Department of Oncology-Pathology, Cancer Center Karolinska Institutet, S-17176 Stockholm, Sweden
* Author for correspondence (e-mail: lars.holmgren{at}cck.ki.se)
Accepted 29 May 2003
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Summary |
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Key words: Endothelium, Neovascularization, Migration, Plasminogen, Receptor
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Introduction |
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We have recently identified a novel gene, angiomotin, via its interaction
with angiostatin in the yeast two-hybrid system
(Troyanovsky et al., 2001).
Angiomotin belongs to a new protein family with only two additional members
characterized by conserved coiled-coil domains and C-terminal PDZ binding
motifs (Bratt et al., 2002
;
Nishimura et al., 2002
). One
of the members of this family, JEAP/AmotL-1, was identified by its
localization to tight junctions in exocrine epithelial cells
(Nishimura et al., 2002
). The
expression pattern of angiomotin differs from that of JEAP as it is expressed
in endothelial cells of tissues undergoing angiogenesis such as
extra-embryonic tissues and human tumors. Angiomotin is also expressed in
cytotrophoblasts of the placenta as well as in polymorphic mononuclear cells
[the latter of which was recently reported to respond to angiostatin
(Benelli et al., 2002
)]. At a
cellular level, endogenous as well as transfected angiomotin localizes to the
leading front of lamellipodia of migrating cells. A role in cell migration is
suggested by the findings that overexpression of angiomotin in endothelial
cells resulted in increased cell-migration. Furthermore, angiostatin inhibited
migration and tube formation in angiomotin-transfected cells whereas control
cells were unaffected. These data suggest that angiostatin is an antagonist of
angiomotin activity in endothelial cells.
In the present study, we found that the putative PDZ binding motif of angiomotin serves as a protein recognition site and binds to a 90 kDa protein. Deletion of three amino acids in the C-terminal binding domain abrogated protein-protein interaction and resulted in inhibition of endothelial migration in vitro and in vivo.
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Materials and Methods |
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Peptide affinity columns
The following peptides were synthesized: CTLERKTPIQ ILGQEPDAEM VEYLI,
CTLERKTPIQ ILGQEPDAEM VE (Innovagen AB, Lund, Sweden). The first cysteines
were added to facilitate immobilization of the peptides to the sulfolink
coupling gel (Pierce, according to the protocol of the manufacturer). For
peptide affinity purification, mouse aortic endothelial cells were grown to
80% confluency in 14.5 cm Petri dishes. Before harvesting, cells were washed
twice in cold PBS and scraped off in 3 ml lysis buffer (20 mM Hepes, 140 mM
KCl, 5 mM MgCl, 10 mM ß-glycerophosphate and 3% thesit and proteinase
inhibitor cocktail pH 7.4; Sigma). The lysates were spun at 30,000
g for 25 minutes at 4°C. The supernatants were loaded to
the peptide affinity column that had been equilibrated with lysis buffer. The
column was washed with eight volumes of lysis buffer. Bound protein was eluted
with 100 mM glycine buffer at pH 3.0. The volume of the collected fractions
was reduced in a speedvac before analysis by SDS-PAGE. Protein bands were
visualized by Coomassie staining.
Generation of deletion mutants
The angiomotin-pBabe vector was generated by blunt-end cloning a 3054 bp
fragment of angiomotin cDNA generated by HindIII digestion into the
EcoRI site of the pBabe vector. Deletion variants were created by
PCR, using the angiomotin cDNA as template. The following primers were used:
forward: 5'-CGGAATTCAGGCCAGCGCAGGACATC-3'
Reverse primer for the 3' of: Del 1,
5'-GGTTATTCCAGAGTATTGGAGT-3', Del 2,
5'-GGTTAGGGAGTTTTTCTTTCCAGAGT-3', Del 3,
5'-GGTTAAGGCTCTTGTCCC-AGGATCT-3', Del 4,
5'-GGTTATTCCACCATCTCTGC-ATCA-3'. The PCR-product was cloned using
the AdvanTAge PCR cloning Kit (Clontech), then cloned as an EcoRI
fragment into the pBabe vector. To direct expression to endothelial cells
during development we used the TIE-1 promoter (kindly provided by Dr K.
Alitalo). Angiomotin (wild type and 4) were blunt-end cloned into the
ApaI site of the TIE-vector using either the HindIII
fragment (wild type angiomotin) or the EcoRI fragment (
4
angiomotin).
Western blotting
Parental MAE cells, transfected with pBabe angiomotin deletions were
subjected to electrophoresis in SDS-PAGE gel under non-reducing conditions,
transferred onto nitrocellulose membrane and non-specific binding was blocked
overnight in PBS containing 5% dried milk. Angiomotin polyclonal antibodies
were added for 1 hour at room temperature, washed three times for 5 minutes
each in PBS and then incubated for 60 minutes with horseradish peroxidase goat
anti-rabbit antibodies (Amersham Life Science). Bands were visualized using
the ECL detection system (Amersham Life Science).
Migration assay
Migration assays were performed in a modified Boyden chamber using a
48-well chemotaxis chamber (Neuroprobe Inc., Gaitherburg, MD). Eight-micron
Nucleopore polyvinylpirrolidine-free polycarbonate filters were coated with
100 µg/ml of collagen type 1 (Cohesion, Palo Alto, USA) in 0.2 N acetic
acid for 24 hours and air-dried. The filter was placed over the bottom chamber
containing basic fibroblast growth factor (bFGF; Pharmacia and Upjohn),
vascular endothelial growth factor (VEGF; R&D systems) or lyso
phosphatidic acid (LPA; Calbiochem). MAE cells were suspended in DMEM and
30,000 cells in 50 µl were added to each well in the upper chamber. In
order to test the inhibitory activity of angiostatin on angiomotintransfected
cells, MAE cells were pre-incubated with 2.5 µg/ml of angiostatin for 1
hour. The assembled chemotaxis chambers were incubated for 5 hours at 37°C
with 10% CO2 to allow cells to migrate through the collagen-coated
polycarbonate filter. Non-migrated cells on the upper surface of the filter
were removed by scraping with a wiper tool (Neuro Probe, Inc., Gaitherburg,
MD) and cotton swab, and the filter was stained with Hematoxylin (Mayer). The
total number of migrated cells per field was counted at 200x
magnification; each sample was tested in quadruplicate.
In vitro tube formation assay
MAE cells, transfected with angiomotin and angiomotin deletions
(1.5x105 cells) were seeded on a layer of polymerized
Matrigel as previously described
(Troyanovsky et al., 2001).
Matrigel cultures were incubated at 37°C. After 16 hours changes in cell
morphology were captured through a phase-contrast microscope and
photographed.
Immunofluorescent stainings
MAE transfected cells were fixed in 4% paraformaldehyde for 7 minutes at
room temperature and blocked with 5% horse serum in PBS for 30 minutes. Cells
were then incubated with angiomotin antibodies for 1 hour at room temperature.
Antibody binding was detected with FITC-labeled anti-rabbit antibodies (Dako).
F-actin was visualized with rhodamine-phalloidin (Molecular Probes). Images
were collected using a Hamamatsu CCD camera and the Openlab software.
Transgenic animals
Transgenic mice were generated as described previously
(Hogan et al., 1995). Briefly,
TIE-angiomotin or TIE-
4 DNA constructs were injected into the
pronucleus of fertilized C57BL/CBA mouse eggs. The eggs were then transferred
into pseudopregnant mothers and transgenic embryos and adult mice were
identified by Southern blot analysis. Genomic DNA was cleaved with
SspI, generating a 1.8 kb fragment containing both the TIE promoter
and approx. 1 kb of the 5' part of angiomotin, which is the same in both
wild-type and
4 constructs. Positive transgenes were verified using a
probe spanning the same region, labeled by random priming (Megaprime kit,
Amersham).
PECAM whole mount staining and sectioning
Both TIE-angiomotin and TIE-4 embryos were dissected at E9.5 and
fixed in 4% paraformaldehyde for 2 hours on ice. After washing in PBS, the
embryos were incubated in 17% sucrose overnight, washed again and then
dehydrated stepwise in methanol (25%, 50%, 75% and 100% for 15 minutes each)
at room temperature. The embryos were bleached in 5%
H2O2 for 5 hours, followed by stepwise rehydration to
PBS. To block non-specific binding the embryos were incubated with 0.5% bovine
serum albumin (BSA; Sigma) and 1% Triton X-100 (Sigma) for 1 hour at room
temperature. Next they were incubated overnight at 4°C with rat anti-mouse
PECAM1 antibody (Pharmingen, cat no. 553370) diluted 1:250 in 0.5% BSA, 0.5%
Triton X-100 (BSA-Tx). Following washing in PBS/0.1% Triton X-100 five times
for 1 hour each the embryos were incubated with a secondary antibody
(biotinylated goat
-rat IgG, Vector laboratories) overnight at a
dilution of 1:200 in BSA-Tx. The embryos were then washed again and incubated
overnight with the ABC complex (Vector laboratories) diluted to 1:500 in
BSA-Tx. After washing five times for 1 hour each the embryos were developed
with diaminobenzidine tetrahydrochloride (DAB; Sigma). The embryos were then
embedded in paraffin and 5 µm sections were stained with Hematoxylin and
Eosin.
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Results |
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Angiomotin promotes, whereas C-terminal deletion mutants inhibit, the
response to chemotactic factors
In order to test the importance of the putative PDZ-binding motif in the
control of endothelial cell migration we generated a series of deletions in
the C terminus. The angiomotin cDNA contains a 2025 bp open reading frame,
predicted to encode a 675 amino acid protein. The last 21 amino acids from the
COOH end of angiomotin were divided into four parts: deletion 1 encoded amino
acids (aa) 1-654; deletion 2, aa 1-658; deletion 3, aa 1-66; deletion 4, aa
1-672 (Fig. 1C). Wild-type
angiomotin or one of these angiomotin mutant cDNAs were inserted into pBabe
vector and used to generate retroviruses. MAE cells were infected with
retroviruses, containing vector, wild-type angiomotin, or angiomotin mutants.
Protein expression was verified by western blot analysis of cell lysates
(Fig. 1D). No significant
difference in proliferation rate could be detected as analyzed in a 3-day
cell-doubling assay (data not shown). We have previously shown that angiomotin
can promote cell migration in vitro. We therefore tested the migratory
response of the cells transfected with the C-terminal angiomotin deletions
using the Boyden chamber assay. Angiomotin-expressing cells exhibited
increased migration towards bFGF, VEGF and LPA, all of which have been shown
to induce migration in endothelial cells. In contrast, expression of the delta
1-4 mutant cDNAs in MAE cells resulted in inhibition of the migratory response
to bFGF (Fig. 2A). Peptides
lacking the last YLI amino acids of angiomotin do not bind the p90 protein as
shown in figure 1. Furthermore,
the 4 mutants did not exhibit any significant increase in migration in
response to VEGF or LPA (Fig. 2B and
C). These data show that deletion of as little as 3 amino acids of
the C-terminal region of angiomotin results in repression of migration. We
chose to focus on mutation 4 as we wanted to correlate cell migration to the
functionality of the C-terminal protein interaction site.
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Next we investigated the behavior of the angiomotin transfectants in an in
vitro tube formation assay. When plated on matrigel, extracellular matrix MAE
cells attach, migrate and form multicellular capillary-like tubular
structures. No significant difference in the capacity to form tubes in vitro
could be detected between angiomotin and vector-transfected MAE cells
(Fig. 3). However, mutants
1-3 all showed perturbed tube formation in that tubes were formed but
collapsed resulting in a lower total tube length
(Fig. 3 and data not shown). A
more dramatic effect was observed after plating
4-transfected cells.
Here, the cells did not spread and migrate to form tube structures but
remained as single cells. These data show that removal of the C-terminal three
amino acids of angiomotin results in repression of cell migration and tube
formation.
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The putative PDZ-binding motif of angiomotin is not required for
localization to lamellipodia
Angiomotin localizes to lamellipodia of migrating cells. Therefore we
wanted to assess whether the three amino acid deletion of the angiomotin C
terminus affected the sub-cellular localization of the protein. It is possible
that removal of the C-terminal end would either change protein conformation or
remove a targeting signal that confers localization to lamellipodia. The
angiomotin antibodies preferentially stained the lamellipodia of motile MAE
angiomotin-transfected cells (Fig.
4). No difference in the cellular localization of the
4-angiomotin could be detected indicating that the last three amino
acids are not required for angiomotin localization to the lamellipodia.
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The angiomotin 4 mutant inhibits embryonic angiogenesis
Next we wanted to assess whether the 4 mutation could inhibit
endothelial cell migration and angiogenesis in vivo. For this purpose, we
generated transgenic mice expressing wild-type angiomotin or the
4
mutant driven by the endothelial cells-specific TIE promoter that is activated
at embryonic day 8 (Fig. 5A).
Expression of transgenic constructs was detected by Southern blot and RT-PCR
analysis from DNA and RNA extracted from the tails. The DNA analysis revealed
that 15% (8/57) of the offspring from TIE-angiomotin injections carried the
transgene (Fig. 5B). No
aberrant phenotype could be detected in these animals that were fertile when
crossed with wild-type C57B6 mice. Two independent TIE-angiomotin transgenic
lines were generated. Analysis of transgenic embryos derived from these lines
at E 9.5, E13.5 and at birth did not reveal any detectable vascular defects
(Table 1 and
Fig. 5C,D).
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In contrast, injections of the TIE-4 construct resulted in no live
transgenic mice (0/38). We therefore analyzed embryos at E9.5 for possible
vascular abnormalities. Embryos carrying the transgene were identified by
RT-PCR analysis of mRNA extracted from the placenta
(Fig. 5B). A total of 14
transgenic embryos were identified, each embryo representing an individual
transgene with a unique integration site. Analysis of these embryos revealed
that 13 out of 14
4 transgenes displayed severe vascular deficiencies
at this time (Fig. 5H). The
non-transgenic littermates (n=86) had no detectable vascular
insufficiencies (Table 1). The
transgenic embryos 1-9 were similar in size to wild-type embryos but suffered
from severe bleeding in the brain as well as from intersomitic vessels
(Table 2). The other embryos
were smaller than the wild-type littermates. These embryos did not suffer from
any detectable bleeding. We visualized the vascular endothelial cells in
wild-type and TIE-
4 mice by whole-mount immunostaining using antibodies
against mouse PECAM1. The TIE-
4 embryos had a defective cranial
vascularization (Fig. 6). The
localization of the vessels in the TIE-
4 angiomotin mice was assessed
histologically. In normal brains at E9.5, capillaries were juxtaposed or
migrating into the neuroepithelium (Fig.
7A,C). The TIE-
4 mice had cranial aneurysms and dilated
vessels were found in the cephalic mesenchyme
(Fig. 7B,D). These vessels did
not align properly and fewer capillaries could be observed within the
neuroepithelium. In addition, some of the dilated vessels showed leakage of
nucleated blood cells into the surrounding mesenchyme
(Fig. 7D). A more severe
phenotype is illustrated in Fig.
7E,F in which no capillaries migrated into the neuroepithelium and
the vessels adjacent to the neuroepithelium were grossly dilated.
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Discussion |
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Angiomotin belongs to a novel protein family with two additional members,
AmotL1 and Amotl2 (Bratt et al.,
2002). These proteins are characterized by coiled-coil domains and
contain a consensus motif for the binding of PDZ domains in the C terminus.
The interaction between PDZ binding motifs and PDZ-containing proteins has
been shown to play an important role in the targeting of proteins to specific
membrane compartments (reviewed by Sheng
and Sala, 2001
). However, immunofluorescent stainings of
angiomotin and the
4 mutant did not reveal any detectable difference in
cellular localization. Both proteins were localized to areas of lamellipodia
formation. Furthermore, both proteins are accessible to antibody staining and
can be biotinylated on the cell surface (A.B., data not shown). Another role
of PDZ proteins is to organize protein scaffolds for signaling complexes
associated to membrane receptors. There are several examples indicating that
PDZ proteins binding to membrane receptors may control the cellular response
to migratory cues. For example, the cytoplasmic protein PDZ-RGS3 binds to the
C terminus of transmembrane B ephrins and modulates the response of cerebellar
granule cells to chemoattractant signaling via heterotrimeric G proteins
(Lu et al., 2001
).
Furthermore, disruption of the PDZ binding domain of syndecan 4 inhibits
migration, tube formation and exerts a dominant-negative effect on bFGF
signaling in endothelial cells (Horowitz et
al., 2002
). In analogy, we show that the putative PDZ binding
motif of angiomotin binds to a 90 kDa protein. Deletion of the last three
amino acids specifically abrogates both protein-protein interaction and
results in loss of responsiveness to migratory stimulators.
We used the TIE promoter to express the angiomotin gene and the 4
mutant in the endothelial cell lineage in mice. The TIE-angiomotin mice were
viable and fertile and did not exhibit any detectable impairment in blood
vessel formation. Expression of TIE-
4 resulted in embryonic lethality,
as it was thus not possible to generate any viable transgenic offspring. The
TIE-
4 mice undergo normal vasculogenesis as could be expected since the
TIE promoter is activated between E8-8.5 and is expressed in differentiating
angioblasts as well as in endothelial cells. During normal development VEGF
produced by the neuroectoderm stimulates the ingrowth of capillaries from the
perineural vascular plexus (Breier et al.,
1992
). The TIE-
4 mice exhibited severely impaired blood
vessel formation in the brain as well as in inter-somitic regions. The
phenotype varied from cranial vessel dilation and hemorrhage to severe
impairment of blood vessel formation in the embryos resulting in malformation
and reduced size. The dilation and leakage of vessels is usually indicative of
abnormalities in the basement membrane, cell-cell adhesion or other supporting
structures. Several reports show that inactivation of components of the
vascular extra-cellular matrix may cause hemorrhage. For example, inactivation
of laminin
4 or fibulin, both components of the vascular wall, result
in hemorrhage during embryonic development
(Thyboll et al., 2002
;
Kostka et al., 2001
). Another
cause of hemorrhage is the failure of endothelial cells to recruit pericytes
to form a vessel wall characteristic of mature vessels. PDGF-B-deficient mice
exhibit dilation of brain vessels and post-natal hemorrhage. The defects are
probably caused by defective migration of pericytes that depend on the
chemotactic signaling from endothelial cells. The phenotype observed in the
TIE-
4 mice occurs earlier than that observed in PDGF-B mice, that is
before the recruitment of pericytes in the brain
(Lindahl et al., 1997
).
Analysis of vessel morphology by histological sectioning revealed that vessels
in transgenic mesenchyme were dilated and did not align properly with the
neuroectoderm. Furthermore, the ingrowth of capillaries into the neuroectoderm
was inhibited. We therefore suggest that the aberrant vascular phenotypes
detected in TIE-
4 mice are caused by the inability of endothelial cells
to respond to local chemotactic factors.
In conclusion, we provide evidence that inactivation of the putative PDZ domain switches angiomotin from promoting to repressing cell motility. Further understanding of the signaling pathways involved may yield important clues on how to inhibit endothelial cells responding to local chemotactic factors and thereby block the formation of new vessels.
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Acknowledgments |
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