CD44 glycoproteins in colorectal cancer; expression, function and prognostic value - Chapter 6: Heparan sulfate-modified CD44 promotes hepatocyte growth factor/scatter factor-induced signal transduction through the r

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CD44 glycoproteins in colorectal cancer; expression, function and prognostic

value

Wielenga, V.J.M.

Publication date

1999

Link to publication

Citation for published version (APA):

Wielenga, V. J. M. (1999). CD44 glycoproteins in colorectal cancer; expression, function and

prognostic value.

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Chapter 6

HEPARAN SULFATE-MODIFÏED CD44 PROMOTES

HEPATOCYTE GROWTH FACTOR/ SCATTER

FACTOR-INDUCED SIGNAL TRANSDUCTION

THROUGH THE RECEPTOR TYROSINE KINASE

C-MET.

Robbert van der Voort, Taher E.I. Taher , Vera J.M. Wielenga, Marcel

Spaargaren, Remko Prevo, Lia Smit, Guido David , Guido Hartmann,

Ermanno Gherardi, and Steven T. Pals

JBiolChem 1999, 274:6499-6506

Heparan Sulfate-modifîed CD44 Promotes Hepatocyte Growth

Factor/Scatter Factor-induced Signal Transduction through

the Receptor Tyrosine Kinase c-Met*

(Received for publication, October 5, 1998, and in revised form, December 2, 1998)

Robbert van der Voort*, Taher E. I. Tahert, Vera J. M. Wielenga, Marcel Spaargaren, Remko Prevo, Lia Smit, Guido David§, Guido Hartmannljl, Ermanno Gherardj and Steven T. Pals**

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 func-tional 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 me-diates 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 lym-phoma cells were stably co-transfected with c-Met and either CD44v3 or the isoform CD44s, which lacks hepa-ran 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 mu-tant HGF/SF with greatly decreased affinity for heparan sulfate, we show that the enhancement of c-Met signal transduction induced by CD44v3 was critically depend-ent on heparan sulfate moieties. Our results iddepend-entify heparan sulfate-modified CD44 (CD44-HS) as a func-tional 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 col-laboration might be instrumental in promoting tumor growth and metastasis.

The CD44 family of cell surface glycoproteins is broadly

* 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. This article must therefore be hereby marked "advertisemenf in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Contributed equally to the results of this study.

II Present address: Glaxo Wellcome, Stevenage, United Kingdom. ** To whom correspondence should be addressed: Dept. of Pathology, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. Tel.: 20-566-5635; Fax: 31-20-6960389; E-mail: ST.Pals@AMC.UVA.NL.

expressed by cells of epithelial, mesenchymal, and etic origin and is involved in cell-matrix adhesion, hematopoi-esis, 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 JV-hnked and O-linked glycan forms of CD44 in addition to proteoglycan variants containing chondroitin, keratan, or heparan sulfate (11-14). The expres-sion pattern of these CD44 variants is tissue-specific. On lym-phocytes 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 inter-act with the inter-actin cytoskeleton via ankyrin and proteins of the E RM 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 poten-tial 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 recep-tor 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 US side chain attached to the evolutionary conserved consensus motif SGSG encoded by exon v3 (13, 27). Heparan sulfate proteoglycans (HSPGs) are

1 The abbreviations used are: CD44s, CD44 Btandard isoform; CD44-HS, heparan sulfate-modified CD44; CD44v, CD44 variant isoform; ERK, extracellular signal regulated kinase; E RM, ezrin radixin moesin; FGF, fibroblast growth factor; HGF/SF, hepatocyte growth factor/scat-ter factor; HS, heparan sulfate; HSPG, heparan sulfate proteoglycan; MAP, mitogen-activated protein; mAb, monoclonal antibody; PBS, phosphate-buffered saline; HRP, horseradish peroxidase; FACS, fluo-rescence-activated cell sorter; RPE, R-phycoerythrin.

FIG. 1. A, schematic representation of the CD44 gene, and the CD44v3-10, CD44v8-10, and CD44s cDNAs used for transfection. Solid boxes represent con-stant ezons, while open, boxes represent alternative exons. Note that, due to a stop codon the variable ezon 1 (vl) is not trans-lated in the human. UT, untranstrans-lated re-gion; EC, extracellular constant rere-gion;

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 trans-acted (Neo) Namalwa cells, and two in-dependent 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.

1 2 3 4 5 6 7 8 0 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 7 16 19

N M l M t Q J Q D ΠH t t O H i ^ C l " " COW gene

UT EC EV ECTM CT UT

^ W W Y k ^ t f f l l b t i ^ CD44V3-10

dhrtkr EtJWT^D—

CD44V8-10

CD44S

-,

-M^S^*

•too

-J L * " ^

f

» £ • • • • • • • •

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HGF/SF (nM)

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, presum-ably by promoting FGF dimerization required for efficient re-ceptor cross-linking and activation (28-32).

In the present study, we explored the physical and functional interaction between heparan sulfa te-modified forms of CD44 (CD44-HS) and hepatocyte growth factor/scatter factor (HGF/ SF). HGF/SF is a hepahn-binding growth factor (33) that in-duces 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 mi-gration (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 exam-ple, HGF/SF induces epithelial cells to invade collagen matri-ces 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

TABLE I

Surface expression ofCD44 on Namalwa transfectants

CD44 isoform Clone MFI" Positive cells

%

• Mean fluorescence intensity after staining with the anti-pan CD44 roAb NK3-P1 followed by fluorescein iaothiocyanate-conjugated rabbit anti-mouse.

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 path-way, which is demonstrated to occur in a heparan sulfate-de-pendent fashion.

EXPERIMENTAL PROCEDURES

Antibodies—Mouse monoclonal antibodies (mAbe) used were

anti-pan CD44, NKI-P1 (IgGl) (53), and Hermes-3 (IgG2a) (54) (a gift from

] PBS I heparitinase ) chandrdtinasa - " r-i — 2000 1750 g 1500

S

c

karn

^Pw

IP: anti-pan CD44 blot: anti-pan CD44

IP: anti-pan CD44 blot: anti-AHS-stub

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 milhunits/ml heparitinase (solid line), or 25 milliunitsAnl chondroitinase ABC (dotted line) at 37 °C for 3 h. Heparan sulfate was detected by the mAb 10E4, followed by RP E-conjugated goat anti-mouse. B, a Bimilar FACS analysis as shown in A, but with the use of mAb 3G10 which recognizes AHS stubs which remain on HSPG core proteins after treatment with heparitinase. C, WeBtern blot of CD44 immunoprecipitates. CD44 was precipitated from CD44 Namalwa transfec-tants using the anti-pan CD44 mAb HermeB-3. Precipitates were then treated with PBS (-), 200 m il li units/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 AHS stubs after treatment of HS with heparitinase.

S. Jalkanen, University of Turku, Turku, Finland), anti-HGF/SF, 24612.111 (IgGl) (R&D Systems, Abington, United Kingdom), anti-heparan sulfate, 10E4 (IgM) (55), anti-desaturated uronate from hep-aritinaBe-treated heparan sulfate ("AHS stub"), 3G10 (IgG2b) (55), anti-phosphotyrosine, PY-20 (IgG2b) (AfBniti, Nottingham, United Kingdom), and IgGl 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 (Thr^/Tyr30*) (New England Biolabs, Beverly, MA), rabbit anti-ERKl (C-16) and anti-ERK2 (C-14) (Santa Cruz Bio-technology), RPE-conjugated goat anti-mouse (Southern Biotechnology, Birmingham, AL), fluorescein isothiocyanate-conjugated rabbit mouse (DAKO, Glostrup, Denmark), HRP-conjugated rabbit anti-mouse (DAKO), and HRP-conjugated goat anti-rabbit (DAKO).

Celt Lines and Transfectants—The Burkitt's lymphoma cell line

Na-malwa was purchased from American Type Culture Collection (ATCC, Rockville, MD). The cells were cultured in RPMI 1640 (Life Technolo-gies, Breda, The Netherlands) supplemented with 10% Fetal Clone I serum (HyClone Laboratories, Logan, UT), 10% fetal calf serum

(Inte-gra, Zaandam, The Netherlands), 2 mM L-glutamine, 100 IU/ml penicil-lin, 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 Na-malwa cells, expressing either CD44s SM) or CD44v3-10 (Nam-V3 M), with c-Met was performed as described (41).

Purification of Wild Type and Mutant HGFISF—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 celts 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 con-centrations were measured by enzyme-linked immunosorbent assay as described previously (41). In addition, HGF/SF (wt and HP1) was ana-lyzed by Western blotting using goat anti-HGF/SF.

Enzyme Treatments—For enzymatic cleavage of giycosaminoglycans,

s *

1

_ra

1

• •

HP1

-JL

-^

n.

Fia. 3. The role of heparan sulfate in the binding of HGF/SF to CD44 Namalwa transfert an u . 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 a t 37 *C for 2 h prior to incubation with 18 nM HGF/SF a t 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 (MFD (as compared with PBS treated mock transfectants). Error bars represent the standard deviation from three independent experiments.

cells were treated with either heparitinase (Flafobacterium heparinum, EC 4.2.2.8, ICN Biomedicals, Aurora, OH) or chondroitinase ABC (Pro-teus vulgaris, EC 4.2.2.4, Boehringer Mannheim, Almere, The Nether-lands) in PBS a t 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 (Bectan Dickinson, Mountain View, CA). For binding of recombinant human HGF/SF (wild type or HP1) (RAD 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—Immunoprecipita-tion was performed as described (41). The only modificaAnalysis—Immunoprecipita-tions 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 /ig/ml aprotinin (Sigma), 10 /ig/ml leupeptin (Sigma), 1 mM sodium orthovandate (Sig-ma), 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 /igrail aprotinin (Sigma), 10 /ig/ml leupeptin (Sig-ma), 2 mM sodium orthovandate (Sig(Sig-ma), 5 mM EDTA, and 5 mM sodium fluoride.

Western blotting of immuno precipitates and total cell lysates was essentially performed as described previously (23). A single modifica-tion 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 x 10s cells were directly lysed in sample buffer and analyzed by 10% S DS -Polyacrylamide gel electro-phoresis and blotted. Equal loading was confirmed by Ponceau S stain-ing of the blot. The part of the blot below 50 kDa was stained with anti-phospho-MAPK antiserum, the upper part with anti-phosphoty-rosine 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-ERKl 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 transfert ion, 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 bind-ing 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. IB, 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-depend-ent—We next conducted a series of experiments aimed at

de-tennining 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 AHS stubs remaining on HSPG core proteins after treat-ment with heparitinase (55). Both figures show that cells trans-fected 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. 2 0 .

To assess the role of HS in the interaction between HGF/SF and CD44v3-10, we studied the effect of heparitinase treat-ment 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.

95

3A, h e p a r i t i n a s e t r e a t m e n t r e s u l t e d in a n e a r complete loss of

H G F / S F binding, while t r e a t m e n t with chondroitinase ABC h a d no effect. T h e essential role of H S moieties on CD44v3-10 in H G F / S F binding w a s further confirmed by t h e observation t h a t H P 1 did not bind to C D 4 4 v 3 - 1 0 (Fig. 3B). These d a t a d e m o n s t r a t e t h a t CD44v3-10 is a h e p a r a n sulfate-modifîed CD44 isoform (CD44-HS) t h a t b i n d s H G F / S F via its H S side chain.

CD44-HS Promotes c-Met Activation—To explore the func-tional impact of H G F / S F b o u n d to CD44-HS on t h e c-Met signaling p a t h w a y , w e g e n e r a t e d double t r a n s f e c t a n t s express-i n g c-Met express-in combexpress-inatexpress-ion wexpress-ith e express-i t h e r C D 4 4 v 3 - 1 0 or CD44s. We selected stable t r a n s f e c t a n t s expressing equal a m o u n t s of c-Met to be used in t h e s u b s e q u e n t s t u d i e s (Fig. 4). U s i n g t h e s e cell lines, w e assessed in t h e first i n s t a n c e H G F / S F induced c-Met phosphorylation. As shown in Fig. 5, triggering with H G F / S F led to a v a s t a n d r a p i d increase in t h e phosphorylation of cMet on tyrosine residues i n t h e cells expressing C D 4 4 v 3 -10. By contrast, phosphorylation of c-Met w a s only weakly

CD44S CD44v3-10 pre c M e t -C-Met ( p ) _ Mw (kOa) _ 2 0 0

1 - m

FIG. 4. Expression of c-Met in CD44 o r 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 carci-noma cell line A431 was used as a positive control. The c-Met precursor

(pre-c-Met) and ^chain (c-Metiß)) are indicated.

increased in t h e cells with CD44s (Fig. 5) a n d w a s a b s e n t in t h e p a r e n t a l cell line (data not shown), confirming t h e lack of endogenous c-Met in t h e s e cells. T h e dose-response studies d e m o n s t r a t e d t h a t CD44v3-10 promotes c-Met phosphoryla-tion over a broad dose r a n g e (Fig. 5A) w i t h a n approximately 7-fold relative increase a t plateau level. T h e t i m e curve (Fig. 5B) showed t h a t phosphorylation w a s m a x i m a l between 2 a n d 10 m i n after addition of t h e growth factor a n d declined t h e r e -after. Moreover, t h i s strong e n h a n c i n g effect of CD44v3-10 on c-Met phosphorylation w a s dependent on H S moieties since it w a s lost upon h e p a r i t i n a s e t r e a t m e n t (Fig. SA). T h e impor-t a n c e of H S for HGF/SF signaling w a s furimpor-ther s impor-t r e n g impor-t h e n e d by s t u d i e s using t h e HGF/SF heparin-binding d o m a i n m u t a n t H P 1 . This m u t a n t induced an equal (weak) phosphorylation of c-Met i n both t h e CD44v3-10 a n d CD44s t r a n s f e c t a n t s (Fig. 6B). T h u s , t h e s e d a t a suggest t h a t C D 4 4 v 3 - 1 0 b i n d s H G F / S F via its H S side chains a n d t h e n p r e s e n t s i t to t h e h i g h affinity receptor c-Met.

CD44-HS Promotes Downstream Signaling through c-Met in a Heparan Sulfate-dependent Fashion—The pivotal role of

CD44HS in promoting t h e action of H G F / S F w a s f u r t h e r s u p -ported by analyzing t h e cell lysates of H G F / S F - s t i m u l a t e d cells for tyrosine-phosphorylated proteins. We observed tyrosine phosphorylation of several s u b s t r a t e s , t h e two most p r o m i n e n t phosphoproteins of u n k n o w n identity a r e found a t 115-125 kDa. A minor phosphoprotein is found a t 145 k D a which likely r e p r e s e n t s c-Met (Fig. SC). In addition, several s m a l l e r phos-phoproteins of u n k n o w n origin were observed (not shown) in-cluding a 42-kDa phosphoprotein which m a y r e p r e s e n t t h e p42 ERK2 MAP kinase.

In order to establish w h e t h e r signal t r a n s d u c t i o n by c-Met is potentiated by t h e H S moieties on C D 4 4 v 3 - 1 0 , we further investigated t h e activation of d o w n s t r e a m t a r g e t s of c-Met signaling. Since H G F / S F h a s been shown to activate t h e E R K

X CD44W5-10 0 2 4 6 8 H O F / S F ( « * l ) H G F / S F 0 0 3 0 6 1 1 2 2 4 4 8 8 0 0 3 0 6 1 1 2 2 4 4 1 pre c-Met _ c-Met ifi) _ pre cMet -c-Met (P)_ (nM) Mw (kDa)

•

IP: anti-c-Met blot: anti-PY

*» «. m m •»«* m m m m • > » • *•

IP: anti-c-Met blot: anti-c-Met

B

IP: anti-c-Met blot: ant -PY

*• «•«•«•••••

* . • «•

-IP: anti-c-Met blot: anti-c-Met

Fie. 5. CD44v3-10 strongly p r o m o t e s 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-phoßphotyrosine 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-HRP-conjugated goat anti-rabbit (lower panel). The ratios of tyrosine-phosphorylated c-Met to precipitated c-Met, as determined by denaitometric 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 CD44a/c-Met double transfectants that were stimulated with 2.2 nu 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

ipre-c-Met) and ^chain (c-Met(ß)) are indicated. Several independent clones were tested and gave comparable results.

H G F / S F ;

heparitinase HGF/SF HP1

cells v3 s v3 s v3 s v 3 s

IP.anli-c-Mel Wol: anti-PY

c Met (|i) — cells: S v3 s v3 s v3 Mw (kDa) 200 Mw (kDa) 200

•

pre c-Met —

«»» .*

c-Met (fij —

mm mm m

' onb-o-MO biot ais-c-Met

HGF/SF: - - + + - - + + heparitinase: - + - + - + - + cells: s s s s v3 v3 v3 v3 Mw(kDB — 200 E R K 1 -E R K 2 -blot. anti-phospho-MAPK

FIG. 6. HGF/SF b i n d i n g to h e p a r a n sulfate moieties on CD44v3-10 p o t e n t i a t e s signal t r a n s d u c t i o n t h r o u g h 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(ß)) 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

(HPJ) 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-phoBpho-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.

MAP kinases in Madin-Darby canine kidney, HT29, a n d A549 cells ( 6 0 - 6 4 ) , we assessed w h e t h e r H G F / S F is also able to induce MAP k i n a s e activation in N a m a l w a B cells. For t h i s purpose we u s e d a n antibody recognizing only t h e active, phos-phorylated form of t h e E R K 1 a n d -2 (p44 a n d p42) MAP ki-n a s e s . As showki-n iki-n Fig. 6C, H G F / S F t r e a t m e ki-n t results iki-n phosphorylation of t h e MAP k i n a s e s E R K 1 a n d -2 in N a m a l w a t r a n s f e c t a n t s expressing c-Met. T h e phosphorylation of t h e ERK2 MAP k i n a s e upon H G F s t i m u l a t i o n of t h e cells w a s also confirmed by MAP k i n a s e gel-shift analysis.2 We observed stronger phosphorylation of E R K 1 a n d -2 in t h e CD44v3-10 expressing cells a s compared with t h e CD44s expressing cells (Fig. 6C, bottom panel). Moreover, h e p a r i t i n a s e t r e a t m e n t re-sulted in a decrease of HGF/SF-induced ERK phosphorylation in t h e CD44v3-10 cells, r e s u l t i n g in a level of ERK phospho-rylation t h a t is similar to t h e level of HGF/SF-induced E R K phosphorylation in CD44s t r a n s f e c t a n t s . HGF/SF-induced phosphorylation of t h e E R K s in CD44s t r a n s f e c t a n t s r e m a i n e d unaffected by h e p a r i t i n a s e t r e a t m e n t . T a k e n together, our d a t a d e m o n s t r a t e t h a t signal t r a n s d u c t i o n elicited by HGF/SF-induced c-Met activation is strongly promoted by CD44-HS, and depends on t h e presence of t h e H S moiety on CD44-HS.

1 M. Spaargaren and G. J. T. Zwartkruis, unpublished observation.

We observed t h a t cells transfected w i t h C D 4 4 v 3 - 1 0 effi-ciently bind H G F / S F (Fig. 1) a n d t h a t t h i s CD44 isoform is decorated with H S moieties (Fig. 2). By c o n t r a s t , t r a n s f e c t a n t s t h a t express CD44s or C D 4 4 v 8 - 1 0 , CD44 isoforms which a r e n o t modified w i t h H S (Fig. 2), w e r e not able to bind H G F / S F above background (parental) levels (Fig. 1). This selective H S modification of CD44v3-10 is in line with t h e recent s t u d y by J a c k s o n et al. (13) which d e m o n s t r a t e d t h a t HS side chains bind to CD44 a t t h e SGSG motif encoded by exon v3. Indeed, we d e m o n s t r a t e d t h a t t h e interaction of H G F / S F w i t h CD44v3-10 is HS-dependent. Binding was completely abrogated by hep-a r i t i n hep-a s e t r e hep-a t m e n t , hep-a n d H P 1 , hep-a H G F / S F m u t hep-a n t with grehep-atly decreased affinity for h e p a r a n sulfate a n d h e p a r i n (57), failed to bind C D 4 4 v 3 - 1 0 (Fig. 3). Interestingly, it h a s been demon-s t r a t e d t h a t demon-specific chemical modificationdemon-s of H S demon-side chaindemon-s on proteoglycans a p p e a r to r e g u l a t e t h e i r affinity for selected heparin-binding growth factors, including H G F / S F a n d FGF-2, a n d h e n c e d e t e r m i n e growth factor binding specificity ( 6 5 - 6 9 ) . This suggests t h a t t h e HS moiety covalentiy a t t a c h e d to C D 4 4 v 3 - 1 0 contains specific binding sites for H G F / S F .

T h e key finding of our study is t h a t CD44-HS h a s a major functional effect on HGF/SF-induced signal transduction. Ex-pression of CD44-HS a t t h e cell surface led to a v a s t increase in HGF/SF-induced phosphorylation of c-Met on tyrosine residues

97

FIG. 7. Model for t h e 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/metastaüc advantage. Presumably, CD44 acts by concen-trating 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.

(Fig. 5). F u r t h e r m o r e , it resulted in a s t r o n g tyrosine phospho-rylation of two a s yet unidentified 115-125-kDa proteins t h a t w e r e h a r d l y phosphorylated in t h e absence of CD44-HS (Fig. 6C). One of t h e s e proteins m i g h t represent pllO/115Grb2 a s -sociated b i n d e r (Gab)-1, an a d a p t o r protein t h a t h a s recently been found to associate w i t h t h e multifunctional docking site of cMet (70). Alternatively, t h e observed b a n d s m i g h t be p l 2 0 -Cbl and/or pl25-FAK. Both protein tyrosine k i n a s e s partici-p a t e in signal t r a n s d u c t i o n via recepartici-ptor partici-protein tyrosine ki-n a s e s a ki-n d i ki-n t e g r i ki-n s (71, 72). This is p a r t i c u l a r l y i ki-n t e r e s t i ki-n g given our previous r e s u l t s t h a t H G F / S F stimulation of N a m a l -w a B u r k i t t ' s l y m p h o m a cells results in e n h a n c e d integrin

o4ßl-mediated adhesion (41) a n d t h e recent observation t h a t

Cbl is involved in integrin activation a n d s p r e a d i n g of macro-phages (73). F u r t h e r m o r e , Cbl w a s recently reported to be required for efficient cellular transformation t h r o u g h t h e Tpr-Met oncoprotein (74). In addition to t h e 1 2 0 - 1 2 5 - k D a proteins, w e d e m o n s t r a t e d for t h e first t i m e t h a t H G F / S F induces phos-phorylation of t h e MAP k i n a s e s E R K 1 a n d -2 in B cells (Fig. 6C). Even m o r e i n t r i g u i n g w a s t h e observation t h a t CD44-HS promoted t h e HGF/SF-induced phosphorylation of ERK1 a n d -2. ERK1 a n d -2 a r e i n t e r m e d i a t e s in signaling p a t h w a y s link-ing extracellular signals to gene t r a n s c r i p t i o n in t h e nucleus and h a v e been implicated in a wide variety of biological sponses including cell proliferation. Interestingly, several re-cent s t u d i e s h a v e implicated t h e E R K s in integrin activation (75) as well a s in HGF/SF-induced motility (i.e. scattering), a n d tubulogenesis of t h e epithelial Madin-Darby canine kidney cell Une (60, 62, 63). Because of our previous d a t a concerning t h e involvement of H G F / S F i n integrin-mediated adhesion of B cells (41), w e a r e c u r r e n t l y investigating t h e possible role of t h e ERKs in B cell adhesion a n d migration.

We d e m o n s t r a t e d t h a t t h e e n h a n c i n g effects of CD44-HS on signal t r a n s d u c t i o n via c-Met were critically d e p e n d e n t on t h e interaction of H G F / S F w i t h t h e HS moieties on CD44-HS, a s t h e y w e r e not observed after h e p a r i t i n a s e t r e a t m e n t , or w h e n t h e cells w e r e triggered w i t h t h e heparin-binding domain H G F / S F m u t a n t H P 1 (Fig. 6). Importantly, t h e specific effects

of t h e h e p a r i t i n a s e t r e a t m e n t a n d t h e m u t a t i o n s in H P 1 on HGF/SF-induced signal t r a n s d u c t i o n in t h e C D 4 4 v 3 - 1 0 ex-pressing cells a s compared w i t h t h e CD44s cells d e m o n s t r a t e s t h a t t h e difference in HGF/SF-elicited responses in t h e s e cells is n o t due to a n y possible clonal variation in these s t a b l e cell Unes. We speculate t h a t CD44-HS promotes t h e action of H G F / S F t h r o u g h concentration of H G F / S F on t h e cell surface a n d by presenting it to t h e high affinity receptor c-Met (Fig. 7). Similar m e c h a n i s m s w e r e proposed for t h e role of high a n d low affinity receptors in F G F functioning (32, 76, 77). In addition, CD44-HS might also protect H G F / S F from proteolytic degra-dation a s endothelial cell-derived H S w a s shown to do for FGF-2 (78).

It should be noted, t h a t , a p a r t from growth factor p r e s e n t a -tion, CD44 m a y h a v e additional functions in HGF/SF-c-Met m e d i a t e d signaling. For example, CD44 might recruit molecu-l a r p a r t n e r s into a mumolecu-lti-momolecu-lecumolecu-lar compmolecu-lex with c-Met. T h i s possibility is suggested by t h e fact t h a t two recently identified cytoplasmic molecules associated with CD44 h a v e also been implicated in c-Met signaling. First, studies by Ponzetto et al. (64) h a v e shown t h a t c-Met is a s u b s t r a t e for Src family tyro-sine k i n a s e s , while o u r own s t u d i e s have revealed a physical a n d functional association between CD44 a n d Src family mem-b e r p5Gfck (23). Second, s t u d i e s by J i a n g et al. (79) a n d Crepaldi

et al. (80) h a v e d e m o n s t r a t e d t h a t H G F / S F s t i m u l a t e s t h e

tyrosine phosphorylation of t h e ERM protein ezrin. As reported by T s u k i t a et al. (22), E R M proteins serve a s molecular linkers between CD44 a t t h e cell surface a n d t h e actin cytoskeleton. This interaction is believed to be involved in t h e regulation of cell s h a p e a n d motility.

We propose t h a t collaboration between CD44-HS a n d growth factor receptors, viz. c-Met, a s shown in our p r e s e n t study, might be an i m p o r t a n t factor in t u m o r growth a n d m e t a s t a s i s . By overexpressing CD44-HS, t u m o r cells would acquire a strongly increased sensitivity to HGF/SF-mediated growth sig-nals, leading to a growth a d v a n t a g e a n d promoting m e t a s t a s i s (Fig. 7). This hypothesis is supported by t h e fact t h a t c-Met a n d H G F / S F a r e (over)expressed in conjunction with CD44 in sev-eral types of t u m o r s . In colorectal cancer, for example, c-Met is frequently overexpressed (48, 49, 81), while H G F / S F is ex-pressed within t h e t u m o r t i s s u e microenviroment.3 I n t e r e s t -ingly, in these t u m o r s CD44 splice v a r i a n t s , including v a r i a n t s decorated with H S , a r e often overexpressed a n d predict meta-static s p r e a d a n d t u m o r related d e a t h (82, 83). A similar sce-nario m a y hold for b r e a s t cancer a n d non-Hodgkin's lymphoma, a s in t h e s e t u m o r types overexpression of CD44v3 a s well a s c-Met h a s also been reported (19, 42, 5 1 , 84).

In conclusion, we d e m o n s t r a t e d t h a t t h r o u g h binding a n d p r e s e n t i n g H G F / S F , CD44-HS promotes signal t r a n s d u c t i o n via t h e receptor tyrosine k i n a s e c-Met. Consequently, overex-pression of CD44-HS m i g h t give t u m o r cells a growth a n d m e t a s t a t i c a d v a n t a g e and, in t h i s way, might influence disease outcome.

Acknowledgments—We thank Dr. M. Snoek for critical reading of the

manuscript and Drs. C. Figdor and S. Jalkanen for mAbs.

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