Induction of HIF-1␣ in response to hypoxia
is instantaneous1
URSULA R. JEWELL, IVICA KVIETIKOVA, ANNETTE SCHEID, CHRISTIAN BAUER,
ROLAND H. WENGER,2 MAX GASSMANN3
Institute of Physiology, University of Zürich, CH-8057 Zürich, Switzerland
SPECIFIC AIMS
Despite the pivotal role the hypoxia-inducible factor 1␣
(HIF-1␣) plays in physiological and pathological pro-
cesses, 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 kinet-
ics of HIF-1␣ induction and degradation, and compar-
ison with its dimerization partner ARNT (aryl hydro-
carbon 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 contin-
ued 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 (t1/2max). Exposure to 0% and 0.5%
oxygen resulted in t1/2max 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 accu-
mulation 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 con-
centration 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%
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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 thresh-
old, thereby limiting the initial protein accumulation
process to below 2 min.
Also after 2 minutes the onset of HIF-1 DNA-binding
can be weakly observed and becomes more pro-
nounced 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 concentra-
tion of any of these redox factors is unlikely to deter-
mine 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 reoxy-
genation, 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␣ pro-
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 Univer-
sity of Lübeck, D-23538 Lübeck, Germany.
3
Correspondence: Institute of Physiology, University of
Zürich, Winterthurerstr. 190, CH-8057 Zürich, Switzerland.
E-mail: maxg@access.unizh.ch
0892-6638/01/0015-1312 © FASEB

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).
tein 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. Measure-
ments 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 reoxy-
genation from hypoxia.
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
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.
INSTANTANEOUS INDUCTION OF HIF–1␣ IN HYPOXIA
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 approx-
imately 5 min in normoxic conditions. Here we show
for the first time that HIF-1␣ protein is already detect-
able 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 translo-
cated to the nucleus. This immediate response tempo-
rally 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 bind-
ing, 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,
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 transcrip-
tion of EPO (erythropoietin), VEGF (vascular endothelial
growth factor), Tf (transferrin), and other oxygen-regulated
genes. Upon reoxygenation, HIF-1␣ proteins undergo effi-
cient proteasomal degradation within 16 min.
1313
which might indicate an inhibitory feedback mecha-
nism. The HIF-1␣ dimerization partner, ARNT, is al-
ready present in the nucleus of normoxic cells, and the
total amount of ARNT within the whole cell is not
affected by hypoxia.
HIF-1 DNA binding is lost rapidly during reoxygen-
ation 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% oxy-
gen) 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 en-
sure sufficient up-regulation of HIF-1 target genes. The
suggestion that the degree of hypoxia affects the reoxy-
genation response is complemented by recent findings
that the duration of hypoxia prior to reoxygenation
influences the response to reoxygenation. In vivo stud-
ies of reoxygenation have also shown that the mecha-
nisms 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 suf-
ficient 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 reoxygen-
ation 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 patho-
logical examples that demonstrate severe tissue injuries
resulting from reoxygenation, e.g., after organ trans-
plantations, bypass operations, and ischemia-reperfu-
sion 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 con-
sumed oxygen is turned into superoxide during mito-
chondrial respiration. In hypoxic conditions, mito-
chondrial respiration is likely to decrease as there is
1314 Vol. 15 May 2001 The FASEB Journal
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 con-
stant during anoxia/hypoxia but increased transiently
upon reoxygenation together with Trx levels, suggest-
ing a rise in reactive oxygen species only during reoxy-
genation. 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␣ degrada-
tion.
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 mech-
anisms do not prevent protein and DNA damage en-
tirely, so that there are further measures in place:
specific enzymes restore damaged DNA, and ubiquiti-
nation processes cause damaged proteins to be de-
graded. 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 patho-
logical implications demonstrate the vital medical
importance to further our understanding of the molec-
ular mechanisms operating in hypoxic and reoxygen-
ation 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.
JEWELL ET AL.