Neurotrophic Action of Gliostatin on Cortical Neurons - The Journal of ...

VOl. 267. N o 28, Issue of October 5, PP . 20311-20316,1992 Printed in U.S.A.

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1992 by The American Society for Biochemistry and Molecular Biology, Inc.

Neurotrophic Actionof Gliostatin on Cortical Neurons IDENTITY OFGLIOSTATIN

AND PLATELET-DERIVEDENDOTHELIAL CELL GROWTH FACTOR* (Received for publication, February 4, 1992)

Kiyofumi Asai, Keiko Nakanishi, IchiroIsobe, Yaman Z. Eksioglu, AtsushiHiranoS, Kazuaki HamaS, Tsumoru MiyamotoS, and TaijiKat08 From the Department of Bioregulation Research, Nagoya City University Medical School, Mizuho-Ku, Nagoya 467, Japan and SMinase Research Institute, Ono Pharmaceutical Company, 3-1-1 Sakurai, Shimamoto-Cho, Mishima-Gun, Osaka 618, Japan

Gliostatin is a polypeptide growth inhibitor of ap- tion is a local phenomenon, guiding axons through asequence parent M, = 100,000 with a homodimeric structure of interactions prior to target innervation (1).In the light of comprising two 50-kDa subunits, acting on astrocyte neurotrophic neuron-glia interaction, the local action of neuas well as astrocytoma cells (Asai, K., Hirano, T., Ka- rotrophicfactors maybe mediated by regional glial cells neko, S . , Moriyama, A., Nakanishi, K., Isobe, I., Ek- adjacent to targetneurons. In support of this, thereis increassioglu, Y. Z., and Kato, T. (1992) J. Neurochem., 59, ing evidence that certain neurotrophic factors, such as nerve 307-3 17). The amino acid sequences of 13 tryptic growth factor (NGF)’ (2), ciliary neurotrophic factor (CNTF) peptides including the amino terminus were completely (3, 4) and glia maturation factor (5),are produced by astroidentical to those of platelet-derived endothelial cell cytes during development and regeneration of the nervous growth factor(PD-ECGF) (Ishikawa, F., Miyazono, K., system. Furthermore, Engele and Bohn (6) have recently Hellman, U.,Drexler, H., Wernstedt, C., Hagiwara, K., Usuki, K., Takaku, F., Risau, W., and Heldin, C.-H. demonstrated that acidic and basic fibroblast growth factors (aFGF and bFGF)affect survival and differentiation of cen(1989) Nature 338, 557-562).GliostatinandPDECGF, purified from human placenta, shared growth tral neurons through an unknown neurotrophic factor produced by glial cells in growing phase. It is still somewhat inhibition on glial cells and growth promotion on endocontroversial, however, as to whether the actions of these thelial cells, and exhibited similar values for half-maximal dose of glial growth inhibition (ID50 = 1.3 nM) and neurotrophic factors except for NGF are direct on neurons or the half-maximal concentration of endothelial growth indirect through the modulation of glial cells by the producpromotion (ECao= 1.0nM), suggesting that both factors tion of unknown neurotrophic factors. Recent research on evoke thebiological actions throughan identical recep- neurotrophic factors, therefore, has become increasingly fotor on each cell surface. We have further demonstrated cused on growth-related glial functions, which may promote evidence of a novel neurotrophic action of a neuronalsurvival and neuritogenic guidance. In thisrespect, gliostatin/PD-ECGF towardembryonic rat cortical we have exerted an effort to find diffusible protein factors for neurons in culture. The half-maximal concentration of glial growth and/or differentiation produced by glial cells (7, gliostatin/PD-ECGF for neurotrophic action was 0.3 8). Our recent study demonstrated anovel glial growth inhibnM. All actions on glial, endothelial, and neuronal cells, itor, gliostatin, which is a protein factor with a molecular were abolished by a monoclonal antibody againstglios- mass of 50 kDa, in central glia (astrocytes) as well as in tatin.Thesedataindicatethat gliostatin/PD-ECGF neurofibroma (neurofibromatosis type 1,NF1) rich in periphmay play important roles on development and regen- eral glia (Schwann cell) (9, 10). In this article, we report the eration of the central nervous system and may also chemical and biological identity of gliostatin and plateletinvolve theinduction of angiogenesis for the formation derived endothelial cell growth factor (PD-ECGF), and the of bloodbrain barrier. potent neurotrophic action of both factors on neurons from the central nervous system. Neurotrophic factors are known to act on specific classes of neurons, by supporting their survival and differentiation during development and by promoting regeneration after brain damage. It is generally accepted that neurotrophic ac-

* This work was supported by a Grant-in-Aid for Scientific Research (Cancer Research, Scientific Research on Priority Areas, Developmental Scientific Research, and Encouragement for Young Scientists) from the Ministry of Education, Science and Culture, Japan, by a Research Grant 3A-2 for Nervous and Mental Disorders from the Ministry of Health and Welfare, by the Japan Health Sciences Foundation, and by the Suzuken Memorial Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18U.S.C. Section 1734 solely to indicate this fact. 3 To whom correspondenceshould be addressed Dept. of Bioregulation Research, Nagoya City University Medical School, Mizuho-Ku, Nagoya 467, Japan. Tel.: 81-52-851-5511 (ext. 2256); Fax: 81-52-842-3316.

MATERIALS AND METHODS

Cultivations of Glial, Neuronal, and Endothelial Cells-The rat astrocytoma cell line, C6, originally derived from a rat brain tumor induced by methylnitrosourea (ll),was obtained from American Type Culture Collection (ATCC) and maintained in Ham’s F-10 medium (F-10, GIBCO) containing 10% fetal bovine serum (FBS, Microbiological Associates). Cortical neuron cultures were prepared from rat embryonalcortex (E16) according tothe method of Bankerand The abbreviations used are: NGF, nerve growth factor; PD-ECGF, platelet-derived endothelial cell growth factor; CNTF, ciliary neurotrophic factor; aFGF and bFGF, acidic and basic fibroblast growth factor; NF1, neurofibromatosis type 1; FBS, fetalbovine serum; CMFTyrode, Ca2+- and M e - f r e e Tyrode’s solution; TIPPS, transferrin, insulin, progesterone, putrescine, and sodium selenite; PBS, phosphate-buffered saline; PVDF,polyvinylidene difluoride; SDS, sodium dodecyl sulfate; BSA, bovine serumalbumin; EC,,, half-maximal effective concentration; IDs0, half-maximal inhibition dose; DMEM, Dulbecco’s modified Eagle’s medium; PAGE, polyacrylamide gel electrophoresis.

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Neurotrophic Gliostatin

Cowan (12). Cerebral cortices were dissected and digested with papain (2 mg/ml), bovine serum albumin (2 mg/ml) and cysteine (2 mg/ml) in Ca2+-and MP-free Tyrode's solution (CMF-Tyrode) for 15 min. The dissociated cells were collected by centrifugation and resuspended in Dulbecco's modified Eagle's medium (DMEM, GIBCO) supplemented with 5% newborn bovine serum (Microbiological Associates), 5% heat-inactivated horse serum (Microbiological Associates), and 1 mM sodium pyruvate. After a gentle mechanical trituration through a siliconized Pasteur pipette, cells were filtered through a sterile lens paper filter. The filtrated cells were collected by centrifugation, resuspended in Ham'sF-12 medium (F-12,GIBCO)/ DMEM medium ( l / l ; v/v) supplemented with 100 pg/ml transferrin, 5 pg/ml insulin, 20 nM progesterone, 100 p M putrescine, and 30 nM sodium selenite (TIPPS), and then plated on a poly-L-lysine-coated 48-well dish (1.0 cm2/well, Costar 3548) a t a density of 6 X lo4 cells/ well. Endothelial cells were obtained from a bovine aorta in accordance with the method described by Booyse et al. (13), and maintained in DMEM medium supplemented with 10% FBS. All media were routinely supplemented with penicillin (100 units/ml) and streptomycin (100 pg/ml), and cultures were maintained in an atmosphere of 5% COZ in air and saturatedhumidity. Bioassays-Glial growth inhibitory assays were performed in 96well microtiter tissue culture plates (0.33 cm2/well, Nunc 1-67008) (10). C6 cells (1 X lo6cells/well) in 100 pl of F-10 medium containing 10% FBSwere plated and incubated for 4 hin microtiter wells. Either 10 pl of 20 mM Tris-HC1 buffer, pH 7.5, or the test sample, which was purified by Blue-Toyopearl, DEAE-Sephacel, Butyl-Toyopearl and hydroxylapatite columns (lo), was added to each well. The cells were further cultured for 16 h following the addition of 740 Bq/well ["HIdThd in CMF-Tyrode and then harvested on glass fiber filters by the use of a multiple cell harvester (Lab0 mash). Filters were airdried and placed in scintillation vials containing 5 ml of AL-1 (Dojindo Laboratories) scintillation fluid. The cell-associated 3H radioactivity was determined on a model 5801 liquid scintillation counter (Beckman). Triplicates for each experimental treatment were performed, and the mean values and standard errors were determined. The reduction in DNA synthesis was estimated by the difference of radioactivity between untreated control and positive control treated with 10 p1 of crude neurofibroma extracts. Growth-promoting activity on endothelial cells was estimated as the incorporation of [3H]Thd into bovine endothelial cells (14). Endothelial cells (lo' cells/well) were seeded in 96-well dishes, and incubated for 24 h in F-10 medium containing 0.5% FBS. The cells were stimulated by the test samples for 16 h and were pulse-labeled with 740 Bq of [3H]Thd/well for 4 h. The incorporation rate was measured by the sameprocedure for gliostatin assay. Neurotrophicaction on corticalneurons was estimated by two indicators, the number of survivingneurons and the increase in neurofilament protein. Surviving and neurite-bearing neurons were quantified by counting in 4 visual fields/well (200-fold magnified) in 48-well dish (1.0 cmz/well). Neurofilament protein was quantified according to the method by Doherty et al. (15). Following fixation with 4% paraformaldehyde for at least 4 h at 4 "C, the cultures were permeabilized with 0.1% Triton X-100 in phosphate-buffered saline (PBS) for 1 h. The cultures were then incubated with mouse antineurofilaments (210, 160, and 70 kDa) IgG (IgGlk, 1:1000, Immunotech S.A.) for 1 h at room temperature, washed twice with PBS containing 10% FBS, and incubated with secondary antibody (horseradish peroxidase-conjugated goat anti-mouse IgG (Zymed Laboratories Inc.) at 1:500 dilution) for 1 h. After exhaustive washing with PBS and water, the cultures were incubated with 0.2% O-phenylenediamine and 0.02% HzO, in 50 mM citrate buffer, pH 5.0, for 30 min. The reaction was stopped by adding an equal volume of 4.5 M HzSO,. Product formation was quantified by reading the absorbance of an aliquot of the reaction product at 492 nm using a UV spectrophotometer (DU-68, Beckman). SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)-SDSPAGE was performed by the use of 7.5% gels according to themethod of Laemmli (16). Reduction of disulfide bonds priorto electrophoresis was accomplished by heating samples at 100 "C for 5 min in the presence of 0.1 M 2-mercaptoethanol, 2% SDS, and 15% glycerol. 10 p1 of heated sample was applied to each slot in a gel slab, 50 X 60 X 0.75 mm in dimension. The stackinggel, separation gel, and electrode buffer, all containing 0.1% SDS, were prepared according to Laemmli (16). Electrophoresis was conducted at a constant current of 20 mA for 1 h. Protein bands were visualized by staining with Ponceau 3R (Wako Pure Chemical Industries, Osaka, Japan) for successive sequence analysis after blotting or with Coomassie Blue R-250 (Sigma)

for ordinary protein detection. Low molecular weight protein standard comprising phosphorylase b (97.4 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45 kDa), and bovine carbonic anhydrase (31 kDa) (Bio-Rad) was used. Western Blotting-Samples to be analyzed by Western blotting were electrophoresed on 7.5% SDS-polyacrylamide gels andthen transferred to polyvinylidene difluoride (PVDF) membrane (Immobilon P, Millipore) by using standard methods. Electroblotting was conducted at a constant current of 180 mA for 60 min in a Tefco transfer chamber, with the PVDF membrane facing the anode. After transfer, the gel was stained with Coomassie Blue to determine the efficiency of transfer. PVDF membranes were processed under the protocols by using an immuno-detection kit (Amersham) for mouse antibodies, employing, as a primary antibody, purified monoclonal antibody against gliostatin following passage through a protein Acolumn at a final concentration of 2 pg IgG/ml in the immunoreaction. Monoclonal antibodies against gliostatin were prepared as previously described (10). Neutralization of Gliostatin by Monoclonal Antibody-Constant amounts (60 pg) of monoclonal antibody (16D8, IgGlk) were incubated with gliostatin or PD-ECGF at the concentration of 45 nM for gliostatin and neurotrophic assays or 45 nM for PD-ECGF assay in a total volume of 40 pl in polypropylene tubes. The initial incubation was carried out on ice for 1 h in 20 mM Tris-HC1 buffer, pH 7.5. Subsequently the mixture was incubated at room temperature for 1 h with 20 p1 of a 20% suspension of Immusorbin precoated with excess rabbit anti-mouse IgG antibody (Seikagaku Kogyo). After removal of immusorbin by centrifugation (1,500 X g for 30 min at 4 "C), 10pl of the supernatant was assayed for gliostatin, PD-ECGF, and neurotrophic activities. Carboxymethylation and in Situ Enzymatic Fragmentation of Gliostatin-Gliostatin and PD-ECGF were purified from neurofibroma (von Recklinghausen neurofibromatosis type 1) by the method previously described (10). Gliostatin or PD-ECGF blotted on PVDF membrane (ProBlot, ABI) was cut out aftervisualization by Ponceau 3R solution, and then directly reduced for 16 h a t 37 'C in 500 p1 of 6 M guanidine hydrochloride in 0.5 M Tris-HC1 buffer, pH 8.5, containing 0.3% EDTAand 2.0% acetonitrile with dithiothreitol added to a final concentration of 30 mM under nitrogen gas. Samples were subsequently S-carboxymethylated for 20 min at room temperature under nitrogen gas by addition of iodoacetic acid in 0.5 M NaOH to make a concentration of70mM. After washing with water, the sample was immersed into 100 mM acetic acid containing 0.5% polyvinylpyrrolidone for 30 min and used directly for trypsin digestion at an enzyme/substrate ratio of 1/60 in ammoniumbicarbonate buffer, pH 7.8, containing 8% acetonitrile. Narrow-bore Reversed-phose High Performance Liquid Chromatography (HPLC)-Peptide mixtureresulting from in situ enzymatic fragmentation was chromatographed on a SMART system (Pharmacia) equipped with a reversed-phase (pRPC C,/C,,, Pharmacia) column (2.1 X 100 mm). Thecharged peptides were eluted by a linear gradient of acetonitrile from 0 to 50% inthe presence of 0.1% trifluoroacetic acid, with a flow rate of 150 pllmin. Fractions were manually collected and immediately stored without drying at -80 "C in preparation for microsequencing. Microsequencing of Peptides-Peptide samples for sequence analysis were applied to a Polybrene precycled glass fiber filter and placed in thereaction cartridge of an Applied Biosystems protein sequenator (model 470A). The samples were subjected to automated Edman degradation using the standard program. The resultant phenylthiohydantoin amino acidfractions were identified by on-line HPLC (Applied Biosystems, model 120A) and manual interpretation of the chromatographic data. Preparation of Enzyme Immunoassay System-Anti-PD-ECGF antibody was raised by immunizing 40 pg of purified sample into a New Zealand albino rabbit. Booster immunizations of20pg were given after 2 and 5 weeks. A sandwich enzyme immunoassay system for gliostatin/PD-ECGF was performed by immobilizing the antibody (Fab')z (17) on a 96-well Immunoplate (Maxisorp, Nunc 446612) and by using the monoclonal antibody against gliostatin (16D8, IgGlK), which was labeled with p-galactosidase from Escherichia coli bymeans of N-hydroxysulfosuccinimide ester (18).50 pl of (Fab'L fragment of anti-PD-ECGF antibody was incubated in each well of the 96-well plate for 1 h at 4 "C. After removal of the antibody, the plate was filled with 10% skim milk (commercially available) in 0.1 M sodium phosphate buffer, pH 7.5, containing 0.1 M NaCl, 1 mM MgCL, 1% bovine serum albumin, and 0.1% sodium azide (Buffer A) to block the unoccupied surface of plate. The plate was washed with 150 pl of

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Neurotrophic Gliostatin cold Buffer A two times after discarding the skim milk. 30 p1 of standard gliostatin, PD-ECGF (0-10 ng), or test sample was added and incubated at room temperature for 2 h with gentle shaking. The monoclonal antibody-0-galactosidase complex containing 0.5 milliunit (1unit = pmol of product/min) in 50 pl of Buffer A was added and incubated for 16 h a t 4 "C after washing three times with 150 pl of cold Buffer A. The plate was thoroughly washed with 150 pl of Buffer A. Successively, the enzyme reaction at 30 "C was started by adding 25 pl of 0.3 mM 4-methylumbelliferyl-~-~-galactopyranoside (Koch-Light, Suffolk, United Kingdom) as substrate, and stopped after 1 h by adding 100 p1 of0.1 M glycine-NaOH buffer, pH 10.3. The fluorescence intensity of 4-methylumbelliferone in the reaction mixture was measured with a fluorescence spectrophotometer (Hitachi, model 650-40) at 360 nm excitation and 450 nm emission, against 100 nM of 4-methylumbelliferone (Wako Pure Chemical Industries) as standard. This enzyme immunoassay system was mainly used to standardize the gliostatin/PD-ECGF concentration in the samples for bioassays. It provided identical standard curves of gliostatin and PD-ECGF enabling detection of 50 pg of protein.

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RETENTION TME (min)

(2)PDICGF

RESULTS

Determination of Partial Sequence of Gliostatin-To determine a partial amino acid sequence, purified gliostatin was run on an SDS-PAGE and transferred toPVDF membrane. The protein band visualized by Ponceau 3R staining was excised from the membrane and digested in situ with trypsin after carboxymethylation. The eluted peptideswere separated on a reversed-phase column. Thirteen peptide sequences including the undigested protein were defined by microsequence analysis (Fig. L4).According to thesequence homology search by NBDF Data Base, all detectable alignments were completely identical to those of human PD-ECGF (Fig. 1B). Purification of PD-ECGF-PD-ECGF with gliostatic activity on C6 cells and immunoreactivity to anti-gliostatin was purified by the established processes for gliostatin purification, most of which were incidental to those for PD-ECGF except for gel filtration chromatography by TSK-G4000 SW and Superose 12 columns (19), using four steps of column chromatographs by DEAE-Sephacel, Butyl-Toyopearl, hydroxylapatite, and Mono Q as previously described (10).Since thiol-reducing reagent is known to preserve or activate PDECGF as well as gliostatin (10, 19), 1 mM dithiothreitol was routinely added in the sample and buffers throughout the whole chromatographs. Purifiedgliostatin andPD-ECGF were run together on an SDS-PAGE under identical conditions in the presence of thiol-reducing reagent. They were stained by Coomassie Brilliant Blue and separately immunoblotted with anti-gliostatin monoclonal antibody (16D8, IgGlk). Both protein factors migrated in the identical position corresponding to 50 kDa on either SDS-PAGE or Western blot analysis (Fig. 2). Furthermore, sequence analysis of purified PD-ECGF attested to their chemical identity (Fig. L4). Functional Identity on Glial Cells and Endothelial Cells in Culture-To assess the functional identity of PD-ECGF and gliostatin, purified PD-ECGF from human placenta which was originally described as a growth factor on endothelial cells (14) was tested in the gliostatic assay, and conversely gliostatin was assayed in anendothelial cell growth-promoting assay. Fig. 3 shows that PD-ECGF and gliostatin elicited identical dose-dependent growth inhibition curves on C6 cells and both factors provide a comparable half-maximal inhibition dose (ID50 = 1.3 nMof gliostatin/PD-ECGF). Gliostatin as well as PD-ECGF showed asimilar profile of growthpromotionon bovine endothelial cells. The half-maximal growth-promotionconcentration of both factors was also identical (1.0 nM).Both growth-inhibitory activity onC6 cells and growth-promoting activity on endothelial cells were neutralized by monoclonal antibody (16D8, IgGlk) as shown in Fig. 3.

FIG. 1. A, tryptic peptide map of gliostatin and PD-ECGF developed on a SMART system equipped with reversed-phase column. Peptide mixture from enzymatic fragmentation of gliostatin (1) or PD-ECGF (2), theprocedure of which is described under "Materials and Methods," was applied to a reversed-phase column (pRPC C2/ C18 SC 2.1/10) and eluted by a linear gradient of acetonitrile from 0 to 50% in the presence of 0.1% trifluoroacetic acid, with a flow rate of 150 pl/min. Fractions were collected manually. B, amino acid alignment of human PD-ECGF reported by Ishikawa et al. (19). The single-letter code for amino acids was used. Determined sequences from tryptic fragments of gliostatin (-) or PD-ECGF (- - -) are underlined. The numbers with the underlines indicate the peak numbers in panel A. Bores indicate the nucleotide binding motif. The NH2-terminal amino acids of both placental and platelet PD-ECGFs from the data of Ishikawa et al. are Thr' and Ala", respectively.

Neurotrophic Action of Gliostatin and PD-ECGFon Central Neurons-Fig. 4 demonstrated for the first time the neurotrophic action of gliostatin/PD-ECGF. To further verify the identity of both factors, we assessed whether neurotrophic action on central neurons observed with gliostatin was also detected using purified PD-ECGF. Rat cortical neurons from E16 rat embryos, when plated on polylysine-coated tissue culture dishes, exhibited neurite outgrowth by 24 h in culture in the F/D medium supplemented with TIPPS. Although spontaneousneuriteextension was detectable for48 hin control culture, thereafter there was a large decrease in the number of neurons in control cultures.The addition of gliostatin or PD-ECGF not only ameliorated the neuronal cell death but also promoted the neurite outgrowth after 8 days in culture as shown in Fig. 4. When compared with untreated


20314 (A) 1

(B) 1 2

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3K-

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~

FIG. 2. A, SDS-polyacrylamide gel electrophoresis of purified gliostatin and PD-ECGF. One pl of each purified sample roughly corresponding to 2.0 pg ofgliostatin (1) or PD-ECGF (2) was analyzed individually under reducing conditions. Molecular mass standards comprising phosphorylase b (97.4 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45 kDa), and bovine carbonic anhydrase (31 kDa) were used. E, Western blot analysis of purified gliostatin and PDECGF. Purified gliostatin (1)or PD-ECGF (2) corresponding to about 50 ng of each factor was electrophoresed and transferred to PVDF membrane. The membrane was processed using the monoclonal antibody (16D8, IgGlk) against gliostatin.

FIG. 4. Neurotrophic effect of gliostatin on rat cortical neurons. Rat cortical neurons were dissociated by papain and seeded on poly-L-lysine-coated 48-well dishes a t a density of 6 X lo4 cells/well. After 24-h incubation, PBS ( A ) ,gliostatin (9 nM) ( B ) or PD-ECGF (9 nM) (C) was added. Photographs were taken after 72 h of testing under 200-fold magnification.

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1 o4 Concentration of GliostatinlPD-ECGF (pM) 1 02

FIG.5. Dose-response curves onratcortical neurons of gliostatin and PD-ECGF. Rat cortical neurons were seeded on poly-L-lysine-coated 48-well dishes a t a density of 6 X lo4 cells/well in the F/D serum free medium containing TIPPS. After 24-h incuConcentration of GliostatinlPD-ECGF (pM) bation, various amounts of gliostatin (0) or PD-ECGF (0)were FIG.3. Dose-dependent curves on bovine aortic endothelial added. Squares indicate the neutralization of neurotrophic action of cells and rat astrocytoma C 6 cells to gliostatin and PD-ECGF. gliostatin (0)or PD-ECGF (U).The number of surviving neurons Various amounts of gliostatin (0)or PD-ECGF (0)were analyzed by ( A ) and neurofilament protein levels ( E ) were measured after 8 days both bioassay systems in endothelial cells ( A )or astrocytoma C6 cells testing by the methods described under “Materials and Methods.” ( B ) as described under “Materials and Methods.” Squares indicate Values of surviving neuron number are expressed as mean values and the neutralization of gliostatin (0) or PD-ECGF (U) activity by standard error(n = 6). Values of neurofilament proteinare expressed monoclonal antibody (16D8, IgGlk) against gliostatin. The methods as mean values from duplicate experiments. of the assay for neutralization are described under “Materials and Methods.” All values are expressed as mean values and standarderror (n = 3). (data notshown). Simultaneously, the neurofilament protein

controls following 8 days in culture, the number of surviving neurons increased in a dose-dependent manner with gliostatin or PD-ECGF treatment (Fig. 5). Both factors elicited virtually identical half-maximal effective concentration (E& = 0.3 n M ) , which were lowerthan the half-maximal value of gliostatin assayed on C6 cells (ID5o= 1.3 nM), or of PD-ECGF assayed on endothelial cells (EC5,, = 1.0 nM). This surviving activity by either factor was also suppressed by adding monoclonal antibody (16D8, IgGlk). The effects of gliostatin on neuronal survival and neurite outgrowth were essentially identical regardless of the platingdensity in neuronal culture

content in separate cultures of the same experiment were measured by an enzyme immunoassay, using the monoclonal antibody recognizing the 210-, 160-, and 70-kDa neurofilament forms, to quantify the neuritogenic activity of factors in culture. Gliostatin and PD-ECGF produced, respectively, a 240 and 200% increase in neurofilament protein level in a dose-dependent fashion by day 8 in culture. However, there was no detectable saturation level of neurofilament contents in eithercase, whereas neuronal survival reached a saturation of 1.0 nM or higher concentrations. The results imply a further neurite outgrowth of surviving neurons without alterations of cell numbers.

Gliostatin

Neurotrophic

DISCUSSION

This study revealed interesting chemical and biological features about gliostatin. The native form of gliostatin is a M , = 100,000 dimer comprised of two 50-kDa subunits (10). It is noteworthy that in a low concentration of dithiothreitol (up to 5 mM) gliostatin is stable or even reactivated. This unique stability to thiol-reducing reagents had also been described in the report by Miyazono et al. (19) on the purification of PD-ECGF (19). Biologically, gliostatin is a potent growth inhibitor of astrocytes and glial tumor cell lines and is expressed and produced in only quiescent astrocytes (type 1 astrocytes) but not in growing astrocytes (10). When these dataaretaken together,gliostatin could be an autocrine regulator of glial growth that may mediate contact inhibition of astrocytes. Furthermore, considering that gliostatin could evoke neuronal survival and neuritogenic actions on central neurons as shown in the currentstudy, it is most likely that this factor plays an essential role in the neuron-glial interaction during development or regeneration of the central and peripheral nervous system. Incontrast, PD-ECGF, which promotes the proliferation and chemotactic migration of endothelial cells resulting in angiogenesis, was initially purified from human platelets as a 45-kDa single chain polypeptide by Miyazono et al. (19). Usuki et al. (20) also recently purified it from human placenta as a 47-kDa protein, the size difference of which is due to a differential processing in the amino-terminal part of nascent PD-ECGF. It is nowwell documented that theabove protein factors, gliostatinand PDECGF, are chemically and immunochemically identical and both inhibit glial growth and promote endothelial cell proliferation.Furthermore, the angiogenic action of PD-ECGF demonstrated by Ishikawa et al. (21), besides these actions, suggests that gliostatin/PD-ECGF might have a role in the formation of blood brain barrier during brain development and regeneration. The half-maximal concentration (ECbO= 1.0 nM) of both gliostatin and PD-ECGF in endothelial cell growth promotion assay was virtually identical to their IDs0 (1.3 nM) in the gliostatin assay. Theseresults imply that gliostatin/PD-ECGF elicit both of these actions through identical receptors on the plasma membrane of each cell. The neurotrophic action of NGF (22-24), CNTF (25-27), brain-derived neurotrophic factor (28, 29), or neurotrophin 3 (30, 31) had been studied in the peripheral nervous system. However, there is lately increasing evidence that neurotrophic factors such as NGF, CNTF, and other unidentified factors, which are found to originate from growing astrocytes induced by certain mitogens (aFGF, bFGF, platelet-derived growth factor, orinterleukin l), affect survival and neurite outgrowth of central neurons. We have now provided evidence of a novel neurotrophic action of gliostatin/PD-ECGF toward embryonic rat cortical neurons and not toward peripheral neurons such as dorsal root ganglionic, parasympathetic ciliary ganglionic neurons, and pheochromocytoma cell (PC12D)(9). Although most of these neurotrophic factors areproduced by astrocytes in the growing phase (2, 6), gliostatin is unique in that it is expressed and produced by quiescent astrocytes in the stationary phase (10). This implies that gliostatin may act differently from other factors. Most neurotrophic factors derived from growing astrocytes probably show their actions at the early stage of gliosis: a physiological glial proliferation during development and a reactive gliosis during woundhealing or regeneration after brain damages. In contrast, itis conceivable that gliostatin derived from quiescent astrocytes displays the neurotrophic and gliostatic actions at two different time points duringreactive gliosis induced by brain ischemia, bleeding, or mechanical damage. At the first stage, just

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before the initiation of reactive gliosis, gliostatin released by mechanical destruction from matured astrocytes, maximally expressing gliostatin, may not only promote the neuronal regeneration but also arrest theinitial gliosis interfering with neurite-outgrowth of neurons. Subsequently, gliostatin secreted from quiescent astrocytesmay also function at thelate stage of reactive gliosis to abolish the aberrant growth of reactive glial cells in order to strengthen contact inhibition and simultaneously support neuronal regeneration. Although the lack of a signal peptide does not support the physiological release of gliostatin from glia (21), there may be alternative mechanisms to release the protein factor from the producing cells, as speculated in aFGF, bFGF, CNTF, and glia maturation factor. In fact, Usuki et al. (32) demonstrated that PDECGF lacking hydrophobic signal sequence was released, but not in a large quantity, from A431, human epidermoid carcinoma cells (32). However, we cannot rule out the possibility of simple leakage or release of the factor by lysis associated withnormal cellular turnover.Furthermore, this research group has recently proposed a plausible mechanism that the intracellular PD-ECGF was linked with nucleotide ATP, and this nucleotidylation might be of importance in the secretion of PD-ECGF from cells in a signal sequence-independent manner (33). In support of these results, Furukawa et al. (34) have recently reported thatPD-ECGF is very similar to pyrimidine nucleoside phosphorylase (thymidine phosphorylase) (34),so that gliostatin/PD-ECGF as a metabolic enzyme may be involved in this nucleotidylation. We have not, however, determined whether gliostatin from NF1 was nucleotidylated. Taking into consideration that we could sequence 10 peptide fragments in the carboxyl-terminal half of gliostatin molecule, and only two tryptic peptides from the aminoterminal half where two sites of consensus sequence (GIy-XGly-X-X-Gly) for nucleotide binding are located (35, 36), it may be possible that gliostatin purified from NF1 is nucleotidylated and shows insusceptibility, due to the steric hindrance by nucleotide moiety, to trypsin digestion at the amino-terminal half including two consensus sequences (GlyZ0-GlyZ5 and Gly'47-Gly'52). Biologicalsignificance of nucleotidylation related to neurotrophic, gliostatic, andPDECGF action remains to be determined. REFERENCES 1. Easter, S. S., Jr., Rurves, D., Rakic, P., and Spitzer, N. C. (1985) Science 230,507-511 2. Furukawa, S., Furukawa, Y., Satoyoshi, E.,and Hayashi, K. (1986)Biochem. Biophys. Res. Commun. 136,57-63 3. Rudge, J. S., Davis, G. E., Manthorpe, M., and Varon, S. (1987) Deu. Brain Res. 3 2 , 103-110 4. Lilien, L. E., Sendtner, M., Pohrer, H., Hughes, S., and Raff, M. C. (1988) Neuron 1 , 485-494 5. Lim, R., Hicklin, D. J., Ryken, T. C., and Miller, J. F. (1987) Deu. Bruin Res. 33.49-57 6. Engele, J., and Bohn, M. C. (1991) J. Neurosci. 11,3070-3078 7. Horiuchi, I., Kato, T., Sasaki, S., Kato, H., Naganawa, N., Masaoka, A,, Tsunooka, H., Ito, J., Okumura-Noji, K., Kano-Tanaka, K., Kato, K., and Tanaka, R. (1985) Neurochem. Int. 7 , 497-504 8. Ito, J., Kato, T., Hara, F., Kano-Tanaka, K., and Tanaka, R. (1987) Neurochem. Int. 11,331-337 9. Asai, K., Hotta, T., Nakanishi, K., Ito J., Tanaka R., Otsuka, T., Matsui, N., and Kato, T.(1991) Brain Res. 666,344-348 10. Asai, K., Hirano, T., Kaneko, S., Moriyama, A., Nakanishi, K., Isobe, I., Eksioglu, Y.Z., and Kato, T. (1992) J. Neurochem., 59,307-317 11. Benda, P., Lightbody, J., Sato, G., Levine, L., and Sweet, W. (1968) Science 161,370-371 12. Banker, G. A., and Cowan, W. M. (1977) Bruin Res. 1 2 6 , 397-425 13. Booyse, F. M., Sedlak, B. J., and Wasteson, A. (1975) Thrornb. Diath. Haemorrh. 34.825-836 14. Miyazono, K., Okabe, T., Urabe, A,, Yamanaka, M., and Takaku, F. (1985) Biochem. Biophys. Res. Commun. 126,83-88 15. Doberty, P., Dickson, J. G., Flanigan, T. P., and Walsh, F. S. (1984) J. Neurochem. 42,1116-1122 16. Laemmli, U. K. (1970) Nature 227,680-685 17. Kato, K., Suzuki, F., and Umeda, Y.(1981) J . Neurochem. 3 6 , 793-797 18. Hashida, S., Imagawa, M., Inoue, S., Ruan, K.-H., and Ishikawa, E. (1984) J. Appl. Biochem. 6,56-63 19. Miyazono, K., Okabe, T., Urabe, A., Takaku, F., and Heldin, C.-H. (1987) J. Biol. Chem. 262,4098-4103 20. Usuki, K., Norberg, L., Larsson, E., Miyazono, K., Hellman, U., Wernstedt,

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C., Rubin, K., and Heldin, C.-H. (1990) Cell Regul. 1,557-596 21. Ishikawa, F., Miyazono, K., Hellman, U., Drexler, H., Wernstedt, C., Hagiwara, K., Usuki, K., Takaku, F., Risau, W., and Heldin, C.-H. (1989) Nature 338,557-562 22. Levi-Montalcini, R.,and Angelletti, P. U. (1968) Physiol. Reu. 4 8 , 534-569 31. 23. Thoenen, H., and Barde, Y.-A. (1980) Physiol. Rev. 60,1284-1335 24. Thoenen, H., Bandtlow, C., and Heumann, R. (1987)Reu. physiol. Biochern. Pharmacol. 109,146-178 25. Barbin, G., Manthowe, M., and S. (1984)J . Neurochem. 43,1468-

.

,"A

29. Leibrock, J., Lottspeich, F., Hohn, A,, Masiakowski, P., Thoenen, H., and Barde, Y.-A. (1989) Nature 341, 149-152 30. Hohn, A., Leihrock, J., Bailey, K., and Barde, Y.-A. (1990) Nature 3 4 4 , 339-341 Maisonpierre, p. c., Belluscio, L., S uinto, S. P., Ip, N. Y., Furth, M. E., Lindsay, R. M., and Yancopoulos,%. D. (1990) Science 247,1446-1451 32. Usuki, K.3 Heldin, N. E., Mi azono, K.9 Ishikawa, F., Takaku, F., Westermark, B., and Heldin, C.-&. (1989) Proc. Natl. Acad. Sei. U. S. A. 86, 7427-7431 33, Usuki. K,. Mivazono. K,. and Heldin. C,-H, (1991) J, BioE, Chern, 266.