA levels. HO-2 mRNA
expression was not altered by hypoxia. Figure 2B summarizes
the data from these experiments. Densitometric analysis shows that
reduction of PO2 in the culture medium for
2 h significantly increased mRNA levels of HIF-1
(n = 6) and HO-1 (n = 6) in RMICs, but
it had no effects on HO-2 mRNA expression (n = 6).
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Effect of HIF-1 induction on HO mRNA expression in RMICs.
We performed the experiments to observe whether HIF-1
inducers
desferrioxamine (DFX) and Cobalt chloride (CoCl2) increase the levels of HO-1 and HO-2 mRNA in RMICs. These cells were incubated with 260 µM DFX and 150 µM CoCl2 for 6 h, and then
total RNA was extracted. Figure
3A shows a typical gel
document of HIF-1
, HO-1, and HO-2 expression by Northern blot
analysis. A significant increase in HIF-1
mRNA was found in DFX- or
CoCl2-treated RMICs. In parallel, HO-1, but not HO-2, mRNA
levels were increased in these cells. These results are summarized in
Fig. 3B. HIF-1
inducers DFX and CoCl2
markedly increased mRNA expression of HIF-1
(n = 6)
and HO-1 (n = 6). Both HIF-1
inducers were without
effect on the levels of HO-2 mRNA.
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Effect of HIF-1 expression inhibition on HO mRNA levels in
RMICs.
It has been reported that ferrous ammonium sulfate (FAS) is an
inhibitor of HIF-1
expression, which blocked DFX-mediated induction
of HIF-1
(27). The present study determined the effect of FAS on HIF-1
expression and HO mRNA levels during cell hypoxia. In these experiments, RMICs were incubated in a hypoxic chamber for
2 h in the presence or absence of 200 µM of FAS. As shown in
Fig. 4A, RMICs subjected to
hypoxia more abundantly expressed HIF-1
and HO-1 compared with
control cells. In the presence of FAS, both HIF-1
and HO-1 mRNAs
were not altered in response to cell hypoxia. These results are
summarized in Fig. 4B. Hypoxia-induced upregulation of
HIF-1
(n = 5) and HO-1 (n = 5) was
inhibited by FAS. However, FAS was without effect on HO-2 mRNA levels.
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Effect of ubiquitin-proteasome inhibition on HO mRNA expression in
RMICs.
Recent studies have indicated that HIF-1 is degraded via the
ubiquitin-proteasome pathway (9, 10). Therefore,
alteration of HIF-1
protein levels by blocking the aforementioned
protease pathway may change HO-1 mRNA expression. In the present study, N-carbobenzoxyl-L-leucinyl-L-leucinyl-L-norvalinal
(CBZ-LLL) was used to specifically inhibit ubiquitin-proteasome
(9), and then the effect on HO mRNA expression was
observed. Pretreatment of RMICs with 10 µM CBZ-LLL for 6 h
dramatically increased HO-1 mRNA levels in these cells, even under
normoxic conditions. However, CBZ-LLL had no effect on the mRNA levels
of HO-2 (Fig. 5A). Because CBZ-LLL primarily acted to block the degradation of HIF-1
protein, HIF-1
mRNA levels were not altered by this protease inhibition. The
above results are summarized in Fig. 5B. CBZ-LLL
significantly increased HO-1 mRNA expression (n = 6),
but it had no effect on the mRNA levels of HO-2 and HIF-1
.
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Effect of hypoxia, CoCl2, and CBZ-LLL on HIF-1
protein expression in RMICs.
By Western blot analysis, HIF-1
protein expression was also found to
increase significantly in RMICs exposed to hypoxia for 2 h,
suggesting that increased transcripts during hypoxia are used to
produce HIF-1
protein. Similarly, the treatment of RMICs with 150 µM CoCl2 for 6 h produced a significant increase in
HIF-1
protein levels in these cells. Moreover, inhibition of
proteasome by 10 µM CBZ-LLL markedly increased HIF-1
levels in
RMICs. All of these results are summarized in Fig.
6. It should be noted that care was taken
to calculate and load nuclear protein amount in each lane, because
there is no positive control to confirm the loading in these nuclear
extracts with very little
-actin.
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Effect of HIF-1 decoy on HO mRNA expression in RMICs.
As shown in Fig. 7, under a fluorescent
microscope, introduction of fluorescein dsODN produced intensive
fluorescence in RMICs. Using this liposome from GTS, 60-80% of
the RMICs were transfected by fluorescein dsODN (Fig. 7C).
These cells were used to determine the effects of dsODNs on HO mRNA
expression in response to hypoxia or CoCl2. As shown in
Fig. 8A, hypoxia induced HO-1
mRNA expression. However, the hypoxia-induced increase in HO-1 mRNA
levels was depressed in the cells transfected with specific dsODN
[dsODN(+)] containing 5'-CGTG-3'. The decoy of HIF-1 had
no effect on the response of HO-2 mRNA levels to hypoxia in these
cells. As a control, scrambled dsODN [dsODN(
)] without
5'-CGTG-3' was used to transfect RMICs, but it had no effect
on the increase in HO-1 mRNA levels induced by hypoxia (Fig.
8B). Figure 8C summarizes the results of these
experiments. Clearly, the decoy of HIF-1 by its specific binding dsODN
blocked the induction of HO-1 mRNA.
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HIF-1-mediated HO-1 transcription induction in vitro.
Using the nuclei prepared from RMICs treated with hypoxia, HIF-1
inducer, inhibitor, or decoy oligodeoxynucleotides, we performed a
nuclear run-on assay in vitro to determine whether HO-1 is
transcriptionally activated by an HIF-1
-mediated mechanism. HO-1
mRNA formed during the nuclear run-on reaction was found to increase
markedly in RMICs exposed to hypoxia for 2 h, but
-actin
transcription was not altered. In the nuclei from the cells treated
with 150 µM CoCl2 and 10 µM CBZ-LLL, newly formed HO-1
mRNA was also markedly increased (Fig.
10A). However, the nuclei
from RMICs transfected with dsODN(+) lost the ability to produce HO-1
mRNA in response to hypoxia. In the cells transfected with dsODN(
),
however, hypoxia still stimulated the production of HO-1 mRNA from
their nuclei (Fig.10B).
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DISCUSSION |
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In the present study, HO-1 and HO-2 mRNAs were detected in cultured RMICs. Under control conditions, the mRNA levels of HO-2 were higher than HO-1. When these cells were exposed to hypoxia for 2 or 4 h, HO-1 mRNA levels were markedly increased but HO-2 mRNA was not significantly altered. These results suggest that HO-1 and HO-2 are present in RMICs and HO-1 is a hypoxia-inducible gene in these cells, which is consistent with the view that HO-1 is inducible and HO-2 is constitutive.
In parallel to the upregulation of HO-1, the mRNA and protein levels of
HIF-1 were also found to increase in response to hypoxia, suggesting
that this transcription factor is probably involved in the
transcriptional activation of HO-1. The increase in HIF-1
mRNA in
RMICs in response to hypoxia indicates that the regulatory response of
HIF-1
may occur at the mRNA level, which is consistent with the
results obtained in in vivo experiments showing the high levels of
HIF-1
mRNA in the renal medulla with low PO2
(34). Although some studies have indicated that HIF-1
is regulated at the post-mRNA level (7, 8, 29), HIF-1
mRNA was also found to increase in response to hypoxia in vivo in mice
or rats (2, 31, 32). It seems that HIF-1
mRNA is
constitutively expressed in transformed cell lines (8,
29), but its expression can be induced by hypoxia or
ischemia in vivo (2, 31, 32). The present study
provides the first evidence that freshly-isolated and cultured RMICs
behave more like cells in vivo, which exhibited elevation of mRNA in
response to hypoxia.
To further determine the role of HIF-1 in the transcriptional
activation of genes in RMICs, additional experiments were performed to
examine the effects of HIF-1
inducers on the expression of HO genes.
It was found that induction of HIF-1
by DFX and CoCl2 significantly increased the HIF-1
mRNA and protein levels and simultaneously increased HO-1 mRNA. In contrast, inhibition of HIF-1
production by an iron donor, FAS, substantially blocked the
hypoxia-induced increase in HIF-1
and HO-1 mRNA in RMICs. Unlike
HO-1, HO-2 mRNA levels were not altered by DFX, CoCl2, and
FAS. It is HO-1 that is activated by pharmacologically increased or
decreased HIF-1
levels in RMICs.
Recent studies have demonstrated that ubiquitin-proteasome is a primary
protease system responsible for the degradation of HIF-1, which may
importantly contribute to the regulation of intracellular HIF-1
levels and thereby to the transcriptional activation of downstream
genes (9, 10). In the present study, HO-1 transcripts were
found to increase markedly even under control conditions, but HO-2 was
not altered, when RMICs were incubated with a selective inhibitor of
ubiquitin-proteasome, CBZ-LLL. This increase in HO-1 mRNA levels
associated with the increase in HIF-1
protein suggests that
transcriptional regulation of the HO-1 gene primarily requires a
structural or functional integrity of HIF-1
protein in RMICs.
In addition to these pharmacological interventions, we also used a
molecular decoy approach to determine the role of HIF-1 in the
transcriptional regulation of HO genes. This anti-gene therapy strategy
can decoy and thereby block the binding of transcription factors to
their binding sites in promoter or enhancer regions by introducing a
synthesized dsODN containing a binding cis element (18). We demonstrated that both hypoxia and HIF-1
induction by CoCl2 increased the mRNA levels of HO-1 to a
much lesser extent in RMICs transfected with a dsODN containing an
HIF-1 binding site, 5'-CGTG-3', compared with control cells.
These results provide evidence that specific blockade of HIF-1
binding decreases an increase in HO-1 mRNA in RMICs, which further
supports our hypothesis that HIF-1 mediates the transcriptional
activation of the HO-1 gene.
To further confirm that the actions of hypoxia and HIF-1 alterations
on HO-1 mRNA expression are due to the changes in newly formed mRNA, in
vitro nuclear run-on assays were performed. Using the nuclei prepared
from RMICs treated with different compounds or hypoxia, newly formed
HO-1 and
-actin mRNAs were detected. It was found that HO-1 mRNA
formation remarkably increased in the nuclei prepared from RMICs
exposed to hypoxia, the HIF-1
inducer CoCl2, and the
ubiquitin-proteasome inhibitor CBZ-LLL, but
-actin mRNA did not
change. Furthermore, the hypoxia-induced increase in HO-1 mRNA
formation was significantly attenuated in the nuclei from the cells
with the decoy of HIF-1
by HRE dsODN. Taken together, these results
further confirm that HO-1 is transcriptionally activated by an
HIF-1
-mediated mechanism. A previous study has demonstrated a
hypoxia-response element in the 5'-flanking region of the mouse HO-1
gene, which contains two potential binding sites for HIF-1
(13). Whether the same binding sites exist in the 5'-flanking region of the rat HO-1 gene remains to be further identified.
In summary, the present studies provided several lines of evidence
indicating that HO-1 expression in RMICs is transcriptionally regulated
via an HIF-1 pathway under normal and hypoxic conditions. First,
alterations of HIF-1 formation or degradation by pharmacological interventions changed HO-1 mRNA levels. Second, the decoy of HIF-1
by transfection of synthesized dsODNs containing 5'-CGTG-3' blocked the hypoxia- or CoCl2-induced increase in HO-1
mRNA levels. Finally, an in vitro transcription assay
demonstrated that hypoxia or induction of HIF-1
increased, but the
decoy of HIF-1
decreased, newly formed HO-1 mRNA in the nuclei of RMICs.
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ACKNOWLEDGEMENTS |
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This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-52112 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 Rd., 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.
Received 7 September 2000; accepted in final form 6 July 2001.
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