From the Medizinische Klinik mit Schwerpunkt
Hepatologie, Gastroenterologie, Endokrinologie und Stoffwechsel,
Charité, Campus Virchow-Klinikum, 13353 Berlin, Germany, the
§ Institut für Molekularbiologie und Tumorforschung,
Philipps-Universität Marburg, Marburg, Germany, and the
¶ Klinik für Chirurgie und Chirurgische Onkologie,
Robert-Rössle-Klinik, Max-Delbrück-Zentrum für
Molekulare Medizin, Charité, Campus-Buch,
Berlin, Germany
Received for publication, November 25, 2002, and in revised form, December 23, 2002
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ABSTRACT |
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Enhanced VEGF-A
(vascular endothelial growth
factor A) gene expression is associated with
increased tumor growth and metastatic spread of solid malignancies
including gastric cancer. Oxidative stress has been linked to
tumor-associated neoangiogenesis; underlying mechanisms, however,
remained poorly understood. Therefore, we studied the effect of
oxidative stress on VEGF-A gene expression in gastric
cancer cells. Oxidative stress generated by
H2O2 application potently stimulated VEGF-A
protein and mRNA levels as determined by enzyme-linked
immunosorbent assay and real-time PCR techniques, respectively, and
elevated the activity of a transfected ( Reactive oxygen species
(ROS),1 such as superoxide
O The gastric mucosa is permanently exposed to luminal oxidants generated
from ingested food, bacteria, and shed mucosal cells, and together with
the surface mucous layer, the gastric epithelium represents the first
line of defense against luminal oxidative alterations. Increased ROS
levels have been linked to peptic lesions of the gastric mucosa
triggered by ethanol, nonsteroidal anti-inflammatory drugs, stress,
ischemia-reperfusion, and Helicobacter pylori infection (10-12). In addition to these deleterious effects, ROS are also capable of influencing mucosal repair processes by stimulating epithelial proliferation, production, and release of mucosal growth factors as well as activation of proangiogenic pathways (13). Moreover,
several studies have suggested that oxidative stress is also involved
in gastric carcinogenesis (14, 15). Accordingly, epidemiological
studies indicated that application of antioxidants can significantly
reduce the risk of gastric cancer (15) and lead to regression of
gastric epithelial metaplasia (16), a lesion believed to reflect
premalignant states of the gastric mucosa.
Neoangiogenesis is a general pathophysiological mechanism critically
involved in healing of inflammatory and ulcerative epithelial lesions
as well as tumor growth and metastasis (17). Among the proangiogenic
factors identified so far, VEGF-A (vascular
endothelial growth factor
A) represents one of the most potent stimuli of neoangiogenesis (17, 18). In the stomach, enhanced VEGF-A gene expression has been identified to critically contribute to peptic
ulcer healing (13, 19-21). In addition, gastric adenocarcinomas were
found to frequently display high levels of VEGF-A expression accompanied by increased intratumoral microvessel density (22, 23),
whereas injection of VEGF-A-specific neutralizing antibodies were
capable of potently inhibiting the growth of gastric cancer xenotransplants in rodents (24). Despite the fact that these studies
clearly established an important role of VEGF-A-dependent angiogenesis in mucosal regeneration, peptic ulcer healing, and gastric
cancer, the pathways controlling VEGF-A gene expression in
these settings have not yet been defined.
Previous studies revealed that VEGF-A gene expression can be
influenced by extracellular growth factors, cytokines, and genetic alterations or hypoxia (17, 25). Moreover, current studies indicated
that angiogenic processes may be influenced by cellular redox processes
(26-30); potential mechanisms linking oxidative stress to
proangiogenic factors like VEGF-A, however, remained unclear (29).
Transcriptional activation of the VEGF-A promoter represents
a core mechanism through which expression of the VEGF-A gene
can be regulated (17, 18, 25). Several cis-acting promoter elements including a hypoxia-responsive site at Here we demonstrate that oxidative stress generated by the model
oxidant H2O2 at submillimolar concentrations
potently stimulates release and production of VEGF-A in gastric cancer
cells and provides evidence that transcriptional activation of the
VEGF-A promoter represents the underlying core mechanism.
Moreover, we identify the zinc finger transcription factors Sp1 and Sp3
as molecular mediators of the VEGF-A redox response and show
that Sp1/Sp3-activated GC-boxes located in the proximal
VEGF-A promoter differentially participate in oxidative
stress-dependent VEGF-A gene regulation. Furthermore, we show that MAPK ERK-related and to a lesser extent JNK-related signaling pathways are crucial for transmission of oxidative stress effects on the human VEGF-A gene in gastric
cancer cells.
Cell Culture--
AGS human gastric adenocarcinoma cells were
grown in Dulbecco's modified Eagle's medium (Invitrogen) supplemented
with 2 mM glutamine (Invitrogen), 100 units/ml penicillin,
100 µg/ml streptomycin (Biochrom KG, Berlin, Germany), and 10%
bovine calf serum (Biochrom KG) in a humidified atmosphere (5%
CO2, 95% air). Cultures of Drosophila
melanogaster Schneider cell line 2 (SL-2) were maintained in
Schneider's Drosophila medium (Invitrogen), supplemented
with 10% fetal bovine serum (Invitrogen), 100 units/ml penicillin, and
100 µg/ml streptomycin at 25 °C and atmospheric
CO2.
VEGF-A Enzyme-linked Immunoprecipitation Assay Determinations in
Cell Cultures--
2 × 104 AGS cells were plated in
24-well plates in growth medium overnight and then switched to
serum-free UltraCulture® medium (BioWhittaker Inc., Walkersville, MD)
containing 2 mM glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin. Conditioned medium was collected after incubation
with or without 1 mM hydrogen peroxide (Sigma) for various
times. To determine cellular VEGF-A production, cultured cells were
treated with lysis buffer containing 2 mM EDTA, 20 mM Tris (pH 7.8), 150 mM NaCl, 50 mM TaqMan® Quantitative Real Time Reverse Transcription-PCR
Analysis--
Primers were designed using Primer Express software
(PerkinElmer Life Sciences). Expression levels of human
VEGF-A and housekeeping gene Transfection Studies--
Transient transfections of cultured
AGS cells were carried out using the calcium phosphate precipitation
technique (DNA transfection kit; 5 Prime DNA Constructs and Reporter Plasmids--
VEGF-A
5'-deletion luciferase constructs have previously been reported (33).
To study VEGF-A regulatory elements in a heterologous promoter system, wild type and GC-box-mutated
VEGF-A(88/ Electrophoretic Mobility Shift Assays--
EMSA analysis of AGS
nuclear extracts was performed as previously described (38). In brief,
nuclear protein extracts (5 µg) were incubated with
[ Immunoblotting--
For signaling studies, AGS cells were
exposed to 1 mM H2O2, and whole
cell lysates were prepared in 20 mM Tris (pH 7.9), 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 10 mM K2HPO4,
1 mM Na3VO4, 10 mM NaF,
1.25% Nonidet P-40, and 10% glycerol as described (38). Following
SDS-PAGE and transfer to nylon membranes, Western blot analysis of
proteins was performed as described (38) using antibodies specifically
recognizing phosphorylated ERK1/2, p38, JNK, or MEK1/2 (New England
Biolabs, Beverly, MA) or corresponding nonphosphospecific antibodies to
demonstrate equal protein loading.
Oxidative Stress Stimulates Production and Release of VEGF-A
Protein and Increases VEGF-A mRNA Levels in AGS Cells--
To
determine the effects of oxidative stress on VEGF-A gene
expression, AGS cells were exposed to H2O2 and
analyzed for VEGF-A protein and mRNA levels.
H2O2 time- and dose-dependently
stimulated production and release of VEGF-A protein, having a maximum
(~3-fold elevation) at 12 h (Fig.
1, A and B). These
responses were redox-specific, as shown by their complete reversal
through application of the antioxidant N-acetylcysteine. To
investigate potential cell lysis due to H2O2
treatment, lactate dehydrogenase activity in cell culture supernatants
was analyzed in parallel (Fig. 1C) and found to be unaltered
(Fig. 1C). Similar to the changes observed on the protein
level, H2O2 treatment increased
VEGF-A mRNA levels (maximum at 8-12 h), suggesting that
enhanced transcription represents the mechanism underlying oxidative
stress responsiveness of the VEGF-A gene (Fig.
1D).
Oxidative Stress Potently Transactivates the VEGF-A Gene
Promoter--
To determine transactivating effects of oxidative stress
on the VEGF-A gene, functional transfection studies using
VEGF-A luciferase reporter gene constructs were performed.
H2O2 treatment of AGS cells potently stimulated
the VEGF-A promoter in a dose-dependent manner,
with maximal stimulation observed at 750 µM
H2O2 (Fig. 2A). Analysis of time-response
relationships demonstrated maximal VEGF-A transactivation
after 4-8 h of H2O2 exposure (Fig.
2B), which corresponds well with the results obtained in
VEGF-A mRNA studies (Fig. 1D).
Oxidative Stress Regulates the VEGF-A Promoter through a Proximal
39-bp Promoter Element--
To identify promoter regions mediating
oxidative stress responsiveness of the human VEGF-A gene,
functional 5' deletion analysis was performed. These studies revealed
that loss of the region spanning Oxidative Stress Stimulates Binding of Sp1 and Sp3 to the VEGF-A
Promoter--
To further characterize the nuclear factors binding to
the VEGF-A( Sp1 and Sp3 Potently Transactivate the VEGF-A( Oxidative Stress Stimulates Sp1 but Not Sp3 Transactivating
Capacity--
To investigate the influence of oxidative stress on the
transactivating capacity of Sp1 and Sp3, we cotransfected AGS cells with Gal4-Sp1 or Gal4-Sp3 expression constructs and the 5×Gal4-Luc reporter plasmid (Fig. 6). Whereas
transfection of 5×Gal4-Luc or Sp1/Sp3 transactivator plasmids alone
produced reporter gene activity close to background levels,
cotransfection of either Gal4-Sp1 or Gal4-Sp3 along with the
5×Gal4-Luc reporter plasmid significantly increased basal promoter
activity (~50-fold). Moreover, oxidative stress clearly increased Sp1
transactivating capacity (~3-fold) but did not significantly
influence Sp3. These data confirm the regulatory influence of oxidative
stress on Sp1 and suggest potential differences in the regulatory
mechanisms controlling the activity of Sp1 and Sp3 in response to
cellular oxidative stress in gastric cancer cells.
Proximal GC-boxes Differentially Contribute to VEGF-A Promoter
Regulation--
To elucidate the functional importance of individual
GC-boxes located within the Analysis of Signaling Cascades Mediating Oxidative Stress
Responsiveness of the VEGF-A Promoter--
To explore the signaling
pathways activated by oxidative stress, the influence of
H2O2 on the phosphorylation status of key kinases related to redox-triggered signaling was investigated. Exposure
of AGS cells to oxidative stress led to a rapid increase in ERK1/2,
JNK, and p38 kinases phosphorylated (Fig.
8A), reaching maximal effects
after 5-10 min and sustained hyperphosphorylation throughout the
entire experimental period of 120 min. Similarly, the upstream ERK1/2
kinase MEK1 was hyperphosphorylated in response to oxidative stress,
showing a similar time-response relationship. After identification of
MEK1/2/ERK1/2, JNK, and p38 as signaling targets of oxidative stress,
we examined the functional role of these kinases for VEGF-A
promoter regulation in transfection assays employing DN kinase mutants
(Fig. 8B). Application of DN ERK1 and/or DN ERK2 decreased
H2O2 responsiveness of the VEGF-A
promoter by 40-50% (Fig. 8B), whereas the PMA response was
almost abolished. Similarly, functional impairment of Raf-1 or Ras,
both of which have been located upstream of the MEK1/2 In the present study, we demonstrate oxidative
stress-dependent stimulation of VEGF-A production and
release in gastric cancer cells and provide clear evidence that
oxidative stress regulates VEGF-A gene expression through
transcriptional mechanisms. Previous studies in epithelial and
nonepithelial cell models suggested a link between the cellular redox
status and enhanced VEGF-A production and/or secretion (26-30);
underlying molecular determinants, however, have not yet been
clarified. To provide a detailed functional analysis of molecular
pathways mediating the effects of oxidative stress on the human
VEGF-A gene, we initially investigated participating cis- and trans-activating factors employing 5'
deletion analysis of the VEGF-A promoter, DNA element
transfer studies, and systematic core promoter mutagenesis. These
studies revealed that a region spanning Transcription factor Sp1 belongs to the superfamily of Sp-like zinc
finger proteins and has been implicated in the regulation of
constitutively expressed "housekeeping genes" as well as genes influencing growth and differentiation (for a review, see Ref. 43). In
addition, current studies demonstrated that Sp1 also participates in
the regulation of inducible gene expression and that interaction of Sp1
with other transcription factors and/or cofactors such as CREB-binding
protein, p300, or CRSP 84 may represent an important transcriptional
control mechanism (43). More recently, it became clear that Sp1 can
also be regulated through changes of its phosphorylation state (44),
and subsequently, different signaling pathways including
Ras-dependent activation of the MEK1/ERK cascade
have been identified to target Sp1 (45-47). Similar to Sp1, Sp3
represents a zinc finger transcription factor comprising highly
conserved DNA-binding domains, but analysis of their functional properties revealed significant differences between these two proteins
(42, 43). Sp3 activates GC-rich DNA elements with affinities similar to
other Sp proteins (42, 43). In some systems, however, Sp3 has been
described to lack intrinsic activity and accordingly can act as
transcriptional repressor of other transcription factors binding to the
same element (42, 43). As demonstrated by experiments in SL-2 Schneider
cells, which represent the appropriate cell model for analysis of Sp
factor-dependent effects, Sp1 and Sp3 exert equipotent
transactivating properties on the VEGF-A promoter, without
any indication of Sp3 acting as an inhibitor of
Sp1-dependent effects (Fig. 5). To explore the influence of
oxidative stress on the transactivation capacity of Sp1 and Sp3, we
employed appropriate Gal4-Sp1- or Gal4-Sp3/Gal4-luciferase cotransfection systems (48, 49). Interestingly, we observed that
application of oxidative stress enhanced the transactivating capacity
of Sp1 but not of Sp3, suggesting potential differences in the
regulatory mechanisms controlling the activity of these transcription
factors in response to cellular oxidative stress (Fig. 6). Recent
studies demonstrated that the transactivating capacity of Sp3 can be
suppressed through SUMO-dependent modification of the
transcription factor (49, 50). To what extent this mechanism also
contributes to regulation of Sp3 in context of VEGF-A gene
regulation remains to be explored in future studies.
Previous studies demonstrated that depending on the cellular context
and/or the stimulus investigated, the VEGF-A promoter can
also be bound and transactivated by non-Sp transcription factors (30,
34). In contrast to these studies, in gastric cancer cells, no
transcription factors other than Sp1 and Sp3 were detected to bind to
the VEGF-A( After identifying the proximal GC-rich site at 2018) VEGF-A
promoter reporter gene construct in a time- and
dose-dependent manner (4-8-fold). These effects were
abolished by the antioxidant N-acetylcysteine,
demonstrating specificity of oxidative stress responses. Functional 5'
deletion analysis mapped the oxidative stress response element of
the human VEGF-A promoter to the sequence
88/
50, and a
single copy of this element was sufficient to confer basal promoter
activity as well as oxidative stress responsiveness to a heterologous
promoter system. Combination of EMSA studies, Sp1/Sp3 overexpression
experiments in Drosophila SL-2 cells, and systematic
promoter mutagenesis identified enhanced Sp1 and Sp3 binding to two
GC-boxes at
73/
66 and
58/
52 as the core mechanism of
oxidative stress-triggered VEGF-A transactivation.
Additionally, in Gal4-Sp1/-Sp3-Gal4-luciferase assays, oxidative stress
increased Sp1 but not Sp3 transactivating capacity, indicating
additional mechanism(s) of VEGF-A gene regulation.
Signaling studies identified a cascade comprising Ras
Raf
MEK1
ERK1/2 as the main pathway mediating oxidative
stress-stimulated VEGF-A transcription. This study for the
first time delineates the mechanisms underlying regulation of
VEGF-A gene transcription by oxidative stress and thereby
further elucidates potential pathways underlying redox control of neoangiogenesis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
975, proximal GC-rich
elements, and Egr-1 and AP2 recognition motifs were found to
participate in VEGF-A gene regulation (31-35). Depending on the cellular context and/or the stimulus investigated, these
recognition motifs and/or their respective binding factors variably
contribute to VEGF-A expression control (31-35). Moreover,
VEGF-A gene expression has been shown to be regulated
through several signaling cascades comprising MAP kinase ERK-, JNK-,
and p38-dependent cascades as well as NF-
B pathways (17,
18, 25, 32). In contrast, signaling pathways mediating the effects of
oxidative stress on the VEGF-A gene await clarification.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-glycerolphosphate, 0.5% Nonidet P-40, 1% glycerol,
1 mM Na3VO4, 1 mM
dithiothreitol, 5 µg/ml aprotinin, 10 mM NaF, 2 µM leupeptin, and 2 mM phenylmethylsulfonyl fluoride. Protein concentrations were determined using the Protein Assay Kit® (Bio-Rad). VEGF-A concentrations were assessed
using a commercial enzyme-linked immunoprecipitation assay
(QuantikineTM; R&D Systems, Minneapolis, MN), according to
the manufacturer's instructions, and normalized to protein content. To
exclude cell damage after H2O2 treatment,
lactate dehydrogenase activity in cellular supernatants was measured
using the CytoTox96® nonradioactive cytotoxicity assay kit (Promega,
Mannheim, Germany).
-actin were determined using
the following primer pairs: forward VEGF-A
(5'-cttgccttgctgctctacc-3') and reverse VEGF-A (5'-cacacaggatggcttgaag-3'); forward
-actin
(5'-tcagcaagcaggagtatgacga-3') and reverse
-actin
(5'-cgcaactaagtcatagtccgcc-3'). Specificity of the products was
demonstrated for each fragment by melting curve analysis, gel
electrophoresis, and sequencing. The SYBR Green I assay and the ABI
Prism 7700 sequence detection system (Applied Biosystems) were used for
detecting real-time quantitative PCR products from 0.25-2.5 ng of
reverse-transcribed cDNA samples. SYBR Green I dye intercalation
into the minor groove of double-stranded DNA reaches an emission
maximum at 530 nm. PCRs for each sample were done in triplicate for
both target gene and
-actin control. Quantitation of mRNA
expression was carried out by relating the PCR threshold cycle obtained
from tissue samples to a cDNA standard curve. In the case of
-actin, the PCR threshold cycle was related to the number of AGS
cells from which
-actin was extracted. The normalized amount of
VEGF-A expression was obtained by dividing the averaged
sample value by the averaged
-actin value of AGS cells and given in
copies of VEGF-A per AGS cell.
3 Prime, Inc., Boulder,
CO) as previously described (36-38). Briefly, AGS cells were
transfected with 0.5 µg of reporter gene plasmid per well unless
otherwise indicated. To correct for transfection efficiency, 50 ng/well
of Renilla luciferase construct pRL-TK (Promega) were
cotransfected. After transfection, cells were maintained in serum-free
UltraCulture® (BioWhittaker) for 24 h. Unless otherwise
indicated, cells were stimulated for 6 h with 500 µM
hydrogen peroxide or 10
8 M phorbol
12-myristate 13-acetate (PMA; Biomol, Plymouth Meeting, PA). To block
p38 MAP kinase, incubations were performed in the presence of 25 µM SB 202190 (Calbiochem). For transfection of SL-2
cells, 106 cells/well were transfected with 3 µg of
VEGF-A(
88/
50)-Luc along with 1 µg of expression
constructs (Sp1, Sp3, Sp1-DBD, Sp3-DBD) or empty vectors. Luciferase
activities were detected in a monolight Luminometer (EG Berthold, Bad
Wildbach, Germany) using the "Dual Luciferase Reporter Assay"
(Promega). Incubations were performed in triplicates, and results were
normalized for transfection efficiency and calculated as means ± S.E. Values were expressed as arbitrary light units or -fold increases
in luciferase activity compared with controls. Statistical
significances were calculated using Student's t test (*,
p < 0.05; **, p < 0.01; ***,
p < 0.001).
50) oligonucleotides (Table
I) were subcloned at HindIII
(5') and XhoI (3') restriction sites into construct pT81-Luc
(39). Constructs were confirmed by restriction analysis and
dideoxysequencing. Reporter plasmid Gal4-Luc, in which the luciferase
gene is driven by a multimer of the Gal4 yeast transcription
factor-binding element (40), as well as transactivator constructs
Gal4-Sp1 and Gal4-Sp3 have been described before (41). Expression
constructs encoding wild type Sp1 or Sp3 or corresponding mutants
lacking their transactivation domain (pPACSp1-DBD and pPACSp3-DBD) have
also been described (42). Constructs encoding human ERK1, ERK2, MEK1,
or Raf-1 have been used before (36, 38). Expression constructs encoding dominant-negative mutants of ERK1 (DN ERK1(K71R)), ERK2 (DN
ERK2(K52R)), MKK4 (DN MKK4), Raf-1 (DN Raf-1), and Ras (DN Ras15(G15A))
have previously been employed (36, 38).
Oligonucleotide sequences for generation of heterologous reporter
constructs and EMSA
-32P]ATP-radiolabeled double-stranded
oligonucleotides. DNA binding reactions were performed in a buffer
containing 20 mM HEPES (pH 8.4), 1 µg of poly(dI-dC), 10 µg of bovine serum albumin, 60 mM KCl, 5 mM
dithiothreitol, 1 mM ZnCl2, and 10% glycerol
for 30 min at 30 °C. For competition experiments, nuclear extracts
were incubated with a 100-fold molar excess of double-stranded
competitor oligonucleotides. For supershift experiments, nuclear
extracts were incubated with 1 µl of anti-Sp1, anti-Sp3, anti-Sp4,
anti-AP2, and anti-Egr-1 antibodies (Santa Cruz Biotechnology, Inc.,
Santa Cruz, CA). DNA-protein complexes were electrophoresed in 6%
nondenaturing polyacrylamide gels containing 0.5× TBE (50 mM Tris, 50 mM borate, 2 mM EDTA).
Gels were dried and exposed to Eastman Kodak Co. BioMax MR films
(Amersham Biosciences) using an intensifying screen.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Oxidative stress stimulates production and
release of VEGF-A protein as well as VEGF-A mRNA
levels in AGS cells. AGS cells exposed to 1 mM
H2O2 for various time spans were assayed for
cellular VEGF-A production (A) or VEGF-A release into cell
culture supernatants (B). To investigate potential cell
damage after H2O2 stimulation, cell culture
supernatants were assayed for the release of cellular lactate
dehydrogenase (C). Results are expressed as mean ± S.E. of three separate experiments (asterisks indicate
statistically significant differences; *, p < 0.05).
D, TaqMan real time reverse transcription-PCR analysis of
H2O2-treated AGS cells. Given are the
expression levels of human VEGF-A versus
-actin mRNA levels in multiples of the basal level. Data shown
represent a typical result obtained from a series of three independent
experiments.
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Fig. 2.
Oxidative stress potently transactivates the
VEGF-A gene promoter. A, AGS cells
transiently transfected with VEGF-A ( 2018/+50)-Luc were
stimulated with increasing concentrations of hydrogen peroxide.
B, for time course experiments, cells were treated with 500 µM H2O2 or left untreated for the
indicated time points. Luciferase activity is expressed as -fold
increase relative to unstimulated controls for each time point. Under
all conditions investigated, responses to H2O2
were statistically significant.
2018 to
85 had no substantial
influence on VEGF-A promoter activity. In contrast, removal
of an additional 33 nucleotides abolished basal and
H2O2-stimulated VEGF-A promoter
activity (Fig. 3A), suggesting
that
85/
52 comprises essential regulatory elements. To investigate
the enhancer properties of this promoter region in detail, it was
transferred to a heterologous promoter system (pT81-Luc) and
functionally analyzed (Fig. 3B). The presence of the
88/
50 element conferred elevation of basal transcriptional activity
(10-15-fold) as well as H2O2 responsiveness to
the per se redox-insensitive pT81-Luc vector (Fig.
3B). Redox responses of VEGF-A
88/
50 were
abolished by the antioxidant N-acteylcysteine (Fig.
3B). Analysis of the
88/
50 promoter region in EMSA
studies showed that under basal and H2O2-
stimulated conditions, two major complexes were bound to this element
(Fig. 3C) and that H2O2 treatment of
AGS cells resulted in a parallel increase of complex I and II (Fig.
3C, lanes 3 and 4). Only
infrequently we observed a third complex (complex III), which was not
influenced by any oligonucleotides used in EMSA studies (data not
shown) and therefore must be regarded as nonspecific.
H2O2 treatment maximally enhanced complex I and II after 10 min, whereas prolonged exposure decreased complex formation
(lanes 5-8). Under identical conditions,
H2O2 did not increase the binding of CREB,
USF-1, and USF-2 transcription factors to a minimal cox-2
(cyclooxygenase 2) promoter
fragment,2 demonstrating that
oxidative stress-triggered protein/DNA interaction at the
VEGF-A promoter is not a random nonspecific process.
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Fig. 3.
Oxidative stress regulates the
VEGF-A promoter through a proximal 39-bp promoter
element. A, AGS cells were transiently transfected with
VEGF-A 5' deletion constructs, stimulated with 500 µM H2O2 or left untreated, and
assayed for luciferase activity after 6 h. Results are expressed
as arbitrary light units, whereas -fold increases in response to
H2O2 are additionally given on the
right. B, the 88/
50 fragment was transferred
into the enhancerless vector pT81-Luc and used in transfection
experiments. To confirm specificity of oxidative stress responses, the
antioxidant N-acetylcysteine (20 mM) was used.
Results are expressed as mean ± S.E. of three separate
experiments (asterisks indicate statistically significant
differences; *, p < 0.05). C, crude nuclear
protein extracts from unstimulated or
H2O2-stimulated (500 µM) AGS
cells were prepared after the indicated time points and subjected to
EMSA analysis using the VEGF-A(
88/
50) sequence as
32P-labeled probe. Data shown represent a typical result
obtained from a series of three independent experiments.
88/
50) element, EMSA competition
studies were carried out (Fig. 4). The
selection of consensus oligonucleotides used in these studies (Sp1,
AP2, and Egr-1) was based on the presence of these sequences within the
88/
50 sequence. We found that complex I and II, which were found in
unstimulated (Fig. 4A, lanes 1-7) as
well as stimulated samples (lanes 8-14) were
completely competed out by an excess of Sp1 consensus sequence
(lanes 2 and 9). In contrast, AP2
consensus sequence was clearly less effective (lanes
6 and 13), whereas Egr-1 consensus
oligonucleotides had no effect (lanes 4 and
11). To confirm these results, EMSA supershift experiments
using specific antibodies recognizing Sp1, Sp3, Sp4, AP2, or Egr-1 were
performed. These studies identified complex I as containing Sp1 protein
(Fig. 4, B (lanes 3 and 8)
and C (lanes 2, 5,
6, and 8)), whereas complex II was shown to
consist of Sp3 (Fig. 4C, lanes 3,
5, 7, and 8). In contrast, zinc finger
protein Sp4 as well as AP2 and Egr-1 were not detected. In control
EMSAs, the specificity of the AP2 antibody was confirmed using HeLa
cell nuclear extracts together with a labeled AP2 consensus site as a
probe (data not shown). Functionality of the Egr-1 antibody was
confirmed in supershifts employing AGS nuclear extracts together with a
proximal fragment of the mouse chromogranin A gene promoter as
radiolabeled probe (38).
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Fig. 4.
Oxidative stress stimulates binding of
Sp1 and Sp3 to the VEGF-A promoter. A,
nuclear extracts of untreated (lanes 1-7) and
stimulated AGS cells (500 µM H2O2
for 10 min) (lanes 8-14) were incubated with the
probe VEGF-A( 88/
50) in the absence or presence of
unlabeled double-stranded oligonucleotides representing
VEGF-A(
88/
50) as well as Sp1, Egr-1, or AP2
consensus sequences. Application of mutant oligonucleotides served as a
control. B and C, specific antibodies recognizing
Sp1, Sp3, Sp4, Egr-1, or AP2 were incubated with unstimulated
(lanes 1-5) or
H2O2-stimulated nuclear extracts
(lanes 6-10) and radiolabeled
VEGF-A(
88/
50) probe. The arrows indicate
specific complexes. Data shown represent a typical result obtained from
a series of three independent experiments.
88/
50)
Element--
After identification of Sp1 and Sp3 binding to the
VEGF-A(
88/
50) element, we next investigated the
functional impact of both transcription factors on this element in
transient transfections. For this purpose, we employed
Drosophila SL-2 cells, a cell model lacking endogenous Sp
factors and therefore allowing investigation of gene regulation without
interference of endogenous Sp proteins (41). Overexpression of either
Sp1 or Sp3 potently stimulated the VEGF-A(
88/
50) element
(7-8-fold), demonstrating that the VEGF-A promoter is
highly reactive to both transcription factors (Fig.
5). Cotransfection of both factors did
not yield in higher effects compared with the expression of the
individual factors. Sp1 and Sp3 mutants lacking their DNA-binding
domain exhibited virtually no transactivating effect on the
VEGF-A(-88/-50) element (Fig. 5), confirming the specificity
of results obtained with wild type expression constructs.
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Fig. 5.
Sp1 and Sp3 potently transactivate the
VEGF-A( 88/
50) element. Drosophila SL-2
cells were transiently transfected with
VEGF-A(
88/
50)-Luc or empty vector pT81-Luc along with
expression constructs encoding Sp1 and/or Sp3. Cells were harvested
after 24 h and assayed for luciferase activity. Results are
expressed as -fold increase of control and represent mean ± S.E.
of three separate experiments. The asterisks indicate
statistically significant differences. **, p < 0.01.
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Fig. 6.
Oxidative stress stimulates Sp1 but not Sp3
transactivating capacity. AGS cells were transiently transfected
with Gal4-Luc alone or Gal4-Luc along with Gal4-Sp1 or Gal4-Sp3
constructs, respectively. Cells were treated with
H2O2 or PMA at the indicated concentrations or
left untreated. Results are expressed as arbitrary light units
(A.L.U.) and represent mean ± S.E. of three separate
experiments. The asterisks indicate statistically
significant differences. *, p < 0.05; **,
p < 0.01.
88/
50 region, a series of
VEGF-A(
88/
50) mutants was characterized in transfection
assays and EMSA studies (Fig. 7).
Compared with the wild type VEGF-A(
88/
50) element, mutation of GC-box I (Mut 1) did not affect oxidative stress-triggered activation of this sequence (Fig. 7A). Functional loss of
GC-box II or GC-box III, however, significantly inhibited basal and
stimulated VEGF-A promoter activity (Mut 4, Mut 6),
suggesting that oxidative stress responsiveness of the
VEGF-A(
88/
50) region requires the functional integrity
of these two elements. In line with this view, simultaneous
inactivation of GC-box II and III in the presence of an intact GC-box I
(Mut 5), reduced basal and stimulated promoter activity to background
levels, a result that was also observed after mutation of all three
GC-boxes (Mut 3). Furthermore, when GC-box I was mutated together with
either GC-box II (Mut 2) or GC-box III (Mut 7), promoter activity was
decreased to a similar degree as observed after individual mutagenesis
of GC-boxes II or III (~50%), further confirming the lack of
functional relevance of GC-box I in VEGF-A(
88/
50)
regulation. EMSA studies using mutated VEGF-A(
88/
50)
oligonucleotides either as competitors (Fig. 7B) or as
radiolabeled probes (Fig. 7C, data only shown for mutants
Mut 1-3) revealed that mutation of individual GC-boxes I, II, or III
had little or no effect on factor binding, supporting the concept that
the loss of single GC-boxes can be compensated regarding Sp1/Sp3
binding by those GC-boxes remaining intact (Fig. 7, B
(lanes 2, 5, and 7) and
C (lane 2)). Examination of double mutations VEGF-A(
88/
50) Mut 2, Mut 5, and Mut 7 showed
reduction of Sp factor binding to variable degrees (Fig. 7,
B and C, Mut 2), whereas alteration of all three
GC-boxes abolished the ability of VEGF-A(
88/
50) to bind
nuclear proteins (Fig. 7, B and C, Mut 3).
Interestingly, mutation of GC-box I together with either GC-box II (Mut
2) or GC-box III (Mut 7) showed only little effect on factor binding
(Fig. 7B and data not shown), which correlates with the
retained function of these mutants in transfection assays (Fig.
7A). In contrast, double mutation of GC-boxes II and III clearly reduced Sp1/Sp3 binding (Fig. 7, B (lane
5) and C (lane 3)), being in
accordance with the reduced functional activity of this mutant.
Together, these data strongly suggest that GC-boxes II and III are
indispensable for basal and oxidative stress-triggered activity of the
VEGF-A(
88/
50) element, whereas GC-box I, which also
possesses the capacity to bind Sp1/Sp3 transcription factors, has
obviously no functional importance in this context.
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Fig. 7.
Proximal GC-boxes differentially contribute
to VEGF-A promoter regulation. GC-box mutations
were introduced into the promoter fragment 88/
50, subcloned into
the enhancerless reporter vector pT81-Luc, and used in transfections.
AGS cells were stimulated with H2O2 (500 µM) or left untreated. Data are expressed as arbitrary
light units, and -fold increases in response to
H2O2 are additionally given on the
right. Results represent means ± S.E. of three
separate experiments, and asterisks indicate statistically
significant differences. *, = p < 0.05. B,
EMSA competition studies were performed with AGS cell nuclear extracts
and VEGF-A(
88/
50) as radiolabeled probe. Mutant
VEGF-A(
88/
50) oligonucleotides were used as cold
competitors as indicated. Data shown represent a typical result
obtained from a series of three independent experiments. C,
crude nuclear protein extracts of AGS cells were incubated with
double-stranded, radiolabeled DNA probes representing
VEGF-A(
88/
50) mutants Mut 1, Mut 2, and Mut 3 (lanes 2-4) or wild type sequence
(lane 1). Supershift studies employed anti-Sp1 or
anti-Sp3 antibodies as indicated. Data shown represent a typical result
obtained from a series of three independent experiments.
ERK1/2
signaling module, potently inhibited H2O2- and
PMA-dependent VEGF-A promoter activation (~60-70% reduction). In contrast, application of a
p38-specific kinase inhibitor (SB 202190) had no effect. Interruption
of JNK-related signaling by transfection of a dominant-negative MKK4
mutant resulted in modest but reproducible inhibition of
H2O2-dependent VEGF-A transactivation (~30% inhibition), however, without reaching
statistical significance. PMA responsiveness of the
VEGF-A promoter was not altered by DN MKK4. To further
explore the role of the Raf
MEK1/2
ERK1/2 pathway in
VEGF-A regulation, ectopic expression of these kinases was
performed. Forced expression of ERK1 or ERK2
dose-dependently transactivated the VEGF-A
promoter, whereas co-expression of both kinases led to additive effects
(Fig. 8C). Similar to ERKs, overexpression of MEK1 or Raf-1
potently stimulated VEGF-A promoter activity.
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Fig. 8.
Analysis of signaling cascades mediating
oxidative stress responsiveness of the VEGF-A
promoter. A, phosphorylation of ERK1/2, p38, JNK,
and MEK1/2 in response to H2O2 (500 µM) was investigated using phosphospecific antibodies in
Western blot analysis (upper panels). As a
loading control, blots were stripped and reprobed with corresponding
nonphosphospecific antibodies (lower panels).
Data shown represent a typical result obtained from a series of three
independent experiments. B, AGS cells were transfected with
construct VEGF-A( 88/
50)-Luc along with various dominant
negative kinase mutants or the corresponding empty vectors and exposed
to H2O2 (500 µM) or PMA (10 nM). Additionally, inhibitor SB 202190 was applied to
inhibit MAPK p38-dependent effects. Results represent
means ± S.E. of three separate experiments, and
asterisks indicate statistically significant differences. *,
p < 0.05; **, p < 0.01. C,
AGS cells transfected with overexpression constructs encoding wild type
ERK1/2, MEK1, or Raf-1 along with VEGF-A(
88/
50)-Luc were
analyzed for luciferase activity. Results were expressed as -fold
increase of control transfectants, and data shown represent a typical
result obtained from a series of three independent experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
88 to
50 is indispensable
for basal as well as oxidative stress-triggered VEGF-A
promoter activity. This GC-rich promoter region, which comprises
consensus binding elements for Sp-like zinc finger proteins as well as
AP2 and Egr-1 transcription factors, has been demonstrated to be
involved in VEGF-A gene regulation in various cell systems
including fibroblasts (32, 33), keratinocytes (34), and glioma cells
(35). Our study revealed that in gastric cells the
88/
50 element is
bound by the zinc finger transcription factors Sp1 and Sp3 (Fig. 4).
Application of oxidative stress increased DNA-protein complex formation
at the
88/
50 site, strongly suggesting that enhanced binding of
Sp1/Sp3 to the VEGF-A promoter represents an important
mechanism through which oxidative stress transactivates the
VEGF-A gene (Fig. 3). This mechanism is clearly different
from previous observations made in platelet-derived growth
factor-stimulated human fibroblasts and tumor growth
factor-
-stimulated skin keratinocytes, showing constitutive
binding of Sp1 and/or Sp3 to the proximal VEGF-A promoter
without detectable changes upon stimulation (33, 34). Enhanced binding
of Sp1 to the proximal VEGF-A promoter has been observed
after activation of the MAPK/ERK signaling pathway in hamster
fibroblasts (32). In this cell type, however, Sp1 was found to
functionally cooperate with the transcription factor AP2, whereas Sp3
was not involved (32). Therefore, oxidative stress-triggered
transactivation of the VEGF-A promoter through enhanced
binding of both Sp1 and Sp3 represents a novel molecular mode of
VEGF-A transactivation.
88/
50) region. Initial competition studies using unlabeled oligonucleotides representing consensus binding sites
for candidate transcription factors binding to the
88/
50 region
suggested that AP2 may be interacting with this element (Fig.
4A, lane 13). Detailed analysis of
this phenomenon, however, revealed that in AGS cells the AP2 consensus
sequence binds Sp transcription factors (data not shown), and
therefore, its effect in EMSA competition studies reflects interaction
with Sp1 and Sp3 binding.
88/
50 as the
critical promoter element mediating VEGF-A oxidative stress responsiveness, we aimed to define the functional properties of three
GC-boxes as putative Sp1/Sp3 binding sites located within the
88/
50
region. For this purpose, a systematic mutational analysis of these
elements was performed in functional transfection studies. In parallel,
the influence of these mutations on transcription factor binding was
investigated in EMSAs. We found that the structural integrity of the
two most 3'-located GC-boxes II and III was indispensable for full
basal and oxidative stress-stimulated VEGF-A promoter activity, whereas the 5'-located GC-box I has only minor importance for
VEGF-A gene regulation in gastric cancer cells (Figs. 7 and 9). To our knowledge, this mapping of
structure/function relationships within the human VEGF-A
core promoter employing a combination of EMSA techniques and functional
promoter studies represents the first detailed evaluation of Sp/XKLF
binding sites for basal and stimulated transcriptional regulation of
the human VEGF-A gene. A similar approach had currently been
used to characterize the importance of the proximal Sp factor binding
sites for basal VEGF-A gene regulation in pancreatic cancer
cells (31). This study, however, focused entirely on
stimulus-independent VEGF-A transcription and GC-box
mutagenesis, was less complete, not clearly allowing the determination
of how individual promoter elements contribute to VEGF-A
transcription (31).
View larger version (50K):
[in a new window]
Fig. 9.
Regulation of VEGF-A
transcription by oxidative stress. Oxidative stress potently
up-regulates release and production of human VEGF-A in gastric cancer
cells, and transcriptional activation of the VEGF-A gene
promoter represents the underlying core mechanism. Enhanced binding of
Sp1 and Sp3 to two GC-boxes at 73/
66 and
58/
52 represents the
core mechanism of oxidative stress-triggered VEGF-A
transactivation. In addition, redox-triggered posttranslational
modification of Sp1, but not Sp3, represents an additional mechanism of
VEGF-A regulation. Activation of Ras
Raf
MEK1
ERK1/2 signaling and to a lesser extent JNK-related pathways are
crucial for transmission of oxidative stress effects on the human
VEGF-A gene.
Epithelial oxidative stress responses comprise activation of various
signaling cascades including the proliferation-associated MAPK-ERK
pathway as well as stress-related MAPK-JNK and -p38 kinase cascades (2,
5-7). In gastric cancer cells, Ras-dependent activation of
the Raf MEK1
ERK1/2 kinase module has been shown to be a major
pathway through which oxidative stress can influence transcriptional
responses (37). Our current study shows that H2O2 treatment of gastric cancer cells at a
concentration of 1 mM led to increased phosphorylation of
ERK1/2, MEK1, p38, and JNK, indicating activation of associated
upstream kinases (Fig. 8A). The finding that JNK is
hyperphosphorylated in response to H2O2 differs
from our previous observations (37) and may be explained by the fact
that H2O2 concentrations required for
VEGF-A transactivation were at least 2,5-fold lower than the
doses at which stimulation of HDC promoter activity was
obtained (2.5-10 mM). In full agreement with our previous
work, functional studies revealed that the Raf-1/MEK1/ERK1/2 kinase
module represents the major pathway through which oxidative stress
transactivates the VEGF-A promoter. This view is supported
by the finding that interruption of this cascade at different levels
resulted in substantial impairment of
H2O2-triggered VEGF-A promoter
activity (70-80% inhibition; Fig. 8B). Conversely,
activation of the signaling cascade upstream of ERKs by overexpression
of Raf or MEK, respectively, resulted in robust transcriptional
VEGF-A responses, which were also obtained after ERK1 and/or
ERK2 overexpression (Fig. 8C). Additionally, application of
an inhibitory Ras mutant (N15) substantially impaired the effect of
H2O2 on the VEGF-A promoter,
confirming our previous observation that oxidative stress activates the
Raf-1/MEK1/ERK1/2 kinase cascade in gastric cancer cells through (a)
Ras-dependent mechanism(s) (37). These findings are
compatible with the concept of the Raf/MEK1/ERK1/2 cascade being a
critical signaling in VEGF-A gene regulation as shown in
other experimental systems including acidic pH challenge of human
glioma cells (51), insulin-like growth factor-I stimulation of NIH3T3
fibroblasts (52), and hamster fibroblast models (30). The finding that
H2O2 stimulated JNK phosphorylation, whereas
functional interruption of JNK signaling by application of a dominant
negative MKK4 mutant reduced VEGF-A promoter activity by
30% (Fig. 8B), is well compatible with the MKK4/JNK
module acting as additional signaling route mediating VEGF-A
oxidant responsiveness in gastric cells (Fig. 9). Similar to these
findings, evidence for dual regulation of VEGF-A expression through both MAPK-ERK and -JNK pathways has been observed in primary rat astrocytes and glioblastoma cells exposed to ionizing
radiation (53) as well as up-regulation of the GADD45 gene
in response to UV radiation (54).
Together, identifying oxidative stress as a potent stimulus of
VEGF-A gene expression in gastric cancer cells, our results support the concept that changes in the cellular redox status can
directly exert regulatory effects on proangiogenic processes. Furthermore, the detailed delineation of pathways and structural elements mediating oxidative stress responsiveness of the
VEGF-A gene can help to develop novel therapeutic approaches
to target VEGF-A gene expression in benign and/or malignant
(patho)biological settings.
![]() |
ACKNOWLEDGEMENTS |
---|
We express gratitude to G. Finkenzeller, B. Simon, R. Davis, M. Naumann, and M. Karin for providing plasmid DNA and D. Pfander for helpful discussions.
![]() |
FOOTNOTES |
---|
* This work was supported by Deutsche Forschungsgemeinschaft Grant HO1288/6-1 and BMBF Grant NBLIII-Schwerpunkt Tumormetastasierung (to M. H.).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.
To whom correspondence should be addressed. Tel.:
49-30-450-559709; Fax: 49-30-450-559989; E-mail:
hoecker@charite.de.
Published, JBC Papers in Press, December 30, 2002, DOI 10.1074/jbc.M211999200
2 M. Höcker, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: ROS, reactive oxygen species; EMSA, electrophoretic mobility shift assay; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; AP2, activator protein 2; DN, dominant negative; PMA, phorbol 12-myristate 13-acetate; MAP, mitogen-activated protein; CREB, cAMP-response element-binding protein.
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