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J Biol Chem, Vol. 274, Issue 10, 6499-6506, March 5, 1999
From the Department of Pathology, Academic Medical Center,
University of Amsterdam, 1105 AZ Amsterdam, The Netherlands, the
§ Center for Human Genetics and Flanders Interuniversity
Institute for Biotechnology, University of Leuven, B-3000 Leuven,
Belgium, and the ¶ Growth Factors Group, Department of
Oncology, University of Cambridge,
Cambridge CB2 2QH, United Kingdom
CD44 has been implicated in tumor progression and
metastasis, but the mechanism(s) involved is as yet poorly understood.
Recent studies have shown that CD44 isoforms containing the
alternatively spliced exon v3 carry heparan sulfate side chains and are
able to bind heparin-binding growth factors. In the present study, we
have explored the possibility of a physical and functional interaction
between CD44 and hepatocyte growth factor/scatter factor (HGF/SF), the
ligand of the receptor tyrosine kinase c-Met. The HGF/SF-c-Met pathway
mediates cell growth and motility and has been implicated in tumor
invasion and metastasis. We demonstrate that a CD44v3 splice variant
efficiently binds HGF/SF via its heparan sulfate side chain. To address
the functional relevance of this interaction, Namalwa Burkitt's
lymphoma cells were stably co-transfected with c-Met and either CD44v3
or the isoform CD44s, which lacks heparan sulfate. We show that, as
compared with CD44s, CD44v3 promotes: (i) HGF/SF-induced
phosphorylation of c-Met, (ii) phosphorylation of several downstream
proteins, and (iii) activation of the MAP kinases ERK1 and -2. By
heparitinase treatment and the use of a mutant HGF/SF with greatly
decreased affinity for heparan sulfate, we show that the enhancement of
c-Met signal transduction induced by CD44v3 was critically dependent on
heparan sulfate moieties. Our results identify heparan sulfate-modified CD44 (CD44-HS) as a functional co-receptor for HGF/SF which promotes signaling through the receptor tyrosine kinase c-Met, presumably by
concentrating and presenting HGF/SF. As both CD44-HS and c-Met are
overexpressed on several types of tumors, we propose that the observed
functional collaboration might be instrumental in promoting tumor
growth and metastasis.
The CD44 family of cell surface glycoproteins is broadly expressed
by cells of epithelial, mesenchymal, and hematopoietic origin and is
involved in cell-matrix adhesion, hematopoiesis, and lymphocyte homing
and activation (1). Furthermore, a large body of experimental and
clinical studies support a role for CD44 in tumor progression and
metastasis (2-4). The CD44 gene consists of 19 exons (5). Due to
alternative splicing, which involves at least 10 exons encoding domains
of the extracellular portion of the CD44 molecule, a large number of
CD44 isoforms is generated (6-10). Post-translational modification
generates further diversity, yielding both N-linked and
O-linked glycan forms of CD44 in addition to proteoglycan
variants containing chondroitin, keratan, or heparan sulfate (11-14).
The expression pattern of these CD44 variants is tissue-specific. On
lymphocytes the short 80-90-kDa standard form of CD44
(CD44s)1 is most abundant,
while larger variants (CD44v) predominate on some normal and neoplastic
epithelia and are also found on activated lymphocytes and on malignant
lymphomas (15-19). This selective expression suggests specific
biological functions for the various splice variants, but at present,
these are poorly defined. Similarly, the mechanism(s) through which
CD44 functions in tumorigenesis is not known.
An obstacle toward understanding the functions of the CD44 family is
the limited knowledge of its molecular partners. The cytoplasmic tail
of the CD44 molecule has been shown to interact with the actin
cytoskeleton via ankyrin and proteins of the ERM family, and is
associated with Src family tyrosine kinases (20-23). This suggests a
role in signaling as well as in the regulation of cell shape and
motility. Although several potential CD44 ligands have been identified,
the only interaction of the extracellular domain of CD44 that has been
extensively studied is that with hyaluronate. CD44s acts as a major
receptor for this glycosaminoglycan which is highly abundant in
mesenchymal tissues and is believed to play a role in cell migration
and differentiation (24, 25).
A novel and potentially highly significant function of CD44 is its
ability to interact with heparin-binding growth factors (26, 27). These
growth factors bind to a HS side chain attached to the evolutionary
conserved consensus motif SGSG encoded by exon v3 (13, 27). Heparan
sulfate proteoglycans (HSPGs) are believed to play an important
regulatory role in cell growth and motility by binding growth factors
and by presenting these factors to their high affinity receptors. This
process has been particularly well explored for the fibroblast growth
factors 1 and 2 (FGF-1 and -2). For these factors, binding to HSPGs has
been shown to be required for their biological function, presumably by
promoting FGF dimerization required for efficient receptor
cross-linking and activation (28-32).
In the present study, we explored the physical and functional
interaction between heparan sulfate-modified forms of CD44 (CD44-HS) and hepatocyte growth factor/scatter factor (HGF/SF). HGF/SF is a
heparin-binding growth factor (33) that induces growth, motility, and
morphogenesis of target epithelial and endothelial cells by binding to
the receptor tyrosine kinase c-Met (34, 35). In addition, recently
HGF/SF was shown to be involved in hematopoiesis, and lymphocyte
adhesion and migration (36-42). Apart from these physiological
functions, there is ample evidence for a key role of the HGF/SF-c-Met
pathway in tumor growth, invasion, and metastasis. For example, HGF/SF
induces epithelial cells to invade collagen matrices in
vitro, and NIH 3T3 cells co-transfected with c-met and
HGF/SF acquire an invasive and metastatic phenotype (43-45). Furthermore, in HGF/SF transgenic mice, tumors develop in many different tissues including mammary glands, skeletal muscles, and
melanocytes (46). In human cancer, both HGF/SF and c-Met are often
overexpressed, and in hereditary renal cancer germline mutations in the
c-met gene have recently been reported (47-52). Here, we
show that CD44-HS strongly promotes signal transduction through the
HGF/SF-c-Met pathway, which is demonstrated to occur in a heparan
sulfate-dependent fashion.
Antibodies--
Mouse monoclonal antibodies (mAbs) used were
anti-pan CD44, NKI-P1 (IgG1) (53), and Hermes-3 (IgG2a) (54) (a gift
from S. Jalkanen, University of Turku, Turku, Finland), anti-HGF/SF, 24612.111 (IgG1) (R&D Systems, Abington, United Kingdom), anti-heparan sulfate, 10E4 (IgM) (55), anti-desaturated uronate from
heparitinase-treated heparan sulfate (" Cell Lines and Transfectants--
The Burkitt's lymphoma cell
line Namalwa was purchased from American Type Culture Collection (ATCC,
Rockville, MD). The cells were cultured in RPMI 1640 (Life
Technologies, Breda, The Netherlands) supplemented with 10% Fetal
Clone I serum (HyClone Laboratories, Logan, UT), 10% fetal calf serum
(Integro, Zaandam, The Netherlands), 2 mM
L-glutamine, 100 IU/ml penicillin, and 100 IU/ml
streptomycin (all Life Technologies). Namalwa cells transfected with
CD44s (Nam-S), CD44v8-10 (Nam-V8), or CD44v3-10 (Nam-V3) were
described previously (56). A second transfection of Namalwa cells,
expressing either CD44s (Nam-SM) or CD44v3-10 (Nam-V3 M),
with c-Met was performed as described (41).
Purification of Wild Type and Mutant HGF/SF--
The
construction of pVL1393 vectors (Pharmingen, San Diego, CA) containing
wild type or mutant HGF/SF (HP1) cDNA was described elsewhere
(57).
HGF/SF (wild type and HP1) was produced in a baculovirus system as
described previously (58). In brief, Sf9 insect cells were
transduced with an amplified virus stock and after 3 days media were
pooled and analyzed for scattering activity in the Madin-Darby canine
kidney cell dissociation assay (59). Then, HGF/SF was purified with
Ni-NTA resin from the QIAexpress system (Qiagen, Hilden, Germany).
HGF/SF concentrations were measured by enzyme-linked immunosorbent
assay as described previously (41). In addition, HGF/SF (wt
and HP1) was analyzed by Western blotting using goat anti-HGF/SF.
Enzyme Treatments--
For enzymatic cleavage of
glycosaminoglycans, cells were treated with either heparitinase
(Flafobacterium heparinum, EC 4.2.2.8, ICN Biomedicals, Aurora, OH) or
chondroitinase ABC (Proteus vulgaris, EC 4.2.2.4, Boehringer Mannheim,
Almere, The Netherlands) in PBS at 37 °C for the periods indicated.
Enzyme treatments were followed by FACS analysis or immunoprecipitation.
FACS Analysis--
For FACS analysis cells were blocked with
10% pooled human serum (CLB, Amsterdam, The Netherlands), 1% bovine
serum albumin (Fraction V) (Sigma, Bornem, Belgium) in PBS at 4 °C
for 15 min and washed with FACS buffer (1% bovine serum albumin in
PBS), respectively. Then, the cells were incubated with the primary antibodies for 1 h, washed, and incubated with the secondary
antibody for 30 min. Incubations were in FACS buffer at 4 °C, and
cells were analyzed by using a FACScan (Becton Dickinson, Mountain
View, CA).
For binding of recombinant human HGF/SF (wild type or HP1) (R&D Systems
or our own product), cells were incubated with this protein (18 nM or as indicated) for 1 h, prior to the antibody incubations. This step was followed by washing with FACS buffer.
Immunoprecipitation and Western Blot
Analysis--
Immunoprecipitation was performed as described (41). The
only modifications were that, for precipitation of CD44, cells were
lysed in lysis buffer containing 50 mM Tris-HCl (pH 8), 150 mM NaCl, 1% Nonidet P-40, 10 µg/ml aprotinin (Sigma), 10 µg/ml leupeptin (Sigma), 1 mM sodium orthovandate
(Sigma), 2 mM EDTA, and 5 mM sodium fluoride.
For precipitation of c-Met, cells were lysed in 10 mM
Tris-HCl (pH 8), 150 mM NaCl, 10% glycerol, 1% Nonidet
P-40, 10 µg/ml aprotinin (Sigma), 10 µg/ml leupeptin (Sigma), 2 mM sodium orthovandate (Sigma), 5 mM EDTA, and
5 mM sodium fluoride.
Western blotting of immunoprecipitates and total cell lysates was
essentially performed as described previously (23). A single
modification was that, for analysis of phosphorylated proteins, membranes were blocked and stained in 2% bovine serum albumin, 20 mM Tris-HCl, 150 mM NaCl (pH 7.5), and 0.05%
Tween 20 (Sigma). Films were scanned with an Eagle Eye II video system
(Stratagene, La Jolla, CA) and band intensities were determined with
ONE-Dscan software (Stratagene). c-Met phosphorylation was expressed as the ratio of phosphorylated c-Met to c-Met precipitated.
For analysis of phosphorylation of the ERK1 and -2 MAP kinases, after
the indicated treatments, 5 × 105 cells were directly
lysed in sample buffer and analyzed by 10% SDS-polyacrylamide gel
electrophoresis and blotted. Equal loading was confirmed by Ponceau S
staining of the blot. The part of the blot below 50 kDa was stained
with anti-phospho-MAPK antiserum, the upper part with
anti-phosphotyrosine PY-20. Primary antibodies were detected by
HRP-conjugated goat anti-rabbit and HRP-conjugated rabbit anti-mouse,
respectively. Identification of the ERKs was confirmed by staining with
anti-ERK1 or anti-ERK2.
Binding of HGF/SF to CD44 Isoforms--
Binding of HGF/SF to
different CD44 isoforms was assessed by using a panel of Namalwa
Burkitt's lymphoma cell lines stably transfected with cDNAs
encoding CD44s, CD44v8-10, or CD44v3-10 (Fig.
1A) (56). Prior to
transfection, the cells were negative for CD44 and c-Met expression at
both the protein and mRNA level (data not shown). All transfectants
used for HGF/SF binding studies expressed comparable levels of CD44
(Table I). HGF/SF binding to the CD44
transfectants was measured by FACS analysis using an anti-HGF/SF mAb,
an approach that avoids chemical modification of the ligand. As shown
in Fig. 1B, CD44 negative control cells as well as CD44s and
CD44v8-10 transfectants showed a low saturable binding of HGF/SF. In
contrast, cells expressing CD44v3-10 bound much larger quantities of
HGF/SF. These results suggest that CD44v3-10 contains a binding
site(s) for HGF/SF.
Binding of HGF/SF to CD44 Is Heparan
Sulfate-dependent--
We next conducted a series of
experiments aimed at determining the role of HS side chains in the
binding of HGF/SF. First, the presence of total HS on the different
transfectants was assessed by FACS analysis using the HS-specific mAb
10E4 (Fig. 2A), and the mAb
3G10 (Fig. 2B) which recognizes the
To assess the role of HS in the interaction between HGF/SF and
CD44v3-10, we studied the effect of heparitinase treatment and
performed binding studies with HP1, a HGF/SF mutant which has a greatly
decreased (more than 50-fold) affinity for heparan sulfate and heparin
(57). As shown in Fig. 3A,
heparitinase treatment resulted in a near complete loss of HGF/SF
binding, while treatment with chondroitinase ABC had no effect. The
essential role of HS moieties on CD44v3-10 in HGF/SF binding was
further confirmed by the observation that HP1 did not bind to
CD44v3-10 (Fig. 3B). These data demonstrate that CD44v3-10
is a heparan sulfate-modified CD44 isoform (CD44-HS) that binds HGF/SF
via its HS side chain.
CD44-HS Promotes c-Met Activation--
To explore the functional
impact of HGF/SF bound to CD44-HS on the c-Met signaling pathway, we
generated double transfectants expressing c-Met in combination with
either CD44v3-10 or CD44s. We selected stable transfectants expressing
equal amounts of c-Met to be used in the subsequent studies (Fig.
4). Using these cell lines, we assessed
in the first instance HGF/SF induced c-Met phosphorylation. As shown in
Fig. 5, triggering with HGF/SF led to a
vast and rapid increase in the phosphorylation of c-Met on tyrosine
residues in the cells expressing CD44v3-10. By contrast, phosphorylation of c-Met was only weakly increased in the cells with
CD44s (Fig. 5) and was absent in the parental cell line (data not
shown), confirming the lack of endogenous c-Met in these cells. The
dose-response studies demonstrated that CD44v3-10 promotes c-Met
phosphorylation over a broad dose range (Fig. 5A) with an approximately 7-fold relative increase at plateau level. The time curve
(Fig. 5B) showed that phosphorylation was maximal between 2 and 10 min after addition of the growth factor and declined thereafter.
Moreover, this strong enhancing effect of CD44v3-10 on c-Met
phosphorylation was dependent on HS moieties since it was lost upon
heparitinase treatment (Fig.
6A). The importance of HS for
HGF/SF signaling was further strengthened by studies using the HGF/SF
heparin-binding domain mutant HP1. This mutant induced an equal (weak)
phosphorylation of c-Met in both the CD44v3-10 and CD44s transfectants
(Fig. 6B). Thus, these data suggest that CD44v3-10 binds
HGF/SF via its HS side chains and then presents it to the high affinity
receptor c-Met.
CD44-HS Promotes Downstream Signaling through c-Met in a Heparan
Sulfate-dependent Fashion--
The pivotal role of CD44-HS
in promoting the action of HGF/SF was further supported by analyzing
the cell lysates of HGF/SF-stimulated cells for tyrosine-phosphorylated
proteins. We observed tyrosine phosphorylation of several substrates,
the two most prominent phosphoproteins of unknown identity are found at
115-125 kDa. A minor phosphoprotein is found at 145 kDa which likely
represents c-Met (Fig. 6C). In addition, several smaller
phosphoproteins of unknown origin were observed (not shown) including a
42-kDa phosphoprotein which may represent the p42 ERK2 MAP kinase.
In order to establish whether signal transduction by c-Met is
potentiated by the HS moieties on CD44v3-10, we further investigated the activation of downstream targets of c-Met signaling. Since HGF/SF
has been shown to activate the ERK MAP kinases in Madin-Darby canine
kidney, HT29, and A549 cells (60-64), we assessed whether HGF/SF is
also able to induce MAP kinase activation in Namalwa B cells. For this
purpose we used an antibody recognizing only the active, phosphorylated
form of the ERK1 and -2 (p44 and p42) MAP kinases. As shown in Fig.
6C, HGF/SF treatment results in phosphorylation of the MAP
kinases ERK1 and -2 in Namalwa transfectants expressing c-Met. The
phosphorylation of the ERK2 MAP kinase upon HGF stimulation of the
cells was also confirmed by MAP kinase gel-shift
analysis.2 We observed
stronger phosphorylation of ERK1 and -2 in the CD44v3-10 expressing
cells as compared with the CD44s expressing cells (Fig. 6C,
bottom panel). Moreover, heparitinase treatment resulted in a
decrease of HGF/SF-induced ERK phosphorylation in the CD44v3-10 cells,
resulting in a level of ERK phosphorylation that is similar to the
level of HGF/SF-induced ERK phosphorylation in CD44s transfectants. HGF/SF-induced phosphorylation of the ERKs in CD44s transfectants remained unaffected by heparitinase treatment. Taken together, our data
demonstrate that signal transduction elicited by HGF/SF-induced c-Met
activation is strongly promoted by CD44-HS, and depends on the presence
of the HS moiety on CD44-HS.
We observed that cells transfected with CD44v3-10 efficiently
bind HGF/SF (Fig. 1) and that this CD44 isoform is decorated with HS
moieties (Fig. 2). By contrast, transfectants that express CD44s or
CD44v8-10, CD44 isoforms which are not modified with HS (Fig. 2), were
not able to bind HGF/SF above background (parental) levels (Fig. 1).
This selective HS modification of CD44v3-10 is in line with the recent
study by Jackson et al. (13) which demonstrated that HS side
chains bind to CD44 at the SGSG motif encoded by exon v3. Indeed, we
demonstrated that the interaction of HGF/SF with CD44v3-10 is
HS-dependent. Binding was completely abrogated by
heparitinase treatment, and HP1, a HGF/SF mutant with greatly decreased
affinity for heparan sulfate and heparin (57), failed to bind
CD44v3-10 (Fig. 3). Interestingly, it has been demonstrated that
specific chemical modifications of HS side chains on proteoglycans appear to regulate their affinity for selected heparin-binding growth
factors, including HGF/SF and FGF-2, and hence determine growth factor
binding specificity (65-69). This suggests that the HS moiety
covalently attached to CD44v3-10 contains specific binding sites for
HGF/SF.
The key finding of our study is that CD44-HS has a major functional
effect on HGF/SF-induced signal transduction. Expression of CD44-HS at
the cell surface led to a vast increase in HGF/SF-induced phosphorylation of c-Met on tyrosine residues (Fig. 5). Furthermore, it
resulted in a strong tyrosine phosphorylation of two as yet unidentified 115-125-kDa proteins that were hardly phosphorylated in
the absence of CD44-HS (Fig. 6C). One of these proteins
might represent p110/115-Grb2 associated binder (Gab)-1, an adaptor protein that has recently been found to associate with the
multifunctional docking site of c-Met (70). Alternatively, the observed
bands might be p120-Cbl and/or p125-FAK. Both protein tyrosine kinases participate in signal transduction via receptor protein tyrosine kinases and integrins (71, 72). This is particularly interesting given
our previous results that HGF/SF stimulation of Namalwa Burkitt's
lymphoma cells results in enhanced integrin We demonstrated that the enhancing effects of CD44-HS on signal
transduction via c-Met were critically dependent on the interaction of
HGF/SF with the HS moieties on CD44-HS, as they were not observed after
heparitinase treatment, or when the cells were triggered with the
heparin-binding domain HGF/SF mutant HP1 (Fig. 6). Importantly, the
specific effects of the heparitinase treatment and the mutations in HP1
on HGF/SF-induced signal transduction in the CD44v3-10 expressing
cells as compared with the CD44s cells demonstrates that the difference
in HGF/SF-elicited responses in these cells is not due to any possible
clonal variation in these stable cell lines. We speculate that CD44-HS
promotes the action of HGF/SF through concentration of HGF/SF on the
cell surface and by presenting it to the high affinity receptor c-Met
(Fig. 7). Similar mechanisms were
proposed for the role of high and low affinity receptors in FGF
functioning (32, 76, 77). In addition, CD44-HS might also protect
HGF/SF from proteolytic degradation as endothelial cell-derived HS was
shown to do for FGF-2 (78).
It should be noted, that, apart from growth factor presentation, CD44
may have additional functions in HGF/SF-c-Met mediated signaling. For
example, CD44 might recruit molecular partners into a multi-molecular
complex with c-Met. This possibility is suggested by the fact that two
recently identified cytoplasmic molecules associated with CD44 have
also been implicated in c-Met signaling. First, studies by Ponzetto
et al. (64) have shown that c-Met is a substrate for Src
family tyrosine kinases, while our own studies have revealed a physical
and functional association between CD44 and Src family member
p56lck (23). Second, studies by Jiang et al. (79)
and Crepaldi et al. (80) have demonstrated that HGF/SF
stimulates the tyrosine phosphorylation of the ERM protein ezrin. As
reported by Tsukita et al. (22), ERM proteins serve as
molecular linkers between CD44 at the cell surface and the actin
cytoskeleton. This interaction is believed to be involved in the
regulation of cell shape and motility.
We propose that collaboration between CD44-HS and growth factor
receptors, viz. c-Met, as shown in our present study, might be an important factor in tumor growth and metastasis. By
overexpressing CD44-HS, tumor cells would acquire a strongly increased
sensitivity to HGF/SF-mediated growth signals, leading to a growth
advantage and promoting metastasis (Fig. 7). This hypothesis is
supported by the fact that c-Met and HGF/SF are (over)expressed in
conjunction with CD44 in several types of tumors. In colorectal cancer,
for example, c-Met is frequently overexpressed (48, 49, 81), while
HGF/SF is expressed within the tumor tissue
microenviroment.3
Interestingly, in these tumors CD44 splice variants, including variants
decorated with HS, are often overexpressed and predict metastatic
spread and tumor related death (82, 83). A similar scenario may hold
for breast cancer and non-Hodgkin's lymphoma, as in these tumor types
overexpression of CD44v3 as well as c-Met has also been reported (19,
42, 51, 84).
In conclusion, we demonstrated that through binding and presenting
HGF/SF, CD44-HS promotes signal transduction via the receptor tyrosine
kinase c-Met. Consequently, overexpression of CD44-HS might give tumor
cells a growth and metastatic advantage and, in this way, might
influence disease outcome.
We thank Dr. M. Snoek for critical reading of
the manuscript and Drs. C. Figdor and S. Jalkanen for mAbs.
*
This work was supported by the University of Amsterdam,
Dutch Cancer Society Grant 98-1712, Het Praeventiefonds Grant 28-2575, and Human Capital and Mobility Program Grant BI02-CT94-7535 from the
European Community.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
**
To whom correspondence should be addressed: Dept. of Pathology,
Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. Tel.: 31-20-566-5635; Fax: 31-20-6960389; E-mail: S.T.Pals{at}AMC.UVA.NL.
2
M. Spaargaren and G. J. T. Zwartkruis,
unpublished observation.
3
V. J. M. Wielenga, unpublished observation.
The abbreviations used are:
CD44s, CD44 standard
isoform;
CD44-HS, heparan sulfate-modified CD44;
CD44v, CD44 variant
isoform;
ERK, extracellular signal regulated kinase;
ERM, ezrin radixin
moesin;
FGF, fibroblast growth factor;
HGF/SF, hepatocyte growth
factor/scatter factor;
HS, heparan sulfate;
HSPG, heparan sulfate
proteoglycan;
MAP, mitogen-activated protein;
mAb, monoclonal antibody;
PBS, phosphate-buffered saline;
HRP, horseradish peroxidase;
FACS, fluorescence-activated cell sorter;
RPE, R-phycoerythrin.
Heparan Sulfate-modified CD44 Promotes Hepatocyte Growth
Factor/Scatter Factor-induced Signal Transduction through the
Receptor Tyrosine Kinase c-Met*
,,
,
ABSTRACT
Top
Abstract
Introduction
References
INTRODUCTION
Top
Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
HS stub"), 3G10 (IgG2b)
(55), anti-phosphotyrosine, PY-20 (IgG2b) (Affiniti, Nottingham, United
Kingdom), and IgG1 and IgM control antibodies (ICN, Zoetermeer, The
Netherlands). Polyclonal antibodies used were rabbit anti-c-Met, C-12
(IgG) (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit
anti-phospho-p44/42 MAP kinase (Thr202/Tyr204)
(New England Biolabs, Beverly, MA), rabbit anti-ERK1 (C-16) and
anti-ERK2 (C-14) (Santa Cruz Biotechnology), RPE-conjugated goat
anti-mouse (Southern Biotechnology, Birmingham, AL), fluorescein isothiocyanate-conjugated rabbit anti-mouse (DAKO, Glostrup, Denmark), HRP-conjugated rabbit anti-mouse (DAKO), and HRP-conjugated goat anti-rabbit (DAKO).
RESULTS
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Fig. 1.
A, schematic representation of the CD44
gene, and the CD44v3-10, CD44v8-10, and CD44s cDNAs used for
transfection. Solid boxes represent constant exons, while
open boxes represent alternative exons. Note that, due to a
stop codon the variable exon 1 (v1) is not translated in the human.
UT, untranslated region; EC, extracellular
constant region; EV, extracellular variable region;
TM, transmembrane region; CT, cytoplasmic region.
B, binding of HGF/SF to CD44 Namalwa transfectants. Using a
FACS flow cytometer, one clone of mock transfected (Neo) Namalwa cells,
and two independent clones of CD44s, CD44v8-10 or
CD44v3-10-transfected Namalwa cells, were analyzed for their binding
of HGF/SF. Bound HGF/SF was detected with mouse anti-HGF/SF followed by
RPE-conjugated goat anti-mouse.
Surface expression of CD44 on Namalwa transfectants
HS stubs remaining on
HSPG core proteins after treatment with heparitinase (55). Both figures
show that cells transfected with CD44v3-10 express approximately
20-fold higher levels of HS compared with those transfected with other
CD44 isoforms. Next, we investigated the presence of HS on CD44 itself.
This was done by using mAb 3G10. With this mAb, a single major HS band
was detected in Western blots of CD44 precipitates from the CD44v3-10
cells, but not from the other transfectants (Fig. 2C). Staining the
blot with an anti-pan CD44 mAb demonstrated that this band corresponded
to CD44v3-10 (Fig. 2C).
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Fig. 2.
Presence of heparan sulfate on CD44
isoforms. A, FACS analysis of heparan sulfate expressed
on representative mock, CD44s, CD44v8-10, or CD44v3-10 Namalwa
transfectants that were treated with PBS (filled histogram),
25 milliunits/ml heparitinase (solid line), or 25 milliunits/ml chondroitinase ABC (dotted line) at 37 °C
for 3 h. Heparan sulfate was detected by the mAb 10E4, followed by
RPE-conjugated goat anti-mouse. B, a similar FACS analysis
as shown in A, but with the use of mAb 3G10 which recognizes
HS stubs which remain on HSPG core proteins after treatment with
heparitinase. C, Western blot of CD44 immunoprecipitates.
CD44 was precipitated from CD44 Namalwa transfectants using the
anti-pan CD44 mAb Hermes-3. Precipitates were then treated with PBS
(
), 200 milliunits/ml heparitinase (HT), or 1 unit/ml
chondroitinase ABC (CH) at 37 °C for 2 h. The
Western blot was stained with the anti-pan CD44 mAb Hermes-3
(upper panel), stripped, and re-stained with the mAb 3G10
(lower panel) which recognizes HS stubs after treatment
of HS with heparitinase.
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Fig. 3.
The role of heparan sulfate in the binding of
HGF/SF to CD44 Namalwa transfectants. A, FACS analysis
to detect HGF/SF bound to CD44 Namalwa transfectants that were treated
with PBS, 10 milliunits/ml heparitinase, or 50 milliunits/ml
chondroitinase ABC at 37 °C for 2 h prior to incubation with 18 nM HGF/SF at 4 °C for 1 h. B, FACS
analysis of wild type or mutated (HP1) HGF/SF bound to CD44
Namalwa transfectants. HGF/SFs were detected with mouse anti-HGF/SF
followed by RPE-conjugated goat anti-mouse. Results are expressed as
relative mean fluorescence intensity (MFI) (as compared with
PBS treated mock transfectants). Error bars represent the
standard deviation from three independent experiments.
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Fig. 4.
Expression of c-Met in CD44 or CD44/c-Met
Namalwa transfectants. CD44s and CD44v3-10 Namalwa transfectants
with or without c-Met were lysed and analyzed for the expression of
c-Met by Western blotting. The Western blot was stained with rabbit
anti-c-Met followed by HRP-conjugated goat anti-rabbit. The epidermoid
carcinoma cell line A431 was used as a positive control. The c-Met
precursor (pre-c-Met) and 
-chain (c-Met())
are indicated.
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Fig. 5.
CD44v3-10 strongly promotes c-Met
activation. A, dose kinetics of the tyrosine
phosphorylation of c-Met in CD44v3-10/c-Met and CD44s/c-Met double
transfectants. Transfectants were stimulated with increasing
concentrations HGF/SF for 10 min at 37 °C. c-Met was
immunoprecipitated with rabbit anti-c-Met and the Western blot was
stained with the anti-phosphotyrosine mAb PY-20 followed by
HRP-conjugated rabbit anti-mouse (upper panel). Then, the
blot was stripped and re-stained with rabbit anti-c-Met followed by
HRP-conjugated goat anti-rabbit (lower panel). The ratios of
tyrosine-phosphorylated c-Met to precipitated c-Met, as determined by
densitometric scanning of the blots, are shown in a diagram.
B, time kinetics of the tyrosine phosphorylation of c-Met in
CD44v3-10/c-Met and CD44s/c-Met double transfectants that were
stimulated with 2.2 nM HGF/SF for increasing periods at
37 °C. c-Met was precipitated and analyzed as in A. The
ratios of tyrosine-phosphorylated c-Met to precipitated c-Met, as
determined by densitometric scanning of the blots, are shown in a
diagram. The c-Met precursor (pre-c-Met) and -chain
(c-Met()) are indicated. Several independent clones were
tested and gave comparable results.
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[in a new window]
Fig. 6.
HGF/SF binding to heparan sulfate moieties on
CD44v3-10 potentiates signal transduction through c-Met.
A, CD44v3-10/c-Met (v3) and CD44s/c-Met (s) double
transfectants were treated with 10 milliunits/ml heparitinase at
37 °C for 3.5 h, and subsequently incubated in the presence or
absence of 2.2 nM HGF/SF. Then, c-Met was precipitated with
rabbit anti-c-Met and the Western blot was stained with
anti-phosphotyrosine (PY-20) followed by HRP-conjugated rabbit
anti-mouse (upper panel). Next, the blot was stripped and
stained with rabbit anti-c-Met followed by HRP-conjugated goat
anti-rabbit (lower panel). The c-Met precursor
(pre-c-Met) and -chain (c-Met()) are
indicated. B, CD44v3-10 does not promote c-Met
phosphorylation by a HGF/SF heparin-binding domain mutant. CD44s/c-Met
(s) and CD44v3-10/c-Met (v3) double transfectants were incubated in
the presence or absence 2.2 nM wild type HGF/SF or with the
heparin-binding domain mutant HGF/SF (HP1) for 10 min at
37 °C. Then, c-Met was precipitated with rabbit anti-c-Met and the
Western blot was stained with anti-phosphotyrosine (PY-20) followed by
HRP-conjugated rabbit anti-mouse (upper panel). Next, the
blot was stripped and re-stained with rabbit anti-c-Met followed by
HRP-conjugated goat anti-rabbit (lower panel). C,
Western blot from total cell lysates from equal numbers of the cells
described in A. The upper part of the blot was
stained with the anti-phosphotyrosine mAb PY-20, followed by
HRP-conjugated rabbit anti-mouse. The lower part of the same
blot was stained with anti-phospho-MAPK antibody, followed by
HRP-conjugated goat anti-rabbit. The arrows indicate a
phosphorylated protein at 145 kDa and two major phosphoproteins at
115-125 kDa (upper panel), and the phosphorylated ERK1 and
ERK2 MAP kinases (lower panel). Several independent clones
were tested and gave comparable results.
DISCUSSION
41-mediated adhesion
(41) and the recent observation that Cbl is involved in integrin
activation and spreading of macrophages (73). Furthermore, Cbl was
recently reported to be required for efficient cellular transformation
through the Tpr-Met oncoprotein (74). In addition to the 120-125-kDa
proteins, we demonstrated for the first time that HGF/SF induces
phosphorylation of the MAP kinases ERK1 and -2 in B cells (Fig.
6C). Even more intriguing was the observation that CD44-HS
promoted the HGF/SF-induced phosphorylation of ERK1 and -2. ERK1 and -2 are intermediates in signaling pathways linking extracellular signals
to gene transcription in the nucleus and have been implicated in a wide
variety of biological responses including cell proliferation.
Interestingly, several recent studies have implicated the ERKs in
integrin activation (75) as well as in HGF/SF-induced motility
(i.e. scattering), and tubulogenesis of the epithelial
Madin-Darby canine kidney cell line (60, 62, 63). Because of our
previous data concerning the involvement of HGF/SF in integrin-mediated
adhesion of B cells (41), we are currently investigating the possible
role of the ERKs in B cell adhesion and migration.
View larger version (37K):
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Fig. 7.
Model for the presentation of HGF/SF to
c-Met. A, HGF/SF molecules, which are largely monomers,
only weakly activate the c-Met pathway in (tumor) cells that lack cell
surface expression of CD44-HS. B, by up-regulating CD44-HS,
(tumor) cells acquire a greatly increased sensitivity to HGF/SF, which
might result in a growth and motogenic/metastatic advantage.
Presumably, CD44 acts by concentrating HGF/SF at the cell surface and
by presenting HGF/SF to c-Met. This presentation may involve ligand
multimerization by HS side chains, resulting in increased c-Met
dimerization. Alternatively, HGF/SF-CD44-HS interaction might lead to a
conformational change of the c-Met receptor promoting signal
transduction.
ACKNOWLEDGEMENTS
FOOTNOTES
Contributed equally to the results of this study.
Present address: Glaxo Wellcome, Stevenage, United Kingdom.
ABBREVIATIONS
REFERENCES
Top
Abstract
Introduction
References
mann, I.,
Matzku, S.,
Wenzel, A.,
Ponta, H.,
and Herrlich, P.
(1991)
Cell
65,
13-24[CrossRef][Medline]
[Order article via Infotrieve]
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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