Hepatocyte growth factor transduces different intracellular signals in aortic and umbilical venous endothelial cells.

Abstract

Endothelial cells are important for maintenance of vascular integrity by producing a variety of bioactive molecules such as nitric oxide (NO). Recent evidence has suggested that there are some differences in characteristics between endothelial cells from different origins. Here we examined responses of two typical endothelial cells to hepatocyte growth factor (HGF), which induces endothelium-dependent relaxation of microvessels. Stimulation of human umbilical vein endothelial cells (HUVEC) and bovine aortic endothelial cells (BAEC) with HGF increased endothelial NO synthase activity, accompanied with an increase of activity-related site-specific phosphorylation of protein kinase B/Akt. However, HGF stimulated phosphorylation of p38 mitogen-activated protein kinase (MAPK) only in HUVEC, but not in BAEC, while it induced phosphorylation of p44/p42 MAPK in both cells. These results suggest that HGF transduces different intracellular signals between aortic and umbilical venous endothelial cells, and that the differences might represent divergent endothelial responses to growth factors, especially those that activate receptor-tyrosine kinases.

Full text

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Instructions for use
Title
Hepatocyte growth factor transduces different intracellular
signals in aortic and umbilical venous endothelial cells
Author(s)
Makondo, Kennedy; Kimura, Kazuhiro; Kitamura, Takanori;
Yamaji, Daisuke; Saito, Masayuki
CitationJapanese Journal of Veterinary Research, 51(2): 105-112
Issue Date2003-08-29
Doc URLhttp://hdl.handle.net/2115/2971
Right
Typebulletin
Additional
Information
Hokkaido University Collection of Scholarly and Academic Papers : HUSCAP

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Jpn. J. Vet. Res. 51 ( 2) : 105-112,2003
FULL PAPER
Hepatocyte growth factor transduces different intracellular signals in
aortic and umbilical venous endothelial cells
Kennedy Makondo, Kazuhiro Kimura, Takanori Kitamura, Daisuke Yamaji and
Masayuki Saito
(Accepted for publication: June 18,2003)
Abstract
Endothelial cells are important for maintenance of vascular integrity by
producing a variety ofbioactive molecules such as nitric oxide (NO). Recent
evidence has suggested that there are some differences in characteristics be-
tween endothelial cells from different origins. Here we examined responses of
two typical endothelial cells to hepatocyte growth factor (HGF) , which induces
endothelium-dependent relaxation of microvessels. Stimulation of human um-
bilical vein endothelial cells (HUVEC) and bovine aortic endothelial cells
(BAEC) with HGF increased endothelial NO synthase activity, accompanied
with an increase of activity-related site-specific phosphorylation of protein
kinase B/Akt. However, HGF stimulated phosphorylation of p38 mitogen-
activated protein kinase (MAPK) only in HUVEC, but not in BAEC, while it
induced phosphorylation of p44 /p42 MAPK in both cells. These results sug-
gest that HGF trans duces different intracellular signals between aortic and
umbilical venous endothelial cells, and that the differences might represent
divergent endothelial responses to growth factors, especially those that acti-
vate receptor-tyrosine kinases.
Key words: Akt, endothelial cell, HGF, MAPK, nitric oxide synthase.
Introduction
Vascular endothelial cells playa crucial
role in many physiological functions such as
the transport of substances between blood
and tissues, the modulation of the vascular
tone, the activation and migration of white
blood cells, the control of blood coagulation
and fibrinolysis3). All endothelial cells are de-
rived from an identical embryonic origin, but
Laboratory of Biochemistry, Department of Biomedical Sciences, Graduate School of Veterinary Medicine,
Hokkaido University, Sapporo 060-0818, Japan.
Corresponding author: Kazuhiro Kimura, D.V.M., Ph.D. Laboratory of Biochemistry, Department of Bio-
medical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, JAPAN.
Tel & Fax: +81-11-757-0703, E-mail: k-kimura®Vetmed.hokudai.ac.jp

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106
Divergence in endothelial response to HGF
recent evidence has demonstrated that endo-
thelial cells show definite morphological and
molecular differences not only among organs
but also in vascular segments (arterial/capil-
lary/venous) in the same organ4,6,17,lS). For ex-
ample, endothelial cells from lung, heart and
brain express different types of lectin-binding
proteins on their plasma membranes4), and
those from lung, kidney and brain bind selec-
tively to structurally different peptidesl7). In
addition, Wang et al. have reported differ-
ences in cell-adhesion-mediated activation of
intracellular signals between lung microvas-
cular and arterial endothelial cells20) , implying
divergent endothelial responses to extracellu-
lar stimuli including angiogenic factors.
Hepatocyte growth factor (HGF) , also
known as Scatter factor, is a mesenchyme-
derived multifunctional cytokine with a pleth-
ora of biological effects including mitogenesis,
motogenesis, morphogenesis, and organogene-
sis5), and possibly involved in tumor invasion
and metastasis13). HGF also stimulates endo-
thelial cell motility, proliferation and organi-
zation into capillary-like tubes19). In addition
to these angiogenic effects, HGF acts as a va-
sorelaxation factor of microvessels, possibly
through nitric oxide (NO) production14). More-
over, we lately demonstrated HGF stimula-
tion of endothelial NO synthase (eNOS) activ-
ity by phosphoinositide 3 -kinase (PI3 K) /
AId-dependent phosphorylation in a Ca2+-
sensitive manner12) •
Bovine aortic endothelial cells (BAEC )
and human umbilical vein endothelial cells
(HUVEC) are most commonly used for vari-
0us experiments as typical endothelial cells.
However, it remains to be elucidated whether
there are differences in the responses to angi-
0genic growth factors, possibly reflecting the
characteristics attributed to the origins and
whether HGF transduces divergent signals.
To test these hypotheses, in the present study,
we compared HGF -induced intracellular sig-
nals between the two types of endothelial cells.
Materials and Methods
Materials
Human recombinant HGF was a gener-
ous gift of Mitsubishi Pharma Co. (Tokyo, Ja-
pan). Antibodies against phospho-specific
p44/p42 MAPK (Thr-202/Tyr-204), p44/p42
MAPK, phospho-specific p38 MAPK (Thr -180
/Tyr -182), p38 MAPK, phospho-specific Akt
(Thr-308), and Akt were purchased from Cell
Signaling Technology (Beverly, MA, USA) .
Anti-eNOS antibody and calmodulin were
bought from Santa Cruz Biotechnology (Santa
Cruz, CA, USA) and Wako Pure Chemicals Co.
(Osaka, Japan), respectively.
Cell culture and treatment
BAEC from Cell Systems (Kirkland, WA,
USA) were maintained in CS-C Complete Me-
dium Kit (Cell Systems) on Type I-collagen-
coated plates (Asahi Techno Glass Co., Tokyo,
Japan) at 37°C and 5 %C02 under humidified
conditions. HUVEC obtained from ATCC
(Manassas VA, USA) were cultured on the
collagen-coated plates in MCDB -104 medium
(Morinaga Institute of Biological Science,
Yokohama, Japan) containing 10% bovine calf
serum and 12. 5 mM HEPES, supplemented
with brain-extracted growth factors (Mori-
naga Institute of Biological Science). Mter
these cells were grown to confluence, they
were serum-starved in phenol red-free Me-
dium 199 (Sigma-Aldrich, St. Louis, MO,
USA) supplemented with 2 mM L-glutamine
and O. 2% bovine serum albumin (BSA) over-
night prior usage. All the experimental treat-
ments were carried out using fresh serum-
starvation medium.
Measurement of NOS activity
The eNOS activity was quantified as the
conversion of L- [U _14C] arginine to L- [U _14
C] citrulline as previously described7) with mi-

Page 4

Kennedy Makondo et al.
107
nor modifications. Briefly, confluent cells were
serum-starved and the medium replaced be-
fore the treatments. Following the experimen-
tal treatments, cells were harvested in ice-
cold homogenization buffer containing 50mM
Tris-HCI, pH 7. 4, 250 mM sucrose, 0.1 mM
EGTA, O. 1 mM EDTA, 1 mM dithiothreitol,
and a protease inhibitor cocktail, Complete ( 1
tablet I 50 mL, Roche Diagnostics, GmbH,
Mannheim, Germany). For each sample lOflL
of homogenate was incubated in duplicate at
37
containing 50 mM HEPES, pH 7.9,1 mM
dithiothreitol, 1mM CaCh, o. 1mM tetrahydro-
L-biopterin (BH4) , 1 mM NADPH, 10 flM
FAD, 10 flg/mL calmodulin and 1. 43flM L- [U
_14C] arginine (Amersham Pharmacia Biotech,
Buckinghamshire, UK). The reaction was ter-
minated by addition of 200flL of a stop solu-
tion containing 100 mM HEPES and 10 mM
EDTA, pH 5. 2 . The reaction mixture was
then applied to O. 5mL neutralized AG 50 w-x
4 resin ( N a + form 200 -400 mesh, Bio-Rad
Labs, Hercules, CA, USA) column to separate
L - [U _14C ] citrulline. The flow through was
analyzed by liquid scintillation counting, and
enzyme activity expressed as fmol of L- [U _14
C] citrulline produced/mg protein ofhomogen-
ate/10min. Protein concentration was deter-
mined by the Lowry method10) using BSA as a
standard.
Western blot analysis
Following the experimental treatments,
cells were washed with ice-cold PBS and
scraped in ice-cold lysis buffer (50mM HEPES,
pH 7. 5, 150mM NaCI, 5 mM EDTA, 20mM so-
dium fluoride, 10mM sodium pyrophosphate,
2 mM sodium vanadate, 1 %NP 40 and a pro-
tease inhibitor cocktail, Complete). Harvested
cells were incubated on ice for 30min followed
by centrifugation at 12, OOOx g for 20min at
4 DC to obtain cell lysate. Aliquots of the cell
lysate (30flg of each sample) were resolved on
DC for 10min in a100flL of reaction mixture
SDS-PAGE under reducing conditions and
protein electroblotted onto polyvinylidene
difluoride membrane (Immobilon TM; Millipore,
MA, USA). The membrane was blocked in
5 % skim milk overnight at 4 DC followed by
incubation with a primary antibody overnight
at 4 DC, and then exposure to a horseradish
peroxidase-conjugated secondary goat anti-
rabbit antibody (Zymed Lab. Inc., San Fran-
cisco, CA, USA) for 1 h at room temperature.
Visualization was performed using the en-
hanced chemiluminescence ECL (Amersham)
detection system according to the instructions.
Intensities of immunoreactive bands in the
Western blots were densitometrically ana-
lyzed on a Macintosh computer using the pub-
lic domain NIH Image program (U.S. National
Institutes of Health; available on the Inter-
net at http://rsb.info.nih.gov/nih-image/) .
Statistical Analysis
Results are expressed as means -±: S. E. of
3 - 4 independent experiments. Statistical
analysis was done using analysis of variance
(ANOVA) and Fischer's test at p< 0.05.
Results
We first tested the expression of eNOS
protein in HUVEC and BAEC, and compared
RLMEC BAEC Swiss3T3
LEII
------.
==-
------....
RAEC HUVEC
eNOS
Figure 1. Western blot analysis of eNOS protein in
various types of endothelial cell.
Celllysates were prepared from HlNEC,
BAEC, rat aortic and lung microvascular
endothelialcells (RAEC and RLMEC) ,
mouse lung microvascular endothelial cell
line (LEn) and Swiss 3 T 3 fibroblast as
negative control, and 40 Ilg of each cell
lysate was separated by SDS-PAGE and
examined for eNOS protein expression by
Western blot. Shown are representative
blots of three independent experiments.

Page 5

108
Divergence in endothelial response to HGF
Table 1. HGF increases eNOS activity in HUVEC and BAEC
Cell line Basal activity
HGF stimulation Fold
HUVEC 1063. O± 164.1 2336. O± 171. 9*
2.4±0.5
BAEC 3554. 7±779. 7 6708. 0±964.1 * 2.1±0.3
Confluent HUVEC and BAEC were serum-starved overnight and stimulated with HGF (40ng/ml) for 20min. The
eNOS activity in the cell homogenate was determined as the conversion ofL- [U _14CJ arginine to L- [U _14CJ citrul-
line, and expressed as fmol of citrulline produced/mg protein of homogenate/ lOmin and the mean±S.E. of four
independent experiments. *, p< 0.05 vs. basal activity.
A
HUVEC
BAEC
HGF
+
+
Phospho-Akt
Akt
=
.!
~
0
~
200
*
0 :c
Q..
fIl
0
~
100
Q..
Ct-I
0
~
S
fIl
~ :c
~ -
~
Q,I
0

HGF
+
+
HUVEC
BAEC
=
.!
~
0 . .,. ..

0
-=
~
fIl
0
.=
~


~
0
.f:!
S
fIl
~ =
Q,)
~
= -
B
10
8
6
4
2
0
HUVEC
HGF-
+
BAEC
+
Phospho-p44/p42 MAPK
p44/p42 MAPK
*
HGF
± ±
HUVEC
BAEC
• P-p44
• P-p42
Figure 2. HGF induces Akt and p44/p42MAPK phosphorylation in HUVEC and BAEC.
Serum-starved HUVEC and BAEC were stimulated with HGF (40ng/ml) forlOmin. The cell
lysate (30llg/lane) was resolved on 10% SDS-PAGE, and examined for activation ofp44/p42
MAPK and Akt with anti-phospho-p44/p42 MAPK (Thr 204/Tyr 202) and anti-phospho-Akt
(Thr-308) antibodies and for their protein content (total) with respective antibodies. Shown
are representative blots of three independent experiments for Akt (A) and p44/p42MAPK(B) ,
respectively. Densitometric analyses as fold increase relative to controls (untreated). Re-
sults represent the mean±S.E. of three independent experiments. *, p<0.05 vs. without
HGF addition.

Page 6

A
=
.....
.s
~
0
..c=
Q,.
rIl
0
..c=
Q,.
~
0 ...
0
~
.....
S
rIl
~
...


~
..... = -
~
~
Kennedy Makondo et al.
HUVEC
B
Cont.
HGF
,,' ,y'L", , t ' n:
Phospho-p38MAPK
I
,_?VY1><"
,
p38MAPK
6
*
5
4
3
2
1
0
Control HGF
BAEC
1.2
1.0
~
~ 0.8
~ .. b 0.6
:s
-< 0.4
0.2
Cont.
HGF
Phospho-p38MAPK
p38MAPK
0 .................... --...... _-
Control
HGF
Figure 3. HGF induces p38 MAPK phosphorylation in HUVEC, but not in BAEC.
Serum-starved HUVEC (A) and BAEC (B) were stimulated with either HGF
(40ng/ml) or H202 (1 mM) for 10 min. The cell lysate (30llg/lane) was resolved
on 10% SDS-PAGE, and examined for activation of p38MAPK with anti-
phospho-p38 MAPK (Thr-180/Tyr-182) antibodies and for p38 MAPK protein
content (total). Shown are representative blots of three independent experi-
ments. Densitometric analyses are expressed as fold increase relative to con-
trols (A). As no signal for phospho-p38 MAPK was detected in control and HGF
-stimulated BAEC, densitometric value for H202 - stimulated cells is shown as
arbitrary units (B). Results represent the mean±S.E. of three independent ex-
periments. *, p< 0.05 vs. without HGF addition.
109
with those in other endothelial cells, primary
cultured rat aortic and lung microvascular en-
dothelial cells (RAEC and RLMEC) 11) and
mouse lung microvascular endothelial cell
line (LEII) 1) and Swiss 3T3 fibroblast as nega-
tive control. As shown in Fig. 1 , all of the en-
dothelial cells tested, but not Swiss 3T3 cells,
expressed eNOS protein, but the levels of the
protein in HUVEC and BAEC were much
higher than those of primary cultured endo-
thelial cells and LEII cells. We also examined
eNOS activity in HUVEC and BAEC as the
conversion of arginine to citrulline. Basal
eNOS activity in HUVEC was relatively lower
than that in BAEC, and the stimulation of the
cells with HGF for 20min significantly in-
creased eNOS activity by almost the same
magnitude (Table 1 ) .
HGF acts on endothelial cells through ty-
rosine kinase receptor, c-Met, and subsequent
activation of both PI3K and p44/ p42MAPK2,16) ,
and Akt which lies down-stream of PI 3 K di-

Page 7

110
Divergence in endothelial response to HGF
rectly regulates eNOS activity by phospho-
rylation12). We next examined the effects of
HGF stimulation on Akt and p44/p42MAPK
phosphorylation, using the antibodies to de-
tect activity-related site-specific phosphoryla-
tion. As shown in Figs. 2 A and 2 B, HGF po-
tently stimulated phosphorylation of Akt (Thr
308) and p44/p42MAPK (Thr-202/Tyr204)
without affecting their total protein contents,
indicating activation of both protein kinases.
There was no apparent difference in these re-
sponses to HGF between HUVEC and BAEC.
Recently, differences in p38 MAPK activa-
tion have been reported between lung mi-
crovascular and arterial endothelial cells
treated with anti-intercellular adhesion mole-
cule - 1 (ICAM - 1) antibody for its cross-
linkintO). To test whether HGF activates p38
MAPK, we examined activity-related site-
specific p38 MAPK phosphorylation. Stimula-
tion ofHUVEC with HGF induced p38 MAPK
phosphorylation (Thr-180/Tyr-182), while it
did not cause any noticeable changes in total
p38 MAPK (Fig. 3 A). In contrast, stimula-
tion of BAEC with HGF failed to induce p38
MAPK phosphorylation, although significant
amount ofp38MAPK was detected (Fig. 3B).
Moreover, hydrogen peroxide (H202) induced
phosphorylation similarly in HUVEC and
BAEC (Fig. 3 ) .
Discussion
We here demonstrated that HGF/c-Met
signaling stimulated pathways leading to
eNOS activation, Akt and p44/p42 MAPK
phosphorylation in the two types of endothe-
lial cells, HUVEC and BAEC, but p38 MAPK
activation only in HUVEC. Our findings are
in accordance with HGF activation of Akt and
p44/p42MAPK pathways in human aortic en-
dothelial cells, necessary for HGF-induced mi-
togenic and antiapoptotic actions16) .
Recently we have demonstrated that
HGF stimulates eNOS activity through PI 3 K
I Akt-dependent eNOS phosphorylation in
BAEC12) : that is, HGF initially activates
PI3K to produce 3 -phosphoinositide, leading
to Akt phosphorylation at Thr308 by 3 -phos-
phoinositide-dependent protein kinase - 1
(PDK - 1 ), and consequently to Akt-mediated
eNOS phosphorylation and activation. Stimu-
lation of HUVEC with HGF similarly in-
creased eNOS activity as in BAEC, possibly
through Akt-mediated pathway, as the activa-
tion of eNOS was accompanied with an in-
crease of Akt phosphorylation. The eNOS acti-
vation obviously indicates enhancement of
NO production, and might explain the mecha-
nism of HGF-induced vasorelaxation14) and
some other HGF-induced changes including
angiogenesis19), limiting of neointimal prolif-
eration2
cardial ischemic damage15). Furthermore, our
results suggest conserved eNOS activation,
Akt and p44/p42 MAPK pathways by HGF in,
at least HUVEC and BAEC, vascular endo-
thelial cells of divergent origins.
As mentioned above, although HGF acti-
vated Akt, p44/p42MAPK and eNOS in both
HUVEC and BAEC, it stimulated activity-
related site-specific p38 MAPK phosphoryla-
tion only in HUVEC. In BAEC, HGF failed to
induce p38 MAPK phosphorylation even tho-
ugh p 38 MAPK protein is present and H2 02
did induce its phosphorylation. These findings
suggest that some machinery leading to p38
MAPK activation through c-Met, a tyrosine
receptor kinase, are lacking in BAEC. Simi-
larly, even in the same species, treatment of
arterial endothelial cells with anti-ICAM - 1
antibodyO) or proinsulin C-peptide (Kitamura,
T. et al. manuscript in submission) fails to ac-
tivate the p38 MAPK pathway, while treat-
ment of lung microvascular endothelial cells
does. Collectively, the deficiency of the ma-
chinery to activate p38 MAPK seems to be due
1) and tissue regeneration after myo-

Page 8

Kennedy Makondo et al.
111
to the origin of endothelial cells, especially
those derived from major arteries.
The activation of p38 MAPK has been
shown to be elicited by a series of cytokines,
growth factors, and autonomic neurotransmit-
ters and also stress factors such as UV irra-
diation and H202, leading to phosphorylation
of some transcription factors9). Actually, we
have recently shown that stimulation of LEU
mouse lung capillary endothelial cells with
proinsulin C-peptide increased p38 MAPK ac-
tivity, thereby leading to phosphorylation of
cyclic AMP responsive element (CRE) binding
protein (CREB) / activating transcription fac-
tor 1 (ATF 1 ) and consequent binding of these
transcription factors to CRES). Therefore, it is
likely that HUVEC, a venous type of endothe-
lial cell, is controlled differently from BAEC
at transcription levels by HGF and possibly
by other growth factors that activate receptor-
tyrosine kinases.
In summary, we have demonstrated that
HGF trans duces different intracellular sig-
nals between aortic and umbilical venous en-
dothelial cells, and the difference, at least in
terms of p38 MAPK activation, might be wor-
thy consideration prior to experimental usage.
Acknowledgements
This work was supported by grants from
the Ministry of Education, Science and Cul-
ture of Japan and PROBRAIN from the Bio-
oriented Technology Research Advancement
Institution, Japan. The work was also sup-
ported by Research Fellowships of the Minis-
try of Education, Science and Culture of Ja-
pan for Foreign Students to K. M. and of the
Japan Society for the Promotion of Science
(JSPS) for Young Scientists to T.K.
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