From the Department of Molecular Biology and Applied
Physiology, Tohoku University School of Medicine, Sendai 980-8575, Japan, the § Laboratory of Environmental Biology, Hokkaido
University School of Medicine, Sapporo 060-8638, Japan, the
Department of Medical Chemistry, Hiroshima University School of
Medicine, Hiroshima 734-8551, Japan, the ** Health
Administration Center, Tohoku University, Sendai 980-8576, Japan, and
the Departments of
Respiratory and
Infectious Diseases and §§ Cardiovascular
Medicine, Tohoku University School of Medicine, Sendai 980-8574, Japan
Received for publication, September 27, 2002, and in revised form, December 27, 2002
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ABSTRACT |
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Heme oxygenase 1 (HO-1) catalyzes heme breakdown,
eventually releasing iron, carbon monoxide, and bilirubin IX Heme oxygenase (EC 1.14.99.3) is the rate-limiting enzyme in heme
catabolism that cleaves heme at the Induction of HO-1 has been extensively studied for the last few decades
by many investigators. In contrast, repression of HO-1 expression has
been largely ignored, despite its physiological importance (3). We have
shown that HO-1 is not induced or rather reduced by heat shock in human
cells (19), whereas rat HO-1 is a heat shock protein (20, 21). The
expression levels of HO-1 are also decreased in human glioblastoma
cells by the treatment with interferon- The inter-species variations in the hypoxic response are of clinical
significance because hypoxia is involved in the pathophysiology of
various disorders, including ischemic heart disease, cerebrovascular disease, cancer, sleep apnea syndrome, and chronic obstructive pulmonary disease, which account for common causes of death and disability in the developed world. Mammalian cells respond to hypoxia
in part by increased expression of several genes coding for
erythropoietin (30), vascular endothelial growth factor (31),
adrenomedullin (32, 33), and glycolytic enzymes (34, 35), all of which
cooperate to protect cells and tissues against the hypoxic state.
Hypoxia-inducible factor-1 (HIF-1) serves as a key regulator that
induces the expression of most of these genes (36).
Transcription repressor Bach1 is a basic leucine zipper (bZip) protein
and forms heterodimers with one of the small Maf proteins (i.e. MafK, MafF, and MafG) that bind to the Maf recognition
element (MARE) (37, 38), which may repress transcription of certain target genes. Recently, Sun et al. (39) have shown that ho-1 is constitutively expressed at higher levels in many tissues of bach1-deficient mice, indicating that Bach1 acts as a
negative regulator of transcription of the mouse ho-1 gene.
In fact, the Bach1-MafK heterodimer binds to the MAREs of the
ho-1 gene enhancers, thereby repressing transcription (39).
Importantly, heme abrogated the repressor function of Bach1 by
inhibiting its binding to the MAREs of the ho-1 gene, which
is consistent with the notion that the in vitro DNA-binding
activity of Bach1 is negatively regulated by direct Bach1-heme
interaction (40). Furthermore, Bach1 is widely expressed in human
tissues that have been analyzed (41).
This study was aimed to clarify the mechanism by which hypoxia
represses the HO-1 expression in human cells. Here we show that hypoxia
induces the expression of Bach1, which is consistently associated with
the repression of HO-1 expression in human cells. Furthermore, we have
identified a MARE in the human HO-1 gene enhancer that is
responsible for the repression by hypoxia and is targeted by Bach1.
Thus, Bach1 is a newly recognized hypoxic regulator and functions as an
inducible repressor for the HO-1 gene in several human cell types.
Cell Cultures--
A human glioblastoma cell line, T98G, was
obtained from the American Type Culture Collection and was cultivated
in minimum essential medium supplemented with 5% fetal bovine serum
(FBS) at 37 °C under 5% CO2/95% room air. To examine
the effect of hypoxia on the expression of heme oxygenase-1, T98G
glioblastoma cells were placed in a chamber filled with 5%
CO2/94% N2/1% O2, as previously reported (33). The cells were cultivated for 6-48 h and were harvested
for RNA extraction. In parallel, T98G cells were cultivated under 5%
CO2/95% room air for the same periods and used as normoxia control. In another series of experiments, T98G cells were exposed to
260 µM desferrioxamine or 150 µM
CoCl2 under normoxia for 6, 12, 18, and 24 h, and were
harvested for total RNA extraction. A549 human lung cancer cells were
obtained from Cell Resource Center for Biomedical Research, Institute
of Development, Aging and Cancer, Tohoku University (Sendai,
Japan), and were cultured in RPMI 1640 medium containing 10%
FBS. HUVECs were obtained from Kurabo and cultured in EGM-2
medium (Takara) containing 2% FBS. C6 rat glioma cells were obtained
from Human Science Research Resource Bank and cultured in Ham's F10
medium containing 15% horse serum and 2.5% FBS. Bovine brain
microvascular endothelial cells (BBMVECs), which are primary
endothelial cells derived from microvessels of the bovine brain, were
obtained from Cell Applications, Inc. (San Diego, CA). BBMVECs were
cultivated in the BBMVEC growth medium (Cell Applications, Inc.)
following the manufacturer's protocol. COS7 monkey kidney cells were
cultured in Dulbecco's modified Eagle's medium, containing 10% FBS,
2 mM L-glutamine, and 4500 mg/liter glucose.
These cell cultures were maintained for 6-48 h at 37 °C under 5%
CO2/95% room air (normoxia) or in a chamber with 5%
CO2/94% N2/1% O2. The number of
viable cells was determined with Cell Counting Kit-8 according to the
manufacturer's protocol (Dojindo).
Northern Blot Analysis--
Total RNA was extracted from
cultured cells by the guanidinium thiocyanate-cesium chloride method
and subjected to Northern blot analysis (42). The northern probe used
for heme oxygenase-1 mRNA was the XhoI/XbaI
fragment ( Western Blot Analysis--
T98G human glioblastoma cells or C6
rat glioma cells were lysed in triple detergent lysis buffer containing
50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.02%
sodium azide, 0.1% SDS, 100 µg/liter phenylmethylsulfonyl
fluoride, 1 µg/ml aprotinin, 1 µg/ml Nonidet P40, and 0.5% sodium
deoxycholate. The cell lysates were centrifuged at 15,000 × g for 10 min, and the supernatant (10 µg of protein) was
analyzed on a SDS-polyacrylamide gel (10%). Nuclear extracts (5 µg
of protein) of T98G cells were also used for Western blot analysis to
confirm the expression of Bach1 protein. The proteins in the gel were
treated with 20% methanol buffer containing 48 mM Tris, 39 mM glycine, and 0.037% SDS and electrophoretically transferred to a polyvinylidene difluoride membrane (Immobilon-P, Millipore Corporation), which was pretreated with the same buffer. Expression of HO-1 and Bach1 was determined with anti-HO-1 antibody (6)
and with anti-Bach1 antibody (37), respectively. The specific
immunocomplexes were detected with a Western blot kit (ECL Plus,
Amersham Biosciences).
HO-1 mRNA Stability Assays--
T98G cells and C6 rat glioma
cells were incubated for 12 h in fresh medium under hypoxia or
normoxia in the presence or absence of CoCl2, followed by
addition of actinomycin D (1 µg/ml). The cells were further incubated
for 2, 4, and 6 h after the addition of actinomycin D and
harvested at each time point for RNA extraction.
Transient Transfection Assays--
A549 lung cancer cells were
seeded in a 6-cm diameter dish 24 h before DNA transfection. Cells
were transfected by the calcium phosphate method with modifications, as
described previously (43). Reporter plasmids used were pHHOL constructs
containing the promoter region of the human HO-1 gene
upstream from the Firefly luciferase gene (43). Two constructs, pHHOL15
and pHHOL14, contain the (GT)15AT(GT)14
repeat in the proximal promoter region (44, 45). The internally deleted
construct pHHOL20 and its derivatives were described previously (43).
Base changes were introduced into the MARE of pHHOL20 by the method
based on polymerase chain reaction (46), yielding pHHOL Electrophoretic Mobility Shift Assay (EMSA)--
T98G
glioblastoma cells were incubated under hypoxia or normoxia for 3-24
h, and their nuclear extracts were prepared as described previously (43). A probe for EMSA was a synthetic
double-stranded hHO-1 MARE (5'-GATTTTGCTGAGTCACCAGTGCCTCCTCAG-3'),
end-labeled with [
EMSA was also performed with a recombinant Bach1 protein, BA1G174-739,
carrying amino acid positions 174-739 of mouse Bach1, fused to
glutathione S-transferase (40). In this series of EMSA, the
hHO-1 MARE was labeled with digoxigenin using the DIG Oligonucleotide 3'-End Labeling kit (Roche Molecular Biochemicals). The
competitors used were chicken Statistics--
Data are shown as mean ± S.E., unless
otherwise stated. The statistical analysis was performed by one-way
analysis of variance and Fisher's protected least significant
difference multiple comparison test.
Repression of Heme Oxygenase-1 by Hypoxia Preceded by Induction of
Bach1 in Human Cells--
We analyzed the effect of hypoxia on the
expression of HO-1 and Bach1 mRNAs in T98G human glioblastoma cells
by Northern blot analysis, because T98G cells are able to respond to
hypoxia by inducing adrenomedullin and express mRNAs for HIF-1
To explore whether the repression of HO-1 expression and induction of
Bach1 expression by hypoxia seen in T98G glioblastoma cells represents
a general response, we repeated similar experiments in A549 human lung
cancer cells and primary culture of HUVECs. It is noteworthy that HIF-1
is functionally activated in HUVECs under our hypoxic conditions (23).
In both cell types, the reduction of HO-1 mRNA levels was
detectable at 12 h after exposure to hypoxia, when the levels of
Bach1 mRNA were higher than those under normoxia (Fig.
2, A and B). The
expression levels of HO-1 mRNA were reduced to the lowest level by
48 h after exposure to hypoxia, whereas the expression levels of
Bach1 mRNA were maintained at higher levels under hypoxia. The
effect of hypoxia on expression profiles of HO-1 and levels of Bach1
mRNA were similar to the results observed in T98G cells although
less marked. Thus, hypoxia induces expression of Bach1 mRNA in
three types of human cells, which is consistently associated with the
hypoxia-mediated repression of HO-1 mRNA expression.
Hypoxic Induction of HO-1 mRNA Expression in Animal
Cells--
We then analyzed the effects of hypoxia on the expression
of HO-1 mRNA in C6 rat glioma cells, BBMVECs, and COS7 monkey
kidney cells (Fig. 3) as well as in
NIH3T3 mouse fibroblasts (data not shown). Hypoxia consistently induced
the HO-1 mRNA expression in these cells, suggesting the species
difference in the regulation of HO-1 expression by hypoxia. Expression
levels of HO-1 mRNA in C6 rat cells were noticeably increased by 48 h of incubation even under normoxia, which may be due to a
certain type of metabolic stress generated during the long-term
culture. In fact, rat HO-1 is a heat shock protein (20). Bach1 mRNA
expression was induced by hypoxia in C6 rat cells and COS7 monkey
kidney cells (Fig. 3, A and C). Bach1 mRNA
expression was undetectable in BBMVECs with a human cDNA probe.
Induction of Bach1 Expression by Other Repressors for HO-1
Expression--
We next examined the effects of interferon- Stability of HO-1 mRNA under Hypoxia--
We then analyzed the
effects of hypoxia on the stability of HO-1 mRNA in T98G human
glioblastoma cells and that in C6 rat glioma cells (Fig.
5), because the hybridization signal
representing human HO-1 gene transcripts is too low under
the basal conditions to detect its reduction by nuclear run-on assays
(43, 50). In T98G cells, the half-life of HO-1 mRNA was about
3 h under normoxia, which is similar to the value determined in
other human cells (12, 23). Hypoxia appeared to reduce the half-life of HO-1 mRNA, and CoCl2 prolonged the half-life of HO-1
mRNA, although these effects were not statistically significant
(Fig. 5B). Thus, the observed repressive effect of hypoxia
on HO-1 mRNA expression is mainly due to the decreased
transcription of the HO-1 gene. It is noteworthy that the
half-life of Bach1 mRNA is much shorter than 2 h.
In C6 rat glioma cells, the half-life of HO-1 mRNA was about
2.5 h under normoxia. CoCl2 induced HO-1 mRNA
expression in C6 cells, as did hypoxia (Fig. 5C). There was
no significant change in the half-life of HO-1 mRNA under hypoxia,
but CoCl2 prolonged the half-life of HO-1 mRNA to
~4.5 h. Thus, the induction of HO-1 mRNA expression in C6 cells
by hypoxia is mainly due to the increased transcription, and the
induction by CoCl2 is in part due to the reduced
degradation rate.
Bach1 as a Repressor for the Human HO-1 Gene--
We next studied
the role of Bach1 in the promoter activity of the human HO-1
gene by transient co-transfection assays in A549 human lung cancer
cells. Co-expression of Bach1 caused significant reduction of the
expression of a reporter plasmid pHHOL15 carrying the 4.5-kb upstream
region (Fig. 6A). Deletion
studies have localized the cis-acting region (positions
Repression of the Human HO-1 Gene Promoter Activity by
Hypoxia--
We then studied the effect of hypoxia on the promoter
activity of the human HO-1 gene by transient expression
assays. Hypoxia significantly reduced the expression of pHHOL15 but not
pHHOL14 (Fig. 7A), whereas
hypoxia consistently induced expression of a construct containing four
copies of hypoxia-response element by more than 3-fold (data not
shown). Thus, the cis-acting region (positions Induction of the MARE-binding Activity by Hypoxia--
Hypoxia
increased the amounts of immunoreactive Bach1 protein in nuclear
extracts of T98G glioblastoma cells in a time-dependent manner (Fig. 8A).
Unexpectedly, Bach1 protein was also increased after a 12-24-h
incubation even under normoxia, which may reflect a preferential
nuclear translocation of Bach1 under a certain metabolic stress
generated during the long-term culture of T98G cells. Bach1 binds to a
MARE as heterodimers with small Maf proteins (37) that are ubiquitously
expressed. EMSA showed that a MARE of the human HO-1 gene
was bound in vitro by nuclear proteins prepared from the
T98G cells exposed to hypoxia (Fig. 8B), indicating that the
nuclear extracts also contain a member of Maf proteins. Importantly,
the MARE-binding activities increased in parallel with the hypoxic
induction of Bach1 protein. In addition, the MARE-binding activities
increased in nuclear extracts prepared from cells after the 12-h
incubation under normoxia, which appears in parallel with the induction
of Bach1 protein. Attempts to demonstrate the supershifted complex of
the detected band with the anti-Bach1 antibody were unsuccessful,
probably due to the properties of the antibody or inaccessibility of
the antibody to the Bach1·Maf complex on the DNA under the conditions
employed. Consequently, we used recombinant Bach1 and MafK proteins to
confirm the involvement of Bach1 in the binding to the MARE of the
human HO-1 gene, because a heterodimer of Bach1 and MafK
binds to a MARE consensus in vitro (37, 40). As shown in
Fig. 8C, the MARE was specifically bound by the complex of
recombinant Bach1 and MafK.
The expression levels of HO-1 mRNA are decreased in human
cells by the treatment with hypoxia, desferrioxamine, or
interferon- The MARE located at An important question that remains to be answered is the physiological
implication for the hypoxia-mediated repression of HO-1 expression that
is usually maintained at relatively low levels. We provide three
explanations that are not mutually exclusive. First, the repression of
HO-1 expression reduces energy expenditure consumed for heme catabolism
because the reaction catalyzed by HO-1 requires at least 3 mol of
oxygen and 4 mol of NADPH to cleave 1 mole of heme (1, 2). Second, it
prevents the local accumulation of carbon monoxide, iron, and bilirubin
IX The inter-species variations in the hypoxic regulation of
HO-1 gene expression are of particular significance, even
though we cannot generalize experimental findings in cultured cells or animal models to the human condition. A simple question has been provoked why hypoxia induces HO-1 expression in animal cells, if the
repression of HO-1 is so important in the host defense, as discussed
for human cells. Many studies have established that hypoxia induces
HO-1 expression in animal cells (see Introduction). Here we also show
the hypoxic induction of HO-1 in rat, bovine, and monkey cells (Fig.
3). Likewise, hypoxia induces HO-1 mRNA expression in NIH3T3 mouse
fibroblasts (data not shown). Thus, the differential regulation of HO-1
expression between human and these animal cells may reflect the
differences in the availability of certain transcription factors for
the HO-1 gene. The inter-species variation might reflect the
defense strategy uniquely developed in humans (19), and its
implications have been discussed in the relevance to the pathogenesis
of cerebral malaria (3).
Importantly, there is at least one report that shows the induction of
HO-1 expression by hypoxia in cultured human dermal fibroblasts (61).
In this case, dermal fibroblasts were exposed to hypoxia in the culture
medium containing 0.5% FBS instead of the maintenance culture medium
that contains high glucose and 10% FBS, suggesting that these cells
might be exposed to the combined stresses of nutrients deprivation and
hypoxia. Moreover, the stabilization of HO-1 mRNA appears to be
responsible for the hypoxic induction of HO-1 in these human dermal
fibroblasts. It is therefore conceivable that expression of HO-1
mRNA is differentially regulated in human cells by hypoxia
depending on cell types.
In summary, hypoxic repression of HO-1 gene expression is
consistently associated with the induction of Bach1 expression in several human cell types. Induction or down-regulation of HO-1 in human
cells by pharmacological means will be a promising strategy for the
treatment of various disorders. Bach1 is an alternate target to
modulate the expression of HO-1. The present study suggests that Bach1
serves as a component of hypoxia-inducible repressor in human cells.
Future studies will be aimed at analyzing whether expression of the
Bach1 gene is under the regulation of HIF-1.
. HO-1
is induced by its substrate heme and various environmental factors,
which represents a protective response against oxidative
stresses. Here we show that hypoxia represses HO-1 expression in
three human cell types but induces it in rat, bovine, and monkey cells,
indicating the inter-species difference in the hypoxic regulation of
HO-1 expression. The hypoxia-mediated repression of HO-1 expression is
consistently associated with the induction of Bach1, a heme-regulated transcriptional repressor, in human cells. Bach1 is a basic leucine zipper protein, forming a heterodimer with a small Maf protein. Expression of HO-1 was also reduced in human cells when exposed to
interferon-
or an iron chelator desferrioxamine, each of which induced Bach1 expression. In contrast, induction of HO-1 expression by
CoCl2 is associated with reduced expression of Bach1
mRNA. Thus, expression of HO-1 and Bach1 is inversely regulated. We have identified a Maf recognition element in the human HO-1
gene that is required for repression of a reporter gene by hypoxia and
targeted by Bach1. Therefore, Bach1 functions as a
hypoxia-inducible repressor for the HO-1 gene, thereby
contributing to fine-tuning of oxygen homeostasis in human cells.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-methene bridge to form
biliverdin IX
, carbon monoxide, and iron (1, 2). Biliverdin IX
is
immediately converted by biliverdin reductase to bilirubin IX
that
is transported to the liver for conjugation and excretion into bile
(3). There are two isozymes of heme oxygenase, heme oxygenase-1
(HO-1)1 and heme oxygenase-2
(HO-2) (4, 5). HO-1 is inducible whereas HO-2 is constitutively
expressed in human cells (6). Expression of HO-1 mRNA is highly
increased in human cells by the substrate heme (7), heavy metals (8,
9), UV irradiation (10), and nitric oxide donors (11-14). Because
bilirubin IX
functions as a natural radical scavenger (15, 16),
induction of HO-1 probably represents a protective response against
oxidative stress. The physiological importance of HO-1 has been
confirmed by the phenotypic consequences of the HO-1-deficient mice
(17) and a patient with HO-1 deficiency (18).
(22). In addition, hypoxia
represses HO-1 mRNA expression in primary cultures of human
umbilical vein endothelial cells (HUVECs), human astrocytes, and human
coronary arterial endothelial cells (23). On the other hand, hypoxia
increased HO-1 expression in rat liver (24) and heart (25) and in
various cultured animal cells, including Chinese hamster ovary cells
(26), rat ventricular smooth muscle cells (27, 28), and rat myocytes (29). These results suggest the inter-species difference in the
regulation of HO-1 gene expression by hypoxia between human and animal cells.
EXPERIMENTAL PROCEDURES
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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64/923) derived from the human heme oxygenase-1 cDNA,
pHHO1 (7). The northern probe used for human Bach1 mRNA was the
PstI fragment of human Bach1 cDNA (41). The rat Bach1
cDNA segment was prepared from C6 glioma RNA by reverse-transcription and polymerase chain reaction (RT-PCR)
using a forward primer (5'-ACAAGGACGGAGCCCTGGCACTGCC-3') and a reverse primer (5'-GAGTCCGTGCTGCAAATGTCACTCC-3'). The primer set was
designed from the rat genome data base (clone AC095903), and the region covered by this primer set shows 89% identity to the mouse Bach1 cDNA sequence (37). The rat Bach1 cDNA fragment of 598 bp was cloned into pCR-BluntII-TOPO (Invitrogen), yielding pCR-rBach1. The
nucleotide sequence of the cDNA insert is identical to that of the
rat genome data base (AC095903). The expression of
-actin mRNA
was examined as an internal control. The probe for
-actin mRNA
was the SmaI/ScaI fragment (nucleotides
124-1050) of a human
-actin cDNA provided by Dr. T. Yamamoto
(Tohoku University). These DNA fragments were labeled with
[
-32P]dCTP (Amersham Biosciences) by the random
priming method and were used as hybridization probes. Total RNA (15 µg per sample) was electrophoresed on 1.0% agarose gels containing 2 M formaldehyde, transferred to nylon membranes filter
(Zeta-probe membrane; Bio-Rad), and fixed with a UV-linker
(Stratalinker 1800; Stratagene). The RNA blot was hybridized with each
32P-labeled probe, as detailed previously (33). Radioactive
signals were detected by exposing the filters to x-ray films (X-AR5;
Kodak) or with a Bioimage Analyser (BAS 1500; Fuji Film Co. Ltd.). The exposure time to x-ray films varied depending of the experiments: about
12 h for the induction of HO-1 and at least 48 h for the repression of HO-1 expression. The intensity of hybridization signals
was determined by photo-stimulated luminescence with a Bioimage Analyzer.
20 (see Fig.
7B). A reporter plasmid, pSV40 promoter-Epo HRE-Luc,
contains four copies of hypoxia-response element
(5'-GATCGCCCTACGTGCTGTCTCA-3'; the core sequence
underlined) and was used as a positive control for hypoxic induction
(47). A549 cells were incubated for 24 h with 8.0 µg of reporter
plasmid DNA and 0.5 µg of internal control DNA (pRL-TK), then
re-fed with fresh medium and incubated for 12 h. The pRL-TK
contains the thymidine kinase promoter region upstream of Renilla
luciferase (Promega). Following the 12-hour incubation, cells were
treated for 12 h under hypoxia or normoxia. Soluble extracts were
prepared from the transfected cells and assayed for luciferase activity
by Dual-Luciferase reporter assay system (Promega). The luciferase
activity was measured with Lumat LB9507 (Berthold).
-32P]ATP (ICN Moravek). Nuclear
extracts (1.0 µg of protein) were incubated with the labeled probe at
37 °C for 10 min in the 40-µl reaction mixture containing 20 mM HEPES (pH 7.9), 0.1 µg/µl poly(dI-dC), 0.1 µg/µl bovine serum albumin, 0.5 mM dithiothreitol, 9 mM MgCl2, 1 mM EDTA, 20 mM KCl, and 4% glycerol (40). The binding reaction was
also performed with the nuclear extracts prepared from untreated T98G
cells in the presence of unlabeled competitors, the probe (hHO-1 MARE),
or hHO-1-MARE mut
(5'-GATTTTTCTTAGTTACCAGTGCCTCCTCAG-3') that is carried by pHHOL
20. The resulting mixture was
separated on a 4% polyacrylamide gel in 0.5× Tris-borate-EDTA buffer
(89 mM Tris, 89 mM borate, and 2 mM
EDTA) buffer at 200 V for 2 h at 4 °C.
-globin enhancer (C
E)
(5'-TCGACCCGAAAGGAGCTGACTCATGCTAGC-3') (48) and control MARE
(5'-GTGGTGCTGAGTCATAGGAGAAG-3') (49). BA1G174-739 (180 ng) and
FLAG-tagged MafK (20 ng) were left on ice for 20 min in 40 µl of the
reaction buffer (40). The digoxigenin-labeled hHO-1 MARE was added to
the above solution with or without an unlabeled competitor, and the
reaction mixture was incubated at 37 °C for 10 min. The
protein·DNA complexes, separated on a 4% polyacrylamide gel,
were transferred onto nylon membranes and were detected using the DIG
Luminescent Detection kit (Roche Molecular Biochemicals).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
and HIF-
that constitute HIF-1 (33). Hypoxia decreased expression
levels of HO-1 mRNA and conversely increased the expression of
Bach1 mRNA (Fig. 1A). In
contrast, hypoxia had no noticeable effect on the expression levels of
-actin mRNA. Under the hypoxic conditions employed, the growth
or survival rate of T98G cells remained unchanged by 48 h (data
not shown). The levels of HO-1 mRNA were decreased by 12 h
after exposure to hypoxia and reached the lowest level by 48 h
(about 36% of the parallel 48-h control) (Fig. 1B). The hypoxia-mediated reduction of HO-1 mRNA expression is associated with the reduction of HO-1 protein levels (data not shown), as observed
in HUVECs (23). Induction of Bach1 mRNA expression was detectable
at 2 h after exposure to hypoxia (Fig. 1A) and reached
the maximum by 12 h (Fig. 1C). The expression levels of Bach1 mRNA remained at the increased levels by 48 h.
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Fig. 1.
Effects of hypoxia on expression of HO-1 and
Bach1 mRNAs in T98G glioblastoma cells. A, Northern blot
analysis. T98G cells were cultivated under normoxia (20% oxygen;
N) or hypoxia (1% oxygen; H) for the indicated
hours and then harvested for RNA preparation. The lane
labeled with 0 contained RNA prepared from the untreated
cells. Top panel, HO-1 mRNA; middle panel,
Bach1 mRNA; bottom panel, -actin mRNA as an
internal control. The data shown are from one of four independent
experiments with similar results. B, relative expression
levels of HO-1 mRNA (means ± S.E., n = 4).
The intensity of hybridization signals in the right panel of
A was quantified with a Bioimage analyzer, and the intensity
representing HO-1 mRNA was normalized with respect to the intensity
for
-actin mRNA in each experiment. The ratio of each normalized
value to that of the control (indicated with 0) is shown as
the relative expression levels of HO-1 mRNA. *, p < 0.05; **, p < 0.01; $$, p < 0.001. C, relative expression levels of Bach1 mRNA (means of
two experiments with similar results). The intensity of hybridization
signals of Bach1 mRNA was quantified as described in
B.
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Fig. 2.
Effects of hypoxia on expression of HO-1 and
Bach1 mRNAs in other human cells. A549 human lung cancer cells
(A) and HUVECs (B) were cultivated under normoxia
(N) or hypoxia (H) for the indicated hours. Shown
are the Northern blot analyses, as described in the legend to Fig.
1.
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Fig. 3.
Induction of HO-1 mRNA expression by
hypoxia in animal cells. C6 rat glioma cells (A),
bovine primary culture of BBMVECs (B), and COS7 monkey
kidney cells (C) were cultivated under normoxia
(N) or hypoxia (H) for the indicated hours. Shown
are the Northern blot analyses.
and an
iron chelator, desferrioxamine, on Bach1 mRNA expression in T98G
glioblastoma cells, as both reagents repress the HO-1 expression in
human cells (22, 23). We also included CoCl2 as a positive
control for the induction of HO-1. Hypoxia and CoCl2
exerted the opposing effects on the HO-1 mRNA expression in HUVECs,
despite that both activated the function of HIF-1 (23). HO-1 mRNA
expression levels were decreased time-dependently by the
treatment with interferon-
(Fig.
4A) or desferrioxamine (Fig.
4B), but remarkably increased by the treatment with
CoCl2 (C). The repression of HO-1 mRNA expression by
interferon-
or desferrioxamine was consistently associated with the
induction of Bach1 mRNA. Thus, interferon-
and desferrioxamine exhibited the effects similar to those of hypoxia on the expression of
HO-1 and Bach1 mRNAs (see Fig. 1). In contrast, Bach1 mRNA levels were reduced time-dependently during the treatment
with CoCl2 and reached the lowest levels at 24 h.
Thus, the inducion of HO-1 mRNA expression by CoCl2 was
associated with the repression of Bach1 mRNA expression. Taken
together, these results indicate that the expression levels of HO-1 and
Bach1 mRNA are inversely regulated.
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Fig. 4.
Effects of
interferon- , desferrioxamine, and cobalt
chloride on HO-1 and Bach1 mRNA expression. T98G human
glioblastoma cells were treated with interferon-
(INF-
, 100 units/ml) in A, desferrioxamine
(DFX, 260 µM) in B or cobalt
chloride (CoCl2, 150 µM) in
C for the indicated hours. Shown are the Northern blots of
HO-1 mRNA (upper panel), Bach1 mRNA (middle
panel), and
-actin mRNA (lower panel). The data
represent one of two experiments with similar results.
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Fig. 5.
Effects of hypoxia and CoCl2 on
the stability of HO-1 mRNA. T98G human glioblastoma cells
(A and B) or C6 rat glioma cells (C
and D) were incubated for 12 h under hypoxia or under
normoxia in the absence or presence of 150 µM
CoCl2. Cells were further incubated for 2, 4, or 6 h
after addition of actinomycin D (1 µg/ml) and harvested for RNA
preparation. Other conditions are the same as in Fig. 1. A
and C, Northern blot analysis (left panel,
normoxia; center, hypoxia; right,
CoCl2). The data shown are from one of three independent
experiments with similar results. B and D,
relative expression levels of HO-1 mRNA. The intensities
representing HO-1 mRNA at the time of addition of actinomycin D
under each condition were considered to be 100%. The data shown are
mean ± S.E. (n = 3). In D, *,
p < 0.05; **, p < 0.01 compared with
normoxia.
4.5 to
4 kb) that is required for the basal promoter
activity and for the Bach1-mediated repression. Incidentally, this
cis-acting region contains the composite enhancer (14) that
consists of the cadmium-responsive element (43, 51) and a putative
MARE. To localize a cis-acting element that is responsible
for the Bach1-mediated repression we used the internally deleted
construct pHHOL20 that contains the upstream cis-acting region (
4.5 to
4 kb) at the position
283 of the human
HO-1 gene (Fig. 6B). Bach1 reduced the expression
levels of pHHOL20 and its derivative pHHOL23 carrying the MARE, but not
a construct pHHOL22, lacking the MARE. Together with the fact that
Bach1 represses the mouse ho-1 gene transcription through
MAREs (39), these results suggest that the identified MARE may be
involved in the repression of the human HO-1 gene by Bach1.
It is noteworthy that the basal expression level of pHHOL23, lacking
the 5' portion of the upstream region, is lower than that of pHHOL20,
suggesting that the entire upstream region is required for the
efficient transcription of the HO-1 gene.
View larger version (26K):
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Fig. 6.
Role of Bach1 in the promoter activity of the
human HO-1 gene. The human HO-1 gene
promoter is schematically shown at top. The composite
enhancer constitutes the cadmium-responsive element (CdRE)
and MARE, and the proximal promoter region contains the potential heat
shock element (HSE) (19), the E box motifs (MTE
and E) (9, 50), and the (GT)n repeat (44, 45).
Note that the internally deleted constructs contain the (GT)n repeat.
A549 lung carcinoma cells were transfected with each reporter construct
together with Bach1 expression plasmid or vector plasmid. Relative
luciferase activity is shown as the ratio to the normalized luciferase
activity obtained with a mock plasmid and pHHOL15 (A) or
pHHOL20 (B). The data are means ± S.D. of five
independent experiments.
4.5 to
4
kb) is required for the reduction of reporter expression by hypoxia.
Moreover, expression of the internally deleted constructs, pHHOL20 and
pHHOL23, carrying the MARE was reduced under hypoxia, but not pHHOL22
(Fig. 7B). Importantly, the hypoxia-mediated repression was
not detectable with a construct, pHHOL
20, carrying a mutated MARE,
suggesting that the MARE located at
4 kb may be required for the
hypoxic repression of HO-1 gene expression. It is also
noteworthy that the basal promoter activity of pHHOL
20 is lower than
that of pHHOL20. Thus, the MARE is also required for the efficient
transcription of the HO-1 gene under basal conditions.
View larger version (31K):
[in a new window]
Fig. 7.
Hypoxia-mediated repression of the human
HO-1 gene promoter function. A549 lung carcinoma
cells were transfected with each reporter construct and incubated under
normoxia or hypoxia. Relative luciferase activity is shown as the ratio
to the normalized luciferase activity obtained with pHHOL15
(A) or pHHOL20 (B) under normoxia. The base
changes introduced into the MARE are shown in bold. The data
are means ± S.D. of five independent experiments.
View larger version (61K):
[in a new window]
Fig. 8.
Induction of the MARE-binding activity by
hypoxia. A, Western blot analysis of the Bach1 protein. Each
lane contained nuclear extracts prepared from T98G
glioblastoma cells exposed to normoxia or hypoxia for the indicated
hours. B, EMSA with nuclear extracts. Nuclear extracts of
untreated T98G cells were incubated with the 32P-end
labeled probe in the absence or presence of an indicated competitor.
The competitors used are the probe itself (hHO-1 MARE) and a mutated
hHO-1 MARE. The first lane shows the MARE-binding activity
detected in untreated T98G cells (0 h). An arrow indicates
the specific protein·DNA complex. C, EMSA with recombinant
Bach1 and MafK. Lane 1 represents a buffer control lacking
proteins. The digoxigenin-labeled hHO-1 MARE was incubated with
recombinant Bach1 and MafK in the absence (lane 2) or the
presence of indicated competitors (lanes 3-10). The
competitors used were the probe itself (lanes 3 and
4), the mutated hHO-1 MARE (lanes 5 and
6), chicken -globin enhancer (C
E)
(lanes 7 and 8), and a control MARE (lanes
9 and 10). The protein·DNA complex is indicated with
an arrow.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, each of which consistently induces Bach1 mRNA
expression. In addition, expression levels of Bach1 mRNA are
decreased by the treatment with CoCl2 that remarkably
induces HO-1 expression. Thus, there is an inverse relationship in the
expression levels between HO-1 and Bach1. These results suggest that
Bach1 may function as a metabolic sensor. Importantly, the present
study identifies Bach1 as a hypoxia-inducible regulator that represses
the transcription of the HO-1 gene in human cells; Bach1
represents a component of the hypoxia-inducible repressor because Bach1
functions as heterodimers with one of small Maf proteins (37-40).
Under our hypoxic conditions, HIF-1, a key regulator in hypoxic
response, is functionally activated in A549 human lung cancer cells as
well as in T98G glioblastoma cells (33) and HUVECs (23). Human cells
therefore fine-tune oxygen homeostasis by inducing Bach1 and HIF-1
in response to hypoxia.
4 kb is required for the hypoxic repression of
the human HO-1 gene expression and is bound by Bach1. Importantly, MARE is also bound by nuclear factor erythroid 2 (NF-E2)
that is a heterodimer of an erythroid-specific subunit (p45) and the
small Maf proteins (48, 52-54). Furthermore, heterodimers of
NF-E2-related factor 2 and one of the small Maf proteins activate transcription of the mouse ho-1 gene by binding to the MAREs
(55-57). Taken together with the overexpression of ho-1 in many
tissues of the bach1-deficient mouse (39), these results
suggest that transcription of the ho-1 gene may vary
depending on the availability of transcriptional activators, such as
NF-E2-related factor 2, and the repressor Bach1. In fact, expression of
HO-1 mRNA is induced by hypoxia in C6 rat glioma cells (Fig.
3A) and monkey kidney cells (Fig. 3C), in which
Bach1 mRNA is also induced by hypoxia.
beyond certain threshold levels in the HO-1-expressing cells and
their surroundings, thereby preventing the tissue damage, as the heme
breakdown products are potentially toxic to cells (58). For example,
bilirubin IX
causes bilirubin encephalopathy in certain newborns
(3). The repression of HO-1 expression could also restrict iron supply to cancer cells or certain pathogens that might be carried by a host
because HO-1 is important in the turnover of iron that is an essential
requirement for cell proliferation. This possibility is plausible for
the repression of HO-1 expression mediated by interferon-
and
desferrioxamine. Third, it represents a mechanism by which the
intra-cellular heme level is maintained at the narrow range by the
balance between HO-1 and Bach1. Importantly, Bach1 functions as a
heme-regulated transcriptional repressor and loses its DNA-binding
activity when bound by heme (39, 40); namely, heme inhibits the
DNA-binding activity of Bach1 (40), thereby leading to de-repression of
the downstream target genes of Bach1. Consistent with this unique
property of Bach1, heme oxygenase activity is inducible by hemin in all
the cultured cells examined, including human, monkey, mouse, pig, and
rat cells (59, 60). Identification of the target genes of Bach1
other than HO-1 will give further insights into the host
defense mechanism against metabolic stress.
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ACKNOWLEDGEMENTS |
---|
We thank Y. Fujii-Kuriyama and E. Ito for the HRE constructs and human Bach1 cDNA, respectively, and M. Yoshizawa for technical assistance.
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FOOTNOTES |
---|
* This study was supported by grants-in-aid for Scientific Research (B), for Exploratory Research, and for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports and Culture of Japan, and by a grant provided by the Uehara Memorial Foundation.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.
This paper is dedicated to the late emeritus Prof. Tamotsu Takishima who was instrumental in initiating the collaborative work on hypoxic response.
¶ Present address: Laboratory of Molecular Pharmacology, Tohoku University School of Medicine, Sendai 980-8575, Japan.
¶¶ To whom correspondence should be addressed: Dept. of Molecular Biology and Applied Physiology, Tohoku University School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan. Tel.: 81-22-717-8117; Fax: 81-22-717-8118; E-mail: shibahar@mail.cc.tohoku.ac.jp.
Published, JBC Papers in Press, January 2, 2003, DOI 10.1074/jbc.M209939200
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ABBREVIATIONS |
---|
The abbreviations used are: HO, heme oxygenase; BBMVEC, bovine brain microvascular endothelial cell; HUVECs, human umbilical vein endothelial cells; HIF, hypoxia-inducible factor; MARE, Maf recognition element; FBS, fetal bovine serum; EMSA, electrophoretic mobility shift assay; NF-E2, nuclear factor erythroid 2.
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