237
Journal of Cell Science 111, 237-247 (1998)
Printed in Great Britain © The Company of Biologists Limited 1998
JCS9693
Agonistic monoclonal antibodies against the Met receptor dissect the
biological responses to HGF
Maria Prat1,*,†, Tiziana Crepaldi1,*, Selma Pennacchietti1, Federico Bussolino2 and Paolo M. Comoglio1
1Institute for Cancer Research (IRCC) and 2Department of Genetics, Biology and Biochemistry, University of Torino, Medical
School, 10060 Candiolo, Italy
*These two authors contributed equally to this work
†Author for correspondence (e-mail: [email protected])
Accepted 5 November 1997: published on WWW 23 December 1997
SUMMARY
Hepatocyte growth factor, also known as scatter factor, is
a pleiotropic cytokine, which stimulates cell motility,
invasion, proliferation, survival and morphogenesis, and
induces the expression of specific genes by activating its
receptor tyrosine kinase. In this work we have isolated,
characterized and used as agonists two monoclonal
antibodies (mAbs) directed against the extracellular
domain of HGF receptor to investigate the requirements for
receptor activation and for the different biological
responses. The two mAbs display similar affinities, react
with epitopes different from the hepatocyte growth factor
binding site, and behave as either full or partial agonists.
The full agonist mAb (DO-24) triggers all the biological
effects elicited by hepatocyte growth factor, namely
motility, proliferation, cell survival, invasion, tubulogenesis
and angiogenesis. The partial agonist mAb (DN-30) induces
only motility. Only the full agonist mAb is able to induce
and sustain the expression of urokinase-type plasminogen
activator receptor for prolonged periods of time, while both
mAbs up-regulate the constitutive expression of urokinasetype plasminogen activator. Both mAbs activate receptor
phosphorylation, which, being strictly dependent on mAb
bivalence, requires receptor dimerization. Since simple
receptor dimerization is not sufficient to trigger full
biological responses, we propose that the region on the β
chain of the receptor recognized by the full agonist mAb is
crucial for optimal receptor activation.
INTRODUCTION
liver and placenta development (Schmidt et al., 1995; Uehara
et al., 1995) and migration of myoblast precursors from the
limb buds (Bladt et al., 1995; Maina et al., 1996).
HGF is a heterodimeric protein composed of an α chain,
containing an N-terminal hairpin loop and four kringle
domains, and a serine protease-like β chain (Ebens et al., 1996;
Mizuno and Nakamura, 1993; Nakamura, 1991). Structurally,
HGF has a 38% amino acid sequence homology to
plasminogen, a five kringle-containing heterodimeric serine
protease, but it is devoid of proteolytic activity because three
critical amino acids in the catalytic domain are mutated. HGF
is synthesized and secreted as a biologically inactive singlechain 92 kDa precursor, which is converted into the bio-active
dimer in the extracellular environment either by serine
proteases activated during the coagulation process (Miyazawa
et al., 1994), or by urokinase type plasminogen activator (uPA)
bound to its high affinity receptor (uPA-R) at the cell surface
(Naldini et al., 1992). On the other hand HGF stimulation
induces, as an early event, the expression of both uPA and its
receptor (Boccaccio et al., 1994; Pepper et al., 1992). uPA
plays a central role in catalyzing extracellular matrix
degradation (Blasi, 1993). Thus, the coordinated activity of the
HGF/HGF-R and uPA/uPA-R couples is critically involved in
cell associated proteolytic processes required for tissue
The hepatocyte growth factor (HGF) and its receptor (HGF-R)
stand out from other ligand-receptor couples because of the
distinctive property to elicit a wide spectrum of biological
responses from target cells. HGF, a cytokine of mesenchymal
origin, stimulates proliferation not only of hepatocytes, but also
of other epithelial cell types, as well as endothelial cells,
melanocytes, hemopoietic and bone cells (Tamagnone and
Comoglio, 1997). HGF is also a cell survival factor (Amicone
et al., 1996; Bardelli et al., 1996). Moreover, it promotes the
dissociation of epithelial and endothelial sheets, it induces cell
motility and it stimulates the invasive growth and cellular
polarization required for tubular morphogenesis. The latter
biological activity is a peculiarity shared only with the other
ligand-receptor couples belonging to the Met family
(Comoglio and Boccaccio, 1996). In vivo, HGF plays a role in
neural system (Streit et al., 1995; Ebens et al., 1996), kidney
(Santos et al., 1994; Wolf et al., 1995) and mammary gland
(Niranjan et al., 1995; Soriano et al., 1995; Yang et al., 1995)
development, tissue regeneration (Matsumoto and Nakamura,
1993), angiogenesis (Bussolino et al., 1992), and tumor
invasion and metastastis (Giordano et al., 1993; Rong et al.,
1994). During embryogenesis HGF signalling is essential for
Key words: Agonist mAb, Growth factor receptor, Invasive growth,
HGF
238
M. Prat and others
remodelling, cell migration and cell invasion during wound
healing, angiogenesis and tumor metastasis.
HGF-R is a heterodimer of 190 kDa composed of an α chain
of 50 kDa, exposed at the cell surface and linked by disulfide
bonds to a β chain of 145 kDa, consisting of the extracellular
ligand binding domain, the transmembrane anchoring segment
and the cytoplasmic tyrosine kinase domain (Comoglio and
Vigna, 1995). Both chains derive from a single-chain 170 kDa
precursor. The pleiotropy of the biological effects of HGF is
based on the activation of multiple intracellular signalling
pathways, consequent to the phosphorylation of two tyrosines
(Y1349, Y1356) embedded within a degenerate consensus
sequence acting as a double docking site for recruitment and
activation of Src homology 2 (SH2) domain containing
transducers or adaptors (Bardelli et al., 1992; Graziani et al.,
1991, 1993; Ponzetto et al., 1994). Among the many signalling
molecules recruited at the plasma membrane, the Ras protein
and the PI3 kinase have been shown to play a critical role in
the control of HGF-dependent cell growth and motility
(Derman et al., 1995; Hartmann et al., 1994; Ponzetto et al.,
1994; Ridley et al., 1995; Royal and Park, 1996). Concomitant
activation of both (Ras and PI3K) signalling pathways is
mandatory for invasive growth (Giordano et al., 1997).
Recently, an IRS-like molecule (Gab-1) was shown to interact
with the HGF-R and to mediate the morphogenic response
(Weidner et al., 1996).
The multiple activities elicited by HGF-R stimulation can be
partially dissociated, by acting at the level of both the ligand
and the receptor. In fact, a motogenic, but not a mitogenic
response, is induced by stimulating cells with a truncated
isoform of HGF (K2; Hartmann et al., 1992), as well as by
transfecting a chimeric HGF-R mutated in the tyrosine residue
Y1356 (Ponzetto et al., 1994).
Anti-receptor monoclonal antibodies (mAbs) represent
versatile probes to study protein structure (Prat et al., 1991)
and, if agonists, can be used to dissect the different biochemical
and biological responses associated with receptor activation
(Bellot et al., 1990; Fuh et al., 1992; Spaargaren et al., 1990,
1991; Taylor et al., 1987; Yarden, 1990). In the present report,
we describe the different agonistic activities of mAbs against
the extracellular domain of HGF-R. A full agonist mAb evokes
the complete range of biological responses required for
invasive growth, while a partial agonist mAb can trigger only
motility. The phenotype of invasive growth correlates with the
ability to induce the expression of an integral uPA/uPA-R
proteolytic network. HGF-R phosphorylation is induced by
both mAbs and requires antibody bivalence, which shows that
dimerization is essential for any effect. It is hypothesized that
the interaction of the full agonist mAb with a critical epitope
is necessary to trigger all the biological responses other than
motility.
MATERIALS AND METHODS
Reagents, cells and antibodies
All reagents, unless specified, were purchased from Sigma. As a
source of HGF we used either dia-filtered conditioned medium from
MRC-5 human fibroblasts or recombinant human HGF purified by
heparin affinity chromatography from the baculovirus expression
system (Naldini et al., 1995), kindly provided by Dr C. Stella (Institute
for Cancer Research (IRCC), University of Torino). The two
preparations yielded 3 and 0.9 µg/ml purified protein, respectively,
with 3 pM corresponding to1 scatter unit/ml.
GTL-16, a clonal cell line derived from a poorly differentiated
gastric carcinoma (Giordano et al., 1989a) and A549 lung carcinoma
cells (ATCC CCL 185) were grown in RPMI-1640 medium,
supplemented with 10% fetal calf serum (FCS). Madine Darby canine
kidney cells (MDCK, ATCC CCL 34) were grown in DMEM medium
supplemented with 5% FCS. Human endothelial cells were prepared
from umbilical cord vein as previously described (Bussolino et al.,
1989) and grown in M199 medium supplemented with 20% FCS,
endothelial cell growth factor (100 µg/ml) and porcine heparin (100
µg/ml) and used at early passages. NIH-3T3 fibroblastic stable
transfectants expressing the β-chain alone of HGF-R were produced
as described by Zhen et al. (1994), and grown in DMEM medium
supplemented with 10% FCS. All cells were maintained at 37°C in
5% CO2.
Murine monoclonal antibody DL-21 against the HGF-R was
prepared as described (Prat et al., 1991). Murine monoclonal antibody
R1.30 against β2 microglobulin was previously obtained (Crepaldi et
al., 1991). Anti-phosphotyrosine monoclonal antibody was purchased
from Upstate Biotechnology (New York, US).
Production and selection of monoclonal antibodies
Living GTL-16 cells, in which the c-MET proto-oncogene is amplified
and overexpressed (Ponzetto et al., 1991), were used as immunogens
to produce mAbs against native epitopes of HGF-R. Immune spleen
cells from Balb/c mice (Charles River) were fused with
P3.X63.Ag8.653 myeloma cells. More than 3,000 independent
hybridomas were obtained from three fusions and their supernatants
screened using a stringent selection strategy. Hybridoma supernatants
were incubated with GTL-16 cell extracts, immunoprecipitates were
recovered on Protein A-Sepharose and goat anti-mouse Ig (GaMIg)
and phosphorylated in the presence of 2.5 µCi [γ-32P]ATP (specific
activity 7,000 Ci/mM; Amersham) in an in vitro kinase assay (Prat et
al., 1991). Labelled proteins were analyzed by SDS-PAGE, run in
parallel under non-reducing and reducing conditions. The shift in
electrophoretic mobility of the radiolabelled HGF-R β chain, which
is retarded by the disulfide-linked α chain in non-reducing conditions,
readily allowed the identification of hybridomas secreting anti-HGFR antibodies. Supernatants from 8-10 hybridomas were initially
pooled and screened in the kinase assay; those scoring positive were
then tested separately. Four hybridomas were selected, successfully
established after two rounds of cloning by limiting dilution and grown
as ascites. Two of these were used in this study.
mAb isotyping, mAb and Fab purification,
immunofluorescence and ELISA
Immunoglobulin subtypic classes were determined using the mouse
monoclonal antibody isotyping kit purchased from Amersham. mAbs
were purified from ascitic fluid by ammonium sulfate precipitation
and affinity chromatography on Protein A-Sepharose 4B (Pharmacia)
with elution at pH 3.5. Fab fragments were obtained by digestion of
purified antibodies by immobilized papain (Pierce) and purified by
negative selection on Protein A-Sepharose 4B.
In indirect immunofluorescence studies, GTL-16 living cells grown
on coverslips were incubated with mAbs for 1 hour at 4°C, fixed with
3% paraformaldehyde and then incubated with rhodamine-conjugated
secondary antibodies. Samples were viewed with Zeiss epifluorescence optics.
For ELISA, GTL-16 cells were plated on polylysine-coated 96-well
microplates (Costar), fixed with paraformaldehyde, incubated with
mAbs and horseradish peroxidase-labelled GaMIg (Kpl,
Gaithersburg, MD). The reaction was visualized by adding
orthophenylendiamine as chromogen (Sorin Biomedica, Saluggia,
Italy) and read at 490 nm in a Microplate Reader (Model 3550, BioRad).
Biological effects of agonist mAbs to HGF-R
HGF, mAbs radioiodination and binding assays
Pure recombinant human HGF (1 µg) was radio-labelled with carrier
free 125I (1 mCi; Amersham) in the presence of 10 µg Iodogen (Pierce)
at 4°C, as described by Naldini et al. (1991). The labelled ligand was
recovered on a heparin-Sepharose affinity column (Pierce), eluted
with 1.3 M NaCl and stored at 4°C. The specific activity was
approximately 0.25 Ci/mg, corresponding to a 125I/HGF molar ratio
of approximately 11/1. Purified antibodies (10 µg) were radio-labelled
with carrier free 125I (1 mCi) and Iodogen with the above procedure.
The labelled mAbs were separated from the free 125I by gel filtration
on Sephadex G50 (Pharmacia). The specific activity of the tracer was
approximately 1 Ci/mg, corresponding to an I/mAb molar ratio of
approximately 20/1.
For binding displacement studies, GTL-16 cell monolayers were
seeded at low density in polylysine-coated microwells, fixed with
paraformaldehyde, saturated with BSA and incubated with 20-50 nM
125I-mAb, with or without the indicated concentration of unlabelled
competitor, for 3 hours at 4°C. The extensively washed monolayers
were then extracted with 2% SDS and counted in a Packard γ-counter.
Total binding was below 10% of the added dpm and specific binding,
calculated by subtracting from the total the dpm bound after
incubation with a 300-fold excess of unlabelled ligand, was
approximately 75%. Specific ligand binding studies were performed
by using increasing concentrations of 125I-mAbs in the presence of
50-fold excess of cold ligand at 4°C for 3 hours in the same
conditions. The mAb Kd was estimated by using a Graphit assisted
algorithm.
For double determinant immunoradiometric assay (DDIA)
polyvinyl microwells were coated with the purified DL-21 mAb (Prat
et al., 1991), which reacts with an independent epitope of the HGFR extracellular domain, saturated with 0.2% BSA and then incubated
with soluble HGF-R. As source of soluble HGF-R, the 5-fold
concentrated culture supernatant from GTL-16 cells was used (Prat et
al., 1991). After overnight incubation, the antigen was washed away
and the microwells were incubated with 1 nM 125I-HGF, with or
without the indicated concentrations of unlabelled competitor, for 3
hours at 4°C. The washed microwells were extracted with 2% SDS
and the soluble radioactivity was counted in a Packard γ-counter.
Cell treatments, metabolic labelling, immunoprecipitation
and western blot
For experiments on glycosylation inhibition, subconfluent GTL-16
cells were pre-treated with 1 µg/ml tunicamycin for 1 hour prior to
and during labelling with [35S]methionine (400 µCi/ml; Amersham)
for 18 hours in methionine-free medium (ICN) supplemented with
10% FCS, as described by Giordano et al. (1989b). Cells were then
washed at 4°C and lysed in RIPA buffer (20 mM Tris-HCl, pH 7.4,
150 mM NaCl, 0.1% SDS, 1% Triton X-100, 1% DOC, 5 mM EDTA,
1 mM Na3VO4), and inhibitors of proteases (2 mM PMSF, 50 µg/ml
pepstatin, 50 µg/ml chymostatin and 10 µg/ml antipain; Chemicon)
for 10 minutes on ice. Clarified cell extracts were rotated 2 hours at
4°C with antibodies covalently linked to Sepharose Protein A by
cross-linking with dimethyl pimelimidate (Pierce). Beads were
washed 3 times with lysis buffer and samples were eluted by boiling
in Laemmli buffer containing 100 mM dithiothreitol. Eluted proteins
were electrophoresed on 8% SDS-PAGE, fixed, fluorographed, dried
and exposed to Amersham Hyperfilm for autoradiography with
intensifying screens.
For stimulation experiments, subconfluent cells were grown for
three days in spent medium, and then stimulated in DMEM containing
25 mM Hepes, 100 µg/ml bacitracin, 0.05% BSA, with or without the
indicated concentrations of factors for 10 minutes at 37°C, as
described by Naldini et al. (1991). Cell lysates were
immunoprecipitated as described above. In western blotting, eluted
immunoprecipitates and total cell extracts solubilized in boiling
Laemmli buffer, in the presence of 100 mM dithiothreitol (100 µg
protein/lane), were run in SDS-PAGE gels, transferred to
239
nitrocellulose filters (Hybond, Amersham), probed with antibodies
using the enhanced chemiluminescence kit (ECL, Amersham) and
visualized on Kodak XOMAT AR film.
Biological assays
For the dissociation and motility assay A549 and MDCK cells were
seeded at 6×103/cm2 in 24-well plates and exposed overnight to the
indicated concentrations of factors in fresh medium. The scattering
effect was monitored by light microscopy.
For the endothelial cell growth assay, 2.5×103 human endothelial
cells were plated in 96-well plates coated with 0.05% gelatin (Difco
Laboratories, Detroit, MI; 1 hour at 22°C) in M199 medium
containing 20% FCS, as described by Bussolino et al. (1992). After
24 hours the medium was removed and replaced with M199
containing 5% FCS with or without factors. Fresh medium plus and
minus stimulating factors was replenished every two days for 8 days.
Endothelial cell number was estimated after staining with crystal
violet by a colorimetric assay. Briefly, the cells were fixed for 20
minutes at room temperature with 2.5% glutaraldehyde and then
stained with 0.1% crystal violet in 20% methanol. The stained cells
were solubilized with acetic acid (10%) and read at 595 nm in a
Microplate Reader (Model 3550, Bio-Rad). A calibration curve was
set up with a known number of cells. Proportionality between
absorbance and cell counts exists up to 8×104 cells.
For the tubulogenesis assay in three-dimensional collagen gels, the
trypsinized MDCK cells were suspended at a final concentration of
1×105 cells/ml in gelling solution prepared in the following
proportions: type I collagen S (3 mg/ml, Collaborative Biomed),
DMEM 10× (Gibco BRL), NaHCO3 (37 g/l), 0.2 M Hepes (7:1:1:1),
at room temperature (Montesano et al., 1991). 100 µl of this mixture
were seeded into microtiter plate wells and allowed to gel for 5
minutes at 37°C, before adding 100 µl DMEM containing 10% FCS.
After 3 days, fresh medium plus and minus stimulating factors was
added. Cysts and tubules were observed after a further 3-5 days, at
which time images were taken by a computer assisted telecamera.
For the proliferation assay of MDCK cells, cells were cultured in
collagen gels as above and [3H]thymidine (Amersham; 1.5-2
µCi/well) was added at the fourth day in the presence of absence of
freshly added stimulating factors. After 20 hours, collagen was
digested with collagenase (2 mg/ml) for 30 minutes at 37°C. Cells
were recovered by centrifugation and TCA-precipitable radioactive
samples were counted in a liquid scintillation Packard β-counter.
In vivo angiogenesis was assayed as growth of blood vessels from
subcutaneous tissue into a solid gel of Matrigel containing the test
sample, as described by Grant et al. (1993). Matrigel (8 mg/ml) in
liquid form at 4°C was mixed with the indicated amounts of
stimulating factor and 10 U/ml heparin, and injected into the
abdominal subcutaneous tissue of female DBA2 mice, along the
peritoneal midline. Matrigel rapidly forms a solid gel at body
temperature, trapping the factors to allow slow release and prolonged
exposure to surrounding tissues. After 10 days mice were killed and
the recovered gels were photographed.
For the invasion assay, polycarbonate filters (8 µm pore size) of
Transwell chambers (6.5 mm, Costar Corporation, Cambridge,
Massachusetts) were coated with 2 µg Matrigel (Collaborative
Research Incorporated, Waltham, MA). MDCK cells (105) in DMEM
5% FCS were seeded on the matrigel coated polycarbonate membrane
in the upper compartment. 500 µl of DMEM and 5% FCS, alone or
containing the stimulating factors, was added to the lower
compartment. The plates were incubated at 37°C for 72 hours. At the
end of incubation, the cells and matrigel on the upper side of the
polycarbonate filter were mechanically removed. Cells that had
invaded the matrigel and migrated to the lower side of the filter were
stained with Crystal Violet and absorbance read as already described.
For the apoptotic assay, 1.7×104 MDCK cells/cm2 in 24-well plates
were incubated with 0.1 µM staurosporin for 6 hours, in the presence
or absence of stimulating factors. Apoptotic cells were detected by
240
M. Prat and others
immunofluorescence staining using the TUNEL (TdT-mediated dUTP
nick end-labelling) assay (Boehringer) and counted (N). The
percentage of protection from apoptosis was calculated as follows:
N in the absence of factor − N in the presence of factor
N in the absence of factor
× 100 .
Northern blot analysis and gene probes
After cell stimulation, total cellular RNA was prepared using the
single-step method of extraction described by Chomczynski and
Sacchi (1987). For northern blot analysis, 20 µg of total RNA were
separated by electrophoresis on 0.8% denaturing agarose gels,
transferred to nylon membranes (Hybond-NTM, Amersham) by
capillary action and fixed according to the manufacturer’s
instructions. The gene probes used for hybridization were as follows.
Glyceraldehyde-3-phosphate dehydrogenase (GADH) was a human
full-length cDNA kindly provided by Dr M. Pierotti. UPA and uPAR probes were human cDNA fragments (1.5 kb and 0.6 kb,
respectively) generously provided by Dr F. Blasi. Probes were labeled
using the Ready-To-GoTM DNA labeling system with [α-32P]dCTP
(specific activity 3,000 Ci/Mm; Pharmacia). Hybridization was
carried out in the Rapid-hyb buffer (Amersham) at 65°C for 2 hours.
Nylon membranes were washed twice under highly stringent
conditions (2× SSC, 0.1% SDS at 65°C for 20 minutes) and
underwent autoradiography using intensifying screens. Filters were
stripped and reprobed in multiple cycles, according to the
manufacturer’s instructions.
RESULTS
The monoclonal antibodies react with the protein
moiety of the HGF-R β chain extracellular domain
Previous work from this laboratory showed that in the human
gastric carcinoma cell line GTL-16 the MET proto-oncogene
is amplified and overexpressed (Ponzetto et al., 1991). Living
GTL-16 cells were thus used as immunogen to produce mAbs
against native epitopes of the HGF-R, as described in Materials
and Methods. The two selected anti-HGF-R mAbs (DO-24 and
DN-30) were tested for their reactivities in different assay
formats, as summarized in Table 1. Briefly, the two mAbs were
found to react with native epitopes of the extracellular domain
of the receptor, since they stained living GTL-16 cells in
immunofluorescence, while neither of the two reacted with the
denatured form of HGF-R in western blotting.
The mAbs identify epitopes localized on the β chain of the
HGF-R. In fact, as shown in Fig.1A, they were able to
precipitate the p145 β chain from cells transfected with the
cDNA encoding the β chain of the MET gene.
The two mAbs recognize epitopes localized in the protein
Table 1. Properties and reactivities of anti-HGF-R
antibodies on GTL-16 cells
Ig isotype and subclass
Immunofluorescence
Immunoprecipitation
ELISA
Western blot
Kd (nM)
DO-24
DN-30
γ2a/k
+
+
+
−
0.69
γ2a/k
+
+
+
−
2.64
All the assays were performed as described in Materials and Methods.
Fig. 1. (A) The anti-HGF-R mAbs react with the receptor β chain.
NIH-3T3 fibroblasts transfected with the cDNA encoding the β chain
of HGF-R were lysed and immunoprecipitated with DO-24 (lane 1),
DN-30 (lane 2) and R1.30 (lane 3, negative control) mAbs. Western
blot analysis was performed with anti-HGF-R DL-21 mAb. The p145
β chain is decorated. (B) The anti-HGF-R mAbs react with the
receptor protein moiety. GTL-16 cells were metabolically labelled
with [35S]methionine either in the absence (lanes 1, 2) or in the
presence (lanes 3, 4) of tunicamycin. Soluble proteins were
precipitated with DO-24 (lanes 1, 3) and DN-30 (lanes 2, 4) and
separated in SDS-PAGE under reducing conditions. In these
conditions the p50 α chain and the p145 β chain are precipitated
from the untreated cells, while the unglycosylated precursor (150
kDa) is precipitated from tunicamycin-treated cells.
moiety of the HGF-R. Immunoprecipitations were performed
with extracts from GTL-16 cells, metabolically prelabelled
with [35S]methionine in the absence or presence of
tunicamycin, a drug inhibiting N-linked glycosylation. In the
latter conditions, both mAbs precipitated a molecule of 150
kDa, corresponding to the non-glycosylated precursor (Fig.
1B).
The reactivity of these mAbs was also tested in species other
than the human, in view of their possible use in different
biological assays on cells from different species. They were
found to cross-react with the HGF-R of the canine and murine
species, with similar affinities (data not shown).
The monoclonal antibodies recognize independent
epitopes on the HGF-R which are different from the
ligand binding site
The spatial relationship of the epitopes defined by the two
mAbs was analyzed in competition experiments, where the
ability of one unlabelled mAb to displace the binding of the
other 125I-mAb to paraformaldehyde-fixed GTL-16 cells was
evaluated. The two mAbs did not reciprocally interfere, while
each one blocked the binding of itself (results with 125I-DO-24
are shown in Fig. 2A). It is thus concluded that they identify
two independent epitopes.
Since HGF binds not only to the high affinity HGF-R but
also, although with lower affinity, to heparin sulfate
proteoglycans associated with the cell membrane (Naldini et
al., 1991), inhibition assays performed on the binding of 125IHGF to tissue culture cells were not perfectly suited. We thus
developed a double determinant radioimmunometric assay,
using the soluble HGF-R isoform. Briefly, an antibody
recognizing a different epitope on the HGF-R (DL-21 mAb)
was used to coat polyvinyl hydrochloride microtiter wells and
catch the soluble HGF-R isoform, followed by incubation with
125I-HGF in the absence or presence of unlabelled competitors.
None of the mAbs was found to compete for the binding with
Biological effects of agonist mAbs to HGF-R
241
Fig. 2. The anti-HGF-R mAbs recognize different epitopes and these
are different from the ligand binding site. (A) Increasing amounts of
unlabelled competitors (DO-24: d; DN-30: s; HGF: j) were used
to displace the binding of 125I-DO-24 mAb to living GTL-16 cells.
Only unlabelled DO-24 mAb is able to compete, while unlabelled
DN-30 mAb and HGF have no effect. (B) Increasing amounts of
unlabelled competitors (DO-24: d; DN-30: s; HGF: j) were used
to displace the binding of 125I-HGF to p130MET HGF-R isoform
bound to DL-21 anti-HGF-R mAb in a solid phase DDIA. None of
the mAbs compete for binding with radiolabelled HGF.
125I-HGF
(Fig. 2B). In the mirror experiments, HGF could not
displace the binding of radiolabelled mAbs to HGF-R (not
shown and Fig. 2A).
The two monoclonal antibodies stimulate cell motility
Both mAbs efficiently scattered the human A549 and canine
MDCK cells, at nM concentrations (Fig. 3), in a manner
indistinguishable from HGF. For the DO-24 mAb the minimal
concentrations required were 1.5 nM (A549) and 0.2 nM
(MDCK). For the DN-30 mAb the minimal effective
concentrations were 12 nM (A549) and 2.5 nM (MDCK). The
difference between the two mAbs probably reflects their
relative affinities (see Table 1). The lower amount of mAbs
required to scatter MDCK cells, as compared to A549 cells,
probably reflects a better susceptibility of the former cells to
scatter. Fab fragments prepared from the two mAbs were
ineffective in scattering these cells, inducing only a slight cell
spreading (not shown). A mAb directed against β2
Fig. 3. The anti-HGF-R mAbs stimulate cell dissociation of A549 (A,B,C,D) and MDCK (E,F,G,H) cells. Cells were plated at low density and
incubated for 18 hours in the absence (A,E) or in the presence of 0.03 nM HGF (B,F), 5 nM purified DO-24 mAb (C,G), 30 nM purified DN-30
mAb (D,H). Bar, 20 µm.
242
M. Prat and others
microglobulin, used as negative control, was ineffective (not
shown).
Monoclonal antibody DO-24, but not DN-30,
stimulates the full array of HGF-dependent
biological effects
The mitogenic activity of the anti-HGF-R mAbs was evaluated
on cultures of human endothelial cells. The different antibody
concentrations chosen were added every 2 days for 8 days. DO24 induced proliferation of human endothelial cells, causing a
3-fold increase in cell number, while DN-30 was ineffective
(Fig. 4A). The stimulatory activity of DO-24 was dose
dependent, with maximal effect in the nM range, which
corresponds to the mAb affinity, and it was comparable to that
induced by 0.02 nM HGF. DN-30 was unable to induce
proliferation also when used at very high concentrations (up to
900 nM; Fig. 4A and data not shown). The anti-β2
microglobulin mAb was negative in this assay at any
concentration used (not shown). Fab fragments prepared from
the two mAbs were ineffective, and the biological response was
reconstituted for DO-24 Fab upon the addition of a secondary
anti-mouse Ig antibody (Fig. 4A). These data indicate that mAb
bivalence is necessary for receptor-mediated cell proliferation,
thus suggesting that HGF-R dimerization is an essential
requirement for this biological effect.
It was reported that proliferation assays on MDCK cells can
be performed by culturing them between sheets of collagen
gels (Weidner et al., 1993). On the basis of this finding, MDCK
cells were cultured within collagen matrices; DO-24 mAb, but
not DN-30 mAb, stimulated [3H]thymidine incorporation, in a
dose-response manner (Fig. 4B). The stimulatory activity was
maximal in the nM range. The HGF and DO-24 mAb doses
required to give maximal effect were higher than that required
in the proliferation assay performed on endothelial cells; this
likely reflects the different conditions of the assays or the better
susceptibility of endothelial cells to proliferate.
The morphogenic activity of the anti-HGF-R mAbs was
visualized as the ability to induce the formation of tubular
structures in MDCK cells cultured in collagen matrices. DO24 mAb (Fig. 5), but not DN-30 mAb, induced the outgrowth
Fig. 5. Branching tubulogenesis activity of anti-HGF-R
mAbs. MDCK cells were grown in collagen gels for 6
days in the absence (A) or in the presence of 0.2 nM HGF
(B), 20 nM DO-24 mAb (C) and 100 nM DN-30 mAb (D).
Phase-contrast microscopy. Bar, 100 µm.
Fig. 4. Mitogenic activity of anti-HGF-R mAbs. (A) Mitogenic
activity in human endothelial cells was determined by measuring cell
growth. (B) Mitogenic activity in MDCK cells grown in collagen
gels was determined by measuring DNA synthesis.
of tubules within a few days, in a dose-response manner,
closely paralleling the dose-response curve observed in the
Biological effects of agonist mAbs to HGF-R
Fig. 6. In vivo angiogenesis effects of anti-HGF-R mAbs. Matrigel in
liquid form was mixed with PBS (A), 0.5 nM HGF (B), 16 nM DO24 mAb (C), 100 nM purified DN-30 mAb (D) and injected into the
abdominal subcutaneous tissues of mice. After 10 days, mice were
sacrificed and the Matrigel plugs were excised and photographed.
Bar, 2.5 mm.
DNA synthesis assay. In parallel experiments, the DN-30 mAb
was unable to antagonize HGF-induced tubulogenesis, even
when used at very high doses (data not shown).
To assess a morphogenic effect of the anti-HGF-R mAbs in
vivo, we performed a murine angiogenesis assay. mAbs and
HGF were incorporated in Matrigel, a matrix of reconstituted
basement membrane, and injected subcutaneously into mice.
Fig. 7. Matrix invasion activity of antiHGF-R mAbs. MDCK cells were grown
on Matrigel coated filters in the absence
(A) or in the presence of 0.1 nM HGF
(B), 20 nM DO-24 mAb (C) and 100 nM
DN-30 mAb (D). After 72 hours
incubation, the non-invasive cells on the
upper side of the filter were removed and
the invasive cells on the lower side of the
filter were stained. Bar, 40 µm.
243
After 10 days, mice were sacrificed and plugs analyzed. DO24 mAb clearly induced plug vascularization (Fig. 6), although
it did not achieve the HGF maximal effect. DN-30 was
ineffective at any concentration tested. No evidence of
inflammation was observed in any case.
The morphogenic assays reported are the best suited assays
to detect an invasive growth phenomenon, which results from
the combination of proliferation, motility and extracellular
matrix/basal lamina (ECM/BM) degradation and cell survival.
To detect the ability of the mAbs to stimulate invasiveness we
performed an in vitro invasion assay that detected cell ability
to migrate through a BM(Matrigel)-coated Transwell filter.
MDCK cells were allowed to invade the filter for 72 hours. No
invasive cells were found in the absence of treatment or in the
presence of DN-30 mAb (Fig. 7). In the presence of either HGF
or DO-24 mAb a large number of invasive cells migrated
through the filter.
HGF is known to protect cells from apoptosis induced by
disruption of cell adhesion, as it occurs during invasive growth.
We thus tested the ability of the two mAbs to protect MDCK
cells from staurosporin induced apoptosis, as measured in a
TUNEL assay. 20 nM DO-24 mAb, as well as 0.3 nM HGF,
gave 50% protection, while 200 nM DN-30 mAb was
ineffective.
From all these data it is clear that in no case could DN-30
mAb induce an invasive growth response, even when used at a
concentration 100 times higher than the one required by DO24 mAb.
Only the full agonist mAb induces an early and
prolonged expression of uPA-R
The main differential biological effect triggered by the two
mAbs was represented by the invasive growth. The uPA/uPAR proteolytic network plays a crucial role in cell surface
associated degradation of the pericellular matrix. We thus
244
M. Prat and others
Fig. 8. Effects of anti-HGF-R mAbs on uPA and uPA-R mRNA
expression. Quiescent MDCK cells were stimulated with 0.3 nM
HGF, 50 nM DO-24 and 300 nM DN-30, lysed after the indicated
times and analyzed by northern blot. The same filter was hybridized
repeatedly with cDNA probes for the genes uPA, uPA-R and GADH.
The uPA and uPA-R probes hybridized with single transcripts of
about 2.5 kb and 1.4 kb, respectively.
investigated the ability of the two mAbs to induce mRNA
expression of uPA and its receptor in MDCK cells. We found
that DO-24 mAb stimulation induced uPA-R expression after
1 hour, and that the response was sustained for 24 hours, with
a kinetics overlapping the one observed in the case of HGF
stimulation (Fig. 8). By contrast DN-30 mAb induced a weaker
uPA-R expression only after 9 hours of treatment, and this
response had already decayed at 24 hours. With all stimulating
treatments, uPA expression was induced later, being evident
after 3 hours of stimulation and increasing further in the
following 24 hours. Thus the prolonged and sustained
induction of uPA-R correlates with the invasive growth
phenotype triggered by both HGF and DO-24 mAb.
The two monoclonal antibodies trigger tyrosine
phosphorylation of the HGF-R
We tested the ability of the anti-HGF-R mAbs to mimic ligandinduced phosphorylation on A549 and MDCK cells. Quiescent
cells were incubated with 100 nM mAbs for 10 minutes at
37°C, extracted in RIPA buffer and precipitated with a cocktail
Fig. 9. The anti-HGF-R mAbs
stimulate tyrosine phosphorylation of
receptor β chain and antibody
bivalence is essential. Quiescent A549
(A) and MDCK (B) cells were
incubated in the absence (lane 1) or in
the presence of 1 nM HGF (lane 2), 50
nM DO-24 mAb (lane 3), 300 nM DN30 mAb (lane 4) for 10 minutes at
37°C. Cells were solubilized in RIPA
buffer and soluble proteins were
precipitated with a cocktail of antiHGF-R mAbs (DO-24 and DN-30)
cross-linked to Sepharose Protein A,
separated in SDS-PAGE, blotted on
nitrocellulose paper and probed with
anti-phosphotyrosine mAbs. A549 cells (C) were incubated with 600
nM Fab prepared from DO-24 (lanes 1, 3) and DN-30 (lanes 2, 4)
mAbs in the absence (lanes 1, 2) or in the presence of rabbit antimouse Ig (lanes 3, 4) for 10 minutes at 37°C and treated as above.
of anti-HGF-R mAbs (DO-24 and DN-30) cross-linked to
Sepharose Protein A, followed by western blot with antiphosphotyrosine mAbs. As depicted in Fig. 9, the two antiHGF-R mAbs, as well as HGF, induced phosphorylation of
tyrosine residues of the receptor β subunit. A mAb directed
against β2 microglobulin (not shown), as well as medium alone
were ineffective. Under the assay conditions used, mAbdependent HGF-R phosphorylation was observed only when
stimulating mAbs were incubated with living cells, but not
when they were added to cell extracts (not shown). Fab
fragments prepared from the two mAbs were ineffective as
stimulators of phosphorylation at any dose tested, up to 600
nM (Fig. 9). The ability to activate the HGF-R was recovered
upon addition of a secondary anti-mouse Ig antibody. These
data indicate that HGF-R phosphorylation requires
dimerization.
DISCUSSION
Differential effects of anti-HGF-R monoclonal
antibodies on motility and invasive growth
We have found that a mAb directed against an epitope of the
extracellular domain of the human HGF-R behaves as full
agonist, evoking all the HGF-triggered biological effects
tested, namely cell motogenesis, mitogenesis, survival,
invasiveness, morphogenesis and angiogenesis. This mAb is
cross-reactive among different species and this finding allowed
us to analyze the different biological responses in well
established models, such as in vitro morphogenesis with the
canine MDCK cells and in vivo angiogenesis in the mouse.
The full agonist mAb interacts at the cell surface only with
the high affinity MET-encoded HGF-R. One of the important
conclusions that can be drawn from this work is that the
information necessary to transmit the HGF-mediated signal is
entirely contained in this receptor and does not involve the low
affinity HGF receptors heparansulfate and sulfoglycolipids.
The latter could play an indirect role in recruiting the ligand or
stabilizing it (Kobayashi et al., 1994; Mizuno et al., 1994;
Schwall et al., 1996). Data from other laboratories already
suggested the unique role of the high affinity HGF-R. In fact,
cells transfected with hybrid cDNA molecules encoding the
transmembrane and intracellular domains of the HGF-R fused
to different ligand binding domains respond to the cognate
ligand with scattering, matrix invasiveness, proliferation and
tubulogenesis (Komada and Kitamura, 1993; Weidner et al.,
1993; Zhu et al., 1994).
One mAb (DN-30) was found to be only partial agonist,
being able to trigger cell motility, but not all the other HGFmediated biological responses. The motogenic function has
been separated by other HGF-induced biological functions also
at the ligand level. The K2 truncated variant of HGF,
containing only the N-terminal hairpin and the first two kringle
domains, is sufficient to mediate high affinity binding to HGFR, but it promotes only motogenesis and not mitogenesis
(Hartmann et al., 1992; Lokker et al., 1992), tubulogenesis
(Sachs et al., 1996), invasion (Jeffers et al., 1996) and
angiogenesis (Silvagno et al., 1995). In this paper we have
shown that protection from apoptosis also can be dissociated
from motility and, similarly to the other biological effects, has
further requirements. Although the partial agonist mAb
Biological effects of agonist mAbs to HGF-R
behaves like the α chain isoform of HGF, the mechanism of
action must be different. In fact, it does not compete with the
ligand and it does not act as antagonist in mitogenesis, while
the K2 variant contains the receptor binding site and acts as an
antagonist in mitogenesis (Chan et al., 1993; Lokker and
Godowski, 1993).
Invasive growth is the distinguishing feature elicited by HGF
and the full agonist mAb, but not the partial agonist mAb. This
phenomenon likely involves the induction of proteases that
mediate the degradation of the extracellular matrix/basal
membrane (ECM/BM) proteins. UPA can catalyze this
degradation by virtue of its ability to convert plasminogen to
active plasmin. The latter, in fact, can activate the
metalloproteinases, a class of proteases with potent ECM/BMdegrading action. The binding of uPA to its high affinity
receptor uPA-R at the cell surface enhances the rate of
plasminogen activation and localizes the uPA activity, therefore
enhancing the ECM/BM degradation and cell invasion. Also
HGF can bind uPA at the cell surface (Naldini et al., 1995) thus
providing an external cue for activation and localization of the
invasive response. HGF is also able to increase the expression
and synthesis of both uPA and its receptor (Boccaccio et al.,
1994; Jeffers et al., 1996; Pepper et al., 1992). In this work we
have found that the full agonist mAb induces the uPA and uPAR mRNA expression with kinetics similar to that of HGF,
therefore reinforcing the correlation between activation of
HGF-R and induction of the uPA proteolysis network. More
interestingly, we have found that the partial agonist mAb is
unable to induce a prolonged expression of uPA-R and it is able
to induce uPA expression with a delayed kinetics. Thus in this
case the uPA/uPA-R proteolytic network is not efficiently
activated. It has been shown that the K2 variant is unable to
induce the invasion of human sarcoma cells and concomitantly
the expression of both uPA and uPA-R proteins (Jeffers et al.,
1996). From this aspect the partial agonist mAb and the K2
variant differ, the mAb being able to up-regulate uPA
expression. It has recently been shown that uPA-R can function
as an adhesion receptor for vitronectin and cause
destabilization of integrin-dependent adhesion (Wei et al.,
1996). Therefore, increased expression of uPA-R may be
meaningful for efficient invasiveness not only by virtue of
enhanced ECM/BM degradation but also for loss of stable
cellular adhesion.
Mechanisms of activation of HGF-R
Ligand-induced oligomerization is the most likely mechanism
responsible for activation of growth factor receptors. This can
be achieved through: (i) the action of a dimeric ligand, such as
PDGF (Kelly et al., 1991); (ii) the binding of a monomeric
ligand that stabilizes pre-associated receptors, such as EGF
(Hurwitz et al., 1991; Yarden and Schlessinger, 1987); (iii) the
binding of a monomeric ligand that contains two receptor
binding sites, such as GH (Fuh et al., 1992). The results
obtained with the agonist mAbs allow us to draw some
conclusions on the mechanisms underlying HGF-R activation.
This clearly involves receptor dimerization, since the
monovalent Fabs were completely ineffective in inducing
receptor phosphorylation and hence activation of HGF
signaling. However, the differential behaviour of the two
mAbs, which have similar affinities and can both induce
tyrosine receptor phosphorylation, suggests that simple
245
dimerization is not sufficient. Among the many mAbs
produced against the extracellular domain of tyrosine kinase
receptors, only a few have been reported to mimic both early
and delayed ligand-mediated cellular responses (Nagy et al.,
1990; Harwerth et al., 1993; Schreiber et al., 1981; Schreurs et
al., 1989; Stancovski et al., 1991; Yarden, 1990). and most of
them recognize the ligand binding site. In our case, the full
agonist mAb defines an epitope present on the protein moiety
of the HGF-R β chain, where the ligand binding site is
localized, but does not compete with it. A likely explanation is
that the DO-24 mAb recognizes and stabilizes a
conformationally active form of the receptor. Indeed the
antibody was raised against cells which express a constitutively
activated receptor, as a consequence of the MET oncogene
amplification and overexpression (Ponzetto et al., 1991). This
indicates that another region of HGF-R β chain is involved in
optimal receptor activation. We thus propose that full HGF-R
activation by mAb DO-24 results from the cooperative action
of dimerization and binding to an epitope critically required to
gain full biological response.
The molecular mechanisms by which full activation of HGFR leads to the invasive growth phenotype are largely unknown.
Substantial evidence supports the idea that it depends on the
integrity of the multifunctional docking site and requires
concomitant activation of multiple signalling pathways
(Giordano et al., 1997). The two mAbs efficiently activate the
intrinsic tyrosine kinase activity of the receptor, which is
mainly directed on the two major phosphorylation sites
mapped at Y1234 and Y1235 (Longati et al., 1994). It is possible
that receptor activation induced by the two mAbs ends up with
qualitative and/or quantitative differences in phosphorylation
of the docking site and thus different recruitment of transducers
at the plasma membrane. It is also possible that the tyrosine
kinase activity towards various substrates is different. The two
mAbs described here will be powerful tools to identify the
signalling pathways and substrates differentially activated to
reach the different biological responses.
The technical assistance of Rita Callipo, Raffaella Albano and
Laura Palmas is gratefully acknowledged. This work was supported
by grants from the Associazione Italiana Ricerca Cancro (A.I.R.C.),
and the National Research Council (C.N.R.: P.F.-A.C.R.O. no.
96.00556.PF39). S. Pennacchietti was supported by a fellowship from
the Associazione Italiana Ricerca Cancro (A.I.R.C.).
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