THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 50, pp.

4286342872, December 16, 2011

2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

Interplay between Vascular Endothelial Growth Factor (VEGF)

and Nuclear Factor Erythroid 2-related Factor-2 (Nrf2)
IMPLICATIONS FOR PREECLAMPSIA *
S

Received for publication, July 27, 2011, and in revised form, October 24, 2011 Published, JBC Papers in Press, October 27, 2011, DOI 10.1074/jbc.M111.286880

Nisreen Kweider, Athanassios Fragoulis, Christian Rosen, Ulrich Pecks, Werner Rath, Thomas Pufe,
and Christoph Jan Wruck1
From the Department of Anatomy and Cell Biology, Faculty of Medicine, RWTH Aachen University and the Department of
Obstetrics and Gynecology, University Hospital RWTH, 52074 Aachen, Germany
Background: Several studies have suggested that decreasing VEGF levels might result in placental oxidative stress in
preeclampsia.
Results: VEGF activates Nrf2 in an ERK1/2-dependent manner, protecting against oxidative stress, and, in turn, up-regulates
VEGF expression.
Conclusion: Reduced VEGF bioavailability may lead to aggregation of oxidative stress and result in preeclampsia.
Significance: Nrf2 activation might be considered as an adjunct therapeutic strategy to combat preeclampsia.

Preeclampsia is unique to human pregnancy and is characterized by proteinuric hypertension in up to 35% of all pregnancies (1). In developing countries where access to health care

* This work was supported by Deutsche Forschungsgemeinschaft Grants Pu

214/3-2, Pu 214/4-2, Pu 214/5-2, and SFB617; the START Program of the
Faculty of Medicine, RWTH Aachen University; and the Department of Histology, Faculty of Medicine, Damascus University.

S
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Fig. 1.
1
To whom correspondence should be addressed: Dept. of Anatomy and Cell
Biology, Faculty of Medicine, RWTH Aachen University, Wendlingweg 2,
52074 Aachen, Germany. Tel.: 49-241-80-88-470; Fax 49-241-80-82-431;
E-mail: cwruck@ukaachen.de.

DECEMBER 16, 2011 VOLUME 286 NUMBER 50

is limited, preeclampsia is a leading cause of maternal mortality,

with estimates of 60,000 maternal deaths per year (2). Despite
intensive research, the etiology of preeclampsia still remains
unclear. Pathogenic factors affecting both fetus and mother are
currently under intensive investigation: in the fetus, the principal pathology appears to be placental oxidative stress resulting
from insufficient uteroplacental blood supply (3), whereas the
underlying maternal pathology is a severe systemic inflammatory response involving leukocyte and endothelial cell activation (4 6).
VEGF expression takes place in the villous trophoblast
(79). Upon gene activation, the resulting VEGF receptor
initiates a cascade of cellular protein phosphorylation reactions by several protein kinases and leads to a variety of
cellular responses (10 12). One family of these downstream
kinases includes MAPKs p42 and p44, also referred to as
ERK1 and ERK2. ERK1 and ERK2 are phosphorylated and
activated by MAPK kinase (MAPKK or MEK) in the cytosol,
translocate to the nucleus, and subsequently stimulate transcription of early response genes regulating cell proliferation
and survival (10 12).
Maynard et al. (13) reported that the soluble VEGF receptor
sFlt-1 (soluble fms-like tyrosine kinase-1; soluble VEGF receptor-1) is involved in the pathophysiology of preeclampsia. More
recently, it was reported that elevated maternal sFlt-1 and
decreased VEGF concentrations result in increased oxidative
stress, which contributes to vascular dysfunction during pregnancy, although the question as to how decreased VEGF concentrations increase oxidative stress still remains unanswered
(14).
Oxidative stress in syncytiotrophoblasts of women with preeclampsia is well documented (3, 6). These cells are especially
sensitive to oxidative stress partly because of their location
in the outermost layer of placental villi, where they are
exposed to high oxygen concentrations, and partly because
they contain surprisingly low concentrations of antioxidant
enzymes (15, 16).
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Several recently published studies have suggested that

decreasing VEGF levels result in placental oxidative stress in
preeclampsia, although the question as to how decreased VEGF
concentrations increase oxidative stress still remains unanswered. Here, we show that VEGF activated Nrf2, the main regulating factor of the intracellular redox balance, in the
cytotrophic cell line BeWo. In turn, this activated the production of antioxidative enzymes thioredoxin, thioredoxin reductase, and heme oxygenase-1, which showed a decrease in their
expression in the placentas of preeclamptic women. Nevertheless, this activation occurred without oxidative stress stimulus.
As a consequence, the activation of Nrf2 protected BeWo cells
against H2O2/Fe2-induced oxidative damage. We further show
that VEGF up-regulated the expression of itself. A positive feedback loop was described in which VEGF activated Nrf2 in an
ERK1/2-dependent manner; the up-regulation of HO-1 expression by Nrf2 augmented the production of carbon monoxide,
which in turn up-regulated VEGF expression. In conclusion,
VEGF induces the Nrf2 pathway to protect against oxidative
stress and, via a positive feedback loop, to elevate VEGF expression. Therefore, decreased VEGF bioavailability during preeclampsia may result in higher vulnerability to placental oxidative cell damage and a further reduction of VEGF bioavailability,
a vicious circle that may end up in preeclampsia.

VEGF Activates Nrf2

MATERIALS AND METHODS

Cell Culture and StimulationHuman choriocarcinoma
BeWo cells were obtained from American Type Culture Collection. The cells were cultured in Hams F-12 medium (PAA
Laboratories GmbH) with 10% FBS (Invitrogen), 100 units/ml
penicillin, and 100 g/ml streptomycin (Invitrogen) and incubated at 37 C. The cells were seeded into Petri dishes (10 cm,
2 106 cells/dish), 6-well plates (5 105 cells/well), or 96-well
plates (10 104 cells/well) for subsequent culture. The cells
were then cultured in 20% O2 for 24 h and incubated overnight
with 50 M vitamin C (Sigma-Aldrich) to attenuate prestimulation of Nrf2.
These cells were subsequently stimulated with 10 ng/ml
human VEGF165 (R&D Systems) or were left unstimulated as a
control. VEGF165, an isoform of VEGF-A, was used in this study
because it is supposed be one of the most potent angiogenic
isoforms (26). For positive control of Nrf2 activation, some cells
were treated with 1 M sulforaphane (Sigma-Aldrich) (27, 28).
Oxidative stress was induced with hydrogen peroxide (Carl
Roth GmbH) in the presence of 10 M iron(II) sulfate (SigmaAldrich) (29).
For experiments with MAPK, p38, c-JNK, and PI3K inhibitors, the cells were seeded into 6- or 96-well plates. After reaching confluence, the cells were incubated with 50 M vitamin C
2

The abbreviations used are: ROS, reactive oxygen species; ARE, antioxidant
response element; HO-1, heme oxygenase; DPI, diphenyliodonium chloride; CORM-2, tricarbonyldichlororuthenium(II) dimer; PMA, phorbol
12-myristate 13-acetate.

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before they were exposed to kinase inhibitors. The inhibitor

concentrations used were as follows: 150 M MEK1 inhibitor
PD98059 and 110 M MEK1/2 inhibitor U0126 (Cell Signaling
Technology) and 1 40 M p38 inhibitor SB203580, 150 M
c-Jun N-terminal kinase inhibitor SP600125, and 0.15 M
PI3K inhibitor wortmannin (Calbiochem). Diphenyliodonium
chloride (DPI) at concentrations of 120 M was used as a specific inhibitor of NAD(P)H oxidase (Sigma-Aldrich). The cells
were pretreated for 30 min with inhibitors before VEGF was
added for an additional 1 h prior to the assay.
To test the effect of carbon monoxide, the tricarbonyldichlororuthenium(II) dimer (CORM-2; Sigma-Aldrich) was applied.
The cells were incubated in Hams F-12 medium containing
10% FCS and the indicated concentration of CORM-2 for 24 h.
To scavenge CO, human Hb (Sigma-Aldrich) was first dissolved in PBS under an argon atmosphere and agitated at room
temperature for 2 h prior to filter sterilization. Various doses of
Hb were added to Hams F-12 medium at the same time as the
administration of VEGF165. After the stimulation, the cells were
lysed, and VEGF protein concentrations were measured by
ELISA.
Cell Lysate Preparation and Immunoblot AnalysisCells
were lysed in buffer containing 10 mM HEPES (PromoCell
GmbH), 1.5 mM MgCl2 (Sigma-Aldrich), 10 mM KCl, 0.5 mM
DTT, 0.05% Nonidet P-40 (pH 7.9), and protease inhibitors,
and the lysate were left on ice for 10 min. Cytosolic fractions
were obtained as the supernatant after centrifugation at 950
g for 10 min at 4 C. The pellets were resuspended in nuclear
extraction buffer containing 5 mM HEPES, 1.5 mM MgCl2, 0.2
mM EDTA (Invitrogen), 0.5 mM DTT, 26% glycerol, and protease inhibitors (pH 7.9). NaCl (4.6 M) was added, followed by a
30-min incubation on ice. Nuclear proteins were found in the
supernatant after a 20-min centrifugation at 24,000 g at 4 C
(following the Abcam protocol). The protein (6 g) from the
nuclear fractions and the protein (15 g) from the cytosolic
fractions were subjected to 12.5% discontinuous SDS-PAGE,
separated by electrophoresis, and then electroblotted onto
PVDF membranes (Millipore GmbH).
The immunoblot analysis was performed with specific antibodies and an enhanced chemiluminescence-based detection
kit (Millipore GmbH). Antibodies against Nrf2, HO-1, thioredoxin, TXNRD1, and p38 were purchased from Abcam. Antibodies against -actin, phospho-ERK, and ERK were purchased
from Santa Cruz Biotechnology. Antibodies against histone H3,
Akt, phospho-Akt (Ser-473), and phospho-p38 were brought
from Cell Signaling Technology).
The densities of the bands were measured using PCBAS 2.0
software, and the ratio between the protein and the corresponding loading control (histone H3 and -actin for Nrf2;
-actin for thioredoxin, TXNRD1, and HO-1; total ERK1/2 for
phospho-ERK1/2; p38 for phospho-p38; and Akt for phosphoAkt) was calculated.
Dual-Luciferase AssayARE from the rat NQO1 gene was
cloned into the pGL3 promoter vector (Promega, Madison, WI)
as described previously (21). The pGL3-ARE vector was
cotransfected with the Renilla luciferase plasmid phRL-TK
(Promega) using jetPRIMETM transfection reagent (Polyplus
Transfection) according to the manufacturers recommendaVOLUME 286 NUMBER 50 DECEMBER 16, 2011

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A battery of genes encoding antioxidant enzymes is orchestrated upon exposure to reactive oxygen species (ROS).2 This
coordinated response is regulated via the antioxidant response
element (ARE) contained within the regulatory regions of socalled safeguard genes such as NQO1 (NAD(P)H:quinone
oxidoreductase-1), TXNRD1 (thioredoxin reductase-1), glutathione peroxidase, and heme oxygenase-1 (HO-1) (17, 18).
Activation of Nrf2 (nuclear factor erythroid 2-related factor-2)
as a consequence of oxidative stress initiates and enhances transcription of these safeguard genes, thus protecting cells against
oxidative stress as well as a wide range of other toxins (19 23).
Mann et al. (24) were the first to discuss a link between Nrf2,
vascular homeostasis, and preeclampsia. Recently, Wruck et al.
(25) provided the first experimental data that Nrf2 is active
exclusively within cytotrophoblasts of preeclamptic placenta,
strongly suggesting that these cells suffer from oxidative stress
caused by ROS. In this work, we hypothesized that oxidative
stress during preeclampsia results in increased expression and
transfer of antioxidant enzymes from cytotrophoblasts into
syncytiotrophoblasts via enhanced syncytial fusion, thereby
increasing cytotrophoblast proliferation and syncytial knot
formation (necrotic and aponecrotic subcellular syncytial
fragments).
Thus, the principal aim of this study was to investigate
whether VEGF activates Nrf2, which counters oxidation stress.
The second objective was to study whether the activation of
Nrf2, which leads to an increase in cellular carbon monoxide,
raises VEGF levels.

VEGF Activates Nrf2

DECEMBER 16, 2011 VOLUME 286 NUMBER 50

version of WST-1 to formazan was measured at 450 nm by

microplate spectrophotometry (Model 680, Bio-Rad). This reaction reflects the reductive capacity of the cells, which represents the viability of the cells, which is expressed as a value of 1.
This value of 1 represents the reductive capacity of the untreated control.
Cytotoxicity AssayThe CytoTox-GloTM cytotoxicity assay
(Promega) was used to measure cytotoxicity. The cells were
seeded in 96-well plates and starved overnight by adding FCSfree medium supplemented with vitamin C. The cells were
treated with or without 10 ng/ml VEGF165 for 3 h, and all cells
were then incubated with 500 M H2O2 and 10 M Fe2 for 6 h.
The CytoTox-GloTM cytotoxicity kit was used according to the
manufacturers recommendations. The luminescence was
measured in a 96-well plate reader (GloMax-96 microplate
luminometer).
ELISAThe cells were seeded in 6-well plates; after reaching
confluence, the cells were treated with VEGF165 for 6 h. Subsequently, the cells were washed twice with PBS and lysed or
supplied with new medium (without FCS) for 24 h. The levels of
VEGF protein in the supernatant or in the cell lysate were
assessed by a sandwich ELISA (R&D Systems) that detects all
VEGF splice forms. Human recombinant VEGF165 was used for
standard curve determination.
Statistical AnalysisAll measurements were performed at
least in triplicates, and the results are expressed as the mean
S.E. Statistical analyses were performed using Students
unpaired t test for dual comparisons. Mean differences were
considered to be significant when p was 0.05. All statistical
graphs and analyses were created with GraphPad Prism 5.0
(GraphPad Software, La Jolla, CA).

RESULTS
VEGF165 Increases Nrf2 Activation and Nrf2 Target Gene
ExpressionTo investigate the efficacy of VEGF165 to activate
the cis-acting element ARE, a Dual-Luciferase reporter
gene assay was performed with the ARE from the NQO1 gene.
ARE activation was determined in a dose-response assay at concentrations up to 10 ng/ml VEGF165 in BeWo cells after 6 h of
incubation. According to Fig. 1A, the relative ARE activity of
VEGF165 (10 ng/ml) was approximately twice that of the control. This was also true for the positive control, 1 M
sulforaphane.
We then tested whether this ARE activation is Nrf2-dependent. Therefore, an shRNA against mRNA coding for human
Nrf2 was designed, whereby a BeWo cell line containing Nrf2
shRNA (BeWo-shNrf2) and a control BeWo cell line containing
a scrambled control shRNA (BeWo-shControl) were produced
and stably transfected in BeWo cells. Real-time RT-PCR was
used to test the efficacy of the shRNA against Nrf2. Real-time
RT-PCR analysis showed an Nrf2 knockdown of 90% in both
untreated and 1 M sulforaphane-treated BeWo-shNrf2 cells.
In contrast, sulforaphane treatment showed a significant activation of Nrf2 mRNA in BeWo-shControl cells (Fig. 1B, inset).
The Nrf2-specific shRNA totally abolished the VEGF165- and
sulforaphane (positive control)-dependent ARE activation.
These results demonstrate that Nrf2 is the transcription factor
that activates ARE expression in response to VEGF165 (Fig. 1B).
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tions. Twenty-four hours after transfection, the cells were

seeded in a 96-well plate. Firefly and Renilla luciferase activities
were determined 48 h after transfection with the Dual-Luciferase reporter gene assay system (Promega) in a 96-well plate
reader (GloMax-96 microplate luminometer, Promega). The
firefly luminescence signals were normalized to the corresponding Renilla luciferase signals acting as an internal control.
Nrf2 SilencingBeWo cells were transfected with 10 g of
control shRNA and Nrf2 shRNA. The plasmids were part of the
MISSION shRNA assortment purchased from SigmaAldrich. These aforementioned cells were transfected using
jetPRIMETM transfection reagent according to the manufacturers instructions. After 24 h, the medium was replaced with
fresh Hams F-12 medium containing 10% FBS; puromycin
(Carl Roth GmbH) was added 48 h after transfection. The cells
were grown in the medium containing 1 g/ml puromycin to
obtain a sufficient selection. Stable cell lines were established
once all of the cells in the negative control plate were killed.
These stable cell lines were continuously grown in the medium
containing 1 g/ml puromycin. After 10 passages, the gene
expression levels of Nrf2 were measured by quantitative RTPCR to check the knockdown from Nrf2 transcription in these
cells.
RT-PCRCells were harvested in peqGOLD TriFastTM
(PEQLAB Biotechnologie GmbH) for extracting RNA according to the manufacturers protocol. The RNA concentration
was determined by photometric analysis with the NanoDrop
1000 system (PEQLAB Biotechnologie GmbH), and 1 g of
total RNA was then digested with DNase I (Roche GmbH)
and transcribed into cDNA by reverse transcription with
RevertAidTM reverse transcriptase (Fermentas Life Sciences).
Real-time PCRs were processed in triplicate using the ABI
StepOnePlusTM apparatus (Applied Biosystems) in a total volume of 20 l containing 100 ng of cDNA, gene-specific primers,
and SYBR Green I reagent (Applied Biosystems). Gene expression was determined by normalizing the target gene Ct values to
the expression of the housekeeping gene, which was 18 S in this
study (Eurofins MWG Operon). The sequences for the 18 S
primer were 5-TCAACTTTCGATGGTAGTCGCC-3 (forward) and 5-ATGTGGTAGCCGTTTCTCAGGC-3 (reverse), with an annealing temperature of 61.5 C, and those for
the Nrf2 primer (Eurofins MWG Operon) were 5-TCCAGTCAGAAACCAGTGGAT-3 (forward) and 5-AATGTCTGCGCCAAAAGCTG-3 (reverse), with an annealing temperature
of 60.5 C.
Assay for Intracellular Redox StateIntracellular redox state
levels were measured using the fluorescent dye 2,7-dichlorofluorescein diacetate (Invitrogen) as described previously (30).
Briefly, cells were washed once with PBS and incubated in the
same buffer containing 10 g of 2,7-dichlorofluorescein
diacetate for 30 min at 37 C. Intracellular fluorescence was
detected at Ex485/Em530 using a SpectraMax Gemini EM system (Molecular Devices).
Cell Viability Assay (WST-1 Assay)For the WST-1 (4-[3(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) assay, the media were supplemented with 10
l/well (96-well plate) WST-1 (Roche GmbH). The spectrophotometric evaluation was performed after 1, 2, and 4 h. Con-

VEGF Activates Nrf2

Nrf2 is a critical regulator of the intracellular antioxidants

and phase II detoxification enzymes as an adaptive response to
oxidative stress or pharmacological stimuli. To investigate
whether VEGF165 induces Nrf2 activation, 10 ng/ml VEGF165
was administered to the cells at various time points. As early as
1 h following the administration of VEGF165, Nrf2 accumulated
in the nucleus of the BeWo cells and remained at an elevated
level for at least for 6 h (Fig. 1C). Fig. 1E presents the mean of
three independent experiments. Subsequently, the protein
expression of three Nrf2 target genes, i.e. thioredoxin,
TXNRD1, and HO-1, was elevated 3 h after the administration
of VEGF165 (Fig. 1D). The mean of three independent experiments is shown in Fig. 1F.
Nrf2 Activation by VEGF165 Is ERK1/2-dependentTo
address the role of MAPK pathways in ARE gene regulation by

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VEGF165, the effects of various kinase inhibitors were examined. Hence, the activation of ERK1/2 was found to be a prerequisite for the activation of Nrf2 by VEGF165 because VEGF165mediated Nrf2 activation was exclusively inhibited by the
MEK1/2 inhibitor PD98059 (50 M) as well as the MEK1/2
inhibitor U0126 (10 M) (Fig. 2A). The c-Jun N-terminal kinase
inhibitor SP600125 (150 M), the p38 MAPK inhibitor
SB203580 (1 40 M), and the PI3K inhibitor wortmannin
(0.15 M) did not affect ARE activation in VEGF165-stimulated BeWo cells (Fig. 2A).
To confirm these results, the effect of VEGF on both p38 and
Akt activation were tested. VEGF stimulation did not up-regulate p38 phosphorylation over time. In contrast, phorbol
12-myristate 13-acetate (PMA), which was used as a positive
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FIGURE 1. VEGF165 activates the Nrf2/ARE system. A, Dual-Luciferase assay with BeWo cells transfected with the ARE-dependent luciferase reporter gene
plasmid. VEGF165 activated Nrf2 in a dose-dependent manner. Sulforaphane (SFN; 1 M) stimulation was used as a positive control. B, VEGF stimulation of BeWo
cells with Nrf2 knockdown failed to activate the Nrf2/ARE system in an ARE Dual-Luciferase assay, as did sulforaphane as the positive control. Inset, Nrf2 was
down-regulated by 90% compared with the control via stably transfected Nrf2-specific shRNA. C, Western blot analysis to measure Nrf2 activation in response
to VEGF stimulation in BeWo cells. Nrf2 translocated into the nucleus in response to VEGF stimulation in BeWo cells. D, Western blot analysis to measure the
effects of VEGF on the expression of antioxidative stress genes in BeWo cells. The Western blot shows up-regulation of HO-1, thioredoxin (Trx), and TXNRD1
protein levels. E, the densities of corresponding bands (Nrf2 and the loading control histone H3) were measured, and the ratio of Nrf2 to histone H3 was
calculated. The mean of three independent experiments is shown. F, the densities of corresponding bands and the loading control -actin were measured, and
the ratio was calculated. The mean S.E. of three independent experiments is shown. Data are presented as -fold induction after treatment compared with
untreated cells (control 1). *, p 0.05; **, p 0.005; ***, p 0.001; #, p 0.0001 versus the control (one representative Western blot is shown; n 3).

VEGF Activates Nrf2

lation was blocked by 20 M SB203580 (supplemental Fig. 1, A

and B). On the other hand, hydrogen peroxide was used as a
positive control for Akt activation (31). VEGF stimulation did
not up-regulate Akt phosphorylation over time. In contrast,
hydrogen peroxide induced phosphorylation of Akt, and this
phosphorylation was blocked by 1 M wortmannin (supplemental Fig. 1, C and D).
Next, DPI was used as an NAD(P)H oxidase inhibitor to elucidate the role of NAD(P)H oxidase and ROS generation in the
signal transduction of VEGF (Fig. 2A). To test if stimulation
DECEMBER 16, 2011 VOLUME 286 NUMBER 50

with VEGF induces ROS production, we monitored the levels of

ROS with the fluorescent dye 2,7-dichlorofluorescein diacetate. VEGF treatment showed no induction of ROS production
up to 6 h after stimulation (Fig. 2F). We used the phorbol ester
PMA as a positive control for ROS production (32) and to test
the effectiveness of the NAD(P)H oxidase inhibitor DPI (Fig.
2F). The results clearly showed that NAD(P)H oxidase and ROS
generation are not involved in VEGF-induced Nrf2 activation.
To determine the role of ERK1/2 in VEGF165-mediated Nrf2
activation, the effect of VEGF on ERK1/2 activity was examJOURNAL OF BIOLOGICAL CHEMISTRY

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FIGURE 2. VEGF165 activation of Nrf2 is ERK1/2-dependent. A, Dual-Luciferase assay to analyze cell signaling of VEGF-mediated Nrf2/ARE activation. VEGF
was added to the cell culture 30 min after the addition of the kinase or NAD(P)H oxidase inhibitors (MEK1 inhibitor PD98059 (150 M), MEK1/2 inhibitor U0126
(110 M), p38 inhibitor SB203580 (1 40 M), PI3K inhibitor wortmannin (0.15 M), and NAD(P) oxidase inhibitor DPI (120 M)). Data are presented as
percentage -fold induction of luciferase activity after treatment compared with the control (treated cells without inhibitor; control 100%). B, immunoblot analysis with anti-phospho-ERK1/2 antibodies (pERK1/2) was used to measure ERK1/2 activation. BeWo cells were pretreated with either
PD98059 or U0126 for 30 min, followed by treatment with VEGF165 (10 ng/ml) for 1 h. The membrane was stripped and probed with anti-ERK1/2
antibodies as a loading control. C, the densities of corresponding phospho-ERK1/2 and ERK1/2 bands were measured, and the ratio was calculated. The
mean of three independent experiments is shown. Data are presented as -fold induction after treatment compared with the vehicle control (control
1). Pos., positive. D, role of ERK1/2 in Nrf2 activation in response to VEGF treatment measured by Western blotting. BeWo cells were pretreated with
PD98059 or U0126 for 30 min, followed by treatment with VEGF165 (10 ng/ml) for 1 h. The immunoblots of BeWo nuclear extracts with anti-Nrf2
antibodies were used to analyze Nrf2 nuclear translocation. After Nrf2 immunoblotting, the membrane was stripped and probed with anti--actin
antibodies as a loading control. E, band intensity was calculated as the ratio of untreated control cells after normalization to the amount of the loading
control. F, intracellular redox state levels of BeWo cells were measured using the fluorescent dye 2,7-dichlorofluorescein diacetate. PMA (1 M) was
used as a positive control. The positive effect of PMA was decreased using DPI (10 M) 30 min before the addition of PMA. Data represented are the
mean S.E. of three separate experiments. *, p 0.05; **, p 0.005; ***, p 0.001; #, p 0.0001.

VEGF Activates Nrf2

ined. ERK1/2 was activated by dual phosphorylation of threonine and tyrosine residues located in the activation lip of the
conserved core kinase sequence, and the activated species
could be detected by antibodies against phosphorylated peptides contained in these residues. BeWo cells were treated with
10 ng/ml VEGF165, and cell extracts were analyzed for phosphorylated and total ERK1/2 by Western blotting. The loading
control via total ERK1/2 Western blotting showed no significant differences (Fig. 2B). The mean of three independent
experiments is shown in Fig. 2C. The results clearly show that
VEGF165 activates ERK1/2 and that 50 M PD98059, as well as
10 M U0126, inhibits this ERK1/2 activation in comparison
with cells treated with VEGF165, which was supposed to be the
positive control.
Consequently, inhibition of ERK1/2 either by 50 M
PD98059 or by 10 M U0126 significantly blocked the
VEGF165-induced nuclear accumulation of Nrf2 (Fig. 2D), and
the cells treated with VEGF165 without inhibitor were considered to be the positive control. The mean of three independent
experiments is shown in Fig. 2E.
Nrf2 Activation by VEGF165 Confers Cytoprotection against
Oxidative StressThe hypothesis that activation of Nrf2 could
protect BeWo cells from lesions caused by oxidative stress via
H2O2 treatment was tested. BeWo cells were preincubated with
10 ng/ml VEGF165 for 3 h and then treated with 500 M H2O2
and 10 M Fe2. Cell viability was measured by the WST-1
assay, and cell death was measured by the CytoTox-GloTM
cytotoxicity assay 6 h after H2O2 administration. Preincubation
of BeWo cells with 10 ng/ml VEGF165 effectively protected the

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cells from H2O2-induced toxicity in both the WST-1 assay (Fig.

3A) and the CytoTox-GloTM cytotoxicity assay (Fig. 3C).
A causal relationship between Nrf2 activation and cytoprotection mediated by VEGF165 was further investigated. As
shown in Fig. 3 (AD), VEGF165 could protect BeWoshControl (but not BeWo-shNrf2) cells from oxidative toxicity.
After incubation with 500 M H2O2 and 10 M Fe2, the cell
viability of BeWo-shNrf2 cells declined by 50% (Fig. 3B) versus
the 20% decline that was found with BeWo-shControl cells (Fig.
3A). Consistently, BeWo-shControl cells showed a significant
30% decrease in cell death after treatment with 500 M H2O2
and 10 M Fe2 in the presence of VEGF (Fig. 3C), whereas
VEGF treatment had no effect in BeWo-shNrf2 cells (Fig. 3D).
VEGF Up-regulates VEGF via Nrf2/HO-1/COTo evaluate
whether treatment with VEGF165 has an effect on the production of VEGF protein itself in BeWo cells, both BeWoshControl and BeWo-shNrf2 cells were treated with 10 ng/ml
VEGF165 for 6 h. 24 h after stimulation, both cell lines showed
elevated levels of VEGF protein itself in the supernatants (Fig.
4A, black bars). Nrf2 silencing reduced the VEGF-induced
increase in VEGF itself by 30% in comparison with BeWoshControl cells, which infers that partial up-regulation of VEGF
protein expression is attributed to at least one Nrf2-mediated
product (Fig. 4A, stippled bars). We next tested if Nrf2 activators induce VEGF expression in our system. BeWo-shControl
cells stimulated with 1 M sulforaphane and 1 mM bipyridyl (33)
showed a 2-fold increase in VEGF expression compared with
control cells. In contrast, BeWo-shNrf2 cells were not able to
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FIGURE 3. Protective effect of VEGF165 on H2O2-induced cytotoxicity via Nrf2/ARE pathway activation. BeWo cell lines stably transfected with control
shRNA (shControl; A and C) or shRNA against Nrf2 (shNrf2; B and D) were stimulated with or without 10 ng/ml VEGF165 for 3 h and exposed to 500 M H2O2 and
10 M Fe2 for 6 h. Cell viability (WST-1 assay; A and B) and cell toxicity (CytoTox-GloTM assay; C and D) assays were prepared. The graphs shows the mean S.E.
(n 8). ***, p 0.001; #, p 0.0001 between the two groups using Students t test; ns, not significant.

VEGF Activates Nrf2

up-regulate VEGF expression in response to sulforaphane

(Fig. 4B).
Gene expression for the VEGF protein is induced by carbon
monoxide produced by exogenous sources or the degradation
of heme by HO-1 (34). Therefore, the effect of CO on VEGF
expression in BeWo cells was examined. CORM-2 treatment
significantly increased VEGF protein levels in a low dose-dependent manner (Fig. 4C, left y-axis) in comparison with
untreated cells. Because a higher dose of CORM-2 (50 M) was
toxic, it did not further induce VEGF expression (Fig. 4C, right
y-axis).
We then examined whether CO is essential for VEGF-induced VEGF up-regulation. Cell treatment with the CO scavenger Hb (10 100 M) showed a dose-dependent inhibition of
VEGF-induced VEGF expression (Fig. 4D). These results indicate that VEGF-up-regulated VEGF expression depends on
Nrf2 and HO-1.

DISCUSSION
Several recently published studies have suggested that
decreasing VEGF levels might result in placental oxidative
stress in preeclampsia (13, 35, 36). Experimental animal studies
have demonstrated that elevated maternal sFlt-1 and decreased
VEGF concentrations result in increased oxidative stress (14).
The question as to how decreased VEGF concentrations
increase oxidative stress still remains unanswered.
DECEMBER 16, 2011 VOLUME 286 NUMBER 50

A recent study suggested a possible role of Nrf2 in mechanisms underlying preeclampsia. The study used genome-wide
transcriptional profiling of preeclamptic and normal pregnancies and showed that the Nrf2-mediated oxidative stress
response is overrepresented in preeclampsia (37). Thus, these
data strengthen our previous published results (25) and our
hypothesis that Nrf2 has a critical role in the etiology and progression of preeclampsia.
Nrf2 plays a key role in the adaptive response to oxidative and
electrophilic stress by maintaining the cellular self-defense. In
this study, we have provided substantial experimental evidence
that VEGF activates Nrf2 and up-regulates Nrf2-dependent
gene products within the well established cytotrophic cell line
BeWo (Fig. 1) (38).
Several studies have shown that protein kinases are involved
in the activation of Nrf2 (39). Because it is also known that
binding of VEGF to VEGF receptor-2 activates downstream
effectors, including the PKC, Raf, and ERK1/2-PI3K-focal
adhesion kinase pathways. Therefore, we examined whether
kinases are involved in the signal transduction of VEGF leading
to Nrf2 activation. Thus, the effects of various kinase inhibitors
on Nrf2 activation were tested. Of all tested inhibitors, only the
MEK1/2 inhibitors PD98059 and U0126 inhibited Nrf2 activation. Consequently, ERK1/2 activation is a prerequisite for Nrf2
activation by VEGF. However, Nrf2 might not be a direct subJOURNAL OF BIOLOGICAL CHEMISTRY

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FIGURE 4. Sulforaphane raises VEGF levels in BeWo cells. A, BeWo cell lines stably transfected with control shRNA and shRNA against Nrf2 were treated with
10 ng/ml VEGF165 for 6 h and then washed twice with PBS and supplied with FCS-free medium for 24 h. The levels of VEGF165 protein in the supernatants were
determined by ELISA. B, the cells were also treated with 1 M sulforaphane for 6 h, washed with PBS, and supplied with FCS-free medium for 24 h. The levels of
VEGF165 protein in the supernatants were determined by ELISA. Bipyridyl (1 mM) was used as a positive control. C, cells were exposed to the indicated
concentrations of CORM-2 for 24 h. VEGF165 levels in the supernatants were determined by ELISA (left y-axis), and cell viability was measured by WST-1 assay
(right y-axis). D, cells treated with 10 ng/ml VEGF165 were co-incubated with various doses of Hb (10 100 M) for 6 h, and the levels of VEGF in the cell lysate were
assessed by ELISA. **, p 0.005; ***, p 0.001; #, p 0.0001 versus cells treated with VEGF165 or sulforaphane alone.

VEGF Activates Nrf2

strate of ERK1/2. Instead, it has been suggested that ERK1/2

phosphorylates the nuclear transcription co-activator CBP
(cAMP-responsive element-binding protein-binding protein)
and that CBP enhances the Nrf2 transcriptional response (35,
36). In addition, BeWo cells were stimulated with VEGF, and
the phosphorylation status of ERK1/2 was analyzed to substantiate the activation status of the kinases (Fig. 2A).
It is generally accepted that oxidative or electrophilic stress is
a requirement for Nrf2 activation (19, 20). Thus, it was assumed
that this activation may occur via VEGF-induced NAD(P)H
oxidase activation because VEGF supposedly utilizes ROS as a
second messenger after VEGF receptor-2 stimulation (40).
However, NAD(P)H oxidase inhibition had no effect on the
VEGF-triggered Nrf2 activation (Fig. 2, A, , and F), so oxidative stress can be excluded here. In this work, we have shown
that VEGF induced Nrf2 activation and that this activation
occurred without oxidative stress stimulus. This means, in
effect, that VEGF stimulation prevents oxidative stress. Hence,
we hypothesized that decreased VEGF bioavailability during
preeclampsia results in reduced basal defense against oxidative
stress and in a higher vulnerability of the cells to oxidative
damage.
To test this hypothesis, BeWo cells were stimulated with
VEGF before challenge with H2O2, implying oxidative stress.
As expected, VEGF-stimulated cells showed less cell death and
lower vulnerability after oxidative stress treatment with H2O2
compared with unstimulated control cells (Fig. 3, A and C).

42870 JOURNAL OF BIOLOGICAL CHEMISTRY

These results clearly show a protective effect of VEGF against

oxidative stress, comparable with the described effects of EGF
(41). To elucidate the role of Nrf2 in the protective effect of
VEGF, a BeWo cell line carrying a stably transfected shRNA
against Nrf2 mRNA (BeWo-shNrf2) was utilized. This cell line
was no longer able to activate the Nrf2/ARE system (Fig. 1B).
Thus, shRNA technology instead of dominant-negative Nrf2
overexpression was applied because shRNA knocks down only
Nrf2. By contrast, dominant-negative Nrf2 blocked binding to
the ARE by competitive inhibition and thus blocked all factors
with ARE affinity. In these Nrf2-deficient BeWo cells, VEGF no
longer protected against oxidative stress (Fig. 3, B and D), which
infers that Nrf2 has a pivotal function in cytoprotection mediated by VEGF. These results indicate that cell death may be
induced in villous trophoblasts following ROS exposure and
demonstrate the placental protective effect of VEGF.
As a transcription factor, Nrf2 unfolds its protective effect via
up-regulation of genes coding for antioxidant enzymes. Therefore, we studied whether VEGF up-regulates the expression of
Nrf2 target genes in the applied system, particularly thioredoxin, TXNRD1, and HO-1 because these enzymes have been
shown to be expressed at lower levels in the placentas of women
suffering from preeclampsia (42 44). Supporting the proposed
hypothesis, all three corresponding Nrf2 target genes were upregulated in response to VEGF treatment (Fig. 1, D and F).
In particular, decreased gene expression for HO-1 and the
resultant decline in CO production have been shown to initiate
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FIGURE 5. Postulated model of the signal transduction of VEGF165-induced Nrf2 activation. VEGF receptor (VEGF-R) activation-induced ERK1/2
phosphorylation leads to Nrf2 liberation from the Nrf2 inhibitor Keap1. In the nucleus, Nrf2 binds to the cis-acting ARE and up-regulates the transcription
of cytoprotective genes: HO-1 and other antioxidant enzymes. HO-1 then contributes via biliverdin to protect the cell against oxidative damage (49) and
via CO to promote VEGF expression by increasing hypoxia-inducible factor-1 (HIF1) protein levels as shown elsewhere (#) (34, 50). HRE, hypoxia
response element.

VEGF Activates Nrf2

AcknowledgmentsWe thank Christiane Jaeschke, Susanne Echterhagen, Angela Rben, and Lian Shen for excellent technical assistance. We thank Wolfgang Graulich for production of the illustration
in Fig. 5.
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decreased VEGF bioavailability during preeclampsia results in
insufficient Nrf2 activation, reduced basal defense against oxidative stress, and higher vulnerability to placental oxidative cell
damage. The resulting damage to the placenta causes disproportionate release of toxic placental factors that are manifested
as preeclampsia and endanger maternal health. Specific
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against oxidative stress with an Nrf2 inducer, i.e. sulforaphane,
methsyticine, or andrographolide, at an early gestation stage
could prove to be a possible therapeutic option and may, in
turn, reduce the risk of preeclampsia and associated perinatal
complications.

VEGF Activates Nrf2

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Data supplement. VEGF stimulation had no effect either on p38 nor Akt kinase pathways.
(A) Immunoblot analysis utilising phospho-specific anti-p38 antibodies (p38) was used to
measure p38 activation. BeWo cells were treated with VEGF165 (10 ng/ml) in time dependent
manner, 10 M PMA was used as positive control for 1 h either alone or followed the
treatment with the inhibitor SB 203580 (20 M) for 30min. The membrane was stripped and
probed with anti-p38 antibodies for loading control. (B) The densities of corresponding p-p38
and p38 bands were measured and the ratio was calculated. The mean of three independent
experiments is shown. Data are presented as fold induction after treatment compared with
vehicle control (control = 1). (C) Immunoblot analysis utilising phospho-specific anti-Akt
antibodies (p-Akt) was used to measure Akt activation. The cells were either treated with
VEGF165 (10 ng/ml) in time dependent manner, or with 50 M H2O2 for 1 h which was used as
positive control either alone or followed the treatment with the inhibitor Wortmannin (1 M)
for 30min. The membrane was stripped and probed with anti-Akt antibodies for loading
control. (D) The densities of corresponding p-Akt and Akt bands were measured as described
in material and methods. (E) Dual luciferase Assay to analyze cell signalling of H2O2mediated Nrf2/ARE activation. The cells were pre-treated with 50 M PD 98059, 25 M SP
600125 or 1 M wortmannin for 30 min before the administration of 50 M H2O2. Data are
presented as x-fold induction of luciferase activity after treatment compared to control
(untreated cells, control = 1). Data represented are the average of three separate experiments
(mean SEM. ** p < 0.005, *** p < 0.001.

Interplay between Vascular Endothelial Growth Factor (VEGF) and Nuclear

Factor Erythroid 2-related Factor-2 (Nrf2): IMPLICATIONS FOR
PREECLAMPSIA
Nisreen Kweider, Athanassios Fragoulis, Christian Rosen, Ulrich Pecks, Werner Rath,
Thomas Pufe and Christoph Jan Wruck
J. Biol. Chem. 2011, 286:42863-42872.
doi: 10.1074/jbc.M111.286880 originally published online October 27, 2011

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