From the Turku Centre for Biotechnology, University
of Turku, Åbo Akademi University, FIN-20521 Turku, Finland,
§ Department of Biology, Laboratory of Animal Physiology,
University of Turku, FIN-20014 Turku, Finland, ** Department of Biology,
Åbo Akademi University, FIN-20521 Turku, Finland, and
Institute
of Physiology, University of
Zürich-Irchel, CH-8057, Switzerland
Received for publication, October 4, 2000, and in revised form, February 27, 2001
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ABSTRACT |
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The mammalian hypoxia-inducible factor-1 (HIF-1)
is a heterodimeric transcription factor that controls the induction of
several genes involved in glycolysis, erythropoiesis, and
angiogenesis when cells are exposed to hypoxic conditions. Until now,
the expression and function of HIF-1 The oxygen content, especially in freshwater environments, varies
markedly daily, seasonally, and spatially. Due to the low oxygen
capacitance of water, respiration of organisms and breakdown of organic
material can cause large decreases in oxygen tensions especially during
the night when oxygen-producing photosynthesis does not occur. It is
thus not surprising that the large variations in environmental oxygen
levels have played a significant role in the evolution of fishes.
Therefore, fish have developed various physiological and biochemical
adaptations to enable survival in hypoxic and anoxic environment,
including air breathing organs (1), specialized metabolic pathways
enabling long term anoxic survival (2), and modifications of the
hemoglobin molecules to optimize oxygen transport (3).
Due to the variability of oxygen content in water and the pronounced
role that oxygen has played in the evolution of structure and function
of fishes, they present a unique opportunity to study the evolution,
function, and regulation of oxygen-dependent genes and
their role in the environmental adaptation. Up to the present, this
possibility has been poorly utilized. In mammals, more than 40 hypoxically regulated genes have been characterized (4), including
those for the glucose transporter, several enzymes of the glycolytic
pathway, erythropoietin, transferrin, and the vascular endothelial
growth factor. In contrast, although up-regulation of the synthesis of
several proteins in hypoxic fish has been described (5), the identity
of these hypoxia-inducible proteins is not known.
In mammals, oxygen-dependent gene expression is
transcriptionally regulated by the hypoxia-inducible factor-1
(HIF-1),1 a heterodimer that
consists of two subunits initially called HIF-1 Since hypoxia-inducible transcription activators have not been
characterized in fish, it is obvious that the mechanisms and conditions
by which the up-regulation of protein synthesis occurs in hypoxic fish
have not been elucidated. In mammals, the activation of hypoxic gene
expression occurs at various levels. Although it appears that the
mRNA for HIF-1 Cell Culture and Hypoxia Treatment--
Rainbow trout
hepatocytes were isolated according to Råbergh et al. (24).
The number of cells was calculated using a hemocytometer, and viability
was tested with trypan blue solution (Sigma). The cells were diluted to
a density of 5 × 106 cells/ml. Ten ml of hepatocyte
suspension was incubated overnight at 18 °C before hypoxia
treatments. The human epitheloid carcinoma cell line HeLa was cultured
in Dulbecco's modified Eagle's medium (high glucose; Life
Technologies, Inc.) supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. The rainbow trout
gonad (RTG-2 (25)) and chinook salmon embryonic (CHSE-214 (26))
cells were cultured in HEPES-buffered Dulbecco's modified
Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml
penicillin, 2 mM L-glutamate, 100 µg/ml
streptomycin, and 0.075% NaHCO3. All fish cells were grown
at 18 °C under air or air supplemented with 1% CO2,
whereas human cells were grown at 37 °C in a multi-gas incubator
under normal gas pressure of 21% O2 supplemented with 5%
CO2. In most experiments, fish cells were exposed to
hypoxia (1% O2, 1% CO2, 98% N2)
for 4 h at 18 °C and HeLa cells at 37 °C (1%
O2, 5% CO2, 94% N2). When the
effect of oxygen tension on HIF-1 Cloning of Rainbow Trout HIF-1 Generation of Polyclonal Antibodies against Rainbow Trout
HIF-1
The lyophilized N- and C-terminal RT-HIF antigens (divided into
portions of 0.1 mg) were dissolved in PBS, mixed with an equal volume
of complete Freund's adjuvant, and injected subcutaneously into
rabbits (immunizations were performed in Cancer Research Center,
Russian Academy of Medical Sciences, Moscow, Russia). After 1 month, the immunization was boosted with 0.1 mg of antigen every other
week for 3-6 months. The collected serum was tested with Western blot
analysis. Sera recognizing rainbow trout HIF-1 Preparation of Nuclear Extract and Electrophoretic Mobility Shift
Assay (EMSA)--
Nuclear extracts of cells treated under normoxic and
hypoxic conditions were prepared according to Semenza and Wang (30). Electrophoretic mobility shift assay was performed according to Kvietikova et al. (31). Since no fish sequences were
available, the sense and antisense strands for the HIF-1 Southern and Northern Blot Analysis--
Genomic DNA was
isolated from rainbow trout liver according to Boyle (32). The DNA was
digested with two different restriction enzymes, EcoRI and
HindIII. Digested DNA was run on a 0.7% agarose gel,
blotted to a Hypond-N (Amersham Pharmacia Biotech) filter, and
hybridized with a 32P-labeled SacI DNA fragment
of rainbow trout HIF-1
Northern blot analysis was performed after denaturation of total RNA
using glyoxal (33). Denaturated RNA was run on a 1.0% agarose gel,
blotted to a Hypond-N filter, and hybridized with a
32P-labeled HincII DNA fragment of rainbow trout
HIF-1 Western Blot Analysis--
Protein samples (10-30 µg/well)
were resolved by denaturing electrophoresis on 7.5% SDS-polyacrylamide
slab gels (34) and transferred to a nitrocellulose membrane (Schleicher
& Schuell) according to Sambrook et al. (33). The membrane
was blocked for 2 h in 3% nonfat dry milk in PBS, 0.3% Tween 20, rinsed, and subsequently incubated with the primary antibody diluted in
1% bovine serum albumin, PBS ranging from 1:100 to 1:1000 for 1 h at room temperature. The membrane was washed with PBS, 0.3% Tween 20 and incubated with the secondary antibody (horseradish
peroxidase-conjugated anti-rabbit antibody, Amersham Pharmacia Biotech)
diluted 1:10,000 in 3% nonfat milk in PBS, 0.3% Tween 20. After
washing the membrane, the rainbow trout HIF-1 Cloning of Rainbow Trout HIF-1
Total cDNA of rainbow trout (rt) HIF-1
Although the overall similarity of the predicted amino acid sequence of
rtHIF-1
The cloning of rtHIF-1 Rainbow Trout HIF-1 The HIF- Characterization of Antibodies Produced against Rainbow Trout
HIF-1
To investigate whether the C-terminal rainbow trout anti-HIF-1 Stabilization of HIF-1
Consequently, the antibodies were used to investigate how oxygen levels
affect the amount of HIF-1
In mammals, the rapid degradation of HIF-1
In conclusion, our results show that HIF-1 have not been studied in fish,
which experience wide fluctuations of oxygen tensions in their natural
environment. Using electrophoretic mobility shift assay, we have
ascertained that a hypoxia-inducible factor is present in rainbow trout
cells. We have also cloned the full-length cDNA (3605 base pairs)
of the HIF-1
from rainbow trout with a predicted protein sequence of
766 amino acids that showed a 61% similarity to human and mouse HIF-1
. Polyclonal antibodies against the N-terminal part (amino acids 12-363) and the C-terminal part (amino acids 330-730) of rainbow trout HIF-1
protein recognized rainbow trout and chinook salmon HIF-1
protein in Western blot analysis. Also, the human and
mouse HIF-1
proteins were recognized by the N-terminal rainbow trout
anti-HIF-1
antibody but not by the C-terminal HIF-1
antibody. The
accumulation of HIF-1
was studied by incubating rainbow trout and
chinook salmon cells at different oxygen concentrations from 20 to
0.2% O2 for 1 h. The greatest accumulation of
HIF-1
protein occurred at 5% O2 (38 torr), a typical
oxygen tension of venous blood in normoxic animals. The protein
stability experiments in the absence or presence of a proteasome
inhibitor, MG-132, demonstrated that the inhibitor is able to stabilize
the protein, which normally is degraded via the proteasome pathway both
in normoxia and hypoxia. Notably, the hypoxia response element of
oxygen-dependent degradation domain is identical in
mammalian, Xenopus, and rainbow trout HIF-1
proteins,
suggesting a high degree of evolutionary conservation in degradation of
HIF-1
protein.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
and HIF-1
(6, 7).
HIF-1
is unique for the hypoxia response, whereas HIF-1
turned
out to be identical to the aryl hydrocarbon nuclear translocator
(ARNT), which acts as a dimerization partner also for other
transcription factors, among them the aryl hydrocarbon receptor (AhR,
dioxin receptor (8)). Both HIF-1
and ARNT belong to the
basic-helix-loop-helix (bHLH)-PAS family of proteins. All of these
proteins have characteristic N-terminal bHLH and PAS domains. The bHLH
domain is required for DNA binding and dimerization, whereas PAS domain
is involved in heterodimerization, DNA binding, transactivation, and
probably also in HSP90 ligand binding (9, 10). Several other members of
bHLH-PAS family of proteins in mammals have recently been cloned that
show a high homology to the HIF-1
such as HIF-2
, also termed EPAS
(endothelial PAS domain protein (11)), HLF (HIF-1
-like factor (12)),
HRF (HIF-related factor (13)), MOP2 (member of PAS superfamily 2 (14)),
and HIF-3
(15). Hitherto, there are no reports on a
hypoxia-inducible factor in fish. The previously characterized bHLH-PAS
family proteins in fish play a role in transcriptional regulation
induced by xenobiotics. To date, fish aryl hydrocarbon receptor (AhR)
and at least two isoforms of its dimerization partner ARNT have been
cloned (see Ref. 16).
is constitutively expressed (17, 18), the levels
of HIF-1
protein are markedly higher in hypoxic than in normoxic
conditions. Under normoxic conditions the HIF-1
protein is rapidly
ubiquitinated and degraded by the 26 S proteasome, the half-life being
less than 5 min (19, 20). However, a shift of cells to hypoxic
conditions stabilizes and enables HIF-1
protein to translocate from
the cytoplasm to the nucleus, where it heterodimerizes with HIF-1
(21). In addition, hypoxic conditions enhance the transactivating
function of HIF-1
(22, 23). Although the stabilizing effects of
hypoxia on the HIF-1
protein have been clearly demonstrated, the
exact relationship between oxygen tension and the stability of the
protein has not been elucidated. Furthermore, the possible differences
in the oxygen thresholds of the hypoxia response between different cell types have not been investigated, and the possible effects on the HIF-1
response of previous acclimation to hypoxic conditions have remained
unclarified. These questions are conveniently investigated using fish,
which experience large variations in the ambient oxygen levels in their
normal life. To enable such investigations, we have in the present
study cloned and characterized the first fish HIF-1
protein from
rainbow trout. Using recombinant fish HIF-1
protein, we have
generated polyclonal antibodies recognizing fish HIF-1
protein.
These antibodies have been utilized to investigate the oxygen tension
dependence of HIF-1
protein levels in several types of fish cells.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
stability was studied, the fish
cells were exposed to different concentrations of oxygen (20, 10, 5, 2.5, 1, and 0.2%) supplemented with 1% CO2 for 1 h
at 18 °C. For studying the degradation of HIF-1
in fish cells by
the 26 S proteasome, a proteasome inhibitor MG-132 (Peptide Institute,
Inc.) was used. Fish cells were first exposed to hypoxia (1%
O2, 1% CO2, 98% N2) for 4 h
at 18 °C in the absence or presence of 10 µM MG-132.
For re-oxygenation experiments, a portion of the cells already treated under hypoxia in the presence or absence of MG-132 were transferred to
air supplemented with 1% CO2 and incubated for 30 min.
cDNA--
Total RNA was
isolated from RTG-2, CHSE-214 cells, and freshly isolated rainbow trout
hepatocytes using the RNAzolTMB method (TEL-TEST Inc.)
based on the method described by Chomczynski and Sacchi (27). cDNA
synthesis was performed with 5 µg of total RNA using avian
myeloblastosis virus reverse transcriptase (Promega) and oligo(dT)
primers (Invitrogen) according to the manufacturer's recommendations.
This cDNA was used as a template in reverse transcription-PCR, in
which the primers were based on the mouse HIF-1
sequence. The primer
pair of the ba1 primer within the basic region of HIF-1
and
the hif375 primer distal to the PAS B region of HIF-1
sequence (Table I) produced two 1100-base pair
RT-PCR fragments. The nucleotide sequence of these two products (termed
rba2 and rba5) differed only by 1%. Both PCR fragments were then
cloned into a pGEM-T vector (Promega) according to manufacturer's
instructions. The rba5 DNA fragment was used as a probe to screen a
juvenile rainbow trout UNI-ZAPTMXR cDNA library (a kind
gift of Dr. Thomas Chen, University of Connecticut). To find the
correct 5' and the 3' ends of the cDNAs, we used 5'-3' rapid
amplification of cDNA ends (RACE) according to Frohman et
al. (28) using the primers listed in Table I. Briefly, 5 µg of
total RNA from rainbow trout hepatocytes was reverse-transcribed either
with the XSC-dT17 primer (for 3' RACE) or the RAC1
primer (for 5' RACE). In 3' RACE, the cDNA was then amplified by
PCR using the XSC-dT17 and PAC3 PCR primers. In 5' RACE,
the RAC1 primer was removed using 30-kDa Centricon concentrators (Millipore Inc.), and the cDNAs were polydedioxyadenylic
acid-tailed using terminal deoxynucleotidyltransferase. The cDNAs
were first amplified with the XSC-dT17 and RAT5 primer pair
followed by a second round of amplification with the XSC and ATF3
primer pair.
PCR primers used in RT-PCR (ba1 and hif375) and 5'-3' RACE
DNA sequence
(GenBankTM accession number x95580). Adaptor primers
xsc-dT17 and XSC have been previously published by Frohman
et al. (28). Primers RAC1, PAC3, RAT5, and ATF3 for 5'-3'
RACE were designed based on rainbow trout HIF-1
DNA sequence (newly
deposited as a result of this work, GenBankTM accession
number AF304864).
--
Two different peptides were constructed to produce
antibodies against both the N-terminal and the C-terminal part of
HIF-1
. The rainbow trout HIF-1
cDNA, called r33a, cloned into
a Bluescript KS+ vector (Stratagene) was cut with either
SacI or HincII. The SacI fragment of
the rainbow trout HIF-1
cDNA spanning the N-terminal amino acids
12-363 was cloned in-frame into the SacI site of the bacterial overexpression vector pET-22b(+) (Novagen). Similarly, the
HincII fragment of rainbow trout HIF-1
cDNA spanning
the C-terminal amino acids 330-730 was cloned in-frame into the Klenow blunt-ended NcoI-EcoRI site of the expression
vector pET-22b(+). This HincII fragment was also in-frame
with the His tag present in the pET-22b(+)vector. The bacterial strain
BL21 (DE3) plysS (Novagen) was transformed with the plasmids. Cells
were cultured in the presence of 1 mM
isopropyl-1-thio-
-D-galactopyranoside for 3 h and
collected by centrifugation. The overexpressed proteins were extracted
according to Soncin et al. (29). The N-terminal RT-HIF12-363 antigen was further purified with preparative SDS-polyacrylamide gel electrophoresis, excised, pressed with a syringe
through a hypodermic needle, and lyophilized. Due to the formation of
inclusion bodies, the C-terminally His-tagged RT-HIF360-730 antigen was further purified by first
washing with 0.2% Triton X-100 in buffer A (20 mM KCl, 100 mM K2HPO4, pH 7.4). The
His6-tagged protein was then purified using metal (Ni2+) chelate affinity chromatography under denaturating
(6 M urea) conditions according to the manufacturer's
recommendations (Novagen). The affinity chromatography-purified protein
was finally desalted and lyophilized.
protein were further
purified. The purification was performed using
N-hydroxysuccinimide-activated SepharoseTM high
performance 1- and 5-ml columns (Amersham Pharmacia Biotech) according
to the manufacturer's recommendations. The serum was first negatively
purified against total protein extract of the Escherichia
coli strain BL21 (DE3) plysS. Further purifications were carried
out using C-terminal HIF-1
protein. This protein could be used for
purification of both antibodies, because the N-and C-terminal HIF-1
antigens overlapped by 30 amino acids, thus producing antibodies that
partially included the same epitope recognition site.
binding
sites in the promoter region of the human erythropoietin gene were used (31). For competition experiments, a 100- or 500-fold excess of
unlabeled oligonucleotides was added before the addition of the labeled
probes. For supershift experiments, 1 µl of preimmunization serum or
serum containing the polyclonal antibody was added to the completed
electrophoretic mobility shift assay reaction mixtures and incubated an
additional 30 min on ice before electrophoresis.
cDNA using standard methods (33).
cDNA using standard methods (33).
protein was detected
using enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
cDNA--
RT-PCR was used to
generate rainbow trout-specific HIF-1
gene fragments. Two isoformic
cDNAs (rba2 and rba5) with sizes of 1100 base pairs were identified
(Table II). These cDNA fragments had
66 and 67% nucleotide sequence identity to human HIF-1
,
respectively, and were therefore considered to be the first fish
HIF-1
sequences. The rba5 DNA fragment that had slightly higher
sequence homology to human HIF-1
cDNA was used as a probe in
screening a cDNA library from juvenile rainbow trout. As shown in
Fig. 1A, four cDNA clones were obtained that had similar and overlapping cDNA sequences. These cDNA sequences were missing the 5' end of the cDNA,
including ATG, the start codon. The missing 5' end of cDNA sequence
was isolated by 5'-3' RACE. Furthermore, the previously found 3' end of
the cDNA could be verified. The 3' end had an atypical
polyadenylation signal (CATAAA) 5' to the normal site of
polyadenylation. In addition, the 3'-untranslated region contained only
three AUUUA mRNA instability elements in rainbow trout HIF-1
,
whereas human HIF-1
has been shown to contain eight such elements
(7).
A cDNA sequence comparison between two rainbow trout RT-PCR
products (rba2 and rba5) and human HIF-1 cDNA (nucleotides
345-1383, GenBankTM accession number U22431)
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Fig. 1.
cDNA cloning of rainbow trout
HIF-1 . A, schematic presentation of
different rainbow trout HIF-1
cDNA clones. The length of
different cDNAs is related to the length of HIF-1
mRNA,
where +1 corresponds to the 5' end of the transcript. B,
comparison of the amino acid identities between rtHIF-1
, human (h)
HIF-1
, Xenopus (x) HIF-1
, and human
HIF-2
. Amino acid identities are presented as percentages. The
number of amino acid changes by insertions (ins) or
deletions (del) compared with rtHIF-1
is indicated below
the amino acid identities. C, comparison of the predicted
amino acid sequences of rainbow trout HIF-1
(GenBankTM
accession number AF304864) to Xenopus laevis HIF-1
(GenBankTM accession number CAB96628), human
HIF-1
(GenBankTM accession number AAC68568), rat
(r) HIF-1
(GenBankTM accession number
AAD24413), and mouse (m) HIF-1
(GenBankTM
accession number CAA70306). Nuclear localization signals
(NLS-N, NLS-C (21)), bHLH and
PAS A/B regions, transactivation domains
(TAD-N, TAC-C), and the
oxygen-dependent degradation domain (ODD) are
indicated above the sequence comparisons. The comparison is made using
ClustalW multiple sequence alignment and BOXSHADE programs (BCM).
was 3605 base pairs long,
with an open reading frame of 766 amino acids. Thus, the rainbow trout
HIF-1
was slightly smaller in size than the corresponding HIF-1
in human (826 amino acids), mouse, or rat (810 amino acids). The
predicted rtHIF-1
amino acid sequence (Table
III) had a 61% similarity to the human,
mouse, and rat HIF-1
, about 52% similarity to the human and mouse
HIF-2
, and 46% similarity to the human and mouse HIF-3
.
Amino acid comparison between different HIFs
was much closer to human HIF-1
than HIF-2
, the
sequence corresponding to the bHLH domain had a similar homology to
both human HIF-1
and to HIF-2
(Fig. 1B).
Interestingly, the homology to the human proteins was greater than to
the HIF-1
from the amphibian Xenopus laevis. However, the
bHLH/PAS A/B regions appear to be relatively well conserved, with
70-90% similarity between the different proteins. Another conserved
sequence was the hypoxia response element of the
oxygen-dependent degradation domain, which comprises of
amino acids 557-571 in human HIF-1
protein (35): it was identical
in all the HIF-1
proteins examined. In contrast, the transactivation
domains (TAD), especially the TAD-N (20, 22) sequence, varied
considerably between the different proteins; there was less than 40%
identity in the TAD-N sequence between the proteins (Figs. 1,
B and C).
indicates that the encoding gene is present
in rainbow trout genome. Its presence was further ascertained using
Southern blot analysis (Fig. 2). The
cleavage of rainbow trout genomic DNA with either EcoRI or
HindIII restriction enzymes produced a band of 8-kilobase
pairs in the rainbow trout genome (Fig. 2).
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Fig. 2.
HIF-1 gene is
present in rainbow trout genomic DNA. A, Southern blot
analysis was performed on genomic DNA isolated from rainbow trout using
the SacI fragment of rainbow trout cDNA as a probe. The
genomic DNA was cleaved using two restriction enzymes, EcoRI
and HindIII. The DNA marker indicates the size of genomic
DNA fragments in kilo base pairs (kbp).
mRNA Is Constitutively
Expressed--
Using Northern blot analysis, the expression pattern of
HIF-1
was determined in rainbow trout gonad (RTG-2) and chinook
salmon embryonic (CHSE-214) cells exposed to hypoxia for 2 and 4 h
(Fig. 3). The levels of HIF-1
mRNA, normalized to 18 S rRNA levels, showed no change during a 2-h
hypoxia treatment and only a slight decrease after a 4-h hypoxia
treatment. This is in agreement with the notion that HIF-1 activation
during hypoxia is due to posttranslational mechanisms (17, 18, 36).
Increased mRNA levels have also been reported (37- 39), possibly
as a result of increased stability of mRNA in hypoxia (4).
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Fig. 3.
HIF-1 mRNA is
constitutively expressed. The amount of HIF-1
mRNA was
quantified using Northern blot analysis after treatment of rainbow
trout gonad (RTG-2) and chinook salmon embryonic (CHSE-214) cells under
normoxia (0 h) and hypoxia (1% O2) for 2 and 4 h. The
level of HIF-1
mRNA was normalized to the ribosomal 18 S rRNA
(lower panel). The level of HIF-1
mRNA under normoxia
was compared with the level of HIF-1
mRNA under hypoxia for 2 and 4 h. The level of HIF-1
mRNA under normoxia was used as
control (defined as 1.0). Numerical data are based on four independent
experiments.
DNA Binding Complex in Rainbow Trout Is Smaller than in
Mammals--
Since the hypoxia response elements (HREs) of
hypoxia-inducible genes have not been characterized in rainbow trout,
we performed electrophoretic mobility shift assay using the HRE of the
human erythropoietin gene (Fig.
4A). As a control for HIF-1
DNA binding, we used nuclear extracts from hypoxia-treated HeLa cells
(Figs. 4, A and C). In fish RTG-2 cells, the
induction of the HIF-1
complex in nuclear extracts was detected
after 2 h of hypoxia treatment (1% O2), and the
amount of HIF-1
complex was further increased after a 4-h treatment
(Fig. 4A). Interestingly, the HIF-1
complex of nuclear
extracts isolated from RTG-2 cells migrated faster in a 4%
nondenaturing polyacrylamide gel than that isolated from HeLa cells,
suggesting a lower molecular weight of the HIF-1 complex in rainbow
trout. This is in agreement with data obtained from the HIF-1
cDNA cloning, suggesting that HIF-1
protein in rainbow trout has
smaller molecular weight than the corresponding human HIF-1
protein.
Both isoforms of HIF-1
(ARNT) in rainbow trout are also smaller (70 and 79 kDa (40)) than the corresponding mammalian proteins (91 to
94 kDa (6)). The formation of the HIF-1
-HRE complex was
also observed after 4 h of hypoxia in primary-cultured rainbow
trout hepatocytes (Fig. 4B). The specificity of the HIF-1
DNA binding was demonstrated for the hepatocytes by adding a 500 M excess of unlabeled erythropoietin HRE probe before
adding the 32P-end-labeled erythropoietin HRE probe. In
the presence of unlabeled HRE, the HIF-1
binding to the radiolabeled
DNA was inhibited. In the presence of serum containing the rainbow
trout HIF-1
antibody, the mobility of the HIF-1
-HRE complex was
further slowed down, indicating that the antibody was specific to the
C-terminal part of HIF-1
bound to the HRE-HIF complex (Fig.
4B).
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Fig. 4.
Electrophoretic mobility shift analysis of
HIF-1 DNA binding in rainbow trout. A, a comparison of HIF-1
complex induction between nuclear extracts isolated from HeLa or
rainbow trout gonad (RTG-2) cells after hypoxic (1% O2)
treatment of 2 and 4 h. In addition to HIF-1, activating
transcription factor-1 (ATF-1), cAMP-responsive element
binding-1 (CREB-1), nonspecific band (NS), and
free probe are indicated by lines. B and
C, to confirm the specificity of HIF-1 binding of rainbow
trout to HRE of human erythropoietin gene, competition and supershift
experiments were performed using nuclear extracts from primary-cultured
rainbow trout hepatocytes (B) or HeLa cells (C)
after treatment under normoxia ( ) or hypoxia (+) for 4 h
(1). A 500 M excess (rainbow trout hepatocytes)
or 100 M excess (HeLa cells) of unlabeled human HRE of
erythropoietin gene was used in competition experiment (2).
For supershift experiment (3), nuclear extracts of rainbow
trout hepatocytes were incubated together with either preimmunization
serum (P) or serum containing C-terminal rainbow trout
anti-HIF-1
antibody (S), whereas nuclear extracts of HeLa
cells were incubated together with preimmunization serum or serum
containing mouse anti-HIF-1
(IgY) antibody (48). A supershifted band
is indicated by an asterisk.
Protein--
Two polyclonal antibodies were generated against
the rainbow trout HIF-1
protein, one against the N-terminal part and
another against the C-terminal part. Initial characterization of these antibodies using crude serum indicated that the antibody produced against the N-terminal part of the recombinant rainbow trout HIF-1
protein recognized a hypoxia-inducible protein both in HeLa cells and
in rainbow trout hepatocytes (Fig.
5A). In contrast, the antibody produced against the C-terminal part only recognized fish proteins in a
hypoxia-dependent manner (Figs. 4B and
5A). This result is expected, since the N-terminal part of
the protein shows more homology across the animal groups than the
C-terminal part.
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Fig. 5.
Characterization of antibodies raised against
N- or C-terminal part of rainbow trout HIF-1
protein. A, Western blot analysis was performed using
nuclear cell extracts from HeLa cells or primary cultured rainbow trout
hepatocytes after treatment of cells under normoxia or hypoxia (1%
O2) for 4 h. 30 µg of nuclear cell extracts was
loaded into each well. Purified N- or C-terminal rainbow trout
anti-HIF-1
antibodies were used as primary antibodies (1:500).
B, induction of HIF-1
protein in two trout cell lines,
rainbow trout gonad (RTG-2) and chinook salmon embryonic (CHSE-214)
cells, after hypoxia (+) treatment for 4 h. Unpurified C-terminal
rainbow trout anti-HIF-1
antibody (1:1000) was used as primary
antibody.
antibody recognized a HIF-1
protein in fish cell lines, two trout
cell lines were exposed to hypoxia for 4 h. The antibody recognized a hypoxia-inducible protein of 95 kDa in both rainbow trout
gonad (RTG-2) and chinook salmon embryonic (CHSE-214) cells (Fig.
5B) based on calculation of the mobility of HIF-1
protein to the standard molecular weights.
Protein Occurs under Physiological Oxygen
Concentrations--
Since the initial screening indicated that the
crude serum containing antibodies against both N-and C-terminal parts
of the HIF-1
recombinant protein reacted with hypoxia-inducible
proteins in fish cells, the antisera were further purified in order to study the behavior of HIF-1
protein in normoxic and hypoxic
conditions (for details, see "Experimental Procedures"). After the
purification, the specificity of antibodies was greatly increased, and
apart from the hypoxia-specific recognition, only some unspecific bands remained that could be used as markers for equal loading in Western blot analysis after normoxic and hypoxic treatments (Fig.
6).
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Fig. 6.
The stabilization of HIF-1
protein in rainbow trout and chinook salmon under different
O2 concentrations. A, Western blot analysis of
nuclear extracts isolated after treatment of rainbow trout gonad
(RTG-2; upper panel) and chinook salmon embryonic (CHSE-214;
lower panel) cells under different O2
concentrations for 1 h. 30 µg of nuclear cell extracts was
loaded into each well. Purified C-terminal rainbow trout anti-HIF-1
antibody was used as primary antibody (1:500 dilution).
protein in rainbow trout gonad (RTG-2)
and chinook salmon embryonic (CHSE-214) cells. Since the mRNA
levels were not affected by hypoxia (Fig. 3), changes in the HIF-1
protein levels would indicate increased stability of the protein. In
CHSE cells, the HIF-1
protein accumulated already at 10% oxygen (76 torr) (Fig. 6). The accumulation of HIF-1
protein was maximal at 5%
O2 (38 torr) in both cell types. Below 5% oxygen (38 torr), the HIF-1
protein levels tended to decrease, and there was a
pronounced drop in the level of HIF-1
at the lowest oxygen level
(0.2% = 1.5 torr). Thus, the fish HIF-1
appears to accumulate at
much higher oxygen levels than mammalian HIF-1
, which shows a
half-maximal response between 1.5 and 2% O2 and maximal
response at 0.5-1% O2 (41, 42). Ambient oxygen levels of
55-60 torr are easily tolerated by the active rainbow trout, resulting
in arterial oxygen tension of 35-45 torr and venous oxygen tensions of
15-25 torr (43). In normoxic conditions (140-150 torr), the arterial
and venous oxygen tensions are around 100 and 30-40 torr, respectively
(43). Thus, oxygen levels under which HIF-1
protein accumulates
during the in vitro incubation are routinely experienced by
fish cells in vivo. Consequently, it is possible that
oxygen-dependent gene regulation forms an important
component of regulation of gene expression in fishes, not only in
extreme conditions and during environmental hypoxia but also in more or
less normoxic conditions.
protein occurs by a
ubiquitin-proteasome pathway that is inhibited by hypoxia (19, 20, 44).
To study whether this applies also to fish, we carried out experiments
with proteasome inhibitor MG-132. Treatment with MG-132 slowed down the
degradation of fish HIF-1
protein under normoxia, hypoxia, and
during re-oxygenation (Fig. 7). The mechanism of degradation and stabilization of HIF-1
protein is therefore most likely the same in man and fish. Interestingly, although
the oxygen-dependent degradation domain of HIF-1
protein generally shows only 47% similarity between rainbow trout and man, a
critical hypoxia response element of oxygen-dependent
degradation domain (35) is identical. This element appears to bind von
Hippel-Lindau tumor repressor protein (VHL), which functions as a
ubiquitin ligase, directing HIF-1
protein degradation (45-47).
View larger version (34K):
[in a new window]
Fig. 7.
Accumulation of HIF-1
protein in rainbow trout and chinook salmon cells under normoxic
and hypoxic conditions in the presence of proteasome inhibitor
MG-132. Rainbow trout gonad cells were treated under normoxia
(21% O2) for 4 h in the absence (
) or presence (+)
of MG-132 (10 µM) (upper panel). Rainbow trout
cells (middle panel) and chinook salmon cells (lower
panel) were first treated under hypoxia (1% O2) for
4 h in the absence (
) or presence (+) of MG-132 (10 µM). Cells were then transferred to normoxic conditions
for 30 min in the absence (
) or presence (+) of MG-132. Purified
C-terminal rainbow trout anti-HIF-1
was used as the primary antibody
(1:500).
is present in fish. The
predicted amino acid sequence shows high level of conservation at the
bHLH/PAS A/B region, whereas there are large variations in the
transactivation domains among vertebrates. The HIF-1
levels in fish
cells are regulated via the ubiquitin-proteasome pathway as has been
shown in mammals, and the protein shows similar
oxygen-dependent DNA binding as other known
hypoxia-inducible transcription factors. However, the HIF-1
of
rainbow trout and chinook salmon cells is stabilized at much higher
oxygen levels than previously reported for mammals, suggesting a role
for oxygen-regulated gene expression in the normal physiology of these fish.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Andrey Mikhailov for technical
instructions concerning production and purification of rainbow trout
HIF-1 antibodies and the Cancer Research Center, Russian Academy of
Medical Sciences, Moscow, Russia for immunizations. Dr. Harry
Björklund and Tove Johansson are acknowledged for providing the
fish cell lines. We also thank Jonna Sonne and Annukka Palomäki
for excellent technical assistance.
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FOOTNOTES |
---|
* This work was supported by the Academy of Finland.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF304864.
¶ To whom all correspondence should be addressed: Arto Soitamo, Dept. of Biology, Laboratory of Animal Physiology, University of Turku, FIN-20014 Turku, Finland. Tel.: 358-2-333-5780; Fax: 358-2-333-6598; E-mail: arto.soitamo@utu.fi.
Published, JBC Papers in Press, March 9, 2001, DOI 10.1074/jbc.M009057200
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ABBREVIATIONS |
---|
The abbreviations used are: HIF-1, hypoxia-inducible factor-1; ARNT, aryl hydrocarbon nuclear translocator; bHLH, basic-helix-loop-helix; RACE, rapid amplification of cDNA ends; rt, rainbow trout; RTG, rainbow trout gonad cells; CHSE, chinook salmon embryonic cells; PBS, phosphate-buffered saline; RT, reverse transcription; PCR, polymerase chain reaction; HRE, hypoxia response element; XSC, adapter primer used in 5'-3' RACE that contains XhoI, SalI, and ClaI restriction enzyme recognition sites (28).
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