 |
INTRODUCTION |
Angiogenesis, the formation of new blood vessels, is a multistep
process that includes endothelial cell proliferation, migration, capillary tube assembly, and recruitment of perivascular support cells
to form mature and functional vessels (1, 2). This process is not only
critical for embryogenesis and the normal function of the female
reproductive tract but also plays an essential role under many
pathological conditions such as wound healing, tumor growth and
metastasis, and rheumatoid arthritis. Over the past decade, tremendous
progress has been made in dissecting the molecular mechanisms
underlying this important biological process. Receptor tyrosine kinases
have emerged as critical molecules in regulating many aspects of
angiogenesis (reviewed in Refs. 3 and 4). At least three families of
receptor tyrosine kinases have been implicated in angiogenesis: the
VEGF1 family, the
angiopoietin/Tie2 family, and the ephrin/Eph family. VEGFs are
vascular endothelial cell growth factors that promote endothelial cell
proliferation, migration, and vessel assembly. Members of the VEGF
receptor family are crucial to de novo blood vessel
formation during embryonic development and mediate angiogenesis in a
number of diseases including inflammation and cancer. Members of the
angiopoeitin/Tie2 family of receptor tyrosine kinases function in blood
vessel remodeling, maturation, and stabilization. A third family, the
Eph family, has recently been shown to significantly regulate angiogenesis.
The Eph family comprises the largest subfamily of receptor tyrosine
kinases, including at least 14 receptors and 8 ligands. The binding of
Eph ligands to their receptors is governed by cell-cell contact,
because the Eph ligands, known as ephrins, are anchored to the cell
surface (5, 6). Ephrins can be further divided into two groups, ephrin
A and ephrin B subclasses, according to how they are anchored to the
cell membrane (7-9). The ephrin A ligands are membrane-bound through
glycosylphosphatidyl linkage, whereas ephrin B ligands are
anchored to the membrane through transmembrane domains. The ephrin A
subclass exhibits rather general and promiscuous binding to the EphA
receptors, and the ephrin B subclass has a general binding preference
for the receptors of the B subclass. Another unique feature of subclass
B is that EphB receptors can activate ephrin B ligands through
phosphorylation of their cytoplasmic domains (7, 8). Such bidirectional signaling may also exist between ephrin A ligands and Eph receptors, as
documented in the case of ephrin A5 (9).
Functional evidence for involvement of Eph family receptor tyrosine
kinases in angiogenesis came from both in vitro studies and
gene knockout experiments. Targeted gene disruptions of ephrin B2
ligand and EphB4 receptor in mice revealed that the expression of
ephrin B2 was restricted to arterial endothelium, whereas EphB4 was
expressed in the venous endothelium during early vascular development.
Embryos lacking ephrin B2 or EphB4 displayed severe defects in vascular
remodeling in both arterial and venous domains (10, 11). Furthermore,
EphB2 and B3 double knockout mice exhibited similar vascular phenotypes
(12). Another ligand-receptor pair, ephrin A1 and EphA2, has also been
implicated in embryonic and adult angiogenesis. During early
embryogenesis, ephrin A1 was expressed in developing vasculature (13),
and ephrin A1 promoted angiogenesis in cornea in adult animals (14).
Although ephrins generally do not induce endothelial cell
proliferation, ephrin A1 could promote human umbilical vein
endothelial cell (HUVEC) assembly into a capillary-like structure in
Matrigel assays (15). Expression of ephrin A1 in HUVEC is
regulated by the multifunctional proinflammatory cytokine TNF-
(16).
Neutralizing antibodies against ephrin A1 inhibited TNF-
-induced
angiogenesis in rabbit cornea assays (14), suggesting that ephrin A1
was a key mediator of TNF-
-induced angiogenesis.
TNF-
is a multifunctional cytokine that induces a broad spectrum of
responses including angiogenesis. It is thought that TNF-
promotes
angiogenesis through its ability to up-regulate the expression of
various angiogenic factors. Multiple signaling pathways have been
connected to TNF-
-dependent gene induction. Two forms of
TNF receptor have been identified, TNFR1 and TNFR2. A current model
postulates that TNF binding triggers trimerization of TNFRs (TNFR1/2)
and recruitment of adaptor proteins. The death domain protein
TNFR-associated death domain protein binds to TNFR1 and serves
as a platform for further recruitment of receptor-interacting protein, Fas-associated death domain, and TRAF2. Whereas Fas-associated death domain signals to the apoptotic protease cascade,
receptor-interacting protein mediates p38 MAPK and NF-
B activation,
and TRAF2 mediates JNK activation. Activation of NF-
B, p38 MAPK, and
JNK can, in turn, regulate a variety of cellular processes, including
TNF-
-dependent gene induction (17, 18). To investigate
how ephrin A1 is regulated by TNF-
, we investigated the signaling
pathways involved in TNF-
-dependent up-regulation of
ephrin A1 in endothelial cells. Here we report that, in contrast to the
TNF-induced expression of other angiogenic factors, NF-
B is not a
prime regulator of ephrin A1 expression. Rather, activation of p38 MAPK
and SAPK/JNK pathways by TNF-
leads to ephrin A1 expression in endothelium.
 |
EXPERIMENTAL PROCEDURES |
Endothelial Cell Culture--
HUVEC were purchased from
Clonetics and maintained in 10-mm dishes in endothelial cell
basal medium with growth factors (Clonetics). Cells were subcultured up
to passage 5. Human endothelial cell line ECV304 was maintained in
Dulbecco's modified Eagle's medium, 10% fetal bovine serum in 10 cm
dishes as described (19). Endothelial cells were cultured to 80%
confluence, starved for 16 h in medium deficient in growth
factors, preincubated with cycloheximide (CHX) at 10 µg/ml for 30 min, and then stimulated with recombinant human TNF-
(R & D
Systems) at 20 ng/ml (unless otherwise indicated in figure legends).
ECV cells were starved in serum-free Dulbecco's modified
Eagle's medium overnight prior to various treatments as described below.
To inhibit NF-
B activity, HUVEC were infected with adenoviruses
overexpressing a dominant negative form of mutant I
B-
protein (AdmI
B
) or with control virus expressing
-galactosidase
(Ad-
gal) (generously provided by Dr. L. Kerr) at 108
plaque-forming units/ml for 24-40 h, prior to the addition of CHX and
TNF-
. For chemical inhibition studies, endothelial cells were
preincubated with the following chemical inhibitors prior to CHX and
TNF-
stimulation: p42/44 MAPK-specific inhibitor PD98059 (New
England Biolabs) at 50 µM for 1 h, p38
MAPK-specific inhibitor SB20358 (Sigma) at 2.5 µM, and
JNK inhibitor DMAP (Sigma) at 1 mM for 15 min. To
inhibit p38 MAPK and SAPK/JNK, cells were infected with adenoviruses
overexpressing a dominant negative form of mutant p38 MAPK protein
(Ad-p38MAPK.DN) or a trans-dominant inhibitory isoform of mutant TRAF2
protein (Ad-TRAF2.DN) for 24-40 h at 108 plaque-forming
units/ml prior to stimulation with TNF-
(see below). To inhibit
TNF-
receptor function, neutralizing antibodies (R & D Systems) to
TNFR1 or TNFR2 were added to endothelial cell cultures at 5 µg/ml for
1 h prior to stimulation with TNF-
.
Northern Blot Analysis--
RNA from stimulated HUVEC and ECV304
cells were isolated using Trizol reagent and chloroform (Life
Technologies, Inc.). 10 µg of total RNA were resolved on a 1%
agarose gel containing 2.2 M formaldehyde, transferred to a
nylon membrane (Hybond N+, Amersham Pharmacia Biotech), and
hybridized for 16 -24 h to an ephrin A1 cDNA probe. Membranes were
washed once with 2× SSC at room temperature, followed by two stringent
washes (1× SSC at 65 °C for 10 min and 0.5× SSC at 65 °C for 10 min), and exposed to Biomax film (Eastman Kodak Co.). The RNA blots
were stripped and reprobed with a glyceraldehyde-3-phosphate dehydrogenase cDNA probe as loading control. Ephrin A1 RNA levels were quantified using the Scion Image 1.62 software program.
Whole Cell Extract Preparation and Western Blot
Analysis--
HUVEC and ECV304 cells were lysed in SDS sample buffer,
and cell extracts were cleared by sonication and centrifugation. 50 µg of protein from cell lysates were fractionated on a 12%
SDS-polyacrylamide gel electrophoresis gel and transferred to a
nitrocellulose membrane (ECL+, Amersham Pharmacia Biotech). Membranes
were blocked for 1 h at room temperature with Tris-buffered saline
containing 0.1% Tween 20 and 5% powdered nonfat milk and then
incubated with antibodies against phosphorylated forms of JNK/SAPK, p38
MAPK, p42/44 MAPK, or ATF2 according to the manufacturer's
instructions (New England Biolabs). Immunoreactive proteins were
detected with secondary antibodies (Santa Cruz Biotechnologies and
Promega) conjugated to horseradish peroxidase using enhanced
chemiluminescence (Amersham Pharmacia Biotech).
Construction of Adenoviruses--
Adenoviruses expressing
dominant negative forms of mutant TRAF2 (Ad-TRAF2.DN) or p38 MAPK
(Ad-p38MAPK.DN) were constructed according to the methods of Becker
et al. (20). Briefly, TRAF2 cDNA truncated at the
NH2 terminus at codon 241 was generated by
polymerase chain reaction using Pfu DNA polymerase
(Stratagene) and subcloned into BamHI and HindIII
sites of pAC-CMV vector to create pACCMVTRAF2(241-501).DN. p38 MAPK
cDNA containing Thr-180
Ala and Tyr-182
Phe substitutions
was cloned from pCMV5 vector (a gift from Dr. R. Davis) into
HindIII and XbaI sites of pAC-CMV to create
pACCMVp38MAPK.DN. The plasmid constructs pAC-CMVTRAF2(241-501).DN or
pAC-CMVp38MAPK.DN were cotransfected with pJM17 (a gift from Dr. L. Kerr) into the 293T cell packaging line for viral production. The
resulting virus was further amplified in 293T cells and purified by a
PD10 column (Centricon).
Electrophoretic Mobility Shift Assay--
HUVEC were infected
with AdmI
B
or Ad-
gal at 108 plaque-forming
units/ml for 24-48 h, followed by the addition of TNF-
for 2 h. Nuclear fractions were prepared by high salt extraction in the
presence of protease inhibitors (tosylphenylalanine chloromethyl ketone
and tosyllysine chloromethyl ketone at 100 µM,
phenylmethylsulfonyl fluoride, aprotinin, and leupeptin at 100 µM). Gel mobility shift assays were performed by using a
32P-labeled oligonucleotide duplex derived from
B
enhancer sequences in the interleukin-2 receptor-
promoter region
(5'-CAACGGCAGGGGAATTCCCCTCTCTT-3') (21). The DNA binding reaction mix
containing 5 µg of nuclear extracts, 2 µg of double-stranded
poly(dI-dC), 10% Nonidet P-40, 0.1 M dithiothreitol, and
10 µg of bovine serum albumin were buffered in 10× HGE (200 mM HEPES, 50% glycerol, 1 mM EDTA). The
resultant binding complex was resolved on a native 5% acrylamide gel
and visualized by autoradiography (22).
Kinase Assay--
In vitro kinase assay was performed
as described (17). Briefly, cell extracts were prepared in Triton lysis
buffer (20 mM Tris-HCl (ph 7.5), 137 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 5 mM EDTA, 2 mM EGTA, 1 mM Na3VO4,
25 mM
-glycerophosphate, 50 mM NaF, 10 mM sodium pyrophosphate, 15% (v/v) glycerol, and 1% (v/v)
Triton X-100). 900 µg of protein were immunoprecipitated with
0.8 µg/ml anti-p38 MAPK antibodies (Santa Cruz Biotechnology). For
kinase reaction, 1.5 µg of the glutathione
S-transferase-c-Jun (New England Biolabs) and 20 µM ATP were incubated with the immunoprecipitated p38
MAPK in kinase reaction buffer for 20 min at 30 °C. The reaction mix
was resolved via 15% SDS-polyacrylamide gel electrophoresis and
visualized by autoradiography.
 |
RESULTS |
Kinetics and Concentration Dependence of TNF-
-induced Ephrin A1
Expression in Endothelial Cells--
To determine the kinetics of
ephrin A1 regulation, HUVEC were stimulated with 20 ng/ml TNF-
for
15 min to 16 h. As shown in Fig.
1A, unstimulated cells did not
express a significant amount of ephrin A1 mRNA. However, upon
treatment with TNF-
, induction was seen as early as 15 min after
stimulation. Maximum induction was reached in 2 h, and mRNA
levels began a steady decline thereafter. TNF-
was also active on
the human endothelial cell line ECV304, with a time course similar to
that of HUVEC. The induction of ephrin A1 by TNF-
was
dose-dependent, with maximum induction at 20-40 ng/ml
(Fig. 1B).

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 1.
Kinetics and concentration dependence of
TNF- -induced ephrin A1 expression in
endothelial cells. A, HUVEC and EV304 cells were
starved overnight, pre-incubated with 10 µg/ml CHX for 30 min, and
stimulated with 20 ng/ml TNF- over a period ranging from 15 min to
16 h. Total RNAs (10 µg) were isolated, separated by
formaldehyde-agarose gel eletrophoresis, transferred onto a nylon
membrane, and probed with an ephrin A1 cDNA fragment. The blots
were stripped and reprobed for glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) as a loading control. B, HUVEC and ECV304
cells were starved, pre-incubated with 10 µg/ml CHX for 30 min, and then stimulated with TNF- ranging from 5 to 40 ng/ml for
2 h. 10 µg of total RNA were analyzed by Northern
analysis for ephrin A1 expression as described above.
|
|
Induction of Ephrin A1 Expression by TNF-
Is Mediated through
Both TNFR1 and TNFR2--
TNF-
induces the expression of many genes
through two different TNF receptors. TNFR1 (p55) and TNFR2 (p75) bind
to TNF-
with different affinities and have been shown to activate
distinct, as well as overlapping, pathways (23, 24). To determine which receptor is involved in regulation of ephrin A1 expression, endothelial cells were treated with neutralizing monoclonal antibodies specifically against either TNFR1 or TNFR2 (25). As shown in Fig.
2, neutralizing antibodies against either
TNFR1 or TNFR2 inhibited ephrin A1 expression. These results indicate
that both TNF-
receptors are involved in regulating ephrin A1
expression.

View larger version (31K):
[in this window]
[in a new window]
|
Fig. 2.
Induction of ephrin A1 expression by
TNF- is mediated through both TNFR1 and
TNFR2. A, HUVEC and ECV304 cells were
pre-incubated with 5 µg/ml neutralizing antibodies to TNFR1 or TNFR2
for 1 h and then stimulated with TNF- . 10 µg of total
RNA were used for Northern blot analysis. B, levels of
ephrin A1 induction in triplicate experiments were quantified using the
Scion Image 1.62 software program and calculated as the ephrin
A1/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) ratio
(*, p < 0.05 versus TNF- positive
control; Student's t test).
|
|
Inhibition of NF-
B Activation Does Not Affect Ephrin A1
Expression--
The NF-
B family of transcription factors is one of
the main mediators of the intracellular functions of TNF-
. In the
quiescent state, NF-
B proteins are sequestered in the cytoplasm by
binding to a family of inhibitory proteins including I
B
. During
TNF-
-induced cell activation, I
B
undergoes signal-induced
phosphorylation and ubiquitin-proteasome-mediated degradation,
permitting the nuclear import of NF-
B to activate transcription.
Several TNF-
-induced angiogenic molecules such as VEGF, VEGFR2
(flk-1), E-selectin, and ICAM-1 (26-29) have been shown to be
regulated through an NF-
B-dependent mechanism. Because
ephrin A1 is also a TNF-
-induced angiogenic factor, we determined
whether NF-
B proteins are also involved in mediating ephrin A1 expression.
To inhibit the NF-
B signaling pathway, HUVEC were infected with an
adenovirus overexpressing a trans-dominant inhibitory isoform of
I
B
(AdmI
B
). This mutant I
B-
contains serine to alanine point mutations at positions 36 and 40 that confer resistance to signal-dependent phosphorylation and degradation, thus
functioning as a constitutive repressor of multiple NF-
B proteins
(30, 31). Gel mobility shift analyses were performed with nuclear extracts from TNF-
-stimulated cells infected with either AdmI
B
or a control virus expressing
-galactosidase (Ad-
gal). As shown in Fig. 3A, TNF-
stimulated
high levels of nuclear NF-
B activity in uninfected cells or cells
infected with Ad-
gal, whereas the nuclear levels of NF-
B were
undetectable in cells infected with AdmI
B
. These results suggest
that overexpression of dominant negative I
B
inhibited NF-
B
activation in endothelial cells. However, although AdmI
B
-treated
cells no longer demonstrated detectable induction of NF-
B by
TNF-
, the induction of ephrin A1 by TNF-
was not affected (Fig.
3B), indicating that NF-
B is not a prime regulator of
TNF-
-induced ephrin A1 expression in endothelial cells.

View larger version (32K):
[in this window]
[in a new window]
|
Fig. 3.
Inhibition of NF- B
activation does not affect ephrin A1 expression. A,
HUVEC were infected with adenoviruses overexpressing a trans-dominant
inhibitory isoform of I B- protein (AdmI B ) or
-galactosidase (Ad- gal) at 108 plaque-forming
units/ml for 24-40 h, prior to the addition of CHX and TNF- .
Nuclear extracts were isolated and subjected to electrophoretic
mobility shift assay analysis. One of the two representative
experiments is shown. B, HUVEC were infected with
Ad-mI B- or Ad- gal for 40 h, stimulated with TNF- for
2 h, and analyzed for changes in ephrin A1 expression by Northern
analysis. One of the four representative experiments is shown.
GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
|
|
Inhibition of p38 MAPK and SAPK/JNK Activation Blocks
TNF-
-induced Ephrin A1 Expression--
In addition to the NF-
B
pathway, TNF-
binding to its receptors activates multiple pathways
that culminate in altered activity of transcription factors. One of the
central mediators that propagate signals from the cell membrane to the
nucleus is the MAP kinase superfamily. At least three different
subtypes of MAP kinases are known: p42/44 MAPK, p38 MAPK, and SAPK/JNK.
To further investigate whether the MAP kinases mediate TNF-
-induced
ephrin A1 expression in endothelial cells, selective chemical
inhibitors PD98059, SB20358, and DMAP were used to inhibit p42/44 MAPK,
p38 MAPK, and SAPK/JUNK, respectively. DMAP has been shown to inhibit
phosphorylation and activation of JNK in endothelial cells (32).
PD98059 has been shown to selectively inhibit phosphorylation of p42/44
MAPK but not p38 MAPK or SAPK/JNK at the concentration of 50 µM (33). In contrast, SB20358 does not inhibit the
phosphorylation of p38 MAPK; rather, it binds to p38 MAPK and
inhibits the ability of p38 MAPK to phosphorylate downstream target
proteins (34).
Endothelial cells were preincubated with the selective MAPK inhibitors
DMAP, SB20358, or PD98059 and stimulated with TNF-
, and ephrin A1
mRNA induction was analyzed by Northern blot analysis. Duplicate
plates were treated under the same conditions, and cell extracts were
prepared for Western blot analysis to determine the levels of MAPK
inhibition. As shown in Fig.
4A, cells pretreated with
either DMAP or SB20358 resulted in a significant reduction of ephrin A1
expression. The inhibition of ephrin A1 induction was also
dose-dependent, because 7 µM SB20358
significantly suppressed the induction, and 100 µM DMAP
completely blocked the induction (Fig. 4B). In contrast,
ephrin A1 expression was not affected in the presence of the p42/44
MAPK inhibitor PD98059 (Fig. 4A). Western blot analysis
(Fig. 4C) confirmed previous investigations showing that
PD98059 selectively inhibits phosphorylation of p42/44 MAPK but does
not affect p38 MAPK or SAPK/JNK. Although SB20358 does not prevent
phosphorylation of p38 MAPK, it inhibits the ability of p38 MAPK to
phosphorylate downstream effector ATF2. The specificity of DMAP was
demonstrated by inhibition of phosphorylation of SAPK/JNK. Although
DMAP had been shown to also inhibit p38 MAPK in mouse Sertoli cells
(35), in the present experiments there was no detectable effect on p38
MAPK or p42/44 MAPK in endothelial cells at a concentration of 1 mM (Fig. 4C). Taken together, these data suggest
that induction of ephrin A1 expression by TNF-
was mediated through
p38 MAPK and SAPK/JNK pathways but was independent of p42/44 MAPK.

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 4.
Chemical inhibition of p38 MAPK and SAPK/JNK
activation blocks TNF- -induced ephrin A1
expression. A, ECV304 cells were pre-incubated
with selective MAPK inhibitors (1 mM DMAP, 50 µM PD98059, or 5 µM SB20358) prior to
stimulation with TNF- . 10 µg of total RNA were isolated and
subjected to Northern analysis for ephrin A1 expression, as described
in the legend to Fig. 1A. B, ECV304 cells were
pre-incubated with increasing doses of SB20358 or DMAP and stimulated
with TNF- , and RNAs were subjected to Northern blot analysis.
C, whole cell extracts were prepared from ECV304 cells
pre-incubated with chemical inhibitors and stimulated with TNF- . 50 µg of protein/lane were resolved via 12% SDS- polyacrylamide gel
electrophoresis, transferred onto a nitrocellulose membrane, and
blotted with antibodies to phosphorylated forms of p38 MAPK, SAPK/JNK,
p42/44 MAPK, or ATF2. The membranes were then stripped and reblotted
with antibodies to the unphosphorylated forms of proteins for loading
controls. GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
unstim, unstimulated.
|
|
Ephrin A1 Induction Is Mediated through p38 MAPK- and
TRAF2-dependent Mechanisms--
Because chemical
inhibition might affect other unknown downstream effectors of TNF
receptors, we used an independent approach to address whether p38 MAPK
and SAPK/JNK mediate ephrin A1 induction by TNF-
. Recombinant
adenoviruses expressing dominant negative p38 MAPK (Ad-p38MAPK.DN) and
dominant negative TRAF2 (Ad-TRAF2.DN) were generated and used for
infection of HUVEC and ECV cells. Dominant negative p38 MAPK contains
Thr-180
Ala and Tyr-182
Phe substitutions, resulting in
resistance to activation-induced phosphorylation. TRAF2 is an adaptor
protein that binds to the TNFR/TRADD complex and has been shown to
specifically mediate JNK activation (17, 36). Dominant negative TRAF2
truncated at codon 241 at the NH2 terminus and was
previously shown to inhibit JNK activity (36, 37). As shown in Fig.
5D, overexpression of dominant
negative p38 MAPK significantly inhibited the ability of p38 MAPK to
phosphorylate c-Jun, a downstream effector of p38 MAPK. Furthermore,
expression of dominant negative TRAF2 did not affect the
phosphorylation status of p38 MAPK but did inhibit JNK phosphorylation
(Fig. 5C), suggesting that TRAF2 specifically mediated JNK
activation but not p38 MAPK in endothelial cells. Consistent with our
chemical inhibitor data, TNF-
-induced ephrin A1 expression was
inhibited by both Ad-p38MAPK.DN and Ad-TRAF2.DN, whereas Ad-
gal did
not affect ephrin A1 expression (Fig. 5, A and
B). These results provide direct evidence of the involvement of p38 MAPK and SAPK/JNK pathways in TNF-
-induced ephrin A1
expression and further indicated that TRAF2 acted upstream of JNK to
activate the JNK pathway.

View larger version (35K):
[in this window]
[in a new window]
|
Fig. 5.
Dominant negative forms of TRAF2 and p38 MAPK
inhibit TNF-dependent ephrin A1 induction.
A, HUVEC and ECV304 cells were infected with
adenovirus overexpressing dominant negative TRAF2 (Ad-TRAF2.DN),
dominant negative p38 MAPK (Ad-p38MAPK.DN), or Ad- gal for 24-40 h
and stimulated with TNF- . 10 µg of total RNA were
subjected to Northern analysis for ephrin A1 expression. One of the
three representative experiments is shown here. B, levels of
ephrin A1 induction in triplicate experiments were quantified using the
Scion Image 1.62 software program and calculated as the ephrin
A1/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) ratio
(*, p < 0.05 versus TNF- positive
control; Student's t test). C, Western blot
analysis showing that dominant negative TRAF2 inhibits the
phosphorylation of JNK but not p38 MAPK. D, cell lysates
prepared from endothelial cells unstimulated ( ) or
stimulated (+) with TNF (10 ng/ml) in the presence
(+) or absence ( ) of either Ad-p38MAPK.DN or
Ad- gal were used to measure p38 MAPK activity by immunocomplex
kinase assay with GST-c-Jun as a substrate.
|
|
 |
DISCUSSION |
TNF-
is a multifunctional cytokine that induces a broad
spectrum of responses, including cell growth, apoptosis, induction of
other cytokines, and angiogenesis. TNF-
apparently promotes angiogenesis through indirect effects, mediated by its regulation of
angiogenic factors. In addition to inducing ephrin A1 expression, TNF-
up-regulates the expression of VEGFR2 (flk-1), basic fibroblast growth factor, platelet activating factor, tissue factors,
E-selectin, and ICAM-1 (26, 35, 38, 39). However, signaling pathways mediating TNF-
-dependent induction in endothelial cells
have not been thoroughly investigated. In this report we studied the signaling mechanisms that mediate TNF-
-induced ephrin A1 expression in endothelial cells. We showed that (1) both TNFR1 and TNFR2 are
involved in regulating ephrin A1 expression in endothelial cells, (2)
the induction of ephrin A1 is TRAF2-dependent, (3) activation of p38 MAPK or SAPK/JNK, but not p42/44 MAPK, signaling pathways lead to ephrin A1 induction, and (4) activation of NF-
B is
not required for TNF-
-induced ephrin A1 expression.
The many functions of TNF are mediated by two cell surface receptors,
TNFR1 and TNFR2. TNF binding induces receptor aggregation, resulting in
the recruitment of different types of intracellular signal transducers
to the TNFR complexes. One of these signaling transducers is TRAF2,
which interacts with the cytoplasmic tails of both TNFRs and serves as
an adaptor protein to recruit downstream signal transducers. Recent
gene disruption and dominant negative transgenic studies demonstrate
that TRAF2 is required for JNK activation but not NF-
B activation
(17, 18) in lymphocytes and embryonic fibroblasts. However, it is not
clear what downstream kinase cascades are mediated by TRAF2 in
endothelial cells. Here we show that as in lymphocytes and embryonic
fibroblasts, TRAF2 mediates JNK activation in HUVEC and ECV
cells. However, in contrast to published reports in 293 cells
(36, 40), TRAF2 apparently does not mediate p38 MAPK activation in
endothelial cells, because the phosphorylation of p38 MAPK was not
affected by a dominant negative TRAF2. Thus, inhibition of ephrin A1
induction by dominant negative TRAF2 is mediated by the JNK pathway.
In endothelial cells, at least three different subtypes of MAP kinases
are known to be activated by TNF-
: p42/p44 MAPK, p38 MAPK, and
SAPK/JNK. p38 MAPK and SAPK/JNK have been shown to regulate expression
of gene products involved in stress-related events (41). In particular,
p38 MAPK activation mediates actin reorganization and cell migration in
human endothelial cells. In contrast, p42/44 MAPK has been shown to be
involved in cell proliferation (42). Here we show that inhibition of
p38 MAPK and SAPK/JNK, but not p42/44 MAPK, either by selective
chemical inhibitors or dominant negative p38 MAPK or TRAF2 blocked
ephrin A1 induction by TNF-
. Regulation of ephrin A1 expression
through SAPK/JNK and p38 MAPK is consistent with the functional role of
ephrin A1, which has been shown to induce cell migration and vessel
assembly (14) but not cell proliferation.
NF-
B proteins are key proinflammatory transcription factors that
mediate TNF-
-dependent gene induction events. NF-
B
proteins have been implicated in the induction of several angiogenic
molecules. For example, VEGFR2 (flk-1/KDR) expression is
induced by TNF-
, and this induction is mediated through NF-
B in
combination with cAMP-response element-binding protein and
histone acetylases (27). NF-
B is also involved in the regulation of
E-selectin and VCAM-1; the soluble forms of the encoded
proteins induce angiogenesis (28, 43). However, our gel mobility shift
studies and Northern blot analysis data showed that TNF-
-induced
ephrin A1 expression is not mediated through an
NF-
B-dependent mechanism. These results are consistent
with our chemical inhibition studies in which curcumin inhibited
NF-
B activation but did not affect ephrin A1 induction (data not
shown). It is currently not known what transcription factors are
required for ephrin A1 induction in endothelial cells, and the ephrin
A1 promoter elements have not been identified. However, HoxB3, a
TNF-
-inducible homeobox transcription factor that promotes capillary
morphogenesis and angiogenesis, induces ephrin A1 expression in
endothelial cells (44), suggesting that HoxB3 may mediate
TNF-
-induced ephrin A1 expression. Because activation of p38
MAPK and SAPK/JNK leads to ephrin A1 induction, AP-1 or ATF2
transcription factors downstream of p38 MAPK and SAPK/JNK may also be
involved in regulation of ephrin A1.
In summary, our data indicate that induction of ephrin A1 in
endothelial cells by TNF-
is mediated through both p38 MAPK and
SAPK/JNK (Fig. 6), but not p42/p44 MAPK
or NF-
B, pathways. The TNF-
-mediated up-regulation of ephrin A1
is likely to play an important role in angiogenesis under physiological
conditions and in pathological diseases such as tumor growth and
metastasis, rheumatoid arthritis, and diabetic retinopathy. Thus
characterizing the signaling mechanisms that regulate
TNF-
-dependent ephrin A1 induction may elucidate new
targets for therapeutic intervention.

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 6.
Proposed model of signaling pathways
mediating TNF- -induced ephrin A1 expression in
endothelial cells. Activation of p38 MAPK and JNK (shown in
black) are required for TNF- -dependent ephrin
A1 induction. The signaling molecules tested in this study are
boxed.
|
|