From the Institut de Pharmacologie Moléculaire et Cellulaire du CNRS, 660 route des Lucioles, Sophia-Antipolis, 06560 Valbonne, France
Received for publication, November 5, 2002, and in revised form, February 12, 2003
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ABSTRACT |
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The mechanism by which hypoxia induces gene
transcription involves the inhibition of hypoxia-inducible factor
(HIF)-1 Cells respond to low oxygen tensions by up-regulating the
expression of genes involved in angiogenesis (e.g.
vascular endothelial growth factor), erythropoiesis (e.g.
erythropoietin), and glycolysis. The transcriptional activation of
target genes is induced by a common transcription factor,
hypoxia-inducible factor-1
(HIF-1).1 HIF-1 was first
identified as a heterodimeric transactivator that recognizes a specific
DNA sequence, termed hypoxia-responsive element (HRE) in the
3'-untranslated region of the erythropoietin gene (1). HIF-1 consists
of two subunits, HIF-1 Cyclosporin A (CsA) is a potent immunosuppressive agent used after
organ transplantation and in the treatment of several autoimmune diseases. CsA is well known to be nephrotoxic probably as a consequence of the constriction of renal vessels and of the resulting hypoxia. Circumstantial evidence further suggests that CsA interferes with the
hypoxic signaling cascade. (i) Maruyama et al. (9) reported that CsA inhibited vascular endothelial growth factor production by
human lymphocytes. (ii) CsA inhibits erythropoietin production in
anemic mice (10). (iii) Kang et al. (11) reported that vascular endothelial growth factor reverses some of the
post-CsA-mediated hypertension and nephropathy in the rat. (iv)
Erythropoietin deficiency has been proposed as a cause of the anemia
observed in children following cardiac transplantation (12). These
would suggest that CsA inhibits the cellular responses that are
mediated by HIF-1 In the present study, we examine the possible influence of CsA on the
hypoxic signaling. Results show that CsA inhibits hypoxia-induced gene
transcription by preventing hypoxia-induced and
ODD-dependent stabilization of HIF-1 Materials--
Culture medium, restriction, and DNA-modifying
enzymes were from Promega. Culture media and fetal calf serum were
from Invitrogen. All chemicals were purchased from Sigma. Mouse
monoclonal antibodies were obtained from the following sources:
HIF-1 Cell Lines and Culture Conditions--
C6 glioma cells were
obtained from the American Type Culture Collection. Cells were grown in
Dulbecco's modified Eagle's medium supplemented with 10% fetal calf
serum, 2 mM L-glutamine, 100 units/ml
penicillin, and 100 µg/ml streptomycin. Cells were maintained at
37 °C in 5% CO2, 95% air in a humidified incubator.
Hypoxic incubations (2% O2) were performed using a
CO2 water incubator (Forma Scientific, Labtech, Model 3110).
Reporter Plasmid Constructs--
The pHRE4-Luc plasmid (13) was
kindly provided by Dr. Y Fujii-Kuriyama. It was originally described as
four tandemly repeated HRE motifs (5'-GATCGCCCTACGTGCTGTCTCA-3').
Sequence analysis indicated, however, that it formed a perfect
palindromic structure that consists of four tandemly repeated HRE
motifs followed by the complementary sequence inserted in the reverse orientation.
The pSV40-ODD-Luc vector containing the ODD domain of human
HIF-1
The pCMV-ODD-eGFP vector was prepared as follows. The same 222-bp
PCR-amplified fragment of HIF-1
The pCMV-eGFP vector was prepared as follows. The pCI-eGFP2 plasmid was
digested by EcoRI and NotI. The
EcoRI-eGFP-NotI fragment was purified and
inserted into the EcoRI/NotI-digested pCDNA
3.1 (+) vector. Sequences of all products were confirmed by DNA
sequencing using the dideoxy terminator method.
Transient Transfections and Luciferase Assays--
Cells were
transfected using LipofectAMINE (Invitrogen). They were incubated with
a mixture of 10 µg of plasmid DNA and 10 µg of LipofectAMINE
dissolved in 400 µl of OPTI-MEM for 5 h at 37 °C. After a
16-h incubation time, cells were dissociated and seeded into 12-well
tissue culture clusters. Eight h after seeding, cells were submitted to
hypoxic conditions in the presence or absence of CsA as indicated.
Luciferase assays (Promega) were performed according to the
manufacturer's protocol. Luciferase activity was normalized by the
total amount of cellular protein as assayed by the Bradford protein
assay (Bio-Rad). Stable GFP and ODD·GFP cells were selected using 1.5 mg/ml G418 and cloned by limited dilution.
Cell Lysis, Immunoblotting, and Immunoprecipitation--
Cells
were washed twice in ice-cold PBS and lysed at 4 °C in lysis buffer
(20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 5 mM MgCl2, 0.5% Nonidet P-40, 1 mM
sodium orthovanadate, 5 mM sodium fluoride, 50 µM leupeptin, 50 µg/ml aprotinin, and 1 mM
phenylmethylsulfonyl fluoride) for 20 min with continuous rocking and
then centrifuged at 12,000 × g for 10 min. The
supernatants were collected, and protein concentrations were determined
by the Bradford protein assay. Protein extracts were electrophoresed
and transferred to nitrocellulose membranes (Schleicher and Schuell)
using standard procedures. For immunoprecipitation, lysis was performed
in lysis buffer. After clearance by centrifugation, 250-µg aliquots
of cell lysate were incubated for 1 h at 4 °C with 5 µg of
anti-GFP monoclonal antibody followed by addition of protein
A-Sepharose beads (10 µl) and an overnight incubation.
Immunoprecipitates were washed four times with lysis buffer, eluted
with sample buffer, and immunoblotted. The membranes were blocked with
5% (w/v) instant non-fat milk powder in PBS, 0.1% Tween 20 for 1 h at room temperature followed by overnight incubation at 4 °C with
the primary monoclonal antibodies against HIF-1 HIF Peptide Hydroxylation Assay--
Rat kidneys were
homogenized at 4 °C in 250 mM sucrose, 50 mM
Tris-HCl (pH 7.5), 1 µM leupeptin, 1 µM
bacitracin, and 0.1 mM phenylmethylsulfonyl fluoride. The
homogenate was centrifuged at 1,000 × g for 10 min to
remove cellular debris and nuclei. The supernatant was recovered and
centrifuged at 3,000 × g for 10 min. The supernatant
was recovered and centrifuged at 18,000 × g for 10 min. The pellet, usually referred to as the light mitochondrial fraction, was suspended in 40 mM Tris-HCl buffer at pH 7.5. Kidney extracts (0.3 mg of protein/ml) were incubated at 37 °C for
30 min in 40 mM Tris-HCl (pH 7.5), 0.5 mM
dithiothreitol, 50 µM ammonium ferrous sulfate, 1 mM ascorbate, 2 mg/ml bovine serum albumin, 0.4 mg/ml
catalase, 50,000 dpm of [5-14C]2-OG, 0.1 mM
unlabeled 2-OG, and 100 µM of HIF peptide. HIF peptides used were: Pro-402 (DALTLLAPAAGDTIISLDFK) and Pro-564 (LDLEALAPYIPADDDFQLRS), where the bold letters indicate Pro-402 and Pro-564. They corresponded to sequences 395-413 and 557-576 of human HIF-1 Cloning, Mutagenesis, and Production of Glutathione
S-transferase (GST)·ODD Fusion Proteins--
The 222-bp DNA fragment
(nucleotides 1616-1837) of human HIF-1 GST Pull-down Assay--
Glutathione-purified GST·ODD and
GST·ODD P564A fusion proteins were in vitro hydroxylated
using a kidney homogenate prepared as described above. The reaction
products were incubated at 4 °C in 200 µl of buffer (50 mM Tris-HCl (pH 8), 120 mM NaCl, and 0.5%
Nonidet P-40) supplemented with glutathione-Sepharose beads and 50,000 dpm of 35S-labeled human vHL. After 2 h of incubation
time, beads were washed three times with cold buffer (20 mM
Tris-HCl (pH 8), 100 mM NaCl, 1 mM EDTA, 0.5%
Nonidet P-40). The bound proteins were eluted in SDS running buffer and
analyzed by SDS-PAGE followed by autoradiography.
[35S]vHL was synthesized by using pcDNA3.1/V5-His-vHL
as a template (a gift of Dr. S. L. McKnight) and the
TNT-coupled reticulocyte lysate system (Promega).
CsA Inhibits HRE-mediated Transcription and Prevents the Hypoxic
Stabilization of HIF-1 CsA Abrogates ODD-mediated Protein Stabilization--
The ODD
domain of HIF-1
GFP is well known to be highly stable in mammalian cells with an
estimated half-life of 24 h. Addition of an ODD domain to GFP was
expected to destabilize the protein. The stability of ODD·GFP was
determined after blocking protein synthesis with 100 µg/ml
cycloheximide. Fig. 2, B and C, shows that
ODD·GFP was degraded with a half-life of about 6 h. The
expression of the control GFP reporter was hardly affected after 8 h of exposure to cycloheximide. These results first confirmed the
stability of GFP. They also indicated that the ODD sequence
destabilized the GFP protein under normoxic conditions.
We next exposed the cells to hypoxia. Fig. 3A shows that
hypoxia increased the expression of ODD·GFP but not that of GFP. The
time course of hypoxia-induced protein expression is analyzed in Fig.
3B by using an ODD-luciferase reporter assay. Expression of
luciferase reached a maximum after 4-6 h of hypoxia. An identical result was obtained with an ODD·GFP reporter or endogenous
HIF-1
Fig. 4A shows that CsA
inhibited hypoxia-induced ODD·GFP protein stabilization. The
dose-response curve for the action of CsA is shown in Fig.
4B. CsA did not decrease expression of GFP (Fig.
4C). Thus, CsA required the presence of an ODD domain to be
active. Note that the extent of the inhibitory effects of CsA on
hypoxia-induced ODD·GFP fusion protein stabilization was comparable with those of HRE-mediated reporter gene transcription (Fig.
1B) and of hypoxia-induced HIF-1 CsA Activates in Vitro Prolyl Hydroxylase Activity--
The
oxygen-dependent destabilization of HIF-1
Experiments were first performed with extracts from normoxic cultured
cells. We observed a degradation of 2-OG by cell extracts, but this
activity was not stimulated by the HIF peptide (data not shown). It
represents undefined enzymatic activities that are unrelated to PHDs.
The absence of peptide-stimulated activity does not mean that PHDs were
not present. It could be that the specific activity of 2-OG was too low
to detect low levels of activity.
We then prepared homogenates from rat tissues and detected a large,
peptide-dependent activity in whole kidney homogenates. The
homogenate was fractionated by differential centrifugations. Activity
was recovered in a light mitochondrial fraction. The peptide-dependent activity did not require Fe(II) or
ascorbate. It required 2-OG. The dose-response curve for 2-OG-induced
activation of the enzyme is presented in Fig.
5A. Half-maximal activation was observed at 0.3 mM 2-OG. We also noticed that the
peptide-independent enzymatic activity was more sensitive to 2-OG. As a
consequence, large concentrations of 2-OG (0.3-1
mM) provide a better signal-to-noise ratio and should be
used to monitor activity with precision.
Fig. 5B documents the influence of different concentrations
of the Pro-564 peptide on the enzymatic activity. It also shows that
CsA increased the degradation of 2-OG. CsA did not modify the basal,
peptide-independent activity. The stimulating action of CsA was
dose-dependent (Fig. 5C). Two other
immunosuppressive drugs, FK506 and rapamycin, were inactive (data not shown).
We then performed experiments to demonstrate that 2-OG degradation was
indeed associated to hydroxylation of Pro-564 by substituting the
Pro-564 residue by a 4-hydroxyproline residue. This peptide was largely
devoided of activity. We generated GST·ODD and GST·ODD P564A fusion
proteins. GST·ODD promoted degradation of 2-OG to the same extent as
the Pro-564 peptide (data not shown). The reaction products were probed
with [35S]vHL. Fig. 5D shows that (i)
degradation of 2-OG was accompanied by an increased
[35S]vHL binding to ODD·GFP; (ii) CsA increased
[35S]vHL binding, as expected if it had stimulated PHD
activity; and (iii) [35S]vHL did not bind to GST·ODD
P564A. Taken together, these results strongly support the hypothesis
that CsA inhibited hypoxic responses by promoting hydroxylation of
Pro-564 in the ODD domain.
Another substrate for PHDs is Pro-402. It is located in the N-terminal
part of the ODD domain. The ODD domain used in the previous experiments
comprised the 530-603 sequence of HIF-1 CsA Activates in Vivo Prolyl Hydroxylase Activity--
The
previous results indicated that CsA increases in vitro PHD
activity when experiments were performed at ambient oxygen tensions. An
obvious hypothesis for the inhibitory action of CsA on
ODD-dependent protein stabilization is that CsA also
increased PHD activity under conditions of reduced oxygen supply. To
test this hypothesis, the ODD·GFP fusion protein was
immunoprecipitated from normoxic or hypoxic cell extracts, and the
bound, endogenous vHL was detected by Western blots using an anti-vHL
antibody. Fig. 5E shows that hypoxia-stabilized ODD·GFP
did not bind to vHL as expected since it was not hydroxylated. Addition
of CsA destabilized ODD·GFP and increased vHL association. This is a direct demonstration that CsA promoted hydroxylation of ODD·GFP by
activating Pro-564 PHD under hypoxic in vivo conditions.
The mechanism by which hypoxia induces gene transcription involves
the inhibition of HIF-1 This report also describes an assay for PHDs. It was based on the
capacity of peptides to stimulate the degradation of labeled 2-OG.
Results indicated the presence in kidney extracts of enzymatic activities that were stimulated by Pro-402 and Pro-564 peptides. Both
activities were enriched in light mitochondrial fractions and required
exogenous 2-OG. They did not require Fe(II) or ascorbate, probably
because cofactors were tightly bound to the enzymes. Based on our
results, we cannot conclude that different PHDs mediate Pro-402 and
Pro-564 hydroxylations. The same enzyme could mediate both reactions;
however, only Pro-564 hydroxylation is activated by CsA. Four different
PHDs are now identified (18, 19, 21). The identity of the enzyme(s)
present in our light mitochondrial kidney fractions is not known.
However, it is of interest to note that SM-20 contains a mitochondrial
targeting sequence (22) and that the newly described PHD-4 isoform is
highly expressed in kidney tissues and associates to light microsomes
(21).
CsA is a well known immunosuppressive drug that binds to cyclophilins,
a family of ubiquitous and conserved proteins with peptidyl-prolyl
cis-trans isomerase and molecular chaperone activities (23,
24). We now show, for the first time, that CsA specifically stimulates
Pro-564 hydroxylation. A hypothesis could be that hydroxylation of
Pro-564 requires a specific configuration of the proline residue and
that a cyclophilin controls in some way the presentation of the peptide
to the enzyme. This hypothesis is unlikely, however, since CsA should
not be active in hypoxic cells in which PHD activity is limited by
oxygen availability. Indeed, in agreement with this observation, we
were unable to show by immunoprecipitation experiments, using whole
cell and kidney protein extracts, a direct interaction of cyclophilins
(Cyp A and Cyp 40) with ODD substrates (GST·ODD, ODD·GFP, or
endogenous HIF-1 Rapamycin and FK506 recognize and bind to another class of
peptidyl-prolyl isomerases, termed FK-binding proteins (FKBP). Rapamycin, like CsA, inhibits cellular responses to hypoxic stresses by
destabilizing the ODD domain of HIF-1 CsA induces renal and systemic vasoconstrictions that are consecutive
to changes in the production of vasoactive substances, such as
endothelin-1 and nitric oxide. This study shows that CsA prevents
HRE-mediated hypoxic adaptation. It is important to note that such a
mechanism would exacerbate the toxicity of hypoxia that results from
the vasoconstriction and may well contribute to the severity of hypoxic
nephropathies. It is of interest to note that FK506, which is less
nephrotoxic than CsA, does not promote PHD activity in kidney microsomes.
In conclusion, this report shows that CsA increases
peptidyl-prolyl hydroxylase activity in kidney extracts and hypoxic
cells. By this mechanism, CsA abrogates hypoxic stabilization of
HIF-1 prolyl hydroxylase activity, which prevents von
Hippel-Lindau (vHL)-dependent targeting of HIF-1
to the
ubiquitin-proteasome pathway. HIF-1
is stabilized, translocates to
the nucleus, interacts with hypoxia-responsive elements, and promotes
the activation of target genes. This report shows that cyclosporin A
(CsA) interferes with the hypoxic signaling cascade in C6 glioma cells.
CsA inhibits hypoxia-dependent gene transcription in a
reporter gene assay and prevents the hypoxic accumulation of HIF-1
.
Addition of the 530-603 C-terminal oxygen-dependent degradation (ODD) domain of HIF-1
to the green fluorescent
protein (GFP) destabilized the protein in an
oxygen-dependent manner. CsA prevented the hypoxic
stabilization of an ODD·GFP fusion protein. An assay for
2-oxoglutarate-dependent dioxygenases was developed using a
light mitochondrial kidney fraction as a source of enzyme. It uses the
capacity of specific peptides to stimulate the degradation of
[14C]2-oxoglutarate. CsA stimulated the enzymatic
activity in the presence of a peptide that mimicked the 557-576
sequence of HIF-1
. The enzyme promoted [35S]vHL
binding to glutathione S-transferase (GST)·ODD fusion
protein. This association increased in the presence of CsA. CsA effects were not observed when the proline residue corresponding to Pro-564 in
the HIF-1
sequence was replaced by a hydroxyproline or an alanine
residue. Finally, CsA increased vHL-ODD interaction during hypoxia. We
conclude that CsA destabilizes HIF-1
by promoting hydroxylation of
Pro-564 in the ODD domain. Such a mechanism may prevent hypoxic
adaptation during CsA-induced nephrotoxicity and contribute to the
adverse effects of this drug.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and the aryl hydrocarbon receptor nuclear
translocator, both of which belong to the large family of basic
helix-loop-helix-per-arnt-sim transcription factors (2, 3).
Under normoxic conditions, HIF-1
subunits are unstable, being
rapidly targeted to the ubiquitin-proteasome pathway. Degradation is
mediated by a ubiquitin-protein isopeptide ligase (E3) complex, in
which the von Hippel-Lindau protein (vHL) binds to a specific
hydroxylated proline residue (Pro-564) within the
oxygen-dependent degradation (ODD) domain of HIF-1
(4, 5). A major action of hypoxia is to suppress prolyl hydroxylation and
degradation of HIF-1
by the proteasome. As a consequence, the
protein accumulates, migrates to the nucleus, and associates with the
aryl hydrocarbon receptor nuclear translocator, and the complex
interacts with the HRE of target genes (6, 7). The importance of this
mechanism is ascertained by the facts that vessel formation is hampered
in HIF-1
/
knock out mice (8) and that mutations in the
vHL gene cause hereditary cancer syndrome associated with
dysregulated angiogenesis and up-regulation of hypoxia-sensitive genes.
and HRE.
. We also show that
CsA stimulates a kidney prolyl hydroxylase activity that specifically
modifies Pro-564 in the ODD domain of HIF-1
.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(Novus Biologicals), GFP (Roche Molecular Biochemicals),
-tubulin (Sigma), vHL (Pharmingen). HIF peptides were synthesized by
Genaxis Biotechnology (Nîmes, France). Horseradish
peroxidase-coupled sheep anti-mouse antibodies were from Jackson
ImmunoResearch Laboratories. [5-14C]2-OG (2.07 GBq/mmol) and L-[35S]methionine (37 TBq/mmol)
were purchased from Amersham Biosciences.
(GenBankTM accession number U22431) fused in-frame
to the 5' end of the luciferase was prepared as follows. A 222-bp DNA
fragment corresponding to amino acids 530-603 (nucleotides 1616-1837)
of human HIF-1
(pcDNA3-HIF-1
vector kindly provided by Dr.
P. J. Ratcliffe) was PCR-amplified using the sense primer
5'-CCGCTCGAGGCCACCATGGAATTCAAGTTGGAATTGGTA-3', corresponding to
positions 1616-1636, and the reverse primer
5'-GCTCTAGACTGGAATACTGTAAC-3', corresponding to positions
1837-1823. The sequence includes the Pro-564 residue that is
hydroxylated by prolyl hydroxylases (PHDs) (4, 5). The PCR
product was digested by XbaI, blunted, and digested by
XhoI. The resulting fragment was inserted into a
XhoI/NcoI-blunted pSV40-Luc plasmid.
was digested by XhoI and XbaI and fused in-frame with the eGFP of a
XhoI/XbaI-digested pCI-eGFP2 plasmid (vector
kindly provided by Dr. F. Lesage). The generated product,
pCI-ODD-eGFP2, was then digested by BglII and NotI. The BglII-ODD-eGFP-NotI fragment
was finally inserted into the BglII/NotI-digested
pCDNA 3.1 (+) vector (Invitrogen).
(1:1,000), GFP
(1:1,000), or
-tubulin (1:5,000). Monoclonal antibodies against GFP
(1:1,000) or against vHL (1:2,000) were used to probe the
immunoprecipitates. Blots were washed three times in PBS, 0.1% Tween
20, and incubated for 1 h at room temperature with horseradish
peroxidase-conjugated sheep anti-mouse antibodies (1:10,000) in PBS,
0.1% Tween 20. After extensive washes in PBS, 0.1% Tween 20, chemiluminescence detection was performed by incubating the membranes
with 100 mM Tris-HCl (pH 8.5), 2.65 mM
H2O2, 0.45 mM luminol, and 0.625 mM coumaric acid for 1 min followed by exposure to x-ray films.
. Methionine residues were converted to
alanines to avoid spurious oxidation events. At the end of the
reaction, the radioactivity associated to succinate was determined as
described previously (14). The basal, peptide-independent, enzymatic
activity was determined in each experiment. It represented less than
50% of total activity and was subtracted from all counts.
(pcDNA3-HIF-1
) was
PCR-amplified with the forward 5'-GGAATTCAAGTTGGAATTGG-3' and reverse
5'-CCGCTCGAGCTGGAATACTGTAACTGT-3' set of primers. The P564A mutant was
generated following three rounds of PCR amplification. (i) The
1616-1741 fragment was PCR-amplified with the forward 5'-GGAATTCAAGTTGGAATTGG-3' and the reverse
5'-GTCATCATCCATTGGGATATAGGCAGCTAACATCTCCAAGTCTAA-3' set of primers.
(ii) The 1697-1837 fragment was amplified with the forward
5'-TTAGACTTGGAGATGTTAGCTGCCTATATCCCAATGGATGATGAC-3' and the
reverse 5'-CCGCTCGAGCTGGAATACTGTAACTGT-3' set of primers. (iii) The
final PCR amplification was initiated in the presence of an equimolar
ratio of the two PCR-purified products and followed by the addition of
the forward 5'-GGAATTCAAGTTGGAATTGG-3' and the reverse
5'-CCGCTCGAGCTGGAATACTGTAACTGT-3' primers. The ODD and the P564A ODD
fragments were purified, digested with EcoRI and
XhoI, and ligated into pGEX-4T-1 (Amersham Biosciences) to generate N-terminal GST-tagged fusion protein. Both plasmids were authenticated by DNA sequencing. The constructs (pGEX-GST·ODD and
pGEX-GST-P564A) were transformed into Escherichia coli
BL21(DE3), grown at 37 °C in LB media containing 100 µg/ml
ampicillin and induced with 0.1 mM isopropyl
-D-thiogalactoside for 4 h. Cells were harvested by
centrifugation at 4,000 × g for 15 min and pelleted. Cells were sonicated on ice in lysis buffer (20 mM Tris-HCl
(pH 7.5), 250 mM NaCl, 2 mM MgCl2,
1 mM dithiothreitol, 10% glycerol) supplemented with a
mixture of protease inhibitors (Roche Molecular Biochemicals). After
centrifugation at 100,000 × g for 30 min, the
clarified sonicates were incubated with glutathione-Sepharose 4B
(Amersham Biosciences) for 1 h at 4 °C. After four washes in PBS, the GST-tagged fusion proteins were eluted in 50 mM
Tris-HCl, 10 mM reduced glutathione (pH 8). The integrity
and yield of purified GST fusion proteins were assessed by SDS-PAGE
followed by Coomassie Blue staining.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
--
HRE-mediated transcriptional activation
was analyzed using the pHRE4-Luc construct. The kinetics of
transcriptional activation reveals that luciferase expression reached a
4-fold increase after 16 h of hypoxia (data not shown). Fig.
1A shows that hypoxia-induced luciferase expression was inhibited by CsA in a
dose-dependent manner. In contrast, CsA did not alter
reporter gene expression under normoxic conditions. Previous studies
have demonstrated that HIF-1
is rapidly degraded by
ubiquitin-proteasome pathway under normoxic conditions (15). Activation
of HIF-1
to a functional form requires protein stabilization.
Expression of HIF-1
was analyzed using Western blots. Fig.
1B shows that a 4-h hypoxia induced a large accumulation of
endogenous HIF-1
protein. Stabilization of HIF-1
was also
observed under hypoxia in the presence of the proteasomal inhibitor
MG132. CsA prevented most of the hypoxia-induced HIF-1
stabilization, but in contrast, it was inactive in proteasomally blocked hypoxic cells. These data thus indicated that the inhibitory action of CsA requires a functional proteasomal machinery.
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Fig. 1.
CsA inhibits HRE-mediated
transcriptional activation and prevents hypoxia-induced accumulation of
HIF-1 . A, dose-response curve
for the inhibitory action of CsA on hypoxic response. Cells were
transfected with pHRE4-Luc reporter plasmid and incubated under
normoxic or hypoxic conditions for 16 h in the presence or absence
of the indicated concentrations of CsA. This time of treatment
corresponds to the peak of hypoxia-induced luciferase expression.
Experiments were performed under hypoxic (filled symbols) or
normoxic (open symbols) conditions. Luciferase expression
was determined and normalized to protein content. Means ± S.E.
(n = 6) are indicated. Essentially identical results
were obtained in two other experiments. As shown in B, CsA
inhibits the hypoxic expression of HIF-1
. Cells were exposed to
normoxic or hypoxic conditions for 4 h in the presence of 10 µM CsA or 10 µM MG132 as indicated. Whole
cell extracts (80 µg of protein) were resolved by SDS-PAGE and
analyzed by immunoblotting using an anti-HIF-1
antibody. The
irrelevant cross-reacting band of higher molecular weight serves as
internal and loading control.
is responsible for the proteasomal degradation of
HIF-1
in normoxic cells. It has been located to the amino acid
residues 401-603 of human HIF-1
. This region overlaps with a vHL
binding domain (526-641) (16, 17) and comprises two essential proline
residues (Pro-402 and Pro-564) that are hydroxylated by PHDs under
normoxic conditions (4, 5). To gain further insight into the mechanism
of ODD-dependent protein stabilization, we generated
chimeric proteins in which the C-terminal part of the ODD region of
human HIF-1
(residues 530-603) was fused to the GFP or luciferase
open reading frames (Fig. 2A). Stable transfectants expressing either ODD·GFP or control GFP were
prepared. Expression of both proteins was analyzed by Western blots
using an anti-GFP antibody. As expected, the antibody recognized a
27-kDa protein in GFP-expressing cells and a larger, 35-40-kDa, protein in ODD·GFP-expressing cells (Fig.
3A).
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Fig. 2.
An ODD domain destabilized GFP.
A, schematic representations of HIF-1 , pCMV-ODD·GFP,
and pSV40-ODD-Luc constructs. Functional domains of HIF-1
are
indicated as follows: bHLH, basic-helix-loop-helix domain
(residues 17-70); PAS, per-arnt-sim homology
domain with internal A (residues 106-156) and B (residues 249-299)
repeats; TAD-N (residues 531-575) and TAD-C
(residues 786-826), N- and C-terminal transactivation domains;
ODD, oxygen-dependent degradation domain
(residues 401-603). CMV, cytomegalovirus. As shown in
B and C, stable GFP or ODD·GFP transfectants
were incubated in the presence of 100 µg/ml cycloheximide to block
protein synthesis. Expressions of GFP, ODD·GFP, or
-tubulin were
analyzed after different times using Western blots. B,
representative blots showing the degradation of ODD·GFP.
C, normalized ODD·GFP/
-tubulin ratio. Means of
triplicates are shown. Essentially identical results were obtained in
four independent experiments.
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Fig. 3.
Hypoxia stabilizes ODD·GFP and
ODD-Luciferase fusion proteins. As shown in A, hypoxia
stabilizes ODD·GFP but not GFP. ODD·GFP and GFP cells were
incubated under normoxic or hypoxic conditions. Whole cell extracts (20 µg of protein) were resolved by SDS-PAGE and immunoblotted with
anti-GFP (top) or anti -tubulin (bottom)
antibodies. B, time course of ODD-induced protein
stabilization. Cells were transfected with a pSV40-ODD-Luc reporter
plasmid. 24 h after transfection, cells were exposed to 2%
O2 (closed circles) or maintained at 21%
O2 (open circles) for the time period indicated.
Results show normalized luciferase activities (Means ± S.E.,
n = 6) and are representative of three independent
experiments.
.
protein expression (Fig.
2B). All together, these observations indicated that
CsA prevented the ODD-dependent stabilization of HIF-1
in hypoxic cells.
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Fig. 4.
CsA prevents the hypoxic stabilization of
ODD·GFP fusion protein. ODD·GFP (A and
B) and GFP (C) cells were incubated under
normoxic or hypoxic conditions in the presence or absence of the
indicated concentrations of CsA. Whole cell extracts (20 µg of
protein) were resolved by SDS-PAGE and immunoblotted with anti-GFP
(A) or anti -tubulin (C) antibodies.
A, CsA inhibits hypoxia-induced stabilization of ODD·GFP.
B, dose-response curve for the action of CsA using
normalized ODD·GFP/
-tubulin signal ratios. C, GFP
expression is insensitive to CsA. Results shown are representative of
3-5 independent experiments.
is governed by a
novel family of PHDs that specifically modify HIF-1
at two conserved
proline residues (Pro-402 and Pro-564) located in the ODD domain of
HIF-1
. One hypothesis for the previous results could be that CsA
increased activity of PHDs. We therefore developed an assay for PHDs.
Prolyl hydroxylations are mediated by 2-OG-dependent dioxygenases (18, 19) and can be followed by measuring the conversion
of [5-14C]2-OG into [14C]succinate.
Experiments were performed in the absence or presence of a HIF peptide
that reproduced the 557-576 sequence of the ODD domain and that
included Pro-564.
View larger version (31K):
[in a new window]
Fig. 5.
CsA activates Pro-564 hydroxylase. A
kidney light mitochondrial fraction was used as a source of enzyme.
Activity was defined as the Pro-564 peptide-dependent
degradation of 2-OG. The peptide-independent activity was determined in
each experiment and was substrated from the data. Means ± S.E.
from triplicate measurements are shown. A, 2-OG dependence.
Experiments were performed in the presence of 100 µM
Pro-564 peptide. B, dependence on Pro-564 peptide.
Experiments were performed in the absence ( ) or the presence (
)
of 10 µM CsA. C, dose-response curve for the
stimulating action of CsA. Experiments were performed in the presence
of 50 µM Pro-564 peptide. D, vHL binding.
GST·ODD (170 µg) was incubated for 30 min at 37 °C in the
presence of the kidney extract and cofactors. Glutathione-Sepharose
beads and 50,000 cpm of [35S]vHL were then added. The
bound vHL was recovered after 2 h at 4 °C, subjected to 15%
SDS-PAGE, and visualized by phosphorimaging. The left lane
shows a band from directly loaded [35S]vHL. E,
in vivo hydroxylation of ODD·GFP. ODD·GFP-expressing
cells were incubated under normoxia or hypoxia in the presence or
absence of 10 µM CsA. ODD·GFP was immunoprecipitated by
using an anti-GFP monoclonal antibody, resolved by SDS-PAGE, and probed
with an anti-vHL (upper panel) or an anti-GFP (lower
panel) monoclonal antibodies. Note than in this experiment, the
expression of ODD·GFP was stabilized under hypoxia and reversed by
addition of CsA. Results shown are representative of two other
independent experiments.
and did not include
Pro-402. We therefore synthesized a peptide that mimicked the 395-413
sequence of HIF-1
and contains the Pro-402 residue. This peptide
stimulated the degradation of 2-OG to the same extent as the Pro-564
peptide. Although we did not demonstrate hydroxylation of Pro-402, it
would indicate that a light mitochondrial kidney fraction supported
hydroxylations of both Pro-564 and Pro-402 peptides. Yet the
Pro-402-dependent 2-OG degradation was insensitive to CsA.
All together, our data suggest that CsA specifically promoted
hydroxylation of Pro-564. Finally we tested
poly(L-Pro-Gly-L-Pro), a well known substrate
of procollagen prolyl hydroxylase(s). It was inactive in this assay.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
prolyl hydroxylase activity, which prevents
vHL-dependent targeting of HIF-1
to the
ubiquitin-proteasome pathway. HIF-1
is stabilized, associates with
the aryl hydrocarbon receptor nuclear translocator, and interacts with
HRE to promote the activation of target genes (see the Introduction).
This study demonstrates that CsA inhibits the hypoxic signaling pathway
by activating Pro-564 hydroxylation. (i) CsA prevents
HRE-dependent gene expression in a
dose-dependent manner. (ii) CsA blocks hypoxia-induced HIF-1
protein accumulation, and this blockade is overcome by the
proteasomal inhibitor MG132. (iii) CsA inhibits hypoxia-induced stabilization of an ODD·GFP chimeric protein, indicating that its
action requires the C-terminal part of ODD. This sequence comprises the
Pro-564 residue that is hydroxylated by PHDs and that is recognized by
vHL (4, 5). (iv) CsA increases a kidney PHD activity that selectively
hydroxylates Pro-564 in the ODD domain. (v) Finally, we present
experimental evidence that CsA increases PHD activity in hypoxic cells.
Inhibitors of PHDs have been developed (20) to promote hypoxic
responses. CsA is the first known activator of a PHD activity. The
major consequence of this stimulating action is an inhibition of
hypoxic responses.
). Another hypothesis could be that a cyclophilin
associates to one PHD and controls its oxygen sensitivity. This
hypothesis would agree with the recent evidence suggesting the
existence of more than one oxygen sensor (18).
(25). Yet the molecular mechanisms involved are different. CsA promotes hydroxylation of the
Pro-564 peptides. Rapamycin and FK506 do not. Hudson et al.
(25) further provided evidence that rapamycin acts via mammalian target of rapamycin, a downstream target of the phosphatidylinositol 3-kinase. All together, these findings further support the idea that
HIF-1
protein stabilization can be achieved by
PHD-dependent and -independent mechanisms (26).
and HIF-1
-mediated cellular responses in glioma cells. CsA
can thus be considered as an oxygen sensitizer that limits adaptative responses to hypoxia.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Dr. S. L. McKnight
for the pcDNA3.1/V5-His-vHL vector, to Dr. P. J. Ratcliffe for
the pcDNA3-HIF-1 vector, to Dr. Y. Fujii-Kuriyama for the pHRE4-Luc
plasmid, and to Dr. F. Lesage for pCI-eGFP2 vector. We thank F. Aguila,
N. Boyer, N. Leroudier, and J. Kervella for technical assistance.
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FOOTNOTES |
---|
* This work was supported by the CNRS (UMR 6097), Association pour la Recherche sur le Cancer, Ligue Nationale contre le Cancer, the Fondation pour la Recherche Médicale, and the Fondation de France.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.
These authors contributed equally to this work.
§ To whom correspondence should be addressed. Tel.: 33-4-93-95-77-51; Fax: 33-4-93-95-77-08; E-mail: dangelo@ipmc.cnrs.fr.
Published, JBC Papers in Press, February 13, 2003, DOI 10.1074/jbc.M211293200
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ABBREVIATIONS |
---|
The abbreviations used are: HIF, hypoxia-inducible factor; 2-OG, 2-oxoglutarate; CsA, cyclosporin A; GFP, green fluorescent protein; GST, glutathione S-transferase; HRE, hypoxia-responsive elements; ODD, oxygen-dependent degradation; PHD, prolyl hydroxylase; vHL, von Hippel-Lindau; Luc, luciferase; PBS, phosphate-buffered saline.
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