EXPERIMENTAL

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RESEARCH

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ctin Is the Major Serum Protein Neurite Outgrowth fro PETERW.GRABHAM,J~PHILLIP

H. GALLIMORE,*AND

*Department of Cancer Studies, Cancer Research Campaign Laboratories, University of Birmingham, P.O. Box 363, Birmingham B15 ZTT, United Kingdom; and tDepartment of Microbiology and Molecular Genetics, College of Medicine, University of California, Irvine, California 92717

The rat pheocbromacytoma cell line PC12 can be induced to differentiate in response to nerve growth factor (NGF) in the presence of 1% fetal calf serum (FCS). Using a novel assay procedure we have developed a purification protocol which has allowed the isolation of the protein in serum responsible for neurite outgrowth after NGF treatment. FCS has been fractionated using four ehromatograpbic procedures and in each case the peah of biologieait activity copurified with vitronectin. We have concluded, therefore, that vitronectin is the protein present in FCS which can mediate NGF-dependent neurite outgrowth in PC12 cells. Vitronectin and fibroneetin from FCS have been chromatographically separated and only the former is capable of inducing neurite outgrowth. We have also shown that vitronectin utilizes the RGD amino acid sequence in binding to the 0 1992 Academic Press, Inc. surface of PC12s.

INTRODUCTION

The differentiation of the nervous system is a highly complex process that depends largely on the interaction of cells with molecules in their environment. Using tissue culture systems a number of molecules that influence many aspects of differentiation have been identified, including soluble neurotrophic factors such as nerve growth factor (NGF) (reviewed in [l]), membrane bound cell adhesion molecules (CAMS) (reviewed in [a]), and molecules of the extracellular matrix (ECM) (reviewed in 13, 41). In recent years much progress has been made in the identification both of the ECM molecules in the neuron’s environment that regulate the outgrowth of neurites and of the normal cell surface receptors that recognize these molecules. Several ECM constituents such as laminin [5-71, fibronectin [8, 91, and collagens [8, lo] have now been shown to be proteins that facilitate the outgrowth of neurites in neuronal cultures.

1 To whom dressed.

correspondence

and reprint

requests

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be ad-

The rat pheochromacytoma ce to be a particularly useful model for the study of many aspects of neuronal differentiation. After long term posure to NGF, PC12 cells undergo mikotie arrest differentiate both morphologically and bioehemic into cells that display many properties rentiated sympathetic neurons [ll, 12]. Since reatment also permits PC12 cell survival in the ence of serum [13], it has been possible to examine the efkect ofindividual ECM proteins on cell adhesion an growth in defined media. A number o shown that following NGF treatment, PC12 cells adhere efficiently to, and exten laminin, collagens I and IV, and fibronect ~wever, after NGF treatment in the prese the cells have been observed to extend neurites on uncoated tissue culture plastic 1121, suggesting that serum contains an EC protein capable of med It is now known that by at least one member of a family of structurally related receptors (integrins) which are ~~t~~~~~rneric proteins with two membrane spanning subunits (cy and ,L3) in [I73 ). Within the EC proteins several cd attachment domains that bin integrins bave been t prominent among identified (reviewed in [4] ). these domains in ECM glyco s is the fibroneetin type III repeat, one of which contains the sequence RGDS as the major cell attachment site in fibronectin. This sequence is also thought to be the integrin attachment site for won Willebrand factor, and vitronectin [IS]. In PC12 cells, shown to be the major binding outgrowth on fibronectin since a ing this sequence inhibits morpholo ells on fibroneetin e ubiquity of the proteins known to be present in serum it is reasonable to assume that there are several ca ates for serum protein capable of me& ;e outgro tbe present study, we in NGF-treated PC12 cells [la]. have developed a biological assay to monitor the purification to homogeneity of a serum protein cap mediating neurite outgrowth in N~F-t~eat~~ 337

0014-4827/92 $5.00 Copyright 0 1932 by Academic Press, Inc. All rights of reproductior! in any form reserved.

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cells. We have identified this as vitronectin and present evidence to show that in our system, serum fibronectin does not mediate neurite outgrowth and that vitronectin utilizes the RGD sequence to bind to PC12 cells. MATERIALS

AND

METHODS

PC12 cells were obtained from the American Tissue Culture Collection. Bovine vitronectin was purified as described and purchased from Calbiochem Corp. 75-NGF and fibronectin (rat, human, bovine) were purchased from Sigma Chemical Co. Fibronectin was also purified from FCS by affinity chromatography using gelatin-Sepharose eluted with NaBr. Cell culture and neurite assays. PC12 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 8% heat-inactivated horse serum and 4% FCS. For the examination of the influence of serum proteins on neurite outgrowth, cells were washed twice in DMEM then plated onto 5-cm uncoated plastic tissue culture dishes (5 X 10’ cells/dish) in serum-free defined DMEM (DMEM supplemented with 5 pg/ml insulin, 10 pg/ml transferrin, 6.6 fig/ml progesterone, 8.8 ng/ml putrescine, and 4 ng/ml sodium selenite). Treatments of 50 ng/ml NGF were made at 24-h intervals. After 5 days of treatment cells were observed using phase-contrast microscopy. For neurite outgrowth assays during the purification procedure and for peptide inhibition studies, cells were initially primed by culture in DMEM supplemented with 8% FCS and 4% horse serum and then treated daily with 50 ng/ml NGF for a period of 1 week. Primed cells were washed twice in DMEM, mechanically divested of their neurites, pipette dispersed, and plated at a density of 2 X lo4 cells per well into 24-well multidishes in serum-free defined DMEM and 10 ng/ml NGF. Twenty-four hours after the addition of test substances, neurite outgrowth was observed under phase contrast microscopy and determined numerically by counting those neurites longer than twice the diameter of the cell body (>25 pm). Outgrowth was assessed in two different ways. First, as the total number of neurites per 100 cells and second, as the percentage of neurite bearing cells. In both cases over 400 cells were scored per well. Each method gave essentially similar results. This assay was adopted for quantification of neurite outgrowth activity at each purification step. All procedures were carried out at 4°C. Purification of vitronectin. FCS (60 ml) was diluted lo-fold with 10 mM Tris-HCl, pH 7.2, and ammonium sulfate added to 50% saturation. The precipitated protein was removed by centrifugation at 10,OOOgfor 20 min and discarded. Ammonium sulfate was added to the supernatant to increase the concentration to 75% saturation. Precipitated protein was recovered by centrifugation at 10,OOOgfor 20 min, redissolved in water (20 ml), and dialyzed overnight against 10 mM Tris-HCl, pH 7.2. Ammonium sulfate precipitation was repeated and the 50-75% pellet was redissolved in water (100 ml) and dialyzed against 50 mMTris-HCl, pH 7.5,0.1 M NaCl, 1 mM CaCl,, 1 mM MnCl,. An aliquot (25 ml) of this solution was chromatographed on a column (3 X 7 cm) of conconavalin ASepharose 4B and bound protein was eluted with a gradient (0 + 0.3 M) of methyl a-D-mannopyranoside. Fractions (6.5 ml) were collected and aliquots tested for their ability to differentiate primed PC12 cells. Samples were also subjected to Western blotting to allow the detection of vitronectin. Fractions which were biologically active (i.e., positive for differentiation) were pooled, dialyzed against 10 m&4 Tris-HCl, pH 7.2, and then chromatographed on a column (2 x 7 cm) of DEAE-cellulose anion exchanger. Bound proteins were eluted with a gradient of NaCl(0 + 0.3 M) in 10 n~&4Tris, pH 7.2. Again, aliquots from the fractions were tested for the presence of vitronectin and their ability to differentiate PC12 cells. Positive fractions were pooled and dialyzed against 5 mM Tris-HCl, pH 7.2, before chromatography on a column (1.5 X 7 cm) of heparin-agarose equilibrated with the same buffer. Bound proteins were eluted with a gradient of NaCl(0 -f

AND GRAND

0.3 M) in 5 mM Tris-HCl, pH 7.2. Fractions were assayed for their ability to differentiate PC12s and for vitronectin by Western blotting. Positive fractions were pooled and concentrated by using a small column (1 X 2.5 cm) of DEAE 52 eluted with 0.5 M NaCl. This material was used for most of the studies described in this report but the final impurities could be removed by chromatography on a column (1 X 30 cm) of Ultragel AcA34 equilibrated and eluted with 25 mM Tris-HCl, pH 7.5, 0.1 M NaCl. Peptides were synthesized by Alta Bioscience Synthetic peptides. using FMOC or tBOC solid-state chemistry and purified by chromatography on a column of Sephadex G15 eluted with 50 mM NH,HCO, or on a Vydac Cl8 reverse-phase HPLC column eluted with an acetonitrile gradient (0 + 65%). Polyacrylamide gel electrophoresis and Western blotting. Protein samples were electrophoresed on 12% polyacrylamide gels in the presence of 0.1 M Tris, 0.1 M bicine, 0.1% SDS, pH 8.3, and stained with 0.1% PAGE 83 or subjected to Western blotting. Vitronectin was detected using the monoclonal antibody Mab 27 directed against bovine vitronectin (GIBCO BRL) and visualized with the Amersham biotinstreptavidin-peroxidase system. Fibronectin was detected using a monoclonal antibody raised against human fibronectin (Telios Pharmaceuticals). RESULTS

Serum Requirement for Neurite Extension of PC1 2 Cells in the Presence of NGF When PC12 cells are cultured in serum-free defined medium and treated with 50 pg/ml NGF at daily intervals for a period of 1 week, they appear loosely attached and do not extend neurites on uncoated tissue culture plastic (Fig. la). In the presence of 1% FCS, however, PC12 cells treated with NGF display an appreciable level of differentiation as judged by the extension of neurites over 100 /*rn long (Fig. lb). Neurite counts (see Materials and Methods) revealed that the concentration of FCS required for optimum neurite outgrowth ranges from 0.1 to 1%. At these concentrations the average number of neurites (longer than twice the cell diameter) scored per 100 cells was 56 and 94, respectively. At concentrations of FCS greater than 1% the number of neurites per 100 cells was reduced significantly, such that at 10% FCS there were only 40 neurites per 100 cells. Horse serum, at concentrations from 0.1 to lo%, does not appear to facilitate neurite outgrowth above basal levels (t5 neurites per 100 cells). From these observations we have hypothesized that, first, a serum factor when in suspension can substitute for substrate coated with collagens and other ECM molecules widely used in PC12 differentiation studies [ll, 151. Second, at higher concentrations of serum an additional factor may inhibit the differentiation of PC12 cells. This effect has also been observed for PC12 attachment to uncoated tissue culture plastic in the presence of high concentrations of BSA or horse serum [14]. Copurification of Vitronectin and Differentiation from FCS

Factor

Cells which have been primed, divested of their neurites, and replated in the presence of 10 wg/ml NGF (see

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FIG. 1. The effect of serum on NGF-mediated neurite extension in PC12 cells. Cultures were maintained on uncoated plastic in serum-free defined medium and then treated daily with 50 rig/ml NGF for a period of 5 days. In the absence of serum (a) cells are nonadherent. In the presence of 1% FCS (b) cells are adherent and have extended a large number of neurites. Bar represents 100 +m.

Materials and ethods) will extend neurites in response to FCS with the same frequency as unprimed cells. In this case, however, neurite extension occurs over a period of 24 h. This has enabled us to develop a more rapid biological assay for use in the purification of the serum factor necessary for NGF-mediated neurite outgrowth (see Materials and Methods). Initial studies bad suggested the factor in FCS responsible for the promotion of neurite outgrowth in PC12 cells was a protein rather than a small molecule (such as a peptide growth factor or metal ion). We therefore adopted the purification protocol outlined under Materials and Methods. It soon became apparent that the protein responsible for the biological activity was a glycoprotein that had a number of properties in common with vitronectin (and not, as expected, fibronectin)-therefore all procedures were assayed both for vitronectin (Western blotting) and for the ability, in

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conjunction with NGF, to facilitate neurite o~tg~~wtb in PC12 cells. The major protein present in serum is albumin an therefore in any purification protocol separation of this contaminant is a primary concern. T achieved using two rounds of ammonium sulfate fraetionation with vitronectin and biological vity precipitating between 58 and 75% saturation w most of the BSA remains in solution. Bovine ~~~Q~e~ti:~ was precipitated by ammonium sulfate at 50% sat~ra~~o~. The ammonium sulfate fraction co~tai~i~~ vitroneetin (5875%) was chromatographed on a column of concanavalin A-Sepharose and eluted with a nt of methyl cr-D-mannopyranoside, allowing the aPof much of the unglycosylated ~o~tarninati~~ protein (Fig. 2)~ AImost all of the vitroneetin was bound by th.e gel matrix and coeluted with the biological activity (fractions 6080). After dialysis these fractions were chromatographed on DEAE-cellulose eluted with a gradient of increasing NaCl ~o~~entratio~. A . the biological aetivity and vitronectin coeluted, be ion exchanger over the conduct (data not shown, but profiles obtained for all ofthe chromatographic separations cated tion ofvitronectin with biological activit evide promote neurite outgrowth). The nants were removed by ~hrorna~o~~a~hy on heparinagarose and Ultragel AeA34 (data not shown). Sihwrstained gels of the most highly purified and biologically active fractions showed only a major band of 70 kDa an a minor one of 60 kDa characteristic [19]. In view of the copurification of ability to differentiate NGF-primed that it is reasonable to suggest t at this protein is the major component in FCS res onsible for the o biological activity.

In order to compare the relative contri vitronectin and fibronectin corn ents sf FCS to the neurite outgrowth of PC12 cells matographed on a column of S Aliquots from the fractions w PAGE followed by Western blotting with antibodies against the two proteins as well as to bio can be seen from the data presented in tin and fibronectin are separated under these conditions (compare Figs. 3B and 3C). The peak containing most of the biological activity coeluted with the vit e&in with a molecular weight of approximately 76 k (fractions 64-68). Virtually no activity was present in those fractions containing fibronectin (5 lar weight protein detected in dimer but it does not appear to be present in sufficient quantity to induce neurite outgrowth.

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FIG. 2. Affinity chromatography of the 50-75% ammonium sulfate fraction from FCS on concanavalin A-Sepharose 4B. The protein obtained after ammonium sulfate fractionation of FCS was dialyzed against 5 mM Tris-HCl, pH 7.4, 0.1 M NaCl, 1 mM CaCl,, 1 mM MnCl, and chromatographed on a column (7 X 2.5 cm) of concanavalin A-Sepharose 4B equilibrated with the same buffer. Bound protein was eluted with a gradient (0 + 0.3 M) of methyl a-D-mannopyranoside commencing at fraction 55. Fractions (6.5 ml) were collected. The elution profile (bottom) shows optical density measured at 280 nm (closed diamonds) and neurite outgrowth (open diamonds). Neurite outgrowth was determined by plating 2 X 10“ primed cells into 24-well multidishes followed by treatment with 10 rig/ml NGF plus 50 ~1 of individual column fractions in the absence of serum. Neurite counts were made 24 h later (see Materials and Methods). Photomicrographs (top) show cell morphology after treatment with various fractions. Bar represents 100 pm.

A Comparison of the Ability of Vitronectin and Fibronectin to Induce Neurite Outgrowth in PC12 Cells While the ability of serum fibronectin to facilitate NGF-mediated neurite outgrowth could not be detected in our assay, it is now well established that fibronectin influences neurite outgrowth in mammalian neurons. We have therefore tested purified bovine fibronectin for its ability to promote neurite outgrowth when added in soluble form to “primed” PC12 cultures. It can be seen in Fig. 4c that fibronectin induces a differentiated phenotype similar to that seen following the addition of vitronectin (obtained either from a commercial preparation or purified in the present report [Fig. 4a]). Both matrix proteins induce the extension of long neurites which are branched and possess terminal growth cones. Cell bodies did not flatten. The relative potencies of vitronectin and fibronectin were found to be comparable. Although neurite outgrowth activity varies between commercial preparations and between assays, comparisons within assays consistently showed that measured neurite extension for vitronectin is 100 f 10 neurites per

100 cells and for fibronectin 70 f 10 neurites per 100 cells. The quantity of vitronectin and fibronectin required for half-maximal stimulation was found to be approximately 0.4 and 0.5 pg/ml, respectively. Both vitronectin and fibronectin are known to bind to members of the integrin superfamily of receptors and contain the tetrapeptide L-arginine-L-glycine-L-aspartic acid-L-serine (RGDS) as their cell recognition site (reviewed in [7]). We have therefore investigated the ability of each matrix protein to bind to PC12 cells and facilitate differentiation. This was achieved by utilizing a series of RGDS-containing peptides as competitive inhibitors. Primed cells were preincubated in varying concentrations of each peptide for 1 h followed by the addition of FCS, vitronectin, or fibronectin at concentrations above those required for maximum neurite outgrowth. Neurite counts were made 24 h later (see Materials and Methods). Peptides used were (i) a control hexapeptide L-Gly-L-Arg-L-Gly-L-Ser-L-Pro (GRGESP) which lacks blocking activity [18]. (ii) LGly-L-Arg-L-Gly-L-Asp-L-Ser-L-Pro (GRGDSP) which

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est concentration used (200 pg/ml). G found to be a more selective in growth; at 200 pg/ml this pepti neurite outgrowth mediated by than the GRGESP control) but d not inhibit neurite outgrowth mediated by vitronec less than the GRGDEP contr cases where neurite outgrowth was sig~i~cant~y inhibited, a large proportion of the Cl2 cells also fai adhere to the tissue culture pl tie, appearing as ing clumps of cells (Fig. 4d).

66-e

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FIG. 3. Gel filtration of FCS on Sepharose 6BCl. FCS (4 ml) was chromatograpbed on a column (1.6 X 110 cm) of Sepharose 6BCl eluted with 50 mM Tris-HCl, pH 7.4, 0.1 M NaCI. Three-milliliter fractions were collected and aliquots from each were assayed for their ability to induce neurite outgrowth and for the presence (determined by Western blotting) of vitronectin and fibronectin. A, elution profile; optical density at 280 nm (closed diamonds) and percentage of cells bearing neurites (open diamonds). B and C, Western blots carried out on aliquots of fractions obtained after chromatography. B, probed for vitronectin with Mab A27 and C, probed with a monoclonal antibody to fibronectin. Fraction numbers are shown at the top of the blots and molecular weights at the left.

blocks fibronectin and vitronectin binding but not laminin or collagen IV [15]. (iii) L-Gly-L-Arg-L-Gly-LAsp-D-Ser-L-Pro (the L-serine residue has been substituted with its corresponding D-isomer) which is an ineffective inhibitor of vitronectin but is quite effective in the inhibition of fibronectin [2Q]. The results of this experiment are shown in Figs. 4 and 5. The GRGESP control peptide did not inhibit neurite extension by either matrix protein or FCS. In contrast, GRGDSP inhibited neurite outgrowth by vitronectin, fibronectin, and FCS with an efficiency of 80% over controls at the high-

The purification pr yield of 12.6 mg of virtually pure vit~Qnecti~~ from BOOml of FCS using ammonium sulfate by concanavalin A-Sepharose, heparin-agarose chromatography. An additional purification step of chromatography of Ultragel AcA34 yields appreciably less but homogeneous vi~~~~~~ti~. The molecular weight of the purified protein run on SDSPAGE was observed to be in the range 65 kDa to 70 kDa, corresponding closely to the molecular weight of bovine vitronectin determined in 0th studies [ZI, 221. Since serum contains between 250 a 400 mg vitronectin per 100 ml [19], this purification represents a rather poor yield. However, more efficient ~~rificat~o~ protocols involve irreversible conformational change ofthevitronectin molecule due to prior de~at~~at~o~ with high concentrations of urea [23, 243. Vitronectin treated in t way has been reported to adsorb poorly to tissue culture plastic and is thus less effective in the promotion of cell attachment ]2S] L e have purified the otein under nondenaturing conditions using its abili to facilitate neurite outgrowth on PC12 cells (Fig. 2), each purifiineided with the vitrocation step biological activi nectin peaks as detected by stern blotting. The sons for the poor yields of obta from FCS include its tendency to associate with min, its avidity for glass and plastic surfaces, its ability to multimerize, and its charge beterog~?neity which causes it to elute in many fractions during i.on-exchange chromatography [26--281. The bulk of neurite outgr riments in the present report have involved rig/ml) and FCS or vitronectin m so non form to previously primed PC12 cells. purified vitronectin (2 yg/ml) in solution were added to “unprimed)’ cells and retreated 4-h iatervals, neus and was morphorite outgrowth occurred after 5-7 logically identical to that seen in the presence of 1% FCS or on previously primed cells ( g. t. ). In the absence of NGF, however, only CE cells extend neurites partially in tke presence of FCS or vitronectin in solution (~~~u~l~~~ed data). Thus, the biological ac-

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FIG. 4. Peptide inhibition of vitronectin and fibronectin mediated neurite outgrowth in PC12 cells. Primed cells were plated into 24-well multidishes as for neurite outgrowth assays (see Materials and Methods) followed by a l-h incubation at 37°C in serum-free defined medium in either the presence or the absence of the selective synthetic peptide GRGD D-Ser P. After 1 h incubation vitronectin or fibronectin was added to the cultures. Photomicrographs were taken 24 h later. (a) Vitronectin control; cells were incubated in serum-free defined medium then treated with 2 pg/ml purified bovine vitronectin obtained from the purification described in this report. (b) Cells incubated in serum-free defined medium plus 200 pg/ml GRGD D-Ser P, followed by 2 pg/ml vitronectin as in (a). (c) Control cells incubated in serum-free defined medium and then treated with 2 pg/ml bovine fibronectin (Sigma). (d) Cells incubated in serum-free defined medium plus 200 pg/ml GRGD D-&r P, followed by the addition of 2 pg/ml fibronectin as in (c). Bar represents 100 am.

tion of vitronectin is active on both primed and unprimed PC12 cells but is absolutely dependent on the presence of NGF. Judged by a number of criteria neurite outgrowth facilitated by vitronectin resembles that produced by fibronectin. First, the morphology of primed PC12 cells induced to extend neurites by both proteins either as a substrate (unpublished data) or in solution form (Fig. 4) is indistinguishable. Second, the biological action of fibronectin, like vitronectin, is dependent on the presence of NGF since it does not facilitate neurite outgrowth on unprimed PC12 cells [29] and only partially on primed cells [15]. Third, neurite outgrowth facilitated by both ECM proteins is dependent on the binding of the cell attachment site RGDS, since a hexapeptide GRGDSP has been shown to inhibit neurite formation on fibronectin ([15] and Figs. 4 and 5 in this study) and vitronectin (Figs. 4 and 5). Finally, the minimum concentrations of

vitronectin (0.4 pg/ml) and fibronectin (0.5 pg/ml) required to produce a biological response were found to be comparable in the present study (see Results). Although purified soluble fibronectin can facilitate neurite outgrowth in PC12 cells and is a known constituent of FCS, we have observed that following fractionation of FCS on Sepharose 6BCl the peak corresponding to fibronectin exhibits negligible neurite outgrowth activity. In contrast, virtually all neurite outgrowth activity cochromatographs with vitronectin (Fig. 3). This result reflects those found in studies on nonneuronal cells in which the cell attachment activity of serum has been shown to be mediated by vitronectin and not fibronectin [30, 311. This is possibly caused by reduced fibronectin concentrations in serum due to binding of the fibrin clot during serum formation [32] and further losses that apparently occur in the processing of FCS for tissue culture [33]. However, we were able to detect significant levels of

PC12 DIFFERENTIATION

REQUIRES

nQ. of neurites /loo

c&a

no. of neurites / 100 cells

no. of neurites /loo

csfls

0

100

200

Pepttde concentratcf7 pgfml

FIG. 5. The effect of RGD containing peptides on neurite outgrowth in PC12 cells. Cells were prepared and preincubated with peptides as described in the legend to Fig. 4. Neurite counts were made 24 b after the addition of A, 2 PI/ml FCS; B, 2 pg/ml bovine vitronectin; and C, 2 kg/ml bovine fibronectin (Sigma). Peptides used are GRGDSP (open circles), which inhibits attachment to both vitronectin and fibronectin; GRGESP (open squares), a control peptide; and GRGD D-Ser P (closed circles), a fibronectin-specific peptide. Each point represents the mean count from at least 400 cells.

fibronectin in FCS (Fig. 3). An additional explanation for the lack of biological activity of serum fibronectin in the present study may be its inability to compete with other serum proteins for substrate binding sites [34]. The similarity between the action of fibronectin and vitronectin on PC12 cells raises the question of whether each protein is binding to its own specific receptor and therefore mediating a distinct response or whether both proteins are simply binding to an identical receptor and

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mediating the same response. tudies of nonneuronal cell types have suggested that both interactions are possible. For example, human umbilical vein endotbelial cells respond to each individual matrix protein by clustering of their respective receptors [35], whereas in human melanoma cells each protein can mediate cell spreading by binding to a single integrin receptor vitronectin receptor (a,&) [36]. For neurite outgro in PC12 cells, vitronectin and ~~r~~ec~~~ appear capable of mediating a response via since the fibronectin-specific peptide 1201 inhibited fibronectin but not (Figs. 4 and 5). The relative importance of the i~ter~ct~o~ of fibron tin and vitronectin with neurons in viva remains to elucidated. A large number of studies have examine the role of fibronectin in both the CNS and the PNS promotes the outgrowth of axons from PNS neurons [8, 91 and is present along pathways of neural crest migration (reviewed in [3] ). Fibronectin ~rnrn~~oreact~v~t~ has also been no there is httle ev vitronectin in neural deve protein has recently been embryonic chick neurore to promote the e Other ECM proteins, s and laminin, are ment [3, 41. In PC12 cell shown to induce neurite ou NGF by binding to receptors n ~d~~t~o~, thromtin receptor ([14], reviewed m bospondin has recently bee outgrowth from central a neurons and from PC12 cells [39]. Thu le that e’n viuo neurons possess multiple adhedon systems, each capable of interacting with di molecules which act in concert to influenc Bopment. The results presented in this re e another protein -vitronectin-as an ad protein that influences neural development. In summary, we have show vitronectin can mediate NGF growth in PC12 cells and that nonneural cells, vitronectin is the major serum protein responsible for biological activity ukthermore, vitronectin acts via a receptor that is tinct from that of fibronectin. We are most grateful to Ms. Rachel Bruton and ME:. Abigail Foxley for invaluable technical assistance and to Mrs. Janet Gunn for her secretarial expertise. This research was funded by the Cancer Research Campaign, London, UK.

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7,

Vitronectin is the major serum protein essential for NGF-mediated neurite outgrowth from PC12 cells.

The rat pheochromocytoma cell line PC12 can be induced to differentiate in response to nerve growth factor (NGF) in the presence of 1% fetal calf seru...
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