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First published online August 9, 2006
doi: 10.1242/10.1242/jcs.03087

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A computational model of intracellular oxygen sensing by hypoxia-inducible factor HIF1

Department of Biomedical Engineering, School of Medicine, Johns Hopkins University, 613 Traylor Bldg, 720 Rutland Avenue, Baltimore, MD 21205, USA

View larger version (86K): [in a new window]   Fig. 1. The HIF1 pathway in normoxia (A) and hypoxia (B). (A) HIF1 hydroxylation and degradation in the presence of oxygen involves: (1) the independent oxidation-reduction reactions of ascorbate (Asc) and iron (Fe); (2) and (3) prolyl hydroxlyase 2 (PHD2) binding to Fe, 2-oxoglutarate (2OG), and O2; (4) PHD2 hydroxylation of HIF1; (5) unbound hydroxylated HIF1 moving in the cell cytoplasm; (6) the von Hippel Lindau (VHL)-Elongin B (EB)-Elongin C (EC) complex ubiquitylating HIF1; and (7) HIF1 degradation. A change in shading of HIF1 indicates addition of a hydroxyl group. (B) In hypoxia, HIF1 enters the nucleus, where hydroxylation, but no degradation occurs. (1) and (2) PHD2 binding to Fe, 2OG and Asc, but not O2. (3) The protein inhibitor of growth 4 (ING4) binding to PHD2 may regulate HIF1 transcriptional activity and (4) block HIF1-HIF1ß binding. When HIF1-HIF1ß binding occurs, the HIF1 dimer can transcriptionally activate genes at the hypoxia response element (HRE) site.

View larger version (17K): [in a new window]   Fig. 2. (A) Effect of Fe2+ on the rate of prolyl hydroxylase (PHD2) reaction at different concentrations of 2-oxoglutarate (2-OG). Iron binds uncompetitively to PHD2, as indicated by lines that will intersect in the double reciprocal plot. (B) The double reciprocal plot of ascorbate concentrations at different levels of 2-OG show parallel lines above 1/[Asc]0=0.01 µM, while at higher ascorbate concentrations the lines begin to converge. This indicates ascorbate predominantly reacting with Fe3+, and not binding to the hydroxylases, at low ascorbate concentrations; at higher ascorbate levels, there is significant ascorbate reacting in iron reduction and the overall hydroxylation reaction. (C) The effect of ascorbate on the hydroxylation reaction. When there is no ascorbate, the reaction occurs though it takes more than 30 minutes for HIF1 to be fully degraded.

View larger version (32K): [in a new window]   Fig. 3. Model comparisons with experiments. (A) HIF1 hydroxylation by PHDs is related to cellular O2 levels (1, 2, 10 and 21%) in the model (lines). Results are compared with independent experimental data (Tuckerman et al., 2004), showing HIF1 modification by PHD2 measured by relative VHL capture at 0, 5 and 10 minutes (symbols). (B) Model predictions of the minimum percentage of HIF1 hydroxylated by PHD2 in normoxia (21% oxygen), lines. This is compared with experiments that measured HIF1 half-life in cells that were initially hypoxic, and at time zero in 20-21% oxygen; experimental values: t1/2=5-8 minutes (Berra et al., 2001; Jewell et al., 2001); t1/2<5 minutes (Huang et al., 1998). (C) Comparison of relative HIF1 accumulation predicted by the model at different oxygen levels with in vitro data (Jiang et al., 1996). Data points in Fig. 3C and 3D are the mean of two experiments. [O2]=0-59 µM in the model corresponds to 0-6% in the experiment. Initial model conditions were default values (Table 1). 10, 20, and 60 minutes correspond to the duration of the hydroxylation reaction. Both the experimental data and model results were normalized to the value obtained at 6% O2 ([O2]=59 µM). (D) Comparison with the same experiment, using the time of 20 minutes in the model. A line is provided showing what the model would predict if the lowest hydroxylation rate was set at 0.5% O2 rather than 0% O2. Intranuclear hydroxylation during anoxia is one possible mechanism by which HIF1 nuclear levels decrease below 0.5%, as shown by Jiang et al (Jiang et al., 1996). The model currently does not account for additional changes during anoxia. The delay accounting for the time it takes unhydroxylated HIF1 in the cytoplasm to move into the nucleus is assumed constant across all O2 levels. (E) Model results for HIF1 expression with ascorbate supplementation. The effects of PHD2:HIF1 concentration ratios on HIF1 expression are shown. (F) Model results showing HIF1 expression with iron or ascorbate supplementation after 1 hour and 4 hours of normoxia.

View larger version (31K): [in a new window]   Fig. 4. Model conditions where initial concentrations determine whether there is a switch-like response to O2 levels in the amount of HIF1 hydroxylated, or a gradual one. (A) When all enzymes are in excess, a steep drop in hydroxylation occurs as [O2] falls below 30 µM. For a range of initial unhydroxylated HIF1 between 0 and 1 µM, a steep, switch-like response is present. (B) An example with [HIF1]0=0.1 µM. In comparison, when iron (C), PHD2 (D) or 2-oxoglutarate (E) is limiting, the hydroxylation shows no apparent switch-like behavior. (F) The effect of ascorbate is mixed. In A-E, [Asc]0=1000 µM, in excess. At low levels of the compound (below its Km for HIF1, which is 180 µM), the oxygen response curve shows a more gradual, but non-linear reduction in hydroxylation. [Asc]0=1 µM is shown as an example.

View larger version (14K): [in a new window]   Fig. 5. Comparisons of O2 response curves. When both PHD2 and Fe2+ are limiting reactants ([Fe2+]0=0.05, [PHD2]0=4 nM), the slope of the HIF1 hydroxylation curve (at 20 minutes) is significantly less than when either one of the compounds separately limit the reaction. These lines are compared with the oxygen response when all compounds are in excess. The effects of the changing sensitivity to oxygen are significant in hypoxia (below 30 µM). For each line, [HIF1]0=0.1 µM.

View larger version (18K): [in a new window]   Fig. 6. Effect of chronic hypoxia of 9 to 24 hours on HIF1 hydroxylation. Model results show the amount of hydroxylated [HIF1] per [PHD2] for four different ratios of PHD2 synthesis rate to HIF1 synthesis rate relative to the maximum [HIF1]hydroxylated[PHD2]. HIF1 synthesis is a function of [O2] and duration of hypoxia, whereas PHD2 synthesis is a function of [HIF1] and duration of hypoxia. HIF1 accumulation begins at 4 hours, and measurable PHD2 synthesis follows at 8 hours.

View larger version (21K): [in a new window]   Fig. 7. Testing potential anti-angiogenic strategies targeting HIF1 hydroxylation during normoxia and hypoxia. The effect on hydroxylation by addition of ascorbate (A), PHD2 (B), and iron and ascorbate (C) is shown for [O2]=50 and 100 µM. Initial concentrations of the compounds not shown are default values (Table 1). (D) Effect of doubling ascorbate concentration on HIF1 hydroxylation as a function of iron, at [O2]=50 µM. For [Fe2+]>5 µM, the increase in hydroxylated HIF1 when [Asc]0 is increased from 1000 to 2000 µM, remains 0.02 µM. For each reaction, t=10 minutes. Model predictions are based on in vitro values. Physiological in vivo concentrations are also variable, although in general lower. Ascorbate concentrations are estimated as 25-50 µM (Knowles et al., 2003); tissue Fe2+ levels may be as low as 10-12 µM (Bullen et al., 1978), whereas intracellular iron complexes are 3-200 µM (Arredondo et al., 1997; Cooper et al., 1996; Hirsila et al., 2005), the fraction that is freely available for binding to PHD2 depends on cell type; absolute in vivo PHD2 concentrations are yet unknown - in cell extracts, they are in the nanomolar range.

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