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J. Biol. Chem., Vol. 277, Issue 33, 29936-29944, August 16, 2002
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From the Cell and Cancer Biology Branch, Center for Cancer
Research, NCI, National Institutes of Health, Rockville, Maryland
20850
Received for publication, May 14, 2002, and in revised form, June 3, 2002
HIF-1 Hypoxia-inducible factor-1 HIF-1 Geldanamycin (GA) is a naturally occurring anasamycin antibiotic that
possesses antitumor properties (29, 30) by virtue of its ability to
associate and interfere with Hsp90 function (31). Hsp90 associates with
client proteins in a nucleotide-dependent manner, and GA
interferes with this association by occupying the nucleotide binding
site of Hsp90 (32-34). Hsp90 substrates are numerous and include
multiple transcription factors (aryl hydrocarbon receptor,
glucocorticoid receptor, Myo D, p53) (35-38) and an array of signaling
kinases (Akt, ErbB2, Raf-1, v-Src) (39-42). Hsp90 plays an essential
role in facilitating the proper conformation, localization, and
function of these client proteins (43-47), and GA-mediated Hsp90
dissociation from client proteins results in their ubiquitination and
subsequent degradation by the proteasome (48-50). Although it is known
that HIF-1 Cell Culture and Treatments--
RCC lines: Caki-1 cells were
obtained from ATCC; UMRC2 were provided by Dr. M. I. Lerman (NCI,
National Institutes of Health, Frederick, MD); UMRC6 were obtained from
Dr. B. Zbar (NCI, Frederick, MD); and 786-O were provided by Dr. R. Klausner (NCI, Bethesda, MD). The designations herein of C2 and C6
refer to UMRC2 and UMRC6. All cell lines were cultured in Dulbecco's
modified Eagle's medium/F-12 supplemented with 10% fetal bovine serum
plus 1× nonessential amino acids and penicillin/streptomycin.
Cells were treated, as indicated, with the following agents: 50 µM N-acetyl-Leu-Leu-norleucinal (ALLnL, Sigma
Chemical Co.), 2 µM geldanamycin (GA), and 2 µM 17-allylamino-17-desmethoxygeldanamycin (17-AAG) (both
obtained from NCI), 40 µg/ml cycloheximide (CHX, Sigma), 5 µM PS-341 (Millennium Pharmaceuticals), and 100 µM cobalt chloride (Sigma).
Hypoxic Treatment--
Cells were placed in a 37 °C
pre-equilibrated Bactron II sealed chamber (Sheldon Labs), and premixed
gas (94% N2, 5% CO2, 1% O2), was
infused to create a hypoxic environment. The oxygen content inside the
chamber was constantly monitored by a previously calibrated oxygen
sensor (Animas Corp., Frazer, PA). Cells were manipulated and lysed
inside the chamber, and all buffers were pre-equilibrated to 1%
O2.
Subcellular Fractionation--
Cells were washed with
phosphate-buffered saline and overlaid for 10 min with low salt buffer
(10 mM HEPES, 10 mM KCl, 0.1 mM
EDTA) containing protease inhibitors. The cells were then scraped into
microcentrifuge tubes, and 10% Nonidet P-40 was added (final concentration 0.5%). The tubes were vigorously vortexed for 10 s
and spun at 3000 rpm for 1 min, and nuclear pellets were washed with
low salt buffer prior to lysis in high salt lysis buffer (20 mM HEPES, 400 mM NaCl, 1 mM EDTA).
DNA Manipulation and Transfection--
A CMV-driven HA-tagged
HIF-1 Immunoblot Analysis--
Following cell lysis and clarification
by centrifugation, equal amounts of protein were loaded onto 7.5% PAGE
gels (Bio-Rad). The following antibodies were used: murine HIF-1 Pulse-Chase Analysis--
Logarithmically growing UMRC2 cells
were starved 30 min in methionine and cysteine-free media (Invitrogen)
and 150 µCi/ml methionine/cysteine (Tran35S-label, ICN)
was added for 1 h. After the labeling period, cells were washed
with nonradioactive complete medium and incubated in this medium for
the indicated times. The cells were then lysed and precleared with
protein G beads, and HIF-1 Luciferase Assay--
Cells were cotransfected with 0.4 µg of
iNOS-luciferase plasmid DNA (kindly provided by Dr. G Melillo, NCI,
Frederick, MD), containing three HIF-1 RNA Analysis--
Cells were grown to ~70% confluency and
subjected to the appropriate treatments, and RNA was purified according
to the manufacturer's specifications (Qiagen RNeasy mini kit).
Aliquots of 15 µg of RNA were lyophilized and resuspended in
formaldehyde loading dye (Ambion). The gels were run and transferred by
capillary diffusion onto a nylon membrane. RNA was cross-linked to the
membrane with ultraviolet light, prehybridized 6 h with ready-made
buffer (Ambion) to which 0.2 mg/ml sheared salmon sperm was added, and
hybridized overnight. The membranes were then washed according to the
manufacturer's protocol (Ambion) and exposed to film at Angiogenesis Assay--
Caki-1 cells were seeded in 10-cm plates
(3 × 106 cells/plate) and, 24 h later, cells
were washed with phosphate-buffered saline and re-supplied with
serum-free medium. The plates were then placed under normoxic or
hypoxic conditions for 12 h, and 0.5 µM GA was added
to the treated plate at the time of hypoxia induction. Following the
12-h incubation, conditioned medium was removed and centrifuged twice
in succession through Centricon filters (YM3, Millipore) to remove any
traces of GA. The molecular mass cutoff of the filters was 3 kDa; the molecular mass of GA is 0.56 kDa, so the flow-through containing excess GA was discarded and the retentate was collected. Because GA itself may have an inhibitory effect on this assay, we
confirmed that this approach efficiently removed GA from conditioned medium by subjecting media preparations containing known concentrations of GA to filtration and measuring the drug remaining in the retentate by high-performance liquid chromatography, as previously described (52). It was determined that the amount of GA remaining after two
successive filtration spins was negligible when the starting concentration did not exceed 0.5 µM. The final filter
retentate was concentrated 3-fold for use in the angiogenesis assay.
For the angiogenesis assay, a modified aortic ring method (80)
was used. Briefly, aortic rings were prepared from 13-day-old chicken
embryos. Each aortic ring was placed in the center of a well and
overlaid with Matrigel (BD Biosciences, Bedford, MA) and growth
factor-free human endothelial-SFM basal growth medium (Invitrogen) to
which a 1:1 volume of cellular serum-free conditioned media was added.
For a positive control, 10 nM bFGF was added, while basal
medium alone was used as a negative control. In addition, filtered
conditioned medium from GA-treated cells was added to the positive
control to ensure that potential trace amounts of GA were not
inhibitory in this assay. The aortic rings were incubated at 37 °C
in 5% CO2 for 36 h in the presence of conditioned
medium, and microvessels sprouting from the aortic rings were
photographed with an inverted microscope. All conditions were performed
in duplicate. In each case, the degree of angiogenesis was normalized to the area of the corresponding ring. For relative comparison, the
degree of angiogenesis for each condition was normalized to the
negative control.
GA Promotes the Down-regulation of HIF-1
Under hypoxic conditions, the normally labile HIF-1
To exclude the possibility that the effects of GA on HIF-1 GA Destabilizes HIF-1
To examine whether the GA-mediated degradation of HIF-1
Previous reports have demonstrated that GA-mediated proteasomal
degradation of Hsp90 client proteins is preceded by their ubiquitination (48, 58, 60, 61). Therefore, we tested whether HIF-1
To confirm that this high molecular weight species represented
ubiquitinated HIF-1
Recently, HIF-1 GA Interferes with HIF-1
It remained unclear whether GA affected VEGF transcription
directly via inhibition of HIF-1
VEGF is one of the most potent angiogenic cytokines released by hypoxic
tumors (69, 70). Because we observed a GA-mediated inhibition of
HIF-1 In this study, we have identified the Hsp90 molecular chaperone as
a novel VHL- and oxygen-independent regulator of HIF-1 GA-mediated dissociation of HIF-1 Although the association of HIF-1 One of the most clinically relevant aspects of this study is the
finding that Hsp90 antagonists interfere in a hypoxia-independent manner with HIF-1 Because 17-AAG is currently in clinical trial, these results are of
particular interest, in that they indicate that this drug may have
antiangiogenic activity in cancer patients. This prediction is
supported by our data demonstrating that conditioned medium from
hypoxic cells exhibits potent angiogenic activity, whereas medium from
hypoxic cells exposed to GA does not. Although this inhibitory activity
may not be due solely to effects on VEGF synthesis and secretion, to
our knowledge this is the first report demonstrating an inhibitory role
for GA in hypoxia-induced angiogenesis. The hypoxic state of a tumor
correlates with increased malignancy, metastatic potential, poor
prognosis, and resistance to radiotherapy and chemotherapy (77, 78).
Our findings demonstrate that GA-induced, Hsp90-dependent
elimination of HIF-1 We thank E. Agnew for high-performance liquid
chromatography analyses and W. Xu, V. Leaner, and G. Melillo for
helpful discussions.
A study describing similar destabilizing effects of
geldanamycin on HIF-1 *
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.
Published, JBC Papers in Press, June 6, 2002, DOI 10.1074/jbc.M204733200
The abbreviations used are:
HIF-1
Hsp90 Regulates a von Hippel Lindau-independent
Hypoxia-inducible Factor-1
-degradative Pathway*
ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
is a normally labile
proangiogenic transcription factor that is stabilized and
activated in hypoxia. Although the von Hippel Lindau (VHL) gene
product, the ubiquitin ligase responsible for regulating HIF-1
protein levels, efficiently targets HIF-1 for rapid
proteasome-dependent degradation under normoxia, HIF-1 is
resistant to the destabilizing effects of VHL under hypoxia. HIF-1
also associates with the molecular chaperone Hsp90. To examine the role
of Hsp90 in HIF-1 function, we used renal carcinoma cell (RCC) lines
that lack functional VHL and express stable HIF-1 protein under
normoxia. Geldanamycin (GA), an Hsp90 antagonist, promoted efficient
ubiquitination and proteasome-mediated degradation of HIF-1 in RCC
in both normoxia and hypoxia. Furthermore, HIF-1 point mutations
that block VHL association did not protect HIF-1 from GA-induced
destabilization. Hsp90 antagonists also inhibited HIF-1
transcriptional activity and dramatically reduced both hypoxia-induced
accumulation of VEGF mRNA and hypoxia-dependent angiogenic activity. These findings demonstrate that disruption of
Hsp90 function 1) promotes HIF-1 degradation via a novel, oxygen-independent E3 ubiquitin ligase and 2) diminishes HIF-1 transcriptional activity. Existence of an Hsp90-dependent
pathway for elimination of HIF-1 predicts that Hsp90 antagonists may be hypoxic cell sensitizers and possess antiangiogenic activity in vivo, thus extending the utility of these drugs as
therapeutic anticancer agents.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(HIF-1)1 is a component of
a transcriptional complex that is extremely labile under normoxia but is stabilized and activated under hypoxia (1). Because HIF-1 regulates a variety of processes such as angiogenesis and glucose metabolism, it is acknowledged to be a critically important tumor cell
survival factor that is required for tumorigenesis in many cancer
models and is expressed in a majority of metastases and late stage
tumors. The rapid degradation of HIF-1 in normoxic cells is mediated
by the tumor suppressor VHL, which, together with a multimeric protein
complex, serves as its E3 ubiquitin protein ligase (2-5). Under
hypoxic conditions, the stabilization and activation of HIF-1 is due
to an inability of VHL to associate with and ubiquitinate HIF-1. It
was recently shown that hypoxic conditions impair the ability of a
class of enzymes termed prolyl hydroxylases to modify two separate
consensus proline motifs present on HIF-1 (6-10). These
modifications are required for VHL to associate with and ubiquitinate
HIF-1, thereby targeting the protein for
proteasome-dependent degradation.
is constitutively stabilized in normoxic tumors and in cell
lines that are VHL null or that express a nonfunctional mutant form of
VHL. This occurs in over 50% of sporadic RCCs and clear cell RCCs
(11), the most common malignant neoplasm of the kidney, and one of the
few human tumors known to depend upon VHL inactivation. The importance
of VHL function is further demonstrated in VHL disease, a human cancer
syndrome caused by hereditary loss of VHL gene function, resulting in
constitutive up-regulation of hypoxically induced genes, and
characterized by highly vascular tumors of the central nervous system,
in addition to RCCs (12). The crucial role of HIF-1 in tumor
progression is underscored by its expression in a significant
proportion of breast, colon, prostate, and a variety of other cancers
(13-16). The ability of HIF-1 to promote both tumor cell survival
and angiogenesis (17-20) strongly suggests that HIF-1
overexpression is important for tumor vascularization and metabolic
adaptation to hypoxia (21-23), both essential events for malignant
tumor progression. This hypothesis is strengthened by the observations
that HIF-1 expression correlates with tumor grade and vascularity
(24), and that VHL-inactivated tumors are highly vascular and
overproduce angiogenic factors such as VEGF (25-27). VEGF, one of the
most potent angiogenic cytokines, is transcriptionally regulated in
large part by HIF-1 (28), suggesting that the ability to
down-regulate HIF-1 expression would have a positive impact on
cancer control.
associates with Hsp90 (51), a role for this association
has remained elusive. Here, we demonstrate that disruption of
HIF-1/Hsp90 association promotes the ubiquitination and
proteasome-mediated degradation of HIF-1 in a manner that is both
oxygen- and VHL-independent, thereby delineating a novel pathway that
regulates HIF-1 protein stability and function.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
construct was originally obtained from Dr. D. Livingston
(Dana-Farber Cancer Institute, Boston, MA). Using primers containing
sites for BamHI (5') and NotI (3'), this insert
was PCR-amplified, subcloned into PCDNA3.1 (Invitrogen), and
confirmed by sequencing. This latter construct was used for all wt
HIF-1 transfections and as a template for subsequent mutations. To
make site-specific mutations, complementary primers containing desired
point mutations were constructed, and PCR amplification was performed
in accordance with Promega's XL site-directed mutagenesis kit. For
transfection, cells were seeded at 60% confluency 24 h prior to
transfection, and 3 µg of plasmid DNA was used per 10-cm dish.
Transfections were carried out using FuGENE (Roche Molecular
Biochemicals), according to the manufacturer's specifications.
,
1:300 (Transduction labs); murine HA, 1:2000 (Covance); rat Hsp90,
1:2000 (Stressgen); murine topoisomerase II, 1:500 (Sigma); and rabbit
polyclonal ubiquitin, 1:1000 (Sigma). For protein visualization,
horseradish peroxidase-linked secondary antibodies were used with the
ECL protein detection system (Pierce). For HA immunoprecipitations, lysates were incubated with HA-conjugated Sepharose beads (Covance) and
for HIF-1 immunoprecipitations, lysates were incubated with HIF-1
antibody, followed by addition of protein G-Sepharose beads (Invitrogen). To detect HIF-1/Hsp90 association, a low detergent (0.1% Nonidet P-40) lysis buffer was used and 20 mM
Na2MoO4 was added to stabilize protein
interactions. Beads were washed four times with lysis buffer, boiled in
Laemmli buffer, and processed as described.
was immunoprecipitated from 1 mg
of soluble lysed protein overnight. For GA-treated samples, 2 µM GA was added to the starve medium, retained throughout
the labeling period, and included in the chase medium. Densitometric analysis was performed, and the values (expressed as a fraction of the
unchased control) were plotted semi-logarithmically.
-responsive elements (HREs),
and 4 ng of Renilla luciferase plasmid DNA, which served as
an internal control. Luciferase activities were measured and normalized
to the Renilla activity. The data represent the means of
three separate experiments.
80 °C
with intensifying screens. The VEGF165 probe was kindly
provided by Dr. Shi-Yuan Cheng (University of Pittsburgh, Pittsburgh,
PA) as plasmid DNA. The 0.6-kb insert was removed by digestion with
BamHI and EcoRI, gel-purified (Qiaex II
extraction kit, Qiagen), and radiolabeled with [32P]dCTP
using the Decaprime kit (Ambion), and 2 × 106 cpm/ml
was used in hybridization reactions.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
under Normoxia and
Hypoxia--
GA mediates the dissociation of Hsp90 from client
proteins and promotes their rapid degradation by the proteasome.
Several reports have demonstrated an interaction between HIF-1 and
Hsp90 (51, 53), although a definitive biological role for this
interaction has remained elusive. We therefore investigated whether
Hsp90 might regulate HIF-1 protein stability in the absence of VHL. To address this question, we used the RCC lines C2 and C6 (3, 54) that
lack a functional VHL protein and therefore express an inherently
stable HIF-1 protein under normoxia. As shown in Fig.
1A (upper panel),
HIF-1 protein was highly expressed under normoxia in untreated cells
but was down-regulated in a dose-dependent manner in
response to GA treatment. It appeared that HIF-1 from C2 cells was
less sensitive to the effects of GA when compared with the protein from
C6 cells, prompting us to extend the duration of drug treatment. As
shown in Fig. 1A (middle panel), this prolonged treatment enhanced the sensitivity of HIF-1 to GA, so that the effective GA concentration for both cell lines occurred within the
0.1-0.5 µM range. The lowest panel of Fig.
1A shows that GA promoted the down-regulation of HIF-1 in
a time-dependent manner, with the protein in both cell
lines diminishing over 6-8 h. To verify the ability of GA to
down-regulate HIF-1 levels, 786-O cells deficient for both HIF-1
and VHL (3) were transfected with wild-type HIF-1 and protein levels
were assessed. As shown in Fig. 1B, GA treatment resulted in
a rapid decrease in HIF-1 protein levels, with over 50% of the
protein being eliminated within 1 h of treatment.
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Fig. 1.
GA disrupts HIF-1
association with Hsp90 and stimulates its VHL-independent
down-regulation. A, VHL-deficient RCC lines were
treated for 8 h with the GA concentration indicated. Middle
panel, C2 cells were treated with the indicated doses of GA for
12 h. Lowest panel, RCC cells were treated with 2 µM GA for varying times. The cells were lysed, protein
was quantitated, and nuclear fractions were subjected to
electrophoresis. B, VHL/HIF-1-deficient 786-O cells were
transfected with HIF-1, treated with GA, and harvested after the
indicated times. C, wild-type VHL-expressing Caki-1 cells or
VHL-deficient C6 cells were treated for 12 h with 2 µM GA or 17-AAG under normoxia or hypoxia (1%
O2). For hypoxic treatment, cells were placed under hypoxia
for 4 h to allow for HIF-1 accumulation prior to GA treatment.
The percent decrease in HIF-1 protein levels following GA treatment
is shown, and topoisomerase II expression was measured as a loading
control. D, C2 cells were treated with 2 µM GA
for 1.5 h (0.5 h for Caki-1), HIF-1 was immunoprecipitated, and
resultant blots were probed with anti-Hsp90.
protein is
stabilized in VHL-competent cells. It was therefore of interest to
determine whether hypoxia could antagonize the effect of GA in cells
expressing or lacking functional VHL. Caki-1 is an RCC line that
contains wild-type VHL, and in these cells HIF-1 is induced by
hypoxia (3). Caki-1 cells were treated with GA while under hypoxia,
subsequent to hypoxia-induced stabilization of HIF-1. Similarly,
VHL-deficient C6 cells were treated with GA under normoxia or hypoxia.
As shown in Fig. 1C, treatment of cells with GA or its
similarly acting, clinically administered analog 17-AAG (55-57),
significantly decreased HIF-1 protein levels, regardless of oxygen tension.
were nonspecific, we examined the association of HIF-1 with Hsp90 in
the presence or absence of drug. In accordance with other reports (40,
58), the GA-mediated down-regulation of HIF-1 would be expected to
be preceded by dissociation of the HIF-1·Hsp90 chaperone complex.
As shown in Fig. 1D, treatment of either C2 or Caki-1 cells
with GA led to a marked decrease in the amount of HIF-1 associated
with Hsp90, and GA was equally capable of displacing Hsp90 from
HIF-1 independent of oxygen tension. Importantly, oxygen level had
no effect on the ability of HIF-1 to associate with Hsp90.
Protein via the Proteasomal
Pathway--
To determine whether GA promoted HIF-1 down-regulation
at the RNA or protein level, we pretreated RCC lines C2 and C6 with GA,
and protein synthesis was subsequently inhibited with CHX. The level of
HIF-1 protein in cells exposed to the combination treatment was
compared with that seen in cells exposed to CHX alone. As shown in Fig.
2A, the stability of existing
HIF-1 protein in both cell lines was markedly compromised by GA. To
more clearly determine the effects of GA on the half-life of HIF-1
protein, and to examine the sensitivity of newly synthesized protein to Hsp90 disruption, we metabolically labeled C2 cells with
[35S]methionine/cysteine for 1 h in the presence or
absence of GA and chased for the indicated times (Fig. 2B).
The data obtained confirm the sensitivity of newly synthesized
HIF-1 protein to GA and demonstrate a greater than 4-fold reduction
in HIF-1 half-life in the presence of drug.
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Fig. 2.
GA destabilizes HIF-1
protein. A, RCC lines were treated with the
protein synthesis inhibitor CHX for the indicated times, either alone
or 1 h after treatment with GA. The right-hand panels
show the effects of GA upon HIF-1 steady-state level in the presence
of CHX. B, C2 cells were pulse-labeled with
[35S]methionine and chased in unlabeled medium for the
indicated times. For those cells subjected to drug treatment, GA was
added to the pulse and chase medium. HIF-1 half-life was determined
from the logarithmic values of chased time points compared with
unchased controls.
proceeds via the proteasome, we treated RCC lines for 8 h with the general proteasome inhibitor ALLnL, with GA, or with both agents and
examined HIF-1 levels in nuclear and cytosolic detergent-soluble and
insoluble fractions. As shown in Fig.
3A, treatment with ALLnL resulted in an increase of total HIF-1 levels in the nuclear soluble
fraction, demonstrating that HIF-1 is degraded by the proteasome,
even in the absence of VHL. No HIF-1 protein was detected in the
cytosolic soluble fraction (data not shown). Compared with GA
treatment, the combination treatment stabilized nuclear soluble
HIF-1 levels, but to a degree still significantly less than that
observed with ALLnL alone. Reports have indicated that treatment of
cells with Hsp90 antagonists in combination with proteasome inhibitors
results in detergent-insoluble client proteins (58, 59). Therefore, we
assessed HIF-1 levels in nuclear and cytosolic detergent-insoluble
fractions. As shown in Fig. 3A (middle and
right panels), in the absence of treatment, minimal HIF-1
was detected in either insoluble fraction, and treatment with ALLnL
alone resulted in only a small increase in insoluble HIF-1. Although
no HIF-1 protein was detected in the insoluble fractions subsequent
to GA treatment alone, the combination of GA and ALLnL increased
HIF-1 protein in both nuclear and cytosolic detergent-insoluble
fractions. In sum, these data show that GA-mediated degradation of
HIF-1 occurred by a proteasome-dependent pathway and
shares characteristics with the degradation of other Hsp90 client
proteins.
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Fig. 3.
GA-induced degradation of
HIF-1 proceeds via the proteasome.
A, RCC lines were either left untreated (lane 1)
or treated 8 h with the proteasome inhibitor ALLnL (lane
2), with GA (lane 3), or with a combination of these
two agents (lane 4). Equal amounts of nuclear soluble
fraction proteins for each cell line, and 20% of the total volume of
resuspended nuclear or cytosolic detergent-insoluble fractions, were
loaded onto the gel. Exposure times for the cytoplasmic insoluble
fractions were ~5 times longer than for the other fractions.
B, RCC lines were treated with 2 µM GA for the
indicated times, either in the presence or absence of a 30-min exposure
to the proteasome inhibitor PS-341 (last 30 min of combination
treatments). HIF-1 was immunoprecipitated, and resultant blots were
probed with an anti-ubiquitin antibody. The right-hand panel
in A depicts a shorter exposure of ubiquitinated protein in
PS-341-treated versus combination treated cells.
C, 786-O cells were transfected with HA-tagged HIF-1 and
cells were treated with either 2 µM GA (1 h), PS-341 (0.5 h), or with a combination of these agents. Lysates were
immunoprecipitated with anti-HIF-1 antibody, and resultant blots
were probed with HA antibody. D, constructs encoding either
wild-type HIF-1 or prolyl hydroxylation mutants were transfected
into 786-O cells and subjected to a 4-h treatment with GA, and nuclear
lysates were examined for HIF-1 expression. E, cells were
transfected as in D, treated with GA, PS-341, or a
combination, HIF-1 was immunoprecipitated, and resultant blots were
probed with an anti-ubiquitin antibody. The Control lane
represents untransfected cells subjected to the combination
treatment.
was similarly ubiquitinated in GA-treated cells prior to its
degradation. This was of special interest, because VHL, which serves as
the primary ubiquitin ligase for HIF-1 (3-5), is nonfunctional in
the RCC lines studied. As shown in Fig. 3B, although no
HIF-1-ubiquitin conjugates were observed in untreated cells, these
species were easily detected in HIF-1 immunoprecipitates after
3 h of GA treatment, with maximal ubiquitination occurring by
6 h. To enhance visualization of HIF-1- ubiquitin conjugates, we treated cells for 0.5 h with the specific proteasome inhibitor PS-341 (62), either alone or in combination with GA. The brief exposure
to PS-341 was used to avoid formation of detergent-insoluble HIF-1
that occurs after prolonged treatment with GA in the presence of
proteasome inhibitors (see Fig. 3A). As shown in Fig.
3B, the combination treatment dramatically increased
HIF-1-ubiquitin conjugates (detectable by 1 h, data not shown).
Although PS-341 alone increased the amount of ubiquitinated HIF-1
species, this increase was significantly less (~8-fold) when compared
with that elicited by the combination of GA and PS-341. These data
suggest that the HIF-1 ubiquitination/degradation process set in
motion by GA is relatively rapid and rather efficient. Interestingly, although the molecular mass of HIF-1 is ~116 kDa, the majority of
the ubiquitinated protein in HIF-1 immune precipitates migrated with
an apparent molecular mass of ~180 kDa, suggesting that most of the
ubiquitinated HIF-1 protein was polyubiquitinated.
, 786-O cells were transfected with an HA-tagged
HIF-1 construct and the experiment described in Fig. 3B
was repeated. HIF-1 was immunoprecipitated, and the protein was
visualized with an anti-HA antibody, as shown in Fig. 3C. The time of exposure to GA was shortened because of the ability of GA
to rapidly degrade HIF-1 protein in this cell line (see Fig.
1B). There was virtually no detectable ubiquitinated
HIF-1 in control or GA-treated cells and only a slight increase in
ubiquitinated species after treatment with PS-341 alone. However,
similar to the data in Fig. 3B, a significant increase in
ubiquitinated HIF-1 species was evident in cells treated with both
GA and PS-341, thus confirming that the HIF-1 protein identified
with a ubiquitin antibody in Fig. 3B represented
ubiquitinated HIF-1.
was shown to undergo proline hydroxylation at two
sites (Pro-402 and Pro-564), and these modifications are required for VHL association and thus VHL-mediated degradation of
HIF-1 (6, 8-10). To confirm that GA-induced degradation of HIF-1
was independent of proline hydroxylation, we mutated these two
residues, either independently or together, and assessed the GA
sensitivity of the mutated proteins in VHL-deficient 786-O cells. As
shown in Fig. 3D, the mutant proteins were as sensitive to
GA as was wild-type HIF-1. To determine whether these mutant proteins could be ubiquitinated, we transfected 786-O cells with wild-type or proline-mutated HIF-1 constructs, treated the cells with GA or GA plus PS-341, and probed HIF-1 immune precipitates with
an antiubiquitin antibody. Both ubiquitinated wild-type and proline-mutated HIF-1 species were detected equally well, showing that proline hydroxylation was not a requirement for GA-stimulated ubiquitination (Fig. 3E).
Transcriptional Activity--
The
increased transcriptional activity of HIF-1 that occurs during
hypoxia is associated with a concomitant increase in protein stability
but also depends upon hypoxic inhibition of an asparagine hydroxylation
modification that facilitates the association of HIF-1 with
cofactors such as p300 (63, 64). Once activated, HIF-1
transactivates its target genes by associating with their hypoxia-responsive elements (HREs) (65, 66). One of the numerous transcriptional targets of HIF-1 is VEGF (28), a potent
proangiogenic cytokine. Because GA down-regulated HIF-1 protein
levels, we determined whether GA could also interfere with HIF-1
transcriptional activity by treating both normoxic RCC lines and
hypoxic VHL-competent Caki-1 cells with GA. As shown in Fig.
4A (upper panel),
GA treatment of the RCC lines resulted in a modest decline (33-36%)
in VEGF165 mRNA by 6 h, with more than a 70%
reduction after 16 h (see table in Fig. 4). Similarly,
in Caki-1 cells, a 6-h treatment with GA following a 6-h hypoxic
pretreatment resulted in a 57% decline in VEGF mRNA, which
progressed to a 71% decline after 16 h.
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Fig. 4.
GA interferes with HIF-1
transcriptional activity. A, RCC lines were
treated with GA under normoxic conditions and Caki-1 cells were treated
with GA following a 6-h hypoxic exposure. Total RNA was isolated, as
described, and 15 µg of RNA was separated on a formaldehyde-agarose
gel, transferred to membrane, and incubated with a
32P-labeled VEGF165 probe. Exposure times are
as follows: C2, 0.5 h; C6, 1.5 h; Caki-1, 1 h.
Densitometric analysis of VEGF signal intensity enabled a calculation
of the -fold HIF-1 induction and percent inhibition by GA, as
depicted in the table. The lower panel in
A demonstrates the quality and loading of RNA. B,
C2 or C2 cells stably transfected with VHL were co-transfected with 5 ng of CMV-Renilla luciferase plasmid (an internal standard)
and 0.5 µg of HRE-dependent iNOS luciferase plasmid. For
GA-treated cells, drug was added for the indicated times before
lysis.
transcription or whether VEGF mRNA expression was affected by other means (67). To address this
issue, we performed transient transfection assays using a luciferase
expression plasmid under control of HREs from the iNOS promoter (68).
As shown in Fig. 4B (left panel),
HIF-1-dependent luciferase activity in VHL-deficient C2
cells was 3.5-fold higher than in C2 cells stably transfected with VHL,
thus confirming the HIF-1 dependence of this reporter. In a second
experiment, cells were treated with GA 4 h after transfection, and
drug treatment was continued for an additional 4 or 6 h. As shown
in Fig. 4B (right panel), GA treatment for 4 h resulted in a modest reduction in HIF-1-dependent
luciferase activity, with over a 50% reduction after 6 h.
activity and a corresponding decrease in VEGF transcription,
we wished to determine whether GA could mitigate hypoxia-induced
angiogenesis. To test this possibility, we collected conditioned medium
from VHL-competent Caki-1 cells subjected either to normoxia, 12 h
of hypoxia, or 12 h of hypoxia in the presence of GA. GA was
removed from the conditioned media by size exclusion filtration, and
the drug-free filter retentates were then used in a chick aortic ring
angiogenesis assay, as shown in Fig. 5. The top three panels show various controls. Basic FGF (Fig.
5B) was ~2.5-fold more angiogenic than the negative
control (Fig. 5A). Medium from Caki-1 cells treated with GA
did not inhibit FGF-induced angiogenesis (Fig. 5C),
demonstrating that inhibition of angiogenesis by media from GA-treated
cells was not due to traces of drug remaining in the medium. Panels
D-F depict the angiogenic activity of medium from Caki-1
cells. Medium conditioned by hypoxic Caki-1 cells (Fig. 5E)
was ~3-fold more angiogenic when compared with medium from normoxic
cells (Fig. 5D). Supporting and extending our previous
results, the angiogenic potential of medium conditioned by hypoxic
Caki-1 cells treated with GA (Fig. 5F) was comparable to the
negative control (Fig. 5, A and D).
View larger version (78K):
[in a new window]
Fig. 5.
GA antagonizes hypoxia-induced
angiogenesis. Serum-free conditioned medium was added to
chick aortic rings for 36 h, as described. As a negative control
(A), serum-free medium was used. B, a positive
control with 10 nM bFGF added to the control medium as in
panel A. C, 10 nM bFGF added to the
filtered medium from GA-treated Caki-1 cells (F).
D-F illustrate the angiogenic potential of filtered
conditioned medium obtained from Caki-1 cells in normoxia and hypoxia:
D, medium obtained from cells under normoxia; E,
medium obtained from hypoxic cells (12 h); F, medium
obtained from hypoxic cells in the presence of 0.5 µM
GA.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
protein
stability. This was demonstrated by the ability of the Hsp90 inhibitors
GA and 17-AAG to promote the loss of HIF-1 protein from C2 and C6,
two VHL-deficient RCC lines containing constitutively elevated HIF-1
levels in normoxia. Transfected HIF-1 was also markedly destabilized
by GA in 786-O cells, which lack both VHL and HIF-1 genes. GA
and 17-AAG were equally effective at reducing HIF-1 levels in
hypoxia and in normoxia and in the presence or absence of endogenous
VHL. Although co-precipitation of Hsp90 with HIF-1 was independent
of oxygen tension, this interaction was rapidly disrupted by GA, prior
to loss of HIF-1 protein. As is the case for other Hsp90 client
proteins (40), when Hsp90 was inhibited by GA, both pre-existing and
newly synthesized HIF-1 protein pools became unstable, although the
rate of HIF-1 synthesis remained essentially unchanged. Importantly,
the HIF-1 degradation that was stimulated by the Hsp90 inhibitors GA
and 17-AAG remained proteasome-mediated. As has been reported with
other Hsp90-soluble client proteins (58, 59), the combination of
proteasome inhibition and Hsp90 inhibition resulted in the accumulation
of HIF-1 protein in the detergent-insoluble pellet fraction, where
under normal circumstances it was undetectable. The fact that the
preponderance of HIF-1 was recovered from the nuclear pellet
fraction suggests either that GA-induced HIF-1 degradation occurred
primarily in the nuclear compartment or that proteasome inhibition
interfered with the nuclear export of HIF-1 protein.
from Hsp90 markedly enhances
HIF-1 ubiquitination in VHL-deficient cells, thereby demonstrating an essential function for Hsp90 in maintaining HIF-1 stability. Although a reduction in steady-state levels of HIF-1 protein required exposure to GA for several hours, a significant amount of
HIF-1 ubiquitination can be seen after as little as 1 h of exposure to the drug (data not shown). As a final proof that GA-induced HIF-1 degradation does not proceed via the VHL pathway, we
demonstrate that mutation of prolines 402 and 564 in HIF-1, which
render the protein fully resistant to VHL-mediated ubiquitination and degradation (8-10), fails to protect HIF-1 from GA. These data point to a novel, oxygen-independent E3 ubiquitin ligase, distinct from
VHL, that is recruited to HIF-1 upon dissociation of the HIF-1·Hsp90 complex. Although the mdm2 ubiquitin ligase may
serve as an E3 for HIF-1 under certain conditions (68), and
GA-induced degradation of mutated p53 requires mdm2 (69), this E3 is
unlikely to be the ubiquitin ligase that mediates GA-induced HIF-1
degradation, because dominant negative mdm2 failed to protect HIF-1
protein from the destabilizing effects of GA in RCC cells (data not
shown). Furthermore, the Hsp90/Hsp70-binding ubiquitin ligase CHIP
(carboxyl terminus of Hsc70-interacting protein), which may
mediate the GA-stimulated degradation of some Hsp90 client proteins
(71, 72), also failed to destabilize HIF-1 when the two proteins were co-transfected into either COS7 or 786-O cells (data not shown).
We are currently screening several additional candidate E3 enzymes in
an effort to identify the oxygen-independent ubiquitin ligase
responsible for GA-induced ubiquitination of HIF-1.
with Hsp90 has been previously
documented (51, 73), a physiologically important role for Hsp90 in
HIF-1 function has remained elusive. We have now demonstrated that
Hsp90 plays a pivotal role as a master regulator of HIF-1 protein
stability. In contrast to a previous report (53), we found that Hsp90
retains the ability to associate with HIF-1 under both normoxic and
hypoxic conditions. It is possible that the use of chemical mimetics of
hypoxia by these investigators, instead of reduced oxygen levels, may
explain this discrepancy. Indeed, a recent report demonstrating that
hypoxic accumulation of HIF-1 protein is antagonized by GA (74)
supports the notion that Hsp90 associates with HIF-1 under both
normoxic and hypoxic conditions. However, we propose that, rather than
preventing its up-regulation, GA promotes the degradation of HIF-1
under hypoxia.
transcription, as measured both by reporter assay
and by analysis of VEGF mRNA levels. Although two recent reports
have suggested that Hsp90 antagonism inhibits expression of VEGF (75,
76), the contribution of the chaperone in these studies is unclear due
to the presence of functional VHL in the cells that were used. Although
it remains to be determined whether GA-mediated down-regulation of VEGF
mRNA is due solely to interference with HIF-1 transcriptional
activity, the kinetics of these two events suggest such a relationship.
occurs under normoxic and hypoxic conditions
and is independent of VHL, thus identifying a novel means of treating
tumors overexpressing HIF-1 protein.
ACKNOWLEDGEMENTS
Addendum
protein in prostate cancer cell lines was
published (79) at the time of submission of this report.
FOOTNOTES
To whom correspondence should be addressed: Cell and Cancer
Biology Branch, Center for Cancer Research, NCI, National Institutes of
Health, 9610 Medical Center Dr., Ste. 300, Rockville, MD 20850. Tel.:
301-402-3128 (ext. 318); Fax: 301-402-4422; E-mail:
len@helix.nih.gov.
ABBREVIATIONS
, hypoxia-inducible factor-1 alpha;
Hsp90, heat shock protein 90;
VHL, von Hippel Lindau;
VEGF, vascular endothelial growth factor;
GA, geldanamycin;
RCC, renal carcinoma cell;
CHX, cycloheximide;
HA, hemagglutinin;
ALLnL, N-acetyl-Leu-Leu-norleucinal;
bFGF, basic fibroblast growth factor;
17-AAG, 17-allylamino-17-desmethoxygeldanamycin;
CMV, cytomegalovirus;
nt, nucleotide(s);
HRE, HIF-1-responsive element;
E3, ubiquitin-protein
isopeptide ligase;
mdm2, murine double minute 2;
iNOS, inducible nitric oxide synthase;
CHIP, carboxyl terminus of
Hsc70-interacting protein.
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 N. C Chi and J. S Karliner Molecular determinants of responses to myocardial ischemia/reperfusion injury: focus on hypoxia-inducible and heat shock factors Cardiovasc Res, February 15, 2004; 61(3): 437 - 447. [Abstract] [Full Text] [PDF]
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K. S. Bisht, C. M. Bradbury, D. Mattson, A. Kaushal, A. Sowers, S. Markovina, K. L. Ortiz, L. K. Sieck, J. S. Isaacs, M. W. Brechbiel, et al. Geldanamycin and 17-Allylamino-17-demethoxygeldanamycin Potentiate the in Vitro and in Vivo Radiation Response of Cervical Tumor Cells via the Heat Shock Protein 90-Mediated Intracellular Signaling and Cytotoxicity Cancer Res., December 15, 2003; 63(24): 8984 - 8995. [Abstract] [Full Text] [PDF]
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H.-T. Yuan, X.-Z. Li, J. E. Pitera, D. A. Long, and A. S. Woolf Peritubular Capillary Loss after Mouse Acute Nephrotoxicity Correlates with Down-Regulation of Vascular Endothelial Growth Factor-A and Hypoxia-Inducible Factor-1{alpha} Am. J. Pathol., December 1, 2003; 163(6): 2289 - 2301. [Abstract] [Full Text]
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R. K. Bruick Oxygen sensing in the hypoxic response pathway: regulation of the hypoxia-inducible transcription factor Genes & Dev., November 1, 2003; 17(21): 2614 - 2623. [Full Text] [PDF]
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 P. de Candia, D. B. Solit, D. Giri, E. Brogi, P. M. Siegel, A. B. Olshen, W. J. Muller, N. Rosen, and R. Benezra Angiogenesis impairment in Id-deficient mice cooperates with an Hsp90 inhibitor to completely suppress HER2/neu-dependent breast tumors PNAS, October 14, 2003; 100(21): 12337 - 12342. [Abstract] [Full Text] [PDF]
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 M. P. Goetz, D. O. Toft, M. M. Ames, and C. Erlichman The Hsp90 chaperone complex as a novel target for cancer therapy Ann. Onc., August 1, 2003; 14(8): 1169 - 1176. [Abstract] [Full Text] [PDF]
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S. T. Palayoor, P. J. Tofilon, and C. N. Coleman Ibuprofen-mediated Reduction of Hypoxia-inducible Factors HIF-1{alpha} and HIF-2{alpha} in Prostate Cancer Cells Clin. Cancer Res., August 1, 2003; 9(8): 3150 - 3157. [Abstract] [Full Text] [PDF]
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E. Metzen, J. Zhou, W. Jelkmann, J. Fandrey, and B. Brune Nitric Oxide Impairs Normoxic Degradation of HIF-1{alpha} by Inhibition of Prolyl Hydroxylases Mol. Biol. Cell, August 1, 2003; 14(8): 3470 - 3481. [Abstract] [Full Text] [PDF]
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S.-k. Park, A. M. Dadak, V. H. Haase, L. Fontana, A. J. Giaccia, and R. S. Johnson Hypoxia-Induced Gene Expression Occurs Solely through the Action of Hypoxia-Inducible Factor 1{alpha} (HIF-1{alpha}): Role of Cytoplasmic Trapping of HIF-2{alpha} Mol. Cell. Biol., July 15, 2003; 23(14): 4959 - 4971. [Abstract] [Full Text] [PDF]
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L. E. Huang and H. F. Bunn Hypoxia-inducible Factor and Its Biomedical Relevance J. Biol. Chem., May 23, 2003; 278(22): 19575 - 19578. [Full Text] [PDF]
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E. Metzen, U. Berchner-Pfannschmidt, P. Stengel, J. H. Marxsen, I. Stolze, M. Klinger, W. Q. Huang, C. Wotzlaw, T. Hellwig-Burgel, W. Jelkmann, et al. Intracellular localisation of human HIF-1{alpha} hydroxylases: implications for oxygen sensing J. Cell Sci., April 1, 2003; 116(7): 1319 - 1326. [Abstract] [Full Text] [PDF]
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S. J. Welsh, R. R. Williams, A. Birmingham, D. J. Newman, D. L. Kirkpatrick, and G. Powis The Thioredoxin Redox Inhibitors 1-Methylpropyl 2-Imidazolyl Disulfide and Pleurotin Inhibit Hypoxia-induced Factor 1{alpha} and Vascular Endothelial Growth Factor Formation Mol. Cancer Ther., March 1, 2003; 2(3): 235 - 243. [Abstract] [Full Text] [PDF]
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Y.-J. Jung, J. S. Isaacs, S. Lee, J. Trepel, and L. Neckers Microtubule Disruption Utilizes an NFkappa B-dependent Pathway to Stabilize HIF-1alpha Protein J. Biol. Chem., February 21, 2003; 278(9): 7445 - 7452. [Abstract] [Full Text] [PDF]
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L. E. Huang, E. A. Pete, M. Schau, J. Milligan, and J. Gu Leu-574 of HIF-1alpha Is Essential for the von Hippel-Lindau (VHL)-mediated Degradation Pathway J. Biol. Chem., October 25, 2002; 277(44): 41750 - 41755. [Abstract] [Full Text] [PDF]
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D. A. Chan, P. D. Sutphin, N. C. Denko, and A. J. Giaccia Role of Prolyl Hydroxylation in Oncogenically Stabilized Hypoxia-inducible Factor-1alpha J. Biol. Chem., October 11, 2002; 277(42): 40112 - 40117. [Abstract] [Full Text] [PDF]
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W. Xu, M. Marcu, X. Yuan, E. Mimnaugh, C. Patterson, and L. Neckers Chaperone-dependent E3 ubiquitin ligase CHIP mediates a degradative pathway for c-ErbB2/Neu PNAS, October 1, 2002; 99(20): 12847 - 12852. [Abstract] [Full Text] [PDF]
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M. D. Basson Gut Mucosal Healing : Is the Science Relevant? Am. J. Pathol., October 1, 2002; 161(4): 1101 - 1105. [Full Text] [PDF]
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 J.M. ARBEIT Quiescent Hypervascularity Mediated by Gain of HIF-1{alpha} Function Cold Spring Harb Symp Quant Biol, January 1, 2002; 67(0): 133 - 142. [Abstract] [PDF]
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