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Induction of HIF-1 in response to hypoxia is instantaneous¹

Institute of Physiology, University of Zürich, CH-8057 Zürich, Switzerland

³Correspondence: Institute of Physiology, University of Zürich, Winterthurerstr. 190, CH-8057 Zürich, Switzerland. E-mail: maxg{at}access.unizh.ch

SPECIFIC AIMS

Despite the pivotal role the hypoxia-inducible factor 1 (HIF-1) plays in physiological and pathological processes, little is known regarding the time frame and mechanisms involved in its regulation. The aim of this study was to gain insight into the sequential events occurring in the nucleus immediately after hypoxic exposure and reoxygenation by determining the kinetics of HIF-1 induction and degradation, and comparison with its dimerization partner ARNT (aryl hydrocarbon receptor nuclear translocator) and nuclear levels of NF-B (nuclear factor kappa B), c-Fos, c-Jun, Ref-1 (redox factor 1), and Trx (thioredoxin) over a range of pathophysiological oxygen concentrations.

PRINCIPAL FINDINGS

1. Within 2 min of anoxic/hypoxic exposure, HIF-1 protein accumulates in the nucleus
Tonometers were used to expose HeLaS3 cells to 0%, 0.02%, 0.1%, 0.5%, and 5% oxygen for 0, 2, 5, 10, 32, and 60 min. Western blot analysis of nuclear extracts did not detect HIF-1 protein at any zero time points (equivalent to exposure to 20% oxygen), but showed nuclear HIF-1 protein already 2 min after exposure to any of the anoxic/hypoxic oxygen concentrations (Fig. 1 ). The accumulation of HIF-1 in the nucleus continued rapidly for 30 min in all oxygen concentrations and then proceeded more gradually until a maximum level was reached 60 min after anoxic/hypoxic exposure had begun. The kinetics of nuclear HIF-1 accumulation is well reflected by the short time it took to reach half-maximum levels (t_1/2max). Exposure to 0% and 0.5% oxygen resulted in t_1/2max values of only 13.3 and 12.4 min, respectively. The appearance of an additional, slower migrating HIF-1 protein band between 10 and 30 min of anoxic/hypoxic exposure suggests further protein modification around the time when the accumulation process turns more gradual. Detecting HIF-1 protein inside the nucleus after only 2 min of anoxic/hypoxic exposure led us to measure the oxygen concentration within the medium during this time. We found that it took 2 min for the oxygen concentration in the medium to decrease from 20% to 0% or 0.5% oxygen (Fig. 1) . As we did not detect nuclear HIF-1 in cells exposed to 20% oxygen, HIF-1 proteins can only have started to accumulate inside the nucleus once the oxygen concentration had fallen below a certain threshold, thereby limiting the initial protein accumulation process to below 2 min.

View larger version (16K): [in this window] [in a new window]   Figure 1. HIF-1 kinetics compared with the decrease in oxygen concentration in medium during the first 5 min of hypoxia. First 5 min of HIF-1 protein levels (right y-axis) in nuclear extracts of HeLaS3 cells exposed to 0.5% oxygen for up to 1 h (n=12) and measurements of oxygen concentration (n=3) in the medium (left y-axis).

Also after 2 minutes the onset of HIF-1 DNA-binding can be weakly observed and becomes more pronounced after 4 minutes. It continues to increase up to 60 minutes of hypoxic exposure, at which time it reaches a maximum level which is maintained for up to 4 hours of hypoxic exposure.

2. Nuclear levels of NF-B, c-Fos, c-Jun, and Ref-1 are not influenced by hypoxia during the first hour of hypoxic exposure
Comparison of nuclear c-Fos, c-Jun, and Ref-1 protein levels to nuclear HIF-1 levels in HeLaS3 cells exposed to 0.5% and 20% oxygen for up to 60 min failed to show hypoxic regulation of these nuclear factors on Western blots, indicating that the protein concentration of any of these redox factors is unlikely to determine the mechanism leading to the rapid HIF-1 stabilization.

3. The kinetics of HIF-1 degradation in response to reoxygenation is dependent on the severity of the foregoing hypoxic insult
To obtain a better understanding of the processes involved in HIF-1 protein degradation upon reoxygenation, we exposed HeLaS3 cells to anoxia (0% oxygen) and hypoxia (0.5% oxygen) for 1 h and then reoxygenated the medium with 20% oxygen. During the first 4 min of reoxygenation from anoxia, nuclear HIF-1 protein levels continued to increase slightly but had started to decrease after 8 min (Fig. 2) . This decrease continued and rendered nuclear HIF-1 protein levels undetectable by Western blotting 32 min after reoxygenation. In contrast, reoxygenation from hypoxia showed no initial significant increase in HIF-1 protein levels, but a decrease in HIF-1 levels after only 4 min of reoxygenation. By 16 min, these signals were already barely detectable 32 min after reoxygenation, and nuclear HIF-1 levels had become entirely undetectable. Measurements of the actual oxygen concentrations in the medium provided some explanation for that difference: it took 2 min (n=3) for the oxygen concentration to reach levels above 15% oxygen in the medium, whereas normoxic levels were already achieved only 30 s (n=3) after reoxygenation from hypoxia.

View larger version (50K): [in this window] [in a new window]   Figure 2. HIF-1, ARNT, NF-B, and Trx kinetics upon reoxygenation from anoxia. Western blot showing that after reoxygenation from 0% oxygen (n=3) the down-regulation of HIF-1 is contrasted by a transient up-regulation of NF-B and Trx protein levels. The apparent reduction of ARNT is due to the undimerized ARNT protein being lost during preparation of nuclear extracts.

4. Nuclear NF-B and Trx protein levels increase transiently in response to reoxygenation from anoxia and hypoxia
It has been shown that the oxidoreductive regulation of NF-B involves the cellular reducing catalyst Trx. We show here that both redox-sensitive factors NF-B and Trx increase transiently between 1 and 8 min after reoxygenation from anoxia (Fig. 2) and to a slightly lesser extent after reoxygenation from hypoxia. The rise in Trx levels occurred shortly before the increase in NF-B (Fig. 2) . By showing an up-regulation of NF-B and Trx, we provide indirect evidence that HeLaS3 cells experience oxidative stress during reoxygenation.

CONCLUSIONS

It has been shown that HIF-1 has a half-life of approximately 5 min in normoxic conditions. Here we show for the first time that HIF-1 protein is already detectable in the nucleus of cells after less than 2 min exposure to anoxia or hypoxia (Fig. 3 ). In that time, HIF-1 is protected from ubiquitination and translocated to the nucleus. This immediate response temporally restricts the amount and type of interactions that confer HIF-1 stability. We also discovered that the accumulating HIF-1 proteins undergo some form of modification, possibly phosphorylation, between 10 and 30 min after nuclear HIF-1 accumulation had started. As this modification occurs only after the onset of nuclear HIF-1 accumulation and HIF-1 DNA binding, it cannot be necessary for the initial HIF-1 protein stabilization/activation process. However, it is worth noting that the appearance of the modified HIF-1 protein coincides with the switch from a rapid to a more gradual increase in nuclear HIF-1 proteins, which might indicate an inhibitory feedback mechanism. The HIF-1 dimerization partner, ARNT, is already present in the nucleus of normoxic cells, and the total amount of ARNT within the whole cell is not affected by hypoxia.

View larger version (23K): [in this window] [in a new window]   Figure 3. Schematic diagram of the rapid HIF-1 regulation during normoxia, hypoxia, and reoxygenation. HIF-1 is a bHLH (basic helix-loop-helix) transcription factor that is expressed ubiquitously. In normoxia, the pVHL (von Hippel-Lindau protein) binds to HIF-1 and recruits it to the ubiquitination machinery (Ub) to be degraded. Within 2 min of hypoxia, HIF-1 accumulates to the nucleus, where it heterodimerizes with ARNT. The functional HIF-1 complex binds to a HBS (HIF-1 binding site) located in the HRE (hypoxia response element) and thereby induces transcription of EPO (erythropoietin), VEGF (vascular endothelial growth factor), Tf (transferrin), and other oxygen-regulated genes. Upon reoxygenation, HIF-1 proteins undergo efficient proteasomal degradation within 16 min.

HIF-1 DNA binding is lost rapidly during reoxygenation and HIF-1 proteins are degraded. Our results indicate that the kinetics of HIF-1 degradation in response to reoxygenation depends on the severity of the hypoxic insult experienced prior to reoxygenation. The half-life of HIF-1 proteins was 2.5-fold longer when cells were reoxygenated from anoxia (0% oxygen) compared with hypoxia (0.5% oxygen). Although it took slightly longer for the medium to equilibrate with 20% oxygen after the anoxic insult, in both cases the medium had reached normoxic oxygen levels within 2 min. The delayed onset of HIF-1 protein degradation after exposure to anoxia might indicate a necessity for keeping HIF-1 protein available to ensure sufficient up-regulation of HIF-1 target genes. The suggestion that the degree of hypoxia affects the reoxygenation response is complemented by recent findings that the duration of hypoxia prior to reoxygenation influences the response to reoxygenation. In vivo studies of reoxygenation have also shown that the mechanisms responsible for reperfusion injury are set in motion during the preceding period of hypoxia.

Comparing the kinetics of HIF-1 accumulation and degradation reveals that it took longer for HIF-1 degradation to be initiated than for HIF-1 to start accumulating. The rapid accumulation of HIF-1 in hypoxic oxygen tensions implies urgency: without sufficient amount of HIF-1 protein, the cell’s survival is endangered. When oxygen tensions return to normal levels, the need to degrade HIF-1 proteins seems to be less acute than the expense of keeping HIF-1 levels elevated, i.e., the presence of HIF-1 after reoxygenation is tolerated for longer than its absence during anoxia/hypoxia.

In vivo, reoxygenation occurs when oxygen supply is restored to hypoxic tissue. There are numerous pathological examples that demonstrate severe tissue injuries resulting from reoxygenation, e.g., after organ transplantations, bypass operations, and ischemia-reperfusion injuries. Especially in the setting of ischemia, the most striking tissue injury occurs during reperfusion, when blood cells pour into the previously unperfused zone. The sudden increase in reactive oxygen species associated with reperfusion/reoxygenation is thought to be the underlying cause of these injuries.

In normal physiological conditions, 2% of the consumed oxygen is turned into superoxide during mitochondrial respiration. In hypoxic conditions, mitochondrial respiration is likely to decrease as there is insufficient oxygen available to maintain the level of the Krebs’ cycle and electron transport chain. Hence, one might presume a reduction in reactive oxygen species during hypoxia, when less oxygen is available. Controversially, it has been shown that leakage of reactive oxygen species from mitochondria increases in hypoxic conditions, suggesting an increase in reactive oxygen species also during hypoxia. We investigated the possible involvement of radical oxygen species in the HIF-1 response indirectly by examining levels of redox-sensitive transcription factors during anoxia/hypoxia and reoxygenation. NF-B and Trx have been shown to respond to a rise in reactive oxygen species. In our experiments, nuclear NF-B levels remained constant during anoxia/hypoxia but increased transiently upon reoxygenation together with Trx levels, suggesting a rise in reactive oxygen species only during reoxygenation. In fact, the up-regulation of NF-B and Trx in response to reoxygenation even precedes the onset of HIF-1 degradation. Although the time frame of NF-B and Trx induction would allow their involvement in HIF-1 degradation, it is also possible that reactive oxygen species are directly affecting both mechanisms: NF-B and Trx induction as well as HIF-1 degradation.

Reactive oxygen species are frequently the source of damaged proteins and DNA, and various protective mechanisms have evolved to neutralize these reactive oxygen species. Catalase, superoxide dismutase, and glutathione peroxidase, for example, each target a specific reactive oxygen species. However, these mechanisms do not prevent protein and DNA damage entirely, so that there are further measures in place: specific enzymes restore damaged DNA, and ubiquitination processes cause damaged proteins to be degraded. It has been shown that reactive oxygen species can play a role in protein degradation by specifically attacking certain amino acid sequences, e.g., the PEST (proline, glutamic acid, serine, threonine) sequence. PEST-like sequences within the ODD domain (oxygen dependent degradation domain) of HIF-1 might prime HIF-1 proteins for ubiquitination by reacting with reactive oxygen species present in normoxic and especially reoxygenation conditions.

The instantaneous response of HIF-1 and its pathological implications demonstrate the vital medical importance to further our understanding of the molecular mechanisms operating in hypoxic and reoxygenation conditions. As HIF-1 is the key player in these processes, further studies with respect to its specific role in different organs and discrimination between the roles of HIF-1, HIF-2, and HIF-3 promise exciting revelations in the future.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0732fje ; to cite this article, use FASEB J. (March 28, 2001) 10.1096/fj.00-0732fje

² Present address: Institute of Physiology, Medical University of Lübeck, D-23538 Lübeck, Germany.

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