Sonic Hedgehog Induces Capillary Morphogenesis by Endothelial
Cells through Phosphoinositide 3-Kinase*
Shigeru
Kanda
§¶,
Yasushi
Mochizuki§,
Takashi
Suematsu
,
Yasuyoshi
Miyata§,
Koichiro
Nomata§, and
Hiroshi
Kanetake§
From the
Department of Molecular Microbiology and
Immunology, Division of Endothelial Cell Biology and
§ Department of Urology, Nagasaki University Graduate School
of Medicine, 1-7-1 Sakamoto, Nagasaki 852-8501, Japan, and
Central Electroscope Laboratory, Nagasaki University School of
Medicine, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan
Received for publication, October 17, 2002, and in revised form, December 26, 2002
 |
ABSTRACT |
Sonic hedgehog (Shh) acts as a morphogen in many
cell types. Recent studies have shown that hedgehog signaling is
involved in vascular development as well as postnatal angiogenesis.
However, the direct action of Shh on cultured endothelial cells has not been clearly shown. To address this issue, we examined the effect of
Shh on morphological changes by murine brain capillary endothelial cells (IBE cells) and human umbilical endothelial cells (HUVECs). Shh
induced capillary morphogenesis by these cells. The effect was
inhibited by cyclopamine or pertussis toxin. Shh-induced capillary morphogenesis was also blocked by LY294002, a phosphoinositide 3-kinase
(PI3-kinase) inhibitor. Shh rapidly increased PI3-kinase activity in
IBE cells and HUVECs; this activity was inhibited by cyclopamine.
Nuclear localization of Gli1 was increased in Shh-treated IBE cells,
which was not affected by LY294002. Actinomycin D and cycloheximide
inhibited Shh-induced capillary morphogenesis. In IBE cells expressing
kinase-inactive c-Fes, Shh failed to stimulate PI3-kinase activity and
capillary morphogenesis. Considered collectively, Shh induced capillary
morphogenesis of endothelial cells through both rapid activation of
c-Fes/PI3-kinase pathways and transcriptionally regulated pathways.
 |
INTRODUCTION |
Sonic hedgehog (Shh)1 is
a member of a family of closely related proteins consisting of Shh,
indian hedgehog, and desert hedgehog. These proteins are known to
regulate morphology of many kinds of tissues (1). Initial studies
showed that hedgehog transduced signals via its receptor Patched (Ptc).
It has been shown that Ptc forms complex with seven-pass membrane
protein, smoothened (Smo), which exists as an inactive form.
Hedgehog-bound Ptc dissociates Smo, resulting in the activation.
Activated Smo allows the entry of a transcription factor, Cubitus
interuptus (Ci), into nuclei, which induces the expression of a panel
of downstream target molecules (2). Vertebrate homologs of Ci are Gli1,
2, and 3. However, recent studies addressing the distribution of Ptc
and Smo have presented evidence that Smo is not present in a complex
containing Ptc (3, 4). Although it is still unclear how Ptc regulates Smo activity, the sterol-sensing domain of Ptc seems to be important for regulation (4, 5). The regulation of Ci depends on their phosphorylation status. Ci is first phosphorylated by protein kinase A
(6, 7). Further phosphorylation of Ci by Shaggy/glycogen synthase
kinase 3 and casein kinase 1 leads to its proteolytic degradation (8,
9). However, protein or lipid kinases activated by hedgehog have not
been well characterized to date.
It has been shown that hedgehog signaling is involved during vascular
development (10-12). Shh was also reported recently to induce
postnatal angiogenesis (13). Angiogenesis is composed of a series of
endothelial cellular responses, and maturation of newly formed vessels
is accompanied by branching, capillary-like morphology (14, 15). In a
previous report, there were no direct effects of Shh on cellular
responses by cultured endothelial cells, such as proliferation,
migration, and serum-deprived survival reported (13). Because hedgehog
signaling regulates morphological changes of many types of cells, we
focused on the capillary morphogenesis of cultured endothelial cells
mediated by Shh. We found that Shh induced capillary morphogenesis by
human umbilical vein cord endothelial cells (HUVECs) as well as
immortalized murine brain capillary endothelial (IBE) cells (16). Shh increased phosphoinositide 3- kinase (PI3-kinase) in endothelial cells, and Shh-mediated capillary morphogenesis was PI3-kinase inhibitor-sensitive.
Expression of deleted mutant p85, which cannot associate with p110
catalytic subunit of PI3-kinase, and kinase-inactive c-Fes blocked
Shh-mediated PI3-kinase activation as well as capillary morphogenesis.
These results suggest that Shh activates PI3-kinase in endothelial
cells, followed by the induction of capillary morphogenesis.
 |
EXPERIMENTAL PROCEDURES |
Reagents--
Anti-phosphotyrosine (PY99) antibody, anti-FLAG
goat antibody, anti-Patched-1 antibody, and anti-Gli1 antibody were
purchased from Santa Cruz Biotechnologies, Santa Cruz, CA. Anti-FLAG
monoclonal antibody M2 and anti-
-actin antibody were from Sigma.
Anti-Akt and phospho-Akt (Ser-473) antibodies were obtained from
PerkinElmer Life Sciences. Mouse recombinant Shh N-terminal
peptide, human recombinant angiopoietin 2 (Ang2), human recombinant
vascular endothelial growth factor-A (VEGF-A), and Tie2/Fc chimera were obtained from R & D Systems, Minneapolis, MN. The recombinant Shh
protein used in this study was an N-terminal fragment corresponding to
Cys-25 to Gly-198 of mouse Shh, which has been shown to be an active
form (17). Recombinant Shh protein was expressed in Escherichia
coli and was purified to more than 97% as determined by SDS-PAGE.
The contaminated endotoxin level was less than 1.0 enzyme units/µg of
the Shh with the Limulus amebocyte lysate method, as determined
by the manufacturer. A PI3-kinase inhibitor, LY294002, a Src
family kinase inhibitor, PP2, and a mitogen-activated protein kinase/extracellular signal regulated kinase kinase inhibitor, PD98059,
were from Calbiochem-Novabiochem (La Jolla, CA) and were dissolved in
dimethyl sulfoxide (Me2SO) as a stock solution 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 from
Calbiochem-Novabiochem and was dissolved in Tris-buffered saline.
Cyclopamine was purchased from Toronto Research Chemicals Inc. North
York, ON, Canada and growth factor-reduced Matrigel was from BD
Biosciences, Bedford, MA. Cycloheximide and actinomycin D were obtained
from Sigma.
Cell Culture--
Parental IBE cells obtained from
temperature-sensitive mutant SV 40 large T transgenic mouse brain
capillaries were cultured as reported previously (16). Stable IBE cell
lines expressing either wild-type c-Fes or kinase-inactive c-Fes
(denoted WTFes 6-8 cells and KEFes 5-15 cells, respectively; 18), and a
stable cell line expressing deleted mutant p85 PI3-kinase subunit,
which does not interact with p110 subunit (19) (denoted
p85-8 cells; 20), were described elsewhere. Experiments using IBE cell lines were
performed at 33 °C, because at 39 °C, cells became senescent and
lost responsiveness to extracellular stimuli (16). HUVECs and their
culture medium were obtained from BioWhittaker, Inc., Walkersville, MD,
and cells were cultured according to the protocol recommended by the manufacturer.
Capillary Morphogenesis Assay--
For IBE cells, cells were
suspended in Ham's F-12 medium containing 0.25% bovine serum
albumin and seeded onto cultured growth factor-reduced
Matrigel® in wells of 24-well plates at a density of 1.0 × 104 cells/well. Cells were treated or left untreated with
indicated pharmacological inhibitors or vehicle (0.1%
Me2SO) for 1 h, and then indicated growth factors were
added. Twenty-four hours later, capillary morphogenesis was examined
under a phase-contrast microscopic observation. For HUVECs, cells
suspended in a culture medium containing 0.5% fetal bovine serum were
inoculated onto growth factor-reduced Matrigel® at a density of
3.0 × 104 cells/well of 24-well plates and treated as
indicated (21). Cells were cultured for 24 h, and capillary
morphogenesis was examined. To quantify the length of capillaries,
three different phase-contrast photomicrographs (×10 objectives) per
well were taken, and the length of each capillary was measured using
NIH Image software (version 1.64). Capillary length was expressed as
-fold increase relative to unstimulated cells.
Immune Complex PI3-kinase Assay--
The method used for
determination of PI3-kinase activity in the immunoprecipitates of
anti-phosphotyrosine was described previously (20). In brief,
serum-starved cells were either stimulated or left unstimulated by
indicated cytokines and lysed in Nonidet P-40 lysis buffer, and
tyrosine-phosphorylated proteins were immunoprecipitated. 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 Shh in the presence of orthovanadate (50 µM) for 10 min. Cells were washed and lysed in Nonidet P-40 lysis buffer, and
c-Akt was immunoprecipitated with anti-Akt antibody. Immunoprecipitated
proteins were separated by SDS-polyacrylamide gel electrophoresis.
After electronic transfer onto polyvinylidene difluoride membranes
(Millipore, Bedford, MA), the blots were probed with either
anti-phospho-Akt or anti-Akt antibodies. To examine the expression of
Patched protein, total cell extracts of HeLa cells, IBE cells, and
HUVECs were electrophoresed, and immunoblotting was performed with
anti-Patched 1 antibody followed by anti-
-actin antibody.
In Vitro Kinase Assay for c-Fes--
Cells were starved, either
stimulated with 5 µg/ml Shh for 8 min or left unstimulated, and lysed
in the Nonidet P-40 lysis buffer. Cell lysate was separated into two
portions, 90% and 10%, respectively, and FLAG-tagged c-Fes was
immunoprecipitated with anti-FLAG monoclonal antibody from the 90% of
each lysate. Immunoprecipitates were washed 4 times with lysis buffer,
twice with Tris-buffered saline, and once with kinase buffer (25 mM HEPES, pH 7.4, supplemented with 10 mM
MnCl2 and 2 mM MgCl2).
Immunoprecipitates were incubated with [
-32P]ATP at
30°C for 10 min, and reaction products were run on SDS-PAGE. Polyacrylamide gels were fixed, soaked in 1 M KOH at 55°C
for 30 min to remove phosphorylated serine residues, dried, and exposed on Imaging Plate for the analysis with Image Analyzer BAS 5000 (Fuji),
followed by the exposure on x-ray films (Amersham Biosciences).
Immunofluorescent Staining of Gli1--
IBE cells were cultured
on fibronectin- and gelatin-coated coverslips for 24 h and then
serum-starved for 16 h. Cells were incubated with 0.1%
Me2SO, LY294002, or cyclopamine for 30 min, and then were
treated with Shh for 2 h, 4 h, or left untreated. Cells were
washed with phosphate-buffered saline and fixed with methanol at
20° for 20 min. Cells were washed and incubated with phosphate-buffered saline containing 5% bovine serum albumin, 10%
normal rabbit serum, and 5% nonfat dried milk for 60 min at room
temperature, followed by incubation with anti-Gli1 antibody or normal
goat IgG (negative control) at 8 µg/ml at 4°C overnight. Cells were
washed and incubated with fluorescein isothiocyanate-conjugated anti-goat IgG. After extensive washing, localization of Gli1 protein was examined under a fluorescent microscopic observation. To examine the percentage of cells with nuclear staining of Gli1, more than 500 cells were determined in each treatment.
 |
RESULTS |
Angiogenic cellular responses by endothelial cells involve
capillary morphogenesis. Shh regulates morphological changes of many
cell types. Thus, we first examined the effect of Shh on morphological
change of IBE cells. IBE cells form capillary-like structures in
response to FGF-2 or Ang2 treatment (16, 22). As shown in Fig.
1, Shh dose-dependently
induced capillary morphogenesis of IBE cells, of which morphology was
similar to that of FGF-2 or Ang2 treated cells. Cells treated with Shh
showed extended cytoplasm on Matrigel and contacted each other. Between
these cells, a lumen-like structure (a slit) was observed. In the
absence of Shh, cells failed to organize slit-containing, continuous
cord-like structures. VEGF-A did not induce capillary morphogenesis of
IBE cells, as has been shown previously (16). Several reports have demonstrated that VEGF-A induced capillary morphogenesis through VEGF
receptor 2 (23, 24). VEGF receptor 2, but not VEGF receptor 1, also
transduces signals, leading to proliferation and migration of
endothelial cells (25). Expression of VEGF receptor 2 in IBE cells was
barely detectable, and VEGF-A also failed to stimulate proliferation
and migration of IBE cells (16). It is therefore possible that a
certain level of VEGF receptor 2 expression in endothelial cells seems
to be required to regulate endothelial cell behaviors in response to
VEGF-A-treatment. Fig. 2A
shows that FGF-2-induced capillary morphogenesis was inhibited by the treatment of cells with a Src family kinase inhibitor, PP2, as reported
(26). Neither LY294002 nor PD98059 inhibited FGF-2-induced capillary
morphogenesis by IBE cells. As shown in Fig. 2B,
cyclopamine, a steroid alkaloid that specifically inhibits
signaling via Smo (27, 28), suppressed Shh-induced capillary
morphogenesis. Pertussis toxin, a protein endotoxin that catalyzes
ADP-ribosylation of guanine nucleotide-binding protein Gi, also blocked
Shh-induced capillary morphogenesis. LY294002, but not PD98059 or PP2,
blocked Shh-mediated capillary morphogenesis. Cyclopamine and pertussis toxin did not affect FGF-2-induced capillary morphogenesis. These results suggest that Shh activated different signaling pathways than
those activated by FGF-2. We also examined the capillary morphogenesis
by HUVECs. Fig. 3 shows that FGF-2 as
well as Shh induced capillary morphogenesis by HUVECs. LY294002 also
markedly inhibited Shh-mediated capillary morphogenesis. LY294002 did
not exhibit marked effect on FGF-2-induced capillary morphogenesis. Cyclopamine also blocked Shh-induced capillary morphogenesis by HUVECs
(data not shown). We next examined the role of
Gli1-dependent transcription in Shh-induced capillary
morphogenesis. Receptor for Shh, Ptc protein, was expressed in IBE
cells and HUVECs (Fig. 4A).
Transcription factor, Gli1, was localized in the cytoplasm of untreated
IBE cells (Fig. 4B). Although FGF-2-treatment did not affect
the localization of Gli1 (data not shown), Shh treatment increased
nuclear localization of Gli1 in IBE cells, and cyclopamine inhibited
translocation. However, nuclear localization was not inhibited by the
treatment of cells with LY294002, suggesting that Shh-mediated nuclear
translocation of Gli1 was independent of PI3-kinase activity. We
examined the effects of cycloheximide, which inhibits protein
synthesis, or actinomycin D, which inhibits translation, on Shh-induced
capillary morphogenesis by IBE cells. These compounds at a
concentration of 500 ng/ml exhibited cytotoxic effects on IBE cells. At
100 ng/ml, both cycloheximide and actinomycin D inhibited Shh-induced
capillary morphogenesis (Fig. 4C). These results suggest
that Shh seems to regulate capillary morphogenesis of endothelial cells
through PI3-kinase-dependent pathways as well as
PI3-kinase-independent, Gli1-mediated gene transcription. In the
present study, we did not observe any direct effects of Shh on
proliferation or migration of cultured endothelial cells (data not
shown).

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Fig. 1.
Shh induces capillary morphogenesis by IBE
cells. IBE cells were suspended in serum-free Ham's F-12 medium
and cultured on growth factor-reduced Matrigel in the presence or
absence of indicated cytokines. Twenty four hours later, pictures were
taken under a phase-contrast microscope. At higher magnification,
slit-like structures were observed between extended endothelial cells
(inset). Capillary length was measured and expressed as
-fold increase relative to untreated cells. Reproducible results were
obtained in three independent experiments. Bar, 100 µm.
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Fig. 2.
Effects of signal-transduction pathway
inhibitors on FGF-2- and Shh-induced capillary morphogenesis by IBE
cells. A, capillary morphogenesis induced by FGF-2
was inhibited by PP2. Cells suspended in serum-free medium in the
presence of 0.1% Me2SO (vehicle) or inhibitors were seeded
onto Matrigel. One hour later, cells were either stimulated or left
unstimulated with 10 ng/ml FGF-2. After 24 h, pictures were taken
and capillary length was measured as described in the legend of Fig. 1.
Bar, 100 µm. B, Shh-induced capillary
morphogenesis is blocked by cyclopamine, pertussis toxin, or PI3-kinase
inhibitor LY294002. Cells suspended in serum-free medium in the
presence of 0.1% Me2SO (vehicle) or inhibitors were seeded
onto Matrigel. One hour later, cells were either stimulated or left
unstimulated with 5 µg/ml Shh or 10 ng/ml FGF-2. After 24 h,
pictures were taken and capillary length was measured as described in
the legend of Fig. 1. Reproducible data were obtained from two
independent experiments. Bar, 100 µm.
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Fig. 3.
Shh induces capillary morphogenesis by
HUVECs, and this morphological change is also LY294002-sensitive.
HUVECs were seeded onto Matrigel in the presence of 0.1%
Me2SO or 10 µM LY294002. One hour later,
cells were either stimulated or left unstimulated with 5 µg/ml Shh or
20 ng/ml FGF-2. After 24 h, pictures were taken and capillary
length was measured as described in the legend of Fig. 1. Reproducible
data were obtained from two independent experiments. Bar,
100 µm.
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Fig. 4.
A, Patched 1, a receptor for sonic
hedgehog, is expressed in HUVECs and IBE cells. Total cell extracts
from HeLa cells (as a positive control), HUVECs, and IBE cells were
electrophoresed, and immunoblotting was performed with goat
anti-Patched 1 polyclonal antibody. After stripping, the membrane was
reprobed with anti- -actin antibody. B, Gli1 is present in
nuclei of Shh-treated IBE cells. Cells grown on coverslips were
serum-starved and incubated with Me2SO, LY294002, or
cyclopamine. One hour later, cells were either stimulated or left
unstimulated with 5 µg/ml Shh for 2 or 4 h. Gli1 was visualized
by an indirect immunofluorescent staining technique and cells with
nuclear staining of Gli1 was defined as positive cells. Ratio of
positive cell number/total cell number was calculated. Data were
expressed as means ± S.E. from three independent cultures.
C, actinomycin D and cycloheximide inhibit Shh-induced
capillary morphogenesis of IBE cells. Cells suspended in serum-free
medium in the presence of 0.1% ethanol (vehicle) or chemicals were
seeded onto Matrigel. One hour later, cells were either stimulated or
left unstimulated with 5 µg/ml Shh. After 24 h, pictured were
taken and capillary length was measured as described in the legend of
Fig. 1. Reproducible data were obtained from two independent
experiments. Bar, 100 µm.
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We next examined the mechanisms underlying Shh-induced activation of
PI3-kinase. As shown in Fig.
5A, Shh increased PI3-kinase activity in anti-phosphotyrosine immunoprecipitates of IBE cells and
HUVECs. Stable expression of deleted mutant p85 subunit, which lacks
binding to p110 catalytic subunit (denoted
p85-8 cells), inhibited
Shh-induced increase in PI3-kinase activity. These results suggest that
Shh induced tyrosine phosphorylation of particular proteins, followed
by the association with p85 subunit of PI3-kinase. c-Akt was also
phosphorylated at Ser-473, which is involved in its activation (29),
and such phosphorylation was also LY294002-sensitive (Fig.
5B). Shh failed to phosphorylate c-Akt in
p85-8 cells. c-Fes tyrosine kinase is exclusively expressed in endothelial cells and
hematopoitic cells. c-Fes is activated by the oligomerization, followed
by autophosphorylation. In a recent study, we have shown that
Ang2-mediated activation of PI3-kinase depended on c-Fes tyrosine
kinase activity (22). We tested whether Shh could activate c-Fes.
FLAG-tagged wild-type (from WTFes 6-8 cells; 18) or kinase-inactive c-Fes (from KEFes 5-15 cells; 18) was immunoprecipitated, and kinase
activity was examined by the incorporation of
[
-32P]ATP into precipitated c-Fes. As shown in Fig.
5C, incorporation of [
-32P]ATP into
wild-type c-Fes, but not kinase-inactive c-Fes, was increased by
Shh-treatment, suggesting that c-Fes seemed to be activated by Shh
treatment. We then examined the Shh-induced activation of PI3-kinase in
KEFes 5-15 cells. Fig. 5D shows that Shh failed to activate
PI3-kinase in KEFes 5-15 cells. This result suggests that Shh-mediated
PI3-kinase activation depends on c-Fes kinase activity. We also
examined the Shh-mediated capillary morphogenesis in KEFes 5-15 cells
and
p85-8 cells. As shown in Fig. 6,
FGF-2 as well as Ang2 induced capillary morphogenesis by these cells. However, Shh failed to induce capillary morphogenesis by these cells.
These results strongly suggest that Shh-mediated capillary morphogenesis requires PI3-kinase activation through c-Fes in endothelial cells.

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Fig. 5.
A, Shh increases PI3-kinase activity in
immunoprecipitates of anti-phosphotyrosine antibody. Serum-starved IBE
cells were treated with cyclopamine, LY294002, or 0.1%
Me2SO for 30 min. Cells were then stimulated with either 5 µg/ml Shh or left unstimulated for 5 and 30 min. HUVECs were
serum-starved for 1 h and then stimulated with Shh for 5 min.
PI3-kinase activity in tyrosine-phosphorylated proteins was measured as
described under "Experimental Procedures." Radioactivity of
phosphatidylinositol 3-phosphate was measured by BAS5000 BioImager
(Fuji) and phosphorylation of phosphatidylinositol in untreated cells
was set to 1.00. ori, start point; PIP,
phosphatidylinositol 3-phosphate. Reproducible data were obtained from
two independent experiments. B, Shh phosphorylates c-Akt at
Ser- 473 in IBE cells, but not in p85-8 cells. Cells were
serum-starved overnight and then treated with 10 µM
LY294002 or 0.1% Me2SO for 30 min. Cells were then either
stimulated with 5 µg/ml Shh or left unstimulated for 10 min. c-Akt
was immunoprecipitated with anti-Akt antibody and phosphorylated c-Akt
was detected by anti-phospho-Akt antibody. Reproducible data were
obtained from two independent experiments. C, c-Fes is
activated by Shh-treatment. IBE cell lines expressing either
kinase-inactive or wild-type FLAG-tagged c-Fes, denoted KE Fes 5-15
cells and WTFes 6-8 cells, respectively, were serum-starved overnight
and stimulated or left unstimulated with 5 µg/ml Shh for 5 min and
FLAG-tagged c-Fes was immunoprecipitated, followed by the in
vitro kinase assay. To examine the loaded amount of c-Fes protein,
10% of each lysate was incubated with anti-FLAG antibody, and
precipitated proteins were examined by immunoblotting with anti-FLAG
antibody. D, Shh-mediated PI3-kinase activation requires
c-Fes kinase activity. Parental IBE cells and KEFes5-15 cells were
serum-starved overnight. Cells were then either stimulated or left
unstimulated with 5 µg/ml of Shh and PI3-kinase activity was measured
as described above. Reproducible data were obtained from two
independent experiments.
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Fig. 6.
Shh cannot induce capillary morphogenesis by
KEFes5-15 cells or p85-8 cells. Cell were
treated or left untreated with 5 µg/ml of Shh or FGF-2 and Ang2 (as a
positive controls) and cultured on Matrigel. Capillary length were
measured as described in the legend of Fig. 1. Reproducible data were
obtained from two independent experiments. Bar, 100 µm.
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 |
DISCUSSION |
It has previously been shown that Shh indirectly induces
angiogenesis by up-regulating expressions of VEGF and Ang1 (13). No
direct action of Shh on proliferation, migration, and survival of
cultured endothelial cells was observed in that study (13). Hedgehog
signaling is involved in the morphogenesis of various tissues. We
therefore focused on the effect of Shh on morphological changes of
endothelial cells and found that Shh promoted capillary morphogenesis
by cultured endothelial cells. However, we could not observe
Shh-induced proliferation or migration of HUVECs and IBE cells. These
observations are compatible with the previous report (13).
HUVECs and IBE cells expressed Ptc1 protein. Shh-induced capillary
morphogenesis was blocked by the treatment of cells with cyclopamine.
Cyclopamine inhibits hedgehog signaling through Smo. These results
indicate that Shh induced capillary morphogenesis through Ptc-Smo
system. Smo is a seven-pass G-protein-coupled receptor. Pertussis toxin
inhibited Shh-induced morphological changes. The data also suggest that
signals via Smo seem to be required for Shh-induced capillary
morphogenesis. Shh activated c-Fes/PI3-kinase pathway in IBE cells.
Activation of PI3-kinase was rapid and was sensitive to the
cyclopamine-treatment, indicating that Smo seemed to be involved in
this process. Expressions of dominant-negative c-Fes and p85 subunit of
PI3-kinase inhibited Shh-induced capillary morphogenesis of IBE cells.
These results suggest that Shh promoted capillary morphogenesis of
endothelial cells through Smo/c-Fes/PI3-kinase pathway. Gli1
transcription factor entered into nuclei of Shh-treated IBE cells. This
translocation of Gli was not inhibited by LY294002, indicating that
Gli1 might act independently on PI3-kinase. In addition, cycloheximide
and actinomycin D blocked Shh-induced capillary morphogenesis. These observations support the notion that Gli1-dependent
transcription, followed by the translation of target proteins might be
required for Shh-induced capillary morphogenesis. On the other hand,
IBE cells endogenously secreted VEGF-A, of which amount was measured by
enzyme-linked immunosorbent assay for murine VEGF-A (R & D Systems).
However, Shh did not up-regulate the VEGF-A secretion (data not shown).
The expression of VEGF receptor-2 in IBE cells was extremely low level
(16), and Shh failed to up-regulate the expression, which was examined
by immunoblot analysis. Furthermore, VEGF-A did not induce capillary
morphogenesis of IBE cells (Fig. 1). Also, treatment of cells with Tie
2/Fc chimera (R & D Systems) never decreased Shh-induced capillary
morphogenesis (data not given). Taken together, it seems likely that
Shh stimulated capillary morphogenesis by endothelial cells
independently of VEGF-A or angiopoietins.
In the present study, Shh increased PI3-kinase activity. It has been
shown that glycogen synthase kinase 3 (GSK3) phosphorylates Ci, a
Drosophila homolog of Gli family transcription factors, which in turn allows its proteolytic degradation (8, 9). Downstream of
PI3-kinase, c-Akt is activated. c-Akt phosphorylates GSK3, which
results in down-regulation of GSK3 activity (30, 31). Thus, PI3-kinase
activated by Shh may suppress GSK3 activity, which in turn inhibits the
degradation of Gli proteins by ubiquitin-proteasome system. Recent
studies have also shown that c-Akt was involved in tube-like structure
formation by endothelial cells (32-34). Thus, it seems likely that
activated c-Akt may contribute to Shh-induced capillary morphogenesis
of endothelial cells directly or indirectly, possibly through
Gli1-dependent transcription.
 |
ACKNOWLEDGEMENTS |
We thank T. Shimogama, M. Yoshimoto,
and members of the Nagasaki Radioisotope Center for excellent and
outstanding help.
 |
FOOTNOTES |
*
This work was supported by a grant-in-aid for scientific
research from the Japan Society for the Promotion of Science.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed. Tel.:
81-95-849-7340; Fax: 81-95-849-7343; E-mail:
shigeruk@net.nagasaki-u.ac.jp.
Published, JBC Papers in Press, January 3, 2003, DOI 10.1074/jbc.M210635200
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ABBREVIATIONS |
The abbreviations used are:
Shh, sonic hedgehog;
IBE cells, immortalized murine brain capillary endothelial cells;
HUVECs, human umbilical vein cord endothelial cells;
PI3-kinase, phosphoinositide 3-kinase;
Ptc, patched;
Smo, smoothened;
Ci, Cubitus
interuptus.
 |
REFERENCES |
1.
|
Ingham, P. W.,
and McMahon, A. P.
(2001)
Genes Dev.
15,
3059-3087[Free Full Text]
|
2.
|
Ingham, P. W.
(1998)
EMBO J.
17,
3505-3511[Abstract/Free Full Text]
|
3.
|
Denef, N.,
Neubuser, D.,
Perez, L.,
and Cohen, S. M.
(2000)
Cell
102,
521-531[Medline]
[Order article via Infotrieve]
|
4.
|
Strutt, H.,
Thomas, C.,
Nakano, Y.,
Stark, D.,
Neave, B.,
Taylor, A. M.,
and Ingham, P. W.
(2000)
Curr. Biol.
11,
608-613[CrossRef]
|
5.
|
Martin, V.,
Carrillo, G.,
Torroja, C.,
and Guerrero, I.
(2001)
Curr. Biol.
11,
601-607[CrossRef][Medline]
[Order article via Infotrieve]
|
6.
|
Chen, Y.,
Gallaher, N.,
Goodman, R. H.,
and Smolik, S. M.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
2349-2354[Abstract/Free Full Text]
|
7.
|
Price, M. A.,
and Kalderon, D.
(1999)
Development
126,
4331-4339[Abstract/Free Full Text]
|
8.
|
Jia, J.,
Amanai, K.,
Wang, G.,
Tang, J.,
Wang, B.,
and Jiang, J.
(2002)
Nature
416,
548-552[CrossRef][Medline]
[Order article via Infotrieve]
|
9.
|
Price, M. A.,
and Kalderon, D.
(2002)
Cell
108,
823-835[Medline]
[Order article via Infotrieve]
|
10.
|
Brown, L. A.,
Rodaway, A. R.,
Schilling, T. F.,
Jowett, T.,
Ingham, P. W.,
Patient, R. K.,
and Sharrocks, A. D.
(2000)
Mech. Dev.
90,
237-252[CrossRef][Medline]
[Order article via Infotrieve]
|
11.
|
Dyer, M. A.,
Farrington, S. M.,
Mohn, D.,
Munday, J. R.,
and Baron, M. H.
(2001)
Development
128,
1717-1730[Abstract/Free Full Text]
|
12.
|
Byrd, N.,
Becker, S.,
Maye, P.,
Narasimhaiah, R.,
St.-Jacques, B.,
Zhang, X.,
McMahon, J.,
McMahon, A.,
and Grabel.
(2002)
Development
129,
361-372[Abstract/Free Full Text]
|
13.
|
Pola, R.,
Ling, L. E.,
Silver, M.,
Corbley, M. J.,
Kearney, M.,
Blake Pepinsky, R.,
Shapiro, R.,
Taylor, F. R.,
Baker, D. P.,
Asahara, T.,
and Isner, J. M.
(2001)
Nat. Med.
7,
706-711[CrossRef][Medline]
[Order article via Infotrieve]
|
14.
|
Folkman, J.,
and D'Amore, P. A.
(1996)
Cell
87,
1153-1155[Medline]
[Order article via Infotrieve]
|
15.
|
Darland, D. C.,
and D'Amore, P. A.
(1999)
J. Clin. Invest.
103,
157-158[Free Full Text]
|
16.
|
Kanda, S.,
Landgren, E.,
Ljungström, M.,
and Claesson-Welsh, L.
(1996)
Cell Growth Differ.
7,
383-395[Abstract]
|
17.
|
Marti, E.,
Bumcrot, D. A.,
Takada, R.,
and McMahon, A. P.
(1995)
Nature
375,
322-325[CrossRef][Medline]
[Order article via Infotrieve]
|
18.
|
Kanda, S.,
Lerner, E. C.,
Tsuda, S.,
Shono, T.,
Kanetake, H.,
and Smithgall, T. E.
(2000)
J. Biol. Chem.
275,
10105-10111[Abstract/Free Full Text]
|
19.
|
Kotani, K.,
Yonezawa, K.,
Hara, K.,
Ueda, H.,
Kitamura, Y.,
Sakaue, H.,
Ando, A.,
Chavanieu, A.,
Calas, B.,
Grigorescu, F.,
Nishiyama, M.,
Waterfield, M. D.,
and Kasuga, M.
(1994)
EMBO J.
13,
2313-2321[Abstract]
|
20.
|
Mochizuki, Y.,
Tsuda, S.,
Kanetake, H.,
and Kanda, S.
(2002)
Oncogene
21,
7027-7033[CrossRef][Medline]
[Order article via Infotrieve]
|
21.
|
Kubota, Y.,
Kleinman, H. K.,
Martin, G. R.,
and Lawley, T. J.
(1988)
J. Cell Biol.
107,
1589-1596[Abstract]
|
22.
|
Mochizuki, Y.,
Nakamura, T.,
Kanetake, H.,
and Kanda, S.
(2002)
J. Cell Sci.
115,
175-183[Abstract/Free Full Text]
|
23.
|
Bussolati, B.,
Dunk, C.,
Grohman, M.,
Kontos, C. D.,
Mason, J.,
and Ahmed, A.
(2001)
Am. J. Pathol.
159,
993-1008[Abstract/Free Full Text]
|
24.
|
Velazquez, O. C.,
Snyder, R.,
Liu, Z. J.,
Fairman, R. M.,
and Herlyn, M.
(2002)
FASEB J.
16,
1316-1318[Abstract/Free Full Text]
|
25.
|
Waltenberger, J.,
Claesson-Welsh, L.,
Siegbahn, A.,
Shibuya, M.,
and Heldin, C.-H.
(1994)
J. Biol. Chem.
269,
26988-26995[Abstract/Free Full Text]
|
26.
|
Tsuda, S.,
Ohtsuru, A.,
Yamashita, S.,
Kanetake, H.,
and Kanda, S.
(2002)
Biochem. Biophys. Res. Commun.
290,
1354-1360[CrossRef][Medline]
[Order article via Infotrieve]
|
27.
|
Kim, S. K.,
and Melton, D. A.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
13036-13041[Abstract/Free Full Text]
|
28.
|
Taipale, J.,
Chen, J. K.,
Cooper, M. K.,
Wang, B.,
Mann, R. K.,
Milenkovic, L.,
Scott, M. P.,
and Beachy, P. A.
(2000)
Nature
406,
1005-1009[CrossRef][Medline]
[Order article via Infotrieve]
|
29.
|
Alessi, D. R.,
Andjelkovic, M.,
Caudwell, B.,
Cron, P.,
Morrice, N.,
Cohen, P.,
and Hemmings, B. A.
(1996)
EMBO J.
15,
6541-6551[Abstract]
|
30.
|
Cross, D. A.,
Alessi, D. R.,
Cohen, P.,
Andjelkovich, M.,
and Hemmings, B. A.
(1995)
Nature
378,
785-789[CrossRef][Medline]
[Order article via Infotrieve]
|
31.
|
Shaw, M.,
Cohen, P.,
and Alessi, D. R.
(1997)
FEBS Lett.
416,
307-311[CrossRef][Medline]
[Order article via Infotrieve]
|
32.
|
Lee, M. J.,
Thangada, S.,
Claffey, K. P.,
Ancellin, N.,
Liu, C. H.,
Kluk, M.,
Volpi, M.,
Sha'afi, R. I.,
and Hla, T.
(1999)
Cell
99,
301-312[Medline]
[Order article via Infotrieve]
|
33.
|
Kureishi, Y.,
Luo, Z.,
Shiojima, I.,
Bialik, A.,
Fulton, D.,
Lefer, D. J.,
Sessa, W. C.,
and Walsh, K.
(2000)
Nat. Med.
6,
1004-1010[CrossRef][Medline]
[Order article via Infotrieve]
|
34.
|
Lee, M. J.,
Thangada, S.,
Paik, J. H.,
Sapkota, G. P.,
Ancellin, N.,
Chae, S. S.,
Wu, M.,
Morales-Ruiz, M.,
Sessa, W. C.,
Alessi, D. R.,
and Hla, T.
(2001)
Mol. Cell
8,
693-704[Medline]
[Order article via Infotrieve]
|
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