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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 95 No. 11 (June 1), 2000:
pp. 3403-3411
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Endostatin-induced tyrosine kinase signaling through the Shb
adaptor protein regulates endothelial cell apoptosis
Johan Dixelius,
Helena Larsson,
Takako Sasaki,
Kristina Holmqvist,
Lingge Lu,
Åke Engström,
Rupert Timpl,
Michael Welsh, and
Lena Claesson-Welsh
From the Department of Genetics and Pathology, Rudbeck Laboratory,
Uppsala, Sweden; Max-Planck-Institut für Biochemie, Martinsried,
Germany; and Department of Medical Cell Biology and Department of
Medical Biochemistry and Microbiology, Biomedical Center, Uppsala,
Sweden.
 |
Abstract |
Endostatin, which corresponds to the C-terminal fragment of collagen
XVIII, is a potent inhibitor of angiogenesis. Fibroblast growth
factor-2 (FGF-2)-induced angiogenesis in the chicken chorioallantoic membrane was inhibited by endostatin, but not by an endostatin mutant
R158/270A, lacking heparin-binding ability. Endostatin was internalized
by endothelial cells, but not by mouse fibroblasts. Treatment of murine
brain endothelial (IBE) cells with endostatin reduced the proportion of
cells in S phase, whereas growth-arrested IBE cells in collagen gels
treated with endostatin displayed enhanced tubular morphogenesis. IBE
cells overexpressing Shb, an adaptor protein implicated in
angiostatin-induced apoptosis, displayed elevated apoptosis and
decreased tubular morphogenesis in collagen gels in response to
endostatin when added together with FGF-2. Induction of apoptosis was
dependent on the heparin-binding ability of endostatin and the
expression of Shb with a functional Src homology 2 (SH2)-domain.
Endostatin treatment for 10 minutes or 24 hours induced tyrosine
phosphorylation of Shb and formation of multiprotein complexes. An Shb
SH2 domain fusion protein precipitated a 125-kd phosphotyrosyl protein
in endostatin-treated cells. The 125-kd component either contained
intrinsic tyrosine kinase activity or occurred in complex with a
tyrosine kinase. In conclusion, our data show that endostatin induces
tyrosine kinase activity and enhanced apoptosis in FGF-treated
endothelial cells.
(Blood. 2000;95:3403-3411)
© 2000 by The American Society of Hematology.
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Introduction |
Angiogenesis, formation of new capillaries from
preexisting vessels, is a prerequisite for many physiological
processes, including embryonic development, wound healing, and the
female reproductive functions.1 On the other hand, a number
of pathologic conditions such as cancer, rheumatoid arthritis, and
other chronic inflammatory diseases are characterized by excessive
angiogenesis.2 The concept that progression of these
diseases may be halted by inhibiting the endothelial cell compartment
has raised considerable interest, and a series of antiangiogenic
substances have been described.3 Interestingly, many of
these are fragments of naturally occurring proteins. Thus, a 29-kd
fragment of fibronectin,4 a 16-kd fragment of
prolactin,5 and a 38-kd fragment of
plasminogen6 (angiostatin) have been shown to inhibit
angiogenes in in vivo tumor models.
O'Reilly et al7 purified a potent angiogenesis inhibitor,
termed endostatin, from medium conditioned by a murine
hemangioendothelioma cell line. Endostatin corresponds to a 20-kd
fragment derived from the carboxy-terminal noncollagenous NC1 domain of
collagen  1 (XVIII),8,9 which is present in the basement
membrane zones around blood vessels.10 Boehm et
al11 showed that cyclic treatment with endostatin in an
insoluble form, possibly acting via slow release, efficiently
eradicated a number of different model tumors in mice. The tumors were
reduced to the size of a small nodule, where the tumor remained dormant
after the cessation of the treatment. Recently, soluble recombinant
endostatin produced in yeast was used to treat renal cell cancer in
nude mice, which resulted in the arrest but not the shrinking of the
tumor.12 Furthermore, adenovirus-mediated expression of
endostatin has been shown to lead to the inhibition of endothelial cell
growth, indicating that gene therapy could be a useful approach in, for example, tumor treatment.13
Structural analyses of murine endostatin by x-ray crystallography
showed a compact globular folding, with arginine-rich clusters exposed
on the surface of the molecule,14 mediating the binding of
endostatin to heparin/heparan sulfate proteoglycans,15 and a core structure related to the carbohydrate-recognition domain of
C-type lectins.14 Furthermore, human and murine endostatin were shown to bind Zn2+ at their amino termini, possibly of
importance for the processing of the inactive collagen XVIII
precursor.16 Boehm et al17 reported
Zn2+-binding to endostatin at a 1:1 mmol/L ratio, and
implicated Zn2+ in the antiangiogenic effect of endostatin
during progression of Lewis lung carcinoma. Endostatin is localized in
adult basement membranes and frequently in elastic fibers and
microfibrils, and in the skin, brain, and vascular basement membrane in
the developing embryo.15,18
The molecular mechanisms of endostatin in inhibition of tumor growth
are not yet clear. Endostatin was recently shown to inhibit proliferation and migration of endothelial cells.12,19,20 Moreover, endostatin treatment was shown to induce a block in cell
cycle progression and apoptosis of cells.20,21
In this paper, these data are confirmed and extended in that we show
that endostatin induces tyrosine kinase activity and the formation of
multiprotein signaling complexes in endothelial cells. One component
identified in these complexes is the Shb adaptor protein,22
that previously has been implicated in apoptosis.23-25 Shb
is tyrosine phosphorylated in response to nerve growth factor treatment
of PC12 cells26 and is known to participate in multiprotein signaling complexes in CD3-stimulated Jurkat cells.27
Structurally, Shb consists of a proline-rich amino-terminus, a central
phosphotyrosine-binding (PTB) domain and a carboxy-terminal Src
homology 2 (SH2) domain.22,27 We have previously shown that
cells overexpressing Shb respond with increased apoptosis on treatment
with the angiogenesis inhibitor angiostatin.24 Here we show
that overexpression of Shb augments endostatin-induced apoptosis,
whereas this response is counteracted by the expression of a Shb
SH2-domain mutant. Endostatin-induced inhibition of angiogenesis in the
chorioallantoic membrane assay, activation of tyrosine kinase activity,
and Shb-mediated apoptosis of endothelial cells correlated with the
heparin-binding ability of endostatin.
| |
Materials and methods |
Tissue culture
Murine brain endothelial cells (IBE)28 were cultured on
gelatin-coated dishes in Ham's F12, 10% fetal calf serum (FCS), and
20 U/mL interferon  (IFN-, Peprotech, Rocky Hill,
NJ) at 33°C. Parental and fibroblast growth factor
(FGF) receptor-1 (FGFR-1) transfected porcine aortic
endothelial (PAE)29 were cultured in Ham's F12, 10% FCS.
Generation of murine brain endothelial cells overexpressing
wild-type and R522K-mutant Shb
Wild-type or R522K-mutant (inactivation of the SH2
domain27) Shb cDNA were inserted into the pBABE
vector30 and used to generate retroviruses. IBE cells were
infected with retroviruses containing vector, wild-type Shb, or Shb
R522K, and clones were isolated after selection in the presence of 5 µg/mL puromycin. Overexpression of wild-type or R522K Shb was
verified by Western blot analysis of cell lysates from different
clones, and the Shb and Shb R522K clones displayed approximately 5-fold
increased levels of Shb (L. Lu and M. Welsh, unpublished data).
Expression, purification, and biotinylation of endostatin
The preparation of recombinant mouse endostatin in human
293-Epstein-Barr virus-associated nuclear antigen (EBNA) cells has been
described.15 This endostatin is highly soluble and contains 1.02 zinc atoms per molecule as shown by atomic emission spectroscopy. Expression vectors for 2 different double mutations of endostatin were
made by fusion polymerase chain reaction (PCR), following standard
protocols, and used for production of mutant proteins as
described.15 Mutant R158/270A showed a strongly reduced
heparin-binding affinity (displacement at 0.11 mol/L NaCl) when
compared with the wild-type (0.35 mol/L NaCl). Mutant H134A/D136A
endostatin displayed a reduced zinc content (0.07 atoms per molecule).
A full description of the mutants is given elsewhere.31 The
purified endostatin in ammonium acetate buffer was dried, redissolved
in 4 mol/L GuHCl in phosphate-buffered saline (PBS), and applied on a
Fast Desalting column (Amersham Pharmacia Biotech, Uppsala, Sweden), equilibrated, and eluted in PBS. Biotinylation
was performed by incubation of endostatin with biotinamidocaproic acid
3-sulfo-N-hydroxy-succinimide ester (100:25 w/w) in PBS for 60 minutes.
The reaction mixture was applied onto a Superdex 75 (Amersham Pharmaca
Biotech) column and eluted with PBS. MALDI-MS analysis of the protein
fraction showed that the major fraction contained 1 biotin molecule per endostatin molecule.
Chorioallantoic membrane assay
Angiogenesis in the chicken chorioallantoic membrane (CAM) was
performed as described.31 Fertilized chick embryos were
preincubated for 10 days at 38°C/70% humidity. A hole was drilled
over the air sac at the end of the egg and an avascular zone was
identified on the CAM. A second hole was made over the CAM, which was
separated from the shell by applying vacuum to the first hole. A
1 × 1-cm window in the shell was made to expose the CAM.
Samples were prepared using filter disks (Whatman, Clifton, NJ),
saturated with 3 mg/mL cortisone acetate (Sigma, St Louis, MO), and
soaked in buffer (40 µL for each filter) with or without FGF-2
(Boehringer Mannheim, Mannheim, Germany; 0.2 µg for each filter) and
endostatin or endostatin mutants (3 µg for each filter). The windows
were sealed with tape and the eggs were incubated for 3 more days. The
membrane was cut around the disks, which was turned upside down and
inspected using a light microscope (Nikon Eclipse TE 300; Nikon, Tokyo, Japan; magnification 2.5 or 4).
Tube formation
Parental IBE and IBE/Shb cells cultured sparsely on gelatin-coated
culture plastic were washed and fresh Ham's F12/0.25% bovine serum
albumin (BSA) was added. After 24 hours, the cells were trypsinized and
resuspended in fresh medium with 0.25% BSA, with or without endostatin
at a final concentration of 0.7 µg/mL or FGF-2 (Boehringer Mannheim)
at 5 ng/mL, and seeded on a collagen gel. The collagen gel was prepared
by mixing collagen I (Vitrogen, Palo Alto, CA), 10 × Ham's F12
medium and neutralizing buffer (260 mmol/L NaCO3, 200 mmol/L HEPES, and 50 mmol/L NaOH) at an 8:1:1 ratio. The mixture was
pipetted into 24-well plates (250 µL per well). After solidification,
serum- and IFN--starved IBE cells were seeded in the absence and
presence of FGF-2 and endostatin, as indicated. After 2 hours, a second
collagen layer was added and incubation continued at 33°C. Tube
formation was analyzed with a Nikon Eclipse TE 300 microscope and
photographed using a SPOT 2 digital camera (Diagnostic Instruments,
Sterling Heights, MI).
Endostatin internalization
IBE cells or Swiss 3T3 cells were trypsinized and seeded on
fibronectin-coated (IBE) or uncoated (Swiss 3T3) 8-chamber microscope slides (NUNC, Als Roskilde, Denmark). After 24 hours, the medium was
changed to F12/0.25% BSA and 5 ng/mL FGF-2. Another 24 hours later, 10 µg/mL biotinylated endostatin was added, and at indicated time
points, the cells were put on ice, washed twice in PBS, and fixed in
3% paraformaldehyde for 10 minutes. After 3 washes in PBS, the cells
were permeabilized in  20°C acetone for 5 minutes. The
acetone was removed, the slides were air-dried, and blocked in PBS with
10% FCS for 1 hour at 37°C. Bound endostatin was detected using
Alexa 488-conjugated avidin (Molecular Probes, Eugene, OR) for 45 minutes. Cells were washed 5 times in PBS and, after adding "Slow
fade light" (Molecular Probes, Eugene, OR) antifade and coverslip,
the preparations were examined with a Nikon Eclipse TE 300 microscope
and photographed using a SPOT 2 digital camera (Diagnostic Instruments).
In vitro kinase assay
IBE/Shb R522K cells or PAE cells, either parental or overexpressing
FGFR-1, were seeded on 6-cm dishes in Ham's F12 medium, 10% FCS.
After 24 hours, the cells were washed twice in serum-free medium and
cultured for 24 hours in F12/0.25% BSA. Cells were stimulated with
growth factors and endostatin for 10 minutes, put on ice, washed with
ice-cold Na3VO4 in Tris-buffered saline and
lysed in Nonidet P-40 (NP-40) lysis buffer (1% NP-40, 20 mmol/L HEPES
pH 7.5, 150 mmol/L NaCl, 10% glycerol, 300 µmol/L
Na3VO4, 1% aprotinin, and 1 mmol/L
phenylmethylsulfonyl fluoride (PMSF) for 10 minutes. The cells were
scraped and lysates centrifuged at 18 000 × g for 13 minutes at 4°C. The supernatants were incubated with antibodies
against phosphotyrosine (4G10; Upstate Biotechnology, Lake Placid, NY)
on ice for 2 hours and with immobilized protein A (Immunosorb; EC
Diagnostics, Uppsala, Sweden) for another 30 minutes at 4°C.
Alternatively, lysates were incubated with a GST-Shb SH2 fusion
protein,22 coupled to glutathione-Sepharose (Amersham Pharmacia Biotech, Uppsala, Sweden). Beads were washed 3 times in lysis
buffer and 2 times in kinase buffer (0.05% Triton X-100, 20 mmol/L
HEPES, 10 mmol/L MgCl2, 2 mmol/L MnCl2, 0.185 MBq (5 µCi) [-32P]ATP was added per
sample. Samples were incubated 10 minutes at room temperature and
heated in sample buffer (8% SDS, 0.4 mol/L Tris-HCl, pH 8.0, 1 mol/L
sucrose, 10 mmol/L EDTA, 0.02% bromophenol blue, 4%
 -mercaptoethanol), followed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in 9% gels. The gel was fixed in 2.5% glutaraldehyde for 10 minutes, incubated at 55°C in 1 mol/L KOH, rinsed, dried, and analyzed using a BioImager (Fujifilm, Tokyo, Japan).
Immunoprecipitation, glutathione-S-transferase-fusion
protein precipitation, and immunoblotting
IBE cells, IBE/ShbR552K cells, or PAE/FGFR-1 cells in
75-cm2 flasks were starved over night in Ham's F12,
supplemented with 0.25% BSA, followed by treatment with or without
FGF-2 (100 ng/mL) and endostatin or endostatin mutants (1 µg/mL) for
10 minutes or 24 hours at 37°C. The cells were washed with ice-cold
PBS containing 100 µmol/L Na3VO4 and lysed
for 10 minutes on ice in 0.5% Triton X-100, 20 mmol/L Tris HCl, pH
7.5, 0.15 mol/L NaCl, 1 mmol/L EDTA, 0.1 mmol/L
Na3PO4, 1% aprotinin, 2 mmol/L PMSF, 0.05 mmol/L leupeptin, and 20 µmol/L N-acetyl-leu-leu-norleucinal. Cleared
lysates were incubated with 10 µg/ mL affinity-purified Shb
antibody,23 and protein A-Sepharose, or the
glutathione-S-transferase (GST)-Shb SH2 domain fusion protein, and
glutathione-Sepharose. Samples were heated in sample buffer and
separated by SDS-PAGE. For immunoblotting, proteins were
electrophoretically transferred onto nitrocellulose membranes (Hybond-C
extra; Amersham Pharmacia Biotech). The membranes were
blocked in PBS-T (0.2% Tween-20 in PBS) containing 5% BSA, incubated
with antiphosphotyrosine antibodies (4G10) or Shb antiserum for 1 hour,
followed by washing in PBS. An appropriate secondary antibody was
incubated with the membranes for another hour, and after washing in
PBS, the immunoreactive proteins were visualized by an enhanced
chemiluminescence detection system, based on a protocol described
earlier.32
Transferase-mediated dUTP nick end labeling assay
IBE cells were seeded on fibronectin-coated plastic dishes
and incubated for 24 hours. The cells were washed and starved for 24 hours in F12/0.25% BSA. Then, fresh F12/0.25% BSA was added and the
cells were incubated in the presence or absence of 1 µg/mL endostatin
and 5 ng/mL FGF-2 as indicated at 33°C for 48 hours, harvested,
fixed, and prepared according to the In situ Cell Death Detection kit,
Fluorescein (Boehringer Mannheim), a terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) kit, with fluorescein-conjugated dUTP. Briefly, the cells were fixed in 4%
paraformaldehyde, washed twice in PBS, permeabilized in 0.1% Triton-X-100 and 0.1% sodium citrate, washed twice in PBS and incubated 1 hour at 37°C in the presence of the
fluorescein-conjugated dUTP. The cells were washed once in PBS and
analyzed with a flow cytometer (FACScalibur; Becton Dickinson, Franklin
Lakes, NJ), using a 488-nm laser for excitation. Data for light
scattering and green fluorescence were collected.
Annexin assay
IBE cells were seeded and starved as for the TUNEL assay,
and incubated with and without 1 µg/mL endostatin and 5 ng/mL FGF-2 at 33°C for 24 hours. The cells were prepared according to the Annexin-V-FLUOS Kit (Boehringer Mannheim). Briefly, the cells were
trypsinized, and resuspended in fluorescein-conjugated Annexin-V and
2.5 µg/mL propidium iodide, incubated for 10 minutes and analyzed with a flow cytometer (FACScalibur), with 488-nm excitation and collecting light scatter, green, and red fluorescence. Apoptotic cells
were defined as cells with enhanced Annexin-V fluorescence simultaneously exhibiting normal propidium iodide staining. The frequency of necrotic cells (with strongly elevated propidium iodide
staining) showed no differences between the experimental conditions.
Cell cycle analysis
IBE cells were seeded and starved as described under TUNEL
assay and then incubated with and without 1 µg/mL endostatin and 5 ng/mL FGF-2 as indicated at 33°C for 24 hours. The cells were harvested, fixed in 20°C ethanol for 10 minutes and washed
twice in PBS. RNAse A (1 mg/mL) and propidium iodide (25 µg/mL) were added. The samples were incubated for 1 hour at 37°C and analyzed with a flow cytometer (FACScalibur), with 488-nm excitation and collecting light scatter and red fluorescence.
| |
Results |
Angiogenesis in the CAM is inhibited by endostatin
Angiogenesis in the chicken CAM was induced by treatment with FGF-2
for 3 days on day 10 embryos. Coincubation of FGF-2 with a 10-fold
molar excess of endostatin led to an efficient inhibition of
angiogenesis in this assay (Figure 1). The
effects of mutated forms of endostatin were analyzed by CAM
angiogenesis. The H134A, D136A mutant, which does not bind
Zn2+,31 inhibited FGF-2-induced angiogenesis
to the same extent as wild-type endostatin (Figure 1). In contrast, the
R158/270A mutant, which lacks heparin-binding ability31
failed to suppress FGF-2-induced angiogenesis in the CAM.
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| Fig 1.
Angiogenesis in the CAM is inhibited by endostatin
dependent on its heparin-binding ability.
Ten-day chick embryos were incubated with filter disks saturated with
buffer alone, or with FGF-2 with or without wild-type and mutated
endostatins (ES). The effect of the additions on CAM angiogenesis was
analyzed after 3 days of incubation, by excising the filter and
microscopical examination. The R158/270A mutant lacks heparin-binding
ability and the H134A, D136A mutant lacks Zn2+-binding
ability.
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Endostatin regulates cell cycle progression in endothelial
cells
IBE cells treated with or without FGF-2 and endostatin were
analyzed for DNA content by flow cytometry. After an initial 24-hour serum deprivation, cells were cultured for 24 hours in the presence or
absence of additions indicated in Table 1.
The addition of FGF-2 increased the proportion of cells in the S-phase
in the cell cycle, compared with control cultures. This was evidenced by a significantly increased S/G1 ratio. The addition of endostatin had
no effect when added alone, whereas it decreased the S/G1 ratio when
added together with FGF-2, compared with FGF-2 alone. This suggests
that endostatin reduces the proportion of endothelial cells actively
synthesizing DNA after FGF-2 stimulation.
Tubular morphogenesis of endothelial cells is supported
by endostatin
IBE cells were seeded on solidified collagen in the absence
or presence of FGF-2 and endostatin as indicated, and a second layer of
collagen was added on top. The cultures were incubated 24 or 48 hours
and inspected by light microscopy (Figure
2). The cells maintained in the absence of
FGF-2 underwent apoptosis after 24 hours as previously
reported,33 regardless of whether they were exposed to
endostatin or not. In contrast, FGF-2 treatment led to tubular
morphogenesis, with branching structures (Figure 2). We have previously
shown that IBE-cell DNA synthesis ceases under these
conditions.33 In the combined presence of FGF-2 and
endostatin for 24 hours, the tubular structures were slender and
contained an increased number of branch points, reminiscent of active
angiogenesis. Incubation with FGF-2 alone for 48 hours led to
a collapse of the tubular structures, with evident signs of
cell death. This was in contrast to cultures incubated with FGF-2 and endostatin for 48 hours, in which the cells
remained in good condition, forming a complex pattern of anastomosing
and branching slender tubes (Figure 2). These data indicate
that collagen-cultured, cell-cycle arrested IBE cells are not adversely
affected by endostatin, and that endostatin in fact stabilized the
tubular structures.
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| Fig 2.
Tubular morphogenesis of murine brain endothelial cells
is supported by endostatin.
IBE cells were cultured between 2 layers of collagen gels, in Ham's
F12, 0.25 mg/mL BSA. The cultures were incubated in the absence of
additions (control) or in the presence of 1 µg/mL of endostatin and 5 ng/mL of FGF-2 alone or in combination for 24 or 48 hours. The cultures
were analyzed by light microscopy and photographed (×20
objective).
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Internalization of endostatin
To follow the pattern of internalization of endostatin, IBE cells
were incubated with 10 µg/mL biotinylated endostatin for different
periods at 37°C. Cell-associated endostatin was detected after the
fixation of cells, by use of Alexa 488-conjugated avidin. As seen in
Figure 3, 5 minutes of incubation at
37°C led to a punctate staining of endostatin, indicating
clustering at the cell surface. By 30 minutes of incubation, endostatin
was taken up and distributed evenly in the cell. By 90 minutes of
incubation, staining was considerably weaker, and by 150 minutes, cells
were no longer stained, indicating degradation and clearing of
endostatin. In contrast, there was no uptake of endostatin in similarly
treated mouse Swiss 3T3 fibroblasts (Figure 3). These data are
compatible with an active uptake of endostatin by endothelial cells,
possibly mediated by a cell surface receptor.
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| Fig 3.
Rapid internalization and degradation of endostatin.
IBE cells or Swiss 3T3 cells were incubated in the absence (control) or
presence of 10 µg/mL biotinylated endostatin for different periods
from 5 minutes to 150 minutes. Binding was identified using Alexa
488-labeled avidin l that was added to the control and
endostatin-incubated samples, which subsequently were analyzed
microscopically (Bar, 10 µM). Upper panels show IBE cells cultures;
lower panels show Swiss 3T3 cell cultures.
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IBE cells that had internalized endostatin displayed a different cell
shape, compared with control cells. Figure
4 shows that control IBE cells, incubated
in BSA-containing medium were polygonal and contained long extensions.
The cells in FGF-2-treated cultures were generally elongated, with
long, crossing processes, typical for growth factor-treated cells.
Treatment of BSA or FGF-2 cultures with endostatin for 24 hours led to
a change in cell shape, with flatter, more spread cells that lacked
extensions. Indeed, treatment of IBE cells with endostatin led to a
morphology very similar to that of serum-treated cells (Figure 4).
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| Fig 4.
Cell shape change in endostatin-treated cells.
IBE cells cultured on fibronectin-coated dishes were treated with 1 µg/mL endostatin, 5 ng/mL FGF-2, and 10% FCS in different
combinations as indicated for 24 hours. The cultures were analyzed by
light microscopy and photographed.
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Endostatin-induced apoptosis in murine brain endothelial cells
The antiangiogenic effects of endostatin may partly be the
consequence of endothelial cell apoptosis.20,21,24 To
determine whether endostatin causes apoptosis of IBE cells, labeling
using Annexin-V was performed after 24 hours of incubation in the
presence of endostatin (Figure 5A).
Annexin-labeling detects early plasma membrane changes connected with
the apoptotic process.34 The addition of FGF-2 to the IBE
cell cultures reduced the rate of apoptosis, compared with untreated
cells. Endostatin alone had no effect, but when added together with
FGF-2, endostatin caused a slight (24% ± 8%) but significant
increase of apoptosis (Figure 5A). IBE cells maintained for 48 hours in
the presence of FGF-2 and in the absence or presence of endostatin were
also examined. Under these conditions, endostatin induced a relative
increase in the rate of apoptosis of 51% ± 12% (n = 4,
P < .05) as assessed by the TUNEL-technique35
(data not shown).
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| Fig 5.
Endostatin-induced apoptosis is dependent on the adaptor
molecule Shb.
IBE infected with vector-containing retrovirus (A) or retrovirus
encoding Shb (B) or an Shb SH2-domain mutant R522K (C) were analyzed
with regard to proportion of Annexin V-stained cells by flow cytometry.
* denotes P < .05 when tested against FGF-2 alone using a
paired Student t test.
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We have previously reported that angiostatin-induced apoptosis is
elevated by overexpression of the Shb adaptor protein and decided to
examine the effect of Shb overexpression on endostatin-treated IBE
cells. Five-fold overexpression of Shb in IBE cells (L. Lu and M. Welsh, unpublished data) led to an increase in the basal level of
Annexin V-positive cells after a 24-hour incubation period (Figure 5B),
compared with the vector-transfected IBE cells. FGF-2 caused a
significant inhibition of apoptosis under these conditions. Again,
endostatin alone had little effect, but augmented the rate of apoptosis
when added in the presence of FGF-2. The increase of apoptosis in the
combined presence of FGF-2 and endostatin was 66% ± 20% above
that in the FGF-2-treated IBE/Shb cells. Thus, Shb overexpressing IBE
cells exhibit an exaggerated response to endostatin, compared with the
control cells. The R158/270A heparin-binding defective endostatin
mutant did not mediate increased apoptosis when added in the presence
of FGF-2 (Figure 5B).
To investigate the role of the Shb SH2 domain in
endostatin-induced apoptosis, IBE cells expressing the Shb SH2
domain mutant R522K27 were generated. Neither FGF-2
nor endostatin significantly affected apoptosis in the IBE/Shb R522K
cells (Figure 5C).
Tubular morphogenesis of IBE cells overexpressing Shb in
response to endostatin
As endostatin-induced apoptosis was augmented in Shb-overexpressing
IBE cells, we decided to investigate tubular morphogenesis of these
cells when grown in a collagen matrix. IBE/Shb cell cultures treated
for 48 hours with FGF-2 contained tubular structures (Figure 6). In contrast, and unlike the pattern in
the parental IBE cells, IBE/Shb cell cultures treated with FGF-2 and
endostatin showed deteriorated tubular morphogenesis and contained
numerous clusters of cells lacking distinct morphologic features
(Figure 6). This suggests that the expression of Shb plays a role for
the effect of endostatin on in vitro tube formation of IBE
cells.
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| Fig 6.
Decreased tubular morphogenesis of IBE cells
overexpressing Shb in response to endostatin.
IBE/Shb cells were grown in collagen as in Figure 2 for 48 hours in the
absence of FGF-2, presence of 5 ng/mL FGF-2, or presence of FGF-2 and 1 µg/mL endostatin, as indicated. Scoring of the endostatin-induced
decrease in tubular morphogenesis (defined as tubular structures with a
diameter of 1 cell and a length of more than 7 cells) led to the
estimation that endostatin diminished the number of tubular structures
by 54% ± 1.9% (n = 3, P < .01) in the IBE/Shb
cells.
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Endostatin-induced Shb-signaling in murine brain endothelial
cells
To examine the potential effects of endostatin on Shb-signaling, IBE
cells were treated for 10 minutes with or without FGF-2 and endostatin.
Phosphotyrosine immunoblotting of Shb immunoprecipitated from
endostatin-treated IBE cells showed that 10 minutes of treatment with
endostatin alone induced Shb tyrosine phosphorylation (Figure 7A, top panel). Furthermore, Shb occurred
in complex with a number of tyrosine phosphorylated molecules, eg, of
100, 35, and 32 kd (Figure 7A, lower panel). Longer exposure of the
blot showed endostatin-induced tyrosine phosphorylation of additional
proteins of 200 and 160 kd (data not shown). Treatment with FGF-2 also
increased Shb tyrosine phosphorylation and coimmunoprecipitation of the
32-kd component (Figure 7A). Exposure to the combination of FGF-2 and
endostatin resulted in Shb tyrosine phosphorylation and
coimmunoprecipitation of both the 35- and 32-kd components (Figure 7A).
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| Fig 7.
Endostatin-treatment induces signal transduction in IBE
cells.
IBE cells were incubated with (+) or without () FGF-2 and
endostatin for 10 minutes (A, C) or 24 hours (B). The lower panels in
(A) and (B) represent longer exposures of the immunoblot shown in the
top panel. In (C), R522KShb-IBE cells were stimulated with endostatin
and/or FGF-2. Cells were lysed and immunoprecipitated (IP) using
antiserum against Shb, followed by SDS-PAGE and immunoblotting using
the antiphosphotyrosine antibodies 4G10 or the anti-Shb antiserum, as
indicated. The ratios of densitometric recordings of Shb and tyrosine
phosphorylated Shb are provided in the figure, because the total
amounts of Shb varied slightly between the different lanes in the (B)
and (C) panels. Molecular masses of marker proteins run in parallel and
the migration of Shb are indicated to the right in the panels.
|
|
A similar analysis was performed on IBE cells treated for 24hours.
Figure 7B, top panel, shows an elevated level of tyrosine phosphorylated Shb in cells treated for 24 hours with
endostatin, albeit to a lesser extent than after the 10-minute
stimulation. Shb was in complex with a similar spectrum of tyrosine
phosphorylated molecules, as in the short-term-treated IBE cells. In
cells treated for 24 hours with FGF-2, or FGF-2 and endostatin, Shb
also exhibited an elevated degree of tyrosine phosphorylation, whereas
the tyrosine phosphorylation of proteins in complex with Shb was
reduced and similar to the basal levels of untreated IBE cells.
To assess to what extent endostatin-induced signaling through
Shb required a functional SH2 domain, IBE/Shb R522K cells were analyzed
(Figure 7C). The anti-Shb antiserum immunoprecipitates both the
endogenously expressed Shb and the transfected R522K Shb. When related
to the amount of Shb protein present in the immunoprecipitations, the
basal Shb tyrosine phosphorylation was similar in the parental IBE
cells and in the IBE/Shb R522K cells. Tyrosine phosphorylation of R522K
Shb is likely to be the consequence of non-SH2 domain (proline-rich or
PTB domains) interactions between Shb and tyrosine kinases. Endostatin
and FGF-2 treatment failed to significantly increase the degree of Shb
phosphorylation further. Likewise, no signs of complex formation
between Shb and other phosphotyrosyl proteins could be detected after
exposure of IBE/Shb R522K cells to endostatin or FGF-2 (Figure 7C). The
data thus suggest that endostatin and FGF-2 signaling through Shb
requires a functional Shb SH2 domain.
Endostatin-induced signal transduction in endothelial cells
To identify potential Shb SH2 domain-binding proteins of relevance
for endostatin signaling, precipitation using GST-Shb SH2 domain fusion
protein was performed after endostatin stimulation. Because it is less
likely that the SH2 domain of endogenously expressed Shb is blocking
SH2 domain-binding sites in the IBE/Shb R522K cells, SH2 domain
pull-down using a GST-Shb SH2 domain fusion protein was performed using
these cells. As seen in Figure 8A, GST-Shb
SH2, but not GST alone, specifically retained a component of
approximately 125 kd. The binding or tyrosine phosphorylation of this
component was increased by stimulation for 10 minutes with endostatin
or FGF-2. This protein may be present in the cluster of bands
immunoprecipitated with the Shb antibody in Figure 7A and B.
View larger version (31K):
[in this window]
[in a new window]
| Fig 8.
Endostatin-induced tyrosine kinase activity in complex
with Shb SH2 domain.
(A) IBE or PAE/FGFR-1 cells were incubated with (+) or without
() FGF-2 and endostatin for 10 minutes. Cell lysates were
incubated with the GST Shb SH2 domain fusion protein, and associated
proteins were analyzed by SDS-PAGE and immunblotting with
phosphotyrosine antibodies (blot PY) or by incubation in the presence
of 32P]ATP (kinase assay). The migration rate of the
Shb SH2-associated protein of 125 kd is indicated to the right. (B) PAE
cells or Swiss 3T3 cells were incubated with indicated concentrations
of endostatin and processed for immunoprecipitation using
antiphosphotyrosine antibodies 4G10 (IP PY) and in vitro kinase assay.
PAE cells treated with the endostatin mutant R158/270A were analyzed
similarly. (C) PAE cells were incubated with (+) or without ()
FGF-2 and endostatin for 10 minutes, lysed, and similar protein amounts
from the different samples were separated by SDS-PAGE, transferred to
nitrocellulose and blotted using antiphosphotyrosine antibodies (4G10).
Migration rates of marker proteins are indicated to the right.
Phosphotyrosyl proteins induced by endostatin are indicated by *.
|
|
The 125-kd component was analyzed for intrinsic or associated kinase
activity by the GST-Shb SH2 protein pull-down from IBE/Shb R522K cells,
followed by incubation in the presence of
[-32P]ATP, and analysis by SDS-PAGE. As seen
in Figure 8A, lower panel, 32P was incorporated into the
125-kd protein, in cells treated for 10 minutes with endostatin alone,
as well as in cells treated with FGF-2, or with a combination of the
two. The requirement of the Shb SH2 domain for endostatin-induced
apoptosis (Figure 5C) and increased tyrosine phosphorylation of Shb
(Figure 7C), together with the endostatin-dependent interaction between
the Shb SH2 domain and the 125-kd phosphotyrosyl protein, raises the possibility that this protein is of relevance for the effects of
endostatin in IBE cells.
We examined whether endostatin-treated PAE/FGFR-1 cells also
contained the Shb SH2-binding 125-kd protein (Figure 8A, right). Again,
the GST-Shb SH2, but not GST alone, bound a 125-kd protein after 10 minutes of endostatin treatment. In vitro kinase assay in the presence
of [-32P]ATP showed that the protein associating with
the Shb SH2 domain contained intrinsic or associated kinase activity
(lower panel).
Parental PAE cells were further examined for
endostatin-induced kinase activity. Antiphosphotyrosine antibodies were
used for immunoprecipitation from PAE cells treated for 10 minutes with
different concentrations of endostatin, or with the endostatin mutant
R158/270A. The samples were processed for in vitro kinase assay in the
presence of [-32P]ATP, followed by SDS-PAGE. As seen
in Figure 8B, endostatin treatment increased the
32P-incorporation into a cluster of proteins of about 125 kd, possibly corresponding to the Shb SH2-associated phosphotyrosyl
protein identified above. This induction of kinase activity was not
seen in endostatin-treated Swiss 3T3 cells, or in PAE cells treated with the endostatin mutant R158/270A (Figure 8B).
Phosphotyrosine immunoblots of cell lysates from PAE/FGFR-1
cells treated with endostatin, FGF-2 or a combination of the two, showed induction of a spectrum of tyrosine phosphorylated components with the different treatments (Figure 8C). Treatment with endostatin alone appeared to induce a subset of phosphotyrosyl proteins (indicated by * in Figure 8C), which was different from that observed after treatment with FGF-2. Notably, FGF-2-stimulation induced tyrosine phosphorylation of 160-kd proteins, indicating that endostatin does not
exert its effects by activating the FGF receptor.
| |
Discussion |
In this paper, we show that treatment of FGF-2-induced
angiogenesis in the chicken CAM is efficiently inhibited by endostatin purified from 293 cells. The specificity of this effect was
demonstrated by the lack of inhibition when treating the
FGF-2-stimulated CAM with an endostatin mutant, R158/270A, in which 2 of 4 arginine residues in a region that constitutes a major
heparin-binding site have been replaced with alanine
residues.31 Mutation of H134 and D136 to alanine residues
eliminates Zn2+-binding.31 Because this mutant
retained the capacity to inhibit angiogenesis in the CAM assay, we
conclude that Zn2+-binding is not required for this
particular function of endostatin. Our data therefore indicate that the
ability to bind heparin, but not Zn2+, is an important
factor in the antiangiogenic action of endostatin.
The antiangiogenic effects of endostatin appear to involve
inhibition of cell growth mediated via both reduced G1/S phase transition and increased apoptosis20,21 (Table 1, Figure
5). It should be noted that although the effect was small, it would be
cumulative. Our data show that overexpression of Shb with a functional
SH2 domain can augment the apoptotic response to endostatin when added
together with FGF-2. Likewise, tubular morphogenesis was reduced in the
Shb-overexpressing cells but not in the parental IBE cells, in
agreement with the possibility of an involvement of Shb in the
antiangiogenic effects of endostatin. Our data do not exclude the
significant contribution of other signal transduction molecules to
these effects. To elucidate signaling pathways responsible for the
action of endostatin, tyrosine kinase activity in response to
endostatin was examined. The data show rapid endostatin-induced tyrosine kinase activity in endothelial cells, but not in mouse fibroblasts, in agreement with the specificity of endostatin for endothelial cells reported by O'Reilly et al.7 Treatment
with endostatin alone induced Shb tyrosine phosphorylation and the formation of multiprotein complexes. Although both FGF-2 and endostatin induced tyrosine phosphorylation of Shb, the composition of the multiprotein complexes found associated with Shb varied between the 2 conditions of stimulation. Such differences may be of relevance for the
cellular responses to the 2 ligands; FGF-2 treatment mediated reduction
in apoptosis, whereas endostatin antagonized this effect. Furthermore,
the Shb SH2 domain specifically interacted with a kinase-active, or
kinase-associated, 125-kd protein in an endostatin- and
FGF-2-dependent manner. Kinase activity was not induced by the
endostatin mutant R158/270A. Thus, heparin-binding ability appears to
be a prerequisite for kinase activation by endostatin, possibly by
facilitating activation directly or indirectly of a cell surface
expressed or cytoplasmic tyrosine kinase.
Recent data show that endostatin does not compete with the binding of
FGF-2 to human tissues.36 The fact that FGF-2-binding, but
not endostatin-binding, to the human tissues could be removed by
heparitinase digestion36 indicates clear distinctions in the heparin/heparan sulfate requirement of FGF-2 and endostatin. Moreover, several observations in this study suggest that FGFR-1 signaling is not affected by endostatin treatment. Endostatin did not
prevent FGF-2-induced tube formation in the parental IBE cells (Figure
2), or tyrosine phosphorylation of Shb and a 160-kd phosphotyrosyl
protein observed in the PAE cell lysates (Figure 8C). Thus, it appears
that endostatin exerts its effects via activation of specific signaling
pathway(s), and we favor the notion that this occurs primarily via
activation of a tyrosine kinase. This kinase may in turn activate
phosphatases or other regulatory signaling proteins, which may
interfere with FGF effects on endothelial cells. The
endostatin-activated kinase remains to be identified. It does not
appear to correspond to Src cytoplasmic tyrosine kinases, focal
adhesion kinase (FAK), or to Abl tyrosine kinase, based on
immunoblotting analyses (data not shown). In repeated assays, endostatin-coated plastic wells failed to mediate attachment of various
endothelial cells (T. Sasaki and R. Timpl, unpublished data). This
suggests that endostatin does not bind to integrins and that it does
not act by disrupting integrin-extracellular matrix interactions.
Furthermore, endostatin does not appear to act via FGF receptors.
Although tyrosine phosphorylation of Shb was induced both by FGF-2 and
by endostatin, the number of and molecular masses of phosphotyrosyl
proteins induced were distinct for the 2 conditions.
Angiostatin,6 which is a fragment of plasminogen, was found
to activate FAK in endothelial cells, resulting in an increased apoptosis. The effect was accentuated by overexpression of
Shb.24 Angiostatin binds to a cell surface-exposed ATP
synthase37; it is unclear at this point whether the ATP
synthase couples to FAK and Shb. We have not been able to further
analyze and compare angiostatin- and endostatin-induced signal
transduction, but our present data indicate that these
angiogenesis-inhibitors have different modes of action, although Shb
may be involved in regulation of the apoptotic response to both
inhibitors. In a recent report from the Hanahan and Folkman
laboratories,38 endostatin and angiostatin were shown to
act in a synergistic manner in arresting tumor expansion in an
insulinoma model tumor in mouse, further emphasizing the different
modes of action of these inhibitors.
Taken together, our data indicate that endostatin binds to the
endothelial cell surface via heparan-sulfated proteoglycans, and
thereby directly or indirectly induces tyrosine kinase activity, which
may lead to apoptosis, dependent on the proliferative state of the cells.
| |
Footnotes |
Submitted August 11, 1999; accepted January 26, 2000.
Supported by grants from The Swedish Medical Research Council
(31x-10822) to M.W.; from The Swedish Cancer Foundation (project no.
3820-B98-03XAC), The Novo Nordisk Foundation, The Göran
Gustavsson Foundation to L.C-W.; and by an EC grant BI04-CT96-0537 to
R.T. J.D. was supported by Pharmacia & Upjohn and by the Swedish
Research Council for Engineering Sciences. M.W. and L.C.W. contributed equally to this study.
Reprints: L. Claesson-Welsh, Department of Genetics
and Pathology, Rudbeck Laboratory, S-751 85 Uppsala, Sweden; e-mail: lena.welsh{at}genpat.uu.se.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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The Minimal Active Domain of Endostatin Is a Heparin-Binding Motif that Mediates Inhibition of Tumor Vascularization
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The Adaptor Protein Shb Binds to Tyrosine 1175 in Vascular Endothelial Growth Factor (VEGF) Receptor-2 and Regulates VEGF-dependent Cellular Migration
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The Human Immunodeficiency Virus-1 Tat Protein Activates Human Umbilical Vein Endothelial Cell E-selectin Expression via an NF-kappa B-dependent Mechanism
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Top
Abstract
Introduction