Characterization of a Hypoxia-inducible Factor (HIF-1alpha ) from Rainbow Trout

ACCUMULATION OF PROTEIN OCCURS AT NORMAL VENOUS OXYGEN TENSION*

Arto J. SoitamoDagger §, Christina M. I. RåberghDagger §, Max Gassmann||, Lea SistonenDagger **, and Mikko Nikinmaa§

From the Dagger  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

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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-1alpha 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-1alpha from rainbow trout with a predicted protein sequence of 766 amino acids that showed a 61% similarity to human and mouse HIF-1alpha . Polyclonal antibodies against the N-terminal part (amino acids 12-363) and the C-terminal part (amino acids 330-730) of rainbow trout HIF-1alpha protein recognized rainbow trout and chinook salmon HIF-1alpha protein in Western blot analysis. Also, the human and mouse HIF-1alpha proteins were recognized by the N-terminal rainbow trout anti-HIF-1alpha antibody but not by the C-terminal HIF-1alpha antibody. The accumulation of HIF-1alpha 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-1alpha 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-1alpha proteins, suggesting a high degree of evolutionary conservation in degradation of HIF-1alpha protein.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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-1alpha and HIF-1beta (6, 7). HIF-1alpha is unique for the hypoxia response, whereas HIF-1beta 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-1alpha 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-1alpha such as HIF-2alpha , also termed EPAS (endothelial PAS domain protein (11)), HLF (HIF-1alpha -like factor (12)), HRF (HIF-related factor (13)), MOP2 (member of PAS superfamily 2 (14)), and HIF-3alpha (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).

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-1alpha is constitutively expressed (17, 18), the levels of HIF-1alpha protein are markedly higher in hypoxic than in normoxic conditions. Under normoxic conditions the HIF-1alpha 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-1alpha protein to translocate from the cytoplasm to the nucleus, where it heterodimerizes with HIF-1beta (21). In addition, hypoxic conditions enhance the transactivating function of HIF-1alpha (22, 23). Although the stabilizing effects of hypoxia on the HIF-1alpha 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-1alpha protein from rainbow trout. Using recombinant fish HIF-1alpha protein, we have generated polyclonal antibodies recognizing fish HIF-1alpha protein. These antibodies have been utilized to investigate the oxygen tension dependence of HIF-1alpha protein levels in several types of fish cells.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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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-1alpha 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-1alpha 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.

Cloning of Rainbow Trout HIF-1alpha 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-1alpha sequence. The primer pair of the ba1 primer within the basic region of HIF-1alpha and the hif375 primer distal to the PAS B region of HIF-1alpha 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.

                              
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Table I
PCR primers used in RT-PCR (ba1 and hif375) and 5'-3' RACE
Primers for RT-PCR were based on mouse HIF-1alpha 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-1alpha DNA sequence (newly deposited as a result of this work, GenBankTM accession number AF304864).

Generation of Polyclonal Antibodies against Rainbow Trout HIF-1alpha -- Two different peptides were constructed to produce antibodies against both the N-terminal and the C-terminal part of HIF-1alpha . The rainbow trout HIF-1alpha 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-1alpha 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-1alpha 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-beta -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.

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-1alpha 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-1alpha protein. This protein could be used for purification of both antibodies, because the N-and C-terminal HIF-1alpha antigens overlapped by 30 amino acids, thus producing antibodies that partially included the same epitope recognition site.

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-1alpha 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.

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-1alpha cDNA using standard methods (33).

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-1alpha cDNA using standard methods (33).

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-1alpha protein was detected using enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech).

    RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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Cloning of Rainbow Trout HIF-1alpha cDNA-- RT-PCR was used to generate rainbow trout-specific HIF-1alpha 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-1alpha , respectively, and were therefore considered to be the first fish HIF-1alpha sequences. The rba5 DNA fragment that had slightly higher sequence homology to human HIF-1alpha 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-1alpha , whereas human HIF-1alpha has been shown to contain eight such elements (7).

                              
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Table II
A cDNA sequence comparison between two rainbow trout RT-PCR products (rba2 and rba5) and human HIF-1alpha cDNA (nucleotides 345-1383, GenBankTM accession number U22431)


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Fig. 1.   cDNA cloning of rainbow trout HIF-1alpha . A, schematic presentation of different rainbow trout HIF-1alpha cDNA clones. The length of different cDNAs is related to the length of HIF-1alpha mRNA, where +1 corresponds to the 5' end of the transcript. B, comparison of the amino acid identities between rtHIF-1alpha , human (h) HIF-1alpha , Xenopus (x) HIF-1alpha , and human HIF-2alpha . Amino acid identities are presented as percentages. The number of amino acid changes by insertions (ins) or deletions (del) compared with rtHIF-1alpha is indicated below the amino acid identities. C, comparison of the predicted amino acid sequences of rainbow trout HIF-1alpha (GenBankTM accession number AF304864) to Xenopus laevis HIF-1alpha (GenBankTM accession number CAB96628), human HIF-1alpha (GenBankTM accession number AAC68568), rat (r) HIF-1alpha (GenBankTM accession number AAD24413), and mouse (m) HIF-1alpha (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).

Total cDNA of rainbow trout (rt) HIF-1alpha was 3605 base pairs long, with an open reading frame of 766 amino acids. Thus, the rainbow trout HIF-1alpha was slightly smaller in size than the corresponding HIF-1alpha in human (826 amino acids), mouse, or rat (810 amino acids). The predicted rtHIF-1alpha amino acid sequence (Table III) had a 61% similarity to the human, mouse, and rat HIF-1alpha , about 52% similarity to the human and mouse HIF-2alpha , and 46% similarity to the human and mouse HIF-3alpha .

                              
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Table III
Amino acid comparison between different HIFs
rt, rainbow trout; h, human; m, mouse; r, rat; x, xenopus.

Although the overall similarity of the predicted amino acid sequence of rtHIF-1alpha was much closer to human HIF-1alpha than HIF-2alpha , the sequence corresponding to the bHLH domain had a similar homology to both human HIF-1alpha and to HIF-2alpha (Fig. 1B). Interestingly, the homology to the human proteins was greater than to the HIF-1alpha 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-1alpha protein (35): it was identical in all the HIF-1alpha 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).

The cloning of rtHIF-1alpha 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-1alpha 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).

Rainbow Trout HIF-1alpha mRNA Is Constitutively Expressed-- Using Northern blot analysis, the expression pattern of HIF-1alpha 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-1alpha 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-1alpha mRNA is constitutively expressed. The amount of HIF-1alpha 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-1alpha mRNA was normalized to the ribosomal 18 S rRNA (lower panel). The level of HIF-1alpha mRNA under normoxia was compared with the level of HIF-1alpha mRNA under hypoxia for 2 and 4 h. The level of HIF-1alpha mRNA under normoxia was used as control (defined as 1.0). Numerical data are based on four independent experiments.

The HIF-alpha 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-1alpha 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-1alpha complex in nuclear extracts was detected after 2 h of hypoxia treatment (1% O2), and the amount of HIF-1alpha complex was further increased after a 4-h treatment (Fig. 4A). Interestingly, the HIF-1alpha 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-1alpha cDNA cloning, suggesting that HIF-1alpha protein in rainbow trout has smaller molecular weight than the corresponding human HIF-1alpha protein. Both isoforms of HIF-1beta (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-1alpha -HRE complex was also observed after 4 h of hypoxia in primary-cultured rainbow trout hepatocytes (Fig. 4B). The specificity of the HIF-1alpha 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-1alpha binding to the radiolabeled DNA was inhibited. In the presence of serum containing the rainbow trout HIF-1alpha antibody, the mobility of the HIF-1alpha -HRE complex was further slowed down, indicating that the antibody was specific to the C-terminal part of HIF-1alpha 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-1alpha antibody (S), whereas nuclear extracts of HeLa cells were incubated together with preimmunization serum or serum containing mouse anti-HIF-1alpha (IgY) antibody (48). A supershifted band is indicated by an asterisk.

Characterization of Antibodies Produced against Rainbow Trout HIF-1alpha Protein-- Two polyclonal antibodies were generated against the rainbow trout HIF-1alpha 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-1alpha 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-1alpha 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-1alpha antibodies were used as primary antibodies (1:500). B, induction of HIF-1alpha 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-1alpha antibody (1:1000) was used as primary antibody.

To investigate whether the C-terminal rainbow trout anti-HIF-1alpha antibody recognized a HIF-1alpha 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-1alpha protein to the standard molecular weights.

Stabilization of HIF-1alpha 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-1alpha recombinant protein reacted with hypoxia-inducible proteins in fish cells, the antisera were further purified in order to study the behavior of HIF-1alpha 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-1alpha 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-1alpha antibody was used as primary antibody (1:500 dilution).

Consequently, the antibodies were used to investigate how oxygen levels affect the amount of HIF-1alpha 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-1alpha protein levels would indicate increased stability of the protein. In CHSE cells, the HIF-1alpha protein accumulated already at 10% oxygen (76 torr) (Fig. 6). The accumulation of HIF-1alpha protein was maximal at 5% O2 (38 torr) in both cell types. Below 5% oxygen (38 torr), the HIF-1alpha protein levels tended to decrease, and there was a pronounced drop in the level of HIF-1alpha at the lowest oxygen level (0.2% = 1.5 torr). Thus, the fish HIF-1alpha appears to accumulate at much higher oxygen levels than mammalian HIF-1alpha , 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-1alpha 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.

In mammals, the rapid degradation of HIF-1alpha 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-1alpha protein under normoxia, hypoxia, and during re-oxygenation (Fig. 7). The mechanism of degradation and stabilization of HIF-1alpha protein is therefore most likely the same in man and fish. Interestingly, although the oxygen-dependent degradation domain of HIF-1alpha 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-1alpha protein degradation (45-47).


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Fig. 7.   Accumulation of HIF-1alpha 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-1alpha was used as the primary antibody (1:500).

In conclusion, our results show that HIF-1alpha 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-1alpha 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-1alpha 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.

    ACKNOWLEDGEMENTS

We thank Dr. Andrey Mikhailov for technical instructions concerning production and purification of rainbow trout HIF-1alpha 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.

    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

    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).

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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