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Haptotactic Migration Induced by Midkine

Haptotactic Migration Induced by Midkine THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 19, Issue of May 11, pp. 15868 –15875, 2001 © 2001 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. INVOLVEMENT OF PROTEIN-TYROSINE PHOSPHATASE z, MITOGEN-ACTIVATED PROTEIN KINASE, AND PHOSPHATIDYLINOSITOL 3-KINASE* Received for publication, July 6, 2000, and in revised form, January 31, 2001 Published, JBC Papers in Press, February 13, 2001, DOI 10.1074/jbc.M005911200 Maosong Qi‡, Shinya Ikematsu§, Nobuaki Maeda¶, Keiko Ichihara-Tanaka‡, Sadatoshi Sakuma§, Masaharu Noda¶, Takashi Muramatsu‡, and Kenji Kadomatsu‡i From the ‡Department of Biochemistry, Nagoya University School of Medicine, Nagoya 466-8550, the §Pharmaceuticals Development Department, Meiji Milk Products Co., Ltd., 540 Naruda, Odawara 250-0862, and the ¶Division of Molecular Neurobiology, National Institute for Basic Biology, Okazaki 444-8585, Japan Midkine, a heparin-binding growth factor, plays a toward substrate-bound molecules such as various extracellu- lar matrix proteins. Cell migration is the result of a series of critical role in cell migration causing suppression of neointima formation in midkine-deficient mice. Here we complicated, integrated processes and is controlled by many have determined the molecules essential for midkine- kinds of intracellular molecules (1, 2). These molecules include induced migration. Midkine induced haptotaxis of os- Rho small G protein family members, PI3-kinases, MAP ki- teoblast-like cells, which was abrogated by the soluble nases (Erk1 and Erk2), and protein kinase C. form of midkine or pleiotrophin, a midkine-homologous Midkine (MK) was first identified as the product of a retinoic protein. Chondroitin sulfate B, E, chondroitinase ABC, acid-responsive gene in embryonal carcinoma cells (3, 4). MK B, and orthovanadate, an inhibitor of protein-tyrosine and pleiotrophin (PTN, also called HB-GAM for heparin-bind- phosphatase, suppressed the migration. Supporting ing growth-associated molecule) comprise a family of heparin- z, a recep- these data, the cells examined expressed PTP binding growth/differentiation factors and are not related to tor-type protein-tyrosine phosphatase that exhibits other heparin-binding growth factors such as fibroblast growth high affinity to both midkine and pleiotrophin and har- factor or hepatocyte growth factor (5, 6, 7). MK has been re- bors chondroitin sulfate chains. Furthermore, strong ported to promote neuronal survival and neurite outgrowth (8, synergism between midkine and platelet-derived 9) and to play roles in carcinogenesis (10, 11) and tissue remod- growth factor in migration was detected. The use of eling (12, 13). specific inhibitors demonstrated that mitogen-activated Using MK knock-out mice, it was demonstrated that MK is protein (MAP) kinase and protein-tyrosine phosphatase involved in neointima formation in a model of restenosis after were involved in midkine-induced haptotaxis but not angioplasty (14). Neointima is the basic lesion in both athero- PDGF-induced chemotaxis, whereas phosphatidylinosi- sclerosis and restenosis after angioplasty (15). A variety of tol 3 (PI3)-kinase and protein kinase C were involved in stresses to the arterial endothelium can induce the migration of both functions. Midkine activated both PI3-kinase and smooth muscle cells from the media into the space between the MAP kinases, the latter activation was blocked by a PI3-kinase inhibitor. Midkine further recruited PTP endothelium and internal elastic lamina to form a neointima. and PI3-kinase. These results indicate that PTPz and One of the most important molecules in this process is PDGF- concerted signaling involving PI3-kinase and MAP ki- BB, which is responsible for the migration of smooth muscle nase are required for midkine-induced migration and cells (16). Macrophages recruited into the arterial wall also demonstrate for the first time the synergism between play a critical role in this lesion formation (17). Neointima midkine and platelet-derived growth factor in cell formation and macrophage recruitment to the arterial wall migration. were suppressed in MK-deficient mice (14). Because MK in- duces the migration of both smooth muscle cells and macro- phages in vitro (14), it was concluded that the cell migration- Cell migration plays a key role in a wide variety of biological inducing activity of MK is crucial for the suppression of phenomena (1, 2). There are three main types of cell migration: neointima formation. chemokinesis, chemotaxis, and haptotaxis. Chemokinesis com- These findings also suggest a possible interaction between prises random, non-directional motility in response to a ligand MK and PDGF in smooth muscle cell migration. In addition, without any orienting cues. Chemotaxis is the cell movement remodeling after bone fractures also supports the interaction of toward a positive gradient of soluble stimulants such as che- MK and PDGF, because MK expression and PDGF accumula- mokines and growth factors. Haptotaxis involves cell crawling tion are induced during this process (13). PTN/HB-GAM pro- motes the migration of osteoblast-like cells, including UMR106 cells, which provides further evidence (18). * This work was supported by Grants-in-aid from the Ministry of Education, Science, Sports, and Culture of Japan (10152224 and MK and PTN/HB-GAM induce the migration of cortical neu- 10171210) and grants-in-aid for Center of Excellence Research and rons (19, 20). MK also induces the migration of neutrophils from CREST of the Japan Science and Technology Corporation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “adver- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate The abbreviations used are: PI3-kinase, phosphatidylinositol 3-ki- this fact. nase; MAP, mitogen-activated protein; MK, midkine; PTN, pleiotro- To whom correspondence should be addressed: Dept. of Biochemis- phin; HB-GAM, heparin-binding growth-associated molecule; PDGF- try, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa- BB, platelet-derived growth factor BB; PTPz, protein-tyrosine ku, Nagoya 466-8550, Japan. Tel.: 81-52-7442064; Fax: 81-52-7442065; phosphatase z; PLL, poly-L-lysine; RT-PCR, reverse transcriptase-po- E-mail: [email protected]. lymerase chain reaction. 15868 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. Synergism between Midkine and PDGF in Cell Migration 15869 FIG.2. Competition between MK and PTN for activating cell migration. A, the lower surface of filters was coated with MK (20 mg/ml) (MK-coat) or PTN/HB-GAM (10 mg/ml) (PTN-coat). The migra- tion assay was performed with medium containing MK (20 mg/ml) or PTN/HB-GAM (10 mg/ml) in the lower chamber. The symbol 0 means no addition of these factors. The essential procedure was the same as FIG.1. MK induces haptotactic migration of UMR-106 cells. A, described in the legend to Fig. 1. The mean number of migrated cells the migration assay was performed with filters coated with MK on their was ;70 for 0/MK-coat, and 120 for 0/PTN-coat, which corresponded to lower surface at the indicated concentrations. UMR106 cells (;1 3 10 10 –20% of the total applied cell number. B, the lower surface of filters cells in 100 ml of 0.3% bovine serum albumin/Dulbecco’s modified Ea- was coated with MK or PTN/HB-GAM alone, or together. MK, coated gle’s medium) were added to the upper chamber of Chemotaxicell (a with 20 mg/ml MK; PTN, 10 mg/ml PTN/HB-GAM; P-M10, 10 mg/ml MK modified Boyden chamber), followed by incubation for 4 h. Ten fields and 5 mg/ml PTN/HB-GAM; P-M20, 20 mg/ml MK and 5 mg/ml PTN/ at 3 400 per filter were counted to obtain the migrated cell number (1 HB-GAM. Values are the means 6 S.E.; n 5 3. field 5 1/160 of entire surface of filter). The value shown as the Mi- grated Cell Number is the mean 6 S.E. (n 5 3) per field. A represent- human PTN/HB-GAM with yeast was the same as that for MK de- ative of three independent experiments is shown. B, a filter coated on its scribed previously (14). The polyclonal anti-PI3-kinase (p85a) antibody lower surface with PLL, yeast-produced human MK (y-hMK), chemi- and anti-phospho-AKT-(Ser-473) antibody were purchased from Up- cally synthesized human MK (c-hMK), or baculovirus-produced mouse state Biotechnology and Cell Signaling Technology, respectively. The MK (mMK), at 20 mg/ml, was used for the assay. MK was added to the rabbit anti-phosphorylated Erk antibody was from New England Bio- lower chamber in a soluble form at 100 ng/ml (sol. MK). C, the effect of Labs. The monoclonal anti-Erk2 antibody and monoclonal anti-RPTPb soluble MK at the indicated concentrations on the migration of (PTPz) antibody were purchased from Transduction Laboratories. The UMR106 cells induced by coated MK on the filters was examined. rabbit polyclonal anti-PTPz antibody (anti-6B4) was prepared as de- scribed previously (22). (21). However, only a limited body of information concerning Cell Migration Assay, PTPz Extraction, and Chondroitinase Diges- the signaling involved in MK-mediated cell migration is avail- tion—The migration assay was performed as described previously (14, able. We conducted the present study to elucidate the molecu- 19, 20) using Chemotaxicell (Kurabo, Japan; 8-mm pores). PTPz extrac- lar components essential for MK-mediated cell migration, and tion and chondroitinase digestion were performed as described previ- to test our hypothesis that MK and PDGF could cooperate in ously (23). cell migration. RT-PCR—Five mg of total RNA from 15-day-old rat brain or cultured cells was used for reverse-transcription with TrueScript (Sawady, Japa- EXPERIMENTAL PROCEDURES n). The primers used for PCR were: 59-GTTCTCAACACATCCCTGAA- Cell Line, Reagents, and Antibodies—A rat osteoblast-like cell line, TCCTACTTCCCA-39 and 59-CTTTAGTGATTCTTCTGAACCTGATGG- UMR106 (ATCC No. CRL 1661), was purchased from the American AGCCGA-39. The 474-bp PCR product corresponded to the 1587–2061 TM Type Culture Collection. Heparin, chondroitin sulfate A, C, D, and E, fragment of rat PTPz (GenBank /EBI no. U09357), which was con- chondroitinase ABC, AC II, and B, and heparitinase were purchased firmed by DNA sequencing. from Seikagaku, Japan. Dermatan sulfate, phosphatidylinositol, and Western Blot Analysis—Polystyrene beads (Polysciences) were coated phosphatidylinositol 4-monophosphate were obtained from Sigma. The with MK (20 mg/ml) or PLL (5 mg/ml, plus 15 mg/ml bovine serum inhibitors for Src (PP1), protein kinase C (Ro318220), and a MAP albumin) at 4 °C overnight or room temperature for 2 h. The beads were kinase kinase, MEK (PD98059), were from Alexis Biochemicals, Cal- washed with phosphate-buffered saline four times before use. UMR106 biochem-Novabiochem and Biomol Research Laboratories, respectively. cells starved for 24 h were stimulated with the coated polystyrene The other inhibitors for protein kinase C (H7 and calphostin C), PI3- beads. Cells were lysed in a buffer comprising 10 mM Tris-HCl, pH 7.4, kinase (wortmannin), and phospholipase C (U-73122) were products of 1% Triton X-100, 0.5% Nonidet P-40, 150 mM NaCl, 1 mM EDTA, 0.2 mM Sigma. Recombinant human PDGF-BB was obtained from PeproTech, phenylmethylsulfonyl fluoride, 0.1 mg/ml aprotinin, 40 nM leupeptin, England. The procedure for producing recombinant human MK with and 0.2 mM sodium vanadate. Thirty mg of protein were separated by yeast has already been described (14), and in this paper MK means 10% SDS-polyacrylamide gel electrophoresis and then transferred to a human MK produced by yeast unless specified otherwise. Recombinant nitrocellulose membrane, followed by detection with anti-phosphory- mouse MK was expressed with baculovirus and purified as described lated Erk, anti-Erk2, anti-phosphorylated AKT, or anti-AKT antibody. previously (9). Chemically synthesized human MK was purchased from PI3-kinase Assay—UMR106 cells were treated as described for West- the Peptide Institute, Japan. The procedure for producing recombinant ern blot analysis of Erk phosphorylation. PI3-kinase activity was meas- 15870 Synergism between Midkine and PDGF in Cell Migration FIG.3. Effect of glycosaminogly- cans on MK-induced cell migration. A, UMR106 cells were preincubated at 37 °C for 30 min with glycosaminoglycans at the concentration of 20 mg/ml or that indicated. The migration assay was car- ried out with the same glycosaminoglycan concentration in the lower chamber as in the upper chamber. CS means chon- droitin sulfate. B and C, UMR106 cells were preincubated at 37 °C with hepariti- nase for 90 min (B), or chondroitinase ABC for 30 min (C). D, at 20 milliunits/ml, chondroitinase ABC and B, but not AC II, inhibited the migration. Values are the means 6 S.E.; n 5 3. FIG.4. Possible role of PTP-z in MK- induced cell migration. A, 5 mg of total RNA were used for RT-PCR. L cell, fibro- blast cell line; G401 cell, Wilms’ tumor cell line. Upper panel, PTPz; lower panel, GAPDH. B, Western blot analysis with anti-RPTPb (PTPz) antibody before and after chondrotinase digestion. Arrow- heads and an arrow indicate the positive bands after chondroitinase ABC diges- tion. The bracket indicates the smear de- tected for UMR106 cells. Note the nonspe- cific bands for all lysates of UMR106 and L cells. C, sodium vanadate (NaVa) inhib- ited MK-induced migration of UMR106 cells. D, preincubation with anti-PTPz (anti-6B4) antibodies, but not control rab- bit IgG, enhanced MK-induced migration. Values are the means 6 S.E.; n 5 3. ured by in vitro phosphorylation of phosphatidylinositol, using essen- trix proteins, such as fibronectin, vitronectin, laminin-1, and tially the same method as described previously (24). collagen IV (25). As chemically synthesized human MK and Immunofluorescence Microscopy—UMR106 cells, which had been ex- baculovirus-produced mouse MK showed the same activity as posed to MK- or PLL-beads for 15 min, were fixed with 4% paraform- that of yeast-produced human MK (Fig. 1B), the migratory aldehyde and then stained with anti-PI3-kinase (p85a) antibody/fluo- rescein isothiocyanate-conjugated anti-rabbit IgG and/or anti-RPTPb effect was not caused by impurities in the MK used. MK con- (PTPz) monoclonal antibody/horseradish peroxidase-conjugated anti- tains 30% basic amino acids. However, PLL showed very weak mouse IgG antibody/cyanine 5-labeled tyramide. For PTPz staining, the migratory activity (Fig. 1B), suggesting that the effect of MK TSA system (PerkinElmer Life Sciences) was employed to enhance the was not because of its high basicity. signal. Soluble MK did not induce UMR106 cell migration (Fig. 1B), RESULTS even if the filter was precoated with collagen I (data not shown). On the contrary, when added to the lower chamber, MK Induces Haptotactic Migration of Osteoblast-type Cells— soluble MK inhibited coated MK-induced migration (Fig. 1C). PTN/HB-GAM, another member of the MK family, induces At the concentration of 10 mg/ml, soluble MK completely abol- haptotactic migration of several osteoblast cell lines (18). We ished coated MK-induced migration. The same concentration of first investigated whether or not MK had the same activity. soluble PLL in the lower chamber did not inhibit MK-induced When coated on the lower surface of a filter, MK induced the haptotactic migration of UMR106 cells, with the maximum migration (data not shown). These findings indicate that the substratum-bound form of MK is active in cell migration. Thus, level at the concentration of 20 mg/ml (Fig. 1A). The profile of MK-induced cell migration was similar to that of the colon MK could induce haptotactic, but not chemotactic, migration of carcinoma cell migration induced by several extracellular ma- UMR106 cells. Synergism between Midkine and PDGF in Cell Migration 15871 FIG.5. Effect of different inhibitors on MK-induced cell migra- tion. UMR106 cells were incubated with the indicated inhibitors for 30 min. The migration assay was then performed with both the upper and lower chambers containing the same concentration of the same inhibi- FIG.6. Erk activation after MK stimulation. UMR106 cells stim- tor. A, concentrations of inhibitors: tyrosine kinase-herbimycin A ulated with polystyrene beads coated with PLL (5 mg/ml) or MK (20 (herb), 1 mg/ml; tyrosine kinase-genistein (geni), 50 mg/ml; Src-PP1, 10 mg/ml) for the indicated times were analyzed by Western blotting with nM; protein kinase C-Ro318220 (Ro), 2 mM; PI 3 kinase-wortmannin anti-phosphorylated Erk (P-Erk1/2) and anti-Erk2 (Erk2) antibodies. (wort), 100 nM; MEK-PD98059 (PD), 20 mM; phospholipase C-U73122 The densitometric data obtained on Western blotting are shown in the (U-7), 10 mM. B and C, dose dependence of protein kinase C inhibitors graph.In B, inhibitors were used as described in the legend to Fig. 5. (B) and MEK, PI3 kinase, and phospholipase C inhibitors (C). The units are: PD98059, mM; wortmannin, nM; U73122, mM. Values are the means 6 S.E.; n 5 3. corresponding to PTPz was detected for UMR106 cells and rat brain (Fig. 4A). The PCR product was confirmed by DNA se- Soluble MK and PTN Abrogate Haptotaxis Mediated by MK quencing (data not shown). No positive band was detected for L and PTN/HB-GAM—We next addressed the question of cells or Wilms’ tumor cells (Fig. 4A). This is consistent with the whether or not MK and PTN/HB-GAM share the same cell observation that coated MK did not induce the migration of surface binding site(s). Soluble MK abrogated PTN/HB-GAM- these cells with a 4-h incubation (data not shown). On Western induced haptotaxis and vice versa (Fig. 2A). Coating with a blotting with monoclonal anti-RPTPb (PTPz) antibody, which combination of MK and PTN/HB-GAM did not enhance the recognizes the intracellular domain of PTPz, a smear was de- migration as compared with MK or PTN/HB-GAM alone (Fig. tected for the lysate of UMR106 cells (Fig. 4B, bracket, lane 3). 2B). These data suggest that if the total molar concentration of This changed to one band corresponding to about 240 kDa after MK and PTN/HB-GAM reaches a critical level, the migration chondroitinase ABC digestion (Fig. 4B, lane 4, arrow). For rat activity will be saturated, and MK and PTN/HB-GAM may brain, smears were also detected (Fig. 4B, lane 1), which function through common molecule(s) on the cell surface. shifted to 380 and 220 kDa upon digestion with chondroitinase Proteoglycan Is Involved in MK-induced Migration—Hepa- ABC (Fig. 4B, lane 2, arrowheads), which represent long- and ran sulfate proteoglycans, such as syndecan-1, -3, and -4, and a short-type receptors, respectively (23). The size difference chondroitin sulfate proteoglycan, PTPz, are supposed to act as around 220/240 kDa between the brain and UMR106 cells is a receptor or co-receptor for MK (20, 26, 27, 28). We investi- probably caused by differential glycosylation, which is depend- gated the effects of different glycosaminoglycans on MK-medi- ent on the cell type (30). Chondroitinase AC II and B appeared ated migration. In addition to heparin, dermatan sulfate (chon- to digest the glycosaminoglycan chains of PTPz only partially, droitin sulfate B) and chondroitin sulfate E showed comparable because the band around 240 kDa was broader than the band effects. They abolished the migration at the concentration of 20 digested with chondroitinase ABC (Fig. 4B, lanes 8, 9, 10). This mg/ml (Fig. 3A). At 20 mg/ml, chondroitin sulfate A, C, and D is reasonable because chondroitinase AC II and B recognize showed only minor effects (Fig. 3A). Treatment with hepariti- different structures (31, 32). Nevertheless, only chondroitinase nase to remove the heparan sulfate on the cell surface did not ABC and B, but not AC II, suppressed MK-induced migration affect MK-mediated migration (Fig. 3B). On the contrary, chon- (Fig. 3D), suggesting that structures susceptible to chondroiti- droitinase ABC digestion and chondroitinase B digestion down- nase ABC and B are important. As chondroitinase B can digest regulated MK-mediated migration (Fig. 3, C and D), suggesting both dermatan sulfate and E-type dermatan sulfate (32), and the involvement of cell surface chondroitin sulfate. chondroitinase ABC can digest all chondroitin and dermatan Involvement of PTPz in MK-induced Migration—As PTPz sulfates (33), this is consistent with the finding that MK-in- exhibits high affinity to both MK and PTN/HB-GAM (20, 29), duced cell migration was inhibited by dermatan sulfate (chon- the results shown in Figs. 1, 2, and 3 suggest that PTPz is droitin sulfate B) and chondroitin sulfate E (Fig. 3A). involved in MK-induced haptotaxis. Using RT-PCR, a band PTPz is also known to be a receptor-type protein-tyrosine 15872 Synergism between Midkine and PDGF in Cell Migration FIG.7. PI3-kinase activity after MK and PDGF-BB stimulation. UMR106 cells were stimulated with PDGF-BB, or polystyrene beads coated with PLL or MK. PI 3-kinase activity was measured as described under “Experimental Proce- dures.” A, representative autoradiogra- phy. Lower panel, quantitative presenta- tion (mean 6 S.E.; n 5 3). B, effects of inhibitors. phosphatase. The effect of orthovanadate, an inhibitor of pro- the case of PDGF-BB (Fig. 7A). Neither Ro318220 nor MEK tein-tyrosine phosphatase, was examined. Sodium vanadate inhibitor PD98059 affected MK-induced PI3-kinase activation decreased the migration in a dose-dependent manner (Fig. 4C). (Fig. 7B). Sodium vanadate had no effect on PLL or PDGF-BB-induced MK Recruits PTPz and PI3-kinase—To further confirm MK- migration of UMR106 cells (data not shown). If the cells were induced PI3-kinase activation, phosphorylation of AKT was preincubated with anti-PTPz (anti-6B4) antibodies, which rec- examined. AKT (protein kinase B) plays important roles in ognize the ectodomain of the full-length PTPz (23), MK-medi- many biological phenomena, such as cell survival (34, 35). AKT ated cell migration was enhanced (Fig. 4D). This suggests that contains a pleckstrin homology (PH) domain, and is phospho- ligation of cell surface PTPz with its ligands or antibodies may rylated (activated) at Thr-308 and Ser-473 residues by PDK1, transduce signals essential for the migration of UMR106 cells. which also carries a PH domain. The PH domain recognizes a MK Induces MAP Kinase and PI3-kinase Activity—To iden- phosphoinositide headgroup, and phosphorylation at the 3 po- tify the intracellular molecules that participate in MK-medi- sition of inositol ring of phosphatidylinositol in the cell mem- ated cell migration, various specific inhibitors were screened. brane is critical for AKT- and PDK1-binding and recruitment Tyrosine kinase inhibitors (genistein and herbimycin A), a Src to the cell membrane (35). As this phosphorylation is mediated inhibitor (PP1), protein kinase C inhibitors (H7, Ro318220, and by PI3-kinase, AKT is an important PI3-kinase effector, and its calphostin C), a PI3-kinase inhibitor (wortmannin), a MAP activation is often used as a marker of PI3-kinase activation kinase kinase (MEK) inhibitor (PD98059), and a phospholipase (34, 35). C inhibitor (U-73122) effectively inhibited MK-mediated cell MK enhanced AKT phosphorylation (Fig. 8A, left), which is migration (Fig. 5). This probably indicates that a dynamically consistent with the data for MK-induced PI3-kinase activation concerted signaling interaction is essential for the cell migra- shown in Fig. 7A. When the cells were preincubated with tion induced by MK. anti-PTPz (anti-6B4) antibodies, MK-induced AKT activation When UMR106 cells were stimulated with MK-coated polys- was stronger than that of the cells treated with control IgG trene beads, a transient increase in phosphorylation of MAP (Fig. 8A, right). This was consistent with that anti-PTPz (anti- kinases (Erk1 and Erk2) was detected, the maximum level 6B4) antibodies enhanced MK-induced migration of UMR106 being observed ;20 min after stimulation (Fig. 6A). At 60 min, cells (Fig. 4D). It is of interest that, in addition to the PI3- it was still higher than the basal level (Fig. 6A). PLL also kinase inhibitor wortmannin, the protein-tyrosine phosphatase enhanced Erk phosphorylation, but the increase was less and inhibitor orthovanadate and Src inhibitor PP1 blocked MK- the duration was shorter than in the case of MK (Fig. 6A). induced AKT activation (Fig. 8A, right), but neither protein Interestingly, the PI3-kinase inhibitor wortmannin partly kinase C inhibitor Ro 318220 nor MEK inhibitor PD98059 blocked MK-induced Erk phosphorylation, whereas the protein affected it (data not shown). As the effect of orthovanadate on kinase C inhibitor Ro318220 did not (Fig. 6B). MK also in- MK-induced AKT phosphorylation suggested the possibility of creased PI3-kinase activity, the profile being similar to that in a close connection between PTPz and PI3-kinase, we localized Synergism between Midkine and PDGF in Cell Migration 15873 FIG.8. Involvement of PI3-kinase and PTPz in MK signaling. A, left; UMR106 cells were exposed to MK- or PLL-beads at 37 °C for 15 min. Cell extracts were then analyzed for phosphorylated AKT (Ser- 473) (p-Akt) or AKT expression by Western blotting. The densitometric ratio (p-Akt versus Akt) was calculated for each condition, and the relative density of p-Akt is shown at the bottom. A, right; UMR106 cells were incubated with either anti-PTPz (anti-6B4) antibodies or control rabbit IgG together with the indicated inhibitor at 4 °C for 2 h. The cells were then exposed to MK-beads in the presence of indicated inhibitor at 37 °C for 15 min. B, UMR106 cells were exposed to MK- or PLL-beads at 37 °C for 15 min. The localization of PI3-kinase and PTPz was examined by immunofluorescence microscopy (PI3-kinase, FITC; PTPz, cyanine 5) using a confocal microscope (MRC-1024, Bio-Rad). The lower two panels show the same cell that was double-stained with anti-PI3- kinase and anti-RPTPb (PTPz) antibodies. Arrows and asterisks indi- cate cells examined and beads, respectively. PTPz and PI 3-kinase after MK beads stimulation on UMR106 cells. MK beads induced PI3-kinase recruitment to the sites of the beads (Fig. 8B, left two panels). Furthermore, MK-beads induced colocalization of PI3-kinase and PTPz (Fig. 8B, lower two panels). MK Has a Synergistic Effect with PDGF-BB on Cell Migra- tion—The hypothesis that MK and PDGF can cooperate in cell migration in some in vivo situations was examined next. On filters coated on both their upper and lower surfaces with collagen I, PDGF-BB induced chemotactic migration of UMR106 cells (Fig. 9A). PDGF-induced migration was sup- FIG.9. Synergistic effect of MK and PDGF-BB on cell migra- tion. A, PDGF-BB-induced migration was inhibited by protein kinase C pressed by Ro318220 and wortmannin (Fig. 9A). But PD98059, and PI3-kinase inhibitors. PDGF-BB (20 ng/ml) was added to the lower which inhibited MK-mediated cell migration (Fig. 5), did not chamber. Concentrations of reagents: PD98059, 20 mM; Ro318220, 2 mM; affect PDGF-mediated migration (Fig. 9A). Whereas heparin wortmannin, 100 nM; heparin, 20 mg/ml. B, MK and PDGF-BB showed abolished MK-mediated cell migration (Fig. 3A), it rather en- synergistic effect to induce UMR106 cell migration. The filter of Che- motaxicell was coated with collagen I on both its upper and lower hanced PDGF-mediated cell migration (Fig. 9A). If the lower surfaces or with MK on only its lower surface. Migration was then surface of the filters was coated with MK instead of collagen I, monitored in the presence or absence of PDGF-BB in the lower cham- the chemotactic activity of PDGF-BB was dramatically en- ber. C, MK/PDGF-BB-mediated migration was not affected by MEK hanced (Fig. 9B). The effects of MK and PDGF-BB were syn- inhibitor. PD98059, Ro318220, and wortmannin were used as described in A. ergistic, because the migrated cell number was much higher than the total when MK and PDGF-BB were used separately. 13, 14, 36, 37). In the case of arterial endothelial injury, we The migration induced by the combination of MK and found not only the induction of MK expression in wild-type PDGF-BB was susceptible to inhibitors of PI 3-kinase and mice, but also dramatically suppressed neointima formation in protein kinase C, but not to the MEK inhibitor (Fig. 9C). This MK-deficient mice (14). The administration of the MK protein profile resembles that of the migration induced by PDGF-BB to MK-deficient mice caused resumption of neointima forma- alone, but not that in the case of MK alone. tion (14). Thus, MK seems to be vital for tissue remodeling. The DISCUSSION present study revealed another important aspect of the mode of MK has been reported to be induced in areas of a variety of MK action, namely, that it acts synergistically with PDGF in types of tissue injury, such as cerebral and heart infarction, cell migration. PDGF was first purified from platelet a gran- bone fractures, skin burns, and arterial endothelial injury (12, ules and has several important activities, such as mitogenesis 15874 Synergism between Midkine and PDGF in Cell Migration and chemotaxis. Because tissue injury is usually accompanied that the chondroitin sulfate chain in PTPz is also important in by bleeding and/or cessation of blood flow, an abundance of migration of osteoblast-like cells. Differential susceptibility of PDGF can be found in these areas. PDGF expression is also the migratory activity to chondroitinases with different speci- induced during the process of wound healing (38). Further- ficities gave further insights into the nature of chondroitin sulfate chain in PTPz. Digestion with chondroitinase B abol- more, PDGF induces the migration of smooth muscle cells (16, 39) and osteoblasts (40), and the migration of both is also ished MK-dependent migratory activity, whereas chondroiti- nase AC II did not. The former enzyme acts on chondroitin induced by MK (Ref. 14 and the present study). Taken together, sulfate chain with the L-iduronic acid residue, namely derma- the synergism between MK and PDGF appears to be pivotal in tan sulfate, whereas the latter does not. Thus, it is concluded many in vivo situations. that chondroitin sulfate, which is important in MK-signaling in Several possibilities should be considered for the mechanism PTPz, has a dermatan sulfate domain. The finding that the underlying the synergism between MK and PDGF. The present MK-induced migration is inhibited by dermatan sulfate is con- study revealed a difference in the signaling mechanism be- sistent with the view. The MK activity was also inhibited by tween MK and PDGF. Heparin inhibited MK-induced cell mi- chondroitin sulfate E, which is an oversulfated chondroitin gration but rather enhanced PDGF-induced cell migration sulfate with 4,6-disulfo-N-acetylgalactosamine residue. Taken (Figs. 3A and 9A). MAP kinases were essential for MK-medi- together, most probably the chondroitin sulfate chain in PTPz, ated cell migration but not for PDGF-induced cell migration to which MK binds, has an oversulfated structure in a derma- (Figs. 5 and 9). Orthovanadate inhibited MK-induced cell mi- tan sulfate domain. Indeed, E-type structure with a dermatan gration but not PDGF-induced cell migration (Fig. 4C and data sulfate domain was found in PG-M/versican, which was iso- not shown). Because distinct signaling molecules are used for lated from mouse embryos and has MK binding activity (54). cell migration, depending on the ligand (41, 42), the synergism The characteristics of MK-induced migration of UMR106 between MK and PDGF might be attributed to the integration cells are very similar to those of MK- and PTN/HB-GAM- of distinct signaling pathways. Alternatively, induction of MK induced neuronal migration in that PTPz is involved in hapto- receptor(s) by PDGF or vice versa should also be considered, as tactic migration (19, 20). In this context, the effect of anti-PTPz in the case of the induction of the Interleukin-1 receptor or antibodies on MK-mediated cell migration was unexpected. PDGF receptor by the neuropeptide substance P in their syn- These antibodies effectively inhibited PTN/HB-GAM-mediated ergism in bone marrow fibroblast proliferation (43). In addi- neuronal migration in the previous study, probably because of tion, a third cell surface molecule, such as integrin b , might be competitive inhibition for the PTN/HB-GAM-binding sites of involved in the synergism, like in the case of the synergism cell surface PTPz by the antibodies (20). But in the present between lysophosphatidic acid and epidermal growth factor or study, the antibodies rather enhanced MK-mediated osteo- PDGF in cell migration (44). blast-like cell migration. One possible interpretation of this is MAP kinases (Erk1 and Erk2) can activate myosin light that PTPz on UMR106 cells may physically associate with chain kinase and induce changes in the cytoskeletal structure, another unidentified component necessary for signal transduc- leading to cell migration (45). In this context, it is noteworthy tion and thus can be readily activated by the oligomerization or that MK enhances collagen gel contraction by dermal fibro- conformational change induced by a specific antibody. On nerve blasts (46). MK-induced MAP kinase activation appeared to be cells, PTPz might need MK to associate with such a component. at least partly regulated by PI3-kinase (Fig. 6B). PI3-kinase Supporting our data, Revest et al. (56) recently reported that functions as an early intermediate in Gbg-mediated MAP ki- cross-linking of PTPz with antibodies enhances the protein- nase activation (47). Several papers have reported that PI3- tyrosine phosphatase activity of C6 astrocytoma cells. kinase acts upstream of MAP kinase, e.g. in insulin signaling in In this study, we confirmed the involvement of Erk1 and 2 3T3-L1 adipocytes and PDGF signaling in Swiss 3T3 cells (48, and PI3-kinase in MK-induced cell migration by detecting their 49). active forms or activity induced by MK, in addition to by dem- The present study demonstrated that MK recruited PTPz onstrating the effects of inhibitors of them. Consistent with our and PI3-kinase (Fig. 8B). Furthermore, MK-induced PI3-ki- data, Souttou et al. reported that Erk1 and 2 and PI3-kinase nase activation was inhibited by the Src inhibitor PP1 and are involved in PTN/HB-GAM-mediated cell proliferation (57). protein-tyrosine phosphatase inhibitor orthovanadate (Fig. In addition, Src, JAK1, and 2 and b-catenin have been reported 8A). 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Haptotactic Migration Induced by Midkine

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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 19, Issue of May 11, pp. 15868 –15875, 2001 © 2001 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. INVOLVEMENT OF PROTEIN-TYROSINE PHOSPHATASE z, MITOGEN-ACTIVATED PROTEIN KINASE, AND PHOSPHATIDYLINOSITOL 3-KINASE* Received for publication, July 6, 2000, and in revised form, January 31, 2001 Published, JBC Papers in Press, February 13, 2001, DOI 10.1074/jbc.M005911200 Maosong Qi‡, Shinya Ikematsu§, Nobuaki Maeda¶, Keiko Ichihara-Tanaka‡, Sadatoshi Sakuma§, Masaharu Noda¶, Takashi Muramatsu‡, and Kenji Kadomatsu‡i From the ‡Department of Biochemistry, Nagoya University School of Medicine, Nagoya 466-8550, the §Pharmaceuticals Development Department, Meiji Milk Products Co., Ltd., 540 Naruda, Odawara 250-0862, and the ¶Division of Molecular Neurobiology, National Institute for Basic Biology, Okazaki 444-8585, Japan Midkine, a heparin-binding growth factor, plays a toward substrate-bound molecules such as various extracellu- lar matrix proteins. Cell migration is the result of a series of critical role in cell migration causing suppression of neointima formation in midkine-deficient mice. Here we complicated, integrated processes and is controlled by many have determined the molecules essential for midkine- kinds of intracellular molecules (1, 2). These molecules include induced migration. Midkine induced haptotaxis of os- Rho small G protein family members, PI3-kinases, MAP ki- teoblast-like cells, which was abrogated by the soluble nases (Erk1 and Erk2), and protein kinase C. form of midkine or pleiotrophin, a midkine-homologous Midkine (MK) was first identified as the product of a retinoic protein. Chondroitin sulfate B, E, chondroitinase ABC, acid-responsive gene in embryonal carcinoma cells (3, 4). MK B, and orthovanadate, an inhibitor of protein-tyrosine and pleiotrophin (PTN, also called HB-GAM for heparin-bind- phosphatase, suppressed the migration. Supporting ing growth-associated molecule) comprise a family of heparin- z, a recep- these data, the cells examined expressed PTP binding growth/differentiation factors and are not related to tor-type protein-tyrosine phosphatase that exhibits other heparin-binding growth factors such as fibroblast growth high affinity to both midkine and pleiotrophin and har- factor or hepatocyte growth factor (5, 6, 7). MK has been re- bors chondroitin sulfate chains. Furthermore, strong ported to promote neuronal survival and neurite outgrowth (8, synergism between midkine and platelet-derived 9) and to play roles in carcinogenesis (10, 11) and tissue remod- growth factor in migration was detected. The use of eling (12, 13). specific inhibitors demonstrated that mitogen-activated Using MK knock-out mice, it was demonstrated that MK is protein (MAP) kinase and protein-tyrosine phosphatase involved in neointima formation in a model of restenosis after were involved in midkine-induced haptotaxis but not angioplasty (14). Neointima is the basic lesion in both athero- PDGF-induced chemotaxis, whereas phosphatidylinosi- sclerosis and restenosis after angioplasty (15). A variety of tol 3 (PI3)-kinase and protein kinase C were involved in stresses to the arterial endothelium can induce the migration of both functions. Midkine activated both PI3-kinase and smooth muscle cells from the media into the space between the MAP kinases, the latter activation was blocked by a PI3-kinase inhibitor. Midkine further recruited PTP endothelium and internal elastic lamina to form a neointima. and PI3-kinase. These results indicate that PTPz and One of the most important molecules in this process is PDGF- concerted signaling involving PI3-kinase and MAP ki- BB, which is responsible for the migration of smooth muscle nase are required for midkine-induced migration and cells (16). Macrophages recruited into the arterial wall also demonstrate for the first time the synergism between play a critical role in this lesion formation (17). Neointima midkine and platelet-derived growth factor in cell formation and macrophage recruitment to the arterial wall migration. were suppressed in MK-deficient mice (14). Because MK in- duces the migration of both smooth muscle cells and macro- phages in vitro (14), it was concluded that the cell migration- Cell migration plays a key role in a wide variety of biological inducing activity of MK is crucial for the suppression of phenomena (1, 2). There are three main types of cell migration: neointima formation. chemokinesis, chemotaxis, and haptotaxis. Chemokinesis com- These findings also suggest a possible interaction between prises random, non-directional motility in response to a ligand MK and PDGF in smooth muscle cell migration. In addition, without any orienting cues. Chemotaxis is the cell movement remodeling after bone fractures also supports the interaction of toward a positive gradient of soluble stimulants such as che- MK and PDGF, because MK expression and PDGF accumula- mokines and growth factors. Haptotaxis involves cell crawling tion are induced during this process (13). PTN/HB-GAM pro- motes the migration of osteoblast-like cells, including UMR106 cells, which provides further evidence (18). * This work was supported by Grants-in-aid from the Ministry of Education, Science, Sports, and Culture of Japan (10152224 and MK and PTN/HB-GAM induce the migration of cortical neu- 10171210) and grants-in-aid for Center of Excellence Research and rons (19, 20). MK also induces the migration of neutrophils from CREST of the Japan Science and Technology Corporation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “adver- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate The abbreviations used are: PI3-kinase, phosphatidylinositol 3-ki- this fact. nase; MAP, mitogen-activated protein; MK, midkine; PTN, pleiotro- To whom correspondence should be addressed: Dept. of Biochemis- phin; HB-GAM, heparin-binding growth-associated molecule; PDGF- try, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa- BB, platelet-derived growth factor BB; PTPz, protein-tyrosine ku, Nagoya 466-8550, Japan. Tel.: 81-52-7442064; Fax: 81-52-7442065; phosphatase z; PLL, poly-L-lysine; RT-PCR, reverse transcriptase-po- E-mail: [email protected]. lymerase chain reaction. 15868 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. Synergism between Midkine and PDGF in Cell Migration 15869 FIG.2. Competition between MK and PTN for activating cell migration. A, the lower surface of filters was coated with MK (20 mg/ml) (MK-coat) or PTN/HB-GAM (10 mg/ml) (PTN-coat). The migra- tion assay was performed with medium containing MK (20 mg/ml) or PTN/HB-GAM (10 mg/ml) in the lower chamber. The symbol 0 means no addition of these factors. The essential procedure was the same as FIG.1. MK induces haptotactic migration of UMR-106 cells. A, described in the legend to Fig. 1. The mean number of migrated cells the migration assay was performed with filters coated with MK on their was ;70 for 0/MK-coat, and 120 for 0/PTN-coat, which corresponded to lower surface at the indicated concentrations. UMR106 cells (;1 3 10 10 –20% of the total applied cell number. B, the lower surface of filters cells in 100 ml of 0.3% bovine serum albumin/Dulbecco’s modified Ea- was coated with MK or PTN/HB-GAM alone, or together. MK, coated gle’s medium) were added to the upper chamber of Chemotaxicell (a with 20 mg/ml MK; PTN, 10 mg/ml PTN/HB-GAM; P-M10, 10 mg/ml MK modified Boyden chamber), followed by incubation for 4 h. Ten fields and 5 mg/ml PTN/HB-GAM; P-M20, 20 mg/ml MK and 5 mg/ml PTN/ at 3 400 per filter were counted to obtain the migrated cell number (1 HB-GAM. Values are the means 6 S.E.; n 5 3. field 5 1/160 of entire surface of filter). The value shown as the Mi- grated Cell Number is the mean 6 S.E. (n 5 3) per field. A represent- human PTN/HB-GAM with yeast was the same as that for MK de- ative of three independent experiments is shown. B, a filter coated on its scribed previously (14). The polyclonal anti-PI3-kinase (p85a) antibody lower surface with PLL, yeast-produced human MK (y-hMK), chemi- and anti-phospho-AKT-(Ser-473) antibody were purchased from Up- cally synthesized human MK (c-hMK), or baculovirus-produced mouse state Biotechnology and Cell Signaling Technology, respectively. The MK (mMK), at 20 mg/ml, was used for the assay. MK was added to the rabbit anti-phosphorylated Erk antibody was from New England Bio- lower chamber in a soluble form at 100 ng/ml (sol. MK). C, the effect of Labs. The monoclonal anti-Erk2 antibody and monoclonal anti-RPTPb soluble MK at the indicated concentrations on the migration of (PTPz) antibody were purchased from Transduction Laboratories. The UMR106 cells induced by coated MK on the filters was examined. rabbit polyclonal anti-PTPz antibody (anti-6B4) was prepared as de- scribed previously (22). (21). However, only a limited body of information concerning Cell Migration Assay, PTPz Extraction, and Chondroitinase Diges- the signaling involved in MK-mediated cell migration is avail- tion—The migration assay was performed as described previously (14, able. We conducted the present study to elucidate the molecu- 19, 20) using Chemotaxicell (Kurabo, Japan; 8-mm pores). PTPz extrac- lar components essential for MK-mediated cell migration, and tion and chondroitinase digestion were performed as described previ- to test our hypothesis that MK and PDGF could cooperate in ously (23). cell migration. RT-PCR—Five mg of total RNA from 15-day-old rat brain or cultured cells was used for reverse-transcription with TrueScript (Sawady, Japa- EXPERIMENTAL PROCEDURES n). The primers used for PCR were: 59-GTTCTCAACACATCCCTGAA- Cell Line, Reagents, and Antibodies—A rat osteoblast-like cell line, TCCTACTTCCCA-39 and 59-CTTTAGTGATTCTTCTGAACCTGATGG- UMR106 (ATCC No. CRL 1661), was purchased from the American AGCCGA-39. The 474-bp PCR product corresponded to the 1587–2061 TM Type Culture Collection. Heparin, chondroitin sulfate A, C, D, and E, fragment of rat PTPz (GenBank /EBI no. U09357), which was con- chondroitinase ABC, AC II, and B, and heparitinase were purchased firmed by DNA sequencing. from Seikagaku, Japan. Dermatan sulfate, phosphatidylinositol, and Western Blot Analysis—Polystyrene beads (Polysciences) were coated phosphatidylinositol 4-monophosphate were obtained from Sigma. The with MK (20 mg/ml) or PLL (5 mg/ml, plus 15 mg/ml bovine serum inhibitors for Src (PP1), protein kinase C (Ro318220), and a MAP albumin) at 4 °C overnight or room temperature for 2 h. The beads were kinase kinase, MEK (PD98059), were from Alexis Biochemicals, Cal- washed with phosphate-buffered saline four times before use. UMR106 biochem-Novabiochem and Biomol Research Laboratories, respectively. cells starved for 24 h were stimulated with the coated polystyrene The other inhibitors for protein kinase C (H7 and calphostin C), PI3- beads. Cells were lysed in a buffer comprising 10 mM Tris-HCl, pH 7.4, kinase (wortmannin), and phospholipase C (U-73122) were products of 1% Triton X-100, 0.5% Nonidet P-40, 150 mM NaCl, 1 mM EDTA, 0.2 mM Sigma. Recombinant human PDGF-BB was obtained from PeproTech, phenylmethylsulfonyl fluoride, 0.1 mg/ml aprotinin, 40 nM leupeptin, England. The procedure for producing recombinant human MK with and 0.2 mM sodium vanadate. Thirty mg of protein were separated by yeast has already been described (14), and in this paper MK means 10% SDS-polyacrylamide gel electrophoresis and then transferred to a human MK produced by yeast unless specified otherwise. Recombinant nitrocellulose membrane, followed by detection with anti-phosphory- mouse MK was expressed with baculovirus and purified as described lated Erk, anti-Erk2, anti-phosphorylated AKT, or anti-AKT antibody. previously (9). Chemically synthesized human MK was purchased from PI3-kinase Assay—UMR106 cells were treated as described for West- the Peptide Institute, Japan. The procedure for producing recombinant ern blot analysis of Erk phosphorylation. PI3-kinase activity was meas- 15870 Synergism between Midkine and PDGF in Cell Migration FIG.3. Effect of glycosaminogly- cans on MK-induced cell migration. A, UMR106 cells were preincubated at 37 °C for 30 min with glycosaminoglycans at the concentration of 20 mg/ml or that indicated. The migration assay was car- ried out with the same glycosaminoglycan concentration in the lower chamber as in the upper chamber. CS means chon- droitin sulfate. B and C, UMR106 cells were preincubated at 37 °C with hepariti- nase for 90 min (B), or chondroitinase ABC for 30 min (C). D, at 20 milliunits/ml, chondroitinase ABC and B, but not AC II, inhibited the migration. Values are the means 6 S.E.; n 5 3. FIG.4. Possible role of PTP-z in MK- induced cell migration. A, 5 mg of total RNA were used for RT-PCR. L cell, fibro- blast cell line; G401 cell, Wilms’ tumor cell line. Upper panel, PTPz; lower panel, GAPDH. B, Western blot analysis with anti-RPTPb (PTPz) antibody before and after chondrotinase digestion. Arrow- heads and an arrow indicate the positive bands after chondroitinase ABC diges- tion. The bracket indicates the smear de- tected for UMR106 cells. Note the nonspe- cific bands for all lysates of UMR106 and L cells. C, sodium vanadate (NaVa) inhib- ited MK-induced migration of UMR106 cells. D, preincubation with anti-PTPz (anti-6B4) antibodies, but not control rab- bit IgG, enhanced MK-induced migration. Values are the means 6 S.E.; n 5 3. ured by in vitro phosphorylation of phosphatidylinositol, using essen- trix proteins, such as fibronectin, vitronectin, laminin-1, and tially the same method as described previously (24). collagen IV (25). As chemically synthesized human MK and Immunofluorescence Microscopy—UMR106 cells, which had been ex- baculovirus-produced mouse MK showed the same activity as posed to MK- or PLL-beads for 15 min, were fixed with 4% paraform- that of yeast-produced human MK (Fig. 1B), the migratory aldehyde and then stained with anti-PI3-kinase (p85a) antibody/fluo- rescein isothiocyanate-conjugated anti-rabbit IgG and/or anti-RPTPb effect was not caused by impurities in the MK used. MK con- (PTPz) monoclonal antibody/horseradish peroxidase-conjugated anti- tains 30% basic amino acids. However, PLL showed very weak mouse IgG antibody/cyanine 5-labeled tyramide. For PTPz staining, the migratory activity (Fig. 1B), suggesting that the effect of MK TSA system (PerkinElmer Life Sciences) was employed to enhance the was not because of its high basicity. signal. Soluble MK did not induce UMR106 cell migration (Fig. 1B), RESULTS even if the filter was precoated with collagen I (data not shown). On the contrary, when added to the lower chamber, MK Induces Haptotactic Migration of Osteoblast-type Cells— soluble MK inhibited coated MK-induced migration (Fig. 1C). PTN/HB-GAM, another member of the MK family, induces At the concentration of 10 mg/ml, soluble MK completely abol- haptotactic migration of several osteoblast cell lines (18). We ished coated MK-induced migration. The same concentration of first investigated whether or not MK had the same activity. soluble PLL in the lower chamber did not inhibit MK-induced When coated on the lower surface of a filter, MK induced the haptotactic migration of UMR106 cells, with the maximum migration (data not shown). These findings indicate that the substratum-bound form of MK is active in cell migration. Thus, level at the concentration of 20 mg/ml (Fig. 1A). The profile of MK-induced cell migration was similar to that of the colon MK could induce haptotactic, but not chemotactic, migration of carcinoma cell migration induced by several extracellular ma- UMR106 cells. Synergism between Midkine and PDGF in Cell Migration 15871 FIG.5. Effect of different inhibitors on MK-induced cell migra- tion. UMR106 cells were incubated with the indicated inhibitors for 30 min. The migration assay was then performed with both the upper and lower chambers containing the same concentration of the same inhibi- FIG.6. Erk activation after MK stimulation. UMR106 cells stim- tor. A, concentrations of inhibitors: tyrosine kinase-herbimycin A ulated with polystyrene beads coated with PLL (5 mg/ml) or MK (20 (herb), 1 mg/ml; tyrosine kinase-genistein (geni), 50 mg/ml; Src-PP1, 10 mg/ml) for the indicated times were analyzed by Western blotting with nM; protein kinase C-Ro318220 (Ro), 2 mM; PI 3 kinase-wortmannin anti-phosphorylated Erk (P-Erk1/2) and anti-Erk2 (Erk2) antibodies. (wort), 100 nM; MEK-PD98059 (PD), 20 mM; phospholipase C-U73122 The densitometric data obtained on Western blotting are shown in the (U-7), 10 mM. B and C, dose dependence of protein kinase C inhibitors graph.In B, inhibitors were used as described in the legend to Fig. 5. (B) and MEK, PI3 kinase, and phospholipase C inhibitors (C). The units are: PD98059, mM; wortmannin, nM; U73122, mM. Values are the means 6 S.E.; n 5 3. corresponding to PTPz was detected for UMR106 cells and rat brain (Fig. 4A). The PCR product was confirmed by DNA se- Soluble MK and PTN Abrogate Haptotaxis Mediated by MK quencing (data not shown). No positive band was detected for L and PTN/HB-GAM—We next addressed the question of cells or Wilms’ tumor cells (Fig. 4A). This is consistent with the whether or not MK and PTN/HB-GAM share the same cell observation that coated MK did not induce the migration of surface binding site(s). Soluble MK abrogated PTN/HB-GAM- these cells with a 4-h incubation (data not shown). On Western induced haptotaxis and vice versa (Fig. 2A). Coating with a blotting with monoclonal anti-RPTPb (PTPz) antibody, which combination of MK and PTN/HB-GAM did not enhance the recognizes the intracellular domain of PTPz, a smear was de- migration as compared with MK or PTN/HB-GAM alone (Fig. tected for the lysate of UMR106 cells (Fig. 4B, bracket, lane 3). 2B). These data suggest that if the total molar concentration of This changed to one band corresponding to about 240 kDa after MK and PTN/HB-GAM reaches a critical level, the migration chondroitinase ABC digestion (Fig. 4B, lane 4, arrow). For rat activity will be saturated, and MK and PTN/HB-GAM may brain, smears were also detected (Fig. 4B, lane 1), which function through common molecule(s) on the cell surface. shifted to 380 and 220 kDa upon digestion with chondroitinase Proteoglycan Is Involved in MK-induced Migration—Hepa- ABC (Fig. 4B, lane 2, arrowheads), which represent long- and ran sulfate proteoglycans, such as syndecan-1, -3, and -4, and a short-type receptors, respectively (23). The size difference chondroitin sulfate proteoglycan, PTPz, are supposed to act as around 220/240 kDa between the brain and UMR106 cells is a receptor or co-receptor for MK (20, 26, 27, 28). We investi- probably caused by differential glycosylation, which is depend- gated the effects of different glycosaminoglycans on MK-medi- ent on the cell type (30). Chondroitinase AC II and B appeared ated migration. In addition to heparin, dermatan sulfate (chon- to digest the glycosaminoglycan chains of PTPz only partially, droitin sulfate B) and chondroitin sulfate E showed comparable because the band around 240 kDa was broader than the band effects. They abolished the migration at the concentration of 20 digested with chondroitinase ABC (Fig. 4B, lanes 8, 9, 10). This mg/ml (Fig. 3A). At 20 mg/ml, chondroitin sulfate A, C, and D is reasonable because chondroitinase AC II and B recognize showed only minor effects (Fig. 3A). Treatment with hepariti- different structures (31, 32). Nevertheless, only chondroitinase nase to remove the heparan sulfate on the cell surface did not ABC and B, but not AC II, suppressed MK-induced migration affect MK-mediated migration (Fig. 3B). On the contrary, chon- (Fig. 3D), suggesting that structures susceptible to chondroiti- droitinase ABC digestion and chondroitinase B digestion down- nase ABC and B are important. As chondroitinase B can digest regulated MK-mediated migration (Fig. 3, C and D), suggesting both dermatan sulfate and E-type dermatan sulfate (32), and the involvement of cell surface chondroitin sulfate. chondroitinase ABC can digest all chondroitin and dermatan Involvement of PTPz in MK-induced Migration—As PTPz sulfates (33), this is consistent with the finding that MK-in- exhibits high affinity to both MK and PTN/HB-GAM (20, 29), duced cell migration was inhibited by dermatan sulfate (chon- the results shown in Figs. 1, 2, and 3 suggest that PTPz is droitin sulfate B) and chondroitin sulfate E (Fig. 3A). involved in MK-induced haptotaxis. Using RT-PCR, a band PTPz is also known to be a receptor-type protein-tyrosine 15872 Synergism between Midkine and PDGF in Cell Migration FIG.7. PI3-kinase activity after MK and PDGF-BB stimulation. UMR106 cells were stimulated with PDGF-BB, or polystyrene beads coated with PLL or MK. PI 3-kinase activity was measured as described under “Experimental Proce- dures.” A, representative autoradiogra- phy. Lower panel, quantitative presenta- tion (mean 6 S.E.; n 5 3). B, effects of inhibitors. phosphatase. The effect of orthovanadate, an inhibitor of pro- the case of PDGF-BB (Fig. 7A). Neither Ro318220 nor MEK tein-tyrosine phosphatase, was examined. Sodium vanadate inhibitor PD98059 affected MK-induced PI3-kinase activation decreased the migration in a dose-dependent manner (Fig. 4C). (Fig. 7B). Sodium vanadate had no effect on PLL or PDGF-BB-induced MK Recruits PTPz and PI3-kinase—To further confirm MK- migration of UMR106 cells (data not shown). If the cells were induced PI3-kinase activation, phosphorylation of AKT was preincubated with anti-PTPz (anti-6B4) antibodies, which rec- examined. AKT (protein kinase B) plays important roles in ognize the ectodomain of the full-length PTPz (23), MK-medi- many biological phenomena, such as cell survival (34, 35). AKT ated cell migration was enhanced (Fig. 4D). This suggests that contains a pleckstrin homology (PH) domain, and is phospho- ligation of cell surface PTPz with its ligands or antibodies may rylated (activated) at Thr-308 and Ser-473 residues by PDK1, transduce signals essential for the migration of UMR106 cells. which also carries a PH domain. The PH domain recognizes a MK Induces MAP Kinase and PI3-kinase Activity—To iden- phosphoinositide headgroup, and phosphorylation at the 3 po- tify the intracellular molecules that participate in MK-medi- sition of inositol ring of phosphatidylinositol in the cell mem- ated cell migration, various specific inhibitors were screened. brane is critical for AKT- and PDK1-binding and recruitment Tyrosine kinase inhibitors (genistein and herbimycin A), a Src to the cell membrane (35). As this phosphorylation is mediated inhibitor (PP1), protein kinase C inhibitors (H7, Ro318220, and by PI3-kinase, AKT is an important PI3-kinase effector, and its calphostin C), a PI3-kinase inhibitor (wortmannin), a MAP activation is often used as a marker of PI3-kinase activation kinase kinase (MEK) inhibitor (PD98059), and a phospholipase (34, 35). C inhibitor (U-73122) effectively inhibited MK-mediated cell MK enhanced AKT phosphorylation (Fig. 8A, left), which is migration (Fig. 5). This probably indicates that a dynamically consistent with the data for MK-induced PI3-kinase activation concerted signaling interaction is essential for the cell migra- shown in Fig. 7A. When the cells were preincubated with tion induced by MK. anti-PTPz (anti-6B4) antibodies, MK-induced AKT activation When UMR106 cells were stimulated with MK-coated polys- was stronger than that of the cells treated with control IgG trene beads, a transient increase in phosphorylation of MAP (Fig. 8A, right). This was consistent with that anti-PTPz (anti- kinases (Erk1 and Erk2) was detected, the maximum level 6B4) antibodies enhanced MK-induced migration of UMR106 being observed ;20 min after stimulation (Fig. 6A). At 60 min, cells (Fig. 4D). It is of interest that, in addition to the PI3- it was still higher than the basal level (Fig. 6A). PLL also kinase inhibitor wortmannin, the protein-tyrosine phosphatase enhanced Erk phosphorylation, but the increase was less and inhibitor orthovanadate and Src inhibitor PP1 blocked MK- the duration was shorter than in the case of MK (Fig. 6A). induced AKT activation (Fig. 8A, right), but neither protein Interestingly, the PI3-kinase inhibitor wortmannin partly kinase C inhibitor Ro 318220 nor MEK inhibitor PD98059 blocked MK-induced Erk phosphorylation, whereas the protein affected it (data not shown). As the effect of orthovanadate on kinase C inhibitor Ro318220 did not (Fig. 6B). MK also in- MK-induced AKT phosphorylation suggested the possibility of creased PI3-kinase activity, the profile being similar to that in a close connection between PTPz and PI3-kinase, we localized Synergism between Midkine and PDGF in Cell Migration 15873 FIG.8. Involvement of PI3-kinase and PTPz in MK signaling. A, left; UMR106 cells were exposed to MK- or PLL-beads at 37 °C for 15 min. Cell extracts were then analyzed for phosphorylated AKT (Ser- 473) (p-Akt) or AKT expression by Western blotting. The densitometric ratio (p-Akt versus Akt) was calculated for each condition, and the relative density of p-Akt is shown at the bottom. A, right; UMR106 cells were incubated with either anti-PTPz (anti-6B4) antibodies or control rabbit IgG together with the indicated inhibitor at 4 °C for 2 h. The cells were then exposed to MK-beads in the presence of indicated inhibitor at 37 °C for 15 min. B, UMR106 cells were exposed to MK- or PLL-beads at 37 °C for 15 min. The localization of PI3-kinase and PTPz was examined by immunofluorescence microscopy (PI3-kinase, FITC; PTPz, cyanine 5) using a confocal microscope (MRC-1024, Bio-Rad). The lower two panels show the same cell that was double-stained with anti-PI3- kinase and anti-RPTPb (PTPz) antibodies. Arrows and asterisks indi- cate cells examined and beads, respectively. PTPz and PI 3-kinase after MK beads stimulation on UMR106 cells. MK beads induced PI3-kinase recruitment to the sites of the beads (Fig. 8B, left two panels). Furthermore, MK-beads induced colocalization of PI3-kinase and PTPz (Fig. 8B, lower two panels). MK Has a Synergistic Effect with PDGF-BB on Cell Migra- tion—The hypothesis that MK and PDGF can cooperate in cell migration in some in vivo situations was examined next. On filters coated on both their upper and lower surfaces with collagen I, PDGF-BB induced chemotactic migration of UMR106 cells (Fig. 9A). PDGF-induced migration was sup- FIG.9. Synergistic effect of MK and PDGF-BB on cell migra- tion. A, PDGF-BB-induced migration was inhibited by protein kinase C pressed by Ro318220 and wortmannin (Fig. 9A). But PD98059, and PI3-kinase inhibitors. PDGF-BB (20 ng/ml) was added to the lower which inhibited MK-mediated cell migration (Fig. 5), did not chamber. Concentrations of reagents: PD98059, 20 mM; Ro318220, 2 mM; affect PDGF-mediated migration (Fig. 9A). Whereas heparin wortmannin, 100 nM; heparin, 20 mg/ml. B, MK and PDGF-BB showed abolished MK-mediated cell migration (Fig. 3A), it rather en- synergistic effect to induce UMR106 cell migration. The filter of Che- motaxicell was coated with collagen I on both its upper and lower hanced PDGF-mediated cell migration (Fig. 9A). If the lower surfaces or with MK on only its lower surface. Migration was then surface of the filters was coated with MK instead of collagen I, monitored in the presence or absence of PDGF-BB in the lower cham- the chemotactic activity of PDGF-BB was dramatically en- ber. C, MK/PDGF-BB-mediated migration was not affected by MEK hanced (Fig. 9B). The effects of MK and PDGF-BB were syn- inhibitor. PD98059, Ro318220, and wortmannin were used as described in A. ergistic, because the migrated cell number was much higher than the total when MK and PDGF-BB were used separately. 13, 14, 36, 37). In the case of arterial endothelial injury, we The migration induced by the combination of MK and found not only the induction of MK expression in wild-type PDGF-BB was susceptible to inhibitors of PI 3-kinase and mice, but also dramatically suppressed neointima formation in protein kinase C, but not to the MEK inhibitor (Fig. 9C). This MK-deficient mice (14). The administration of the MK protein profile resembles that of the migration induced by PDGF-BB to MK-deficient mice caused resumption of neointima forma- alone, but not that in the case of MK alone. tion (14). Thus, MK seems to be vital for tissue remodeling. The DISCUSSION present study revealed another important aspect of the mode of MK has been reported to be induced in areas of a variety of MK action, namely, that it acts synergistically with PDGF in types of tissue injury, such as cerebral and heart infarction, cell migration. PDGF was first purified from platelet a gran- bone fractures, skin burns, and arterial endothelial injury (12, ules and has several important activities, such as mitogenesis 15874 Synergism between Midkine and PDGF in Cell Migration and chemotaxis. Because tissue injury is usually accompanied that the chondroitin sulfate chain in PTPz is also important in by bleeding and/or cessation of blood flow, an abundance of migration of osteoblast-like cells. Differential susceptibility of PDGF can be found in these areas. PDGF expression is also the migratory activity to chondroitinases with different speci- induced during the process of wound healing (38). Further- ficities gave further insights into the nature of chondroitin sulfate chain in PTPz. Digestion with chondroitinase B abol- more, PDGF induces the migration of smooth muscle cells (16, 39) and osteoblasts (40), and the migration of both is also ished MK-dependent migratory activity, whereas chondroiti- nase AC II did not. The former enzyme acts on chondroitin induced by MK (Ref. 14 and the present study). Taken together, sulfate chain with the L-iduronic acid residue, namely derma- the synergism between MK and PDGF appears to be pivotal in tan sulfate, whereas the latter does not. Thus, it is concluded many in vivo situations. that chondroitin sulfate, which is important in MK-signaling in Several possibilities should be considered for the mechanism PTPz, has a dermatan sulfate domain. The finding that the underlying the synergism between MK and PDGF. The present MK-induced migration is inhibited by dermatan sulfate is con- study revealed a difference in the signaling mechanism be- sistent with the view. The MK activity was also inhibited by tween MK and PDGF. Heparin inhibited MK-induced cell mi- chondroitin sulfate E, which is an oversulfated chondroitin gration but rather enhanced PDGF-induced cell migration sulfate with 4,6-disulfo-N-acetylgalactosamine residue. Taken (Figs. 3A and 9A). MAP kinases were essential for MK-medi- together, most probably the chondroitin sulfate chain in PTPz, ated cell migration but not for PDGF-induced cell migration to which MK binds, has an oversulfated structure in a derma- (Figs. 5 and 9). Orthovanadate inhibited MK-induced cell mi- tan sulfate domain. Indeed, E-type structure with a dermatan gration but not PDGF-induced cell migration (Fig. 4C and data sulfate domain was found in PG-M/versican, which was iso- not shown). Because distinct signaling molecules are used for lated from mouse embryos and has MK binding activity (54). cell migration, depending on the ligand (41, 42), the synergism The characteristics of MK-induced migration of UMR106 between MK and PDGF might be attributed to the integration cells are very similar to those of MK- and PTN/HB-GAM- of distinct signaling pathways. Alternatively, induction of MK induced neuronal migration in that PTPz is involved in hapto- receptor(s) by PDGF or vice versa should also be considered, as tactic migration (19, 20). In this context, the effect of anti-PTPz in the case of the induction of the Interleukin-1 receptor or antibodies on MK-mediated cell migration was unexpected. PDGF receptor by the neuropeptide substance P in their syn- These antibodies effectively inhibited PTN/HB-GAM-mediated ergism in bone marrow fibroblast proliferation (43). In addi- neuronal migration in the previous study, probably because of tion, a third cell surface molecule, such as integrin b , might be competitive inhibition for the PTN/HB-GAM-binding sites of involved in the synergism, like in the case of the synergism cell surface PTPz by the antibodies (20). But in the present between lysophosphatidic acid and epidermal growth factor or study, the antibodies rather enhanced MK-mediated osteo- PDGF in cell migration (44). blast-like cell migration. One possible interpretation of this is MAP kinases (Erk1 and Erk2) can activate myosin light that PTPz on UMR106 cells may physically associate with chain kinase and induce changes in the cytoskeletal structure, another unidentified component necessary for signal transduc- leading to cell migration (45). In this context, it is noteworthy tion and thus can be readily activated by the oligomerization or that MK enhances collagen gel contraction by dermal fibro- conformational change induced by a specific antibody. On nerve blasts (46). MK-induced MAP kinase activation appeared to be cells, PTPz might need MK to associate with such a component. at least partly regulated by PI3-kinase (Fig. 6B). PI3-kinase Supporting our data, Revest et al. (56) recently reported that functions as an early intermediate in Gbg-mediated MAP ki- cross-linking of PTPz with antibodies enhances the protein- nase activation (47). Several papers have reported that PI3- tyrosine phosphatase activity of C6 astrocytoma cells. kinase acts upstream of MAP kinase, e.g. in insulin signaling in In this study, we confirmed the involvement of Erk1 and 2 3T3-L1 adipocytes and PDGF signaling in Swiss 3T3 cells (48, and PI3-kinase in MK-induced cell migration by detecting their 49). active forms or activity induced by MK, in addition to by dem- The present study demonstrated that MK recruited PTPz onstrating the effects of inhibitors of them. Consistent with our and PI3-kinase (Fig. 8B). Furthermore, MK-induced PI3-ki- data, Souttou et al. reported that Erk1 and 2 and PI3-kinase nase activation was inhibited by the Src inhibitor PP1 and are involved in PTN/HB-GAM-mediated cell proliferation (57). protein-tyrosine phosphatase inhibitor orthovanadate (Fig. In addition, Src, JAK1, and 2 and b-catenin have been reported 8A). 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Published: May 1, 2001

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