Department of Biology, University of Turku, Turku, 20014, Finland
* Author for correspondence (e-mail: miknik{at}utu.fi)
Accepted 26 February 2004
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Summary |
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Key words: Hypoxia, Hypoxia-inducible factor, Rainbow trout, Oxidative stress, Phosphorylation
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Introduction |
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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.
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Materials and Methods |
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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 (ChemiImagerTM4400, 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).
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Results |
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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.
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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.
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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.
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Discussion |
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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.
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Acknowledgments |
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