From the Department of Anesthesia, Kyoto University
Hospital, Kyoto University, Kyoto 606-8507, Japan and the
§ Institute of Genetic Medicine, Department of Pediatrics
and Medicine, The Johns Hopkins University School of Medicine,
Baltimore, Maryland 21287-3914
Received for publication, January 24, 2001, and in revised form, March 22, 2001
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ABSTRACT |
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Hypoxia-inducible factor 1 (HIF-1) is a
transcription factor that mediates cellular and systemic homeostatic
responses (including erythropoiesis, angiogenesis, and glycolysis) to
reduced O2 availability in mammals. Hypoxia induces
both the protein expression and transcriptional activity of the
HIF-1 Mammalian cells exhibit many homeostatic responses to hypoxia,
including transcriptional activation of genes encoding proteins that
function to increase O2 delivery and that allow metabolic adaptation under hypoxic or ischemic conditions. Although a variety of
transcription factors (including AP-1, Egr-1, and nuclear factor Although much has been learned about the role of HIF-1 in controlling
the expression of hypoxia-inducible genes, the underlying mechanisms by
which cells sense a decrease in O2 concentration and
transduce this signal to HIF-1 In addition to changes in cellular redox, hypoxia signal transduction
may also require kinase/phosphatase activity because treatment of cells
with genistein (a tyrosine kinase inhibitor) or sodium fluoride (a
serine/threonine phosphatase inhibitor) blocks hypoxia-induced HIF-1 In this study, we have focused on the Rho family small GTPase Rac1 as a
potential intermediate in the hypoxia signal transduction pathway. Rac1
plays a pivotal role in multiple cellular processes, including
cytoskeletal organization, gene transcription, cell proliferation, and
membrane trafficking, through direct or indirect interactions with
PI3K, p21-activated kinase (PAK), Ras, and p70 S6 kinase (20-23). Rac1
also regulates assembly of the active NAD(P)H oxidase complex (24).
Rac1 is expressed in most cells and is recognized as a critical
determinant of intracellular redox status. We demonstrate here that
Rac1 is activated in response to hypoxia and plays an essential role in
the induction of HIF-1 Cell Culture and Reagents--
Hep3B cells were maintained in
minimal essential medium with Earle's salts and 10% fetal
bovine serum (Life Technologies, Inc.). HEK293 cells were maintained in
Dulbecco's modified Eagle's medium with 10% fetal bovine serum.
CoCl2 and DFX were obtained from Sigma. Rotenone,
diphenyleneiodonium (DPI), LY294002, wortmannin, PD98059, SB203580, and
genistein were obtained from Calbiochem. Rabbit anti-phospho-p38 MAPK
(Thr183/Tyr185) polyclonal antibody was
obtained from New England Biolabs, Inc. (Beverly, MA).
Plasmid Construction--
Expression vectors pCEP4/HIF-1 Hypoxic Treatment--
Tissue culture dishes were transferred to
a modular incubator chamber (Billups-Rothenberg, Del Mar, CA), which
was flushed with 1% O2, 5% CO2, and 94%
N2; sealed; and placed at 37 °C (30).
Reporter Gene Assays--
All reporter assays were performed in
Hep3B cells. Cells were transferred to 24-well plates at a density of
5 × 104 cells/well on the day before transfection.
Fugene-6 reagent (Roche Molecular Biochemicals) was used for
transfection (31). In each transfection, the indicated doses of test
plasmids, 200 ng of reporter gene plasmid, and 50 ng of control plasmid
pTK-RL (containing a thymidine kinase promoter upstream of
Renilla reniformis (sea pansy) luciferase coding sequences;
Promega) were premixed with the transfection reagent. In each assay,
the total amount of DNA was held constant by addition of empty vector.
After treatment, the cells were harvested, and the luciferase activity
was determined using the Dual-LuciferaseTM reporter assay
system (Promega) (17). The ratio of firefly to sea pansy luciferase
activity was determined for each reporter experiment; at least two
independent transfections were performed in triplicate.
Immunoblot Assays of HIF-1 PAK p21-binding Domain (PBD) and Immunoblot Assays--
HEK293
cells were transfected with pBOS-HA-Rac1. After 18 h of serum
starvation, cells were exposed to 1% O2,
CoCl2, or DFX. Then, cells were lysed in Mg2+
lysis/wash buffer (25 mM HEPES, pH 7.5, 250 mM
NaCl, 1% Nonidet P-40, 10 mM MgCl2, 1 mM EDTA, and 2% glycerol) supplemented with EDTA-free
Complete protease inhibitor mixture (Roche Molecular Biochemicals) in a
controlled atmosphere chamber (Plas-Laboratories, Inc.) maintained at
1% O2. Lysates (200 µg) were incubated with 15 µg of
GST-PBD (containing amino acids 69-150 of PAK1), bound to
glutathione-agarose beads for 1 h at 4 °C, and washed three times with Mg2+ lysis/wash buffer. The bead pellet was
finally suspended in 20 µl of Laemmli sample buffer (32). Bound
proteins were fractionated by SDS-polyacrylamide gel electrophoresis
and subjected to immunoblot assay using anti-HA antibody 12CA5 (Roche
Molecular Biochemicals). In each experiment, to confirm that equal
amounts of HA-tagged Rac1 protein were expressed, immunoblot assay of
the starting lysate with the anti-HA antibody was also performed.
Rac1-N17 Suppresses Hypoxia-, CoCl2-, or DFX-induced
HIF-1-dependent Gene Transcription--
To examine the
role of the small GTPase Rac1 in hypoxia-induced HIF-1 activation,
Hep3B cells were cotransfected with reporter p2.1 containing an
HIF-1-dependent HRE and an expression vector encoding
either a dominant-negative (Rac1-N17) or a constitutively activated
(Rac1-V12) form of Rac1. Cells were exposed to 20 or 1% O2
for 16 h and then subjected to luciferase assays. Rac1-N17 expression significantly suppressed hypoxia-induced reporter gene transcription in a dose-dependent manner (Fig.
1A). Rac1-V12 expression had a
small but reproducible stimulatory effect.
In addition to hypoxia, HIF-1 activity is also induced in cells exposed
to CoCl2 or DFX (7). Rac1-N17 expression significantly attenuated reporter gene transcription in response to 100 µM CoCl2 or DFX, although the degree of
inhibition was less than the inhibition of the hypoxic response (Fig.
1B). We next tested two other members of the Rho family of
small GTPases, Rho and Cdc42. As shown in Fig. 1B, Cdc42-N17
suppressed hypoxia-induced gene transcription, whereas Rho-DN did not.
The dominant-negative form of another small GTPase, Ras-N17, also
suppressed hypoxia-induced luciferase expression. Transcription of p2.1
in hypoxic HEK293 and NIH3T3 cells was also inhibited by Rac1-N17 (data
not shown). Moreover, transcription of a reporter gene containing the
HRE from the human VEGF gene in hypoxic Hep3B,
HEK293, and NIH3T3 cells was also inhibited by Rac1-N17 (data not shown).
HIF-1 HIF-1 Rac1 Is Activated in Response to Hypoxia--
Activated GTP-bound
Rac1 regulates distinct downstream signaling pathways by interacting
with specific effector molecules, including the serine/threonine
protein kinase PAK1 (21). Using recombinant GST-PBD, which contains the
Rac1-binding domain of PAK1, we examined whether Rac1 is activated in
response to hypoxia. HEK293 cells overexpressing Rac1-WT or Rac1-V12
(GTP-bound) were lysed and used for an affinity precipitation assay
(Fig. 4A). GST-PBD bound and
precipitated the activated form of Rac1 in lysates from
Rac1-V12-expressing cells (Fig. 4A, lane 3) and
from Rac1-WT-expressing cells incubated with GTP Kinase Inhibitors Inhibit HIF-1 and Rac1 Activation in Response to
Hypoxia--
To investigate potential components of the hypoxia signal
transduction pathway upstream and downstream of Rac1, we first utilized a PI3K inhibitor, wortmannin. As shown in Fig.
5 (A and B,
respectively), treatment with 50 nM wortmannin
significantly attenuated HIF-1- and HIF-1
LY294002, wortmannin, and genistein suppressed hypoxia-induced HIF-1
LY294002 and genistein also inhibited Rac1 activation in response to
hypoxia (Fig. 7). DPI and rotenone also
markedly inhibited hypoxia-induced Rac1 activation. In contrast,
neither PD98059 nor SB203580 attenuated Rac1 activation in
response to hypoxia. These results, which are consistent with the
analysis of HIF-1 p38 MAPK Is Activated in Response to Hypoxia in a
Rac1-dependent Manner--
Because the p38 MAPK inhibitor
SB203580 blocked HIF-1-dependent gene transcription and
HIF-1 Rac1-N17 Suppresses Hypoxia-induced AP-1-dependent Gene
Transcription--
As the results for the ATF2 TAD indicate, HIF-1 is
not the only transcription factor that is activated in response to
hypoxia. We therefore explored the possibility that Rac1 regulates the activation of AP-1 in response to hypoxia. Hep3B cells were
cotransfected with reporter pAP-1-Luc, containing seven copies of an
AP-1-binding site, and expression vector encoding either the
dominant-negative (Rac1-N17) or constitutively activated (Rac1-V12)
form of Rac1. Cells were exposed to 20 or 1% O2 for 8 h and then subjected to luciferase assays. Rac1-N17 significantly
suppressed hypoxia-induced reporter gene transcription (Fig.
9). Furthermore, Rac1-V12 strongly stimulated AP-1-dependent gene transcription in both
non-hypoxic and hypoxic cells.
The O2-dependent regulation of HIF-1
activity occurs at multiple levels in vivo (1). Among these,
the mechanisms regulating HIF-1 Rac1 has been shown to modulate both phosphorylation and redox status
via its binding to protein kinases (20, 39, 40) and to the NAD(P)H
oxidase complex (24), respectively. Our data indicate that Rac1 is
required for the induction of HIF-1 Rac1 and Hypoxia Signal Transduction--
Previous studies have
demonstrated that inhibitors of mitochondrial ETC (1, 13, 15), PI3K
(13, 14, 17-19), serine/threonine protein phosphatase (14, 16), and
protein-tyrosine kinase (16) activities block hypoxia-induced HIF-1 Rac1 and HIF-1 Rac1 and HIF-1 Broader Role for Rac1 in Hypoxia-induced Gene
Transcription--
Hypoxia induces the activity of multiple
transcription factors in addition to HIF-1. Transcription of an
AP-1-dependent reporter gene was induced by hypoxia, and
the induction was specifically blocked by Rac1-N17 (Fig. 9), as in the
case of the HIF-1-dependent reporter gene (Fig. 1).
However, Rac1-V12 markedly induced AP-1-dependent transcription under both hypoxic and non-hypoxic conditions, whereas it
had only a minor effect on HIF-1-dependent transcription.
These data indicate that Rac1 plays an important role in hypoxia signal transduction in other systems, although the specific mechanisms of
transcriptional regulation involved may differ. With these results as a
foundation, future studies will be necessary to further delineate the
mechanisms and consequences of Rac1 activation in response to hypoxia.
subunit. However, the molecular mechanisms of sensing and
signal transduction by which changes in O2 concentration result in changes in HIF-1 activity are poorly understood. We report
here that the small GTPase Rac1 is activated in response to hypoxia and
is required for the induction of HIF-1
protein expression and
transcriptional activity in hypoxic cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B)
mediate hypoxia-inducible gene expression in specific contexts, hypoxia-inducible factor 1 (HIF-1)1 is an essential
global regulator of oxygen homeostasis (1). HIF-1 is a basic
helix-loop-helix/PAS (PER-ARNT-SIM
homology domain) protein consisting of HIF-1
and HIF-1
subunits (2). The mechanism by which HIF-1 activity is induced under
hypoxic conditions remains to be established. HIF-1
and HIF-1
mRNAs are constitutively expressed in cultured cells, indicating
that HIF-1 activity is regulated by post-transcriptional events.
HIF-1
protein expression and HIF-1 transcriptional
activity are precisely regulated by cellular O2
concentration, whereas HIF-1
protein is constitutively expressed
(1). The molecular mechanisms of sensing and signal transduction by
which changes in O2 concentration result in changes in
HIF-1 activity are complex and involve regulation at multiple levels,
including changes in HIF-1
protein stability, nuclear localization,
and transactivation function in response to hypoxia (1). HIF-1
protein expression is negatively regulated in non-hypoxic cells by the
ubiquitin-proteasome system (3). Under hypoxic conditions, HIF-1
protein levels increase, and the fraction that is ubiquitinated
decreases (4). The carboxyl-terminal half of HIF-1
contains a domain
that negatively regulates protein stability (5, 6) and two
transactivation domains that are also negatively regulated under
non-hypoxic conditions (7, 8).
are largely unknown. Presently, four
diverse O2-sensing mechanisms have been proposed to mediate the hypoxic transcriptional response (9). Two of these models postulate
involvement of an iron-containing unit, in the form of either a heme
group or an iron/sulfur cluster, that undergoes a change in activity
(10). These models are supported by the observation that exposure of
cells to cobaltous ion (CoCl2) or the iron chelator
desferrioxamine (DFX) stabilizes HIF-1
under non-hypoxic conditions
(1). However, no specific proteins with this role have been identified
in mammalian cells. Two other models involve the generation of reactive
oxygen intermediates by a flavoprotein-containing NAD(P)H oxidase or by
mitochondria. In the NAD(P)H model, decreased reactive oxygen
intermediate production triggers the transcriptional response to
hypoxia (11, 12), whereas in the mitochondrial model, increased
reactive oxygen intermediate production by the electron transport chain
(ETC) is an initial trigger of the response (13-15). In these latter
two models, O2 signals are converted to redox signals.
expression (16). In certain cell types, phosphatidylinositol 3-kinase
(PI3K) inhibitors such as LY294002 and wortmannin also block
hypoxia-induced HIF-1
expression (14, 17). Reporter assays involving
expression of constitutively activated or dominant-negative forms of
PI3K or Akt (protein kinase B) demonstrate that the PI3K/Akt pathway
modulates hypoxia-induced HIF-1 activation and induces HIF-1 activity
in non-hypoxic cells (17-19). Thus, the signaling pathway from the
putative O2 sensor(s) to HIF-1 may contain several
intermediate molecules.
protein expression and transcriptional activity.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
pGAL4/HIF-1
-(531-826), pGAL4/HIF-1
-(531-575), and
pGAL4/HIF-1
-(786-826) were described previously (7). Expression
vector pFA-ATF2, which encodes the GAL4 DNA-binding domain (amino acids
1-129) fused to the transactivation domain (amino acids 1-96) of the
transcription factor ATF2 under control of the cytomegalovirus
promoter, was obtained from Stratagene (La Jolla, CA). Reporter plasmid
p2.1, harboring a 68-base pair hypoxia response element (HRE) from the
ENO1 gene inserted upstream of an SV40 promoter and
Photinus pyralis (firefly) luciferase coding sequences, and
reporter G5E1bLuc, containing five copies of a GAL4-binding site
upstream of a minimal E1b gene TATA sequence and firefly luciferase
coding sequences, were described previously (7, 25). Reporter plasmid
pAP-1-Luc, which contains seven tandem copies of an AP-1-binding site,
was described previously (26). HA-tagged expression plasmids
pBOS-HA-Rac1 (-WT, -V12, and -N17) and pBOS-HA-Cdc42 (-WT, -V12, and
-N17) (27) were kindly provided by Dr. K. Kaibuchi (Nara Institute of
Science and Technology). Expression vectors encoding Myc-tagged
dominant-negative forms of Rho and Ras (28) were gifts from Dr. K. Irani (The Johns Hopkins University). Plasmid encoding p85
, a
dominant-negative form of the PI3K p85 regulatory subunit (19), was a
gift from Dr. A. J. Giaccia (Stanford University). Expression
vector pSR
-HA-p38 MAPK, encoding HA-tagged p38 MAPK (29), was a gift
from Dr. E. Nishida (Kyoto University).
--
Whole cell lysates were
prepared by incubating cells for 30 min in cold radioimmune
precipitation assay buffer containing 2 mM dithiothreitol,
0.4 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin,
2 µg/ml pepstatin, and 1 mM NaVO3. Samples
were centrifuged at 10,000 × g to pellet cell debris.
Aliquots were fractionated by SDS-polyacrylamide gel electrophoresis
and subjected to immunoblot assay using protein G-purified mouse
monoclonal antibody H1
67 at 1:1000 dilution (4). Signal was
developed using ECL reagents (Amersham Pharmacia Biotech).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Effect of small GTPases on HIF-1-mediated
gene transcription. A, Hep3B cells were transfected
with pTK-RL (50 ng; encoding Renilla luciferase), p2.1 (100 ng; containing an HRE upstream of an SV40 promoter-firefly luciferase
reporter gene), and the indicated amounts of expression vectors
encoding either no protein (Empty vector) or a
dominant-negative (Rac1-N17) or constitutively activated (Rac1-V12)
form of Rac1. The total amount of expression vectors was adjusted to
500 ng with empty vector. Cells were exposed to 20 or 1%
O2 for 16 h. The ratio of firefly to
Renilla luciferase activity was determined and normalized to
the value obtained from non-hypoxic cells transfected with empty vector
to obtain the relative luciferase activity. Results shown represent
means ± S.D. of three independent transfections. Reporter p2.4
contains a 3-base pair mutation that eliminates binding of HIF-1 to the
HRE (30). B, Hep3B cells were transfected with pTK-RL; p2.1;
and expression vector encoding no protein, Rac1-N17, Cdc42-N17, Rho-DN,
or Ras-N17. Transfected cells were exposed to 1% O2, 100 µM CoCl2, or 100 µM DFX.
Relative luciferase activities were determined. Results (means ± S.D. of three independent transfections) are expressed as percent of
the control (empty vector).
Protein Expression in Response to Hypoxia,
CoCl2, or DFX Is Differentially Regulated by Rac1--
The
biological activity of HIF-1 is mainly determined by the expression and
activity of the HIF-1
subunit. HEK293 cells overexpressing Rac1-N17
were exposed to hypoxia to examine whether Rac1 is involved in the
regulation of HIF-1
protein expression. Rac1-N17 completely suppressed the induction of HIF-1
expression in response to hypoxia, whereas expression of Rac1-V12 modestly enhanced the induction of
HIF-1
in hypoxic cells (Fig.
2A). Cdc42-N17 partially
suppressed hypoxia-induced expression of HIF-1
(Fig. 2A,
lane 6), which was consistent with its effects on reporter
gene expression (Fig. 1B). Under non-hypoxic conditions,
neither Rac1-V12 nor Cdc42-V12 had a detectable effect on HIF-1
expression (Fig. 2A, lanes 7 and 8).
Compared with Rac1-N17, the dominant-negative form of Rho did not
affect HIF-1
expression (Fig. 2B). We next examined the
effect of Rac1-N17 on chemical-induced HIF-1
expression. Neither
CoCl2- nor DFX-induced HIF-1
expression was
significantly affected by Rac1-N17 (Fig. 2C).
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Fig. 2.
Effects of small GTPases on
HIF-1 protein expression. HEK293 cells
were transfected with 4 µg of the indicated expression vector and
left untreated (U) or exposed to 1% O2, 100 µM CoCl2, or 100 µM DFX for
4 h before whole cell lysate preparation. 100-µg aliquots were
fractionated by SDS-polyacrylamide gel electrophoresis and subjected to
immunoblot (IB) assay using anti-HIF-1
antibody
(
HIF-1
). The constructs analyzed were as
follows: A, empty vector (EV), Rac1-V12,
Rac1-N17, Cdc42-V12, and Cdc42-N17; B, empty vector, Rho-DN,
and Ras-N17; C, empty vector and Rac1-N17.
-mediated Transactivation in Response to Hypoxia,
CoCl2, or DFX Is Differentially Regulated by
Rac1--
There are two independent transactivation domains (TADs)
present in HIF-1
designated as the amino-terminal (amino acids
531-575) and carboxyl-terminal (amino acids 786-826) TADs (TAD-N and
TAD-C, respectively) (7). Because it has been shown that steady-state levels of fusion proteins consisting of the GAL4 DNA-binding domain fused to HIF-1
TADs are similar under hypoxic and non-hypoxic conditions (7), these GAL4-HIF-1
fusion constructs can be used to
examine the transcriptional activity of HIF-1
independent of its
protein expression level. Rac1-N17 completely blocked hypoxia-induced transactivation mediated by GAL4-HIF-1
-(531-826), which contains both TAD-N and TAD-C (Fig.
3A). Moreover, transactivation
by GAL4-HIF-1
-(531-826) induced by CoCl2 or DFX was
also significantly suppressed by Rac1-N17 expression. It is noteworthy
that CoCl2- or DFX-induced transactivation was attenuated
by Rac1-N17, although CoCl2- or DFX-induced HIF-1
protein expression was not sensitive to Rac1-N17. Rac1-V12 expression stimulated transactivation mediated by GAL4-HIF-1
-(531-826), especially in cells not exposed to inducers (20% O2) (Fig.
3A), in contrast to its lack of effect on HIF-1
protein
expression. Transactivation mediated by GAL4-HIF-1
-(531-575), which
contains only TAD-N, was also blocked by Rac1-N17 (Fig. 3B).
In contrast, GAL4-HIF-1
-(786-826), which contains only TAD-C, was
constitutively activated and not inhibited by Rac1-N17 (Fig.
3C). These results demonstrate that Rac1 activity is
specifically required for HIF-1
transcriptional activity that is
induced by hypoxia or chemical agents.
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Fig. 3.
Effects of Rac1 on HIF-1
transactivation domain function. Constructs encoding the
GAL4 DNA-binding domain (amino acids 1-147) fused to the indicated
amino acids of HIF-1
were analyzed for their ability to
transactivate reporter gene G5E1bLuc, containing five GAL4-binding
sites. Hep3B cells were cotransfected with pTK-RL (50 ng), G5E1bLuc
(100 ng), and expression vectors encoding either no protein
(Empty vector) or the indicated small GTPase (250 ng) and
GAL4-HIF-1
fusion protein (100 ng). Cells were exposed to 20%
O2, 1% O2, 100 µM
CoCl2, or 100 µM DFX for 16 h and then
harvested. The ratio of firefly to Renilla luciferase
activity was determined and normalized to the value obtained from cells
transfected with plasmid encoding GAL4-(1-147) at 20% O2
to obtain the relative luciferase activity (means ± S.D. of three
independent transfections).
S (lane
2). GST-PBD did not interact with Rac1-WT loaded with GDP (Fig.
4A, lane 1) or with Rac1-N17 (data not shown).
Activated Rac1 was also recovered from Rac1-WT-expressing cells treated
with 500 ng/ml epidermal growth factor (Fig. 4A, lane
6) or 100 nM phorbol 12-myristate 13-acetate
(lane 7). Exposure to 1% O2 for 2 h (Fig.
4A, lane 5) or to 15 min of reoxygenation after
2 h of hypoxia (lane 8) also activated Rac1. We next
investigated the time course of Rac1 activation under hypoxic
conditions (Fig. 4B). Rac1 was activated as early as 30 min
after exposure to 1% O2 (Fig. 4B, lane
3), and this activation, although diminished after 2 h,
lasted for at least 16 h of continuous hypoxia (lanes 4-8). This time course differed from that of epidermal growth factor- or phorbol 12-myristate 13-acetate-induced Rac1 activation, which lasted no more than 15 min (data not shown). Exposure of cells to
75 µM CoCl2 also activated Rac1 (Fig.
4C, lane 3). However, exposure to 130 µM DFX did not activate Rac1 (Fig. 4C,
lane 4).
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Fig. 4.
Rac1 activation assay. HEK293 cells
overexpressing HA-tagged Rac1-WT or Rac1-V12 were serum-starved for
16 h. A, after the indicated treatment (lane
5, 1% O2 for 2 h; lane 6, 500 ng/ml
epidermal growth factor (EGF) for 10 min; lane 7,
100 mM phorbol 12-myristate 13-acetate (PMA) for
10 min; lane 8, 1% O2 for 2 h and then
reoxygenation for 15 min (hypoxia-reoxygenation (H/R))),
cells were harvested. In lanes 1 and 2, lysates
were loaded with GDP or GTP S, respectively, prior to affinity
precipitation. B, after exposure to 1% O2 for
the indicated times (lanes 2-8) or to 1% O2
for 2 h followed by reoxygenation for 15 min (lane 9),
cells were harvested. C, after a 30-min treatment with
CoCl2 or DFX, cells were harvested. Cell lysates were
incubated with 15 µg of GST-PBD. Activated Rac1 was detected by
GST-PBD pull-down and immunoblot (IB) assays with anti-HA
antibody (
HA). Equal amounts of HA-tagged Rac1
proteins were detected by immunoblot assay in all lysates prior to
pull-down assay.
TAD-dependent
transcriptional activity in response to hypoxia. Hypoxia-induced
reporter gene transcription was also inhibited by p85
, a
dominant-negative form of the PI3K p85 regulatory subunit (Fig.
5C). 10 µM PD98059, a MEK1 inhibitor, and 25 µM SB203580, a p38 MAPK inhibitor, also reduced
HIF-1-dependent gene transcription (Fig. 5, A
and B).
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Fig. 5.
Effect of kinase inhibitors on HIF-1
transcriptional activity. Hep3B cells were cotransfected with
pTK-RL (50 ng) and either p2.1 (100 ng) (A and C)
or G5E1bLuc and pGAL4/HIF-1 -(531-826) (B). In
C, cells were cotransfected with the p85
expression
vector or empty vector. After transfection, wortmannin (WM),
PD98059 (PD), or SB203580 (SB) was added
(A and B), and cells were exposed to 20 or 1%
O2 (A-C) or 100 µM
CoCl2 or DFX (C) for 16 h.
expression (Fig. 6A). In
contrast, neither PD98059 nor SB203580 affected HIF-1
expression
(Fig. 6A, lanes 3 and 4). Rotenone and DPI, which inhibit the mitochondrial ETC at complex I,
significantly attenuated the expression of HIF-1
in hypoxic cells
(Fig. 6B), as previously reported (1, 13, 15).
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Fig. 6.
Effect of kinase inhibitors on
HIF-1 expression. HEK293 cells were
pretreated with 50 µM PD98059 (PD), 25 µM SB203580 (SB), 100 µM
genistein (GS), 100 µM LY294002
(LY), 100 nM wortmannin (WT),
1 µg/ml rotenone (Rot), or 10 µM DPI and
then exposed to 20 or 1% O2 for 4 h prior to whole
cell lysate preparation. 100-µg aliquots were fractionated by
SDS-polyacrylamide gel electrophoresis and subjected to immunoblot
assay using anti-HIF-1
antibody.
expression and reporter gene transcription above
(Figs. 5 and 6), demonstrate that mitochondrial ETC, tyrosine kinase,
and PI3K activities are required for the activation of both Rac1
and HIF-1 in response to hypoxia.
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Fig. 7.
Effect of inhibitors on hypoxia-induced Rac1
activation. HEK293 cells overexpressing HA-tagged Rac1-WT
were serum-starved for 16 h and pretreated with 1 or 10 µM DPI, 0.5 or 1 µg/ml rotenone (Rot), 5 or
50 µM LY294002 (LY), 10 or 100 µM genistein (GS), 10 or 50 µM
PD98059 (PD), or 5 or 25 µM SB203580
(SB) for 1 h; exposed to 1% O2 for 30 min;
and harvested. Lysates were subjected to GST-PBD pull-down assay.
IB, immunoblot; HA, anti-HA
antibody.
TAD function in a hypoxia-specific manner (Fig. 5), we
examined whether p38 MAPK activation in response to hypoxia is
regulated by Rac1. Hypoxia induced transactivation mediated by
GAL4-ATF2-(1-96), which contains the TAD from the transcription factor
ATF2 that is known to be phosphorylated by p38 MAPK (33). Rac1-N17
blocked hypoxia-induced transactivation, and Rac1-V12 expression
stimulated transactivation mediated by GAL4-ATF2-(1-96) under both
non-hypoxic and hypoxic conditions (Fig.
8A). We next analyzed p38 MAPK
activation using a rabbit anti-phospho-p38 MAPK
(Thr183/Tyr185) polyclonal antibody. Fig.
8B shows that Rac1-N17 completely blocked p38 MAPK
phosphorylation in response to hypoxia.
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Fig. 8.
Effect of Rac1 on p38 MAPK activation.
A, constructs encoding the GAL4 DNA-binding domain (amino
acids 1-147) fused to the transactivation domain (amino acids 1-96)
of ATF2 were analyzed for their ability to transactivate reporter gene
G5E1bLuc, containing five GAL4-binding sites. Hep3B cells were
cotransfected with pTK-RL (50 ng), G5E1bLuc (100 ng), expression
vectors encoding Rac1-V12 or Rac1-N17 (250 ng), and GAL4-ATF2 fusion
protein (100 ng). Cells were exposed to 20 or 1% O2 for
8 h and then harvested. The ratio of firefly to Renilla
luciferase activity was determined and normalized to the value obtained
from cells transfected with GAL4-ATF2 at 20% O2 to obtain
the relative luciferase activity (means ± S.D. of three
independent transfections). B, HEK293 cells were transfected
with pSR -HA-p38 MAPK and either Rac1-N17 or empty vector (pEF-BOS).
Transfected cells were exposed to 20 or 1% O2 for 6 h
or to 100 µM H2O2 for 15 min, and
cell lysates were subjected to immunoprecipitation using anti-HA
antibody matrix. Precipitates were analyzed by Western immunoblotting
(IB) using anti-phospho-p38 MAPK
(
Pp38) and anti-HA
(
HA) antibodies.
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Fig. 9.
Effect of Rac1 on AP-1-dependent
reporter gene expression. Hep3B cells were cotransfected with
pTK-RL (50 ng); pAP-1-Luc (100 ng); and expression vectors (250 ng)
encoding no protein (Empty vector), Rac1-N17, or Rac1-V12.
Cells were exposed to 20 or 1% O2 for 8 h and then
harvested. The ratio of firefly to Renilla luciferase
activity was determined and normalized to the value obtained from cells
transfected with empty vector at 20% O2 to obtain the
relative luciferase activity (means ± S.D. of three independent
transfections).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
protein expression and
transcriptional activity have been most extensively analyzed. An
important recent advance has been the identification of the von
Hippel-Lindau tumor suppressor protein (pVHL) as the HIF-1
-binding
component of the ubiquitin-protein ligase that targets HIF-1
for
proteasomal degradation in non-hypoxic cells (34-38). Hypoxia may
induce changes in the phosphorylation and/or redox status of HIF-1
,
pVHL, or another component of the ubiquitination machinery. Remarkably,
exposure of cells to hypoxia, CoCl2, or DFX induces both
HIF-1
protein stabilization and transcriptional activation (7, 8),
even though these agents are mechanistically distinct. For example,
inhibitors of mitochondrial ETC complex I block hypoxia-induced (but
not CoCl2- or DFX-induced) HIF-1
protein expression (1,
13). For both protein stabilization and transcriptional activation,
hypoxia may induce change(s) in the phosphorylation and/or redox status
of HIF-1
or HIF-1
-interacting protein(s).
protein expression, HIF-1
TAD
function, and HIF-1-dependent gene transcription in
response to hypoxia. Although the dramatic inhibitory effects of the
dominant-negative form of Rac1 (Rac1-N17) under hypoxic conditions
indicate that Rac1 is necessary for these events, the modest
stimulatory effects of its constitutively activated form (Rac1-V12)
under non-hypoxic conditions indicate that Rac1-independent signals are
also required for HIF-1 activation. Below we consider, first, the
relationship of Rac1 to other putative components of the hypoxia signal
transduction pathway and, second, the mechanisms by which Rac1 may
regulate HIF-1
expression and activity.
expression. The inhibitory effects of DPI, rotenone, LY294002,
wortmannin, and genistein on the activation of Rac1 (Fig. 7) indicate
that Rac1 is downstream of these putative components of the hypoxia
signal transduction pathway (Fig. 10).
Hypoxia does not induce PI3K activity (17), and an oxygen-regulated
phosphatase or kinase that is required for HIF-1
expression has not
been identified. Hypoxia-induced hydrogen peroxide generation that is
dependent upon mitochondrial ETC activity has been reported (13, 14),
but how this signal is transduced to HIF-1
is unknown. The present
data suggest that activation of Rac1 may represent an intermediate step
in this process. In contrast, the p38 MAPK activity that is induced by hypoxia is downstream of Rac1 (Fig. 10). HIF-1
protein expression and HIF-1 DNA-binding activity increase exponentially as cellular O2 concentration decreases and rapidly decay upon
reoxygenation (2, 41, 42). In contrast, Rac1 has previously been shown to mediate the effects of hypoxia-reoxygenation on the activity of
transcription factors such as nuclear factor
B and heat shock factor
1 via generation of reactive oxygen intermediates (43, 44). In a recent
study, hypoxia-reoxygenation, but not hypoxia, was shown to induce heat
shock factor 1 activation as a result of Rac1-mediated
H2O2 generation (45). Thus, the involvement of
Rac1 in hypoxia-induced HIF-1 activation represents a novel pathway,
and delineation of both the upstream signal for Rac1 activation in
response to hypoxia as well as the downstream signal leading to HIF-1
activation will require further studies.
View larger version (15K):
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Fig. 10.
Signal transduction pathways leading to
induction of HIF-1 activity.
Protein Expression--
As in the case of ETC
activity, Rac1 activity is specifically required for hypoxia-induced
(but not CoCl2- or DFX-induced) HIF-1
expression. These
results are consistent with data indicating that CoCl2 and
DFX directly disrupt the interaction of HIF-1
with pVHL (3, 4),
i.e. at a step downstream of Rac1.
TAD Function--
The carboxyl-terminal half of
HIF-1
consists of two TADs separated by an inhibitory domain that
represses TAD function especially under non-hypoxic conditions (7, 8).
TAD-N function (either in the presence or absence of the inhibitory
domain) is induced by hypoxia, an effect that is dependent upon Rac1
activity (Fig. 3). In contrast, TAD-C function is independent of both
O2 concentration and Rac1, again demonstrating that Rac1 is
specifically required to transduce hypoxic signals to HIF-1
. Hypoxia
also induced p38 MAPK activity in a Rac1-dependent manner,
and the p38 inhibitor SB203580 attenuated hypoxia-induced TAD function
(Fig. 5). Rac1 is known to interact with the MAPK kinase kinase PAK1
(20), and p38 MAPK has been shown to phosphorylate the HIF-1
inhibitory domain in vitro (46). Taken together, these data
suggest that in response to hypoxia, activated Rac1 induces p38 MAPK
activity, leading to HIF-1
phosphorylation and increased TAD
function. Rac1-N17 completely blocked hypoxia-induced transactivation,
whereas SB203580 had only a partial inhibitory effect, suggesting that in addition to p38 MAPK activation, there may be other pathways by
which Rac1 induces HIF-1
TAD function in response to hypoxia. The
p42/p44 ERK MAPKs phosphorylate HIF-1
and stimulate HIF-1 transcriptional activity (46, 47), but this process in not regulated by
O2 concentration.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. Kozo Kaibuchi, Kaikobad Irani, Amato J. Giaccia, and Eisuke Nishida for providing plasmids and Erik Laughner for excellent technical advice.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants R01-HL55338 and R01-DK39869 (to G. L. S.).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 the Yamanouchi Foundation for Research on Metabolic Disorders and by a fellowship from the Uehara Memorial Foundation.
To whom correspondence should be addressed. Fax: 410-955-0484;
E-mail:gsemenza@jhmi.edu.
Published, JBC Papers in Press, March 30, 2001, DOI 10.1074/jbc.M100677200
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ABBREVIATIONS |
---|
The abbreviations used are:
HIF-1, hypoxia-inducible factor 1;
DFX, desferrioxamine;
ETC, electron
transport chain;
PI3K, phosphatidylinositol 3-kinase;
PAK, p21-activated kinase;
DPI, diphenyleneiodonium;
MAPK, mitogen-activated
protein kinase;
ATF, activating transcription factor;
HRE, hypoxia response element;
WT, wild-type;
HA, hemagglutinin;
PBD, p21-binding domain of PAK;
GST, glutathione S-transferase;
DN, dominant-negative;
TAD, transactivation domain;
GTPS, guanosine
5'-O-(3-thiotriphosphate);
MEK, mitogen-activated protein
kinase/extracellular signal-regulated kinase kinase;
ERK, extracellular
signal-regulated kinase.
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