From the Molecular/Cancer Biology Laboratory and the ¶ Department of Virology, Haartman Institute, PL 21 Haartmaninkatu 3, University of Helsinki, 00014 Helsinki, Finland and the § Department of Gene Expression, GBF, D-38124 Braunschweig, Germany
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ABSTRACT |
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The vascular endothelial growth factor
(VEGF) and the VEGF-C promote growth of blood vessels and lymphatic
vessels, respectively. VEGF activates the endothelial VEGF receptors
(VEGFR) 1 and 2, and VEGF-C activates VEGFR-3 and VEGFR-2. Both VEGF
and VEGF-C are also potent vascular permeability factors. Here we have
analyzed the receptor binding and activating properties of several
cysteine mutants of VEGF-C including those (Cys156
and Cys165), which in other platelet-derived growth
factor/VEGF family members mediate interchain disulfide bonding.
Surprisingly, we found that the recombinant mature VEGF-C in which
Cys156 was replaced by a Ser residue is a selective agonist
of VEGFR-3. This mutant, designated N
C156S, binds and activates
VEGFR-3 but neither binds VEGFR-2 nor activates its autophosphorylation or downstream signaling to the ERK/MAPK pathway. Unlike VEGF-C,
N
C156S neither induces vascular permeability in vivo
nor stimulates migration of bovine capillary endothelial cells in
culture. These data point out the critical role of VEGFR-2-mediated
signal transduction for the vascular permeability activity of VEGF-C
and strongly suggest that the redundant biological effects of
VEGF and VEGF-C depend on binding and activation of VEGFR-2. The
N
C156S mutant may provide a valuable tool for the analysis
of VEGF-C effects mediated selectively via VEGFR-3. The ability of
N
C156S to form homodimers also emphasizes differences in the
structural requirements for VEGF and VEGF-C dimerization.
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INTRODUCTION |
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The PDGF/VEGF1 family of growth factors currently includes seven members: PDGF-A, PDGF-B (1, 2), VEGF (3, 4), placenta growth factor (PlGF) (5), VEGF-B/VEGF-related factor (6, 7), VEGF-C/VEGF-related protein (8, 9), and c-fos-induced growth factor/VEGF-D (10). All members of the family share a common structure in that they contain eight characteristically spaced cysteine residues in the core domain. PDGF-A and PDGF-B promote the growth of several cell types, whereas VEGF, PlGF, and VEGF-C regulate almost exclusively endothelial cells, which express the corresponding receptors. VEGF binds VEGFR-1 (Flt-1) and VEGFR-2 (Flk-1/KDR), whereas PlGF and VEGF-B bind only VEGFR-1 (3, 12, 13).2
VEGF-C and VEGF-D are ligands and activators of VEGFR-3 (8, 9, 11). Mature VEGF-C and VEGF-D, which are generated by proteolytic processing of precursor polypeptides, also activate VEGFR-2. VEGF and VEGF-C are distinct in their specificity toward endothelial cells. VEGF specifically stimulates proliferation of the endothelial cells of blood vessels (14), whereas VEGF-C preferentially promotes growth of lymphatic endothelia (15, 16). On the other hand, there are certain similarities in the biological activities of VEGF and the mature form of VEGF-C in that both factors are potent inducers of vascular permeability (8, 17, 18). In addition, at higher concentrations VEGF-C, similarly to VEGF, also stimulates proliferation and migration of vascular endothelial cells in culture (8, 18). These data addressed a question of whether certain redundancy in VEGF and VEGF-C activities might be mediated via VEGFR-2, which is used by both of these two growth factors.
The previously known members of the PDGF/VEGF family form homodimers
via disulfide bonds between the second and fourth of the eight
conserved cysteine residues. These bonds are crucial for the
dimerization and biological activity of VEGF but not for the activity
of PDGF-BB (19-21). Unlike these factors, the recombinant mature
VEGF-C forms mostly noncovalent homodimers. It also contains an
unpaired extra cysteine residue (Cys137) located between
the first and second conserved cysteine residues (18). Another unpaired
Cys residue (Cys87) is located in the N-terminal VEGF-C
propeptide, which is cleaved off during the proteolytic processing of
VEGF-C precursor. To evaluate the significance of disulfide bonds for
VEGF-C homodimerization and activity, we converted selected cysteine
residues to serine residues either in the VEGF-C precursor or in the
recombinant "processed" VEGF-C (N
C, described in Joukov
et al. (18)) and analyzed these mutants for their abilities
to bind and to activate VEGFR-3 and VEGFR-2. This led to the
identification of a ligand for VEGFR-3 that is devoid of VEGFR-2
stimulating properties and does not possess VEGF-like activities
in vitro or in vivo.
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EXPERIMENTAL PROCEDURES |
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Cell Culture, Transfections, and Antibodies-- 293 EBNA cells were cultured in DMEM/10% FCS, porcine aortic endothelial (PAE)/VEGFR-2 (22), and PAE/VEGFR-3 (23) cells in Ham's F-12 medium/10% FCS. BCE cells (24) were cultured as described in Ref. 25. Cell transfections employing the VEGF-C constructs or the pREP7 plasmid without insert (mock transfections) were carried out using the calcium phosphate precipitation method. Antiserum 882 raised against the N-terminal part of mature VEGF-C was used for the immunoprecipitations and for the detection of VEGF-C on Western blots (18). Immunoprecipitations of VEGFR-3 and VEGFR-2 were carried out using previously described antisera (22, 26).
Generation of VEGF-C Mutants--
The replacement of Cys with
Ser residues was carried out using the Altered Sites II in
vitro Mutagenesis System (Promega) essentially as described (17).
The template was a cDNA encoding either full-length wt or
recombinant mature VEGF-C fused in-frame with the signal peptide and
containing His6 tag at the C terminus (N
C). The
mutant constructs in the pALTER vector were digested with
HindIII and NotI, subcloned into
HindIII/NotI-digested pREP7, and used to
transfect 293 EBNA cells.
Binding, Receptor Autophosphorylation, and MAPK Activation
Studies--
Concentrations of the mutant proteins were estimated from
serially diluted conditioned media by Western blotting and comparison with known amounts of recombinant yeast protein. The ability of VEGF-C
mutants to compete with [125I]N
C for VEGFR-2 and
VEGFR-3 and to stimulate their tyrosine autophosphorylation were
analyzed as described (8, 18). To study ERK/MAPK activation,
PAE/VEGFR-3 and PAE/VEGFR-2 cells were serum starved for 12 h
prior to treatment with equal amounts (~100 ng/ml) of purified factor
or 10% fetal calf serum for 5 min, after which ERK1 and ERK2
phosphorylation was analyzed by Western blotting using phospho-specific
p44/42 MAPK antibodies. Blots were then stripped and reprobed with
p44/42 MAPK antibodies to detect total protein. p44/42 and
phospho-specific p44/42 antibodies were purchased from New England
BioLabs and used according to the manufacturer's instructions.
Analysis of VEGF-C Biological Activity--
To eliminate the
effect of trace amounts of co-purified VEGF, the conditioned media,
purified N
C,
N
C156S, and the material similarly purified
from the mock transfected cells were pretreated for 1 h at room
temperature with anti-human VEGF neutralizing antibody (R & D
systems, 2.5 ng/1 ng of VEGF-C). Analysis of the migration and
permeability assays were carried out as described (8, 18).
Analysis of Dimerization of VEGF-C Mutants-- The ability of VEGF-C mutants to form disulfide linked dimers was analyzed in SDS-PAGE in nonreducing conditions with subsequent Western blotting and detection using the antiserum 882. Chemical cross-linking of the 35S metabolically labeled VEGF-C mutants with disuccinimidyl suberate (Pierce) was carried out as described (18).
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RESULTS |
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Binding of VEGF-C Mutants to VEGFR-3 and VEGFR-2--
Taking
into account the importance of the second and the fourth characteristic
cysteine residues for VEGF activity, we replaced these residues either
separately or in combination with Ser residues in the VEGF-C precursor
and in the recombinant mature VEGF-C (N
C). The mutants were
expressed in 293 EBNA cells, and the conditioned media were studied for
their ability to compete with [125I]
N
C for VEGFR-3
and VEGFR-2 binding. Replacement of Cys165 alone or
together with Cys156 in
N
C or in wt VEGF-C abolished
the ability of the proteins to bind VEGFR-3 or VEGFR-2 (Fig.
1 and data not shown). On the other hand,
the C83S,C137S mutant displaced [125I]
N
C from both
receptors, indicating that these unpaired cysteine residues are not
critical for the receptor binding properties of VEGF-C.
N
C165S
and
N
C156S,C165S also failed to induce tyrosine phosphorylation
of VEGFR-3 or VEGFR-2, whereas C83S,C137S was nearly as active as wt
VEGF-C in this assay (data not shown). Surprisingly, the
N
C156S
growth factor mutant efficiently bound VEGFR-3 but not VEGFR-2 (Fig. 1,
A and B, respectively).
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N
C156S Is a Selective Activator of VEGFR-3--
The
recombinant
N
C and
N
C156S proteins were purified from the
conditioned media, and their abilities to stimulate the tyrosine
phosphorylation of VEGFR-3 and VEGFR-2 were compared. In agreement with
the receptor binding studies,
N
C156S was as active as
N
C in
the stimulation of VEGFR-3 tyrosine phosphorylation (Fig.
2A, upper panel),
but unlike VEGF or
N
C,
N
C156S did not stimulate tyrosine
phosphorylation of VEGFR-2 (Fig. 2A, middle panel). Western blotting analysis confirmed that similar amounts of the
N
C and
N
C156S proteins were used in the analysis
(Fig. 2A, lower panel). These data indicate that
N
C156S is a selective ligand and an activator of VEGFR-3 but that
it lacks activity toward VEGFR-2.
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N
C156S Is a Selective Activator of VEGFR-3 ERK/MAPK
Signaling--
Mitogenic signals from growth factor receptors are
frequently relayed via the ERK/MAPK pathway into the nucleus. Purified recombinant
N
C and
N
C156S produced in a Pichia expression system were used to determine MAPK pathway activation of cells expressing either VEGFR-2 or VEGFR-3 (Fig. 2B). The growth
factor-treated cells were lysed, and activated MAPK was detected using
Western blotting with antibodies against the phosphorylated forms of
ERK1 and ERK2. At a concentration of 100 ng/ml, VEGF-C showed rapid activation of the ERK1 and ERK2 MAPK in VEGFR-2 and VEGFR-3 expressing cells; however,
N
C156S activated these kinases only via VEGFR-3. At the concentrations used, both forms of VEGF-C appeared to be equally
potent in activating the MAPK through VEGFR-3. The amounts of total
MAPK protein were confirmed to be similar in the treated and untreated
cells, as shown by staining the filter with p44/p42 MAPK antibodies
made against a synthetic peptide of rat p42, which in our hands give a
weaker signal from p44.
N
C156S Lacks VEGF-like Effects--
Similar to VEGF, the
mature VEGF-C increases vascular permeability in vivo, and
at higher (compared with VEGF) concentrations also stimulates the
migration and proliferation of BCE cells in vitro (8, 18).
We therefore compared
N
C and
N
C156S for their abilities to
induce these biological responses.
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N
C156S Forms Partially Disulfide-bonded
Homodimers--
Replacement of the second and/or the fourth cysteine
residues of VEGF abolishes its dimer formation and biological activity (21). We investigated the dimeric nature of the VEGF-C mutants. No
homodimers were obtained when
N
C156S,C165S or
N
C165S were chemically cross-linked (Fig.
4A, lanes 1-4). On
the other hand, about half of both cross-linked
N
C (18) and
N
C156S (lane 6) migrated as dimers. This indicates
that
N
C156S forms homodimers. Moreover, unlike
N
C, which
forms preferentially noncovalently bound dimers, a fraction of
N
C156S was disulfide bonded, as detected by SDS-PAGE in
nonreducing conditions (Fig. 4B). These data suggest that
homodimerization is required for VEGFR-3 activation by VEGF-C and
indicate that the inability of
N
C156S to activate VEGFR-2 and to
induce VEGF-like effects is not due to an inability of this mutant to
form homodimers.
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DISCUSSION |
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Here we describe a VEGF-C point mutant that is active toward
VEGFR-3 but, unlike wt VEGF-C, is unable to bind to and to activate signaling through VEGFR-2. This mutant (N
C156S) was generated by
replacement of the second conserved Cys residue of the recombinant processed VEGF-C by a Ser residue.
N
C156S was inactive in the vascular permeability assay and did not increase migration of the
capillary endothelial cells, indicating that these VEGF-like effects of
VEGF-C require VEGFR-2 binding. Interaction with VEGFR-2 has been shown
to be a critical requirement for the full spectrum of biological
responses induced by VEGF (22, 27, 28). Taking into account that
VEGFR-2 is the only known receptor shared by VEGF and VEGF-C, one can
speculate that the ability of VEGF-C to increase vascular permeability
and the ability to stimulate the migration of capillary endothelial
cells are mediated via VEGFR-2 and that the activation of VEGFR-2 is
sufficient to induce these biological effects. Moreover, downstream
signaling from VEGR-2 requires activation of the MAPK pathway through
at least ERK1 and ERK2 (29). However, the possibility remains that
there are additional, as yet unknown receptors for VEGF and VEGF-C, which could mediate the vascular permeability effect of VEGF-C instead
of or in addition to VEGFR-2.
Interestingly, the structural requirements for binding and activation
of VEGFR-2 by VEGF and VEGF-C are different. Despite the prominent
similarity between the mature VEGF-C and VEGF121, none of
the basic amino acid residues shown to be critical for VEGF binding to
VEGFR-2 (28) are conserved in VEGF-C. Unlike fully processed VEGF-C,
which forms noncovalent homodimers (18), VEGF needs to be a covalent
dimer for efficient receptor binding and activation (21). Both the
second and fourth cysteine residues are critical for the dimerization
and biological activity of VEGF. In the case of VEGF-C, only the fourth
conserved cysteine residue (Cys165) is critical, whereas
elimination of the second one (Cys156) did not affect dimer
formation. Instead, a small fraction of the N
C156S molecules
acquired the ability to form disulfide-linked homodimers, which
apparently were also inactive toward VEGFR-2. Taken together, these
data indicate that VEGF-C homodimerization is necessary but not
sufficient for VEGFR-2 activation and for induction of the VEGF-like
effects of VEGF-C. The inability of the
N
C156S,C165S and
N
C165S proteins to form homodimers and to bind and to activate
VEGFR-3 suggests that VEGFR-3 activation also requires the dimerization
of VEGF-C.
VEGF-C, unlike VEGF, stimulates the growth of lymphatic vessels (15).
It would be interesting to know whether the selective activation of
VEGFR-3 is sufficient for this effect or whether activation of both
receptors is required. The N
C156S mutant provides a suitable tool
to answer this question and to delineate the signaling pathways
involved in the various activities of VEGF-C. The ERK/MAPK pathway may
be involved in the permeability, proliferative, and migration-inducing
activities of VEGF-C toward capillary endothelial cells. If both
VEGFR-3 and VEGFR-2 are needed for the full range of biological
activities of VEGF-C toward the lymphatics, one needs to consider the
possibility that
N
C156S would inhibit the effects of wt VEGF-C.
On the other hand, if VEGFR-2 is able to mediate the VEGF-like
responses of VEGF-C independently of VEGFR-3, as seems likely because
BCE cells do not express
VEGFR-3,3 then
N
C156S
would be a valuable and highly selective VEGFR-3 agonist. These issues
will be further studied.
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ACKNOWLEDGEMENTS |
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We thank Dr. Eija Korpelainen, Dr. Ilkka Julkunen, Dmitri Chilov, and Michael Jeltsch for collaboration and helpful discussions and Raili Taavela, Liisa Koivunen, Mari Helanterä, Paula Pietiläinen, and Tapio Tainola for excellent technical assistance.
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FOOTNOTES |
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* This work was supported by the Finnish Cancer Organizations, the Finnish Academy, the Sigrid Juselius Foundation, the University of Helsinki, and the State Technology Development Centre.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.
Present address: Dana-Farber Cancer Inst., Harvard Medical School,
Boston, MA 02115.
To whom correspondence should be addressed. Tel.:
358-9-1912-6434; Fax: 358-9-1912-6448; E-mail:
Kari.Alitalo@Helsinki.Fl.
1 The abbreviations used are: PDGF, platelet-derived growth factor; BCE, bovine capillary endothelial; EBNA, Epstein-Barr nuclear antigen; ERK, extracellular signal-regulated kinases; FCS, fetal calf serum; MAPK, mitogen-activated protein kinase(s); PAE, porcine aortic endothelial; PAGE, polyacrylamide gel electrophoresis; PlGF, placenta growth factor; VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor; DMEM, Dulbecco's modified Eagle's medium; wt, wild type.
2 B. Olofsson, E. Korpelainen, S. Mandriota, M. S. Pepper, K. Aase, V. Kumar, Y. Gunji, M. M. Jeltsch, M. Shibuya, K. Alitalo, and U. Eriksson, submitted for publication.
3 V. Joukov and K. Alitalo, unpublished data.
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REFERENCES |
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