Content uploaded by Jacques Pouysségur
Author content
All content in this area was uploaded by Jacques Pouysségur on Mar 26, 2015
Content may be subject to copyright.
MOLECULAR AND CELLULAR BIOLOGY,
0270-7306/01/$04.00⫹0 DOI: 10.1128/MCB.21.21.7218–7230.2001
Nov. 2001, p. 7218–7230 Vol. 21, No. 21
Copyright © 2001, American Society for Microbiology. All Rights Reserved.
Transforming Growth Factor 1 (TGF-1) Promotes Endothelial Cell
Survival during In Vitro Angiogenesis via an Autocrine
Mechanism Implicating TGF-␣Signaling
FRANCESC VIN
˜ALS* AND JACQUES POUYSSE
´GUR*
Institute of Signaling, Developmental Biology and Cancer Research, CNRS UMR 6543-Centre Antoine Lacassagne,
06189 Nice Cedex 2, France
Received 2 March 2001/Returned for modification 19 April 2001/Accepted 6 August 2001
Mouse capillary endothelial cells (1G11 cell line) embedded in type I collagen gels undergo in vitro
angiogenesis. Cells rapidly reorganize and form capillary-like structures when stimulated with serum. Trans-
forming growth factor 1 (TGF-1) alone can substitute for serum and induce cell survival and tubular
network formation. This TGF-1-mediated angiogenic activity depends on phosphatidylinositol 3-kinase
(PI3K) and p42/p44 mitogen-activated protein kinase (MAPK) signaling. We showed that specific inhibitors of
either pathway (wortmannin, LY-294002, and PD-98059) all suppressed TGF-1-induced angiogenesis mainly
by compromising cell survival. We established that TGF-1 stimulated the expression of TGF-␣mRNA and
protein, the tyrosine phosphorylation of a 170-kDa membrane protein representing the epidermal growth
factor (EGF) receptor, and the delayed activation of PI3K/Akt and p42/p44 MAPK. Moreover, we showed that
all these TGF-1-mediated signaling events, including tubular network formation, were suppressed by incu-
bating TGF-1-stimulated endothelial cells with a soluble form of an EGF receptor (ErbB-1) or tyrphostin
AG1478, a specific blocker of EGF receptor tyrosine kinase. Finally, addition of TGF-␣alone poorly stimulated
angiogenesis; however, by reducing cell death, it strongly potentiated the action of TGF-1. We therefore
propose that TGF-1 promotes angiogenesis at least in part via the autocrine secretion of TGF-␣, a cell
survival growth factor, activating PI3K/Akt and p42/p44 MAPK.
Angiogenesis or the formation of new blood vessels from
preexisting vasculature occurs in normal situations such as
embryonic development, wound healing, and during the fe-
male reproductive cycle. However, activated blood vessel
growth is also found in many diseases, such as tumor progres-
sion, diabetic retinopathy, and arthritis (24, 33). In the last few
years, several studies have led to the discovery of inducers and
inhibitors of the angiogenic process (6, 9, 12). Among the
inducers are factors such as vascular endothelial growth factor
(VEGF) and fibroblast growth factor 1 (FGF-1) and -2, which
induce angiogenesis in vivo and in vitro. They can also induce
the proliferation and migration of endothelial cells in two-
dimensional cultures. In contrast, other factors such as trans-
forming growth factor (TGF-) and tumor necrosis factor
alpha induce angiogenesis in vivo and in vitro but inhibit en-
dothelial cell proliferation in vitro (6, 9, 12).
TGF-1 is a 25-kDa peptide belonging to a family of mul-
tifunctional cytokines that control the development and ho-
meostasis of most tissues by regulating diverse cellular func-
tions, such as proliferation and differentiation (49, 72). The
receptors for this family are basically two transmembrane
serine/threonine kinases, termed receptor type I and type II.
The binding of the ligand causes the heterodimerization of
receptors I and II followed by the activation by phosphoryla-
tion of receptor I. This receptor then phosphorylates and ac-
tivates the Smad family of proteins, which transduce the signal
to the nucleus (19, 36, 49, 72). The role of TGF-in angio-
genesis was first shown by new capillary formation after injec-
tion of the factor into mice (23, 65) and by application of the
factor to the chicken chorioallantoic membrane (80). More-
over, TGF-1 and TGF-2 are expressed during the develop-
ment of angiogenically active tissues (35, 60). This proangio-
genic activity of TGF-has been confirmed by experiments
using knockout mice. The knock out of TGF-1 (20), the type
II receptor (59), and type I receptor activin receptor-like ki-
nase 1 (ALK1) (57, 74) is lethal at 10.5 days of gestation due
to defective vasculogenesis (the initial formation of the prim-
itive vasculature in the embryo), along with defective endothe-
lial cell differentiation and inadequate capillary tube forma-
tion. Moreover, Smad5 knockout mice also die due to defects
in vasculogenesis and angiogenesis (14, 81). Finally, mutations
in the human ALK1 gene and in the endoglin gene, which
encodes a TGF-1-binding protein that presents TGF-1to
the type I and II receptors, all cause hereditary hemorrhagic
telangiectasia, a disease characterized by vascular malforma-
tions (39, 50). Endoglin knockout mice also show a defective
angiogenesis and die at embryonic day 11.5 (44). In vitro,
TGF-inhibits endothelial cell proliferation in two-dimen-
sional cultures (3, 26, 34, 56) but induces tube formation when
endothelial cells are cultured inside three-dimensional colla-
gen gels (45, 53, 73). The differences between these studies
have been attributed to changes in type I and II receptor
expression (66). Finally, TGF-1 promotes the in vitro differ-
entiation of embryonic stem cells into endothelium cells as well
as the formation of cord-like structures (32). However, the
* Corresponding author. Present address for Francese Vin˜als: Fac-
ultad de Odontologia, Universitat de Barcelona, 08907 L’Hospitalet de
Llobregat, Barcelona, Spain. E-mail: fvinals@bell.ub.es. Mailing ad-
dress for Jacques Pouysse´gur: Institute of Signaling, Developmental
Biology and Cancer Research, CNRS UMR 6543-Centre Antoine
Lacassagne, 33 Av. Valombrose, 06189 Nice Cedex 2, France. Phone:
(33) 492 03 1222. Fax: (33) 492 03 1225. E-mail: pouysseg@unice.fr.
7218
basic mechanisms underlying the proangiogenic action of
TGF-are largely unknown. With a mouse vascular endothe-
lial cell model (1G11 cell line) which rapidly forms capillary-
like structures in collagen and responds nicely to TGF-1, we
have investigated the basic signaling action of this angiogenic
cytokine. We show that, surprisingly, TGF-1 can stimulate the
phosphatidylinositol 3-kinase (PI3K)/Akt and the p42/p44 mi-
togen-activated protein kinase (MAPK) pathways; however,
this action is mediated by an autocrine mechanism, implicating
at least the production of TGF-␣. Moreover, TGF-1-medi-
ated stimulation of PI3K and p42/p44 MAPK signaling cas-
cades is essential for cell survival and formation of capillary-
like structures.
MATERIALS AND METHODS
Materials. Human TGF-1 was obtained from R&D Systems, FGF-2 and
platelet-derived growth factor BB (PDGF-BB) were obtained from Pepro Tech,
epidermal growth factor (EGF) and insulin were obtained from Sigma, and
TGF-␣was obtained from Boehringer. LY-294002 was obtained from Alexis;
wortmannin and rapamycin were obtained from BioMol; PD-98059 was obtained
from New England Biolabs; cycloheximide, actinomycin D, brefeldin A, and
tyrphostin AG1478 were also obtained from Sigma. Genistein and SB202190
were obtained from Calbiochem. Cell culture media, fetal calf serum (FCS),
glutamine, and antibiotics were obtained from Gibco-BRL. The most commonly
used chemicals were purchased from Sigma.
Cell culture. Murine lung capillary endothelial cells (1G11 cell line) were
obtained from Alberto Mantovani and Annunciata Vecchi (Instituto Ricerche
Farmacologiche Mario Negri, Milan, Italy) (21). They were cultured in Dulbec-
co’s modified Eagle’s medium (DMEM) containing 20% inactivated FCS; 50 U
of penicillin, 50 g of streptomycin sulfate, 150 g of endothelial cell growth
supplement (Becton Dickinson), and 100 g of heparin (Sigma)/ml; 1% nones-
sential amino acids; and 2 mM sodium pyruvate. Before the incubation with
growth factors, cells were depleted for 24 h in a 1:1 mixture of DMEM and
Ham’s F-12 medium.
Murine heart capillary endothelial cells (H5V cell line) transformed by poly-
oma middle T antigen (27) were obtained from P. Huber (Grenoble, France) and
cultured in DMEM containing 20% FCS.
Human umbilical vein endothelial cells (HUVEC) were obtained as previously
described (5) and were cultivated in SFM (Gibco-BRL) supplemented with 20%
FCS and 100 g of heparin, 20 ng of FGF-2, and 10 ng of EGF (Sigma)/ml.
HEK293 cells were cultured in DMEM containing 7.5% inactivated FCS, 50 U
of penicillin/ml, and 50 g of streptomycin sulfate/ml. Cells were transiently
transfected by the calcium phosphate method. HEK293 cells were seeded at a
density of 700,000 cells per well in six-well plates and transfected with 0.25 gof
the green fluorescent protein (GFP) plasmid only or cotransfected with 0.25 g
of GFP plasmid together with 5 g of expression vector CDM7-IgB-1, which
codes for the extracellular portion of EGF receptor (ErbB-1) fused to an Fc
portion of human immunoglobulin G1 (15). Forty-eight hours after transfection,
the culture medium from transfected HEK293 cells was used to treat 1G11 cells.
Tubulogenesis and cell death counting assay. To induce capillary tube forma-
tion, 1G11 cells grown to confluence were trypsinized and resuspended in 2⫻
DMEM. Cells were added to a type I collagen solution (Becton Dickinson; 4
mg/ml) to achieve a cell concentration of 3 ⫻10
6
cells/ml and a final collagen
concentration of 2 mg/ml. Sixty microliters of this preparation was placed in
24-well plates and incubated for 45 min at 37°C in a humidified incubator to
allow polymerization, and complete 1G11 cell culture medium, DMEM alone, or
DMEM containing 10 ng of TGF-1/ml was added where indicated in the
legends for Fig. 1, 2, 11, and 12.
To extract proteins from the collagen gels, cultures were lysed in radioimmu-
noprecipitation assay buffer (phosphate-buffered saline with 1% NP-40, 0.5%
sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS], 40 mM -glycero-
phosphate, 200 M sodium orthovanadate, 100 M phenylmethylsulfonyl fluo-
ride, 1 M pepstatin A, 1 g of leupeptin/ml and 4 g of aprotinin/ml), homog-
enized with a minipotter homogenizer, and extracted for 15 min at 4°C. Insoluble
material was removed by centrifugation at 12,000 ⫻gfor 10 min at 4°C.
To measure the numbers of live and dead cells after 24 h in collagen gels,
Hoechst stain (50-g/ml bis-benzimide; Sigma) or propidium iodide (1 g/ml;
Sigma) was added and cells were incubated for 30 min at 37°C. The live (nuclei
Hoechst stained) and dead cells (nuclei with propidium iodide stain and con-
densed chromatin) were counted using an immunofluorescence microscope.
Western blot analysis. Cells were washed twice with cold phosphate-buffered
saline and lysed in Triton X-100 lysis buffer (50 mM Tris-HCl [pH 7.5], 100 mM
NaCl, 50 mM NaF, 5 mM EDTA, 40 mM -glycerophosphate, 200 M sodium
orthovanadate, 100 M phenylmethylsulfonyl fluoride, 1 M pepstatin A, 1 g
of leupeptin/ml, 4 g of aprotinin/ml, 1% Triton X-100) for 15 min at 4°C.
Insoluble material was removed by centrifugation at 12,000 ⫻gfor 5 min at 4°C.
For conditioned medium from 1G11 cells, proteins from 10 ml were precipitated
with acetone for1hat⫺20°C and centrifuged at 5,000 ⫻gfor 5 min. The final
pellet was resuspended in 50 l of Laemmli sample buffer (40), followed by
protein separation on an SDS–15% polyacrylamide gel. Western blotting was
performed as described previously (76). The blots were incubated with a poly-
clonal anti-Ets-1 antibody (Santa Cruz Biotechnology), polyclonal antiphospho-
(Ser473)-Akt (New England Biolabs), a polyclonal anti-Akt antibody (New En-
gland Biolabs), a anti-poly(ADP-ribose) polymerase (anti-PARP) monoclonal
antibody (BioMol), a monoclonal antiphospho-Erk1/2 antibody (Sigma), anti-
p42/p44 MAPK antiserum EIB (51), a polyclonal anti-TGF-␣antibody (R&D),
and a polyclonal antiphospho-Smad2 antibody (Upstate Biotechnology) in block-
ing solution overnight at 4°C.
Where indicated (see Fig. 4B), the p70 S6K activity was determined by a
mobility shift assay as described previously (76).
WGL and EGF receptor immunoprecipitation. Quiescent cells stimulated with
or without the different agonists were lysed with Triton X-100 lysis buffer for 15
min at 4°C. Insoluble material was removed by centrifugation at 12,000 ⫻gfor
5 min at 4°C. Protein lysates (500 g) were incubated for1hat4°C with wheat
germ lectin (WGL) preadsorbed to Sepharose beads (Pharmacia) or for3h(1
mg of lysate) with a specific polyclonal anti-EGF receptor (Santa Cruz Biotech-
nology) preadsorbed to protein A-Sepharose beads (Pharmacia). Precipitates
were then washed four times with Triton X-100 buffer. The final pellet was
resuspended in 50 l of Laemmli sample buffer (40), followed by protein sepa-
ration on an SDS–7.5% polyacrylamide gel and Western blotting with an an-
tiphosphotyrosine monoclonal antibody or an anti-EGF receptor antibody (San-
ta Cruz Biotechnology).
Northern blotting. Poly(A)
⫹
RNA was isolated from confluent 1G11 cells
treated for different times with 10 ng of TGF-1/ml or from H5V cells treated for
3 h with TGF-1 using a Micro-FastTack 2.0 kit (Invitrogen). A Northern blot
assay was performed as described previously (77). The mouse cDNA probe for
TGF-␣was a 190-bp PCR-amplified band obtained from 1G11 RNA using
specific oligonucleotide primers for mouse TGF-␣(5⬘-ATGGTCCCCGCGAC
CGGACAGCTC-3⬘; reverse oligonucleotide: 5⬘-ACATGCTGGCTTCTCTTCC
TGCAC-3⬘). The identity of the amplified TGF-␣band was confirmed by se-
quence analysis (Eurogentec).
Reverse transcription-PCR (RT-PCR). Poly(A)
⫹
RNA was isolated from con-
fluent HUVEC treated for 2 h with 10 ng of TGF-1/ml using a Micro-FastTack
2.0 kit (Invitrogen) or not treated. After reverse transcription using an oligo(dT)
primer (Amersham), cDNAs were amplified by PCR (Amersham) using specific
oligonucleotide primers for human TGF-␣(5⬘-TTCTGGAGCTTCTCAAGGG
AT-3⬘; reverse oligonucleotide: 5⬘-CCTGGTAAATCAATGGCTAGA-3⬘) (35
cycles) and mouse actin (5⬘-ATGGATGACGATATCGCTG-3⬘; reverse oligo-
nucleotide: 5⬘-ATGAGGTAGTCTGTCAGGT-3⬘) (33 cycles). The mouse
cDNA probe for TGF-␣was a 330-bp PCR-amplified band, and that for actin
was a 500-bp band.
RESULTS
Inhibition of PI3K or p42/p44 MAPK blocks TGF-1-in-
duced endothelial tube formation and cell survival. Like other
capillary endothelial cell models, as described previously (45,
55, 67), 1G11 lung mouse capillary endothelial cells completely
reorganize when cultured with complete medium in three-
dimensional type I collagen gels. The final result was a network
of tubular structures with multiple cell-cell contacts (Fig. 1),
which caused the retraction of the collagen gel (data not
shown). A lack of this organization could be seen when cells
are cultured in the presence of growth factor-free DMEM (Fig.
1). The same effect obtained in the presence of complete me-
dium was observed when only TGF-1 was added. This TGF-
1-induced tube formation was also observed in primary en-
dothelial cell cultures from mouse lung cultured inside
VOL. 21, 2001 SIGNALING IN VITRO ANGIOGENESIS VIA TGF-7219
FIG. 1. TGF-1 and complete medium stimulate 1G11 capillary endothelial cells to form a tubular network in type I collagen gels. 1G11 cells
were mixed with type I collagen and placed on culture plates to polymerize. After gel formation, DMEM alone (basal), complete medium (20%
FCS and 150 g of endothelial cell growth supplement/ml), or TGF-1 (10 ng/ml) was added for 48 h and gels were examined by phase-contrast
microscopy.
7220 VIN
˜ALS AND POUYSSE
´GUR MOL.CELL.BIOL.
collagen gels (D. Grall and J. C. Chambard, unpublished ob-
servations) and has already been described for other microvas-
cular endothelial cells (45, 53). The tubular-growth remodeling
induced by TGF-1 or complete medium was dependent on
mRNA and protein synthesis, since preincubation in the pres-
ence of their respective inhibitors, actinomycin D and cyclo-
heximide, completely blocked tube formation (data not
shown). In contrast, when added to two-dimensional 1G11 cell
cultures, TGF-1 behaved as a growth inhibitor, as judged by
the severe block in thymidine incorporation stimulated by EGF
or FGF-2 (data not shown). This effect has also been described
for other endothelial cell types (3, 26, 34, 56).
To determine the signal transduction pathways important
for TGF-1-induced tube formation, we preincubated 1G11
cells grown in collagen gels with specific inhibitors before ad-
dition of TGF-1. Rapamycin, a specific inhibitor of p70 S6K
activation that blocks 1G11 vascular endothelial cell prolifer-
ation (76), did not have any effect on tube formation in colla-
gen gels (Fig. 2A). In contrast, preincubation in the presence
of PI3K inhibitors LY-294002 (15 M) or wortmannin (300
nM) blocked tube formation induced by TGF-1 (Fig. 2A and
data not shown). In the presence of PI3K inhibitors cells were
not organized and appeared as isolated and refractile, similar
to control cells cultured in DMEM only (Fig. 1 and 2A, basal).
A similar result was obtained when p42/p44 MAPK activation
was blocked by preincubating cells in the presence of 30 M
PD-98059, an inhibitor of p42/p44 MAPK kinase 1 (MEK1)
(Fig. 2A). In contrast, inhibition of p38 MAPK by SB202190
did not have any effect. This blocking effect of PI3K and p42/
p44 MAPK inhibitors on TGF-1-induced tube formation was
confirmed by the lack of induction of a typical angiogenic
marker, transcription factor Ets-1 (73, 75) (Fig. 2B).
As observed in Fig. 1 and 2A, 1G11 cells cultured in collagen
gels for 24 or 48 h in the absence of exogenous growth factors
presented a rounded aspect, with few cells presenting exten-
sions. To a large extent, cells presented a refractile aspect and
FIG. 2. LY-294002 and PD-98059 prevent TGF-1-induced tube
formation and Ets-1 induction. (A) 1G11 endothelial cells immersed in
collagen gels were cultured for 24 h in the presence of DMEM alone
(basal) or 10 ng of TGF-1/ml either alone or with 10 M SB202190,
15 M LY-294002 (Ly), 10 nM rapamycin (rapa), or 30 M PD-98059
(PD). Cells were examined by phase-contrast microscopy. Magnifica-
tion, ⫻128. (B) 1G11 cells grown in collagen gels for 24 h in the
presence of 10 ng of TGF-1/ml either alone (TGF-1 lane ⫺) or with
15 M LY-294002 (lane ⫹Ly) or 30 M PD-98059 (lane ⫹PD) or in
the absence of TGF-(B lane ⫺) were lysed, and Ets-1 was detected
by immunoblotting with a specific antibody. Identical amounts of pro-
tein were loaded on the gel. The decrease in p42 MAPK content in
lanes ⫹Ly and ⫹PD reflects partial cell death. A representative West-
ern blot of three different experiments is shown.
VOL. 21, 2001 SIGNALING IN VITRO ANGIOGENESIS VIA TGF-7221
condensed chromatin, typical of dying cells. The number of
cells presenting this morphology dropped when growth factors
or TGF-1 was added to the culture medium but reappeared
when cells were pretreated with LY-294002, wortmannin, or
PD-98059 before addition of TGF-1 (Fig. 2A). This result
suggested that one of the effects of TGF-1 on three-dimen-
sional cultures of microvascular endothelial cells was to pro-
vide survival signals. To confirm this, we counted the live and
dead cells (Hoechst stain- or propidium iodide-positive nuclei,
respectively) in the presence and absence of TGF-1. As
shown in Fig. 3A, when cells were incubated for 24 h in
DMEM only, 64% of the cells were dead (live cell/dead cell
ratio of 0.6). Dead cells were round and refractile. In contrast,
the addition of TGF-1 to the cultures doubled the number of
live cells (live cell/dead cell ratio of 1.85). This cell survival
effect of TGF-1 was abolished by preincubation in the pres-
ence of LY-294002 (live cell/dead cell ratio, 0.54) or in the
presence of PD-98059 (live cell/dead cell ratio, 0.7). Preincu-
bation with LY-294002 or PD-98059 alone had no effect on the
basal live cell/dead cell ratio. These results were confirmed by
measuring apoptosis in collagen gels. Apoptosis was measured
by evaluating cleavage of PARP, a well-established substrate of
caspase 3 and a marker of caspase cascade activation during
apoptosis. As observed in Fig. 3B, culture of 1G11 cells in
collagen gels in the presence of growth factor-free DMEM
caused PARP cleavage. Treatment with TGF-1 completely
precluded the apoptotic effect of collagen immersion, and this
effect was reversed by LY-294002 or PD-98059 pretreatment.
TGF-1 stimulates PI3K and p42/p44 MAPK in collagen
gels and in two-dimensional cultures by an autocrine mecha-
nism. The sensitivity of tube formation to PI3K and p42/p44
MAPK inhibitors prompted us to investigate the action of
TGF-1 on these two signaling cascades, in particular during
tubular network formation in collagen gels. When cells were
grown in collagen gels and in the absence of growth factors, we
observed a persistent activation of p42/p44 MAPK (measured
with an antibody against the phosphorylated active form of the
kinases) (Fig. 4A). This stimulation was probably due to inte-
grin activation (8, 37, 69). Interestingly, under the same con-
ditions of culture no activation of PI3K, as indicated by the
phosphorylation of its downstream effector Akt, could be de-
tected at short times; only after 8 h of culture on collagen gels
was a weak increase in phospho-Akt detected (Fig. 4A). In
contrast, the addition of TGF-1 to cells grown in collagen gels
FIG. 4. TGF-1 stimulates PI3K, p70 S6K, and p42/p44 MAPK
activities in endothelial cells grown in collagen gels and in two-dimen-
sional cultures. (A) 1G11 cells were grown in collagen gels for the
periods of time indicated. Cells were lysed, and phospho-Akt, phos-
pho-p42/p44 MAPK (pp42/pp44 MAPK), and p42 MAPK were de-
tected by immunoblotting with specific antibodies. A representative
Western blot is shown. (B) 1G11 cells were grown on collagen- or
gelatin-coated plates until confluence. After depletion of growth fac-
tors, cells were stimulated with 10 ng of TGF-1/ml for the periods of
time indicated or with 20 ng of EGF for 30 min. Cells were lysed, and
Western blotting was performed using anti-phospho-Akt, anti-phos-
pho-p42/p44 MAPK, or p42/p44 MAPK. The same extracts were
loaded on an SDS–9% polyacrylamide gel (shift-up) and blotted with
an anti-p70 S6K antibody. Hyperphosphorylated and active forms of
p70 S6K (arrows) migrated more slowly than hypophosphorylated
forms. The Western blots are representative of three independent
experiments.
FIG. 3. TGF-1 stimulates endothelial cell survival in collagen gels.
(A) 1G11 cells immersed in collagen gels were cultured for 24 h in the
presence of DMEM alone (B), 15 M LY-294002 alone (B⫹Ly), 30
M PD-98059 alone (B⫹PD), 10 ng of TGF-1/ml (TGF1), and
TGF-1 in the presence of 15 M LY-294002 (TGF1⫹Ly) or 30 M
PD-98059 (TGF1⫹PD). After this time, live and dead cells were
counted as described in Materials and Methods. Results are expressed
as the ratios of live cells/dead cells and are averages of eight different
experiments. (B) Proliferative 1G11 cells (0) or cells immersed in
collagen gels for8hintheabsence or presence of TGF-1 (10 ng/ml),
LY-294002 (15 M), or PD-98059 (30 M) were lysed, and PARP was
detected by immunoblotting with a specific antibody. A representative
Western blot is shown. The observed difference in the mobility of
PARP between proliferative cells and cells immersed in collagen gels
is due to the presence of collagen in the SDS-polyacrylamide gel
electrophoresis.
7222 VIN
˜ALS AND POUYSSE
´GUR MOL.CELL.BIOL.
led to a marked activation of Akt (Fig. 4A). Akt activation was
observable at 2 h, peaked at 8 h, and persisted at least until
24 h (data not shown). For p42/p44 MAPK, increased activa-
tion could also be seen after1hofstimulation with TGF-1,
with a maximal difference obtained at 4 h. These results indi-
cate that TGF-1 stimulated PI3K and enhanced p42/p44
MAPK in collagen gels. With the objective to better charac-
terize the observed PI3K and p42/p44 MAPK stimulation by
TGF-1, we evaluated whether the effect of TGF-1 on p42/
p44 MAPK and PI3K activity could also be observed on two-
dimensional 1G11 cell cultures. Therefore, cells were cultured
on plates precoated with type I collagen or gelatin and were
stimulated for different periods of time with TGF-1. The
activities of Akt, p70 S6K, which also depends on the activity of
PI3K, and p42/p44 MAPK were analyzed. As shown in Fig. 4B,
TGF-1 stimulated all these signaling pathways as it did in
cells cultured in a three-dimensional collagen gel, but in a
more transient manner. As was observed in collagen gels, the
effect of TGF-1 was only observed after stimulation periods
of over 1 h. Akt and p70 S6K activation started at 2 h (data not
shown), peaked at 4 h, and started to decrease at 8 h. A weak
and transient stimulation of p42/p44 MAPK, starting at 1 h,
reaching maximum effect at 4 h, and returning to basal levels at
8 h, was also observed.
Given the latency observed for TGF-1-stimulated PI3K
and p42/p44 MAPK activities, we hypothesized that TGF-1
could be having an indirect effect on these pathways. In view of
the signaling action of TGF-1, it was possible that an auto-
crine factor released by 1G11 cells after TGF-1 stimulation
was activating PI3K and p42/p44 MAPK. To validate this hy-
pothesis, we preincubated cells in the presence of different
inhibitors of mRNA synthesis (actinomycin D), protein syn-
thesis (cycloheximide), or vesicular traffic (brefeldin A, a com-
pound that disturbs the trans-Golgi apparatus) before addition
of TGF-1. All these inhibitors completely blocked the effect
of TGF-1 on Akt and p42/p44 MAPK stimulation (Fig. 5 and
data not shown). This result suggested that the effect of
TGF-1 was caused by the synthesis and secretion of an auto-
crine factor. This factor would then activate PI3K and p42/p44
MAPK signaling pathways.
TGF-1 stimulates the synthesis of TGF-␣and EGF recep-
tor phosphorylation. Since this autocrine factor activates the
PI3K and p42/p44 MAPK pathways through cell surface re-
ceptors, we wanted to evaluate the possible activation of the
cell surface tyrosine kinase receptor following TGF-1 stimu-
lation. Therefore, we stimulated cells for different periods of
time with TGF-1 and isolated membrane proteins by using
Sepharose-coupled WGL, which binds glycoproteins. After ex-
tensive washing, purified glycoproteins were separated on a
polyacrylamide gel and phosphotyrosine-containing proteins
were immunodetected with a specific antibody. Incubation
with TGF-1 for 2 or 4 h induced the tyrosine phosphorylation
of a number of proteins on total lysates, some of which were
also detected after stimulation with FGF-2, EGF, and
PDGF-BB (data not shown). Purification of glycoproteins with
lectin elicited the enrichment of cell surface proteins such as
growth factor receptors. The PDGF receptor and EGF recep-
tor, which were nearly undetectable in total lysates, could
clearly be seen after treatment with WGL (Fig. 6A). More
interestingly, extracts from cells stimulated for 2 or 4 h with
TGF-1 increased tyrosine phosphorylation on a 160- to 170-
FIG. 5. Effect of TGF-1 on Akt and p42/p44 MAPK activation
depends on mRNA and protein synthesis and vesicular secretion. Qui-
escent 1G11 cells were preincubated for 15 min in the presence of 5 g
of actinomycin D/ml, 10 g of cycloheximide/ml, or 1 g of brefeldin
A/ml or in the absence of inhibitors (⫺), followed by a 4-h stimulation
with 10 ng of TGF-1/ml, a 30-min stimulation with 10 ng of PDGF-
BB/ml, or no stimulation (basal). Cells were lysed, and phospho-Akt
and Akt were immunodetected as previously (76) described. A West-
ern blot representative of three different experiments is shown.
FIG. 6. Stimulation with TGF-1 causes EGF receptor activation.
(A) Quiescent 1G11 cells were stimulated or not (lane B) for the
indicated times with 10 ng of TGF-1/ml and for 10 min with 25 ng of
FGF-2/ml, 10 ng of EGF/ml, or 10 ng of PDGF-BB/ml. Cells were
lysed, and proteins were incubated with Sepharose-WGL for 1 h. After
being washed, the final pellet was resuspended in Laemmli sample
buffer and loaded on a SDS–7.5% polyacrylamide gel. Phosphoty-
rosine-containing proteins were immunodetected by using a specific
antibody. Arrow, phosphotyrosine-containing protein that appeared
after TGF-1 treatment. A representative Western blot is shown. (B)
Cells were treated as for panel A and lysed, and the EGF receptor
(EGFR) was immunoprecipitated (IP) by incubation with a specific
anti-EGF receptor antibody preadsorbed to protein A-Sepharose
beads. After being washed the pellet was treated as for panel A. WB,
Western blot.
VOL. 21, 2001 SIGNALING IN VITRO ANGIOGENESIS VIA TGF-7223
kDa protein. This molecular mass was identical to that of the
EGF receptor. This result suggested that TGF-1 induces the
phosphorylation of a member of the EGF receptor family. To
confirm this hypothesis, we immunoprecipitated the EGF re-
ceptor with a specific antibody and evaluated its activity status
by phosphotyrosine immunoblotting. As observed in Fig. 6B,
incubation for 2 h with TGF-1 increased the tyrosine phos-
phorylation of the EGF receptor, confirming the implication of
a member of the EGF family of growth factors in the TGF-
1-mediated effect.
The EGF family is composed of at least six members that
can activate ErbB-1 or the EGF receptor (1). Moreover, only
TGF-␣and EGF have been implicated in the angiogenesis
process (58, 68). To identify the factor implicated in the TGF-
1-mediated effect, we performed RT-PCR experiments with
mRNAs obtained from 1G11 cells treated for 2 h with TGF-1
or not treated. A fragment corresponding to TGF-␣could be
amplified in these cells; in contrast there was no transcript
corresponding to EGF (data not shown). We confirmed these
results with Northern blot analysis using poly(A)
⫹
RNA from
1G11 cells and the PCR fragment amplified from 1G11 cells as
a specific TGF-␣probe. As shown in Fig. 7A, we detected a
4.5-kb band corresponding to the TGF-␣mRNA. Treatment
with TGF-1 caused a transient increase in the TGF-␣tran-
script, which peaked at 2 h and returned to nearly basal levels
after 4 h. This result indicates that TGF-1 stimulates the
synthesis of TGF-␣in 1G11 capillary endothelial cells. Next,
we investigated whether this effect of TGF-1 on TGF-␣syn-
thesis is also observed in other vascular endothelial cells. We
performed a Northern blot assay using poly(A)
⫹
RNA from
H5V cells (a mouse heart endothelial cell line transformed by
polyoma middle T antigen [27]) treated with TGF-1ornot
treated. As observed in Fig. 7A, treatment with TGF-1 also
caused an increase in TGF-␣mRNA in this endothelial cell
model. Finally we performed RT-PCR experiments with mR-
NAs obtained from primary cultures of HUVEC treated for
2 h with TGF-1 or not treated. As shown in Fig. 7B, stimu-
lation with TGF-1 for 2 h also increased TGF-␣cDNA in
HUVEC.
Next, we performed Western blot experiments to detect the
TGF-␣protein. As is observed in Fig. 8, the antibody used
clearly detected commercial human TGF-␣as a 6-kDa band in
Western blot assays. However, we have never detected the
mature and secreted form of TGF-␣on conditioned medium
from 1G11 cells treated for 2 (Fig. 8) or 4 h (data not shown)
with TGF-1 or not treated. In contrast, in lysates from 1G11
cells we detected a 19-kDa band that probably corresponded to
an unprocessed form of the TGF-␣protein. This form in-
creased after treatment with TGF-1 for 2 h (Fig. 8). This
result indicates that TGF-1 stimulates the synthesis of the
TGF-␣protein in 1G11 capillary endothelial cells; this form is
then translocated to the membrane where it probably acts in an
autocrine or paracrine (cell-to-cell) manner, stimulating the
ErbB-1 receptor.
EGF receptor mediates TGF-1 stimulation of PI3K and
p42/p44 MAPK and cell survival. We next wanted to deter-
mine whether EGF receptor stimulation was responsible for
PI3K and p42/p44 MAPK stimulation after treatment with
TGF-1. First, we incubated 1G11 cells in the presence of
recombinant TGF-␣to examine if this growth factor stimu-
lated the same signaling pathways as TGF-1. As shown in Fig.
9A, incubation for1hinthepresence of 10 ng of TGF-␣/ml
stimulated both Akt and p42/p44 MAPK phosphorylation. Sec-
ond, prior to treatment with TGF-1, we preincubated cells
with a selective inhibitor of EGF receptor tyrphostin AG1478
(43). This compound specifically inhibited TGF-␣and EGF
signaling in 1G11 cells, while having no effect on PDGF-BB-,
insulin-, or FCS-induced PI3K or p42/p44 MAPK activities
FIG. 7. TGF-1 induces TGF-␣mRNA expression in endothelial
cells. (A) 1G11 and H5V cells stimulated with 10 ng of TGF-1/ml for
the indicated times or not stimulated were lysed, and poly(A)
⫹
mRNA
was isolated. Gels were loaded with 2 g of mRNA, and Northern
blotting was performed using a 190-bp PCR-amplified fragment as a
TGF-␣-specific probe. Rat GAPDH (glyceraldehyde-3-phosphate de-
hydrogenase) was used as a control. A representative result is shown.
(B) HUVEC stimulated with 10 ng of TGF-1/ml for2hornot
stimulated were lysed, and poly(A)
⫹
mRNA was isolated. After cDNA
was obtained, PCR was performed using specific primers for human
TGF-␣or actin (as a control). Samples were loaded on a 2% agarose
gel. 1 and 2, two independent preparations of HUVEC poly(A)
⫹
for
unstimulated and stimulated cells.
FIG. 8. TGF-1 induces TGF-␣protein expression in 1G11 cells.
Quiescent 1G11 cells were stimulated for 2 h with 10 ng of TGF-1/ml
or were not stimulated (lane B). After this time, medium was collected
and proteins were precipitated and loaded on a 15% gel. As a control,
300 ng of human TGF-␣was also loaded. In parallel, cells were lysed
and 100 g was loaded on the gel. TGF-␣was immunodetected by
using a specific antibody. A Western blot representative of three dif-
ferent experiments is shown.
7224 VIN
˜ALS AND POUYSSE
´GUR MOL.CELL.BIOL.
(Fig. 9A) or on the normal TGF-1 signaling, indicated by
Smad2 phosphorylation (Fig. 9B). Even more importantly, pre-
incubation with tyrphostin AG1478 precluded the activation of
p42/p44 MAPK by TGF-1 and decreased Akt phosphoryla-
tion by 50% (Fig. 9). Moreover, the effect of AG1478 was also
observed in three-dimensional collagen gels, where it blocked
TGF-1-stimulated p42/p44 MAPK and Akt activities (data
not shown). Third, to sequester the possible TGF-␣or other
EGF-like members secreted after TGF-1 stimulation, we
used a soluble and extracellularly secreted form of the extra-
cellular portion of the EGF receptor fused in frame to an Fc
portion of human immunoglobulin G1, denoted CDM7-IgB-1
(15). This construction was expressed in HEK293 cells, and the
conditioned medium of these cells was added to 1G11 cells
before TGF-1 treatment. Thus, when 1G11 cells were pre-
treated for 1 h in the presence of the medium from control
transfected HEK293 cells, an incubation for 4 h with TGF-1
stimulated Akt and p42/p44 MAPK (Fig. 10). 1G11 cells pre-
treated with CDM7-IgB-1 supernatants demonstrated a higher
basal activation of Akt and p42/p44 MAPK. However TGF-1
was no longer capable of stimulating these two signaling path-
ways (Fig. 10). These results reinforce the notion that TGF-
1-stimulated Akt and p42/p44 MAPK signaling in 1G11 cells
is mediated mainly through the EGF receptor.
We then wanted to evaluate whether inhibition of the EGF
receptor could affect tubular morphogenesis and cell survival
in collagen gels. As shown in Fig. 11, top, preincubation of cells
in the presence of tyrphostin AG1478 prior to the addition of
TGF-1 inhibited capillary-like formation in collagen gels.
Moreover, preincubation with tyrphostin AG1478 inhibited the
protective effect of TGF-1 on cell survival (Fig. 11, bottom).
Finally, it was possible that the in vitro angiogenic effect of
TGF-1 was wholly mediated by TGF-␣. To evaluate this pos-
sibility, 1G11 cells were incubated in the presence or absence
of TGF-␣alone. In this condition TGF-␣increased cell sur-
vival (Fig. 11, bottom). After a 24-h treatment TGF-␣did not
stimulate tubular network formation; however, long-term
treatment (5 days) elicited tube formation although at a lesser
level than that elicited by TGF-1 (Fig. 12). Moreover, when
TGF-1 was added in the presence of TGF-␣, we noticed a
very marked synergistic effect on cell reorganization at con-
centrations of TGF-1 that alone produced a limited effect
(compare 2 and 5 ng of TGF-1/ml in the presence or absence
of TGF-␣). These results indicate that TGF-␣is necessary but
not sufficient for TGF-1-induced cell reorganization in colla-
gen gels. Taken together, all these results suggest that TGF-␣
secretion induced by TGF-1 is essential for maintaining cell
survival in three-dimensional cell cultures and plays a role in
the TGF-1-induced capillary-like formation in endothelial
cells.
DISCUSSION
In this study we demonstrate that, besides its effects on cell
reorganization, TGF-1 promotes endothelial cell survival
during in vitro angiogenesis in collagen gels. This protective
effect is mediated by the activation of the PI3K and p42/p44
MAPK signaling pathways and is obtained through an auto-
FIG. 9. Tyrphostin AG1478, a specific inhibitor of the EGF recep-
tor, blocks TGF-1 stimulation of p42/p44 MAPK and Akt in the
absence of changes in TGF-signaling. (A) Quiescent 1G11 cells were
preincubated for 15 min in the presence (⫹) or the absence (⫺)of
tyrphostin AG1478 (1 M). After this time, cells were stimulated for
4 h with 10 ng of TGF-1/ml (in duplicate in the presence of tyrphostin
AG1478) or for 1 h with 50 ng of TGF-␣/ml, 10 ng of PDGF-BB/ml, 1
M insulin, or 10% FCS or were left unstimulated (lane B). Cells were
lysed, and phospho-Akt, Akt, and phospho-p42/p44 MAPK were im-
munodetected as described in Materials and Methods. A Western blot
representative of four different experiments is shown. (B) Quiescent
1G11 cells were preincubated for 15 min in the presence (⫹)orthe
absence (⫺) of tyrphostin AG1478 (1 M). After this time, cells were
stimulated for the times indicated with 10 ng of TGF-1/ml or for 15
min with 50 ng of TGF-␣/ml. After lysis, phospho-Smad2, phospho-
Akt, Akt, and phospho-p42/p44 MAPK were immunodetected as de-
scribed in Materials and Methods. A representative Western blot is
shown.
FIG. 10. A soluble EGF receptor (IgB-1) blocks TGF-1 stimula-
tion of Akt and p42/p44 MAPK. Quiescent 1G11 cells were preincu-
bated for 1 h with medium from control or soluble extracellular EGF
receptor IgB-1-transfected HEK293 cells. After this period, cells were
stimulated with TGF-1 for 4 h. Cells were then lysed, and phospho-
Akt, phospho p42/p44 MAPK, and p42/p44 MAPK were immunode-
tected as described in Materials and Methods. A representative West-
ern blot from three different experiments is shown.
VOL. 21, 2001 SIGNALING IN VITRO ANGIOGENESIS VIA TGF-7225
FIG. 11. Inhibition of TGF-␣signaling blocks tube formation and
cell survival induced by TGF-1 in collagen gels. (Top) 1G11 endo-
thelial cells grown in collagen gels were cultured for 24 h in the
presence of DMEM alone (basal) or 10 ng of TGF-1/ml either alone
or supplemented with 1 M tyrphostin AG1478. After this time, cells
were examined by phase-contrast microscopy. Magnifications: left,
⫻90; right, ⫻180. (Bottom) 1G11 endothelial cells grown in collagen
gels were cultured for 24 h in the presence of DMEM either alone
(lane B) or with 1 M tyrphostin AG1478 (B⫹AG), in 10 ng of
TGF-1/ml either alone (TGF) or with 1 M tyrphostin AG1478
(TGF⫹AG), in 50 ng of TGF-␣/ml either alone (TGF␣) or with
tyrphostin AG1478 (TGF␣⫹AG), or in 100 ng of FGF-2/ml either
alone (FGF) or with 1 M tyrphostin AG1478 (FGF⫹AG). Dead and
living cells were counted as described in Materials and Methods. Re-
sults are averages of three different experiments.
7226 VIN
˜ALS AND POUYSSE
´GUR MOL.CELL.BIOL.
FIG. 12. TGF-␣stimulates tube formation in collagen gels in the long term and potentiates TGF-1 action in the short term. 1G11 cells grown
in collagen gels were cultured for 24 h in the presence of DMEM alone or different concentrations of TGF-1 (2 and 5 ng/ml) in the presence
or the absence of TGF-␣(50 ng/ml). In parallel, 1G11 cells were grown in collagen gels for 5 days in the presence or the absence of TGF-1 (10
ng/ml) or TGF-␣(50 ng/ml). Cells were examined by phase-contrast microscopy.
VOL. 21, 2001 SIGNALING IN VITRO ANGIOGENESIS VIA TGF-7227
crine mechanism, which implicates the synthesis and secretion
of TGF-␣and the activation of the EGF receptor.
We show that TGF-1 has a clear effect on 1G11 endothelial
cell reorganization into capillary-like structures when cells are
cultured in collagen gels. However, this effect appears to de-
pend on an indirect antiapoptotic action of TGF-1. If this
prosurvival effect is inhibited by PI3K or p42/p44 MAPK in-
hibitors, cells do not organize but rather die. The proangio-
genic effect of TGF-1reflects its role in the later stages of
angiogenesis, where endothelial cells pass from a migratory
and proliferative state to a more quiescent, mature, and dif-
ferentiated state (6, 64). This is characterized by downregula-
tion of endothelial cell proliferation, increased basement mem-
brane deposition, and final morphological organization of cells
into capillary tubes. These processes have been shown to be
induced by TGF-(45, 53, 61, 80), a key role confirmed by
TGF-1 and TGF-receptor type II knockout mice. Indeed in
both cases these animals die early in embryogenesis due to
defects in vasculogenesis. Endothelial cell proliferation in wild-
type animals was similar to that in knockout animals; however,
a problem in the terminal differentiation and maintenance of
the tube integrity was observed (20, 59). Differentiating cells
entering growth-arrested G
0
need to maintain a good balance
of cell survival signals. Thus, it is not surprising that TGF-1,
which alone induces in vitro angiogenesis, can promote endo-
thelial cell survival. Two pathways, PI3K/Akt and p42/p44
MAPK, capable of inducing survival signals have been well
documented (10, 25, 41, 47). In endothelial cells these signaling
cascades provide a strong antiapoptotic action. VEGF has a
clear cell survival effect through the activation of PI3K/Akt and
stimulation of Bcl2 (28–30, 52). Moreover, endothelial cell-
specific vascular endothelial-cadherin forms a macrocomplex
with the type II VEGF receptor and PI3K, and this interaction
is essential for VEGF antiapoptotic signaling (13). Phorbol
myristate acetate induces tube formation and survival of
HUVEC on collagen gels through activation of p42/p44
MAPK and PI3K (38). In this context, it is interesting that
TGF-1 stimulates PI3K and p42/p44 MAPK (this study) and
induces cell survival. A similar effect of TGF-1 was observed
in macrophages, which TGF-1 protects from apoptosis in-
duced by serum deprivation (16).
In the majority of cells, stimulation by TGF-1 does not
activate PI3K or p42/p44 MAPK. In 1G11 endothelial cells we
do not detect any rapid activation of p42/p44 MAPK. The same
results can be seen in HUVEC (data not shown). In contrast,
we observe a clear stimulation of PI3K and p42/p44 MAPK by
TGF-1 in 1G11 cells, but only 2 h after TGF-1 addition. We
show that this activation is mediated by EGF receptor stimu-
lation, probably through an autocrine loop involving TGF-␣
synthesis and secretion. This type of autocrine system has also
been implicated in TGF-1 induction of connective tissue cell
proliferation, mediated by an autocrine PDGF loop (7, 71).
This mechanism of PDGF production also exists in mouse
embryo AKR-2B cells (42) but is not present in endothelial
cells (6). Moreover, TGF-1 has been shown to induce FGF-2
production in bovine corneal endothelial cells (63). Interest-
ingly, TGF-1 induces VEGF in different cellular types but not
in HUVEC (62). Moreover, it causes the downregulation of
VEGF receptor flk-1 (46). All these results reflect the role of
TGF-1 in the later stages of angiogenesis, where endothelial
cells pass from a proliferative state (stimulated by VEGF) to a
more quiescent and differentiated state (less sensitive to
VEGF). In our study, TGF-␣seems to be the major factor
responsible for TGF-1 stimulation of p42/p44 MAPK and
PI3K, leading to endothelial cell survival. However, we cannot
discard the synthesis of other autocrine factors that could also
contribute to part of the response.
We have shown that TGF-1 stimulates TGF-␣mRNA and
protein synthesis. We have only detected synthesis of a high-
molecular-mass form of the TGF-␣protein in 1G11 cell ly-
sates. TGF-␣is derived from a larger 20- to 22-kDa transmem-
brane precursor that, after shedding, generates the soluble and
mature 6-kDa form (18). Higher-molecular-mass forms of bi-
ologically active TGF-␣are not unusual and have been previ-
ously reported (4). Moreover, transmembrane TGF-␣can ac-
tivate the EGF receptor but only at neighboring cells (11, 79).
We have never detected TGF-␣processing and release to the
medium in our experimental conditions. Moreover, Akt and
p42/p44 MAPK stimulation by TGF-1 increases with cellular
confluence (data not shown). All these results indicate that
probably TGF-␣arrives at the cellular membrane as a high-
molecular-weight precursor and there exerts its effects in an
autocrine or paracrine (from a cell to its neighboring cell)
manner. Further experiments are required to validate or dis-
prove this hypothesis. TGF-␣is an important angiogenic fac-
tor, more effective than EGF, and is highly expressed in neo-
vascularized tumors (68). Thus, it is possible that the effect of
TGF-␣production by TGF-1 is not restricted to the prosur-
vival effect and also may contribute to tube formation. In fact,
different studies support a role for p42/p44 MAPK and PI3K in
the angiogenic process. Sustained integrin-induced p42/p44
MAPK activity is required for FGF-2-induced angiogenesis in
the chick chorioallantoic membrane (22). Virally activated Ras
cooperates with integrins to induce tubulogenesis in sinusoidal
endothelial cells, an effect that is blocked by PD-98059 (48).
Moreover, B-Raf and MEK-1 knockout mice die during em-
bryonic development due to defects in vascular endothelial cell
differentiation and survival (B-Raf knockout) (78) or in pla-
cental angiogenesis (MEK-1 knockout) (31). The block of
PI3K by wortmannin results in partial inhibition of tumor-
induced angiogenesis (2). However, TGF-␣-stimulated signals,
such as p42/p44 MAPK and PI3K, seem to be necessary but not
sufficient for tube formation in collagen gels. TGF-␣alone is
only partially able to reorganize endothelial cells and form
tubes, and only in the long term. This demonstrates that
TGF-1 unchains other signals that are important for the re-
organization of cells on collagen gels. This could be the secre-
tion of compounds of the extracellular matrix (such as fi-
bronectin, collagens, etc.), the stimulation of integrins (8, 17),
or the release of metalloproteinases (54, 70). Future studies
will help us to identify these signals more precisely and under-
stand the mechanisms of TGF-1-induced angiogenesis.
ACKNOWLEDGMENTS
We thank A. Vecchi for 1G11 cells, J. Madri (Yale University) for
protocols for protein analysis in collagen gels; Y. Yarden (Weizmann
Institute, Israel) for CDM7-IgB-1 construction; S. Pagnotta, J. P. Lau-
gier, and G. Nicaise (Centre de Microscopie Applique´e, Universite´de
Nice-Sophia Antipolis) for microscopic studies; and P. Huber for H5V
cells. We particularly thank D. E. Richard and E. Berra for editorial
7228 VIN
˜ALS AND POUYSSE
´GUR MOL.CELL.BIOL.
help and L. Sevilla, R. Busca`, and F. Ventura and all laboratory
members for discussions and technical support.
This work was supported by research grants from CNRS, University
of Nice-Sophia Antipolis, INSERM, Association pour la Recherche
contre le Cancer, Ligue Nationale contre le Cancer, and the European
Community (EC contract B104-CT97-2071). F.V. is the recipient of a
Marie Curie Research Training Grant (EC contract FMBI-CT97-
2706).
REFERENCES
1. Alroy, I., and Y. Yarden. 1997. The ErbB signaling network in embryogenesis
and oncogenesis: signal diversification through combinatorial ligand-recep-
tor interactions. FEBS Lett. 410:83–86.
2. Arbiser, J. L., M. A. Moses, C. A. Fernandez, N. Ghiso, Y. Cao, N. Klauber,
D. Frank, M. Brownlee, E. Flynn, S. Parangi, H. R. Byers, and J. Folkman.
1997. Oncogenic H-ras stimulates tumor angiogenesis by two distinct path-
ways. Proc. Natl. Acad. Sci. USA 94:861–866.
3. Baird, A., and T. Durkin. 1986. Inhibition of endothelial cell proliferation by
type beta-transforming growth factor: interactions with acidic and basic fi-
broblast growth factors. Biochem. Biophys. Res. Commun. 138:476–482.
4. Banerjee, S., P. P. Banerjee, B. R. Zirkin, and T. R. Brown. 1998. Regional
expression of transforming growth factor-alpha in rat ventral prostate during
postnatal development, after androgen ablation, and after androgen replace-
ment. Endocrinology 139:3005–3013.
5. Barbieri, B., G. Balconi, E. Dejana, and M. B. Donati. 1981. Evidence that
vascular endothelial cells can induce the retraction of fibrin clots. Proc. Soc.
Exp. Biol. Med. 168:204–207.
6. Battegay, E. J. 1995. Angiogenesis: mechanistic insights, neovascular dis-
eases, and therapeutic prospects. J. Mol. Med. 73:333–346.
7. Battegay, E. J., E. W. Raines, R. A. Seifert, D. F. Bowen-Pope, and R. Ross.
1990. TGF-beta induces bimodal proliferation of connective tissue cells via
complex control of an autocrine PDGF loop. Cell 63:515–524.
8. Bazzoni, G., E. Dejana, and M. G. Lampugnani. 1999. Endothelial adhesion
molecules in the development of the vascular tree: the garden of forking
paths. Curr. Opin. Cell Biol. 11:573–581.
9. Beck, L., Jr., and P. A. D’Amore. 1997. Vascular development: cellular and
molecular regulation. FASEB J. 11:365–373.
10. Bonni, A., A. Brunet, A. E. West, S. R. Datta, M. A. Takasu, and M. E.
Greenberg. 1999. Cell survival promoted by the ras-MAPK signaling pathway
by transcription-dependent and -independent mechanisms. Science
286:1358–1362.
11. Brachmann, R., P. B. Lindquist, M. Nagashima, W. Kohr, T. Lipari, M.
Napier, and R. Derynck. 1989. Transmembrane TGF-alpha precursors acti-
vate EGF/TGF-alpha receptors. Cell 56:691–700.
12. Bussolino, F., A. Mantovani, and G. Persico. 1997. Molecular mechanisms of
blood vessel formation. Trends Biochem. Sci. 22:251–256.
13. Carmeliet, P., M. G. Lampugnani, L. Moons, F. Breviario, V. Compernolle,
F. Bono, G. Balconi, R. Spagnuolo, B. Oostuyse, M. Dewerchin, A. Zanetti,
A. Angellilo, V. Mattot, D. Nuyens, E. Lutgens, F. Clotman, M. C. de Ruiter,
A. Gittenberger-de Groot, R. Poelmann, F. Lupu, J. M. Herbert, D. Collen,
and E. Dejana. 1999. Targeted deficiency or cytosolic truncation of the
VE-cadherin gene in mice impairs VEGF-mediated endothelial survival and
angiogenesis. Cell 98:147–157.
14. Chang, H., D. Huylebroeck, K. Verschueren, Q. Guo, M. M. Matzuk, and A.
Zwijsen. 1999. Smad5 knockout mice die at mid-gestation due to multiple
embryonic and extraembryonic defects. Development 126:1631–1642.
15. Chen, X., G. Levkowitz, E. Tzahar, D. Karunagaran, S. Lavi, N. Ben-Baruch,
O. Leitner, B. J. Ratzkin, S. S. Bacus, and Y. Yarden. 1996. An immuno-
logical approach reveals biological differences between the two NDF/heregu-
lin receptors, ErbB-3 and ErbB-4. J. Biol. Chem. 271:7620–7629.
16. Chin, B. Y., I. Petrache, A. M. Choi, and M. E. Choi. 1999. Transforming
growth factor beta1 rescues serum deprivation-induced apoptosis via the
mitogen-activated protein kinase (MAPK) pathway in macrophages. J. Biol.
Chem. 274:11362–11368.
17. Collo, G., and M. S. Pepper. 1999. Endothelial cell integrin alpha5beta1
expression is modulated by cytokines and during migration in vitro. J. Cell
Sci. 112:569–578.
18. Derynck, R. 1992. The physiology of transforming growth factor-alpha. Adv.
Cancer Res. 58:27–52.
19. Derynck, R., Y. Zhang, and X. H. Feng. 1998. Smads: transcriptional activa-
tors of TGF-beta responses. Cell 95:737–740.
20. Dickson, M. C., J. S. Martin, F. M. Cousins, A. B. Kulkarni, S. Karlsson, and
R. J. Akhurst. 1995. Defective haematopoiesis and vasculogenesis in trans-
forming growth factor-beta 1 knock out mice. Development 121:1845–1854.
21. Dong, Q. G., S. Bernasconi, S. Lostaglio, C. R. De, P. I. Martin, F. Breviario,
C. Garlanda, S. Ramponi, A. Mantovani, and A. Vecchi. 1997. A general
strategy for isolation of endothelial cells from murine tissues. Characteriza-
tion of two endothelial cell lines from the murine lung and subcutaneous
sponge implants. Arterioscler. Thromb. Vasc. Biol. 17:1599–1604.
22. Eliceiri, B. P., R. Klemke, S. Stromblad, and D. A. Cheresh. 1998. Integrin
alphavbeta3 requirement for sustained mitogen-activated protein kinase ac-
tivity during angiogenesis. J. Cell Biol. 140:1255–1263.
23. Folkman, J., and M. Klagsburn. 1987. Angiogenic factors. Science 235:442–
447.
24. Folkman, J., and Y. Shing. 1992. Angiogenesis. J. Biol. Chem. 267:10931–
10934.
25. Franke, T. F., D. R. Kaplan, and L. C. Cantley. 1997. PI3K: downstream
AKTion blocks apoptosis. Cell 88:435–437.
26. Frater-Schroder, M., G. Muller, W. Birchmeier, and P. Bohlen. 1986. Trans-
forming growth factor-beta inhibits endothelial cell proliferation. Biochem.
Biophys. Res. Commun. 137:295–302.
27. Garlanda, C., C. Parravicini, M. Sironi, M. De Rossi, R. Wainstok de
Calmanovici, F. Corozzi, F. Bussolino, F. Colotta, A. Mantovani, and A.
Vecchi. 1994. Progressive growth in immunodeficient mice and host cell
recruitment by mouse epithelial cells transformed by polyoma middle-sized
T antigen: implications for the pathogenesis of opportunistic vascular tu-
mors. Proc. Natl. Acad. Sci. USA 91:7291–7295.
28. Gerber, H. P., V. Dixit, and N. Ferrara. 1998. Vascular endothelial growth
factor induces expression of the antiapoptotic proteins Bcl-2 and A1 in
vascular endothelial cells. J. Biol. Chem. 273:13313–13316.
29. Gerber, H. P., K. J. Hillan, A. M. Ryan, J. Kowalski, G. A. Keller, L. Rangell,
B. D. Wright, F. Radtke, M. Aguet, and N. Ferrara. 1999. VEGF is required
for growth and survival in neonatal mice. Development 126:1149–1159.
30. Gerber, H. P., A. McMurtrey, J. Kowalski, M. Yan, B. A. Keyt, V. Dixit, and
N. Ferrara. 1998. Vascular endothelial growth factor regulates endothelial
cell survival through the phosphatidylinositol 3⬘-kinase/Akt signal transduc-
tion pathway. Requirement for Flk-1/KDR activation. J. Biol. Chem. 273:
30336–30343.
31. Giroux, S., M. Tremblay, D. Bernard, J. F. Cardin-Girard, S. Aubry, L.
Larouche, S. Rousseau, J. Huot, J. Landry, L. Jeannotte, and J. Charron.
1999. Embryonic death of Mek1-deficient mice reveals a role for this kinase
in angiogenesis in the labyrinthine region of the placenta. Curr. Biol. 9:369–
372.
32. Gualandris, A., J. P. Annes, M. Arese, I. Noguera, V. Jurukowski, and D. B.
Rifkin. 2000. The latent transforming growth factor--binding protein-1 pro-
motes in vitro differentiation of embryonic stem cells into endothelium. Mol.
Biol. Cell 11:4295–4308.
33. Hanahan, D., and J. Folkman. 1996. Patterns and emerging mechanisms of
the angiogenic switch during tumorigenesis. Cell 86:353–364.
34. Heimark, R. L., D. R. Twardzik, and S. M. Schwartz. 1986. Inhibition of
endothelial regeneration by type-beta transforming growth factor from plate-
lets. Science 233:1078–1080.
35. Heine, U., E. F. Munoz, K. C. Flanders, L. R. Ellingsworth, H. Y. Lam, N. L.
Thompson, A. B. Roberts, and M. B. Sporn. 1987. Role of transforming
growth factor-beta in the development of the mouse embryo. J. Cell Biol.
105:2861–2876.
36. Heldin, C. H., K. Miyazono, and P. ten Dijke. 1997. TGF-beta signalling
from cell membrane to nucleus through SMAD proteins. Nature 390:465–
471.
37. Howe, A., A. E. Aplin, S. K. Alahari, and R. L. Juliano. 1998. Integrin
signaling and cell growth control. Curr. Opin. Cell Biol. 10:220–231.
38. Ilan, N., S. Mahooti, and J. A. Madri. 1998. Distinct signal transduction
pathways are utilized during the tube formation and survival phases of in
vitro angiogenesis. J. Cell Sci. 111:3621–3631.
39. Johnson, D. W., J. N. Berg, M. A. Baldwin, C. J. Gallione, I. Marondel, S. J.
Yoon, T. T. Stenzel, M. Speer, M. A. Pericak-Vance, A. Diamond, A. E.
Guttmacher, C. E. Jackson, L. Attisano, R. Kucherlapati, M. E. M. Porteous,
and D. A. Marchuk. 1996. Mutations in the activin receptor-like kinase 1
gene in hereditary haemorrhagic telangiectasia type 2. Nat. Genet. 13:189–
195.
40. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of
the head of bacteriophage T4. Nature 227:680–685.
41. Le Gall, M., J. C. Chambard, J. P. Breittmayer, D. Grall, J. Pouyssegur, and
E. Van Obberghen-Schilling. 2000. The p42/p44 MAP kinase pathway pre-
vents apoptosis induced by anchorage and serum. Mol. Biol. Cell 11:1103–
1112.
42. Leof, E. B., J. A. Proper, A. S. Goustin, G. D. Shipley, P. E. DiCorleto, and
H. L. Moses. 1986. Induction of c-sis mRNA and activity similar to platelet-
derived growth factor by transforming growth factor beta: a proposed model
for indirect mitogenesis involving autocrine activity. Proc. Natl. Acad. Sci.
USA 83:2453–2457.
43. Levitzki, A., and A. Gazit. 1995. Tyrosine kinase inhibition: an approach to
drug development. Science 267:1782–1788.
44. Li, D. Y., L. K. Sorensen, B. S. Brooke, L. D. Urness, E. C. Davis, D. G.
Taylor, B. B. Boak, and D. P. Wendel. 1999. Defective angiogenesis in mice
lacking endoglin. Science 284:1534–1537.
45. Madri, J. A., B. M. Pratt, and A. M. Tucker. 1988. Phenotypic modulation of
endothelial cells by transforming growth factor-beta depends upon the com-
position and organization of the extracellular matrix. J. Cell Biol. 106:1375–
1384.
46. Mandriota, S. J., P. A. Menoud, and M. S. Pepper. 1996. Transforming
growth factor beta 1 down-regulates vascular endothelial growth factor re-
VOL. 21, 2001 SIGNALING IN VITRO ANGIOGENESIS VIA TGF-7229
ceptor 2/flk-1 expression in vascular endothelial cells. J. Biol. Chem. 271:
11500–11505.
47. Marte, B. M., and J. Downward. 1997. PKB/Akt: connecting phosphoinosi-
tide 3-kinase to cell survival and beyond. Trends Biochem. Sci. 22:355–358.
48. Maru, Y., S. Yamaguchi, T. Takahashi, H. Ueno, and M. Shibuya. 1998.
Virally activated Ras cooperates with integrin to induce tubulogenesis in
sinusoidal endothelial cell lines. J. Cell Physiol. 176:223–234.
49. Massague, J. 1998. TGF-beta signal transduction. Annu. Rev. Biochem.
67:753–791.
50. McAllister, K. A., K. M. Grogg, D. W. Johnson, C. J. Gallione, M. A.
Baldwin, C. E. Jackson, E. A. Helmbold, D. S. Markel, W. C. McKinnon, J.
Murrell, et al. 1994. Endoglin, a TGF-beta binding protein of endothelial
cells, is the gene for hereditary haemorrhagic telangiectasia type 1. Nat.
Genet. 8:345–351.
51. McKenzie, F. R., and J. Pouyssegur. 1996. cAMP-mediated growth inhibi-
tion in fibroblasts is not mediated via mitogen-activated protein (MAP)
kinase (ERK) inhibition. cAMP-dependent protein kinase induces a tempo-
ral shift in growth factor-stimulated MAP kinases. J. Biol. Chem. 271:13476–
13483.
52. Meeson, A. P., M. Argilla, K. Ko, L. Witte, and R. A. Lang. 1999. VEGF
deprivation-induced apoptosis is a component of programmed capillary re-
gression. Development 126:1407–1415.
53. Merwin, J. R., J. M. Anderson, O. Kocher, C. M. Van Itallie, and J. A. Madri.
1990. Transforming growth factor beta 1 modulates extracellular matrix
organization and cell-cell junctional complex formation during in vitro an-
giogenesis. J. Cell Physiol. 142:117–128.
54. Miralles, F., T. Battelino, P. Czernichow, and R. Scharfmann. 1998. TGF-
beta plays a key role in morphogenesis of the pancreatic islets of Langerhans
by controlling the activity of the matrix metalloproteinase MMP-2. J. Cell
Biol. 143:827–836.
55. Montesano, R., L. Orci, and P. Vassalli. 1983. In vitro rapid organization of
endothelial cells into capillary-like networks is promoted by collagen matri-
ces. J. Cell Biol. 97:1648–1652.
56. Muller, G., J. Behrens, U. Nussbaumer, P. Bohlen, and W. Birchmeier. 1987.
Inhibitory action of transforming growth factor beta on endothelial cells.
Proc. Natl. Acad. Sci. USA 84:5600–5604.
57. Oh, S. P., T. Seki, K. A. Goss, T. Imamura, Y. Yi, P. K. Donahoe, L. Li, K.
Miyazono, P. ten Dijke, S. Kim, and E. Li. 2000. Activin receptor-like kinase
1 modulates transforming growth factor-1 signaling in the regulation of
angiogenesis. Proc. Natl. Acad. Sci. USA 97:2626–2631.
58. Okamura, K., A. Morimoto, R. Hamanaka, M. Ono, K. Kohno, Y. Uchida,
and M. Kuwano. 1992. A model system for tumor angiogenesis: involvement
of transforming growth factor-alpha in tube formation of human microvas-
cular endothelial cells induced by esophageal cancer cells. Biochem. Biophys.
Res. Commun. 186:1471–1479.
59. Oshima, M., H. Oshima, and M. M. Taketo. 1996. TGF-beta receptor type
II deficiency results in defects of yolk sac hematopoiesis and vasculogenesis.
Dev. Biol. 179:297–302.
60. Pelton, R. W., S. Nomura, H. L. Moses, and B. L. Hogan. 1989. Expression
of transforming growth factor beta 2 RNA during murine embryogenesis.
Development 106:759–767.
61. Pepper, M. S., D. Belin, R. Montesano, L. Orci, and J. D. Vassalli. 1990.
Transforming growth factor-beta 1 modulates basic fibroblast growth factor-
induced proteolytic and angiogenic properties of endothelial cells in vitro.
J. Cell Biol. 111:743–755.
62. Pertovaara, L., A. Kaipainen, T. Mustonen, A. Orpana, N. Ferrara, O.
Saksela, and K. Alitalo. 1994. Vascular endothelial growth factor is induced
in response to transforming growth factor-beta in fibroblastic and epithelial
cells. J. Biol. Chem. 269:6271–6274.
63. Plouet, J., and D. Gospodarowicz. 1989. Transforming growth factor beta-1
positively modulates the bioactivity of fibroblast growth factor on corneal
endothelial cells. J. Cell Physiol. 141:392–399.
64. Risau, W. 1995. Differentiation of endothelium. FASEB J. 9:926–933.
65. Roberts, A. B., M. B. Sporn, R. K. Assoian, J. M. Smith, N. S. Roche, L. M.
Wakefield, U. I. Heine, L. A. Liotta, V. Falanga, J. H. Kehrl, et al. 1986.
Transforming growth factor type beta: rapid induction of fibrosis and angio-
genesis in vivo and stimulation of collagen formation in vitro. Proc. Natl.
Acad. Sci. USA 83:4167–4171.
66. Sankar, S., B. N. Mahooti, L. Bensen, T. L. McCarthy, M. Centrella, and
J. A. Madri. 1996. Modulation of transforming growth factor beta receptor
levels on microvascular endothelial cells during in vitro angiogenesis. J. Clin.
Investig. 97:1436–1446.
67. Schor, A. M., S. L. Schor, and T. D. Allen. 1983. Effects of culture conditions
on the proliferation, morphology and migration of bovine aortic endothelial
cells. J. Cell Sci. 62:267–285.
68. Schreiber, A. B., M. E. Winkler, and R. Derynck. 1986. Transforming growth
factor-alpha: a more potent angiogenic mediator than epidermal growth
factor. Science 232:1250–1253.
69. Schwartz, M. A. 1997. Integrins, oncogenes, and anchorage independence.
J. Cell Biol. 139:575–578.
70. Sehgal, I., and T. C. Thompson. 1999. Novel regulation of type IV collage-
nase (matrix metalloproteinase-9 and -2) activities by transforming growth
factor-beta1 in human prostate cancer cell lines. Mol. Biol. Cell 10:407–416.
71. Soma, Y., and G. R. Grotendorst. 1989. TGF-beta stimulates primary human
skin fibroblast DNA synthesis via an autocrine production of PDGF-related
peptides. J. Cell Physiol. 140:246–253.
72. ten Dijke, I., I. Miyazono, and I. Heldin. 2000. Signaling inputs converge on
nuclear effectors in TGF-beta signaling. Trends Biochem. Sci. 25:64–70.
73. Tsujii, M., S. Kawano, S. Tsuji, H. Sawaoka, M. Hori, and R. N. DuBois.
1998. Cyclooxygenase regulates angiogenesis induced by colon cancer cells.
Cell 93:705–716. (Erratum, 94:271.)
74. Urness, L. D., L. K. Sorensen, and D. Y. Li. 2000. Arteriovenous malforma-
tions in mice lacking activin receptor-like kinase-1. Nat. Genet. 26:328–331.
75. Vandenbunder, B., L. Pardanaud, T. Jaffredo, M. A. Mirabel, and D. Ste-
helin. 1989. Complementary patterns of expression of c-ets 1, c-myb and
c-myc in the blood-forming system of the chick embryo. Development 107:
265–274.
76. Vin˜als, F., J. C. Chambard, and J. Pouyssegur. 1999. p70 S6 kinase-mediated
protein synthesis is a critical step for vascular endothelial cell proliferation.
J. Biol. Chem. 274:26776–26782.
77. Vin˜als, F., J. Ferre´, C. Fandos, T. Santalucia, X. Testar, M. Palacin, and
Zorzano, A. 1997. Cyclin adenosine 3⬘,5⬘-monophosphate regulates GLUT4
and GLUT1 glucose transporter expression and stimulates transcriptional
activity of the GLUT1 promoter in muscle cells. Endocrinology 138:2521–
2529.
78. Wojnowski, L., A. M. Zimmer, T. W. Beck, H. Hahn, R. Bernal, U. R. Rapp,
and A. Zimmer. 1997. Endothelial apoptosis in Braf-deficient mice. Nat.
Genet. 16:293–297.
79. Wong, S. T., L. F. Winchell, B. K. McCune, H. S. Earp, J. Teixido, J.
Massague, B. Herman, and D. C. Lee. 1989. The TGF-alpha precursor
expressed on the cell surface binds to the EGF receptor on adjacent cells,
leading to signal transduction. Cell 56:495–506.
80. Yang, E. Y., and H. L. Moses. 1990. Transforming growth factor beta 1-in-
duced changes in cell migration, proliferation, and angiogenesis in the
chicken chorioallantoic membrane. J. Cell Biol. 111:731–741.
81. Yang, X., L. H. Castilla, X. Xu, C. Li, J. Gotay, M. Weinstein, P. P. Liu, and
C. X. Deng. 1999. Angiogenesis defects and mesenchymal apoptosis in mice
lacking SMAD5. Development 126:1571–1580.
7230 VIN
˜ALS AND POUYSSE
´GUR MOL.CELL.BIOL.
