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INTRODUCTION |
Cellular hypoxia is an important phenomenon in developmental
biology, normal physiology, and many pathological conditions, including
cancer. Hypoxia triggers a multifaceted adaptive response that is
primarily mediated by the heterodimeric transcription factor
hypoxia-inducible factor
(HIF).1 HIF-1 is a
heterodimer composed of two subunits, the rate-limiting factor HIF-1
and the constitutive expressed HIF-1
(1, 2). HIF-1
has been
characterized as an aryl hydrocarbon receptor nuclear translocator, and
this family of proteins has previously been shown to heterodimerize
with the aryl hydrocarbon receptor (3). On the other hand,
HIF-1
specifically mediates hypoxic responses. In normoxia, HIF-1
is maintained at low and often undetectable levels. HIF-1
is
targeted for degradation by the ubiquitination-proteasome pathway
through directly binding to the von Hippel-Lindau tumor suppressor gene
(pVHL), which forms the recognition component of an
E3 ubiquitin-protein ligase leading to ubiquitination of HIF-1
(4-7). Recent reports demonstrate that HIF-1
undergoes an
iron- and oxygen-dependent modification before it
can interact with pVHL. This modification is
catalyzed by a specific family of enzymes termed HIF-1
-proline
hydroxylases (8-10). During hypoxia, this specific hydroxylase is
inactive since it requires dioxygen for its activity. As a result,
HIF-1
accumulates due to the failure of pVHL to
recognize its non-hydroxylated form. HIF-1
then translocates to
the nucleus and dimerizes with the constitutively present HIF-1
(11).
The importance of the HIF-1
response pathway in human tumorigenesis
is underscored by the finding that HIF-1
is overexpressed in
multiple human cancers, because tumor cells, unlike normal cells from
the same tissue, are often chronically hypoxic (12). The tumor
suppressor protein p53 integrates numerous signals that control cell
life and death (13). Wild-type p53 is expressed at low levels in most
cells because of its short half-life under normal conditions. In
contrast, the p53 protein is stabilized, and its level increases in
response to various stresses such as DNA damage, hypoxia, and
inappropriate oncogene signaling (14, 15). In its active form, p53 can
bind DNA in a sequence-specific manner and activate transcription of
target genes. p53 levels are regulated in large part by Mdm2, the
product of a p53-inducible gene. Mdm2 can interact with the N terminus
of p53, which also contains the major acidic transcriptional activation
domain. The interaction between Mdm2 and p53 can inhibit p53
transcriptional activity by interfering with the ability of p53 to
contact transcriptional coactivators such as p300/CBP (16).
Importantly, Mdm2 binding also promotes the ubiquitination of p53 and
its export from the nucleus to the cytoplasm, where p53 is then
degraded by cytoplasmic proteasomes (17).
Hypoxic induction of p53 requires concomitant induction of HIF-1
,
whereby HIF-1
can then bind to and stabilize p53 (18-21). However,
the molecular mechanism by which HIF-1
stabilizes p53 remains
unknown. Moreover, it is not clear whether HIF-1
interacts with p53
directly despite previous indications that p53 associated with HIF-1
in cells. Nevertheless, an array of immobilized peptide assay showed
that the core domain of p53 has an affinity for the oxygen-dependent
degradation domain of HIF-1
(22). As described in this
report, we have found a strong interaction between HIF-1
and Mdm2,
while we failed to detect any direct interaction of p53 with HIF-1
.
Our data demonstrate that HIF-1
regulates p53 activity including
stability and nuclear export through interactions with Mdm2. These
results provide a potential mechanism for p53 stabilization by HIF-1
in response to hypoxia.
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EXPERIMENTAL PROCEDURES |
Cell Culture and Transfection--
p53-null H1299
lung carcinoma cells and mouse embryonic fibroblast cells (MEFs) were
cultured in Dulbecco's modified Eagle's medium (Mediatech,
Richmond, VA) supplemented with penicillin/streptomycin and
10% fetal bovine serum (Mediatech, VA).
1 × 106 or 2 × 105 cells were
plated in 10-cm or six-well plates, respectively, and 24 h later
transfections were done by calcium phosphate precipitation procedures.
After a 24-h incubation with 20% O2, the cells were harvested.
Plasmids--
Full-length HIF-1
DNA was generated by PCR
using hemagglutinin-tagged HIF-1
from David Livingston (Dana Farber
Cancer Institute) and was subcloned into pcDNA3.1/v5-His-Topo
(Invitrogen) or p3×FLAG-CMV-14 (Sigma). Green fluorescence protein
(GFP)-tagged wild-type p53 was obtained from Yanping Zhang (M. D.
Anderson Cancer Center). Plasmid DNA for transfections was isolated
using Qiagen plasmid maxi kit (Qiagen).
Recombinant Protein Preparation and Glutathione S-Transferase
(GST) Pull-down Assay--
GST fusions of p53 and Mdm2 were expressed
in Escherichia coli BL-21 (DE3) (Promega) and induced with
0.1 mM
isopropyl-1-thio-
-D-galactopyranoside. Bacterial pellets
were lysed in BC500 (25 mM Tris, pH 7.8, 500 mM
NaCl, 1 mM EDTA, 1 mM DTT, 10% glycerol, 0.2%
Nonidet P-40, fresh 1 mM PMSF) with sonication. Levels of
expressed GST fusion proteins were estimated by incubation with
glutathione-Sepharose beads, washing, and quantification by SDS-PAGE
followed by staining with Coomassie Brilliant Blue R-250. Known amounts
of bovine serum albumin were used as standards.
35S-Labeled in vitro translated HIF-1
was
prepared by using the TNT system (Promega). Equal amounts (1 µg) of
GST, GST-p53, and GST-Mdm2 immobilized on glutathione-Sepharose beads
were incubated with in vitro translated HIF-1
in BC200
for 3 h at 4 °C. After washing, the bound proteins were eluted
with SDS sample buffer and were separated by SDS-PAGE, followed by autoradiography.
Immunoblotting and Co-immunoprecipitation--
Cells were lysed
in FLAG lysis buffer (50 mM Tris, 137 mM NaCl,
10 mM NaF, 1 mM EDTA, 1% Triton X-100, 0.2%
Sarkosyl, 1 mM DTT, 10% glycerol, pH 7.8) with fresh
protease inhibitors (1 mM PMSF, protease inhibitor mixture
(Sigma)). Aliquots (30 µg) of cell extracts were resolved in SDS, 8%
polyacrymide gels and then transferred to nitrocellulose membranes in
20 mM Tris-HCl, pH 8.0, 150 mM glycine,
20%(v/v) methanol. Membranes were blocked with 5% (v/v) nonfat dry
milk, TBST (20 mM Tris-HCl, pH 7.6, 137 mM
NaCl, 0.1% Tween 20), incubated with
-p53 (DO-1) antibody (Santa
Cruz), or anti-GFP (Clontech), and detected with
ECL reagents (Amersham Biosciences).
Coimmunoprecipitation assay was performed essentially as described
previously (23). In brief, 50 µl of proteasome inhibitor N-acetyl-leucyl-leucyl-norleucinal was added to the
cotransfected culture 6 h before harvest. 24 h after
transfection, the cells were lysed in BC100 (25 mM Tris, pH
7.8, 100 mM NaCl, 1 mM EDTA, 1 mM
DTT, 10% glycerol, 0.2% Nonidet P-40, fresh 1 mM PMSF)
and incubated with anti-FLAG M2 beads (Sigma) overnight at 4 °C. The beads were washed five times with 1 ml of lysis buffer, after which the
associated proteins were eluted with BC100, 0.2% Nonidet P-40 plus 0.2 mg/ml FLAG peptide (Sigma). The eluted proteins were resolved on 8%
SDS-PAGE and Western blot with the anti-p53 or anti-Mdm2 (SMP14, Santa
Cruz) monoclonal antibody.
Luciferase Assay--
Luciferase activity was determined using a
dual luciferase assay system (Promega) following the manufacturer's
protocol. Cells in six-well plates were removed by scraping into
100 µl of reporter lysis buffer. Cell lysate was collected by
centrifugation for 15 min at 12,000 × g. Luciferase
activity was measured using a Lumat LB 9507 luminometer (EG&G Wallac,
Gaithersburg, MD).
In Vitro Ubiquitination Assays--
The in vitro
ubiquitination assay was performed as previously described (24) with
some modifications. For a standard reactions, the purified FLAG-p53
proteins from H1299 cells were mixed with other purified components,
including E1, E2 (GST-UbcH5C), E3 (GST-Mdm2), and His-ubiquitin in
reaction buffer (40 mM Tris, 5 mM
MgCl2, 2 mM ATP, 2 mM DTT, pH 7.6).
The reaction was stopped after 60 min at 37 °C by additions of SDS
sample buffer and subsequently resolved SDS-PAGE gels for Western blot
analysis with
-p53 (DO-1).
Nuclear Export Assay for p53--
H1299 cells were plated
on six-well-containing glass coverslips, and GFP-p53 was transfected as
described. 50 µM proteasome inhibitor
N-acetyl-leucyl-leucyl-norleucinal was added for
6 h before fixation. Twenty-four hours after transfection, cells
on the coverslips were washed three times with phosphate-buffered saline (PBS) and then fixed in 4% paraformaldehyde for 10 min at room
temperature. After fixation, cells were washed in PBS three times and
then permeabilized in ice-cold PBS containing 0.2% Triton X-100 for 10 min. Cells were blocked in PBS containing 1% bovine serum albumin and
1 µg of DAPI (Sigma)/ml at room temperature for 30 min. Cells were
washed three times with PBS, and the stained cells were mounted with
mounting medium (Polysciences, Inc., Warrington, PA) and sealed
with nail polish. Immunofluorescence was recorded using an
immunofluorescence microscope.
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RESULTS |
Interaction between HIF-1
and Mdm2--
Although earlier
studies indicated that p53 can bind HIF-1
in cells, we repeatedly
failed to detect any direct interaction between these two protein in a
GST pull-down assay (data not shown), suggesting that HIF-1
may
interact with p53 through other factors. To examine the notion that p53
may interact with HIF-1
through Mdm2, we tested whether HIF-1
directly binds Mdm2 in vitro by GST pull-down assays. Both
GST-Mdm2 and GST were expressed in bacteria and purified to near
homogeneity. As shown in Fig.
1a, the
35S-labeled in vitro translated HIF-1
strongly bound to immobolized GST-Mdm2 (lane 2) but not to
immobilized GST (lane 3). However, using the same assay, no
significant interaction was detected between GST-p53 and HIF-1
.
Furthermore, we tested the interaction between HIF-1
and Mdm2
in vivo by coimmunoprecipitation assays. FLAG-tagged
full-length HIF-1
was transiently co-transfected with Mdm2 in H1299
cells. Immunoprecipitations were performed with anti-Flag M2 beads, and
the precipitated proteins were analyzed by Western blot with an
anti-Mdm2 antibody. As shown in Fig. 1b, the Mdm2-HIF-1
complexes were readily detected in cells cotransfected with
FLAG-HIF-1
and Mdm2 (lane 4) but not by Mdm2 alone
(lane 3), indicating that there is a specific in
vivo interaction between Mdm2 and HIF-1
.

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Fig. 1.
HIF-1 interacts with
Mdm2 in vitro and in vivo.
a, the in vitro interaction of Mdm2 and
HIF-1 . The GST (lane 3) and GST-Mdm2 (lane 2)
fusion protein were used in a GST pull-down assay with in
vitro translated 35S-labeled full-length HIF-1 .
b, Mdm2 interacts with HIF-1 in vivo. Western
blot analysis of whole cell extracts (lanes 1 and
2) or immunoprecipiates with the anti-FLAG M2 beads
(IP/M2) (lanes 3 and 4) from cells
cotransfected with FLAG-HIF-1 (10 µg) and Mdm2 (10 µg)
(lanes 2 and 4) or Mdm2 alone (lane 1 and 3) by anti-Mdm2 antibody.
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Mdm2 Enhances the in Vivo Binding between p53 and HIF-1
--
To
examine the possibility that the HIF-1
-p53 interaction is mediated
by Mdm2, we tested whether Mdm2 expression is required for the
HIF-1
-p53 interaction in cells. p53 and FLAG-HIF-1
were transiently transfected with or without Mdm2 in H1299 cells.
Immunoprecipitations were carried out from cell extracts using
anti-Flag M2 beads. As shown in Fig. 2,
p53 was barely detectable in the HIF-1
-associated immunocomplexes in
the absence of Mdm2 expression (lane 5), further confirming
the idea that p53 cannot directly bind HIF-1
in vivo. In
contrast, when Mdm2 was expressed in the cells, p53 was efficiently coprecipitated with HIF-1
by the same method (lane 6).
These results indicate that Mdm2 acts as a bridge mediating the binding between HIF-1
and p53.

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Fig. 2.
Mdm2 enhances the binding between p53 and
HIF-1 . Western blot analysis of whole
cell extracts (lanes 1-3) or immunoprecipites with the
anti-FLAG M2 beads (IP/M2) (lanes 4-6) from
cells cotransfected with FLAG-HIF-1 (10 µg) and p53 (5 µg)
(lanes 2 and 5), plus Mdm2 (5 µg) (lanes
3 and 6) or p53 alone (lanes 1 and
4) by anti-p53 monoclonal antibody (DO-1).
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HIF-1
Abrogates Mdm2-mediated p53 Degradation and Transcription
Repression--
Mdm2 is a key regulator of p53 and can both inhibit
p53 transcriptional activity and target it for degradation (24). We tested the possibility whether HIF-1
could protect p53 from
degradation mediated by Mdm2. The p53-null H1299 cells were transfected
with CMV-p53, CMV-Mdm2, CMV-GFP, and pTOPO-HIF-1
. 24 h after
transfection the cells were lysed in a FLAG lysis buffer and analyzed
by Western blot. As shown in Fig.
3a, HIF-1
effectively
rescues p53 from Mdm2-mediated degradation. Overexpression of Mdm2
significantly induced p53 degradation (lane 2 versus lane 1), whereas this degradation was
inhibited in a dose-dependent manner upon overexpression of HIF-1
(lanes 3 and 4 versus
lane 2). Interestingly, HPV E6 protein can induce p53
degradation through the E6/E6 AP ubiquitin ligase complex. However,
overexpression of HIF-1
cannot protect p53 degradation mediated by
E6 (Fig. 3b). Taken together, these results demonstrated an
effect of HIF-1
directly on Mdm2-mediated degradation of p53.
Furthermore, we used p53 transcriptional activity assay to support the
notion. To this end, we cotransfected MEFs
(p53
/
) with vectors expressing p53, Mdm2,
and HIF-1
, along with a reporter construct containing the minimal
p21 promoter (p21min-luc). As indicated in Fig. 3c,
cotransfection of Mdm2 with p53 strongly repressed p21 luciferase
activity due to p53 degradation. However, HIF-1
significantly
abrogated the inhibitory effect of Mdm2 in a dose-dependent
manner. The above data demonstrate that HIF-1
can strongly stabilize
p53 by abrogating Mdm2-mediated effects, which leads to activation of
p53-mediated transcription.

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Fig. 3.
a, protection of p53 from Mdm2-mediated
degradation by HIF-1 . Western blot analysis of extracts from cells
transfected with p53 (lane 1) or cotransfected with p53 (1 µg) and Mdm2 (2 µg) (lane 2) or in combination with
different amounts of HIF-1 (lanes 3 and 4), by
the anti-p53 monoclonal antibody (DO-1). The CMV-GFP expression vector
was included in each transfection as a transfection efficiency control,
and the levels of GFP were detected with the anti-GFP monoclonal
antibody (JL-8, Clontech). b, failure to
protect E6-mediated p53 degradation. Western blot analysis of extracts
from cells transfected with p53 (lane 1) or cotransfected
with p53 (1 µg) and E6 (0.5 µg) (lane 2) or in
combination with different amounts of HIF-1 (lanes 3 and
4). c, abrogation of Mdm2-mediated repression of
p53-dependent transcription activation. MEF
(p53 / ) cells were transfected with p53
alone (0.25 µg) or cotransfected with p53 (0.25 µg) and Mdm2 (0.5 µg) or in combination with indicated amount of HIF-1 expression
vector (µg) together with the p21-Luc reporter construct (2 µg).
d, HIF-1 suppresses Mdm2-mediated p53 ubiquitination. The
ubiquitination reactions were performed in the absence of Mdm2 as
control (lane 1), with Mdm2 (lane 2), and Mdm2
plus HIF-1 (lane 3).
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HIF-1
Suppresses p53 Ubiquitination Mediated by Mdm2--
To
investigate whether HIF-1
can suppress p53 ubiquitination mediated
by Mdm2, we carried out an in vitro ubiquitination assay. As
shown in Fig. 3d, ubiquitinated p53 was easily detected at
the reaction with Mdm2 but no HIF-1
(lane 2 versus lane 1). However, in the presence of
HIF-1
, the levels of ubiquitinated p53 decreased dramatically
(lane 3 versus lane 2). This result demonstrates that HIF-1
can significantly suppress Mdm2-mediated p53 ubiquitination.
HIF-1
Blocks Nuclear Export Mediated by Mdm2--
Current
models indicate that Mdm2 can also induce nuclear export of p53 (25,
26). To test the possibility that HIF-1
can block this export, we
transfected H1299 cells with vectors expressing GFP-p53, Mdm2 or
HIF-1
and examined them by immunofluorescence microscope (Fig.
4a). The distribution of
GFP-p53 was almost entirely nuclear when expressed alone, but it
localized to the cytoplasm to varying extents when coexpressed with
Mdm2. We scored the extent by which p53 localized to the cytoplasm when
expressed alone or when expressed together with Mdm2 (Fig.
4b). Cells cotransfected with p53 and Mdm2 had a 76%
cytoplasmic expression versus 24% when p53 was transfected
alone, indicating that nuclear Mdm2 can promote the cytoplasmic
localization of p53 (25, 26). Interestingly, only 26% of cytoplasmic
expression occurred in cells cotransfected with HIF-1
in addition to
p53 and Mdm2 (Fig. 4, a and b). These results
indicate that HIF-1
can strongly block Mdm2-mediated p53 nuclear
export.

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Fig. 4.
HIF-1 blocks p53
nuclear export mediated by Mdm2. a, subcellular
localization of GFP-p53 in H1299 cells transfected with GFP-p53 alone
(top row), GFP-p53 + Mdm2 (middle row), and
GFP-p53 + Mdm2 + HIF-1 (bottom row). GFP-p53 images are
shown in the left column, costaining with DAPI in the
middle column, and merge of GFP-p53 and DAPI in right
column. b, the proportion of cells expressing
cytoplasmic p53 following transfection of GFP-p53, Mdm2, or HIF-1 in
H1299 cells. 150 cells were scored, and data from three independent
experiments were averaged.
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DISCUSSION |
How cells sense changes in ambient oxygen is a central question in
biology. In mammalian cells, lack of oxygen, or hypoxia, leads to
stabilization of a sequence-specific DNA binding transcriptional factor
called HIF. Downstream genes of HIF are linked to processes such as
angiogenesis and glucose metabolism (20). On the other hand, hypoxia
induces accumulation of the tumor suppressor p53. Earlier studies
indicated that HIF-1
interacts with p53 in vivo (18);
however, the nature of this interaction has not been elucidated. In
this study, we demonstrate for the first time that HIF-1
directly binds to Mdm2 both in vitro and in vivo. In the
absence of Mdm2, the binding between HIF-1
and p53 is almost
undetectable. Furthermore, Mdm2 expression significantly induces the
indirect interaction between p53 and HIF-1
in cells, indicating that
Mdm2 may act as a bridge mediating the p53-HIF-1
interaction. In
this regard, we failed to detect any significant interaction between
p53 and HIF-1
in MEF Mdm2
/
cells, and
stabilization of p53 induced by HIF-1
expression was also severely
abrogated (data not shown). Our findings seem at odds with the recent
report of tight binding between the ODD domain of HIF-1
and
the core domain of p53 by an array of immobilized peptide assay (22).
It is likely that the experiment was based on p53 pepetide fragments,
which may not be equivalent to the native, folded protein. However, the
existence of Mdm2 somehow might cause the conformational change of p53
native protein, thereby favoring the exposure of binding sites of p53
to HIF-1
.
In addition, our results demonstrate that HIF-1
protects p53 from
degradation mediated by Mdm2 and can abrogate p53 transcriptional repression by Mdm2. Considering that p53 degradation is mainly induced
by Mdm2 in normal cells, we also found that Mdm2-mediated ubiqutination
of p53 is significantly inhibited by HIF-1
. Since Mdm2-mediated p53
ubiquitination promotes its nuclear export (25, 26), we further
demonstrate that HIF-1
expression can block Mdm-2-mediated nuclear
export of p53. Thus, these results have significant implications
regarding the molecular mechanism by which HIF-1
modulates p53
function in vivo.
Our results are also consistent with published results indicating
HIF-1
interacts with the wild-type p53 protein but not the
tumor-derived p53 mutant form in cells (18). Since wild-type p53
protein is capable of inducing Mdm2 expression in cells, the observed
interaction between p53 and HIF-1
is most likely mediated by
endogenous Mdm2. In contrast, because the tumor-derived p53 mutant is
completely inactive in transcriptional activation of endogenous Mdm2,
HIF-1
failed to interact with p53 since there is no (or very low
levels) Mdm2 in cells expressing mutated p53. Recent studies also
indicate that Mdm2 is involved in modulating HIF-1
stability under
hypoxic conditions (21), further supporting the notion that HIF-1
directly interacts with Mdm2 but not p53. Mdm2 is a potent E3 ubiquitin
ligase and induces both p53 degradation and nuclear export of p53
through ubiquitination. Therefore, it is possible that Mdm2 also
directly mediates degradation and nuclear export of HIF-1
. Our
study, together with several other studies (15, 18, 21), strongly
implicates the important role of HIF-1
in the regulation of the
p53-Mdm2 pathway in response to hypoxia.