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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

View larger version (35K): [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 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 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.

View larger version (78K): [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).

View larger version (39K): [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.

View larger version (48K): [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 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.

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