Kennedy Institute of Rheumatology Division, Faculty of Medicine, Imperial College of Science, Technology and Medicine, London W6 8LH, UK
* Author for correspondence (e-mail: s.kiriakidis{at}ic.ac.uk)
Accepted 20 November 2002
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Summary |
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Key words: VEGF, Macrophages, Angiogenesis, NF-B, Signalling, CD40 ligand
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Introduction |
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VEGF is produced by many types of cells including fibroblasts
(Pertovaara et al., 1994),
macrophages (McLaren et al.,
1996
), neutrophils (Taichman
et al., 1997
; Webb et al.,
1998
), endothelial cells
(Namiki et al., 1995
) and T
cells (Freeman et al., 1995
).
Although hypoxia is a very potent stimulus for VEGF expression
(Levy et al., 1995
;
Shweiki et al., 1992
), other
factors such as cytokines, oncogenes, NO and modulators of protein kinase C
have been reported to stimulate VEGF production
(Brown et al., 1997
;
Neufeld et al., 1999
). For
example, tumour necrosis factor (TNF)-
upregulates VEGF production by
synovial membrane cells and peripheral blood mononuclear cells from patients
with rheumatoid arthritis (Bottomley et
al., 2000
; Paleolog et al.,
1998
). Although many of the signalling events downstream of VEGF
receptor activation have been elucidated, the stimuli and mechanisms involved
in the production of VEGF are still unclear. There is evidence that
mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase
(PI3-kinase) are involved in the induction of VEGF by growth factors,
cytokines and hypoxia (Clarke et al.,
2001
; Jiang et al.,
2000
; Sodhi et al.,
2001
; Tanaka et al.,
2000
; Wang et al.,
1999
; Yamamoto et al.,
2001
). The downstream events of those pathways generally include
activation of transcription factors such as AP-1 and nuclear factor-
B
(NF-
B).
The NF-B/Rel family includes NF-
B1 (p50), NF-
B2 (p52),
p65 (RelA), RelB and c-Rel. They can all form homo- and heterodimers, with the
most abundant form being p50/p65 (Tak and
Firestein, 2001
). These dimers are present in the cytosol in an
inactive form, complexed with proteins of the I
B family, including
I
B
, I
Bß, I
B
and Bcl3
(Baldwin, 1996
). The
best-characterised mechanism of NF-
B activation involves
phosphorylation of I
B
at Ser32 and Ser36, through the
I
B-kinase (IKK) complex, resulting in ubiquitination and degradation of
I
B
. This enables the release and nuclear translocation of active
NF-
B and its binding to promoters and enhancers, leading to gene
transcription. Two I
B kinases, IKK-1 (IKK
) and IKK-2
(IKKß), have been well described
(DiDonato et al., 1997
;
Mercurio et al., 1997
).
Although IKK-1 and IKK-2 share a high degree of amino acid sequence
similarity, studies in mice indicate that they have non-equivalent functions
and that activation of IKK-2, rather than IKK-1, participates in the primary
pathway by which pro-inflammatory stimuli activate NF-
B
(Baldwin, 2001
;
Hu et al., 1999
;
Li et al., 1999
;
Tak and Firestein, 2001
).
NF-B regulates the expression of many pro-inflammatory cytokines
including TNF-
, interleukin-1ß (IL-1ß) and IL-6
(Baldwin, 2001
;
Bondeson et al., 1999a
;
Bondeson et al., 1999b
;
Tak and Firestein, 2001
).
However, the importance of NF-
B in terms of VEGF expression is
controversial. For example, it was shown that induction of VEGF in
UV-irradiated mouse skin cells can be blocked by treatment with NF-
B
decoy-oligodeoxynucleotides (Abeyama et
al., 2000
). However, administration of NF-
B antisense
oligonucleotides in human endothelial cells only partially abrogated VEGF
expression in response to TNF-
(Yoshida et al., 1997
). More
recently, expression of a mutated I
B
significantly inhibited the
expression of VEGF in human prostate cancer cells
(Huang et al., 2001
;
Huang et al., 2000
) but not in
human head and neck squamous cell carcinomas
(Bancroft et al., 2001
).
In our study we investigated the role of NF-B in the induction of
VEGF in human primary macrophages, which are the main cytokine producers in
chronic inflammatory diseases such as rheumatoid arthritis and a source of
VEGF when stimulated with cytokines and lipopolysaccharide (LPS)
(Itaya et al., 2001
;
Perez-Ruiz et al., 1999
).
However, one of the obstacles in studying biochemical signalling pathways in
primary cells is the difficulty of transfecting DNA, an essential tool in this
process (Stacey et al., 1993
).
To overcome this, we used a very efficient technique of gene transfer, based
on replication-deficient adenoviruses
(Bondeson et al., 1999a
;
Bondeson et al., 1999b
;
Foxwell et al., 1998
), to
express a kinase-negative mutant of IKK-2 (IKK-2dn), as well as the endogenous
NF-
B inhibitor I
B
. We also made use of relatively
specific drug inhibitors of p38 MAPK, p42/44 MAPK and PI3-kinase.
Our results show that several pathways are involved in the induction of
VEGF, in that p38 MAPK and p42/44 MAPK, as well as PI3-kinase, regulate
LPS-induced VEGF production in human primary macrophages. Moreover, we show
for the first time that activation of NF-B plays a key role in VEGF
production by macrophages, as expression of both I
B
and IKK-2dn
could significantly inhibit its production in response to either LPS or CD40
ligand (CD40L), an immune inflammatory stimulus. Our findings show that, like
TNF-
and other pro-inflammatory cytokines, the potent angiogenic factor
VEGF is regulated by NF-
B.
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Materials and Methods |
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Cell culture
Human monocytes were obtained from single-donor plateletphoresis residues
(North London Blood Transfusion Center, London, UK) and differentiated to
macrophages, as described previously
(Foxwell et al., 1998).
Macrophages were cultured in RPMI-1640 medium containing 5% (v/v) fetal calf
serum (FCS) and 100 U/ml penicillin/streptomycin (Bio Whittaker, Rockland, ME)
at 37°C and 5% CO2. Adherent cells were washed twice in
FCS-free RPMI-1640 and removed using Cell Dissociation Medium (Sigma). The
cells were washed twice more and re-plated at 1x106 cells/ml.
Transfected mouse fibroblasts were maintained in RPMI-1640 with 2 mM
L-glutamine containing 1% heat-inactivated FCS, 100 U/ml
penicillin/streptomycin and 2 mg /ml gentamycin (G418). All reagents and media
used in this study were shown to contain <0.1 U/ml LPS, measured using the
Limulus amoebocyte lysate assay (BioWhittaker).
Adenoviral vectors
Recombinant, replication-deficient adenoviral vectors encoding E.
coli ß-galactosidase (Advßgal) or having no insert (Adv0) were
generously provided by A. Byrnes and M. Wood (Oxford University, UK).
Adenoviruses encoding either a dominant form of IKK-2 (single point mutation
of K44A) or porcine I
B
(AdvI
B
) with a
cytomegalovirus promoter and a nuclear localisation sequence were generously
provided by R. de Martin (University of Vienna, Austria). Porcine
I
B
has >95% amino acid homology with the human molecule. All
viruses are (E1/E3) early transcribed regions deleted, belong to the Adv5
serotype and have been previously used in other studies
(Oitzinger et al., 2001
;
Smith et al., 2001
;
Wrighton et al., 1996
).
Viruses were propagated in the 293 human embryonic kidney cell line (American
Type Culture Collection, Rockville, MD) and were purified by ultra
centrifugation through two caesium chloride gradients. The titres of viral
stocks were determined through a plaque assay on 293 cells, as described
previously (Foxwell et al.,
1998
). All viruses used were plaque-purified from a master stock
to prevent contamination with wild-type adenovirus.
Infection techniques
To optimise infection, purified human monocytes obtained by centrifugal
elutriation were seeded at a density of 1x106 per ml in assay
medium in Petri dishes. M-CSF was added to a final concentration of 100 ng/ml.
Cells were cultured for 3 days at 37°C, 5% CO2. This time was
necessary to allow upregulation of integrin Vß5, which we have
previously shown to be essential for adenovirus infection of monocytes)
(Andreakos et al., 2002
;
Bondeson et al., 1999a
;
Bondeson et al., 1999b
;
Ciesielski et al., 2002
;
Foxwell et al., 1998
;
Smith et al., 2001
). Adherent
cells were then washed twice in FCS-free RPMI-1640 and removed from plastic by
Cell Dissociation Medium (Sigma). The cells were washed twice more and
re-plated at 1x106 cells/ml on either 960 mm2
dishes for western blot or electrophoretic mobility shift assay (EMSA)
analysis or in 30 mm2 wells for cytokine analysis. Cells were
infected for 2 hours in serum-free RPMI-1640 with control adenoviruses (Adv0,
Advßgal), AdvI
B
and AdvIKK-2dn, and then incubated in
RPMI-1640 containing 2 mM L-glutamine, 5% (v/v) FCS and 100 U/ml
penicillin/streptomycin for 48 hours to allow for significant overexpression
of proteins. A multiplicity of infection (m.o.i.) of 100:1 was used in all
experiments, consistently giving >95% infection of cells as determined
using Advßgal, and Fluorescein-di-ß-galactopyranoside (Sigma), a
fluorimetric substrate of ß-galactosidase, in accordance with published
data (Bondeson et al., 1999a
;
Bondeson et al., 1999b
;
Foxwell et al., 1998
). Use of
higher m.o.i. did not result in any improvements in infectibility above 95%.
This was supported by data using adenovirus encoding green fluorescent protein
(AdvGFP; Quantum Biotech, Canada), which showed optimal infection at m.o.i.
100:1 (data not shown) (see Bondeson et
al., 1999a
).
Measurement of VEGF production in response to different stimuli
Macrophages were plated at 1x105 cells per
30mm2 well and stimulated for 24 hours with LPS (10 ng/ml), or
sCD40L (1 µg/ml). Additionally, macrophages were seeded in 30
mm2 wells at 1x105 cells/well and CD40L+ or MOCK
cells were added at 1x105 cells/well (1:1 ratio).
Supernatants were collected and analysed for VEGF using an in-house VEGF
ELISA. Briefly, polystyrene plates (Nunc-Immunoplate II, BRL, Middlesex, UK)
were coated with anti-human VEGF antibody (R&D; 100 ng/ml in PBS)
overnight at 4°C. VEGF standard (rhVEGF, R&D) or samples were added
overnight at 4°C. Biotinylated anti-human VEGF antibody (50 µg/ml in
0.5% BSA/PBS) was then added and left at room temperature for 2 hours. The
plates were incubated for 1 hour with streptavidin-horseradish peroxidase
(HRP) (Amersham Pharmacia Biotech, Little Chalfont). After removal of the HRP
conjugate, plates were washed with PBS containing 0.05% Tween 20, and a 1:1
mixture of peroxidase solution (H2O2) and
3,3',5,5'-tetramethylbenzidine (TMB) peroxidase substrate
(Kirkegaard and Perry Laboratories, Gaithesburg, MD) was added for 20 minutes.
After addition of 2M sulphuric acid, plates were read at 450 nm on a
spectrophotometric ELISA plate reader (Labsystems Multiscan Biochromic) and
analysed using Delta Soft II-4 program. In all cases, viability of the cells
was not significantly affected when examined by MTT (3-(4,
5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay (Sigma).
Preparation of cytosolic and nuclear extracts
Two days after adenovirus infection, cells were stimulated for 45 minutes
with either LPS (10 ng/ml) or sCD40L (1 µg/ml), and cytosolic and nuclear
extracts were prepared as described
(Whiteside et al., 1992).
Adherent cells were scraped into ice-cold PBS harvested by centrifugation and
washed once with ice-cold PBS. Cell-pellets were then lysed in hypotonic lysis
buffer (5 mM HEPES, 1 mM MgCl2, 0.2 mM EDTA, 0.5 M NaCl, 25%
glycerol, pH 7.0). After incubation on ice for 10 minutes, lysates were
centrifuged (13,000 g, 5 minutes, 4°C) to remove nuclei
and cell debris. The cleared lysates were then removed to fresh tubes, frozen
and stored at -20°C for subsequent estimation of protein concentration and
use in western blotting. The nuclei pellets were resuspended in hypertonic
extraction buffer (10 mM HEPES, 1.5 mM MgCl2, 10 mM KCl, pH 7.9)
for 1-2 hours at 4°C under agitation. After centrifugation (13,000
g for 10 minutes at 4°C), supernatants containing the
nuclear protein were removed to fresh tubes and stored at -70°C. Protein
concentrations were assessed by reaction with Bradford reagent (0.1% Coomassie
blue G, 5% methanol, orthophosphoric acid)
(Bradford, 1976
).
Western blotting
Antibodies used for western blotting (anti-human IB
; IKK-2
antibodies and
-tubulin) were purchased from Santa Cruz Biotechnology
(Santa Cruz, CA, USA). For western analysis, equal amounts of protein extracts
were separated by SDS/PAGE on a 10% (w/v) polyacrylamide gel, followed by
electrotransfer onto polyvinyl difluoride membrane (Millipore, Watford, UK).
Blots were blocked for 1 hour with blocking buffer (5% (w/v) fat-free milk,
0.1% (v/v) Tween 20 in PBS) followed by overnight incubation with the
antibodies diluted 1:1000 in blocking buffer. Blots were then incubated in
HRP-conjugated anti-mouse IgG (DAKO, Cambridge, UK) diluted 1:2000 in blocking
buffer. Bound antibody was detected using the ECL kit and visualised using
Hyperfilm MP (Amersham Pharmacia Biotech, Little Chalfont, UK).
Electrophoretic mobility shift assay
EMSA reactions were performed as previously described
(Clarke et al., 1995).
Briefly, nuclear protein extracts were incubated with the double-stranded
NF-
B consensus oligonucleotide
(5'-AGTTGAGGGGACTTTCCCAGGC-3'), which was end-labelled with
[
-32P]dATP (3000 Ci/mmol at 10 µCi/µl) using the Gel
Shift Assay System (Promega, Southampton, UK). Formed protein:DNA complexes
were separated on 5% nondenaturing polyacrymide gels and retarded DNA:protein
complexes were analysed by phosphoimaging using the Fuji FLA-2000
phosphoimager (Raytek Scientific, Sheffield, UK).
Statistical analysis
One-way ANOVA with Bonferroni post-test for multiple comparisons was used
to compare the effects of different inhibitors or adenoviruses on VEGF
production. All data presented are from a representative experiment, and the
total number of experiments performed is indicated.
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Results |
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Subsequently, macrophages were treated for 1 hour with different MAPK inhibitors or DMSO as a vehicle control, before stimulation with LPS. The culture supernatants were harvested at 24 hours and then analysed for the presence of VEGF by ELISA. As shown in Fig. 1B,C, treatment of macrophages with the p38 MAPK inhibitor SB203580 (0.002-2 µM) and the p42/44 MAPK inhibitor PD098059 (0.01-10 µM) resulted in dose-dependent suppression of LPS-induced VEGF production, although complete inhibition of VEGF could not be achieved in any experiment. Furthermore, we used two specific inhibitors of the PI3-kinase pathway namely, wortmannin (0.1-100 nM) and LY294002 (0.01-10 µM). Addition of these inhibitors could also reduce LPS-induced VEGF production in a dose-dependent manner (Fig. 1D,E), although complete inhibition of VEGF was again not observed in any experiment. The addition of vehicle alone (DMSO) was without effect (data not shown). There was no significant effect of these inhibitors on cell viability, as determined by MTT, even at the highest concentrations used: 100% viability for 2 µM SB203580, 99% for 10 µM PD09859, 100% for 100 nM wortmannin and 88% for 10 µM LY294002.
IB
overexpression inhibits LPS-induced VEGF release by
human macrophages
To investigate whether LPS-induced VEGF requires NF-B, we infected
macrophages with an adenovirus overexpressing I
B
, the endogenous
inhibitor of NF-
B. As shown in Fig.
2A, by infecting the macrophages with AdvI
B
at a
m.o.i. of 100:1, we were able to induce cytosolic overexpression of
I
B
as assessed by western blotting analysis of the cytosolic
extracts. Stimulation with LPS induced complete degradation of
I
B
, which was prevented in macrophages infected with
AdvI
B
but not in cells infected with control adenovirus (Adv0,
Advßgal). There was no difference between experiments using Adv0 and
Advßgal as controls. In addition, LPS-induced NF-
B activation and
translocation into the nucleus was also inhibited by I
B
overexpression (Fig. 2C). In
parallel experiments, we stimulated macrophages for 24 hours with LPS
following infection, and examined the culture supernatants for VEGF production
using ELISA. In Fig. 2D a
representative of ten independent experiments shows that in
AdvI
B
-infected cells, LPS-induced production of VEGF was
significantly inhibited. The range of inhibition following overexpression of
I
B
was 76-98% (mean 92%).
|
Expression of IKK-2dn does not inhibit LPS-induced NF-B
activation but partially abrogates VEGF production
To examine whether macrophage NF-B activation and VEGF production
requires IKK-2, a kinase-defective (dominant negative) form of IKK-2 was
adenovirally delivered into macrophages. As
Fig. 3A shows, we achieved a
significant expression of IKK-2dn in cells infected with AdvIKK-2dn compared
with the levels in uninfected cells or cells infected with control adenovirus
(Advßgal). However, we unexpectedly found that expression of dominant
negative IKK-2 did not significantly inhibit LPS-induced I
B
degradation (Fig. 3B) and
NF-
B activation (Fig.
3D).
|
Nevertheless, in all our experiments we found consistent inhibition of VEGF
in LPS-stimulated macrophages infected with AdvIKK-2dn, although the extent of
inhibition was less than that observed following overexpression of
IB
. The range of inhibition following overexpression of IKK-2dn
was 38-74% (mean 59%). In comparison, infection with control adenovirus was
without significant effect (Fig.
3E). In the same experiments, infection with AdvI
B
reduced VEGF release by >90% (not shown)
Induction of VEGF by CD40 ligation is NF-B-dependent
Two recently published reports have described that stimulation of
monocytes, endothelial cells and synovial fibroblasts with sCD40L or
CD40L-expressing cells resulted in VEGF production
(Cho et al., 2000;
Melter et al., 2000
).
Therefore, we were interested to examine whether CD40-CD40L engagement on
macrophages induces production of VEGF. As shown in
Fig. 4A from a representative
experiment, stimulation of macrophages for 24 hours with CD40L-expressing
mouse fibroblasts (CD40L+) but not MOCK cells, at a 1:1 ratio, resulted in
marked induction of VEGF.
|
Because we could show that NF-B activation was essential for VEGF
production in response to LPS, we were interested to examine whether the
CD40L-induced VEGF production in those cells follows a similar pathway. We
found that stimulation of macrophages with sCD40L (1 µg/ml) induced
I
B
degradation, which was inhibited by over-expression of both
I
B
or IKK-2dn in macrophages
(Fig. 4B,C) suggesting that
phosphorylation and degradation of I
B
in macrophages through
CD40L requires IKK-2. In addition, as shown in
Fig. 4D, infection of
macrophages with AdvI
B
could strongly inhibit VEGF production in
response to CD40L+, suggesting that I
B
is significantly involved
in the CD40L-induced production of VEGF. However, as was observed for
LPS-induced VEGF release, we could observe only a partial reduction of VEGF
production in response to CD40L+ using AdvIKK-2dn
(Fig. 4D).
Neutralising antibody to TNF- inhibits both LPS- and
sCD40L-induced VEGF release in macrophages
In a previous study we found, using anti-TNF- antibody cA2, that
VEGF production by synovial joint cells, a heterogeneous population of T
cells, macrophages and fibroblasts, is at least in part mediated by
TNF-
(Paleolog et al.,
1998
). Macrophages respond to LPS or CD40L to produce TNF-
.
To examine whether endogenous TNF-
is involved in the induction of VEGF
by these stimuli, we stimulated macrophages with either LPS (10 ng/ml) or
sCD40L (1 µg/ml) in the presence of anti-TNF-
antibody (10
µg/ml). We observed a potent inhibition of VEGF production in macrophages
in response to both LPS (Fig.
5A) and sCD40L (Fig.
5B), suggesting that endogenously produced TNF-
plays an
important role in both LPS- and CD40L-induced VEGF production by macrophages.
Stimulation of macrophages with exogenous TNF-
did not significantly
induce VEGF release (data not shown).
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Discussion |
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LPS is known to bind to CD14 on the surface of macrophages and to signal
through toll-like receptor 4 (TLR4) to activate MAPK and NF-B
(Anderson, 2000
;
Guha and Mackman, 2001
). To
study whether MAPKs are involved in LPS-induced VEGF production by
macrophages, we used specific MAPK inhibitors. SB203580, a specific p38 MAPK
inhibitor, only partially inhibited the LPS-induced VEGF production.
Additionally, we showed that other inhibitors, such as the p42/44 MAPK
inhibitor PD09859 and two specific inhibitors for the PI3-kinase pathway
wortmannin and LY294002 could also partially inhibit the
production of VEGF. These data suggest that signals through those proteins are
involved in the upregulation of VEGF. The inhibitors used in this study might
exert effects on other kinases at higher concentrations; however, we have
previously shown that at the concentrations employed in the present study,
these inhibitors have been shown by in vitro kinase assays to be specific
(Crawley et al., 1996
;
Foey et al., 1998
;
Lali et al., 2000
;
Williams et al., 2000
).
Virtually complete inhibition of VEGF production in response to LPS was
achieved only by overexpression of the endogenous NF-B inhibitor,
I
B
. Ubiquitination and degradation of I
B
enables
the release and nuclear translocation of NF-
B and binding to promoters
and enhancers. Overexpression of I
B
inhibited LPS-induced
degradation of endogenous I
B
and NF-
B nuclear
translocation, and resulted in more than 90% inhibition of VEGF expression.
Expression of IKK-2dn resulted in a significant inhibition of VEGF release,
although complete inhibition of VEGF could not be achieved as it was in the
case of I
B
.
Previous studies on monocytic cell lines showed that IKK-2 rather than
IKK-1 is required for LPS-induced NF-B activation
(Hawiger et al., 1999
).
Expression of dominant negative forms of IKK-2 could inhibit LPS-induced
B-dependent transcription (Fischer
et al., 1999
; O'Connell et
al., 1998
), whereas expression of dominant negative forms of IKK-1
had no effect on LPS-induced
B-dependent transcription in one study
(O'Connell et al., 1998
) but
partly inhibited it in another (Fischer et
al., 1999
). However, we found that IKK-2dn could not prevent
LPS-induced I
B
degradation and NF-
B nuclear translocation
in human primary macrophages. In addition, in a parallel study we found that
IKK-2dn did not block the production of NF-
B-dependent cytokines
namely, TNF-
, IL-6 or IL-8 in response to LPS,
suggesting that LPS-induced NF-
B activation by macrophages does not
require IKK-2 (E.A., C. Smith, S.K. et al., unpublished).
The selective partial inhibition of VEGF release in response to IKK-2dn
suggested that, in macrophages, IKK-2 might be required in the activation of
other signalling pathways contributing to the induction of VEGF. In a previous
study we showed using anti-TNF- antibody that production of VEGF by
synovial joint cells was in part induced by TNF-
(Paleolog et al., 1998
). To
address the possible role of TNF-
in the induction of VEGF, macrophages
were stimulated with LPS in the presence of neutralising antibody to
TNF-
. We found that blockade of endogenous TNF-
activity
resulted in a significant inhibition of VEGF release, although addition of
exogenous TNF-
did not induce VEGF production. As discussed previously,
we have also observed that IKK-2 is essential for TNF-
-induced
NF-
B activation and cytokine production in macrophages, but not for
LPS-induced production of TNF-
(E.A., C. Smith, S.K. et al.,
unpublished). A possible explanation of our data is that LPS activates
macrophages through an IKK-2-independent pathway to induce degradation of
I
B
, nuclear translocation of NF-
B and stimulation of
TNF-
transcription. Endogenously released TNF-
might, in turn
augment VEGF expression through an IKK-2-dependent mechanism. Support for this
hypothesis comes from time-course experiments that showed much slower
induction of VEGF expression, peaking after 24 hours. By contrast, TNF-
expression peaks after 4 hours. However, in addition to TNF-
induction,
other genes induced by LPS might code for proteins that are essential for the
TNF signalling or other events associated with VEGF regulation such as
the stabilisation of VEGF mRNA and they might also contribute
to the end result VEGF induction via the NF-
B
pathway.
The essential role of NF-B in LPS-induced VEGF production prompted
us to investigate its role in CD40L-induced production another major
inflammatory stimulus. CD40-CD40L signalling has been found to have multiple
functions in inflammation, predominantly in the effector phase of the immune
response (Van Kooten and Banchereau,
1996
). In monocytes it was previously shown that CD40L activates
NF-
B and induces the expression of pro-inflammatory cytokines such as
TNF-
, IL6 and IL-8 (Alderson et al.,
1993
; Kiener et al.,
1995
). Recently, it was shown that ligation of CD40 induces the
expression of VEGF by rheumatoid synovial fibroblasts
(Cho et al., 2000
),
endothelial cells and monocytes (Melter et
al., 2000
). In addition, studies in non-haematopoietic cells
showed that CD40 mediates NF-
B mobilisation and IL-6 production
(Hess et al., 1995
). Studies
on human hepatic stellate cells have shown that ligation of CD40 on those
cells induces the activation of important signalling pathways such as
IKK/NF-
B and c-Jun N-terminal kinase following secretion of
IL-8 and MCP-1 (Schwabe et al.,
2001
).
We found that stimulation of human macrophages with a mouse fibroblast cell
line transfected with a plasmid expressing CD40L (CD40L+) resulted in strong
VEGF induction compared with macrophages stimulated with MOCK-transfected
cells. This is, to our knowledge, the first evidence linking CD40-CD40L
interactions on normal human macrophages with VEGF production. We also found
that CD40L stimulation of macrophages induced IB
degradation,
which was inhibited after adenovirus-mediated delivery into the cells of
I
B
or IKK-2dn, showing that IKK-2 is essential for NF-
B
activation in response CD40L. In addition, CD40L induced VEGF production was
also inhibited following I
B
overexpression, and we observed a
50% inhibition of VEGF after expression of IKK-2dn. This is in contrast to the
response of macrophages to LPS, in which VEGF production, but not NF-
B
activation, was inhibited by IKK-2dn, suggesting that the involvement of
components of the NF-
B (I
B
, IKK-2) pathway is stimulus
dependent. The partial inhibitory effect of AdvI
B
and AdvIKK-2dn
on CD40L-mediated VEGF secretion suggests that additional pathways are
involved in the induction of VEGF release by this stimulus.
The involvement of NF-B in VEGF production has been investigated in
other studies, with variable and conflicting results. NF-
B has been
shown to be partly responsible for the upregulation of VEGF mRNA and
development of vessel-like structures in human microvascular endothelial cells
in response to TNF-
(Yoshida et
al., 1997
). In recent studies, expression of mutated
I
B
could not inhibit VEGF production in human head and neck
squamous cell carcinomas (Bancroft et al.,
2001
), but significantly inhibited the expression of VEGF in human
prostate cancer cells (Huang et al.,
2001
; Huang et al.,
2000
). Our results suggest that the NF-
B pathway is
involved in the induction of VEGF release from LPS- and CD40L-stimulated human
macrophages. However, the role of IKK-2 is not fully understood and needs
further study.
Although the human VEGF promoter contains multiple binding sites for
different transcriptional factors such SP-1, AP-1 and hypoxia-regulated
elements, there is no direct evidence to date for the presence of classical
NF-B binding sites (Tischer et al.,
1991
). These are the decameric consensus sequences of NF-
B
(5'-GGGRNNYYCC-3', where R indicates A or G, Y indicates C or T,
and N indicates any nucleotide) and the
B-like motifs
(5'-HGGARNYYCC-3', where H indicates A, C, or T; R indicates A or
G; Y indicates C or T, and N indicates any nucleotide)
(Parry and Mackman, 1994
). Our
finding that both LPS- and CD40L-induced VEGF expression is inhibited by
blocking NF-
B with the specific inhibitor I
B
supports the
hypothesis about the existence of such NF-
B binding sites within the
VEGF gene. Examination of the 5'-regulatory region of the
VEGF gene revealed the presence of several sequences that differ at
the level of one or two positions from the decameric NF-
B and
B-like consensus sequences (Fig.
6). Studies are now underway using self-designed double-stranded
VEGF oligonucleotide-constructs containing those motifs to define NF-
B
binding sites in the promoter region of the VEGF gene.
|
NF-B is an important therapeutic target in chronic inflammatory
diseases, enabling significant downregulation of macrophage-produced
pro-inflammatory cytokines. Our findings show that the most potent angiogenic
factor, VEGF, is among those cytokines regulated by NF-
B. Targeting
angiogenesis using adenovirally delivered inhibitors of VEGF production might
also be applicable to the treatment of angiogenesis-related diseases including
rheumatoid arthritis.
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Acknowledgments |
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References |
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