Interactions with Tenascin and Differential Effects on Cell Adhesion of ...

THEJ O U ~ N A L OF BIOLWICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 269, No. 16, Issue of April 22, pp. 12142-12146, 1994 Printed in U.S.A.

Interactions with Tenascin and Differential Effects on Cell Adhesion of Neurocan and Phosphacan, Two Major Chondroitin Sulfate Proteoglycans of Nervous “issue* (Received for publication, February 14, 1994, and in revised form, February 21, 1994)

Martin Grumet, Peter Milev, Takeshi Sakurai, Laina Karthikeyan, Mario Bourdon, Renee K. Margolis, and Richard U.Margolis4 From the Department of Pharmacology, New York University Medical Center, New York, New York 10016, La Jolla Institute for Experimental Medicine, La Jolla, California 92037, and Department of Pharmacology, State University of New York Health Science Center, Brooklyn, New York 11203

We have studied interactions of tenascin with two Tenascin is a large oligomeric glycoprotein of the extracelluchondroitin sulfate proteoglycans, neurocan and phos- lar matrix that is expressed in a restricted pattern during phacan. Neurocan is a multi-domain proteoglycan with development (1).It is present a t lower levels in most adult a 136-kDa core protein that is synthesized by neurons tissues, but its expression is increased during wound healing and binds to hyaluronic acid, whereas the 173-kDacore and invarious tumors suchas glioblastomas (2,3). Tenascin is protein of phosphacan, which is synthesizedby glia, rep- believed to be important for several cellularprocesses including resents an extracellular variant of the receptor-type adhesion, migration, and proliferation of cells, and different protein tyrosine phosphatase RPTPZJB. Keratan sulfate- regions in theprotein have been identified that eitherpromote containing glycoforms of phosphacan (designated phos- or inhibit some of these activities (4, 5). The protein consists of phacan-KS) are also present in brain. Immunocytoan amino-terminal cysteine-rich region involved in oligomerchemical studies of early postnatal rat cerebellum demonstrated that the localization of neurocan, phos- ization, followed by linear segmentsof EGF1-like and fibronecphacan, and phosphacan-KS all overlap extensively tin-type I11 repeats anda fibrinogen-like region at the carboxyl with that of tenascin, an extracellular matrix protein terminus. Cells in culture differ in theirresponses to tenascin. For example, whereas glioma cells attach and sensory neurons that modulates cell adhesion and migration. Binding studies using purified proteins covalently attached to extend neurites on tenascin-coated substrates, Schwann cells fluorescent microbeads demonstrated that proteogly- avoid such regions (6, 7). These findings and the restricted can-coated beads co-aggregated with differently fluo- expression of tenascin during neural development ( 8 ) suggest rescing beads coated with tenascin. The co-aggregation that it isinvolved in axonal guidance. Chondroitin sulfate proteoglycans are another group of exwas specificallyinhibited by Fab’ fragments of antibodies against tenascin or theproteoglycans and by soluble tracellular matrix proteins that have been suggested to moduneurocan, phosphacan, and tenascin, A solid phase ra- late cell adhesion and direct the growth of neurites. Studies dioligand binding assay confirmedthat neurocan, phos- using antibodies that recognize chondroitin sulfate haveshown phacan, and phosphacan-KS bind to tenascin but not to that it islocated in regions that appear toserve as boundaries laminin and fibronectin. Chondroitinase treatment of for neurites, such as theroof plate at themidline of the develthe proteoglycans or addition of free chondroitin sulfate oping spinal cord (9). Recent studies have also shown that two had no significant effect, indicating that thebinding ac- chondroitin sulfate proteoglycans of brain, neurocan and phostivity is mediated largely via the core glycoproteins. phacan, inhibit neuronal adhesion and neurite growth in culScatchard analysis demonstrated high affinity binding ture in a manner that isonly partially dependent on the presof lZ6I-phosphacan, phosphacan-KS, and neurocan to a ence of chondroitin sulfate chains (10). Neurocan contains an single site in tenascin, and neurocan and various glyco- amino-terminal immunoglobulin-like domain followed by tanforms of phosphacan all inhibited binding of 12sI-phos- dem repeats characteristic of the hyaluronic acid-binding rephacan to tenascin. In studies of cell adhesion to progion of aggregating proteoglycans, a central 595-amino acid teins adsorbed to Petri dishes, phosphacan inhibited adhesion of C6 glioma cells to tenascin whereas neuro- portionwith nohomology with other reportedproteinsecan had noeffect. Our results suggest that tenascin quences, and a COOH-terminal portion with -60% identity to binds phosphacan and neurocan in vivo and that inter- regions in theCOOH termini of versican and aggrecan, includactions between chondroitin sulfate proteoglycans and ing two EGF-like domains, a lectin-like domain, and a completenascin may play important roles in nervous tissue his- ment regulatory protein-like region (11).Phosphacan, previtogenesis, possibly by modulating signal transduction ously designated as the 3F8 proteoglycan (121, contains an amino-terminal carbonic anhydrase-like sequence followed bya across the plasma membrane. fibronectin type I11 repeat and a large (-870 amino acid) cysteine-free domain with no homology with other reported protein sequences, and represents an extracellular variantof the receptor-type protein tyrosinephosphatase RpTPyp(13). Vari* This work was supported by Grants NS-09348, NS-13876, and MH- ous developmentally regulated glycoforms of phosphacan that 00129 from the NationalInstitutes of Health. Thecosts of publication of this article were defrayed in part by the payment of page charges. This contain keratan sulfate in addition to chondroitin sulfate glyarticle must thereforebe hereby marked “advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The abbreviations used are: EGF, epidermal growth factor; PBS, j: To whom correspondence should be addressed: Dept.of Pharmacolphosphate-buffered saline; BSA, bovine serum albumin; DMEM, Dulogy, New York University Medical Center,550 First Ave., New York, NY becco’s modified Eagle’s medium. 10016. Tel.: 212-263-7113;Fax: 212-263-7133.

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Interactions of Neurocan and Phosphacan with Tenascin

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FIG.1. Immunolocalization of neurocan, tenascin, phosphacan, and phosphacan-KS in 7-day postnatal rat cerebellum using the monoclonal antibodies described under “Experim e n t a l Procedures.” Rats were perfusion-fixed with picric acid/paraformaldehyde/glutaraldehyde, and sagittal Vibratomesections of cerebellumwere stainedwithperoxidase-conjugated second antibody a s described (12). M L , molecular layer; EGL, external granule cell layer; WM, white matter. Bar, 50 pm.

cosaminoglycan chains also occur in brain (12) andare designated phosphacan-KS. Although no structural similarity has been found between the core proteins of neurocan and phosphacan, both bind to the neural cell adhesion molecules NgCAM/Ll/NILE and N-CAM and inhibithomophilic binding and cell adhesion mediated by them (10). In the presentstudy, we show that tenascin, neurocan, and phosphacan are expressed in overlapping patterns in developing nervous tissue. Experiments using protein-coated microspheres andradioligand binding assays indicate thatneurocan and various glycoforms of phosphacan bind to tenascin. Phosphacan, which is secreted by C6 glioma cells, also inhibits their adhesion to tenascin. These results suggest that binding of phosphacan to tenascin in the extracellular matrix of the central nervous system can modulate interactions with cells, and raise the possibility that tenascin may be a ligand for the receptor-type protein tyrosine phosphatase RI?TP(/p (14,15) on the surfaceof glial cells.

Covaspheres for 15 min a t 4 “C. Proteoglycans were labeled to a specific activity of 2.5-13 x lo’*c p d m o l with lzsI by the lactoperoxidase/glucose oxidase method, and radioligand binding assays were performed a s described (21). Cells a n d Adhesion Assays-C6 glioma cells were grown in DMEM, 10% fetal calf serum. For adhesion assays, cells were removed from tissue culture dishes by treating briefly with 0.25% trypsin, 0.1 mM EDTA and washed with medium containing10% fetal calf serum. The cells were then washed by centrifugation through 3.5% BSAPBS and resuspended in DMEMATS’. 250 pl of DMEWTS’ containing 5 x 10“ cells were deposited inthe central region of 35-mm polystyrene dishes that had been coated with proteins. Substrates for cell adhesion were prepared by incubating 1.5pl of protein, and the dishes were washed 3 times with PBS andblocked with 1% BSA(10). Fordouble coats, blocking solution was appliedonly once, following the second coating. RESULTS Localization of Tenascin withBrain Proteoglycans-The

3D2 monoclonal antibody to rat tenascin was discovered in the course of preparing antibodies to chondroitin sulfate proteoglycans of brain. Thiswas notsurprising insofar as tenascin binds EXPERIMENTAL PROCEDURES to some of these proteoglycans (see below) and copurifies with Proteins a n d Antibodies-Chondroitin sulfate proteoglycans were ex- them. Moreover, it has previously been reported that a minor tracted from rat brain with PBS and purifiedby ion exchange chroma- 250-kDa form of chick brain cytotactin (tenascin) is a chontography andgel filtration (161, followed byimmunoafinity chromatog- droitin sulfate proteoglycan (22), and we have found that a raphy using the 1D1,3F8, and 3H1 monoclonal antibodies for isolation small portion of rat braintenascin also occurs in a proteoglycan

of neurocan, phosphacan, and phosphacan-KS, respectively (12). Rat chondrosarcoma chondroitin sulfate proteoglycan (aggrecan) was isolated by CsCl density gradient centrifugation (17). Human tenascin wasOverlapping staining patterns of neurocan, tenascin, phosphacan, and phosphacan-KS were observed in 7”day postnatal obtained either from Telios (La Jolla, CA) or prepared as described previously (1, 18). Monoclonal antibodies specific for phosphacan (3F8) rat cerebellum (Fig. 1). In most cases,staining was strongest in andneurocan(1D1)(12)andrabbitantibodiesagainstchicken the molecular layer and absent from the external granulecell cytotactidtenascin (19) were prepared as described previously. layer except along the Bergmann glia fibers. However, certain The 3D2 monoclonal antibody to tenascin is an IgGl that was prodifferences were also evident, such as thecomplete absence of duced using PBS-soluble chondroitin sulfate proteoglycansof rat brain a s immunogen (12). Tenascin copurifies with rat brain chondroitinsul- neurocan from the external granule cell layer and the strong fate proteoglycans and can be isolated on a 3D2 monoclonal antibody staining of both phosphacan-KS and neurocan in the prospecimmunoafinity column. Specificity for tenascin was demonstrated by tive white matter. The staining of tenascin with the 3D2 monothe ability of the 3D2 monoclonal antibody torecognize human tenascin clonal antibody is essentially the same as thatpreviously deon immunoblots, and by the finding that the 13 NH,-terminal amino scribed for chick (19) and mouse (24) cerebellum. In rat acids detected inthe 220-kDa protein immunoafinity-purified with the 3D2 antibody are 79 and 71% identical to the NH,-terminal amino acidsembryos, tenascin was observed in both nervous tissue and in other tissues such as developing cartilage (81, whereas in E13in mouse and human tenascin, respectively? The 4H7 monoclonal antibody to phosphacan is an IgG2a that was E18 embryos phosphacan and neurocan are essentially conalso produced using rat brain chondroitin sulfate proteoglycans a s im- fined to brain,spinal cord, retina, andperipheral ganglia (data munogen.” On immunoblots of these proteoglycans, it recognizes a core not shown). glycoprotein that is seen only after chondroitinase treatment and has P h o s p h a c a n and Neurocan Bind to Tenascin-In view of the an apparentmolecular sizeof -400 kDa, corresponding tothat of phosat phacan. It also recognizes phosphacan purified by immunoafinity chro- colocalization of phosphacan and neurocan with tenascin matography with the 3F8 monoclonal antibody, and when used itself for certain sites during brain development and a previous report immunoafinity chromatography the 4H7monoclonal antibody specifi- that a chick brain chondroitin sulfate proteoglycan binds to cally binds a chondroitin sulfate proteoglycan having the NH,-terminal tenascin (201,we tested whether neurocan and phosphacan amino acid sequence of phosphacan. Covasphere Aggregation a n d Radioligand BindingAssays-Proteins This can be demonstrated both by an increase in3D2 immunostainwere covalently coupled to 0.5-pm Covaspheres, and coaggregation asof says were performed a s described (10, 20) in a totalvolume of 50 pl of ing of the 220-kDa tenascin band after chondroitinase treatment PBS containing 0.1 mg/ml BSA using mixturesof 5 pl of red-fluorescing PBS-soluble rat brain chondroitin sulfate proteoglycansor immunoaftenascin Covaspheres with 2pl of green-fluorescing Covaspheres deri- finity-purified tenascin, and by the appearance after chondroitinase vatized with either phosphacan, neurocan, aggrecan, or BSA. When treatment of a 220-kDa band that reacts with the2B6 and 3B3 monoclonal antibodies (23) to 4- and 6-sulfated unsaturated disaccharide antibodies were tested, Fab’ fragments were preincubated with the “stubs” remaining on the core protein after chondroitinase treatment, confirming that chondroitin sulfate chains are covalently linked to a tenascin core protein (R. K. Margolis and R. U. Margolis, unpublished R. K. Margolis and R. U. Margolis, unpublished results. H. Tekotte, L. Hilgenberg, and R. K. Margolis, unpublished results. results).

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Interactions of Neurocan and Phosphacan with Tenascin TABLEI Quantitative analysisof coaggregution of tenascin-coated Covaspheres with Covaspheres coated with proteoglycans Covaspheres were mixed on ice, dissociated by sonication, and incubated for 2 h at room temperature. 10-plaliquots were diluted into20 ml of PBS, and a Coulter counter was used to measure particles (aggregates of Covaspheres) thatexceeded the threshold usingamplification = Y4 and current = Y4 a s described (20).Normal rabbit (NR) serum was from a pool obtained from four unimmunized rabbits. Fab’ fragments of antibodies and other proteins in solution were used at the concentration in pg/ml indicated in parentheses. 4H7 is a monoclonal lOBll is amonoclonal antibody to fibronecantibody to phosphacan and tin. Each determination is the averageof duplicate samples f S.E. F’roteins on Covaspheres

FIG.2. Coaggregation of tenascin-coated C o v a s p h e r e s w i t h neurocan-coated Covaspheres. Red-fluorescing Covaspheres coated with tenascin were mixed with green-fluorescing Covaspheres coated with neurocan in the absence ( a and b ) or presence (c and d ) of Fab‘ fragments of antibodies to neurocan. Control antibodies did not inhibit (cf. Table I). Identical fields were visualized specifically for red-fluorescing tenascin-Covaspheres ( a and c) and green-fluorescing neurocanCovasphnres ( h and d ) and were photographed using n fluorescence microscope.

Tenascin Tenascin Tenascin

Phosphacan Phosphacan Phosphacan

Tenascin Tenascin Tcnnscin Tenascin

Phosphacan Phosphacan Phosphacan Phosphacan

Soluble protein

Phosphacan (30) PhosphacanKS (30) Tenascin (30) Fibronectin (300) Agqecan (300) Anti-tenascin Fab’ (400) NR Fab’ (400) 4H7 Fab’ (40) lOBll Fab‘ (40)

Particles x IC? 15.5 f 0.9 2.8f 0.4 3.1 f 0.2

2.40.2 13.4 0.5 12.9I O . 1 0.40.1

14.6 f 0.9 Phosphacan Tenascin bind to tenascin. When the proteins were covalently linked to 2.7 * 0.5 Phosphacan Tenascin fluorescent microbeads (Covaspheres), red fluorescing Cova13.0f 0.4 Phosphacan Tenascin spheres coated with tenascin co-aggregated with green fluo13.80.3 Phosphacan-KS Tenascin rescing Covaspherescoated with neurocan (Fig.2, a and b ) ,and 2.7-c 0.2 Phosphacan (30) Phosphacan-KS Tenascin the co-aggregation was strongly inhibited by polyclonal anti3.70.3 Phosphacan-KS PhosphacanTenascin KS (30) bodies against tenascin (Fig. 2, c and d ) . Aggregation was also quantitated using a Coulter counter 12.6 0.8 Neurocan Tenascin Neurocan (30) 3.02 0.3 Neurocan Tenascin (20)set to detect large aggregates but not individual Cova4.4 2 0.3 Phosphacan (30) Neurocan Tenascin spheres and small aggregates (Table I). Covaspheres coated 1.2 t 0.1 Tenascin (30) Neurocan Tenascin with tenascin, neurocan, and phosphacan did not self-aggreFibronectin (300) 9.9= 0.3 Neurocan Tenascin gate (except for tenascin, which self-aggregated weakly), nor Aggrecan (300) 10.7* 0.2 Neurocan Tenascin did they co-aggregate with Covaspheres coated with either BSA Anti-tenascin 2.1 f 0.2 Neurocan Tenascin Fab’ (400) or rat cartilage aggrecan, a chondroitin sulfate proteoglycan Anti-neurocan 4.8 f 0.3 Neurocan Tenascin that is structurally related to neurocan. However, tenascinFab’ (400) coated Covaspheresco-aggregatedextensivelywhen mixed NR Fab’ (400) 10.1 f 0.5 Neurocan Tenascin with beads coated with phosphacan or various forms of phos1.9 f 0.2 Tenascin phacan-KS, and the coaggregation was inhibitedby antibodies 2.0 * 0.2 BSA Tenascin to tenascin and phosphacan. Similarly, neurocan-coated beads 0.3f 0.1 Phosphacan 0.5f 0.0 Neurocan co-aggregated specifically with tenascin-coated beads (Table I). Moreover, the aggregation of mixtures of beads coated with tenascin and eitherof the brainproteoglycans was inhibitedby soluble neurocan or phosphacan but not by otherproteins tested includingaggrecan. These results indicate that both neurocan and phosphacan bind to tenascin. To study these interactionsby a different method, we used a radioligand binding assay. ‘251-Phosphacanand 12sI-neurocan bound to tenascin and to Ng-CAM/NILE/Ll and N-CAM but not to many other cell surface and extracellular matrix proteins FIG.3. Saturation curve and Scatchard analysis of ‘%phostested including laminin, fibronectin, severalcollagens, the my- phacan binding to tenascin. Binding values representspecific binding (total countdmin bound minus countdminbound to BSA). Points in elin-associated glycoprotein, and EGF and fibroblast growth saturation curve are the averages of duplicate determinations, and factor receptors(21,25). When increasing amountsof Iz5I-phos- the the error hars represent mean deviations. Scatchard plots were generphacan weretested for binding to tenascin(applied at 10 pg/ml) ated, and the Kd was determined using the Macintosh version of the the binding was saturable(Fig. 3), indicating thepresence of a Ligand program (31). limited number of phosphacan binding siteson tenascin. Scatchard analysisof these datayielded linear plots consistent with (Life Technologies, Inc.) did not inhibit thebinding when tested a single class of binding sites with an apparent Kd of -3 nM at 10 pg/ml. Neurocan, phosphacan from adult brain (which (Fig. 31, and similar valueswere obtained for binding of phos- differs in carbohydrate composition from the early postnatal phacan-KS and neurocan (data notshown). These resultsdem- form of the proteoglycan), and various glycoforms of phosphaonstrate that phosphacan and neurocan bind to tenascin with can-KS (previously designated the 3H1 proteoglycan (12)) all high affinity. yielded 30-50% inhibition when usedat 10 pg/ml. Treatment of The specificity of binding of 1251-phosphacanto tenascin was the proteoglycans with chondroitinase ABC had little effect on tested using unlabeled phosphacan, neurocan, and other mol- their ability to inhibit binding of 1251-phosphacanto tenascin. ecules as potential inhibitors. Unlabeled phosphacan isolated These results suggest that neurocan, phosphacan, and phosfrom 7-day rat brain was the most potent inhibitor, giving 50% phacan-KS all bindto tenascin through theircore glycoproteins inhibition when usedat 5 pg/ml. In control experiments, struc- and that the chondroitin sulfate chains play relatively little turally unrelated proteins such as aggrecan and vitronectin role in theseinteractions. Theseresults arealso consistent with


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DISCUSSION

We have shown that two major chondroitin sulfate proteoglycans of developing brain, neurocan and phosphacan, bind to tenascin. These interactions may be physiologically relevant insofar as tenascin and theproteoglycans are present in overlapping patterns duringembryonic and early postnataldevelopment of the rat central nervous system. Although both neurocan and phosphacan inhibit adhesion of chick brain neurons to Ng-CAM (101, in certain cases the two proteoglycans may have different effects on cell behavior, insofar as adhesion of C6 by phosphacan but notby glioma cells to tenascin was inhibited neurocan. Neurocan is similar in several respectsto a neuronal chondroitin sulfate proteoglycan from chicken brain called the cytotactin-binding proteoglycan. These similarities include its ability to bind to cytotactidtenascin and copurify with it, its distribution indeveloping cerebellum, the molecular sizes of its core glycoproteins and degradation products on SDS-polyacrylamide gel electrophoresis, and the presence on both proteoglycans of sulfated HNK-1 carbohydrate epitopes (12,20,22). Additional molecular characterization of the cytotactin-binding proteoglycan is needed to determine whether it is the chicken homologue of rat neurocan. Other cell surface proteins of nervous tissue have also been found to bind to tenascin,including the cell adhesion molecule contactidF11 (26) andseveral integrins (18, 27). Although tenascin has been reported to have various and sometimes opposite effects on cells, including promotion or inhibition of cell adhesion, cell spreading, neurite growth, and growth control, the mechanismsresponsible for these effects are poorly understood. It is likely that cell surface receptors mediate cellular responses to tenascin, and it is particularly interesting to consider the possibility that tenascin binds to phosphacan as well as to its transmembrane variant RF'TPsIp. In view of the amino acid sequence identity between the extracellular region of the larger form of RFTPUP and phosphacan (13)and the demonstration that RFTPP can be synthesized as a chondroitin sulfate proteoglycan (281, it is likely that RPTPUP also binds tenascin. RPTPWP and phosphacan have been shown to be associated with developing glial cells at key stages of neural development and in patterns overlapping with those of different ligands including tenascin (Fig. 1)and the neural CAMS Ng-CAM/Ll/ NILE and N-CAM (12, 25, 29). Interactions between these ligands and RFTPSIP could modulate phosphorylation of intracellular substrates by alteringthe activity or the local distribution of the phosphatase. Moreover, soluble neurocan and phosphacan may compete for binding of various ligands to cell surface receptors such as RFTPUP. C6 glioma cells provide a convenient model to study such interactions because they express mRNAs encoding membrane-bound receptor forms of RPTP4.43' and secrete a proteoglycan that is indistinguishable from phosphacan when analyzed on SDS-polyacrylamide gel electrophoresis, following chondroitinase treatment.6 These cells adhered to and spread on tenascin and laminin, and soluble phosphacaninhibited their interactionswith tenascin. Additional studies areneeded to determine which receptors on the C6 glioma cells mediate interactions with tenascin and to explore why, in contrast to non-transformed glial cells (7, 30), glioma cells adhere to and spread on tenascin. In this regard,it is interesting thatglioma cells and human glioblastomas express elevated levels of both RPTPUfllphosphacan and tenascin (2, 3, 14h6 In contrast to phosphacan, which also occurs in RPTPUP

'"3 80

6o

N

1

Neurocafln

15 20 25 30 Protein (pglml) FIG.4. Inhibition by phosphacan of adhesionof C6 glioma cells to tenascin. Substrates were coated first with 10 pdml tenascin (Tn; a and c) or 7.4 pglml laminin (Lm; b and d l , and then with 30 pg/ml '0

5

-E-

10

neurocan or phosphacan. Following incubation with cells for 90 min at 37 "C,unattached cells were removed by washing gently with PBS, and the remaining cells were fixed with 3.5% formalin. Panel e shows the effect of different concentrations of added neurocan and phosphacan on adhesion of C6 glioma cells to tenascin or laminin under the conditions described above. Attached cells were counted under the microscope in one field a t x 200, and the average of duplicate samples is shown.

the observation that neitherfree chondroitin sulfate chains (2 pg/ml) nor chondroitinsulfate disaccharides (10 pg/ml) affected the binding of phosphacan to tenascin and that the core glycoproteins of neurocan and phosphacan co-aggregated with tenascin when tested in theCovasphere co-aggregation assay (data not shown). Adhesion of C6 Glioma Cells to Tenascin Is Inhibited by PhosphacanSubstrate-bound tenascin hasbeen found to have effects on different cell types, including promoting adhesion of human U-251 glioma cells (6). Because we found that rat C6 glioma cells also bound to tenascin and extended processes between tenascin (Fig. 4), we investigated whether interactions and brain proteoglycans affect the adhesion of these cells. C6 glioma cells bound to polystyrene dishes coated with tenascin or laminin but notto dishes coated with phosphacan or neurocan. When tenascin-or laminin-coated areas were treated with a second coating of neurocan (NeurocanITn) there was little or no effect on cell binding, whereas a second coating with phosphacan (Phosphacan1T n )produced a concentration-dependent inhibition of binding to tenascin but not to laminin (Fig. 4e). Using 1251-labeledproteoglycans, we confirmed that neurocan and phosphacanbound equally well to tenascin-coated polystyrene dishes. When the order of protein coating was reversed and theproteoglycans were usedas a first coat, a second treatment with tenascin allowed adhesion of C6 glioma cells, but higher concentrationsof tenascin on phosphacan wererequired to obtain levels of adhesion equal to those obtained on neurocan (data not shown).These results suggest that phosphacan binds to tenascin in a manner that can inhibit interactions of C6 glioma cells with tenascin.

R. K. Margolis, unpublished observations. D. Friedlander and M. Grumet, unpublished observations.

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transmembrane forms (13-151, other proteoglycans such as neurocan have been detected only as secreted molecules that may interact with cell surface receptors (12). Although neurocan binds to tenascin, it does not inhibit adhesion of C6 glioma cells to tenascin. Nevertheless, it is a potent inhibitor of neuronal interactions with Ng-CAM (10).It is clear that additional investigations will be required to define specific receptors and functions of chondroitin sulfate proteoglycans in nervous tissue, but the results of this and related studies suggest that these complex molecules play important roles in guiding and restricting cell migration and axonal growth during development. Acknowledgment-We thank David Friedlander for helpful suggestions.

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