From the Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institutet, S-171 77 Stockholm, Sweden
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ABSTRACT |
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HIF-1 Hypoxia-inducible factor 1 (HIF-1)1 consists of a
heterodimer of two basic helix-loop-helix PAS proteins, HIF-1 Recently, a basic helix-loop-helix PAS protein with high similarity to
HIF-1 For many short-lived eukaryotic proteins, conjugation to the short
polypeptide ubiquitin is a mandatory step in their degradation. Highly
selective degradation of target proteins is a central component of a
great variety of cellular regulatory mechanisms, examples ranging from
the progression of the cell cycle, maintenance of circadian rhythms, to
the pathways controlling signal transduction and metabolism (11, 12).
The advantages obtained by this regulatory mechanism are obvious. In
addition to the irreversibility of the inactivation, the other major
benefit is the speed by which new steady-state levels of regulatory
molecules can be established.
Here we show that the HIF-1 Reagents--
Proteasome inhibitors
N-acetyl-Leu-Leu-norleucinal (LLnL) and clasto-lactacystin
Cell Culture and Transfections--
COS7 cells (from ATCC) were
routinely maintained in Dulbecco's minimal essential medium
supplemented with 10% fetal calf serum plus penicillin (50 IU/ml) and
streptomycin (50 µg/ml). The cells were transfected using Dotap
(Boehringer Mannheim) according to the manufacturer's recommendations.
For ubiquitination assays, 6 µg of FLAG-tagged HIF-1 Immunostaining of HIF-1 Purification of Ubiquitin Conjugates--
Twenty h after
transfection, cells were washed with phosphate-buffered saline and
lysed in 1.3 ml of 6 M guanidinium-HCl in 20 mM
Hepes, pH 7.9, per 60-mm dish. The lysate was passed through a 27-gauge
needle to decrease viscosity, and the protein concentration was
measured using a Bio-Rad protein assay. Twenty µl of Talon metal
affinity resin (CLONTECH) was then added/900 µg
of total protein. The lysate was rotated overnight at 4 °C in the
presence of 10 mM imidazole. The matrix was then washed
with decreasing concentrations of guanidinium-HCl (buffers containing
1:2 and 1:4 dilutions of the original guanidinium-HCl in buffer A (40 mM Hepes, pH 7.9, 100 mM KCl, 15% glycerol)
supplemented with 10 mM imidazole) and then finally washed
with a 1:8 dilution of this buffer before washing with buffer A only.
Remaining proteins were eluted with SDS sample buffer supplemented with
100 mM EDTA and analyzed by immunoblotting.
Immunoblotting and Detection--
The detailed protocol for the
immunoblotting of HIF-1 Inhibition of 26 S Proteasome Activity Leads to Accumulation of
Endogenous and Transiently Expressed HIF-1
To examine whether transiently expressed HIF-1 Up-regulation of HIF-1 Hypoxia-induced Nuclear Translocation of HIF-1 Increase in Protein Levels in Response to Hypoxia Is Mediated by
Structures within the HIF-1 Degradation of HIF-1 Mammalian cells contain two distinct major proteolytic pathways
(reviewed in Refs. 12 and 15), one important nonlysosomal machinery for
degradation of intracellular proteins being the ubiquitin-proteasome
pathway. In this pathway, proteins are recognized by the
ubiquitin-conjugating system in a process where multiple copies of the
76-amino acid ubiquitin polypeptide are assembled in a three-step
mechanism onto the HIF-1 To address the possible involvement of polyubiquitination in targeting
HIF-1 Interestingly, exposure of cells to an hypoxia-mimicking agent
simultaneously with the proteasome inhibitors resulted in marked reduction of ubiquitin-bound HIF-1 (hypoxia-inducible factor 1
) is a
basic-helix-loop-helix PAS
(Per/Arnt/Sim) transcription factor
that, under hypoxic conditions, dimerizes with a partner factor, the
basic-helix-loop-helix/PAS protein Arnt, to recognize
hypoxia-responsive elements of target genes. It has recently been
demonstrated that HIF-1
protein but not mRNA levels are
dramatically up-regulated in response to hypoxia. Here we show that
inhibitors of 26 S proteasome activity produced a dramatic accumulation
of endogenous as well as transfected HIF-1
protein under normoxic
conditions, whereas the levels of Arnt protein were not affected.
HIF-1
was polyubiquitinated in vivo under normoxic
conditions, indicating rapid degradation via the ubiquitin-proteasome
pathway. This degradation process appeared to target a region within
the C terminus of HIF-1
. Importantly, HIF-1
ubiquitination was
drastically decreased under hypoxic conditions. Up-regulation of
HIF-1
protein by proteasome inhibitors did not result in
transcriptional activation of reporter genes, indicating either the
requirement of additional regulatory steps to induce functional
activity of HIF-1
or the inability of polyubiquitinated forms of
HIF-1
to mediate hypoxic signal transduction. In support of
both these notions, we demonstrate that HIF-1
showed
hypoxia-dependent translocation from the cytoplasm to the
nucleus and that this regulatory mechanism was severely impaired in the
presence of proteasome inhibitors. Taken together, these data
demonstrate that the mechanism of hypoxia-dependent
activation of HIF-1
is a complex multistep process and that
stabilization of HIF-1
protein levels is not sufficient to generate
a functional form.
INTRODUCTION
Top
Abstract
Introduction
References
and
Arnt. Upon decrease in oxygen tension, the activated HIF-1
·Arnt
complex functions as a transcription factor to control the expression of genes encoding products aimed at restoring cellular homeostasis such
as erythropoietin, vascular endothelial growth factor, and several
glycolytic enzymes (reviewed in Ref. 1). Both HIF-1
and Arnt
mRNAs are constitutively expressed in a number of mammalian cell
lines under normoxic and hypoxic conditions, suggesting that functional
activity of the HIF-1
·Arnt complex is regulated by some as yet
unknown post-transcriptional mechanism(s). Recently we and others have
shown that HIF-1
protein levels are rapidly and dramatically
up-regulated upon exposure of target cells to hypoxia (2-4). However,
the precise mechanism by which HIF-1
becomes activated during
hypoxia remains unclear. This process may involve either increased
translation or increased stability of the HIF-1
protein or both. It
has previously been suggested that inhibition of protein synthesis by
cycloheximide blocks induction of HIF-1
/Arnt DNA binding activity,
indicating that the rate of synthesis of the HIF-1
subunit may have
been altered (5, 6). In striking contrast to this report, the addition
of cycloheximide after maximal hypoxic induction was reported to have
no effect either on the protein levels of HIF-1
or on the DNA
binding activity by the HIF-1
·Arnt complex (4), suggesting that an
increase in protein stability rather than increased translation may
primarily determine the induction response. Furthermore, it has been
shown that the HIF-1
protein has a rapid turnover with an estimated half-life of 5 to 10 min upon return of target cells to normoxia after
induction (7).
, EPAS1/HLF (endothelial PAS domain protein 1/HIF-1
-like factor), has been identified. After activation by hypoxia, this factor
functions as a heterodimer with Arnt and can bind to the same DNA
elements as the HIF-1
·Arnt complex (8-10). In contrast to the
ubiquitous mRNA expression pattern of HIF-1
, expression of
EPAS1/HLF mRNA is predominantly restricted to endothelial cells (9,
10).
protein is polyubiquitinated under
normoxic conditions and that this ubiquitination activity is
significantly decreased in response to hypoxic conditions. Moreover,
HIF-1
levels were up-regulated under normoxic conditions in the
presence of inhibitors of the proteasome, strongly suggesting that
HIF-1
function is regulated by proteasome-mediated degradation. This
degradation process appeared to target the C terminus of HIF-1
.
Finally, we demonstrate that HIF-1
translocates from the cytoplasm
to the nucleus in response to hypoxia and that this function as well as
the transactivation function of HIF-1
is perturbed in the presence
of proteasome inhibitors.
EXPERIMENTAL PROCEDURES
-lactone were obtained from Sigma and Calbiochem, respectively.
Expression plasmid encoding histidine-tagged ubiquitin (13) was a
generous gift from Dr. Dirk Bohmann (EMBL, Heidelberg). The
iron-chelating agent 2,2'-dipyridyl was purchased from Sigma. pFLAG
CMV2- HIF-1
encoding FLAG epitope-tagged HIF-1
was assembled by
subcloning in-frame the full-length coding sequence of HIF-1
from
pGEX4T3- HIF-1
to BamHI/SmaI-digested pFLAG
CMV2 (Eastman Kodak Co.). The construction of the various HIF-1
deletion mutants fused to the GAL4 DNA binding domain will be presented
elsewhere.2
or the empty
control plasmid in combination with 6 µg of His-tagged ubiquitin (or
the control plasmid) were used per 60-mm dish. When comparing the
expression levels of various constructs, cells were collectively
transfected in a 80-cm2 flask and, after transfection,
split onto 60-mm dishes for treatments. Whole cell extracts were
prepared as described previously (2). Primary mouse brain endothelial
cells (kindly supplied by Dr. Yihai Cao, Karolinska Institute) were
incubated in F12 medium containing 10% fetal calf serum and
antibiotics. For transactivation experiments, cells were transiently
transfected with pCMV-HIF-1
(14) together with a luciferase reporter
gene carrying three tandem copies of the hypoxia response element of
the erythropoietin gene (HRE-luc). Six h after transfection, cells
received fresh medium and were either left nontreated or treated with
100 µM 2,2'-dipyridyl or 35 µM LLnL for
24 h before harvesting.
--
COS7 cells were grown on
fibronectin-coated coverslips in 6-well culture plates and transiently
transfected with pCMV-HIF-1
. After 24 h of expression, the
cells were incubated in the presence or absence of 20 µM
LLnL for 12 h and further incubated with or without 100 µM 2,2'-dipyridyl for 6 h. Thereafter, fixation of the cells was performed with a freshly prepared solution of 4% (w/v)
paraformaldehyde in phosphate-buffered saline overnight at 4 °C. The
fixed cells were incubated with anti-human HIF-1
rabbit antiserum
(2) in phosphate-buffered saline containing 0.1% Triton X-100 for
9 h at 4 °C. Indirect immunofluorescence was obtained by
incubation with biotinylated goat anti-rabbit IgG antibodies and Texas
red-conjugated streptavidin (Amersham Pharmacia Biotech) in
phosphate-buffered saline containing 0.1% Triton X-100. The coverslips
were mounted on glass slides and subjected to microscopic analysis.
and Arnt proteins together with description
of the rabbit antiserum has been described previously (2). The
anti-HIF-1
antiserum used is specific for HIF-1
, because it does
not recognize in vitro translated EPAS/HLF (data not shown).
For the detection of the FLAG epitope-tagged constructs or GAL4 fusion
proteins, proteins were blotted after SDS-polyacrylamide
electrophoresis onto nitrocellulose filters and blocked overnight with
5% nonfat milk in Tris-buffered saline. Either the anti-FLAG M2
antibody (Kodak) or anti-GAL4 antiserum (Upstate Biotechnology, Lake
Placid, NY) were used as a primary antibody in a dilution of 10 µg of protein/ml of Tris-buffered saline containing 1% nonfat milk for 2 h. After several washes, a 1:750 dilution of anti-rabbit
IgG-horseradish peroxidase conjugate (Amersham Pharmacia Biotech) in
Tris-buffered saline, 1% nonfat milk was used as a secondary antibody.
After extensive washing with Tris-buffered saline, the complexes were visualized using enhanced chemiluminescence (Amersham Pharmacia Biotech) according to the manufacturer's instructions.
RESULTS
Levels--
In the case
of a broad number of either inducible or periodically expressed gene
products, a major determinant of the protein levels is the turnover
rate of the polypeptide (12, 15). Previously we and others have shown
that hypoxic stimulation either by lowering the oxygen concentration or
by exposing cells to hypoxia-mimicking (16) chemicals such as
CoCl2 or desferrioxoamine leads to a rapid increase in
cellular HIF-1
levels but not those of the partner factor Arnt (2,
4). Therefore we attempted to investigate whether blocking one of the
major cellular protein degradation pathways, i.e. the
pathway mediated by the 26 S proteasome, would lead to accumulation of
HIF-1
protein also under normoxic conditions. To this end we exposed
mouse brain primary endothelial cells to the peptide aldehyde LLnL, an
inhibitor of the proteasomal pathway, and analyzed whole cell extracts
by immunoblotting using HIF-1
-specific antisera. As shown in Fig.
1, treatment of mouse brain endothelial cells with 35 µM LLnL for 20 h resulted in a marked
increase of endogenous HIF-1
protein levels (whereas most of the
nonspecific cross-reacting signals remained unaffected). Because LLnL
is also known to have an inhibitory effect on some nonproteasomal
degradation pathways, e.g. those mediated by lysosomes or by
calpains and cathepsins (17), we decided to perform the same experiment
using clasto-lactacystin
-lactone, a highly selective inhibitor of 26 S proteasome activity (18). Lactacystin and its more potent derivative, clasto-lactacystin
-lactone, have both been shown to
modify and thereby inactivate the catalytic
-subunits of the proteasome (17). Interestingly, incubation of the brain endothelial cells in the presence of 5 µM clasto-lactacystin
-lactone for 20 h also lead to a marked stabilization of
HIF-1
protein levels as compared with cells treated with vehicle
only (Fig. 1A, compare lanes 1 and 2).
Furthermore, identical results were obtained in the mouse hepatoma cell
line Hepa 1c1c7 (data not shown). In parallel experiments, we examined
the effect of the two proteasome inhibitors, clasto-lactacystin
-lactone and LLnL, on intracellular levels of Arnt in primary mouse
brain endothelial cells. In contrast to HIF-1
protein levels, Arnt
protein levels remained unaffected upon exposure to these inhibitors
(Fig. 1B), in excellent agreement with the observation that
hypoxic treatment does not alter Arnt protein levels (2). Taken
together, these results clearly demonstrate that HIF-1
protein
levels are specifically up-regulated in the presence of proteasome
inhibitors and that this enhanced accumulation of protein is associated
with increased metabolic stability.
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Fig. 1.
HIF-1 levels are
up-regulated in the presence of proteasome-specific inhibitors.
Mouse primary brain endothelial cells were treated with either 5 µM clasto-lactacystin
-lactone (lactac;
lane 2), 35 µM LLnL, or with vehicle only
(lanes 1 and 3) for 20 h before preparation
of whole cell extracts. Twenty-µg aliquots of the extracts were
analyzed by immunoblotting with anti-HIF-1
(A) or with
anti-Arnt antibodies (B). The positions of the molecular
weight mass (in kDa) are indicated on the left.
follows the same mode
of regulation as the endogenous protein, we transiently transfected
COS7 cells with a FLAG epitope-tagged HIF-1
expression vector and,
20 h after transfection, exposed the cells to either inducers of
the hypoxic response or to proteasome inhibitors. These conditions were
identical to those used above when studying endogenous HIF-1
protein
levels. After induction for 24 h, the cells were harvested and
analyzed by Western blotting for the presence of the transfected
protein. As seen in Fig. 2, even the high
level of expression usually obtained after transfection into COS7 cells
did not allow us to detect by immunoblot analysis any significant
levels of FLAG-HIF-1
expression in normoxic cells, suggesting also
that transfected HIF-1
is rapidly degraded. However, in analogy to
the endogenous HIF-1
protein levels (2), exposure of cells to the
hypoxia-mimicking iron chelator 2,2'-dipyridyl leads to a very
significant accumulation of epitope-tagged HIF-1
. Thus, these data
suggest that the increase in HIF-1
protein levels is mediated by
sequences within the coding region of the gene product. Furthermore, in
analogy to the endogenous HIF-1
, exposure of the transfected cells
to proteasome inhibitors, either the peptide aldehyde LLnL or
clasto-lactacystin
-lactone, dramatically increased the levels of
epitope-tagged HIF-1
(Fig. 2; compare lanes 1,
3,and 4), indicating that the transiently
expressed protein is rapidly degraded via the 26 S proteasome.
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Fig. 2.
Regulation of HIF-1
protein levels by hypoxia-mimicking agents and the 26 S
proteasomal complex. COS7 cells were transiently transfected with
expression plasmids encoding epitope-tagged HIF-1
and, 24 h
after transfection, treated for an additional 20 h with either 100 µM 2,2'-dipyridyl (DP; lane 2), 35 µM LLnL (lanes 3 and 5), or 25 µM clasto-lactacystin
-lactone (lactac; lane
4). The cells were subsequently lyzed, and whole cell extracts
were prepared. Eighty-µg aliquots of the cell extracts were then
analyzed by immunoblotting with monoclonal anti-FLAG antibodies.
Lane 5 represents cells transfected with empty expression
vector. The positions of the molecular mass markers are indicated on
the left.
Protein Levels by Proteasome Inhibitors
Does Not Generate a Functional Form of HIF-1
--
Under hypoxic
conditions, up-regulation of HIF-1
protein levels correlates with
induction of hypoxia-regulated genes and activation of reporter genes
(1, 2). Given the up-regulation of both endogenous and transiently
expressed HIF-1
protein levels upon exposure of cells to proteasome
inhibitors under normoxic conditions, we next wanted to examine the
effects of these inhibitors on functional activities of HIF-1
. In
control experiments, treatment of mouse brain endothelial cells with
2,2'-dipyridyl resulted in potent activation of a hypoxia-responsive
reporter gene construct (Fig. 3).
Strikingly, however, exposure of the cells to increasing concentrations
of the proteasome inhibitor LLnL under normoxic conditions did not
result in activation of the reporter gene (Fig. 3). Thus, these results
indicate that mere up-regulation of HIF-1
protein levels by
proteasome inhibitors was not sufficient to elicit a transcriptional
response. In conclusion, under these conditions, HIF-1
may not be
functional, and/or additional regulatory steps may be important to
activate HIF-1
function. In support of this model,
2,2'-dipyridyl-induced transcriptional activation by HIF-1
was
inhibited in a dose-dependent manner by cotreatment of the
cells with LLnL (Fig. 3).
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Fig. 3.
Up-regulation of HIF-1
protein levels by exposure to proteasome inhibitors does not
result in HIF-1
-mediated transcriptional
activation. Mouse brain endothelial cells were transfected with
the pCMV-HIF-1
expression vector (1.0 µg/30-mm dish) together with
the HRE-driven luciferase reporter gene construct (1.5 µg). Six h
after transfection, cells were incubated with fresh medium and treated
with either 100 µM 2,2'-dipyridyl (DP), 5 (+)
or 10 (++) µM LLnL, or with vehicle only for
24 h. Reporter gene activities were standarized to protein content
and expressed as -fold induction relative to the activity under
normoxic conditions of the reporter gene alone in the presence of
transiently expressed HIF-1
.
--
We next
transiently expressed HIF-1
in COS7 cells using a CMV
promoter-driven expression vector and studied the intracellular localization of HIF-1
. Under normoxic conditions, HIF-1
immunoreactivity of rather low intensity was preferentially localized
in the cytoplasmic compartment of the cell, as assessed by
immunostaining using polyclonal anti-HIF-1
antibodies. In contrast,
exposure of the cells to the hypoxia-mimicking agent 2,2'-dipyridyl
resulted in an almost complete nuclear translocation of HIF-1
with
no detectable immunoreactivity remaining in the cytoplasm (Fig.
4). After incubation of the cells with
the proteasome inhibitor LLnL under normoxic conditions, high intensity
HIF-1
immunoreactivity was uniformly distributed throughout the
cell. Strikingly, this intracellular distribution was not altered upon
exposure of the cells to a combination of both LLnL and 2,2'-dipyridyl
(Fig. 4). Thus, hypoxia-dependent nuclear accumulation of
HIF-1
was impaired upon stabilization of HIF-1
protein levels by
LLnL.
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Fig. 4.
Inhibition of hypoxia-inducible nuclear
translocation of HIF-1 in the presence of
proteasome inhibitor. COS7 cells were transiently transfected with
pCMV HIF-1
and, after 24 h of expression, the cells were
incubated for 12 h in the presence or absence of 20 µM LLnL. Subsequently, the cells were treated with or
without 100 µM 2,2'-dipyridyl (DP) for 6 h, as indicated, whereafter the cells were fixed for
immunocytochemistry. Subcellular localization of expressed HIF-1
was
determined by indirect immunofluorescence using anti-HIF-1
rabbit
antiserum. The photographs show immunofluorescence
(
-HIF-1
) and corresponding phase contrast
(phase) pictures.
Protein--
In our initial attempts to
map the putative destabilizing elements within the HIF-1
protein, we
transiently expressed in COS7 cells a set of HIF-1
deletion mutants
fused to the minimal DNA binding domain of the yeast transcription
factor GAL4 (schematically represented in Fig.
5A). In excellent agreement
with the corresponding effects on endogenous HIF-1
levels (Fig. 1),
GAL4-HIF-1
levels were significantly increased upon exposure of the
cells to either 2,2'-dipyridyl, LLnL, or a combination of these two
agents (Fig. 5B; compare lane 1 to lanes
2-4). A very similar mode of regulation of the protein levels was
observed using the HIF-1
deletion mutant GAL4 HIF-1
1-652.
Importantly, the protein levels of GAL4-HIF-1
526-826 spanning the
C terminus of HIF-1
were also stabilized by treatment with either
2,2'-dipyridyl, LLnL, or a combination of both. Interestingly, the C
terminus of HIF-1
(residues 526-826) harbors both an N-terminal and
a C-terminal transactivation domain, which both act in concert with one
another to mediate hypoxia-inducible transcriptional activation (19,
20). In addition, as outlined in Fig. 5A, this region of
HIF-1
contains in the vicinity of the N-terminal transactivation
domain a sequence with strong similarity to a PEST motif, which often
targets proteins for rapid degradation (21). Apart from this motif, the
C terminus of HIF-1
does not display any significant similarity to
previously described protein-destabilizing elements. The C-terminal
transactivation domain of HIF-1
contained in GAL4 HIF-1
786-826
showed significant and stable steady-state levels of expression upon
transient expression in normoxic COS7 cells. These stable protein
levels were not further increased in response to hypoxic stimulation
and only slightly elevated in the presence of proteasome inhibitors
(Fig. 5B; compare lanes 13-16). In conclusion,
these data indicate that the region spanning the N-terminal
transactivation domain of HIF-1
also contains a structural motif
involved in protein destabilization under normoxic conditions and,
thus, shows a complex functional architecture. It is noteworthy that
EPAS/HLF, which also mediates hypoxia-inducible transcriptional
activation (9, 10), contains a PEST motif in a very similar region of
the protein. Consistent with this observation and in analogy to GAL4
HIF-1
, GAL4 HIF-1
1-652, and GAL4 HIF-1
526-826, GAL4 HLF
steady-state protein levels were hardly detectable upon transient
expression in COS7 cells under normoxic conditions and significantly
stabilized after exposure to hypoxic stimulation, proteasome inhibitor,
or both treatments (data not shown). In contrast, in control
experiments, the minimal GAL4 DNA binding domain was stably expressed
under normoxic conditions and showed only modest stabilization in the
presence of proteasome inhibitor (Fig. 5C).
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Fig. 5.
HIF-1 harbors
destabilizing elements in the C-terminal region of the
protein. A, schematic representation showing the
various GAL4 HIF-1
fusion protein constructs. bHLH,
basic-helix-loop-helix. DBD, DNA binding domain. The N-
(N-TAD) and C-terminal (C-TAD) transactivation
domains as well as a PEST (A) sequence motif are indicated.
B and C, COS7 cells were transiently transfected
with cytomegalovirus promoter-driven expression vectors expressing GAL4
HIF-1
deletion mutants (B) or the minimal GAL4 DNA
binding domain (GAL4 DBD) alone (C). After
24 h of expression, the cells were further treated for an
additional 20 h with either vehicle, 100 µM
2,2'-dipyridyl (DP) or 35 µM LLnL or a
combination of DP and LLnL, as indicated. Subsequently, the cells were
lysed, and extracts were prepared. Twenty-five-µg protein aliquots of
cell extracts were then analyzed by immunoblotting with anti-GAL4
antibodies. The positions of the molecular weight markers are indicated
on the left, and the expressed protein is indicated by an arrow
head.
via the Ubiquitin-Proteasome
Pathway--
To directly assess whether HIF-1
can form ubiquitin
conjugates in vivo, COS7 cells were cotransfected with an
HIF-1
expression vector together with a vector expressing
multimerized polyhistidine-tagged ubiquitin or a corresponding control
vector (13). Twenty-four h after transfection, the cells were exposed
to either LLnL, clasto-lactacystin
-lactone, or to vehicle only.
After incubation for 20 h, cells were harvested in a denaturing
buffer to prevent protein degradation and to permit co-purification of
noncovalently associated polypeptides. To this end, equal amounts of
total cellular protein were incubated with a metal-affinity resin, and
ubiquitin-conjugated proteins were affinity-purified and analyzed by
immunoblotting (Fig. 6). These
experiments show that immunoreactivity, either against the epitope tag
(Fig. 6A) or against HIF-1
(Fig. 6B), was
detected only upon transient expression of HIF-1
and
histidine-tagged ubiquitin in the presence of the proteasome inhibitors
(Fig. 6, A and B, lane 6). In control
reactions lacking either transfected His-tagged ubiquitin or HIF-1
,
no signals were seen (Fig. 6, A and B,
lanes 1-4). Thus, no background reactivity was demonstrated under the stringent washing conditions used. By contrast, the reason
for the absence of virtually any immunoreactivity in the lanes containing both transfected HIF-1
and
histidine-tagged ubiquitin lacking proteasome inhibitors (Fig. 6,
A and B, lane 5) is most likely
explained by the extremely rapid turnover of multiubiquitinated
complexes with a half-life being in the range of 1 min (22).
Importantly, simultaneous stimulation of the cells with the
hypoxia-mimicking agent 2,2'-dipyridyl very significantly decreased the
affinity-purified levels of multiubiquitinated HIF-1
as compared
with those detected under normoxic conditions (Fig. 6, A and
B, compare lanes 6 and 7). In
conclusion, the decreased levels of ubiquitinated HIF-1
are
consistent with the elevated protein levels observed under these
conditions (Fig. 2).
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Fig. 6.
HIF-1 is
polyubiquitinated under normoxic conditions. COS7 cells were
transfected either with empty FLAG (lanes 1-3) or with FLAG
HIF-1
expression vector (lanes 4-7) together with an
expression plasmid encoding histidine-tagged ubiquitin
(H6-ub, lanes 2, 3, and
5-7) or with corresponding control vectors (lanes
1 and 4). After transfection, cells were treated for
20 h with 35 µM LLnL (A), 10 µM clasto-lactacystin
-lactone (B)
(lanes 1, 3, 4, 6, and
7), or vehicle (Me2SO) only (lanes 2 and
5). His-tagged proteins were purified from the transfected cell
lysates using a metal-affinity column, and specifically bound material
was then analyzed by immunoblotting using anti-FLAG (A) and
anti-HIF-1
antiserum (B). The sample in lane 7 was treated identically as that in lane 6 except during the
20-h induction period with the chemicals, lane 7 was
simultaneously exposed to 100 µM 2,2'-dipyridyl
(DP), mimiking hypoxic induction. For each panel,
the positions of the molecular weight markers are shown on the
left.
DISCUSSION
-amino groups of lysine residues of the target
protein. The ubiquitinated substrates are then rapidly escorted for
hydrolysis to the 26 S proteasome, whereas the ubiquitin molecules are
recycled via the action of deubiquitinating enzymes present in the 26 S
complex (12, 15).
is among the most short-lived proteins currently known. Its
half-life after induction and subsequent return of the cells to
normoxia is in the range of a few min (7), remarkably short compared
with other stress-activated transcription factors such as c-Jun
(half-life
90 min (23)) or p53 (half-life
7-8 h;
Ref. 24 and references therein). In our efforts to characterize the
mechanisms of activation of HIF-1
function and given the massive
up-regulation of HIF-1
protein levels by hypoxia (2, 4), we
investigated the effect of inhibitors of the 26 S proteasome on
HIF-1
protein levels. Both a wide-specificity inhibitor, LLnL, and a
highly specific lactacystin metabolite, clasto-lactacystin
-lactone,
produced a dramatic accumulation of HIF-1
already under normoxic
conditions. In agreement with these observations, it has recently been
reported that similar proteasome inhibitors induce the DNA binding
activity of the HIF-1
·Arnt complex in normoxic cells (25). Our
experiments indicate that these elevated levels of DNA binding activity
are specifically because of increased levels of HIF-1
protein,
because Arnt levels were unaltered in the presence of proteasome
inhibitors. Although the DNA binding activity of the HIF-1
·Arnt
complex appears to be induced under conditions of proteasome inhibition
(25), this response did not correlate with activation of hypoxia
response element-dependent reporter gene expression,
suggesting that a nonfunctional form of HIF-1
is accumulated.
Moreover, in excellent agreement with the lack of functional activity
of HIF-1
upon stabilization by proteasome inhibition, our data
indicate the failure of HIF-1
to accumulate in the nucleus of
hypoxic cells in the presence of proteasome inhibitors. Taken together,
these data strongly argue that polyubiquitinated forms of HIF-1
that
are generated in the presence of proteasome inhibitors are nonfunctional.
for destruction, we used an in vivo assay (13) where, upon transient expression of affinity-tagged ubiquitin, target
proteins can be isolated using an affinity resin. Employing this assay
in the presence of a proteasome inhibitor to prevent rapid destruction
of the conjugated complexes, HIF-1
was effectively copurified in the
presence of histidine-tagged ubiquitin. This effect was selective for
HIF-1
because no immunoreactivity was detected in the absence of
either His-tagged ubiquitin or proteasome inhibitors. Furthermore, the
finding that HIF-1
is multiubiquitinated under normoxia may be of
potential importance, because it has been demonstrated that not all
proteins targeted to the 26 S proteasome are ubiqutinated; it has
rather been shown that proteasome may have a more general function in
selective removal of short-lived proteins by recognizing degradation
signals other than ubiquitin (26).
, which correlated with elevated cellular protein levels. Thus, as outlined in the model in Fig. 7, HIF-1
is differentially regulated
by ubiquitination in normoxia as opposed to hypoxia, and inhibition of
ubiquitination constitutes an early and critical step in regulation of
HIF-1
function preceding nuclear translocation, recruitment of Arnt,
and ensuing gene activation. Our present experiments indicate that
regulation of HIF-1
protein levels by the proteasome pathway were
mediated by a C-terminal structure of the protein spanning a PEST
sequence motif (21). Interestingly, the same region of HIF-1
has
been shown to harbor the N-terminal transactivation domain of HIF-1
and has thus been implicated to be involved in conditional regulation
of HIF-1
function (19, 20). Thus, these data illustrate the very
complex functional architecture of the C terminus of HIF-1
. In
agreement with the present results, the identification of a broadly
defined oxygen-dependent degradation domain between
residues 401 and 603 was reported during the completion of this study
(27). What is the degradation motif (or motifs) within this C-terminal
region of HIF-1
and what is the nature of the signal and mechanism
that renders the degradation process inactive during hypoxic
conditions? Transplantable sequence elements, destruction boxes,
recognized and targeted by a proteolytic apparatus have been identified
in many short-lived proteins (28), but despite this, the structural features characterizing these elements are largely unknown. Against this background, HIF-1
provides an interesting model system to understand regulation of protein function by protein degradation. In
particular, it will be important to investigate how the process of
HIF-1
protein stabilization is related to the mechanism of conditional regulation of HIF-1
function.
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Fig. 7.
Role of ubiquitin
(Ubq)-mediated proteasomal degradation in activation
of HIF-1 function. HIF-1
is
multiubiquitinated under normoxic conditions and, following
ubiquitination, rapidly degraded via the 26 S proteasome. Hypoxic
stimulation allows HIF-1
to escape ubiquitination, leading to an
increase in protein levels, induced nuclear import, and dimerization
with Arnt, enabling HIF-1
to recognize cognate hypoxia response
elements and thereby activate target gene transcription.
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ACKNOWLEDGEMENTS |
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We thank Dr. Dirk Bohmann for providing us with the His-tagged ubiquitin cDNA construct, Dr. Pascal Coumailleau for the HRE-luc reporter gene construct, and Dr. Yihai Cao for mouse brain endothelial cells.
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FOOTNOTES |
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* This work was supported by grants from the Swedish Cancer Society and the Swedish Medical Research Council.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by a Marie Curie Research Training Grant from the
European Commission.
§ Partly supported by the Uehara Memorial Foundation.
¶ To whom correspondence should be addressed. Tel.: +46-8-728 7330; Fax: +46-8-34 88 19; E-mail: lorenz.poellinger{at}cmb.ki.se.
2 P. Carrero, P. Coumailleau, S. O'Brien, and L. Poellinger, manuscript in preparation.
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ABBREVIATIONS |
---|
The abbreviations used are:
HIF-1, hypoxia-inducible factor 1
;
EPAS1/HLF, endothelial PAS domain
protein 1/HIF-1
-like factor;
PAS Per-Arnt-Sim homology, Dotap
N-[1(2,
3-dioleoyloxy)propyl]N,N,N-trimethylammonium methyl
sulfate;
LLnL, N-acetyl-Leu-Leu-norleucinal;
CMV, cytomegalovirus.
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REFERENCES |
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