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First published online 15 June 2004
doi: 10.1242/jcs.01192

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Redox state regulates HIF-1 and its DNA binding and phosphorylation in salmonid cells

Department of Biology, University of Turku, Turku, 20014, Finland

^* Author for correspondence (e-mail: miknik{at}utu.fi)

Accepted 26 February 2004

	Summary

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Rainbow trout (Oncorhynchus mykiss) hypoxia-inducible factor-1 (HIF-1) is a heterodimeric transcription factor structurally similar to mammalian HIF-1. It consists of HIF-1 and HIF-1ß subunits, of which the HIF-1 subunit confers the hypoxia sensitivity. HIF-1 is rapidly degraded by a proteasome under normal oxygen (21% O₂) conditions, mainly as a result of prolyl hydroxylation needed for protein destabilization. Although prolyl hydroxylation at conserved proline residues is a major factor controlling HIF-1 stability, the redox state of the cells may, in addition, influence the function of HIF-1 like proteins by influencing their stability, DNA binding and phosphorylation. Sensitivity of the protein to oxidation/reduction may be due to cysteine residues at critical positions. The predicted amino acid sequence of rainbow trout HIF-1 contains several unique cysteine residues, notably in the DNA-binding area at position 28 and in the transactivation domain of the molecule in the vicinity of the conserved proline residue at position 564 of mammalian HIF-1. In the present studies we have investigated if the redox state influences HIF-1 stability, DNA binding and phosphorylation in two established salmonid cell lines RTG-2 and CHSE-214. The results indicate that reducing conditions, achieved using N-propylgallate (nPG) or N-acetylcysteine (NAC), stabilize HIF-1, facilitate its DNA binding, and increase its phosphorylation even under normal oxygen conditions. On the other hand, oxidizing conditions, achieved using L-buthionine sulfoximine (BSO) dampen the hypoxia response. Furthermore, the hypoxia-like effect of cobalt is increased in the presence of the reducing agent. On the basis of these results, we suggest that redox state influences the accessibility of the conserved prolyl residues to oxygen-dependent hydroxylation and the accessibility of the residues involved in the phosphorylation of HIF-1.

Key words: Hypoxia, Hypoxia-inducible factor, Rainbow trout, Oxidative stress, Phosphorylation

	Introduction

Top Summary Introduction Materials and Methods Results Discussion References

 
High oxygen concentrations form an oxidizing environment and hypoxia a reducing environment. Because of this, several studies have investigated whether alterations in the cellular redox state (i.e., reduction/oxidation state of cells) are involved in the induction of changes in the function of hypoxia-inducible transcription factors. Although some studies suggest that oxidative conditions, and increased production of reactive oxygen species decrease the activity of HIF-1 protein (Haddad et al., 2000; Yang et al., 2003), others have failed to observe an effect of reactive oxygen species (or cellular redox state) on HIF-1 function (Lando et al., 2000; Srinivas et al., 2001).

In contrast to HIF-1, redox sensitivity of DNA binding has been clearly demonstrated (Lando et al., 2000) for HIF-like factor (HLF), a transcription factor that is structurally very similar to HIF-1. One important residue in the DNA-binding domain of HIF and related proteins is the residue at position 28, which is a serine in mammalian HIF-1 and a cysteine in mammalian HLF. Cysteine residues within proteins can be modified by the cellular redox state. Cysteine-rich glutathione is a major component of cellular redox balance, and cysteine-rich thioredoxins are formed in oxidative stresses (Arrigo, 1999; Powis and Montfort, 2001). Notably, it appears that the serine to cysteine substitution at position 28 confers redox sensitivity to the DNA binding ability of HIF-1 (Lando et al., 2000). Interestingly, residue 28 of HIF-1 in rainbow trout (Oncorhynchus mykiss) is also cysteine (Soitamo et al., 2001), suggesting that the DNA binding of HIF in this fish species may be redox sensitive. If this were the case, then environmental disturbances involving oxidative and reductive stresses would affect hypoxia-responsive gene expression. This could be the case for several organic pollutants and heavy metals with variable valency state. Redox regulation (by for example sulfhydryl modifications of cysteine residues) may also occur in the transactivation domain. This influences the degradation of HIF-1, as the redox sensitive sulfhydryl groups not only affect the activity of proteins, but also the rate of their degradation via the ubiquitin conjugation activity (Obin et al., 1998). In this regard it is interesting that the predicted amino acid sequence of rainbow trout HIF-1 has four cysteine residues in or near the area characterized in the mammalian HIF-1 as the transactivation domain, close to the oxygen-dependent degradation domain (Soitamo et al., 2001).

The present experiments were carried out to evaluate whether redox reagents can regulate HIF-1 protein levels and the DNA binding of HIF-1 protein in salmonids, as the structural properties suggest. During the experiments it became apparent that treatments altering the redox state of the cell also affected the apparent molecular weight of HIF-1, suggesting that the phosphorylation of salmonid HIF-1 may be under redox control. Thus, the other goal of the studies was to investigate whether treatments affecting the redox state of the cell can influence HIF-1 phosphorylation.

	Materials and Methods

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Cell culture and hypoxia treatment
Two established fish cell lines, RTG-2 (ATCC CCL 55) from the gonads of rainbow trout (Oncorhynchus mykiss) and CHSE-214 (ATCC CRL 1681) from Chinook salmon (Oncorhynchus tshawytscha) embryos, were used. The cells were cultured almost to confluence at 18°C under atmospheric air with 1% CO₂ in Eagle's minimum essential medium supplemented with 10% foetal bovine serum, penicillin (100 units/ml), streptomycin (100 µg/ml), L-glutamine (2 mM) and 0.075% NaHCO₃, pH 7.4. All cell culture medium components were from Gibco BRL Life Technologies Ltd (UK). In most experiments fish cells were exposed to hypoxia (1% O₂, 1% CO₂, 98% N₂) for 2 hours at 18°C. To investigate the effect of oxidative stress on the hypoxic induction of HIF-1 protein, the cells were cultivated for 24 hours in the absence or presence of 50 µM L-buthionine sulfoximine (BSO) before the 2-hour hypoxia treatment in the absence or presence of BSO. According to earlier results, this reduces the cellular GSH levels to below 20% of the original value (Haddad and Land, 2000). To evaluate the effect of a reducing agent, N-acetylcysteine (NAC), on the stability of HIF-1 protein in RTG-2 cells under normal oxygen conditions, the cells were treated with 0, 0.1, 1 or 10 mM NAC for 30 minutes, then maintained at 21% oxygen in the absence or presence of NAC. For reoxygenation experiments, a portion of the cells exposed to hypoxia in the presence or absence of 100 µM N-propylgallate (nPG) or 10 mM NAC were transferred to increasing oxygen levels (5, 10 and 21%) supplemented with 1% CO₂ and incubated for 15 minutes at each level. Two different types of antioxidants were used: NAC, a membrane permeable thiol, which buffers reactive oxygen species and nPG, a superoxide dismutase mimic (Reddan et al., 2003). The use of antioxidants with two different mechanisms of action ensures that, if similar results are obtained with these different antioxidants, the actual redox state (of the cysteine residues) is important, and not the mode of action of the antioxidant. To evaluate the possible role of protein kinase C in the responses of HIF-1 protein, observed in the present study, the experiments were carried out in the presence and absence of 10 nM protein kinase C inhibitor bisindolylmaleimide (Calbiochem).

RNA analysis
RNA was isolated from fish cells using the TRI-reagent method according to the manufacturer's (Sigma) instructions. RNA samples were denatured using glyoxal at 50°C for 1 hour, separated (10 µg per lane) on a 1.2% agarose gel and blotted onto nylon filters. Blots were hybridized with a 600-bp [-³²P]dCTP-labelled rainbow trout HIF-1 probe overnight at 42°C. Radiolabelling of the probes was performed using a prime-a-gene kit (Promega). Blots were washed to reach the final conditions of 42°C, 1xSSC, 0.1% SDS and visualized by autoradiography. HIF-1 expression levels in cell samples were analyzed and quantified using a computerized image analysis scanner (ChemiImager^TM4400, Alpha Innotech Corp., USA).

Electrophoretic mobility shift assays (EMSAs)
Electrophoretic mobility shift assays on nuclear extracts were performed as described earlier (Kvietikova et al., 1995; Soitamo et al., 2001). Since no fish sequences are available, the HIF-1 binding site in the promoter region of the human transferrin gene (Rolfs et al., 1997) was used: 5'-TTCCTGCACGTACACACAAAGCGCACGTATTTC-3' (prepared at Institute of Biotechnology, University of Helsinki). The oligonucleotide was 5' end-labelled with [-³²P]dATP (Amersham Pharmacia Biotech, UK) using T4 polynucleotide kinase (Promega). Unincorporated nucleotides were removed using a TE Micro SELECT® D Sephadex-G-25 column (5'-3' PRIME Inc., USA) according to the manufacturer's recommendations.

Immunoprecipitation and immunoblot analysis
Immunoprecipitation studies were performed using whole-cell extracts from cells isolated in RIPA buffer (10 mM Tris/HCl pH 7.4, 150 mM NaCl, 1% sodium deoxycholate, 1% TritonX-100, 1 mM PMSF, 2 µg/ml leupeptin, pepstatin A, antipain and aprotinin, 1 mM Na₃VO₄, 10 mM NaF) (Eriksson et al., 1998). After lysis of cells in RIPA for 5 minutes on ice, the lysate was cleared by centrifugation at 5000 g for 15 minutes at 4°C. The supernatant was added to 5 µl double-purified rainbow trout HIF-1 antibody made against the C-terminal end of the protein (Soitamo et al., 2001). Cell extracts were incubated at 4°C overnight. 20 µl Protein A immobilized on Sepharose CL-4B (1.5 g in 30 ml phosphate buffered saline, P-3391 Sigma, Sweden) were added to the reaction and the tube was incubated for a further hour at 4°C. Immunoprecipitates were collected by centrifugation at 2000 g for 2 minutes at 4°C, and washed twice with RIPA buffer with 0.1% SDS. Samples were resuspended in sample loading buffer containing lithium dodecyl sulphate instead of SDS, subjected to SDS/PAGE (7.5%) and then transferred to nitrocellulose. After transfer samples were analysed by western blotting (ECL).

	Results

Top Summary Introduction Materials and Methods Results Discussion References

 
High oxygen levels form an oxidizing environment and hypoxia results in a reducing environment. As the redox status appears to influence HIF-1 levels in some mammalian cells (Welsh et al., 2003; Yang et al., 2003), we investigated whether oxidative stress destabilizes and antioxidants/reducing agents stabilize HIF-1 protein in salmonid cells. To generate oxidative stress, cells were incubated with L-buthionine sulfoximine (BSO; 50 µM) for 24 hours before hypoxia incubation (Haddad and Land, 2000). As observed earlier (Soitamo et al., 2001), hypoxia caused a clear increase in the level of HIF-1 protein in salmonid cells, although some HIF-1 protein was also observed with normal oxygen levels (Fig. 1). The hypoxic induction of HIF-1 protein was significantly reduced after BSO treatment (Fig. 1) suggesting that an oxidizing environment destabilizes HIF-1 protein. N-propylgallate (nPG) and N-acetylcysteine (NAC) were used as antioxidants. The reducing agent NAC increased the amount of HIF-1 protein under normal oxygen levels (Fig. 2). Furthermore, these antioxidants prevented the breakdown of HIF-1 during re-oxygenation, as shown in Fig. 3 for nPG. Although oxygen had a significant destabilizing effect on HIF-1 protein in the absence of the reducing agents, the effect was absent in the presence of reducing agents. Furthermore, since both antioxidants had a similar effect on HIF-1 protein stability, it is probable that the redox-dependent effect on protein stability is due to the effect of cysteine residues on the accessibility of the oxygen-dependent degradation domain including the hydroxylation sites of the HIF-1 protein. The effect of reducing agents/antioxidants on HIF-1 protein levels was not due to changes in HIF-1 mRNA levels (that remained essentially unchanged regardless of the treatment, data not shown), but indicate that the stability of HIF-1 protein is influenced by the redox state of the cells.

View larger version (35K): [in this window] [in a new window]   Fig. 1. Hypoxia (1% oxygen for 2 hours) causes an increase in the level of hypoxia-inducible factor 1 (HIF-1) in RTG-2 cells as detected by a polyclonal antibody, produced against the HIF-1 protein of rainbow trout (Soitamo et al., 2001). The hypoxia-induced increase in HIF-1 protein level is reduced by previous treatment of the cells under oxidizing conditions (24-hour culture in the presence of 50 µM BSO). (A) A representative gel showing the intensities of HIF-1 protein bands under normal oxygen conditions (21% oxygen), hypoxia (1% oxygen) and hypoxia after culture for 24 hours in the presence of BSO (1% + BSO). (B) Quantification of HIF-1 protein band intensities in normal oxygen before the experiments (21% oxygen), under hypoxia (1% oxygen) and hypoxia after culture for 24 hours in the presence of BSO (1% + BSO). The intensity of HIF-1 protein band in normal oxygen at the onset of the experiments was taken as 100%. BSO treatment to induce oxidative stress decreased the hypoxia-induced induction of HIF-1 protein significantly (P<0.01, paired t-test, SPSS statistical software). Values represent mean±s.e.m. (n=4).

View larger version (19K): [in this window] [in a new window]   Fig. 2. The effect of N-acetylcysteine (NAC) on the stability of HIF-1 protein in RTG-2 cells under normal oxygen conditions. The cells were treated with 0, 0.1, 1 or 10 mM NAC for 30 minutes, then they were maintained at 21% oxygen in the absence (0) or the presence of NAC, and in the absence of NAC at 1% oxygen (H) for 2 hours. HIF-1 protein was detected using immunoprecipitation techniques with HIF-1 antibody produced against rainbow trout protein.

View larger version (42K): [in this window] [in a new window]   Fig. 3. Effects of the antioxidant N-propylgallate (nPG; 100 µM, preincubation for 30 minutes before treatment) on the stability of HIF-1 protein during reoxygenation in the salmonid cell lines RTG-2 and CHSE-214. In both cell lines, hypoxia treatment (1% O2 for 2 hours) caused stabilization of HIF-1 protein. Consequent reoxygenation (15 minutes at 5, 10 and 21% O2) caused a reduction in the level of HIF-1 protein in the absence of the antioxidant, whereas reoxygenation in the presence of nPG prevented the reduction of HIF-1 protein level completely. (A) A representative gel showing the intensities of HIF-1 protein bands at the different conditions. HIF-1 protein was detected using immunoprecipitation techniques with HIF-1 antibody produced against rainbow trout protein. (B) Quantification of HIF-1 protein band intensities in normal oxygen conditions before the experiment (21% oxygen), in hypoxia (1% oxygen), during reoxygenation (5, 10 and 21% oxygen) and during reoxygenation in the presence of nPG (N5%, N10% and N21%). The intensity of HIF-1 protein band in normal oxygen at the onset of the experiments was taken to be 100%. ANOVA, carried out using SPSS statistical software, indicated that oxygen had a significant (P<0.01) effect on the HIF-1 protein band intensity during reoxygenation in the absence but not in the presence of nPG. Values represent mean±s.e.m. (n=4). Similar results were obtained using NAC (10 mM) as the antioxidant.

The increase in the amount of HIF-1 protein under normal oxygen conditions in the presence of antioxidants is expected to facilitate its binding to DNA, as indicated by EMSA. Also, as rainbow trout HIF-1 protein has a cysteine residue at position 28 within the DNA binding domain, reducing agents may facilitate its DNA-binding under normal oxygen conditions as indeed we found to be the case (Fig. 4). This facilitation disappeared under hypoxic conditions.

View larger version (78K): [in this window] [in a new window]   Fig. 4. Electrophoretic mobility shift assay (EMSA) of the DNA binding of HIF-1 protein in control RTG-2 cells (C), in the presence of the antioxidant NAC (1 mM; N) and in the presence of the protein kinase C inhibitor bisindolylmaleimide (10 nM; P). The antioxidant markedly increased the DNA binding of HIF-1 protein in normal oxygen conditions. Hypoxic conditions (1% oxygen) markedly increased the DNA binding of HIF-1 protein in control cells (HC), and in cells treated with the protein kinase C inhibitor (HP), but slightly decreased the DNA binding of cells treated with the antioxidant (HN).

CoCl₂ stabilised HIF-1 under normal oxygen conditions (Fig. 5), as previously shown (Wang and Semenza, 1995). The stabilisation may be due to cobalt inhibiting the interaction between HIF-1 and von Hippel Lindau (VHL) protein (Yuan et al., 2003). Even in this case, treatment of the cells with the reducing agent nPG (20 µM) for 30 min prior to the treatment of the cells with 200 µM CoCl₂ markedly increased the amount of HIF-1 protein (Fig. 5), suggesting that the hypoxia-like effect of cobalt is also influenced by the redox state of the cells. However, the present result cannot exclude the possibility that the reducing agent and cobalt could affect the stability of HIF-1 protein via independent, additive mechanisms.

View larger version (39K): [in this window] [in a new window]   Fig. 5. Pre-treatment of RTG-2 and CHSE-214 cells with the antioxidant N-propylgallate (nPG; 20 µM) for 30 minutes increases the amount of HIF-1 protein after CoCl2 (200 µM) treatment. Cells were first treated in the presence or absence of nPG and then with CoCl2 (2 hours) under normal oxygen conditions (21% O2). Cobalt treatment in the absence of nPG caused an increase in the level of HIF-1 protein. The level of HIF-1 protein was further increased in the presence of nPG. Furthermore, the HIF-1 protein band was broadened in the presence of nPG, a finding compatible with redox-sensitive phosphorylation of the protein. The HIF-1 protein was detected using immunoprecipitation techniques with trout-specific HIF-1 antibody.

When cobalt and nPG acted together, the HIF-1 protein band was clearly broadened, as if the general molecular weight of the protein was increased, compatible with HIF-1 protein phosphorylation (Richard et al., 1999). As the broadening of the HIF-1 protein band was inhibited by protein phosphatase (Fig. 6), it is likely that the higher molecular weight protein is a phosphorylated form of HIF-1 protein. As the apparent increase in the molecular weight of HIF-1 protein occurred in the presence of an antioxidant, the result suggests that the redox state of the cell may influence the phosphorylation of HIF-1 protein. In order to study the effect of antioxidant on the apparent phosphorylation of HIF-1 protein further, two other compounds, phorbol myristate acetate (PMA) and NaVO₃, were tested. Both of these compounds are commonly used in protein phosphorylation studies. Pre-treatment of cells with the antioxidant nPG (50 µM), not only stabilised the HIF-1 protein in the presence of PMA and NaVO₃, but also induced a clear increase in the molecular weight of HIF-1 protein, compatible with its phosphorylation (Fig. 7). These results suggest that phosphorylation of HIF-1 protein in salmonid cells is under redox control. We failed to see any consistent effect of protein kinase C inhibitor bisindolylmaleimide (10 nM) on either the apparent effect of antioxidant on protein phosphorylation (not shown) or the effect of hypoxia on DNA binding (Fig. 4). Thus, the results suggest that the possible redox regulation of HIF-1 protein phosphorylation in salmonid cells is independent of protein kinase C.

View larger version (48K): [in this window] [in a new window]   Fig. 6. The effect of -phosphatase on HIF-1 protein in rainbow trout RTG-2 cells. Total cell protein extract was immunoprecipitated using purified C-terminal HIF-1 antibody in the presence of phosphatase inhibitors. After precipitation, the proteins bound to Sephadex-protein-A were treated with -phosphatase in the absence of phosphatase inhibitors. Notably, phosphatase treatment inhibited the broadening of the HIF-1 protein band, a finding compatible with HIF-1 protein phosphorylation.

View larger version (44K): [in this window] [in a new window]   Fig. 7. A pre-treatment of cells with the antioxidant N-propylgallate (nPG) for 30 minutes increased the level of HIF-1 protein. A higher molecular weight component of HIF-1 protein appeared after a 2-hour treatment at 21% oxygen with the commonly used phosphorylating agents phorbol myristate acetate (PMA; 50 nM) or sodium vanadate (NaVO3; 50 µM). The HIF-1 protein was detected using immunoprecipitation techniques with trout-specific HIF-1 antibody.

	Discussion

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The present results indicate that the stability of HIF-1 protein in salmonid cells can be modified by the redox state of the cell. A reducing environment stabilizes the protein and oxidizing environment destabilizes it. It appears that the DNA binding of HIF-1 protein in salmonid cells is also affected by the redox state of the cells. In addition, the phosphorylation of HIF-1 protein may also be influenced by the redox state of the protein, possibly in a manner independent of protein kinase C function. Furthermore, it appears that the stabilizing effect of cobalt on HIF-1 protein is strengthened in the presence of antioxidants. These findings are compatible with a role for the cysteine residue at position 28 in the DNA binding area in conferring redox sensitivity to DNA binding in the HIF-1 protein (Lando et al., 2000). They also support a role for the unique sulfhydryl groups in the salmonid HIF-1 protein (Soitamo et al., 2001) in regulating HIF-1 protein function. These sulfhydryl groups are located in the area equivalent to the transactivation domain of mammalian HIF-1 protein (Jiang et al., 1996; Semenza et al., 1997; Jiang et al., 1997). The findings are also in accordance with earlier data highlighting the importance of the redox state of the cells in HIF-1 protein function (Haddad et al., 2000).

It is apparent that the stabilization of HIF-1 protein, its DNA binding and its phosphorylation, are all affected by the redox state of the cells and represent different aspects of HIF-1 protein regulation. Increased stability of the protein is essential for induction of HIF-1 protein-regulated genes. With regard to the stabilization of HIF-1 protein, it appears that oxygen-sensitive prolyl hydroxylases, hydroxylating proline residues (at positions 402 and 564 in mammalian HIF-1 protein) play a decisive role in regulating the stability (Ivan et al., 2001; Jaakkola et al., 2001; Semenza, 2001; Huang et al., 2002; Hirsila et al., 2003). As the proline residues are also conserved in rainbow trout HIF-1 protein (Soitamo et al., 2001), it is likely that the stability of the salmonid protein is also regulated by proline hydroxylases, although, as yet, the enzymes involved have not been characterized. The present results suggest that either the accessibility of the two conserved prolines to proline hydroxylases is under redox control or that redox reactions, possibly via reactive oxygen intermediates, are involved in the enzyme mechanism of hydroxylation.

In addition to the direct effect of redox regulation on the DNA binding of HIF-1 protein in salmonid cells and consecutive transcriptional activation of HIF-dependent proteins, it appears that phosphorylated HIF-1 protein, as a dimer with aryl hydrocarbon nuclear translocator (ARNT) and several coactivators such as p300/CBP, is involved in mediating the transcriptional activation of hypoxia-induced genes by HIF-1 (Wenger, 2000). The phosphorylation sites of HIF-1 protein are not unequivocally characterized, but may involve serine/threonine residues at the transactivation domain (Wang et al., 1995; Gradin et al., 2002). MAP kinases probably phosphorylate HIF-1 protein, thus playing a role in its activation (Richard et al., 1999; Hofer et al., 2001; Mottet et al., 2003). The present results suggest that the accessibility of the phosphorylation site(s) in salmonid HIF-1 protein is/are influenced by the redox state. In addition, as we failed to see a consistent effect of protein kinase C inhibition on either the DNA binding of HIF-1 or apparent redox-reagent-dependent phosphorylation of HIF-1 protein, the results suggest that protein kinase C function and redox regulation of HIF-1 protein are independent in salmonid cells. Notably, earlier studies in mammalian systems have observed an HIF-independent protein kinase C effect on genes that are often transcriptionally regulated by HIF (Hossain et al., 2000)

Significantly, our recent results on Baltic salmon indicate that the mortality of yolk sac fry is associated with dysfunction of HIF-1 protein (Vuori et al., 2004). Additionally, several pieces of evidence suggest that the early mortality syndrome of the Baltic salmon is associated with oxidative stress (Lundstrom et al., 1999). As redox reactions play a pronounced role in the stability, DNA binding and phosphorylation of HIF-1 protein in salmonid cells, it is likely that environmental disturbances involving oxidative stress, such as metal or organic pollutant contamination and increased UV radiation, may influence HIF-1 protein function and consequent gene expression.

	Acknowledgments

 
This study was supported by grants from the Academy of Finland.

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