Molecular Biology of the Cell
Vol. 19, 86–94, January 2008
Hypoxia-inducible Factor-1? Stabilization in Nonhypoxic
Conditions: Role of Oxidation and Intracellular Ascorbate
Elisabeth L. Page ´,* Denise A. Chan,†Amato J. Giaccia,†Mark Levine,‡
and Darren E. Richard*
*Centre de recherche de L’Ho ˆtel-Dieu de Que ´bec, Department of Medicine, Universite ´ Laval, Que ´bec, QC,
G1R 2J6, Canada;†Center for Clinical Science Research, Department of Radiation Oncology, Stanford
University, Stanford, CA 94305; and‡Molecular and Clinical Nutrition Section, National Institute of Diabetes
and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892
Submitted June 27, 2007; Revised September 18, 2007; Accepted October 10, 2007
Monitoring Editor: J. Silvio Gutkind
Hypoxia-inducible factor-1 (HIF-1) is a decisive element for the transcriptional regulation of many genes induced under
low oxygen conditions. Under normal oxygen conditions, HIF-1?, the active subunit of HIF-1, is hydroxylated on proline
residues by specific HIF prolyl-hydroxylases, leading to ubiquitination and degradation by the proteasome. In hypoxia,
hydroxylation and ubiquitination are blocked and HIF-1? accumulates in cells. Recent studies have shown that in normal
oxygen conditions G-protein–coupled receptor agonists, including angiotensin (Ang) II and thrombin, potently induce
and activate HIF-1 in vascular smooth muscle cells. The current study identifies HIF-1? protein stabilization as a key
mechanism for HIF-1 induction by Ang II. We show that hydroxylation on proline 402 is altered by Ang II, decreasing
pVHL binding to HIF-1? and allowing HIF-1? protein to escape subsequent ubiquitination and degradation mechanisms.
We show that HIF-1? stability is mediated through the Ang II–mediated generation of hydrogen peroxide and a
subsequent decrease in ascorbate levels, leading to decreased HIF prolyl-hydroxylase activity and HIF-1? stabilization.
These findings identify novel and intricate signaling mechanisms involved in HIF-1 complex activation and will lead to
the elucidation of the importance of HIF-1 in different Ang II–related cell responses.
Oxygen is an essential element in the biology of every aer-
obic organism. Tissue and cellular regulation of oxygen
supply is essential to mediate adaptation mechanisms dur-
ing low oxygen conditions. At the cellular level, the hypoxia-
inducible transcription factor, HIF-1, is a key regulator of
responses in low oxygen conditions. HIF-1 specifically binds
hypoxic response element (HRE)-driven promoters on a num-
ber of genes that include vascular endothelial growth factor
(VEGF), heme oxygenase, glucose transporter-1, and erythro-
poietin (Semenza et al., 1991; Semenza and Wang, 1992; Lee et
al., 1997). HIF-1 is a heterodimeric complex composed of
HIF-1? and HIF-1?. HIF-1? is found in all cells, whereas
HIF-1? is the oxygen-regulated subunit (Wang et al., 1995).
HIF-1? is highly unstable and the mechanisms controlling
HIF-1? degradation in normoxic conditions have been well
described (Schofield and Ratcliffe, 2005). Human HIF-1? is
hydroxylated on two proline residues; Pro402and Pro564.
Both residues are situated in the oxygen-dependent degra-
dation domain (ODDD) of HIF-1?. Three different HIF
prolyl-hydroxylases have been described, termed PHD1,
PHD2, and PHD3 for their prolyl hydroxylase domain
(Bruick and McKnight, 2001; Epstein et al., 2001). PHD en-
zymes hydroxylate HIF-1? using oxygen and 2-oxoglutarate
as substrates and iron and ascorbate as essential cofactors.
Although all three PHDs have been shown to regulate HIF-
1?, the key isoform responsible for HIF-1? regulation in
many cell types is PHD2 (Berra et al., 2003; Appelhoff et al.,
2004). It has been shown that PHD1 and PHD3 also hydroxy-
late HIF-1? in vivo and in vitro (Epstein et al., 2001; Appelhoff
et al., 2004). However, the exact relevance of these two isoforms
in cells remains to be elucidated.
HIF-1? hydroxylation allows the binding of pVHL, the
product of the von Hippel-Lindau tumor suppressor gene.
pVHL is the recognition component of a E3 ligase complex
necessary for ubiquitination and subsequent proteasome-
dependent degradation of HIF-1? (Huang et al., 1998; Maxwell
et al., 1999; Cockman et al., 2000; Ivan et al., 2001; Jaakkola et
al., 2001). In hypoxic conditions, low oxygen leads to HIF-1?
stabilization due to the inhibition of proline hydroxylation and
subsequent decreases in HIF-1? ubiquitination and degrada-
tion. HIF-1? is stabilized and forms, in combination with the
HIF-1? subunit, the active HIF-1 complex.
HIF-1 regulation by nonhypoxic stimuli has gained con-
siderable interest (Feldser et al., 1999; Hellwig-Burgel et al.,
1999; Richard et al., 2000; Gorlach et al., 2001; Laughner et al.,
This article was published online ahead of print in MBC in Press
on October 17, 2007.
Address correspondence to: Darren E. Richard (darren.richard@crhdq.
Abbreviations used: Ang II, angiotensin II; HIF, hypoxia-inducible
factor; HRE, hypoxic response element; GPCR, G-protein–coupled
receptor; ODDD, oxygen-dependent degradation domain; PHD,
HIF prolyl-hydroxylases; pVHL, von Hippel-Lindau protein; ROS,
reactive oxygen species; VEGF, vascular endothelial growth factor;
VSMCs, vascular smooth muscle cells.
86© 2007 by The American Society for Cell Biology
2001). In vascular smooth muscle cells (VSMCs), angiotensin
(Ang) II and thrombin are strong inducers of HIF-1? and
potent activators of HIF-1 (Richard et al., 2000; Gorlach et al.,
2001; Page et al., 2002; Lauzier et al., 2007). Interestingly,
these two stimuli induce HIF-1? in VSMCs through mark-
edly different pathways than hypoxic induction (Page et al.,
2002; BelAiba et al., 2004; Bonello et al., 2007; Lauzier et al.,
2007). Ang II activates at least two separate pathways to
induce HIF-1? protein levels in VSMCs under normoxic
conditions. First, activation of diacylglycerol-sensitive protein
kinase C (PKC) plays an important part in increasing the tran-
scription of HIF-1?. Second, Ang II increases HIF-1? transla-
tion by a reactive oxygen species (ROS)-dependent activa-
tion of the phosphatidylinositol 3-kinase (PI3K)/p70S6
kinase (p70S6K) pathway and through the 5?-untranslated
region (5?-UTR) of HIF-1? mRNA (Page et al., 2002). How-
ever, in these normoxic conditions, the HIF-1? protein deg-
radation system should be at its full capacity. This is not the
case because the induction and activation of HIF-1? during
Ang II treatment is fully comparable to that seen under
hypoxic conditions (Richard et al., 2000). Therefore, the goal
of present study was to investigate the possibility that the
treatment of VSMCs with Ang II may also regulate HIF-1?
Here, we demonstrate that HIF-1? stabilization after Ang
II treatment is similar to that seen in hypoxic conditions. We
show that the stimulation of VSMCs with Ang II stabilizes
HIF-1? through its ODDD and regulates HIF-1? hydroxyla-
tion, leading to changes in HIF-1? ubiquitination and pro-
teasome targeting. Our studies indicate that Ang II regulates
PHD enzyme activity through the modulation of intracellu-
lar ascorbate concentrations, reducing the availability of this
cofactor. Finally, we show that Ang II–induced hydrogen
peroxide (H2O2) production participates in decreasing intra-
cellular ascorbate levels and promotes HIF-1? stability.
These results identify intricate signaling mechanisms in-
volved in HIF-1? protein regulation after the activation of
VSMCs through G-protein–coupled receptors.
MATERIALS AND METHODS
Ang II, thrombin, ascorbate, H2O2and catalase were from Sigma (St. Louis,
MO). MG132 was from Calbiochem (La Jolla, CA). GST-S5a-agarose was from
BIOMOL (Plymouth Meeting, PA). Easytag express protein labeling mix was
from Perkin Elmer (Boston, MA). Anti-HIF-1 antiserum was raised by our
laboratory in rabbits immunized against the last 20 amino acid of the C
termini of human HIF-1? (Richard et al., 1999). Anti-hydroxylated HIF-1?
against hydroxylated Pro402and hydroxylated Pro564of the human sequence
of HIF-1? were obtained as previously described (Chan et al., 2002, 2005). Mono-
clonal anti-phospho-p42/p44 MAPK, anti-tubulin, and the monoclonal anti-?-
actin antibodies were from Sigma. Total polyclonal p42/p44 MAPK antibody
was from Upstate (Lake Placid, NY). Anti-phospho-p70S6K (Thr389) antibody
was from Cell Signaling (Beverly, MA). Anti-GST antibody was from Novus
Biologicals (Littleton, CO). Monoclonal HA.11 antibody was from Convance
(Emeryville, CA). Anti-ubiquitin was from Boston Biochem (Cambridge,
MA). Horseradish peroxidase–coupled anti-mouse and anti-rabbit antibodies
were from Promega (Madison, WI). Glutathione S-transferase (GST)-HIF-1?,
pVHL-hemagglutinin (HA), and luc-HIF-1?-ODDD constructs were kindly
provided by Drs. Jacques Pouysse ´gur (Institute of Signaling, Developmental
Biology and Cancer Research, Universite ´ de Nice), Peter Ratcliffe (University
of Oxford), and Richard K. Bruick (University of Texas), respectively.
VSMCs were isolated from the thoracic aorta of 6-wk-old male Wistar rats by
enzymatic dissociation (Owens et al., 1986). Cells were cultured in Dulbecco’s
modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS),
50 U/ml penicillin, and 50 U/ml streptomycin (Invitrogen, Carlsbad, CA) in
a humid atmosphere (5% CO2, 95% air). Cells were passaged upon reaching
confluence, and all experiments were performed between passages 4 and 12.
Cells were serum-deprived for 16 h before treatment. Hypoxic conditions
were obtained by placing cells in a sealed hypoxic workstation (Ruskinn,
Bridgend, United Kingdom). The oxygen level in this workstation was main-
tained at 1%, with the residual gas mixture containing 94% nitrogen and 5%
Western Blot Analysis
Confluent cells were lysed in 2? Laemmli sample buffer. Protein concentra-
tion was determined by Lowry assay. Samples were resolved in SDS-poly-
acrylamide gels and electrophoretically transferred onto polyvinylidene di-
fluoride membranes (Immobilon-P, Millipore, Bedford, MA). Proteins were
revealed with specific antibodies as indicated and visualized with an en-
hanced chemiluminescence (ECL) system (GE Healthcare Life Sciences, Pis-
cataway, NJ) or with the Odyssey Infrared Imaging System (LI-COR, Lincoln,
NE). Western blots were quantified using Odyssey quantification software or
Scion Image (Frederick, MD; http://www.scioncorp.com).
Northern Blot Analysis
Confluent cells were lysed and RNA was isolated with TRIzol reagent (Invitro-
gen). RNA resolved on 1% agarose/6% formaldehyde gels was transferred to
Hybond N? nylon membranes (GE Healthcare Life Sciences) and hybridized
with a radioactive cDNA probe against the total coding sequence of the mouse
VEGF gene. A probe against 18S rRNA was used as a loading control.
VSMCs, seeded in six-well plates, were transfected with the cytomegalovirus
(CMV)-luc-HIF-1?-ODDD luciferase reporter vector (1 ?g/well). Renilla reni-
formis luciferase expression vector (250 ng/well) was also used as a control for
transfection efficiency. Transfection was performed on 45% confluent cells
with Superfect transfection reagent (Qiagen, Valencia, CA) at a 1:3 DNA/
reagent ratio. Six hours after transfection, fresh medium was added to cells.
Forty-eight hours after transfection, cells were deprived of FBS for 16 h and
stimulated as indicated for 6 h. Cells were washed with cold phosphate-
buffered saline, and luciferase assays were performed with the Dual-Lucif-
erase Reporter Assay System (Promega). Results were quantified with a
Luminoskan Ascent microplate reader with integrated injectors (Thermo
Electron, Milford, CA). Results are expressed as a ratio of firefly luciferase
activity to Renilla reniformis luciferase activity. Experiments are an average ?
SEM of triplicate data representative of three independent experiments per-
formed on different cell cultures.
VSMCs were grown to confluence, serum-deprived for 16 h, and stimulated
as indicated for 4 h. Cells were then washed with phosphate-buffered saline
(PBS) and lysed in lysis buffer [20 mM Tris, pH 7.5, 5% glycerol, 0.1% Triton
X-100, 2 ?g/ml leupeptin, 2 ?g/ml aprotinin, 1 ?g/ml pepstatin, 1 mM
4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF)]. Lysates
were centrifuged (20,000 ? g) for 15 min at 4°C. VSMC extracts (equal
amounts of HIF-1? protein) were incubated with 10 ?g of GST-S5a-agarose
for 2 h at 4°C in S5a buffer (20 mM Tris, 140 mM NaCl, 5% glycerol, 2 ?g/ml
leupeptin, 2 ?g/ml aprotinin, 1 ?g/ml pepstatin, 1 mM AEBSF). Samples
were washed with S5a buffer containing 0,1% Triton and denatured in 2?
Laemmli sample buffer. Samples were resolved in SDS-polyacrylamide gels
(6%) and revealed by Western blot analysis.
In Vitro Ubiquitination Assay
VSMCs were grown to confluence, serum-deprived for 16 h, and stimulated
as indicated for 4 h. Cells were washed once in PBS and twice in HEB buffer
(20 mM Tris, pH 7.5, 5 mM KCl, 1.5 mM MgCl2, 1 mM dithiothreitol). Cells
were then suspended and lysed using a Dounce homogenizer. Cytoplasmic
extracts were cleared by centrifugation (20,000 ? g).35S-radiolabeled HIF-1?,
translated in vitro using the TnT Coupled Reticulocyte Lysate System (Pro-
mega), was incubated for the times indicated with 100 ?g of cytoplasmic
extracts in 0.5 mg/ml ubiquitin, 15 ?M ubiquitin aldehyde, 2 mM Tris, pH 7.5,
1 mM ATP, 1 mM magnesium acetate, 30 mM phosphocreatine, and 0.05
mg/ml creatine phosphokinase. Samples were denatured in 2? Laemmli
sample buffer and resolved in SDS-polyacrylamide gels (6%). Gels were dried
and exposed to Kodak BioMax MS film (Eastman Kodak, Rochester, NY).
pVHL Capture Assay
VSMCs were grown to confluence, serum-deprived for 16 h and stimulated as
indicated for 4 h. Cytoplasmic extracts (250 ?g), prepared as described above,
were incubated with Sepharose-bound GST-HIF-1? (30 ?g) for 1 h at room
temperature, washed with NETN buffer (150 mM NaCl, 0.5 mM EDTA, 20
mM Tris, pH 8.0, 0.5% Igepal, 100 ?M desferioxamine) and incubated over-
night with in vitro–translated pVHL-HA in NETN at 4°C. Samples were
washed with NETN, denatured in 2? Laemmli sample buffer, resolved in
SDS-polyacrylamide gels (12%), and revealed by Western blotting with
anti-HA and anti-GST antibodies.
HIF-1 Stabilization by Ang II
Vol. 19, January 200887
Determination of Intracellular Ascorbate Concentrations
VSMCs were grown to confluence in 100-mm plates in DMEM supplemented
with ascorbate (asDMEM, 250 ?M). Cells were serum-deprived for 16 h, and
fresh asDMEM was added for 1 h before stimulation for 4 h in DMEM. Cells
were then washed in PBS, harvested, centrifuged, and resuspended in a 90%
methanol/1 mM EDTA solution. Samples were vigorously vortexed and
centrifuged at 20,000 ? g. Ascorbate levels were analyzed by spectrophotom-
etry as previously described (Queval and Noctor, 2007). Briefly, samples were
mixed with 0.1 mM NaH2PO4, pH 5.6, and absorbance at 265 nm was
measured by spectrophotometry. Ascorbate peroxidase (0.4 U) was then
added to the samples for 3 min, and absorbance at 265 nm was again
measured. Ascorbate concentrations were determined as the difference in
absorbance prior and after the addition of ascorbate peroxidase. Alternatively,
ascorbate levels were measured by HPLC as previously described (Levine et al.,
2001). A Lowry protein assay was used for normalization of the samples.
Ang II Regulates HIF-1? Protein Degradation
Past work showed that Ang II induced HIF-1? in VSMCs
through transcriptional and translational mechanisms (Page
et al., 2002). Given the elevated levels of HIF-1? protein
induction during Ang II treatment and the high instability of
HIF-1? under normal oxygenation, we undertook studies to
determine if Ang II treatment stabilized HIF-1? protein lev-
els in VSMCs. The half-life of HIF-1? protein was evaluated
using cycloheximide, a general protein synthesis inhibitor.
VSMCs, stimulated with Ang II or hypoxia for 4 h, were
treated with cycloheximide to block all novel HIF-1? protein
synthesis. Half-life of HIF-1? was then evaluated by West-
ern blotting. As seen in Figure 1, the half-life of HIF-1?
protein in Ang II–treated cells is similar to the half-life of
HIF-1? protein under hypoxic conditions. These results in-
dicate that Ang II stimulation of VSMCs causes the stabili-
zation of HIF-1? protein levels.
In normoxic conditions, HIF-1? is rapidly ubiquitinated
and targeted for proteosomal degradation. We attempted to
determine whether Ang II could affect ubiquitination and
proteosomal targeting. To address this, we studied the
HIF-1? ubiquitination potential of VSMC extracts. These
experiments were performed by incubating cytoplasmic
VSMC extracts with
HIF-1? protein. Ubiquitinated HIF-1? was then resolved on
SDS-polyacrylamide gels. As shown in Figure 2A, the ap-
pearance of high-molecular bands denotes the ubiquitina-
tion state of HIF-1?. Interestingly, cell extracts from Ang
II–treated cells have a weaker capacity to ubiquitinate
HIF-1? than unstimulated VSMCs. This is particularly evi-
denced by the weaker intensity of high-molecular bands at
35S-radiolabeled in vitro–translated
all time points during the assay. This result suggests that
HIF-1? ubiquitination is decreased in VSMCs after Ang II
treatment. To demonstrate the effect of Ang II on endoge-
nous HIF-1? ubiquitination, we performed GST pulldown
assays using a GST-S5a fusion protein. The S5a protein is a
non-ATPase subunit of the 26S proteasome that binds polyu-
biquitinated proteins with a high affinity and its utilization
for the purification of polyubiquitinated proteins has been
well described (Layfield et al., 2001; Groulx and Lee, 2002;
Heessen et al., 2002). Total VSMC extracts (equal amounts of
HIF-1? protein) were incubated with GST-S5a protein cou-
VSMCs were maintained under control conditions, in the presence
of Ang II (100 nM) or in hypoxic conditions (1% O2) for 4 h.
Cycloheximide (CHX, 30 ?g/ml) was then added for the indicated
times. Total cell extracts (25 ?g) were resolved by SDS-PAGE (8%)
and immunoblotted using anti-HIF-1? and anti-tubulin antibodies.
Ang II modifies HIF-1? protein half-life. Quiescent
escent VSMCs were maintained under control conditions or in the
presence of Ang II (100 nM) for 4 h. Cytoplasmic extracts were
prepared and incubated with in vitro–translated and35S-radiola-
beled HIF-1? for in vitro ubiquitination (see Materials and Methods).
Samples were then resolved by SDS-PAGE (6%). Gels were dried
and exposed for35S autoradiography. (B) Total cell extracts (1 mg)
maintained in control conditions, in hypoxic conditions (Hyp, 1%
O2), or in the presence of Ang II (100 nM), thrombin (Thr, 5 U/ml),
or MG132 (20 ?M) for 4 h were incubated with 10 ?g GST-S5a-
agarose protein for 2 h. Alternatively, GST-S5a-agarose protein was
incubated without cell extracts (NE). Samples were resolved by
SDS-PAGE (6%) and immunoblotted with anti-HIF-1?, anti-ubiq-
uitin, and anti-GST antibodies.
Ang II modifies HIF-1? protein ubiquitination. (A) Qui-
E. L. Page ´ et al.
Molecular Biology of the Cell 88
pled to agarose beads. The level of bound HIF-1? was then
evaluated by Western blotting. As seen in Figure 2B, extracts
treated with the proteasome inhibitor MG132 demonstrated
increased HIF-1? protein binding to S5a. As expected, when
cells were treated in hypoxic conditions, HIF-1? binding to
S5a was blocked, a hallmark of decreased HIF-1? ubiquiti-
nation in these conditions. More interestingly, VSMCs
treated with Ang II also blocked HIF-1? binding to S5a.
Taken together, these results show that HIF-1? ubiquitina-
tion is decreased after the treatment of VSMCs with Ang II
and suggest that HIF-1? stabilization is the result of an
impairment of HIF-1? ubiquitination. It is important to note
that thrombin, another nonhypoxic activator of HIF-1
(Richard et al., 2000; Gorlach et al., 2001), also blocks HIF-1?
proteasome binding and ubiquitination (Figure 2B and Sup-
plementary Figure S1) through pathways similar to those
that will be described below for Ang II.
Ang II Regulates HIF-1? Protein Hydroxylation
We next attempted to determine the effect of Ang II on
upstream signaling events leading to HIF-1? ubiquitination.
As mentioned previously, HIF-1? ubiquitination and degra-
dation are controlled through its ODDD. To determine
whether Ang II could control the stability of the ODDD, we
utilized a fusion protein construct comprised of amino acids
401-602 of the HIF-1? ODDD and the C-terminal end of the
firefly luciferase protein. This construct generates an unsta-
ble form of luciferase when transfected into cells. The half-
life of this luciferase construct is increased by oxygen depri-
vation and can be quantified by traditional luciferase assays
(Salnikow et al., 2004). VSMCs were transiently transfected
with the CMV-luc-HIF-1?-ODDD vector. As expected, treat-
ment of cells with MG132 or hypoxia increased luciferase
activity in VSMCs by 3.3- and 2.0-fold over basal levels,
respectively (Figure 3). More interestingly, the stimulation
of VSMCs with Ang II also increased luciferase activity by
1.9-fold. This result demonstrates that Ang II targets the
HIF-1? ODDD to increase HIF-1? protein stabilization.
HIF-1? ubiquitination and degradation are controlled by
the hydroxylation of two specific proline residues (Pro402
and Pro564for human HIF-1?) contained in the ODDD.
PHD2 is the prolyl-hydroxylase shown to be the key regu-
lator of HIF-1? protein levels in many cell types (Berra et al.,
2003). The expression of PHD2 has been shown to be
decreased by TGF-?1, a nonhypoxic inducer of HIF-1?
(McMahon et al., 2006). However, in VSMCs we did not
observe any decrease in PHD2 protein expression after Ang
II treatment (Figure 4A), demonstrating that HIF-1? protein
stabilization by Ang II is not a consequence of decreased
To investigate the possibility that the treatment with Ang
II could regulate hydroxylase activity, we evaluated the
level of HIF-1? hydroxylation using two specific antibodies
fected with 1 ?g of CMV-luc-HIF-1?-ODDD encoding a luc-HIF-1?-
ODDD fusion protein and 250 ng of an expression vector encoding
Renilla reniformis luciferase. Six hours after transfection, cells were
serum-deprived for 16 h. Cells were maintained under control con-
ditions or stimulated with Ang II (100 nM) or MG132 (20 ?M) for
6 h. VSMCs were lysed and luciferase activity was measured using
the Dual-Luciferase reporter assay. Results are expressed as a ratio
of beetle luciferase activity to R. reniformis luciferase activity and are
an average ? SEM of at least three independent experiments per-
formed in triplicate.
Ang II regulates the HIF-1? ODDD. VSMCs were trans-
escent VSMCs were maintained under control conditions, in the
presence of Ang II (100 nM) or in hypoxic conditions (1% O2) for
indicated times. Total cell extracts (25 ?g) were resolved by SDS-
PAGE (10%) and immunoblotted using anti-PHD2 and anti-tubulin
antibodies. (B) Quiescent VSMCs were treated with MG132 (20 ?M)
and stimulated with Ang II (100 nM), thrombin (thr, 5 U/ml) or
CoCl2(200 ?M) for 4 h. Total cell extracts (25 ?g) were resolved by
SDS-PAGE (8%) and immunoblotted using anti-hydroxylated-
Pro402(P402-OH), anti-hydroxylated-Pro564(P564-OH), anti-HIF-1?,
and anti-?-actin antibodies. (C) Western blots in B were quantified
with Scion Image or the Odyssey Infrared Imaging System using
?-actin as an internal loading control. Results are expressed as a
percentage of hydroxylated HIF-1? (normalized to total HIF-1?
protein levels) on Pro402(black bars) or Pro564(gray bars) compared
with untreated cells (control) and are an average ? SD of at least
three independent experiments.
Ang II regulates HIF-1? protein hydroxylation. (A) Qui-
HIF-1 Stabilization by Ang II
Vol. 19, January 200889
against hydroxylated proline residues Pro402and Pro564
(Chan et al., 2005). For this assay, cells were treated with
MG132 and maintained with or without Ang II, thrombin, or
CoCl2, a hypoxia mimetic that strongly inhibits HIF-1? hy-
droxylation. As expected, the treatment of VSMCs with
CoCl2led to a near complete inhibition of HIF-1? protein
hydroxylation of both proline residues (Figure 4B). Interest-
ingly, treatment of VSMCs with Ang II also significantly
decreased HIF-1? protein hydroxylation. The effect of Ang II
was predominantly on Pro402hydroxylation, reducing this
modification by 69.2 ? 5.2% compared with control cells
treated only with MG132 (Figure 4C). Alternatively, we
could detect no significant effect of Ang II treatment on
HIF-1? Pro564hydroxylation. Finally, the treatment of cells
with thrombin also blocked HIF-1? hydroxylation in a man-
ner similar to Ang II (Figure 4, B and C).
A key step in HIF-1? protein degradation is its binding to
pVHL, which is a direct consequence of HIF-1? hydroxyla-
tion. To determine the effect of Ang II treatment on pVHL
binding, a pVHL capture assay was used. A GST-HIF-1?
fusion protein, comprised of amino acids 344-582 from hu-
man HIF-1?, was subjected to modification by Ang II–
treated cell extracts, followed by interaction with in vitro–
translated pVHL protein. As seen in the left panel of Figure
5A, the treatment of cells with Ang II decreased pVHL
binding to HIF-1?. As expected, the treatment of VSMCs
with CoCl2completely abolished pVHL binding to HIF-1?.
To control the specificity of pVHL binding to hydroxylated
HIF-1?, a mutated form of the GST-HIF-1? fusion protein
(proline residues Pro402and Pro564to alanine) was used.
Using this mutated construct, no pVHL binding was ob-
served (right panel of Figure 5A). Furthermore, time-course
experiments indicated that the treatment of VSMCs with
Ang II decreased pVHL binding to HIF-1? between 2 and
4 h of stimulation (Figure 5B). This time course is similar to
previous studies on HIF-1? induction, which also showed a
maximal HIF-1? protein induction between 2 and 4 h of Ang
II treatment (Page et al., 2002). Taken together, our results
demonstrate that Ang II modulates hydroxylation of HIF-1?
on Pro402, leading to decreased pVHL binding and increased
Ang II Modifies Cellular Levels of Ascorbate, a PHD
As mentioned previously, PHDs belong to the family of
2-oxoglutarate–dependent dioxygenases and catalyze the
ferrous iron (Fe2?)-dependent hydroxylation of HIF-1?.
Moreover, PHDs are also dependent on the presence of
ascorbate for maximal activity (Kivirikko and Myllyharju,
1998; Knowles et al., 2003). Recent studies have demon-
strated that ascorbate levels can be regulated by certain
stimuli shown to induce HIF-1? (Salnikow et al., 2004; Kac-
zmarek et al., 2007). Therefore, we attempted to evaluate the
possibility that Ang II treatment could regulate the activity
of PHD through the modulation of intracellular ascorbate
levels. We first set out to determine if ascorbate supplemen-
tation could modify HIF-1? induction by Ang II treatment.
We found that ascorbate supplementation in culture me-
dium completely blocked HIF-1? protein induction in
VSMCs after Ang II treatment (Figure 6A). Interestingly,
ascorbate supplementation also decreased the expression of
VEGF, a HIF-1 target gene, after Ang II treatment (Figure 6C).
Ascorbate supplementation did not affect other signaling
pathways activated through the AT1receptor, such as p42/
p44 MAPK or the downstream target of the PI3K pathway,
p70S6K. Ascorbate supplementation also did not affect the
hypoxic induction of HIF-1? (Figure 6B). Finally, ascorbate
supplementation restored the ability of pVHL to bind
VSMCs were stimulated as indicated and cytoplasmic extracts were
incubated with wild-type or mutated (P402/564A) GST-HIF-1? pro-
tein coupled to Sepharose beads for 1 h. Samples were then incu-
bated overnight in the presence of in vitro–translated pVHL and
resolved by SDS-PAGE (12%). Immunoblotting was performed us-
ing anti-HA (pVHL) and anti-GST antibodies.
Ang II regulates pVHL binding to HIF-1?. (A and B)
(A and B) Quiescent VSMCs were pretreated or not with ascorbate
(Asc, 5 ?M or indicated concentrations) for 15 min and maintained
under control conditions, in the presence of Ang II (100 nM) or in
hypoxic conditions for 4 h. Total cell extracts (25 ?g) were resolved
by SDS-PAGE (8%) and immunoblotted using anti-HIF-1?, anti-
phospho-p42/p44 MAPK, anti-p42/p44 MAPK, anti-phospho-
p70S6K, anti-p70S6K, and anti-tubulin antibodies. (C) Quiescent
VSMCs were pretreated or not with 5 ?M ascorbate for 15 min and
maintained under control conditions or in the presence of Ang II
(100 nM) for 4 h. Total RNA was extracted and resolved in formal-
dehyde/agarose gels. Northern blots were performed using a spe-
cific radiolabeled VEGF probe. An 18S RNA probe was used as a
control for gel loading. (D) Quiescent VSMCs were incubated in
ascorbate-supplemented DMEM for 16 h. Cells were then main-
tained under control conditions or in the presence of Ang II (100
nM) in nonsupplemented DMEM. Cells were washed with PBS and
resuspended in 90% methanol/1 mM EDTA solution. Ascorbate
concentrations were measured in samples using HPLC (see Materi-
als and Methods). Results are expressed as the percentage of intra-
cellular ascorbate concentrations compared with untreated cells and
are an average ? SEM of at least three independent experiments
performed in triplicate.
Impact of intracellular ascorbate on HIF-1? stabilization.
E. L. Page ´ et al.
Molecular Biology of the Cell90
HIF-1? (results not shown). Taken together, these results
indicate that the supplementation of VMSC with ascorbate
restores the ability of the PHD enzymes to hydroxylate HIF
during Ang II treatment and inhibits Ang II–induced HIF-1?
Recent studies have demonstrated that cobalt and nickel
ions regulate intracellular ascorbate levels leading to HIF-1?
protein induction (Salnikow et al., 2004). Moreover, it has
been shown that the depletion of intracellular ascorbate
levels leads to HIF-1? protein up-regulation (Vissers and
Wilkie, 2007). Therefore, we determined whether Ang II
treatment could modify intracellular ascorbate levels in
VMSC. Using both HPLC (Figure 6D) and spectrophotomet-
ric (Figure 7C) determinations of intracellular ascorbate lev-
els, our results demonstrated that the treatment of VSMCs
with Ang II decreased intracellular levels of ascorbic acid by
56.3 ? 2.4% compared with nontreated cells. These results
suggest that decreased ascorbate availability in VSMCs dur-
ing Ang II stimulation leads to increased HIF-1? protein
It is well accepted that Ang II activates the production of
reactive oxygen species in VSMCs (Griendling et al., 1994;
Kimura et al., 2005; Lyle and Griendling, 2006; Zhang et al.,
2007). The major ROS products generated by Ang II in
VSMCs are hydroxyl and superoxide radicals. In cells, su-
peroxide radicals are rapidly transformed into H2O2by
action of superoxide dismutase. H2O2has been shown to be
implicated in HIF-1? protein induction (Chandel et al., 2000;
Gerald et al., 2004; Brunelle et al., 2005; Bell et al., 2007; Pan
et al., 2007). We have previously demonstrated that utiliza-
tion of catalase inhibited HIF-1? induction by Ang II in
VSMCs (Richard et al., 1999; Page et al., 2002). To evaluate
the possibility that after Ang II treatment H2O2production
in VSMCs is involved in decreasing PHD activity and
HIF-1? hydroxylation, we studied the effect of H2O2and
catalase on the interaction of HIF-1? with pVHL. As seen in
Figure 7A, the addition of exogenous H2O2to VSMC ex-
tracts also inhibited pVHL binding to HIF-1?. Moreover, the
pretreatment of VSMCs with catalase was able to restore
pVHL binding to HIF-1?, which was decreased after Ang II
treatment (Figure 7B). These results indicate that H2O2is
involved in decreasing HIF-1? hydroxylation and its subse-
quent binding to pVHL. We then hypothesized that the
inhibition of HIF-1? hydroxylation and pVHL binding by
H2O2may be through the modulation of ascorbate levels in
VSMCs observed after Ang II treatment. We therefore de-
termined the effect of H2O2on intracellular ascorbate con-
centrations. As shown in Figure 7C, VSMCs treated with
H2O2demonstrated decreased intracellular ascorbate levels.
Additionally, catalase restored intracellular ascorbate levels
decreased after Ang II treatment of VSMCs (Figure 7C).
Taken together, our results indicate that in VSMCs, Ang II
treatment–induced H2O2production is responsible for de-
creases in intracellular ascorbate levels, which hampers
PHD activity, leading to the stabilization of HIF-1?.
HIF-1 is a key regulator of gene induction by hypoxia in all
mammalian cell types. Studies have clearly delineated that
in VSMCs, different GPCR agonists, including Ang II and
thrombin, are able to strongly increase the formation of the
HIF-1 complex by inducing the HIF-1? subunit (Richard et
al., 2000; Gorlach et al., 2001). Although the hypoxic activa-
tion of HIF-1 mainly implicates the stabilization of HIF-1?
protein levels, GPCR agonists have devised an interesting
and more complex mechanism to induce HIF-1? in nor-
moxic conditions. VSMC stimulation with Ang II and throm-
bin: 1) increases the transcription of the HIF-1? gene, via the
activation of diacylglycerol-sensitive PKC and nuclear factor
(NF)-?B (Page et al., 2002; Bonello et al., 2007); 2) increases
translational regulation of HIF-1? protein, via a ROS-depen-
dent activation of the PI3K/p70S6K pathway (Page et al.,
2002); and 3) permits receptor tyrosine kinase transactiva-
tion that is necessary for increased HIF-1? protein transla-
tion and HIF-1 complex activation (Lauzier et al., 2007).
However, given the high instability of HIF-1? protein in
normoxic conditions, it was unclear whether these effects
alone were sufficient to increase HIF-1 levels. In this work,
we demonstrate that Ang II also increases HIF-1? protein
stabilization in VSMCs. Indeed, our studies clearly demon-
strate that Ang II increases HIF-1? half-life. Ang II–treated
cells increase HIF-1? protein stability by decreasing Pro402
hydroxylation, thereby attenuating pVHL binding to HIF-1?
and diminishing HIF-1? ubiquitination and proteasome
binding. Finally, we identify that Ang II–stimulated gener-
ation of H2O2is responsible for decreasing intracellular
ascorbate levels in VSMCs, an essential cofactor for the
HIF-1? hydroxylation reaction. This work identifies novel
and intricate signaling mechanisms that are intimately in-
volved in HIF-1? protein regulation and activation after
GPCR activation of VSMCs.
Recent evidence has emerged indicating that when in-
duced in normoxic conditions, HIF-1? protein can also be
stabilized. Hydroxylation by PHD enzymes is clearly de-
fined as a major posttranslational modification implicated in
HIF-1? stability (Bruick and McKnight, 2001; Epstein et al.,
2001; Ivan et al., 2001; Jaakkola et al., 2001; Masson et al.,
hydroxylation and ascorbate depletion during Ang II treatment. (A)
Extracts from quiescent VSMCs were treated with or without H2O2
(100 ?M). (B) Quiescent VSMCs were pretreated with or without
catalase (cat, 1000 U/ml) and maintained under control conditions,
in the presence of Ang II (100 nM) or CoCl2(200 ?M). Cytoplasmic
extracts were incubated with GST-HIF-1? protein coupled to Sepha-
rose beads. Samples were then incubated overnight in the presence
of in vitro–translated pVHL and resolved by SDS-PAGE (12%).
Immunoblotting was performed using anti-HA (pVHL) and anti-
GST antibodies. (C) Quiescent VSMCs were incubated in ascorbate-
supplemented DMEM for 16 h. Cells were maintained under control
conditions or in the presence of H2O2(50 ?M), catalase (1000 U/ml)
and/or Ang II (100 nM) in nonsupplemented DMEM. Cells were
washed with PBS and resuspended in 90% methanol/1 mM EDTA
solution. Ascorbate concentrations were measured in samples using
spectrophotometry (see Materials and Methods). Results are ex-
pressed as the percentage of intracellular ascorbate concentrations
compared with untreated cells and are an average ? SEM of at least
three independent experiments performed in triplicate.
Hydrogen peroxide is responsible for decreased HIF-1?
HIF-1 Stabilization by Ang II
Vol. 19, January 200891
2001). Certain normoxic inducers have been shown to reg-
ulate HIF-1? stability by modulating PHD enzyme activity.
McMahon et al. (2006) have shown that PHD2 is down-
regulated after a TGF-?1 treatment on different human cells,
allowing the stabilization of HIF-1?. They demonstrated that
TGF-?1 markedly and specifically decreases both mRNA
and protein levels of PHD2 through the Smad signaling
pathway. In VSMCs, Ang II stimulation has been shown to
activate the Smad pathway (Rodriguez-Vita et al., 2005;
Wang et al., 2006). However, we did not observe decreases in
PHD2 mRNA or protein levels in VSMCs treated with Ang
II. In opposition, PHD2 mRNA levels were even increased
during the same time period (data not shown). This last
result was expected because PHD2 is a HIF-1 target gene
(Epstein et al., 2001; Berra et al., 2003; Metzen et al., 2005).
Knowles et al. (2006) have also demonstrated that normoxic
stabilization of HIF-1? is mediated by a decrease in PHD
enzyme activity. They show that PMA-induced macrophage
differentiation also implicates HIF-1? induction via a down-
regulation of intracellular labile iron pool, an essential co-
factor of PHDs.
Our results clearly show that Ang II regulates HIF-1?
hydroxylation. Studies by Chan et al. (2005) have shown that
both of HIF-1?’s hydroxylated proline residues are differen-
tially regulated in hypoxic conditions. They demonstrated
that Pro564is the first residue to be hydroxylated when
reaction conditions are favorable and that this modification
promotes the subsequent hydroxylation of Pro402. Together,
the two hydroxylated proline residues potentiate the degra-
dation of HIF-1? protein. It has been shown that modifica-
tion of only one of these proline residues is sufficient for
significant stabilization of HIF-1 (Chan et al., 2005). Our
results are in agreement with these observations and show
that the treatment of VSMCs with Ang II decreases hydroxy-
lation on Pro402, resulting in decreased pVHL capture, while
having no significant effect on Pro564hydroxylation. Pro564
being the primary residue hydroxylated when all reaction
conditions are filled, it is reasonable to believe that Pro402
will be the first residue to be regulated during conditions of
cofactor depletion, which lead to decreased PHD activity.
Given the potency of ascorbate to inhibit increases in
HIF-1? protein levels after Ang II treatment, we investigated
the possibility that ascorbate could have inhibitory effects on
mechanisms already described for HIF-1? induction by Ang
II. First, the transcriptional modulations of HIF-1? by Ang II
treatment were not inhibited by addition of ascorbate (see
Supplementary Figure S2). Additionally, ROS production is
an essential step for PI3K/p70S6K pathway activation by
Ang II. PI3K/p70S6K pathway activation is involved in
increasing HIF-1? translation during Ang II treatment (Page
et al., 2002). Although ascorbate is also known as an antiox-
idant compound, it had no inhibitory effect on the ROS-
dependent activation of the PI3K/p70S6K pathway by Ang
II. These observations converge on the potential role of
ascorbate in the regulation of HIF-1? protein stability.
Oxygen and 2-oxoglutarate are cosubstrates, whereas
ascorbate and Fe2?are essential cofactors for PHD enzymes.
Changes in the levels of these cofactors lead to the modula-
tion of PHD enzymatic activity and its downstream effects
on HIF-1?. Studies have shown that stimuli inducing HIF-
1?, such as cobalt and nickel, modulate intracellular ascor-
bate levels to decrease hydroxylation and avoid degradation
(Salnikow et al., 2004; Karaczyn et al., 2006). Vissers and
Wilkie (2007) showed that HIF-1? is up-regulated in ascor-
bate-deficient neutrophils. Ascorbate has also been shown to
enhance PHD enzyme activity and HIF degradation (Gorlach
et al., 2001; Knowles et al., 2003; Vissers et al., 2007). In the
present study, we demonstrate that Ang II depletes VSMCs
of ascorbate. During the hydroxylation reaction, Fe2?bound
to the enzyme is oxidized to Fe3?, leading to the inactivation
of catalytic activity. Ascorbate has been proposed to play the
role of the reducing agent of Fe3?to Fe2?directly in the
active site of the enzyme prolyl-4-hydroxylase, leading to
the reactivation of the enzyme (de Jong et al., 1982; Majamaa
et al., 1986). Decreased intracellular ascorbate may promote
the oxidized Fe3?-enzyme–bound state and cause a de-
crease in PHD enzyme activity.
It is now well documented that Ang II is a prooxidant
hormone that increases ROS production in many cell types
(Sachse and Wolf, 2007), including VSMCs (Kimura et al.,
2005; Zhang et al., 2007; Griendling et al., 1994; Lyle and
Griendling, 2006). In these cells, the superoxide radical is
transformed to H2O2by action of superoxide dismutase
(Clempus and Griendling, 2006). Our study shows that
H2O2contributes to the stabilization of HIF-1? protein
through the regulation of intracellular ascorbate levels. Hy-
drogen peroxide is the major reactive moiety involved in
Fenton reaction, which also implicates the oxidation of fer-
rous iron into ferric iron:
H2O2? Fe2?3 ? OH ? Fe3?? ?OH
Additionally, it has been reported that enhanced ROS pro-
duction promotes the inactivation of PHD and HIF stabili-
zation, most probably via the Fenton reaction and oxidation
of Fe2?into Fe3?(Gerald et al., 2004; Bell et al., 2007). Gerald
et al. also demonstrate that ROS generation interferes with
iron availability at the HIF prolyl-hydroxylase catalytic site,
thereby inhibiting PHD activity in normal oxygen condi-
tions. H2O2has been implicated in the induction of HIF-1
and the transcriptional activation of target genes (Fandrey
and Genius, 2000; Gorlach et al., 2001; Mansfield et al., 2005;
Simon, 2006). Moreover, H2O2has been shown to stabilize
HIF-1? protein (Chandel et al., 2000; Gerald et al., 2004;
Brunelle et al., 2005; Bell et al., 2007; Pan et al., 2007). Because
ascorbate is important in maintaining full activity of the
enzyme by reducing ferric iron, ascorbate depletion in cells
would contribute to HIF-1? stabilization in these conditions.
Ascorbate itself may be consumed by oxidation in condi-
tions where cells are exposed to peroxide or superoxide
(Holmes et al., 2000). In VSMCs, the precise mechanisms
involved in H2O2-mediated decreases in ascorbate levels are
presently unclear. However, our data, along with relevant
published work, suggest that the treatment of VSMCs with
Ang II leads to the generation of H2O2. The higher cellular
level of H2O2contributes to the oxidation of Fe2?into Fe3?
via the Fenton reaction. Decreased Fe2?availability leads to
a decrease in PHD activity, decreased HIF-1? hydroxylation
and increased HIF-1? stability. Increased Fe3?causes aug-
mented consumption of ascorbate for reduction to Fe2?,
further decreasing the ability of the cell to reduce Fe3?.
Supplementation of ascorbate in these conditions reverses
the effect of H2O2on iron availability, blocking the induction
of HIF-1? by Ang II.
In conclusion, our study identifies novel signaling inter-
mediates implicated in the regulation of the HIF-1 complex
through nonhypoxic means. Ang II is a potent inducer of
HIF-1? protein, and the present work identifies stabilization
as a novel mechanism of HIF-1? protein induction by Ang II
in VSMCs. This work contributes to the elucidation of Ang
II–induced mechanisms for the activation of the HIF-1 com-
plex and the subsequent activation of target genes. Given the
importance and the evident implication of Ang II and the
genes activated by the HIF-1 complex in different domains
E. L. Page ´ et al.
Molecular Biology of the Cell92
of vascular biology, we believe that elucidation of pathways
responsible for the regulation of the HIF-1 complex will
have a very strong impact in vascular biology.
We are grateful to Guylaine Soucy and Genevie `ve Robitaille for their excellent
technical assistance. We also thank Dr. Christopher Pugh, Dr. Edurne Berra,
and Amandine Ginouves for help with plasmid constructs. This work was
supported by grants from the Canadian Institutes of Health Research (CIHR,
MOP-49609) and the Heart and Stroke Foundations of Que ´bec and Canada.
D.E.R. is the recipient of a CIHR New Investigator Award. E.L.P. holds a
Canada Graduate Scholarship from the CIHR.
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