Haptotactic migration induced by midkine. Involvement of protein-tyrosine phosphatase zeta. Mitogen-activated protein kinase, and phosphatidylinositol 3-kinase.

Midkine, a heparin-binding growth factor, plays a critical role in cell migration causing suppression of neointima formation in midkine-deficient mice. Here we have determined the molecules essential for midkine-induced migration. Midkine induced haptotaxis of osteoblast-like cells, which was abrogated by the soluble form of midkine or pleiotrophin, a midkine-homologous protein. Chondroitin sulfate B, E, chondroitinase ABC, B, and orthovanadate, an inhibitor of protein-tyrosine phosphatase, suppressed the migration. Supporting these data, the cells examined expressed PTPzeta, a receptor-type protein-tyrosine phosphatase that exhibits high affinity to both midkine and pleiotrophin and harbors chondroitin sulfate chains. Furthermore, strong synergism between midkine and platelet-derived growth factor in migration was detected. The use of specific inhibitors demonstrated that mitogen-activated protein (MAP) kinase and protein-tyrosine phosphatase were involved in midkine-induced haptotaxis but not PDGF-induced chemotaxis, whereas phosphatidylinositol 3 (PI3)-kinase and protein kinase C were involved in both functions. Midkine activated both PI3-kinase and MAP kinases, the latter activation was blocked by a PI3-kinase inhibitor. Midkine further recruited PTPzeta and PI3-kinase. These results indicate that PTPzeta and concerted signaling involving PI3-kinase and MAP kinase are required for midkine-induced migration and demonstrate for the first time the synergism between midkine and platelet-derived growth factor in cell migration.

Effects of midkine during in vitro maturation of bovine oocytes on subsequent developmental competence.

Midkine (MK) is known to be a member of a new family of heparin-binding growth/differentiation factors, together with pleiotrophin, and to be quite rich in bovine follicular fluid. To examine whether treatment with MK during in vitro maturation (IVM) of bovine granulosa-enclosed oocytes affects their nuclear maturation and postfertilization development to the blastocyst stage, bovine granulosa-enclosed oocytes obtained from slaughterhouse-derived ovaries were cultured for 24 h in IVM medium without (control) or with various concentrations (1-500 ng/ml) of MK, followed by in vitro fertilization (IVF) and culturing. Although the MK treatment during IVM did not affect the rate of nuclear maturation or the postfertilization cleavage of oocytes, MK at > or = 10 ng/ml significantly (P: < 0.05) increased the blastocyst yields per tested and per cleaved oocyte compared with the case of the control. Next, the effects of various glycosaminoglycans (heparin, heparan sulfate, chondroitin sulfate A and C, and hyaluronic acid) preincubated with MK at 50 ng/ml were examined. The enhancing activity of MK was completely suppressed by heparin at 600 ng/ml but not by the other compounds. The effects of MK during IVM were also tested on oocytes freed from granulosa cells (GCs). When the denuded oocytes were cultured in IVM medium, no blastocyst formation after IVF was observed, regardless of MK supplementation. However, coculture of the denuded oocytes with isolated GC pellets enhanced the cleavage rates and the blastocyst yield, and these effects were more pronounced with MK supplementation. These results indicate that the presence of MK during IVM of bovine granulosa-enclosed oocytes can enhance their developmental competence to the blastocyst stage after IVF and suggest that the enhancing effects might be mainly mediated by GCs.

cDNA cloning of bovine midkine and production of the recombinant protein, which affects in vitro maturation of bovine oocytes.

In the present study, we cloned bovine midkine (bMK) cDNA by RT- and RACE-PCR, and determined its nucleotide sequence. The nucleotide and deduced amino acid sequences of bMK showed a high degree of homology to those of mouse and human MK. Moreover, a large amount of recombinant bMK (rbMK) was produced using a baculovirus expression system and the protein was purified to homogeneity by heparin affinity chromatography. To examine whether treatment with rbMK during in vitro maturation (IVM) of bovine cumulus-enclosed oocytes affects their nuclear maturation and postfertilization development to the blastocyst stage, bovine cumulus-enclosed oocytes obtained from slaughterhouse-derived ovaries were cultured for 24 hr in IVM medium without (control) or with various concentrations (50-400 ng/ml) of rbMK, followed by in vitro fertilization (IVF) and culture. Although rbMK treatment during IVM did not affect the rates of nuclear maturation and postfertilization cleavage of oocytes, rbMK at all experimental concentrations significantly (P < 0.05) increased the blastocyst yields per tested and per cleaved oocyte compared to the control. Next, it was examined whether heparitinase (HTN) treatment of cumulus-enclosed oocytes would affect the enhancing activity of rbMK during IVM on the developmental competence of oocyte after IVF. The enhancing activity of rbMK was completely suppressed by HTN (1.0 mU/ml) treatment. These results indicate that the presence of rbMK during IVM of bovine cumulus-enclosed oocytes can enhance their developmental competence to the blastocyst stage after IVF and suggest that heparan sulfate chains on the cell surface of cumulus cells and/or oocytes are required for bMK to exert its effect.

Midkine rescues Wilms' tumor cells from cisplatin-induced apoptosis: regulation of Bcl-2 expression by Midkine.

Midkine (MK) is a heparin-binding growth factor involved in diverse biological phenomena, e.g. neuronal survival, carcinogenesis, and tissue repair. MK expression is detected mainly in the kidney in adult mice. In this study, we show that, at a dose that can induce recoverable renal damage and induce apoptosis, cisplatin (CDDP) transiently suppressed MK expression in mouse kidney. In vitro, CDDP suppressed MK expression and induced apoptosis in cultured G401 cells, a Wilms' tumor cell line. Exogenous MK protein partially rescued G401 cells from CDDP-induced apoptosis. MK enhanced the expression of Bcl-2, but not that of Bcl-x(L), in G401 cells in a dose-dependent manner, and it prevented the Bcl-2 reduction due to CDDP. Moreover, Bcl-2 expression in mouse kidney was also transiently suppressed by CDDP treatment, the expression profile being similar to that of MK. These results imply that MK exerts cytoprotective activity toward a damaging insult, presumably at least in part through enhancement of the expression of Bcl-2.

A receptor-like protein-tyrosine phosphatase PTPzeta/RPTPbeta binds a heparin-binding growth factor midkine. Involvement of arginine 78 of midkine in the high affinity binding to PTPzeta.

Midkine is a 13-kDa heparin-binding growth factor with 45% sequence identity to pleiotrophin. Pleiotrophin has been demonstrated to bind to protein-tyrosine phosphatase zeta (PTPzeta) with high affinity. In this study, we examined the binding of midkine to PTPzeta by solid-phase binding assay. Midkine and pleiotrophin binding to PTPzeta were equally inhibited by soluble pleiotrophin and also by some specific glycosaminoglycans. For both bindings, Scatchard analysis revealed low (3.0 nM) and high (0.58 nM) affinity binding sites. These results suggested that PTPzeta is a common receptor for midkine and pleiotrophin. Midkine is structurally divided into the N- and C-terminal halves, and the latter exhibited full activity for PTPzeta binding and neuronal migration induction. The C-terminal half contains two heparin-binding sites consisting of clusters of basic amino acids, Clusters I and II. A mutation at Arg78 in Cluster I resulted in loss of the high affinity binding and reduced neuronal migration-inducing activity, while mutations at Lys83 and Lys84 in Cluster II showed almost no effect on either activity. Chondroitinase ABC-treated PTPzeta exhibited similar low affinity binding both to the native midkine and midkine mutants at Arg78. These results suggested that Arg78 in midkine plays an essential role in high affinity binding to PTPzeta by interacting with the chondroitin sulfate portion of this receptor.

Clusters of basic amino acids in midkine: roles in neurite-promoting activity and plasminogen activator-enhancing activity.

The removal of N-terminally located clusters of basic amino acids (N-tail) or C-terminally located clusters of basic amino acids (C-tail) from the midkine (MK) molecule severely reduced its neurite-promoting activity. However, experiments involving chemically synthesized MK derivatives revealed that the roles of the N-tail and C-tail were mostly indirect ones, i.e. they probably maintain the steric arrangements of the N-terminal and C-terminal halves. In particular, the C-domain, which is the C-terminal half devoid of the C-tail, retained considerable neurite-promoting activity when it was uniformly coated on a dish. The removal of the N-tail or C-tail also reduced the enhancing activity of plasminogen activator (PA) in aortic endothelial cells, although the effect was lower. There are two heparin-binding sites in the C-domain, Clusters I and II. A mutation in Cluster I [R78-->Q] affected the PA-enhancing activity only slightly, and a mutation in Cluster II [K83K84-->QQ] abolished the activity, while both mutations are known to reduce the neurite-promoting activity moderately. Therefore, the two heparin-binding sites in the C-domain play different roles in these two activities. Indeed, heparin exhibited different effects on these two activities. We also observed that intact MK was required for ordered neurite-promotion along the path of MK; one possible interpretation of this is that the N-terminal half is necessary for the stability of the molecule. Furthermore, K76 and K99 were found to be required for the secretion of MK; i.e. mutants in which one of these K residues was changed to Q were produced in the host cells, but not found in the medium.

Identification of heparin-binding sites in midkine and their role in neurite-promotion.

Midkine (MK) is a heparin-binding growth factor, which promotes neurite outgrowth in embryonic neurons and enhances their survival. The three dimensional structure of MK clarified by NMR spectroscopy indicates that several basic amino acids are exposed on the surface of the C-terminal half domain, which retains heparin-binding and neurite-promoting activity. We performed site-directed mutagenesis of these amino acids, and found that mutation of arginine78 reduced the heparin-binding activity. Mutation of either lysine83 or lysine84 scarcely affected heparin-binding activity, while the double mutant involving both lysine residues showed reduction in the activity. Neurite-promoting activity of mutant MKs always correlated with their heparin-binding activity, illustrating the close relationship of the two activities. Thus, the present result verifies the occurrence of two distinct heparin-binding sites involved in neurite-promoting activity of MK molecule.

Expression of syndecan-1 and -3 during embryogenesis of the central nervous system in relation to binding with midkine.

Midkine (MK) is a 13-kDa heparin-binding growth/differentiation factor, and its interaction with heparan sulfate proteoglycan is important in promotion of neurite outgrowth. This study was performed to reveal the species of syndecans that interact with MK during embryogenesis of the central nervous system in the rat. Northern blot analysis and immunohistochemical staining using antibodies to syndecans showed that syndecan-1 was strongly expressed in the brain and spinal cord of the 10-day rat fetus, and the expression became weak as embryogenesis proceeded. On the other hand, syndecan-3 expression was stronger both in the brain and spinal cord in the later developmental period (day 14-16 of gestation). No significant expression of ryudocan (syndecan-4) was detected in fetal brains by Northern blot analysis. Syndecan-3 isolated from 16-day rat fetus bound to MK strongly, as did syndecan-1 isolated from 10-day rat fetus. Thus, both syndecan-1 and syndecan-3 were considered to be involved in interaction with MK during construction of the central nervous system; syndecan-1 is expected to play important roles in the earlier periods, and syndecan-3 in the later periods.

Suppression of transformed phenotypes of human fibrosarcoma cells by overexpression of recombinant fibronectin.

Loss of fibronectin (FN) from the cell surface has been shown to be closely associated with malignant transformation of cells. To elucidate the role of the FN matrix in the modulation of malignant phenotypes, we overexpressed a full-length cDNA encoding plasma-type FN in HT1080 human fibrosarcoma cells. The cells overexpressing FN adopted a more flattened morphology and deposited a moderately developed FN matrix both in vitro and in vivo, although the level of expression of integrin alpha5beta1 remained unchanged. FN-overexpressing cells exhibited a reduced cell motility on the substratum and grew poorly when injected s.c. into nude mice. Overexpression of FN also suppressed the ability of the tumor cells to proliferate in soft agar, whereas the suppression was reversed by inclusion in soft agar of the Arg-Gly-Asp (RGD)-containing peptide and adhesion-blocking antibodies against the central cell-binding domain of FN. Neither cell motility nor growth potential was altered by overexpression of a truncated form of FN lacking the central cell-binding domain. These results, taken together, indicate that increased deposition of FN in the pericellular matrix per se can suppress the motility and growth potential of tumor cells through interaction with RGD-recognizing integrins, most likely alpha5beta1.

Role of the carboxyl-terminal Fib2 domain in fibronectin matrix assembly.

A truncated form of fibronectin consisting of the N-terminal 70 kDa and C-terminal 37 kDa regions, designated r70F2, retained the ability to assemble into the extracellular matrix when expressed in cultured fibroblasts (Ichihara-Tanaka et al. (1992) FEBS Lett. 299, 155-158). To elucidate the role of the C-terminal 37 kDa region in fibronectin matrix assembly, we expressed a panel of mutant forms of r70F2 with various deletions and amino acid substitutions in mouse L cells. Although substitution of Ser for two Cys residues in the C-terminal dimerforming segment led to a marked reduction in the matrix assembly activity of r70F2, the resulting monomeric r70F2 still retained a low, but significant activity to assemble into the matrix. Neither the N-terminal 70 kDa nor the C-terminal 37 kDa regions, when expressed as monomeric forms, exhibited any residual activity, suggesting that the core domain of the 37 kDa region consisting of III15 and I10 through I12 modules, termed Fib2 domain, is actively involved in the matrix assembly of r70F2. In support of the role of Fib2 domain, the proteolytic fragment derived from the 37 kDa region inhibited the assembly of r70F2. Furthermore, en bloc deletion of the Fib2 domain or deletion of the I10 through I12 modules from r70F2 resulted in a marked decrease of the matrix assembly activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Targeting of the immunoglobulin-binding domain of protein A to the extracellular matrix using a minifibronectin expression vector.

A truncated form of fibronectin consisting of the N-terminal 70-kDa and C-terminal 37-kDa regions, referred to as "minifibronectin," retains the ability to assemble into the extracellular matrix, even though it lacks the central approximately 120-kDa region containing most of the type III modules (Ichihara-Tanaka, K., Titani, K., and Sekiguchi, K., FEBS Lett. 299, 155-158, 1992). Taking advantage of the matrix assembly activity of minifibronectin, we developed a novel method to target non-matrix proteins to the extracellular matrix by inserting them between the N-terminal 70-kDa and the C-terminal 37-kDa regions of minifibronectin. Using the immunoglobulin-binding domain of Staphylococcal protein A as a model, we demonstrated that the bacterial protein expressed in mouse L cells as a chimeric protein with minifibronectin is secreted as disulfide-bonded dimers and successfully deposited onto the extracellular matrix of transfected cells. The chimeric protein retained the immunoglobulin-binding activity not only in solution but also after deposition at the matrix. This targeting strategy we developed will provide a means to manipulate the biological functions of the extracellular matrix through targeting of a wide variety of non-matrix proteins.

Matrix assembly of recombinant fibronectin polypeptide consisting of amino-terminal 70 kDa and carboxyl-terminal 37 kDa regions.

Three different forms of recombinant human fibronectin polypeptides consisting of the amino-terminal 70 kDa region, the carboxyl-terminal 37 kDa region, or both, were expressed in mouse L cells. Although either the amino-terminal or the carboxyl-terminal region alone was only poorly incorporated into the extracellular matrix, the fused form of the polypeptide was highly capable of assembling into the matrix. These results indicate that matrix assembly of fibronectin requires both regions and can proceed in the absence of the type III repeats including the one containing the cell adhesive Arg-Gly-Asp sequence.

Rapid purification and characterization of human platelet glycoprotein V: the amino acid sequence contains leucine-rich repetitive modules as in glycoprotein Ib.

Glycoprotein V (GPV) is a membrane-associated, 82 Kd platelet glycoprotein that is hydrolyzed during thrombin activation to yield 69 Kd fragment. We have developed a rapid and simple method for isolation of the protein from platelet extracts using a combination of gel permeation, anion-exchange, and lectin affinity chromatography. The partial amino acid sequence was determined by analysis of peptides generated by digestion of the S-carboxyamido-methylated protein with Achromobacter protease I or cyanogen bromide. The sequence shows a remarkable periodicity of leucine residues, which is homologous to the consensus sequence of a highly diversified protein super-family with a common repetitive module. Thrombin cleavage site was determined to be located at the C-terminal region of GPV by analysis of the products separated by sizing and reversed-phase high performance liquid chromatography. By lectin blot analysis, the existence of mucin-type carbohydrate chains was indicated, as well as the existence of asparagine-linked carbohydrate chains shown by the amino acid sequence analysis. From these data, we report a structural model of GPV that is analogous to glycoprotein Ib.

Recombinant carboxyl-terminal fibrin-binding domain of human fibronectin expressed in mouse L cells.

The carboxyl-terminal fibrin-binding domain, Fib2, of human fibronectin was expressed in mouse L cells as a fusion protein with the signal sequence of human protein C inhibitor. The recombinant Fib2 (rFib2) protein synthesized by transfected cells retained the ability to form dimers with each other or with mouse fibronectin subunits and was secreted to the medium after extensive glycosylation. Only a small fraction of the secreted protein was incorporated into the pericellular matrix. Interestingly, the secreted rFib2 protein displayed a remarkable heterogeneity upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, giving rise to a broad band corresponding to Mr of 60,000-90,000. The heterogeneity was eliminated mostly by treatment with neuraminidase and further by treatment with endo-alpha-N-acetylgalactosaminidase. Treatment with peptide:N-glycosidase F did not alter the heterogeneity of the protein, indicating that differential sialylation of O-linked, but not N-linked, glycans is largely responsible for the apparent heterogeneity. The presence of O-linked but absence of N-linked glycans was further supported by the observations that peanut agglutinin specifically bound to the desialylated rFib2 protein, whereas neither concanavalin A nor lentil lectin bound to the protein irrespective of prior neuraminidase treatment. Since the apparent heterogeneity of the rFib2 protein was only observable with the secreted, but not the cytoplasmic form, sialylation of O-linked glycans may be essential for, or regulate as a rate-limiting step, the transit of the recombinant protein to the extracellular space.

A novel cell adhesive protein engineered by insertion of the Arg-Gly-Asp-Ser tetrapeptide.

A tetrapeptide Arg-Gly-Asp-Ser (RGDS) has been shown to be a versatile cell recognition signal of extracellular matrix components for the interaction with cells. We introduced the RGDS tetrapeptide into a truncated form of protein A, a staphylococcal immunoglobulin-binding protein, by inserting an oligonucleotide cassette encoding the tetrapeptide into the coding region of the protein A expression vector pRIT2T. The mutagenized protein was capable of not only binding to immunoglobulin G but also mediating cell attachment and spreading onto an inert substrate. Cell adhesion mediated by the mutagenized protein was inhibitable by a synthetic peptide Gly-Arg-Gly-Asp-Ser but not by a related peptide Gly-Arg-Gly-Glu-Ser, confirming that the inserted RGDS tetrapeptide served as a recognition signal for cell adhesion. Furthermore, the RGDS-containing protein was capable of adhering cells onto an immunoglobulin-coated surface which could not by itself support cell adhesion. Thus, the cell adhesive and immunoglobulin binding activities of the mutagenized protein appear to function coordinately. The protocol described here is essentially applicable to any protein and, therefore, provides a general principle in tailoring novel multifunctional proteins having cell adhesive activity.