From the Cardeza Foundation for Hematological Research, Departments of Medicine and Physiology, and Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107
Received for publication, September 20, 2002, and in revised form, February 3, 2003
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ABSTRACT |
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Hypoxia-inducible factors (HIF) are a family of
heterodimeric transcriptional regulators that play pivotal roles in the
regulation of cellular utilization of oxygen and glucose and are
essential transcriptional regulators of angiogenesis in solid tumor and ischemic disorders. The transactivation activity of HIF complexes requires the recruitment of p300/CREB-binding protein (CBP) by HIF-1 Hypoxia-inducible factors
(HIF)1 consist of a family of
heterodimeric transcriptional regulators that control the expression of
a series of genes involved in angiogenesis, oxygen transport, and
glucose metabolism (reviewed in Refs. 1-3). Each of the HIF complexes
contains an The activity of HIF- In addition to hypoxia, multiple oncogenic pathways including growth
factor signaling or genetic loss of tumor suppressor genes, like
VHL and PTEN, up-regulate HIF activity (28, 29). Particularly, ERK1/2 (also known as p44/p42), two kinases of the mitogen-activated protein kinase (MAPK) signaling pathway, have been
implicated in HIF activation (30-32). ERK1/2 are activated by
extracellular proliferative signaling triggered by membrane-tyrosine kinases and transduced through the Ras-Raf-MEK pathway by a cascade of
phosphorylation events that can be repressed by specific kinase inhibitors (33). ERK1/2 are serine/threonine kinases that regulate gene
expression by phosphorylating nuclear substrates (33). Enhanced MAPK
signaling is a common event in tumors, and constitutively active MAPK
signaling transforms mammalian cells (34). Therefore, up-regulation of
HIF activity by MAPK signaling may play an essential role in
transformation as well as, in the process of tumor growth and
metastasis that depends on angiogenesis and changes in glucose metabolism (35). In this study, we examined the molecular basis by
which MAPK signaling influences HIF activity. Our data indicate that
MAPK signaling may affect HIF activity by promoting the formation of
the HIF-p300/CBP complex and by modulating the transactivation activity
of p300/CBP.
Plasmids--
Plasmids expressing G4.H1 Special Chemicals--
Genistein and desferrioxamine (Dfx) were
purchased from Sigma. PD98059 (PDx) was purchased from BioMol
(Plymouth, PA). Dimethyoxalylglycine (DOG) was provided by Dr. Peter
Ratcliffe (Oxford University) (12). Genistein, Dfx, and PDx were first
dissolved in Me2SO and used at the final concentration as
indicated in each experiment.
Cell Lines, Cell Culture, and Transfection--
The
establishment of the B1 cell line was described previously (5). All
cells were maintained at a 37 °C humidified incubator in an
atmosphere of 5% CO2. B1 cells and parental Hep3B cells were cultured in minimal essential medium, whereas HeLa cells in
Dulbecco's modified Eagle's medium, both supplemented with 10% fetal
bovine serum, penicillin (100 units/ml), and streptomycin (100 µg/ml)
(Invitrogen). Transient transfection was performed with LipofectAMINE
Plus reagents (Invitrogen) by following the low serum protocol provided
by the manufacturer. The amount of DNA used in transfection is
described in each experiment. Twenty-four hours after transfection, the
cells were trypsinized, pooled, and equally divided into 12-well plates
for luciferase assays or into 60-mm dishes for Western blot analysis.
When needed, 0.5 µg of plasmid expressing Hypoxia Treatments--
Cells were flushed with a gas mixture of
0.47% O2, 5% CO2, and balanced N2
in a sealed chamber as previously described (5). Alternatively, in some
experiments, cells were incubated directly in a hypoxia work station
(IN VIVO2) at the O2 tension specified in each experiment.
Luciferase Assays--
All cell extracts were prepared and
analyzed using the luciferase assay system purchased from Promega
(Madison, WI) following the manufacturer's procedure. Luminescence was
measured in a TD20/20 Luminometer (Promega), and the results were
expressed as relative light units. Protein concentration was determined
by the Bio-Rad method. GST Fusion Protein Expression, Purification, and in Vitro
Pull-down Assays--
Plasmids expressing the indicated GST fusion
proteins were transformed into BL21-competent cells (Stratagene, La
Jolla, CA), and expression was induced by the addition of
isopropyl-1-thio- In Vitro Kinase Assays--
In vitro kinase assays
were performed as described by Pei with minor modifications (38).
Briefly, purified GST, GST fusion proteins, and commercially obtained
myelin basic protein (Sigma) were incubated at 30 °C for 20 min with
activated recombinant MAPK (BioMol) in the presence of 5 µCi of
[ Antibodies, Immunoprecipitation, and Western Blot
Analyses--
Monoclonal antibody against HIF-1 MAPK Signaling Enhances Both Basal and Hypoxia-stimulated HIF-1
Activity--
Previously, we reported the establishment of the
hepatoma-derived B1 cell line that carries a hypoxia-responsive
luciferase reporter gene and showed that this cell line is responsive
to hypoxia and hydroxylase inhibitors (hypoxia mimics) including transition metals, iron chelators, and oxoglutarate analogs (5). It has
been observed that genistein, a tyrosine kinase inhibitor, decreased
the protein levels of both HIF-1 MAPK Signaling Promotes the Transactivation Activity of
HIF-1 Direct Phosphorylation of HIF-1 MAPK Signaling Promotes the Transactivation Activity of
p300--
Since CAD is sensitive to PDx but lacks MAPK phosphorylation
sites, we explored the possibility that a transcriptional coactivator might be involved in MAPK signaling to HIF. Because p300/CBP are the
major coactivators involved in HIF activation, we next tested the
effects of MAPK on p300 transactivation activity. A plasmid expressing
G4.p300 (aa 1-2414) was co-transfected with the pFR-Luc reporter, and
the transfected HeLa cells were treated with Me2SO, PDx, or
genistein and cultured under normoxic or hypoxic conditions. Fig.
4A indicates that whereas
hypoxia and Me2SO had little effect on p300 transactivation
activity, exposure to PDx or genistein significantly repressed this
activity. The effect of PDx was dose-dependent (Fig.
4B) and affected the transactivation domain of p300
(G4.p300TD; aa 1751-2414) (Fig. 4C). Furthermore,
transfection with a plasmid expressing MEK1, the upstream activator of
ERK1/2 (36), enhanced the transactivation activity of p300TD (Fig.
4D). We next tested whether p300TD was a direct substrate of
MAPK. We expressed and purified p300TD (aa 1572-2370) as a GST fusion
protein and used it in in vitro MAPK assays. We observed
that this region of p300 was readily phosphorylated by MAPK in
vitro (Fig. 4E). Finally, when coexpressed with NAD
(G4.H1 MAPK Signaling Facilitates the Interaction between p300 and
HIF-1 MAPK Signaling Is Not a Part of the Oxygen Sensing
Mechanism--
Finally, we investigated whether MAPK signaling is
involved in the oxygen sensing mechanism in HeLa cells. We first
examined whether hypoxia or hydroxylase inhibitors might affect the
levels of activated (phosphorylated) ERK. HeLa cells were cultured in normal medium with 10% fetal bovine serum, and both the total and
activated ERK levels were examined by specific polyclonal antibodies in
immunoblot assays. As shown in Fig.
6A, total and activated ERK1/2
levels were found at rather high levels under normal culturing
conditions and were affected neither by hypoxia nor by the hypoxia
mimics Dfx or DOG. The effect of further activation of MAPK on the
responsiveness of HIF-1 The role of phosphorylation in HIF activation has become a
contentious issue since it was first observed that genistein decreased the protein levels of both HIF-1 Although it is now clear that MAPK signaling facilitates the
transactivation activity of HIF-1 MAPK-dependent phosphorylation of CBP was originally
reported in the regulation of Elk-1 activity (49), and later MAPK
signaling through CBP was found to play a role in T-cell activation
(50). Recently, phenylephrine-mediated stimulation of gene expression in cardiomyocytes has been found to involve MAPK signaling through p300
and CBP (51). Interestingly, overexpression of MEKK1 enhanced the
transcriptional activity of p300 in a c-Jun N-terminal
kinase-independent manner (52). Since MEKK1 is also able to activate
MEK1, the MEK1-MAPK pathway may play a role in the stimulatory effect
of MEKK1. How p300 and CBP integrate the MAPK signaling is currently unclear. Since p300 and CBP are co-factors for a large number of
transcription factors involved in multiple cellular processes (53),
signal-mediated redistribution of p300/CBP among different interacting
partners may differentially regulate p300/CBP-dependent transcriptional factors (54) and the functionality of p300TD, thus
reprogramming gene expression in response to the signal. Therefore, one
possibility is that p300 and CBP are direct substrates of MAPK or a
downstream kinase. Supporting this hypothesis, it has been reported
that the C-terminal transactivation domain of CBP was phosphorylated by
MAPK in vitro (49), and we now demonstrate that the
C-terminal transactivation domain of p300 can be phosphorylated by MAPK
in a similar manner (Fig. 4E). It is equally possible that
MAPK signaling leads to phosphorylation of a pool of
p300/CBP-interacting factors, as shown for several other
transcriptional factors. In either case, the phosphorylation events may
have differential impact on the affinity between p300 and various
protein factors, thus leading to a signal-mediated redistribution of
p300/CBP among interacting partners. Particularly, since MAPK signaling
stimulates the transactivation activity of p300/CBP, phosphorylation
events very likely enhance the interaction between p300/CBP and the
basic transcriptional machinery.
The biological relevance of MAPK signaling in HIF activation is only
partly understood. Previously, ERK activation upon hypoxia was observed
in HMEC cells (55). In our system, however, MAPK inhibitors suppressed
the transcriptional activity of HIF under both normoxic and hypoxic
conditions. Therefore, the MAPK dependence is not specific for hypoxia
or hypoxic mimic-stimulated HIF activity. In cells exposed to MAPK
inhibitors or MEK1 overexpression, the hypoxic response remains.
Furthermore, in the presence of serum, MAPK is constitutively active,
and the levels of activated MAPK are not changed either by hypoxia or
by hypoxia mimics in HeLa cells. Finally, we have also found that FIH
is not a substrate for MAPK, ruling out the possibility that MAPK
signals to HIF by modifying FIH hydroxylase activity (not shown). These
results indicate that MAPK signaling is not part of the hypoxia sensing mechanism in HeLa cells. However, since MAPK signaling facilitates HIF
activation, it may represent an independent pathway that links oncogenesis to HIF activation and angiogenesis and may synergize the
effect of other HIF-activating pathways.
and HIF-2
that undergo oxygen-dependent degradation. HIF activation in tumors is caused by several factors including
mitogen-activated protein kinase (MAPK) signaling. Here we investigated
the molecular basis for HIF activation by MAPK. We show that MAPK is
required for the transactivation activity of HIF-1
. Furthermore,
inhibition of MAPK disrupts the HIF-p300 interaction and suppresses the
transactivation activity of p300. Overexpression of MEK1, an upstream
MAPK activator, stimulates the transactivation of both p300 and
HIF-1
. Interestingly, the C-terminal transactivation domain of
HIF-1
is not a direct substrate of MAPK, and HIF-1
phosphorylation is not required for HIF-CAD/p300 interaction. Taken
together, our data suggest that MAPK signaling facilitates HIF
activation through p300/CBP.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-subunit and a common dimerization partner, HIF-
,
also known as aryl hydrocarbon receptor nuclear translocator. Whereas
both HIF-
and HIF-
are required to form the HIF heterodimer, HIF-
is the key regulatory subunit whose transcriptional activity is
indispensable for HIF complex function (1).
is controlled at the level of protein stability
(1, 2, 4, 5) and transcriptional stimulation (3, 6, 7). The degradation
of HIF-
is mediated by the ubiquitin-proteasome system (5, 8) and
requires the hydroxylation of prolyl residues in the conserved
oxygen-dependent degradation domain (8, 9), a
process carried out by the oxygen, iron, and
oxoglutarate-dependent prolyl-hydroxylase enzymes (10-15). Hydroxylated oxygen-dependent degradation domain recruits the von Hippel-Lindau protein (11, 12, 16), a tumor suppressor protein that
serves as a part of the E3 ubiquitin-ligase complex (17, 18). In
addition to HIF-
stabilization, HIF-
activity is regulated by the
functional stimulation of its transactivation domains, NAD and CAD,
which are separated by a negative regulatory region (6, 7). The
recruitment of p300/CBP plays an essential role in the functional
activation of HIF-
(19). The interaction between HIF-
CAD and the
CH1 domain of p300/CBP involves a hydrophobic interface (20-22) and
thus is disrupted by the hydroxylation of the asparagine residue
(Asn803) in the CAD of HIF-1
under normoxic conditions
(23, 24). Hydroxylation of CAD depends on the negative regulatory
region's recruitment of factor inhibiting HIF (FIH) (24, 25), an
asparagine hydroxylase that serves as an inhibitor of HIF activity (26, 27).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
530-826,
G4.H1
530-778, G4.H1
744-826, and G4.H1
786-826 were described
previously (24). pFR-Luc is a luciferase reporter vector in which the
luciferase gene is under the control of a minimal E1B promoter and
upstream four copies of GAL4 binding sites
(Clontech). pCMV-
-p300, pRSV-
-CBP, pCMV-G4.p300, pCMV.
-gal, and pSV.
-gal were generous gifts from Dr. Antonio Giordano (Temple University). pRL-CMV was obtained from
Promega (Madison, WI). pGEX.H1
530-826, pGEX.H1
530-658, and
pGEX.H1
744-826 were constructed by inserting corresponding PCR
fragments into the EcoRI site of pGEX4T (Amersham
Biosciences). pGEX.H1
757-826 was kindly provided by Dr. Gregg
Semenza (Johns Hopkins University, Baltimore, MD) (25). pGEXp300TD (aa
1572-2370) was kindly provided by Dr. Pier Lorenzo Puri (Salk
Institute). pCMV.MEK1 is a generous gift from Dr. Kun-Liang Guan
(University of Michigan) (36).
-galactosidase were
co-transfected to normalize the transfection efficiency.
-Galactosidase assays were performed for
normalization reasons with the
-galactosidase assay system from
Promega.
-D-galactopyranoside (Promega) to 0.1 mM. The procedures used to perform purification and
in vitro pull-down assays were in general the same as
described previously (37). Briefly, cells were lysed in lysis buffer
(50 mM Tris-HCl, 250 mM NaCl, 1% Triton 100, 5 mM EDTA, 50 mM NaF, 0.1 mM
Na3VO4, 1 mM phenylmethylsulfonyl
fluoride, 1× protease inhibitor mix, pH 7.5). GST and fusion proteins
were first incubated with 3% milk in lysis buffer, washed with lysis
buffer, and then incubated with cell lysates for 1 h on a roller
at 4 °C, followed by three washes with lysis buffer.
-32P]ATP (Amersham Biosciences) in MAPK buffer (25 mM Hepes, 10 mM MgCl2, 1 mM dithiothreitol, pH 7.5). The reaction was stopped by
adding an equal volume of 2× Laemmli sample buffer and by heating at
95 °C for 3 min. After separation through a 4-20% continuous gradient SDS-PAGE (Bio-Rad), the gels were stained with Coomassie Blue
R250 (Sigma), destained, and dried before autoradiography.
(catalog no.
610959) and anti-p300 monoclonal antibody (NM11) were purchased from
Pharmingen. Monoclonal anti-GAL4 DNA binding domain antibody was
purchased from Clontech (catalog no. 5399-1).
Purified polyclonal antibodies against tyrosine-phosphorylated and
total MAPK and horseradish peroxidase-coupled donkey anti-rabbit
polyclonal antibody were purchased from Promega. Horseradish
peroxidase-coupled anti-mouse IgG (Fc fragment) was purchased from
Sigma. Immunoprecipitations were carried out as described previously
with minor modifications (37, 39). Briefly, cells were lysed in 1×
lysis buffer supplemented with 75 µM PDx when needed.
Cell lysates were first incubated with nonspecific normal mouse serum
and killed protein A-positive Staphylococcus aureus cells
(Roche Diagnostics). The precleared lysates were incubated with 2 µg
of monoclonal antibody on ice for 30 min followed by rocking with
immobilized protein A (Pierce) at 4 °C for 45 min. For Western blot
analyses, samples were separated on a 4-20% gradient SDS-PAGE
(Bio-Rad), if not specified, followed by electrotransferring onto
polyvinylidene difluoride membrane. The membrane was blocked with 5%
milk in TBST (24), incubated with specific antibody, washed in
TBST, and incubated with horseradish peroxidase-labeled secondary
antibody. The membranes were finally developed with the ECL Plus system
(Amersham Biosciences).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and HIF-1
and inhibited the
formation of DNA binding complex (40). Previously, we also found that
in B1 cells, genistein inhibited HIF-1 activity and gene expression in
response to hypoxic stimulation (30). However, PDx, a selective MEK
inhibitor (41), inhibited hypoxia-stimulated gene expression but had
little effect on HIF-1
level and the formation of DNA binding
complex (30). Here, we investigated further the role of MAPK signaling
in basal and induced activity of HIF-1
in B1 cells. The MAPK
signaling pathway, the targeting sites of two kinase inhibitors,
genistein (GSN) and PDx, and the experimental system are
schematically represented in Fig. 1,
A and B. Treatment of the B1 cells with genistein
revealed a dose-dependent inhibitory effect on both the
basal and hypoxia-induced HIF-1 activity (Fig. 1, C and
D). Similar inhibition was observed with the use of the
hypoxic mimic Dfx (an iron chelator) and DOG (an oxoglutarate analog)
(not shown). We further examined the effects of PDx both on a basal
level and on a stimulated transactivation activity of HIF-1. Fig. 1,
E and F, shows that PDx inhibited luciferase expression in B1 cells both under normoxic and hypoxic conditions, further confirming that MAPK signaling enhances not only
hypoxia-stimulated HIF-1 activity but its basal activity as well. In
control experiments, PDx showed no effect on the luciferase activity
from a pRL-CMV (not shown). These results indicate that MAPK signaling
has a general role in promoting endogenous HIF activity, regardless of
oxygen concentration.
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Fig. 1.
Role of MAPK signaling in endogenous HIF-1
activity. A, schematic representation of MAPK-signaling
pathway and the sites for inhibitor action. B, model of the
luciferase-reporter system. C and D, effects of
genistein on HIF-1 activity. B1 cells were exposed to various doses of
genistein, a tyrosine kinase inhibitor, and cultured in normoxia
(C) or in a hypoxic work station with 2% O2
(D) for 8 h. E and F, effects of
PDx on HIF-1 activity. B1 cells were exposed to various doses of PDx,
MAPK inhibitor, under normoxic or hypoxic conditions (2%
O2) for 8 h. RLU, relative light
units.
--
We next tested the hypothesis that MAPK signaling
regulates HIF-1 activity through the transactivation domains of
HIF-1
. HeLa cells were transfected with plasmids expressing
G4.H1
530-826 in which the HIF-1
fragment was fused with the DNA
binding domain of the yeast transcription factor GAL4. A luciferase
reporter driven by an E1B minimal promoter and four copies of GAL4
binding sites (pFR-luc) was co-transfected to monitor the
transactivation activity. Fig. 2,
A and B, shows the dose-dependent
inhibitory effect of PDx on the transactivation activity of
G4.H1
530-826, which contains both the NAD and the CAD, under both
normoxic and hypoxic conditions. The protein levels of G4.H1
530-826
were not significantly affected by PDx treatment (Fig. 2, C
and D). Taken together, these data clearly confirm that MAPK
signaling up-regulates the transactivation activity of HIF-1
.
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Fig. 2.
Role of MAPK signaling in the transactivation
activity of HIF-1 . A and B, effects of PDx on
transactivation activity of HIF-1
. HeLa cells were transfected with
a plasmid expressing G4.H1
530-826 (2.5 µg) and the luciferase
reporter pFR-luc (2.5 µg). Following transfection, the cells were
treated with variable doses of PDx and incubated in normoxic
(Nmx) on hypoxic (Hpx) conditions for 8 h
before luciferase measurements. C and D, effects
of PDx on protein levels of G4.H1
530-826 under normoxic and hypoxic
conditions. HeLa cells cultured in 100-mm dishes were transfected with
pG4.H1
530-826 (5 µg/dish). 24 h after the transfection, the
cells were trypsinized, pooled, and evenly reseeded into fresh dishes
and cultured for 12 h. The cells were treated with indicated doses
of PDx and cultured under either normoxic on hypoxic (2%
O2) conditions for 8 h before harvest. RLU,
relative light units.
by MAPK Is Not Correlated to Its
Transactivation Activity--
Whereas previous reports observed that
HIF-1
was directly phosphorylated by MAPK (32), the exact amino acid
residues involved in HIF-1
phosphorylation have not been clearly
identified. Furthermore, there have been inconsistencies between
results of different groups on the role of MAPK-induced direct
phosphorylation of HIF-
in its transactivation activity (31, 32). To
clarify further the role of MAPK in HIF activation, we first attempted
to identify the functional domains that were sensitive to MAPK
inhibitors. G4.HIF fusion constructs containing various segments of the
carboxyl half of HIF-1
were co-transfected with the pFR-Luc
reporter, and the transfected HeLa cells were treated with increasing
doses of PDx. As shown in Fig. 3,
A-F, all of the constructs tested, including
G4.H1
530-778 (which represents the NAD), G4.H1
744-826 (which
represents the hypoxia-responsive CAD), and G4.H1
786-826 (which
represents the constitutive CAD), were inhibited by PDx both under
normoxic and hypoxic conditions. These data suggest at least two
possibilities: 1) there is more than one phosphorylation site, each of
the constructs tested containing at least one site for MAPK-mediated
phosphorylation, or 2) MAPK signaling affects a protein factor(s) that
is required for both NAD and CAD activity. To test the first
hypothesis, we expressed HIF-1
as GST fusion proteins and used them
in in vitro MAP kinase assays (Fig. 3G). Whereas
GST alone was not phosphorylated by active MAPK, GST.H1
530-658 and
GST.H1
530-826 were readily phosphorylated. However, neither GST.H1
757-826 nor GST.H1
744-826, which corresponds to the
hypoxia-responsive CAD (6), was phosphorylated, demonstrating that
whereas both hypoxia-responsive CAD and constitutive CAD are sensitive
to PDx treatment, they do not contain MAPK phosphorylation sites.
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Fig. 3.
Correlation between MAPK phosphorylation and
the transactivation activity of HIF-1 . A-F, effects of
MAPK inhibitor on the transactivation activity of G4.H1
constructs.
Each G4.H1
construct (3 µg) was co-transfected with the pFR-Luc
reporter (2 µg), and 24 h later the cells were trypsinized and
evenly divided into 12-well culture plates. Immediately before the
reporter assays, the indicated doses of PDx were added, and the cells
were exposed to either normoxic (A, C, and
E) or hypoxic (2% O2) conditions (B,
D, and F) for 8 h before luciferase assays.
G, in vitro MAPK assays. HIF-1
fragments were
purified as GST fusion proteins and incubated with activated MAPK and
[
-32P]ATP. The top panel shows
the autoradiography, and the bottom panel shows
the protein input. GST and myelin basic protein (MBP) were
used as negative and positive control, respectively. RLU,
relative light units.
530-778), p300 (pCMV-
-p300) or CBP (pRSV-CBP) enhanced the
transactivation activity of NAD, which was repressible by wild type E1A
but not by an E1A mutant defective in targeting p300/CBP (not shown)
(42), suggesting that p300 is also required for NAD activity.
Therefore, the effect of PDx on both NAD and CAD activity is at least
partly caused by the repression of p300/CBP.
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Fig. 4.
Effects of MAPK on p300 transactivation
activity. A, effect of PDx and genistein on p300
transactivation activity. HeLa cells where co-transfected with a
plasmid expressing G4.p300 and with the GAL4-luciferase reporter
(pFR-luc). The transfected cells were divided evenly and exposed to
Me2SO (DMSO), PDx (75 µM), or
genistein for 6 h in normoxia or hypoxia immediately before the
luciferase assays. B, dose-dependent effect of
PDx on p300 activity. HeLa cells were transfected as in B,
and increasing amounts of PDx were used, as indicated (in
µM). C, dose-dependent effects of
PDx on the transactivation domains of p300TD. HeLa cells were
transfected with plasmid G4.p300TD, expressing the transactivation
domain of p300 (aa 1751-2414), and the cells were exposed to
increasing concentrations of PDx. D, effects of
overexpression of MEK1 on the transactivation activity of p300TD.
Plasmid expressing MEK1 (2 µg) was co-transfected with pG4.p300TD (2 µg) and the luciferase-reporter (pFR-Luc; 2 µg). Luciferase
activity was expressed as -fold increase over the co-transfected vector
control. E, direct phosphorylation of p300TD in
vitro by MAPK. GST.p300TD was expressed in and purified from BL21
cells and used in MAPK kinase assays. RLU, relative light
units.
CAD--
HIF-1
transactivation activity depends on the
recruitment of p300. We next investigated whether MAPK signaling
affected the physical interaction between p300 and HIF-1
CAD. We
exposed HeLa cells to hypoxia in the presence or absence of PDx. As
shown in Fig. 5A, treatment
with PDx had no effect on p300 levels. We incubated the cell lysates
with bacterially expressed GST.H1
530-826 protein in pull-down
assays, and the copurified p300 was detected by immunoblotting with an
anti-p300 monoclonal antibody. As shown in Fig. 5B, whereas p300 in cell lysates from normal or Me2SO-treated cells
readily bound to GST.H1
530-826, little p300 was copurified from
PDx-treated lysates. Similar results were observed when
GST.H1
757-826 was used (not shown). To further confirm that
blocking MAPK signaling disrupts the HIF-1/p300 interaction in
vivo, a plasmid expressing G4.H1
786-826 (constitutive CAD) was
transfected into HeLa cells, and the transfected cells were treated
with Me2SO or PDx and cultured under either normoxic or
hypoxic conditions. Western blot analysis showed that G4.H1
786-826
was expressed in all transfected cells (Fig. 5C). Coupled
immunoprecipitation and Western blot assays with either
anti-p300 or anti-Gal4 antibodies confirmed that G4.H1
786-826 interacted with p300 regardless of oxygen concentration as previously demonstrated (24). This interaction, however, was significantly weakened by PDx treatment (Fig. 5C, bottom
two panels). These results indicate that MAPK
signaling facilitates the interaction between p300 and HIF-1
CAD
in vivo.
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Fig. 5.
MAPK signaling regulates the interaction
between p300 and HIF-1 CAD. A,
PDx treatments do not affect p300 levels. HeLa cells were incubated
with PDx at 100 µM for 6 h, and the cell lysates
were analyzed by immunoblotting using anti-p300 antibodies.
B, GST pull-down assays. GST and GST.H1
530-826 were
incubated with whole cell lysates (WCL) from HeLa that were
untreated or treated with Me2SO (DMSO) or PDx
(100 µM) for 6 h. Precipitates were resolved in an
8% SDS-PAGE. Top panel, Coomassie Blue staining
shows GST and GST.H1
530-826 proteins used in the pull-down assays.
Bottom, Western blotting with anti-p300 monoclonal antibody
demonstrating that GST.H1
fails to pull down p300 from PDx-treated
cell lysates. C, effects of PDx treatment on the interaction
between HIF-1
CAD and p300 in vivo. HeLa cells were
transfected with G4.H1
786-826 and treated with or without PDx and
cultured under either normoxic or hypoxic conditions for 6 h
before harvest. Whole cell lysates (WCL; top
three panels) were assayed for the levels of
HIF-1
, phosphorylated ERK (ERKp), and G4.H1
786-826 by
Western blot (WB). Immunoprecipitations
(I.P.) were performed with anti-p300 and
anti-Gal4 monoclonal antibody, and the immunoprecipitates were detected
with an anti-Gal4 or an anti-p300 monoclonal antibody reciprocally
(bottom two panels).
to hypoxia and Dfx was analyzed by
overexpressing MEK1, an upstream activator of ERK. Overexpression of
MEK1 enhanced HIF-CAD activity under all conditions tested, whereas the
addition of PDx blocked the MEK1 effect (Fig. 6B). Most
importantly, variations in MAPK levels did not affect the
responsiveness of HIF-1
activity to hypoxia or Dfx.
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Fig. 6.
Effect of MAPK signaling in hypoxia
sensing. A, effect of hypoxia and hypoxia mimics on
MAPK activity. HeLa cells were maintained in normoxia or exposed to
hypoxia (2% O2), Dfx (130 µM), or DOG (1 mM) for 6 h. 50 µg of whole cell lysates were
applied to an SDS-PAGE. The total and activated ERK1/2 (pERK1/2) were
measured by immunoblotting. B, effect of overexpression of
MEK1 on HIF-1 activity and its responsiveness to hypoxia. 2 µg of
pCMV.MEK1 or vector alone were co-transfected with 2 µg of
pG4.H1
744-826 and 2 µg of pRR-Luc into HeLa cells in 100-mm
culture dishes. 24 h after transfection, cells were trypsinized
and evenly split into 12-well culture plates. 12 h after seeding,
cells were switched to the indicated culture condition or exposed to
the indicated chemicals for 6 h before analyses. Luciferase
activity was normalized to co-transfected 0.5 µg of pCMV.
-gal.
RLU, relative light units.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and HIF-1
and blocked the
formation of DNA binding complex (40). It was later shown that PDx, a MEK-specific inhibitor, suppressed hypoxia-stimulated HIF activity but
had little effect on the formation of HIF DNA-binding complex (30).
These results suggested that MAPK signaling might play a role in the
regulation of the transactivation activity of HIF-1 but had an
insignificant effect on the protein levels of HIF-1
and
hypoxia-stimulated accumulation of HIF-1
. This notion is supported
by reports indicating that p44/p42 kinases (ERK1/2) directly
phosphorylate HIF-1
and regulate its transcriptional activity with
no effect on HIF-1
protein levels induced by hypoxia (32). This
issue is complicated further by the later finding that both
PTEN/phosphatidylinositol 3-kinase/Akt and MAPK pathways enhance
HIF-
levels in tumor cells or cells stimulated by mitogenic signaling under normoxic conditions (43, 44) and by evidence indicating
that such enhancement involves the control of HIF-1
translation
(45). One recent report, however, demonstrates that the
phosphatidylinositol 3-kinase/Akt pathway is neither required nor
sufficient for stabilization of HIF-1
(46). The data presented here
indicate that the transactivation activity of HIF-1
is affected by
MAPK signaling. Whereas at low dosages (50 µM or less),
PDx was very effective in blocking the transactivation activity of HIF-1
, it showed little or no effect on the protein level. However, higher doses (100 µM or more) of PDx reduced
hypoxia-accumulated HIF-1
levels (not shown). These data are
consistent with the translational role of MAPK signaling and suggest
that, in certain conditions, MAPK signaling may regulate HIF activity,
in part, by affecting the protein levels of HIF-1
.
and it has been suggested that
MAPK directly phosphorylates HIF-
(32), there is no direct evidence
for correlation of the MAPK-mediated phosphorylation to the
transactivation activity of HIF-1
. Moreover, it was reported that
PDx inhibited HIF-2
(EPAS1) transactivation activity but did not
inhibit its phosphorylation (31). Whereas it was demonstrated recently
that the C-terminal domain of HIF-
is phosphorylated at multiple
sites and that mutation of a conserved threonine residue (Thr796 in HIF-1
/T844 in HIF-2
) inhibited the
interactions between HIF-
and CBP/p300, such phosphorylation is not
mediated by MAPK (47). We observed that whereas both responsive CAD and
constitutive CAD of HIF-1
were repressed by PDx exposure, neither
the aa 757-826 nor the aa 744-826 region was phosphorylated by MAPK,
indicating that it is not through direct phosphorylation of HIF-1
that MAPK affects the transactivation activity of HIF-1
CAD. Instead,
we observed that MAPK signaling affected the transactivation activity of p300, the co-activator required for HIF-
CAD, and that the interaction between p300 and HIF-1
was affected by MAPK signaling. Although the aa 530-743 region, encompassing NAD and part of the oxygen-dependent degradation domain, is phosphorylated by
MAPK in vitro (Fig. 3G) (48), because
HIF-1
530-826 and HIF-1
757-826 expressed in Escherichia
coli, thus lacking posttranslational modifications, efficiently
bind p300 in cell lysates, it appears that direct phosphorylation of
HIF-1
is not necessary for the recruitment of p300. Moreover,
whereas PDx does not affect p300 levels, HIF-1
fails to bind p300 in
PDx-treated cell lysates, suggesting that phosphorylation of p300 or
other cellular factor(s) by MAPK or its downstream kinases may regulate
the interaction between p300 and HIF-CAD. Therefore, our data indicate
that p300 and very likely CBP as well serve as integrators of MAPK
signaling in HIF activation.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. A. Giordano (Temple University), P. L. Puri, K. L. Guan (University of Michigan), D. Livingston (Dana-Faber Institute), G. Semenza (Johns Hopkins University), and P. Rattcliffe (Oxford University) for providing plasmids and other reagents essential for this research. We thank Dr. S. McKenzie for support. We appreciate help from A. Likens in preparing the figures.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant RO1CA89212 and American Heart Association Grant 9950122N (to J. C.).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 in part by National Institutes of Health Grant
T32-HL007821. To whom correspondence may be addressed: Cardeza
Foundation and Dept. of Medicine, Thomas Jefferson University, 1015 Walnut St., Curtis Bldg., Rm. 809, Philadelphia, PA 19107. Tel.:
215-955-5118; Fax: 215-923-3836; E-mail: nianli.sang@mail.tju.edu.
§ Supported in part by a Fellowship from Boehringer Ingelheim Fonds, Germany.
¶ Supported in part by American Heart Association Grant 0060194U. Present address: Division of Orthopedic Surgery Research, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA 19107.
To whom correspondence may be addressed: Cardeza Foundation
and Dept. of Medicine, Thomas Jefferson University, 1015 Walnut St.,
Curtis Bldg., Rm. 809, Philadelphia, PA 19107. Tel.: 215-955-5118; Fax:
215-923-3836; E-mail: Jamie.caro@mail.tju.edu.
Published, JBC Papers in Press, February 13, 2003, DOI 10.1074/jbc.M209702200
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ABBREVIATIONS |
---|
The abbreviations used are: HIF, hypoxia-inducible factor; E3, ubiquitin-protein isopeptide ligase; FIH, factor inhibiting HIF; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; CREB, cAMP-response element-binding protein; CBP, CREB-binding protein; aa, amino acids; Dfx, desferrioxamine; PDx, PD98059; DOG, dimethyoxalylglycine; GST, glutathione S-transferase.
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