Received for publication, May 15, 2002, and in revised form, October 29, 2002
Stromal cell-derived factor-1
(SDF-1
) is a
CXC chemokine, which induces tube formation of endothelial cells.
Although SDF-1
transduces signals via CXC receptor 4 (CXCR4),
resulting in activating a panel of downstream signaling
molecules, such as phosphoinositide 3-kinase (PI3-kinase), little
is known about the SDF-1
-mediated signaling pathways leading to tube
formation. Here we examined the signal transduction pathway involved in
SDF-1
-mediated tube formation by primary human umbilical
endothelial cells and murine brain capillary endothelial cell line (IBE
(immortalized murine brain capillary endothelial) cells). SDF-1
stimulated tube formation by IBE cells, which was blocked by LY294002
and pertussis toxin, suggesting that PI3-kinase and Gi
protein were involved in this process. SDF-1 also stimulated tube
formation of human umbilical endothelial cells, and the response was
LY294002-sensitive. SDF-1
activated PI3-kinase in IBE cells. In
stable IBE cell lines expressing either the mutant p85 subunit of
PI3-kinase (denoted
p85-8 cells), which lacks association with
the p110 subunit, or kinase-inactive c-Fes (denoted KEFes 5-15 cells), SDF-1
failed to activate PI3-kinase and to stimulate tube
formation. SDF-1
-induced tube formation was inhibited by an antibody
against murine vascular endothelial cadherin. The antibody as well as
LY294002 attenuated SDF-1
-mediated compact cell-cell contact, which
proceeded to tube formation. Taken together, SDF-1
induces compact
cell-cell contact through PI3-kinase, resulting in tube
formation of endothelial cells.
 |
INTRODUCTION |
CXC chemokines are involved in angiogenesis (1-4). They are
divided into two groups, angiogenic stimulators, which share a
consensus Glu-Leu-Arg-motif preceding the first cysteine residue, and
inhibitors that lack this motif in their sequence. Among angiogenic CXC
chemokines, stromal cell-derived factor-1-
(SDF-1
)1 plays pivotal
roles in inflamed immune responses and angiogenesis. Targeted
disruption of a gene encoding SDF-1
in mouse resulted in impaired
hematopoiesis and lymphopoiesis (5). SDF-1
transduces signals
via its receptor CXCR4. CXCR4 knockout in mice demonstrated impaired vasculogenesis in intestine, suggesting that signals via CXCR4
may contribute to vasculogenesis and angiogenesis. In fact, CXCR4 is
expressed in vascular endothelial cells (6, 7), and its expression is
up-regulated by angiogenic growth factors, fibroblast growth factor-2,
and vascular endothelial growth factor (8, 9). Furthermore, treatment
of endothelial cells by SDF-1
induces tube-like structure formation
and migration (10, 11).
Activation of CXCR4 in lymphatic cells resulted in activation of
Gi protein, the Ras/mitogen-activated protein kinase (MAPK) pathway, phosphoinositide 3-kinase (PI3-kinase), focal adhesion kinase,
SHP2, c-Fyn, c-Lyn, and Pyk2 (12-16). SDF-1
activated PI3-kinase
and SHP2 have been implicated in migration of lymphocytes and leukemia
cells (14, 15). However, little is known about the roles of these
signaling molecules in SDF-1
-mediated angiogenic responses of
endothelial cells to date.
Previously, we have established a murine brain capillary endothelial
cell line from ts-A58-H-2Kb transgenic mice, denoted IBE
(Immortomouse Brain Endothelial)
cells. IBE cells can form lumen-containing tube-like structures in
response to fibroblast growth factor 2 (FGF-2) and angiopoietin 2 (Ang2) treatment (17-19). Using this culture model, we have shown
previously that expression of dominant negative c-Fes caused impaired
chemotaxis toward FGF-2 and Ang2 (19, 20). It has also been shown that
Ang2-induced PI3-kinase activation depended on c-Fes (19). Furthermore,
expression of dominant negative c-Fyn inhibited FGF-2- and
Ang2-mediated tube formation (19, 21).
In the present study, we examined the signal transduction pathways
leading to SDF-1
-mediated tube-like structure formation of IBE cells
and human umbilical cord vein endothelial cells (HUVECs). SDF-1
stimulated tube formation of both cells, and PI3-kinase inhibitor
LY294002 blocked tube formation, suggesting that PI3-kinase might be
involved in this process by these cells. PI3-kinase was activated by
SDF-1
-treatment in IBE cells, and the activation was dependent on
c-Fes kinase activity. These results suggest that PI3-kinase is an
important signaling molecule of SDF-1
-induced tube-like structure
formation of endothelial cells.
 |
EXPERIMENTAL PROCEDURES |
Reagents--
Anti-phosphotyrosine (PY99) antibody was purchased
from Santa Cruz Biotechnology (Santa Cruz, CA). Rat monoclonal
antibody against mouse vascular endothelial cadherin
(VE-cadherin) was from Pharmingen. Mouse recombinant SDF-1
was
obtained from R&D Systems (Minneapolis, MN). LY294002, PP2, PD98059,
and pertussis toxin were from Calbiochem-Novabiochem and were dissolved
in dimethyl sulfoxide (Me2SO) as a stock solution,
except pertussis toxin, and stored at
30 °C until use. Stock
solutions were further diluted with Me2SO and dissolved in
culture medium. Final concentration of Me2SO was 0.1% in
all cases. Pertussis toxin was suspended in Tris-buffered saline and
was kept at 4 °C until use.
Cell Culture--
HUVECs and their culture medium were purchased
from BioWhittaker, Inc. (Walkersville, MD) and cultured in
endothelial cell basal medium supplemented with endothelial cell growth
supplement, dexamethasone, fetal bovine serum, insulin, and epidermal
growth factor as described in the protocol provided by the
manufacturer. Parental IBE cells obtained from temperature-sensitive
mutant SV40 large T transgenic mouse brain capillaries were cultured in
Ham's F-12 medium containing fetal bovine serum, endothelial cell
growth supplement, insulin, interferon-
, and epidermal growth factor
as has been described previously (17). Stable IBE cell lines expressing
kinase-inactive (dominant negative) c-Fes (denoted KEFes 5-15 cells)
were described elsewhere (20). A stable cell line expressing deleted
mutant p85 PI3-kinase subunit, which does not interact with p110
catalytic subunit (22) (denoted
p85-8 cells), was established, which
demonstrated the dominant negative effect on epidermal growth
factor-mediated PI3-kinase activation (23). Experiments using IBE cell
lines were performed at 33 °C rather than at 39 °C because at the
latter temperature, cells became senescent and lost responsiveness to
extracellular stimuli (17).
Tube Formation Assay--
For IBE cells, cells were cultured
between two layers of type I collagen gels in Ham's F-12 medium
containing 0.25% bovine serum albumin with or without indicated
samples as described previously (17). For HUVECs, cells suspended in
endothelial cell basal medium containing 0.5% fetal bovine serum were
inoculated onto growth factor-reduced Matrigel® (BD Biosciences) with
or without indicated samples and cultured for 24 h as described
previously (24). To quantify the length of newly formed tubes, three
random phase-contrast photomicrographs (×10 objectives) per well were taken, and the length of each tube was measured using NIH Image software (version 1.64). Tube length obtained from FGF-2-stimulated cells or SDF-1
-stimulated cells was set to 100 as described in the
respective figure legends.
Immune Complex PI3-Kinase Assay--
The method used for
determination of PI3-kinase activity in immunoprecipitates of
anti-phosphotyrosine was described previously (25). In brief,
serum-starved cells were lysed in Nonidet P-40 lysis buffer and
incubated with anti-phosphotyrosine antibody followed by absorption
with protein A-agarose beads. After extensive washing,
immunoprecipitates were incubated with phosphatidylinositol and
[
-32P]ATP, and reaction products were separated
by thin layer chromatography on silica gel 60 plates. Incorporation of
[
-32P]ATP into phosphatidylinositol was measured by
Image Analyzer BAS 5000 (Fuji) followed by exposure on x-ray films
(Amersham Biosciences).
Immunoprecipitation and Immunoblotting--
Serum- and growth
factor-starved IBE cells were either stimulated or left unstimulated
with 500 ng/ml SDF-1
in the presence of orthovanadate (50 µM) for 10 min. c-Akt was immunoprecipitated with
anti-Akt antibody followed by immunoblotting with either anti-phospho-Akt or anti-Akt antibodies.
 |
RESULTS AND DISCUSSION |
SDF-1
induces tube-like structure formation of primary human
endothelial cells (10, 11). To examine whether SDF-1
also stimulates
tube formation of IBE cells, we tested the effect of SDF-1
on
morphological changes of the cells. IBE cells respond to FGF-2 and
angiopoietin 2 (Ang2) to form lumen-containing tube-like structures
(18, 19). As shown in Fig. 1A,
FGF-2 induced multicellular aggregates of IBE cells followed by
sprouting and fusion of aggregates, resulting in branching tube-like
structures (26). SDF-1
induced the formation of the tube-like
structures of IBE cells, which was similar to the effect of FGF-2.
CXCR4 is a G protein-coupled receptor. We then examined the effect of
pertussis toxin on SDF-1
-induced tube formation. As shown in Fig.
1B, pertussis toxin at 50 ng/ml blocked SDF-1
-induced
tube formation, suggesting that Gi protein was involved in
this process. We then tested the effects of pharmacological inhibitors
on SDF-1
-induced tube formation. Neither Src family inhibitor PP2
nor mitogen-activated protein kinase/extracellular signal regulated
kinase kinase inhibitor PD98059 inhibited SDF-1
-induced tube
formation of IBE cells (data not shown). Treatment of cells with
PI3-kinase inhibitor LY294002 blocked SDF-1
-induced tube formation
(Fig. 1C). PI3-kinase inhibitor could not block FGF-2- and
Ang2-mediated tube formation (19, 25). FGF-2- and Ang2-mediated tube
formation was dependent on c-Fyn kinase activity (19, 21). Ang2 could
not further stimulate FGF-2-induced tube formation (19), suggesting
that Ang2 and FGF-2 utilized a common signaling pathway leading to tube
formation, such as c-Fyn. To test whether SDF-1
-induced tube
formation requires different signaling pathways activated by FGF-2 and
Ang2, we examined the additive effect of SDF-1
on FGF-2- and
Ang2-induced tube formation. As shown in Fig. 1D, SDF-1
further stimulated FGF-2- and Ang2-induced tube formation. These
results suggest that SDF-1
-induced tube formation of IBE cells may
be dependent on PI3-kinase, which is not involved in FGF-2- and
Ang2-mediated tube formation. We also examined the formation of
tube-like structures by primary HUVECs on Matrigel. Fig.
2A shows that FGF-2 as well as
SDF-1
induced the formation of tube-like structures. In addition,
LY294002 markedly inhibited SDF-1
-mediated formation of
tube-like structures of HUVECs (Fig. 2B). LY294002 showed
little effect on FGF-2-induced formation of tube-like structures of
HUVECs.2 Considered
collectively, PI3-kinase may be involved commonly in SDF-1
-mediated
tube formation by endothelial cells from different origins.

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|
Fig. 1.
As shown in A, SDF-1
stimulates tube-like structure formation of IBE cells cultured
between two layers of collagen gels. FGF-2 was used as a positive
control. Tube length obtained from FGF-2-stimulated cells was set to
100. As shown in B, SDF-1 -mediated tube formation is
pertussis toxin-sensitive. IBE cells inoculated on collagen gels were
incubated with 50 ng/ml of pertussis toxin for 1 h and then either
stimulated or left unstimulated with 500 ng/ml SDF-1 . Tube length
obtained from SDF-1 -stimulated cells was set to 100. As shown in
C, SDF-1 -mediated tube formation of IBE cells was
PI3-kinase inhibitor LY294002-sensitive. Cells in the presence of
PI3-kinase inhibitor LY294002 or vehicle (0.1% Me2SO
(DMSO)) were seeded onto the first layer of collagen gels,
and 1 h later, 500 ng/ml of SDF-1 was added. Tube length
obtained from SDF-1 -stimulated cells was set to 100. As shown in
D, SDF-1 synergistically stimulates tube formation by IBE
cells treated with either FGF-2 or Ang2. IBE cells were treated with
FGF-2 or Ang2 in the presence (closed bars) or absence
(open bars) of 500 ng/ml SDF-1 simultaneously. Tube
length obtained from SDF-1 -stimulated cells was set to 100. Data
shown are reproduced from two to three experiments. Bar, 100 µm.
|
|

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|
Fig. 2.
As shown in A, SDF-1 -induces
tube-like structure formation of HUVECs. HUVECs were cultured on to the
surface of Matrigel in the presence or absence of indicated
growth factors. Tube length obtained from FGF-2-stimulated cells was
set to 100. As shown in B, the SDF-1 -induced tube-like
structure formation of HUVECs is LY294002-sensitive. HUVECs were seeded
onto the surface of Matrigel in the presence of either 0.1%
Me2SO (DMSO) or 10 µM LY294002 and
cultured for 24 h. Tube length obtained from SDF-1 -stimulated
cells was set to 100. Data shown are reproduced from two experiments.
Bar, 100 µm.
|
|
Although distinct methods are employed to assess the ability of
cultured endothelial cells to form tube-like structures, common behavior involved in this process includes cell-cell contact. Endothelial cells contact each other to form cellular aggregates followed by lumen formation between aggregated cells (26, 28-30). We
then examined the effect of SDF-1
on endothelial cell-cell contact.
When treated with SDF-1
, IBE cells adhered to neighboring cells and
formed compact aggregation at 8 h, which is characterized by the
disappearance of borders between aggregated cells (Fig. 5A). This tight cell-cell
contact, denoted cell compaction, is observed in
cadherin-dependent cell-cell aggregation (31, 32). Tight
cell-cell contact was not observed in untreated IBE cells (Fig.
5A). As shown in Fig. 5B, cell compaction was
inhibited by anti-VE-cadherin antibody, suggesting that VE-cadherin was required for cell compaction. Consequently, anti-mouse VE-cadherin antibody inhibited SDF-1
-induced tube formation (Fig.
5C), suggesting that VE-cadherin-mediated cell-cell contact
may be involved in this process. As shown in Fig.
6, compaction was hardly observed in
SDF-1
-treated IBE cells in the presence of LY294002. Only loose
cell-cell contact was observed in SDF-1
-treated KEFes 5-15 cells
and in
p85-8 cells as well. These results suggest that SDF-1
-induced tube formation may be regulated by
PI3-kinase-dependent tight cell-cell contact. Considered
collectively, PI3-kinase and its downstream target molecules, such as
c-Akt, seem to be required for SDF-1
-induced tube formation. In
fact, recent reports have demonstrated that c-Akt was involved in tube
formation of endothelial cells (33-35). Although FGF-2 induced cell
compaction of IBE cells as well, PI3-kinase inhibitor did not inhibit
the cell compaction (data not shown). FGF-2 sufficiently promoted tube
formation by KEFes 5-15 cells and
p85-8 cells. FGF-2 activates
PI3-kinase through activated Ras but not through binding to tyrosine
phosphorylated proteins (23). Ang2 activated PI3-kinase through binding
to tyrosine phosphorylated proteins, and the activation was dependent on c-Fes kinase activity (19). Ang2-induced PI3-kinase activity was
involved in chemotaxis of IBE cells. Treatment of cells with LY294002
did not inhibit Ang2-induced tube formation (19), suggesting that
PI3-kinase does not seem to be involved in this response. On the other
hand, FGF-2 and Ang2 activated c-Fyn in IBE cells (19, 21). Treatment
of cells with PP2 or expression of kinase-inactive c-Fyn inhibited
FGF-2- or Ang2-promoted tube formation of IBE cells, suggesting that
signals through receptor tyrosine kinases leading to tube formation
seem to require c-Fyn but not PI3-kinase (19, 21). A previous study has
shown that c-Fyn was activated by SDF-1
treatment in Jurkat T cells
(15). However, we could not detect SDF-1
-induced c-Fyn activation in
IBE cells (data not shown). Additionally, PP2 failed to inhibit
SDF-1
-induced tube formation. Since induction of chemotaxis involves
dissociation of cells, Ang2-mediated PI3-kinase activation would not be
involved in cell compaction. Conversely, SDF-1
-induced PI3-kinase
activation was required for cell compaction. A number of protein
kinases are activated by lipid products of PI3-kinase (36, 37).
Depending on the culture condition (i.e. two dimensional
culture for migration assay and three-dimensional culture for tube
formation), qualitatively different signaling molecules may be
accumulated into focal adhesion complexes or cell-cell contacts, where
cross-talk between growth factor signaling and cell adhesion signaling
exist. Therefore, PI3-kinase activated by Ang2 or SDF-1
may regulate
distinct sets of downstream signaling molecules. In conclusion,
we have shown in the present study that SDF-1
induced tube formation
of endothelial cells through c-Fes-dependent activation of
PI3-kinase, and formation of cadherin-dependent tight
cell-cell contact seemed to be involved in this process.
We are grateful to T. Shimogama, M. Yoshimoto, and members of the Nagasaki University Radioisotope
Center for excellent outstanding help.
Published, JBC Papers in Press, October 31, 2002, DOI 10.1074/jbc.M204771200
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