From the Dipartimento di Biologia Molecolare, Università degli Studi di Siena via Fiorentina 1, 53100 Siena, Italy
Received for publication, October 19, 2000
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ABSTRACT |
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Vascular endothelial growth factors (VEGFs) are a
highly conserved family of growth factors all angiogenic in
vivo with mitogenic and chemotactic activity on
endothelial cells. VEGFs are expressed in
fibroblasts either in hypoxia or in response to growth factors. Here we
report that, differently from the other members of the family,
Vegf-D is induced by cell-cell contact. By in
situ hybridization we demonstrated that noninteracting
fibroblasts express low levels of Vegf-D mRNA, whereas
contacting cells express high levels of Vegf-D transcripts.
By immunostaining we observed that the surface protein cadherin-11 is
localized at the opposite sites of interacting cell surfaces.
Ca2+ deprivation from the culture medium determined the
loss of cadherin-11 from the cell surfaces and down-regulation of
Vegf-D mRNA. Moreover, a cadherin-11 antisense RNA
construct inhibited Vegf-D expression in confluent BALB/c
fibroblasts, whereas in NIH 3T3 cells, which express low levels
of cadherin-11, Vegf-D induction could be obtained by
overexpression of cadherin-11. This suggests that cell interaction mediated by cadherin-11 induces the expression of the angiogenic factor
Vegf-D in fibroblasts.
The VEGF1 family is
composed of several structurally and functionally related growth
factors involved in vascular development. This family includes the
vascular endothelial growth factor (VEGF), the placental growth factor,
VEGF-B, VEGF-C, VEGF-D, and VEGF-E (1-11). All members of this family
are angiogenic in vivo and able to stimulate proliferation
of endothelial cells in vitro. Each member of the family
recognizes and activates specific receptors on endothelial cells: VEGF
recognizes VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1); placental growth
factor and VEGF-B recognize VEGFR-1 (12-14); VEGF-C and VEGF-D
recognize VEGFR-2 and VEGFR-3 (Flt-4) (7, 15, 16). This latter is
almost exclusively expressed in lymphatic vessels, suggesting that
these factors, beside playing a role in angiogenesis, are also involved
in the formation of lymphatic vessels.
Due to the similarity of structure and promiscuity of receptor
recognition, the specific role of each member of the family has not yet
been identified. In differentiating tissues, specific regulation of
each factor may be required to determine the correct succession and
composition of the appropriated angiogenic factors for vessel
formation. VEGF expression has been extensively studied. It
responds to low levels of oxygen with induced transcription and
increasing mRNA stability (17-19). Moreover, VEGF
mRNA expression is up-regulated by epidermal growth factor,
transforming growth factor- Analyzing Vegf-D mRNA expression in mouse fibroblasts we
observed that this growth factor, differently from the other members of
the VEGF family, was induced by calcium-dependent cell-cell interactions. Cell-cell adhesion is mediated by cadherins, a large family of transmembrane calcium-dependent adhesive
glycoproteins that form homotypic binding with their extracellular
domain on adjacent cells (25-27). Although it is generally thought
that cadherin expression results in a tight cell association, this is
not a general principle and mesenchymal cells, which are loosely
associated, express mesenchyme-specific cadherins like cadherin-11
(28-31).
The data presented in this report demonstrate that Vegf-D
messenger is strongly induced by direct cell-cell contact. This induction can be inhibited by depletion of extracellular
Ca2+ from the culture medium. Inhibition of cadherin-11
expression in contacting fibroblasts reduces Vegf-D mRNA
induction, whereas cadherin-11 expression in fibroblasts, that do not
express cadherin-11, restores Vegf-D induction. These
results identify cadherin-11 as a surface molecule involved in
Vegf-D regulation by cell-cell interaction.
Cloning and Cell Culture--
The mouse cadherin-11
full-length cDNA was amplified from a mouse fibroblast cDNA
library (9), using the primers 5'-GAGAGGATCCACCACCATGAAGGAGAACTACTG-3' and 5'-GAGACTCGAGTTAAGAGTCATCATCAAAAGTG-3'. The polymerase chain reaction product was cloned into the plasmid pcDNA3 (Invitrogen Corp.) in the sense (giving MO447) and antisense (giving MO334) orientation under the control of the cytomegalovirus promoter. The
oligonucleotide sequences were obtained from the EBI Nucleotide Sequence Data Base under accession numbers D21253 (OB-cadherin) and
D31963 (cadherin-11). All constructs were checked by automated sequencing.
Mouse embryo fibroblasts were isolated from 14-day CD1 mouse embryos as
described previously (32). Unless otherwise stated, mouse embryo, mouse
3T3-type, NIH 3T3, and BALB/c 3T3 fibroblasts were grown in
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum (Life Technologies, Inc.), 100 units/ml penicillin, and
100 µg/ml streptomycin at 37 °C in a humidified, 5%
CO2 atmosphere. Stable clones expressing mouse cadherin-11 were obtained from NIH 3T3 cells transfected with the
cadherin-11 expression vector MO447 by standard
CaPO4 precipitation procedures (32). Transfectants were
selected using 1 mg/ml G418 (Life Technologies, Inc.). Stable clones
expressing mouse cadherin-11 in the antisense orientation
were obtained from BALB/c 3T3 fibroblasts transfected with the plasmid
MO334, and transfectants were selected using 0.4 mg/ml G418. The same
empty vector was used to generate mock stable clones.
Cell-Cell Contact and Induction Experiments--
Mouse
fibroblasts were plated 14-16 h before day 0 on 10-cm tissue culture
dishes at different density, and starting from day 0, culture medium
was changed every 2 days. Low, medium, and high cell densities
corresponded to cells plated from about 20% to about 70% confluence.
The degree of cell confluence was monitored under an inverted
microscope. The cell cycle was arrested by adding cyclosporin A (0.95 µg/ml), colchicine (0.11 µg/ml), or tunicamycin (0.5 µg/ml) in
the culture medium of subconfluent fibroblasts for 24 h.
Conditioned medium was obtained from the culture medium of fibroblasts
growing at high cell confluence, diluted 1:1 (v/v) with complete
medium, and used to stimulate subconfluent fibroblasts for 33 h.
The heparin wash of confluent fibroblasts was performed by using a
solution of heparin (100 µg/ml, Sigma-Aldrich) in PBS. After taking
off the medium, the heparin solution was left on confluent cells for
2 h at room temperature, collected, centrifuged, diluted in
Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine
serum, and used to stimulate subconfluent fibroblasts for 33 h. As
negative control the heparin solution not left on the cells was used.
To chelate Ca2+ in the culture medium, confluent
fibroblasts were grown for 24 h in the presence of 2.2 mM EGTA. Poly-HEME (Sigma-Aldrich) was used to inhibit cell
adhesion to growth surface in culture dishes. Culture plates were
coated with 6 mg/ml poly-HEME in 95% ethanol and allowed to air dry in
a sterile environment. Fibroblast cells were seeded at high cell
density in plates precoated with poly-HEME and after 48 h cells
were collected by centrifugation and RNA was extracted. To block or
stimulate Ca2+ flux through calcium channels, fibroblasts
were grown either for 18 h at high cell density in the presence of
Ca2+ channel antagonists (10 µM diltiazem, 50 µM amiloride, 20 µM nifedipine, 10 µM verapamil, 1 µM Northern Blot Analysis--
Total cellular RNA was extracted
from cells by the guanidinium thiocyanate method (33). Total RNA (10 µg) was run on denaturing formaldehyde-agarose gel, transferred onto
nylon membranes, and cross-linked by UV irradiation using a
Stratalinker (Stratagene). Filters were hybridized with
32P-labeled probes, washed as described (9), and analyzed
by using a PhosphorImager (Molecular Dynamics). Rat
glyceraldheyde-3-phosphate dehydrogenase (gapdh) was used as
a control for RNA loading.
In Situ Hybridization--
Digoxigenin-labeled Vegf-D
sense and antisense RNA probes were generated from a cDNA fragment
corresponding to the complete coding sequence of the mouse
Vegf-D gene. Mouse fibroblasts were grown on microscopic
slides at different degrees of confluence and fixed for 20 min with 4%
paraformaldehyde in PBS. In situ hybridization was performed
as described previously (34) with minor modifications. Briefly, to
increase permeability cells were treated for 10 min with 0.2 N HCl and for 25 min at 37 °C with 1 µg/ml proteinase
K (Sigma-Aldrich) in 50 mM Tris-HCl, pH 8. Then cells were
washed in PBS and post-fixed for 10 min with 4% paraformaldehyde in
PBS. Cells were hybridized overnight in a humidified chamber at
37 °C with the digoxigenin-labeled probes diluted at 1 µg/ml in
hybridization buffer (60% deionized formamide, 2× SSC buffer, 50 mM sodium phosphate, 5% dextran sulfate, 250 µg/ml yeast
RNA, and 250 µg/ml salmon sperm DNA). The slides were washed, and the
hybridized digoxigenin-conjugated probes were detected by using the
fluorescent antibody enhancer set (Roche Diagnostics) according to
standard procedures. Slides were counterstained with propidium iodide
(Sigma-Aldrich) at 100 ng/ml, mounted in PBS containing 2%
1,4-diazabicyclo[2.2.2]octane (Sigma-Aldrich) and 50%
glycerol, and examined under a Leica TCS confocal laser-scanning microscope. The sense strand gave no signal.
Immunoblotting--
Whole cell extracts were prepared by rinsing
cultures with cold buffer (20 mM HEPES, pH 7.4, 130 mM NaCl, 5 mM KCl, 1 mM
MgCl2, 2 mM EGTA). Cells were harvested with a
rubber policeman, centrifuged, and lysed in 0.5% Nonidet P-40 buffer
(20 mM HEPES, pH 7.4, and 2 mM EDTA) containing
Complete protease inhibitors (Roche Diagnostic). Protein concentration
of cell extracts were determined by using the BCA protein assay reagent
(Pierce). The proteins were separated by 10% SDS-polyacrylamide gel
electrophoresis and transferred to a nitrocellulose membrane. Equal
loading was confirmed by staining in Ponceau S solution
(Sigma-Aldrich). The membranes were blocked for 1 h at room
temperature in PBS containing 3% dry milk and 0.1% Triton X-100 and
incubated with goat polyclonal antibodies against OB-cadherin (Santa
Cruz Biotechnology, Inc.) at 0.4 µg/ml for 2 h at room
temperature. The blots were washed, incubated with horseradish
peroxidase-labeled donkey anti-goat IgG (Santa Cruz Biotechnology,
Inc.) for 1 h at room temperature and washed in PBS, and finally
the bound antibodies were detected by enhanced chemiluminescence
(Amersham Pharmacia Biotech).
Immunofluorescence--
Mouse fibroblasts were seeded onto glass
coverslips, cultured overnight, and fixed with 3% paraformaldehyde in
PBS for 15 min. Cells were then permeabilized in 0.5% Triton X-100 in
PBS for 3 min and blocked for 1 h with 1% bovine serum albumin in PBS. Coverslips were incubated for 1 h at 37 °C with goat
polyclonal anti-OB-cadherin antibodies. After washing, the coverslips
were incubated for 45 min at 37 °C in the presence of donkey
anti-goat IgG labeled with tetramethylrhodamine isothiocyanate
(Jackson ImmunoResearch Laboratories). To localize actin filaments,
fluorescein isothiocyanate-labeled phalloidin (Sigma-Aldrich) was added
along with secondary antibodies at 2 µg/ml. Coverslips were then
mounted in Mowiol 4-88 (Calbiochem) and examined under a Leica TCS
confocal laser-scanning microscope.
Vegf-D mRNA Is Expressed in Confluent Cells--
Unlike
VEGF-C, whose expression is induced by several growth
factors (23, 24), Vegf-D is not induced by cell treatment with platelet-derived growth factor, epidermal growth factor, fibroblast growth factor 4, basic fibroblast growth factor, or transforming growth factor
To test whether Vegf-D mRNA induction may require high
cell density, RNA was collected at various time points from fibroblasts plated at different densities. At day 0, when cells were plated at low
(20% confluence) or medium (40% confluence) density, the expression
of Vegf-D was barely detectable, whereas some
Vegf-D expression could be detected in cells plated at the
highest density (70% confluence) (Fig.
1A, lanes 1-3).
Two days later, after the cells reached a higher confluence, we
observed a correspondent induction of Vegf-D transcripts. In
particular, Vegf-D expression was increased in the cells
originally plated at medium and high density (lanes 5 and
6). In fact, after 2 days, these cells reached about 90 and
98% confluence, respectively, with elevated cell-cell interactions. At
day 2, cells that were originally plated at low density (lane
4) were at about 30% confluence and showed still low
Vegf-D expression. Quantitative analysis revealed that cells plated at higher density reached the highest expression of
Vegf-D between days 4 and 6 from plating, cells plated at
medium density at day 6, and cells plated at low density at about day 8 (Fig. 1B). Thus, the levels of Vegf-D transcripts
and cell density are directly correlated.
Extracellular Ca2+ Is Required for Vegf-D
Expression--
Next we examined whether cell cycle arrest or soluble
autocrine growth factor(s) accumulating in the culture medium, or on the surface of cells growing at high cell density, could be responsible for Vegf-D induction. The treatment of subconfluent cells
with the cell cycle inhibitors cyclosporin A, colchicine, or
tunicamycin did not lead to Vegf-D induction (Fig.
2A). Neither the treatment of
subconfluent cells with conditioned medium nor with a heparin wash from
confluent cells could induce Vegf-D expression, suggesting that autocrine-soluble factors were not involved in Vegf-D
induction (Fig. 2B, compare lane 1 with
lanes 2 and 4). To test whether cell-cell
interaction and/or cell-plate contacts would play a role in
Vegf-D induction, we analyzed Vegf-D mRNA
levels in cells growing either in the presence of EGTA or in plates
precoated with poly-HEME. In the presence of EGTA, cells grew at a
normal rate, acquired a round shape, and lost interactions with each other and with the culture plate. Deprivation of Ca2+ from
the culture medium strongly inhibited Vegf-D mRNA
accumulation (Fig. 2C, lane 1). On the contrary,
culture plates precoated with poly-HEME, which inhibited cell adhesion
to the plate, did not affect Vegf-D induction (Fig.
2C, lane 2), suggesting that cell-matrix interactions are not involved in Vegf-D expression. Because
Ca2+ depletion from the culture medium might affect calcium
influx into the cells, we tested the expression of Vegf-D
mRNA in cells treated with different Ca2+ channel
blockers. The treatment of confluent cells with the calcium channel
antagonists nifedipine, verapamil, amiloride, diltiazem, or
Vegf-D mRNA Is Strongly Induced by Direct Cell-Cell
Interaction--
To directly observe Vegf-D mRNA
expression in contacting cells we performed in situ
experiments with cultured fibroblasts plated at various degrees of
confluence. Hybridization with Vegf-D antisense probe showed
that single cells expressed very poor levels of Vegf-D
mRNA, whereas contacting fibroblasts did express high levels of
Vegf-D messenger. This could be observed even at the level
of two interacting cells (Fig. 3,
A and B).
Taken together, the above experiments demonstrated that both cell-cell
interaction and extracellular calcium ions are required for
Vegf-D up-regulation in mouse fibroblasts. Homophilic
calcium-dependent cell-cell interactions are mediated by
cadherins (25). We therefore tested the hypothesis that direct
interaction between contacting fibroblasts, mediated by cadherins,
could be responsible for Vegf-D up-regulation.
First we examined by Northern blot analysis the expression of cadherins
in sparse and confluent fibroblasts with different cadherin probes. We
observed that, in fibroblasts, grown at different degrees of
confluence, cadherin-11, a mesenchymal-specific cadherin, was strongly induced by cell-cell contact (Fig.
4). Importantly, cadherin-11
mRNA induction was preceding Vegf-D mRNA of 8-12 h, suggesting that cadherin-11 could be involved in Vegf-D
regulation.
Immunostaining of contacting fibroblasts using anti-cadherin-11
antibodies revealed a positive staining at the cell surfaces (Fig.
5). In sparse cells cadherin-11 was
localized at intercellular contacts, whereas it was mostly absent from
surfaces free of cell contact (Fig. 5A). At confluence
cadherin-11 staining was observed at the level of the whole cell
membrane (Fig. 5B). To examine whether the localization of
cadherin in fibroblasts depends on Ca2+, contacting cells
were treated with EGTA. As expected, within the first hour cadherin-11
signal disappeared from the cell surface and became mostly cytoplasmic
(Fig. 5C). Addition of Ca2+ to the media
restored cell-cell contacts, with reappearance of cadherin-11 at the
intercellular contacts and induction of Vegf-D mRNA in
the cells (not shown).
Cadherin-11 Is Required for Vegf-D Induction in Interacting
Cells--
In our study, mouse 3T3 type fibroblasts, derived from mice
strain 129/SvJ × C57BL/6J (129-B6) (35), were used for
comparative analysis of gene expression, because Vegf-D was
strongly expressed in these cells. We tested whether other fibroblasts
showed the same Vegf-D mRNA regulation. Primary embryo
fibroblasts obtained from CD1 mice and BALB/c 3T3 fibroblasts revealed
a Vegf-D mRNA strong induction that correlated with
cadherin-11 high expression in confluent cells. Instead, NIH
3T3 fibroblasts expressed barely detectable levels of both
cadherin-11 and Vegf-D mRNAs (Fig.
6). Thus, extending the correlation
between Vegf-D mRNA induction and cadherin 11 expression in the same cells.
To directly evidence that cadherin-11 is required for Vegf-D
expression, we generated, from BALB/c 3T3 cells, stable cell lines
overexpressing cadherin-11 in the antisense orientation. Analysis of several stable clones revealed a variable level of cadherin-11 measured by Western blot of cell lysates. Two clones expressing low levels of cadherin-11 were analyzed for
Vegf-D expression (Fig.
7A). By Northern blot analysis
of the cadherin-11 antisense clones using Vegf-D
probe, we observed that inhibition of cadherin-11 resulted in a strong
reduction of Vegf-D expression (Fig. 7, compare A
and B).
The converse experiment was performed in NIH 3T3 fibroblasts, because
these cells expressed low levels of cadherin-11. From NIH 3T3 we
generated stable clones expressing, under the control of a constitutive
promoter, cadherin-11 and analyzed Vegf-D mRNA expression levels in contacting cells. Two clones expressing higher levels of cadherin-11 were chosen for Vegf-D expression
analysis (Fig. 7C). In these cells the ectopic expression of
cadherin-11 induced a significant increase of Vegf-D
transcripts (Fig. 7, compare C and D).
In multicellular organisms, intercellular interactions and
inductive signals play a major role in cell fate during development. Cell-cell adhesion, dictated by homophilic surface molecules like cadherins, determine cell patterning establishing the tissue
architecture; however, secreted growth factors, which act at a few cell
diameters, modify the expression pattern of neighboring target cells.
Here we demonstrate that in cultured fibroblasts direct cell-cell
interaction, mediated by the mesenchyme-specific cadherin-11, triggers
Vegf-D mRNA induction, suggesting cross-talk occurs
between cell adhesion and growth factor signaling.
Several lines of evidence support the model that Vegf-D
expression is regulated by direct cell-cell interactions via
cadherin-11. First, Vegf-D is not induced in subconfluent
cells under diverse culture conditions, whereas its expression is
dramatically increased in cells that reach confluence. Second, the
addition of conditioned media from cells highly expressing
Vegf-D does not induce its expression in cells grown at low
density, excluding the possibility that autocrine-diffusible factors
are instrumental in this activation. Third, depletion of extracellular
calcium ions, but not inhibition of cells to signal through calcium
flux, blocks Vegf-D expression, demonstrating that direct
calcium-dependent cell-cell interactions are required.
Fourth, Vegf-D expression directly correlates with cadherin-11 localization on the cell-interacting surfaces. Fifth, down-modulation or overexpression of cadherin-11 in fibroblasts affects
Vegf-D expression in a negative and positive manner,
respectively. Thus, the experiments described in this report provide
evidence that cadherin-11 mediates a cell interaction signaling that
leads to the regulation of Vegf-D in contacting fibroblasts.
Cadherins play an important role in cell recognition and sorting during
development (36, 37; and references therein). Their function has been
perceived to link and stabilize connections between cells through
interaction with the cytoskeleton. However, cytoplasmic domains of
cadherins are highly diversified, and examples of cadherins have been
found associated with signal transduction molecules and/or able to
induce intracellular messengers, suggesting that cadherins mediate
signal transduction pathways upon ligand binding (for review see Ref.
38). During development Vegf-D mRNA expression appears
to be restricted to cadherin-11-positive mesenchymal cells. In the
developing mouse embryo cadherin-11 is expressed in
migratory cells derived from neural crest cells and in cells involved
in mesenchymal condensation (30, 31). Vegf-D expression
shows a striking overlap with cadherin-11 (30, 31, 39).
VEGFs are a family of angiogenic factors that are known to induce
vessels sprouting in vivo and activation of endothelial specific receptors in vitro which are in part overlapping
(7, 12-16). Although it is difficult to assign a specific role to each member of the family, it is likely that a coordinated regulation of
each factor is required for the correct three-dimensional formation of
new vessels. Therefore, the pattern of expression of each factor may
contribute to explain its role in the angiogenic process. Interestingly, VEGF is strongly induced by hypoxia whereas VEGF-B, VEGF-C, and VEGF-D are not (17, 19, 23, 40). In lung fibroblasts VEGF-C
is up-regulated by growth factors (23, 24). By contrast, in mouse
fibroblasts Vegf-D is not induced by growth factor
treatment,2 whereas it is
up-regulated by cadherin-dependent cell-cell interaction. Vegf-D regulation by cell-cell contact is consistent with a model in
which aggregating fibroblasts switch on the expression of a factor that
is chemotactic to endothelial cells and thus could recruit them to the
forming tissue. The cell-cell contact mediated by cadherin-11 in
vitro may mimic, in part, the angiogenic process and suggests that
Vegf-D expression might be involved in the initial steps of the
angiogenic process.
The neovascularization and/or formation of lymphatic vessels in solid
tumors is induced by tumor cells that express inductive angiogenic
factors, which promote vessels sprouting with the recruitment of
endothelial cells to the growing tumor mass (10, 41, 42). However,
tumor vasculature is highly disorganized with vessels being dilated,
tortuous, and fenestrated and having excessive branching and uneven
diameters (43). This could be the result of the imbalance of
angiogenic factors. The fine regulation of VEGF-D by cadherin signaling
is probably compromised in tumor cells especially considering that
cadherin expression is altered during the malignant progression of
tumors (44-49). In this respect it is interesting that the expression
of VEGF-D has been found to correlate inversely with tumor size and
node positivity in lung adenocarcinoma (50).
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, IL-6 in several cell types, and by
IL-1
in smooth muscle cells (20-22). VEGF-C expression
in cultured fibroblasts is induced by serum, phorbol 12-myristate
13-acetate, and several factors, including IL-1
and tumor necrosis
factor
(23, 24). Vegf-D appeared to be differently
regulated, because it was expressed in cells grown in low serum
conditions (9).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-conotoxin GVIA) or
for 24 h at low cell density in the presence of a Ca2+
channel agonist (1.25 µM BAY K-8644). Cyclosporin A,
colchicine, tunicamycin, diltiazem, amiloride, and
-conotoxin GVIA
were purchased from BioMol Research Laboratories, and nifedipine,
verapamil, BAY K-8644 were from Sigma-Aldrich. Cell synchronization
agents and calcium channel modulators were used at concentrations
established from the literature to have maximal effects on their targets.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(data not shown). Moreover, we
previously observed that in low serum conditions fibroblasts expressed
high levels of Vegf-D transcripts (9).
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Fig. 1.
Analysis of Vegf-D
expression in correlation with cell density in cultured mouse
fibroblasts. A, Northern blot analysis using
Vegf-D and gapdh cDNA probes. Fibroblasts
were plated at low (L), medium (M), and high
(H) cell density. After 15 h, corresponding to day 0, cell confluence was monitored and resulted about 20%, 40%, and 70%,
respectively, for plates L, M, and H. Starting from day 0, every 2 days
the culture medium was changed, the degree of confluence was monitored,
and the RNA was collected. Above the lanes are indicated the days from
the time of plating. Vegf-D transcripts were induced when
cells reached confluence at day 2 for M and H, and at day 6 for L
(lanes 5, 6, and 10, respectively).
B, quantitative analysis of Vegf-D mRNA
levels from a representative experiment with cells plated as in
A. The blots were analyzed by using a PhosphorImager, and
the values, normalized to the gapdh mRNA levels, are
expressed as arbitrary units.
-conotoxin GVIA did not inhibit Vegf-D mRNA
expression, and the treatment of sparse cells with the calcium channel
agonist BAY K-8644 did not induce Vegf-D expression (Fig.
2D). Therefore, this excluded that calcium influx plays a
role in the Vegf-D up-regulation.
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Fig. 2.
Vegf-D expression is
Ca2+-dependent in cultured fibroblasts.
A, Northern blot analysis of RNA extracted from subconfluent
fibroblasts treated for 24 h with drugs as indicated. Cell cycle
arrest did not lead to Vegf-D expression in sparse cells.
Total RNA from untreated subconfluent fibroblasts (lane 1).
B, Northern blot analysis of RNA extracted from subconfluent
fibroblasts stimulated for 33 h with conditioned media obtained
from fibroblasts grown at high cell density. Vegf-D was not
induced by autocrine growth factor(s). Subconfluent cells not treated
with conditioned medium (untreated); subconfluent cells
treated with conditioned medium obtained from fibroblasts at confluence
(c.m.); subconfluent cells treated with a solution of
heparin (heparin); subconfluent cells treated with a heparin
wash of fibroblasts at confluence (he-extr.); fibroblasts at
confluence (control). C, Northern blot analysis
of RNA extracted from fibroblasts grown for 24 h in the presence
of 2.2 mM EGTA or for 48 h in culture plates precoated
with poly-HEME (poly-H), which inhibits cell adhesion to the
culture plate. Deprivation of Ca2+ from the culture medium
inhibited Vegf-D mRNA accumulation. Total RNA from
untreated confluent cells (control). D, Northern
blot analysis of RNA extracted from fibroblasts grown in the presence
of calcium channel modulators. Fibroblasts were grown either at high
cell density for 18 h with calcium channel antagonists (diltiazem,
amiloride, nifedipine, verapamil, -conotoxin GVIA) or at low cell
density for 24 h with a calcium channel agonist (BAY K-8644).
Vegf-D expression was not induced by calcium flux through
calcium channels. Control of untreated fibroblasts confluent
(lane 1) and subconfluent (lane 7).
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Fig. 3.
Direct cell-cell contacts induce
Vegf-D mRNA. B, D, and
F, mouse fibroblasts, grown on microscopic slides at
different degrees of confluence, were analyzed by in situ
hybridization with a digoxigenin-labeled probe specific for
Vegf-D transcripts (green). A,
C, and E, nuclei of the same cells as in
B, D, and F were visualized with the
nuclear dye propidium iodide (red). Stainings were analyzed
by confocal microscopy. Single cells expressed very poor levels of
Vegf-D mRNA, whereas contacting fibroblasts expressed
high levels of Vegf-D messengers (E and
F), even at the level of two interacting cells (A
and B). The scale bar represents 30 µm.
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Fig. 4.
Cadherin-11 mRNA is up-regulated in
contacting fibroblasts. Northern blot analysis using Vegf-D,
cadherin-11, and gapdh probes. Fibroblasts were plated
at low cell density, and every 2 days the culture medium was changed,
the degree of confluence was monitored, and the RNA was collected.
Cadherin-11, a mesenchyme-specific cadherin, was induced
when cells established cell-cell contacts (lane 3).
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Fig. 5.
Cadherin-11 is localized at cell-cell
interaction surfaces. A-C, immunofluorescence analysis
using anti-cadherin-11 primary antibodies (red). Mouse
fibroblasts were seeded at different degrees of confluence, grown in
the absence (A and B) or in the presence
(C) of 2.2 mM EGTA in the culture medium and
processed for immunostaining. A', B', and
C', actin filaments of the same cells as in A-C
were stained using fluorescein isothiocyanate-labeled phalloidin
(green). Stainings were analyzed by confocal microscopy.
Cadherin-11 is localized on the cell surface at intercellular contacts
(A), and in the presence of EGTA cadherin-11 signal
disappeared from the cell surface and became mainly cytoplasmic
(C). The scale bar represents 30 µm.
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Fig. 6.
Vegf-D and cadherin-11
mRNA expression correlates in different fibroblasts. RNA
was collected from 3T3-type (129-B6), mouse embryo fibroblasts, BALB/c,
or NIH 3T3 fibroblasts grown at low (NC) and high
(C) cell confluence. Vegf-D and
cadherin-11 expression correlated in confluent fibroblasts
from different origin, whereas in NIH 3T3 fibroblasts low levels of
Vegf-D and cadherin-11 mRNAs were
detected.
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Fig. 7.
Cadherin-11 is required for Vegf-D
induction. A and B, inhibition of
cadherin-11 affects Vegf-D expression in fibroblasts. Stable
cell lines, overexpressing cadherin-11 in the antisense
orientation, were generated in BALB/c 3T3 cells and grown at high cell
confluence for 4 days. A, Western blot analysis of cell
lysates from cadherin-11 antisense clones revealed with
anti-cadherin-11 antibodies. B, Northern blot analysis of
RNA extracted from the same antisense clones as in A, using
Vegf-D and gapdh probes. C and
D, overexpression of cadherin-11 induces Vegf-D
in NIH 3T3 fibroblasts. Stable cell lines, overexpressing cadherin-11,
were generated in NIH 3T3 cells and grown at high cell confluence for 4 days. C, immunoblotting analysis of cell lysates from
cadherin-11 stable clones with anti-cadherin-11 antibodies.
D, Northern blot analysis of RNA extracted from the same
clones as in C, using Vegf-D and gapdh
probes. Mock clones are in lanes C.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Elisabetta Dejana, Giuliano Callaini, and Nicholas Valiante for helpful discussions and Beatrice Grandi for technical assistance.
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FOOTNOTES |
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* This work was supported by grants from Associazione Italiana per la Ricerca sul Cancro, Chiron Biocine Spa, and Biomed II (BMH4-CT98-3380).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: Fax: 39-0577-234929;
E-mail: oliviero@unisi.it.
Published, JBC Papers in Press, December 6, 2000, DOI 10.1074/jbc.M009573200
2 M. Orlandini and S. Oliviero, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: VEGF, vascular endothelial growth factor; IL, interleukin; PBS, phosphate-buffered saline; poly-HEME, poly-2-hydroxyethyl methacrylate.
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