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 (PTP ) with high affinity. In this study, we examined the binding of midkine to PTP by solidphase binding assay. Midkine and pleiotrophin binding to PTP 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 PTP is a common receptor for midkine and pleiotrophin. Midkine is structurally divided into the Nand C-terminal halves, and the latter exhibited full activity for PTP 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 Arg 78 in Cluster I resulted in loss of the high affinity binding and reduced neuronal migration-inducing activity, while mutations at Lys 83 and Lys 84 in Cluster II showed almost no effect on either activity. Chondroitinase ABC-treated PTP exhibited similar low affinity binding both to the native midkine and midkine mutants at Arg 78 . These results suggested that Arg 78 in midkine plays an essential role in high affinity binding to PTP by interacting with the chondroitin sulfate portion of this receptor.PTP /RPTP␤ 1 is a receptor-like protein-tyrosine phosphatase, which is abundantly expressed in the central nervous system as a chondroitin sulfate proteoglycan (1-4). PTP is composed of an N-terminal carbonic anhydrase-like domain, a fibronectin type III domain, a serine, glycine-rich domain that is thought to be chondroitin sulfate attachment region, a transmembrane segment, and two tyrosine phosphatase domains (1, 2). There are three splice variants of this molecule: (a) the full-length PTP (PTP -A); (b) the short form of PTP , in which most of the serine, glycine-rich region is deleted (PTP -B); and (c) the secreted form (PTP -S), which corresponds to the extracellular region of PTP -A and is also known as 6B4 proteoglycan/phosphacan (3, 5). All these splice variants are expressed as chondroitin sulfate proteoglycans in the brain (6), suggesting that chondroitin sulfate plays an essential role in receptor function.Several proteins such as contactin, tenascin, L1, NCAM, and TAG1 have been reported to bind PTP (7-9). Contactin is thought to be a neuronal receptor of PTP expressed on glial cells (7). Recently, we found that PTP binds with pleiotrophin/ heparin-binding growth-associated molecule (10), in that a chondroitin sulfate portion of PTP constitutes a part of the pleiotrophin binding site and regulates the affinity of PTPpleiotrophin binding (10). We further demonstrated that pleiotrophin-induced neurite outgrowth and neuronal migration were suppressed by chondroitin sulfate, polyclonal antibodies against the extracellular domain of PTP , and sodium vanadate, a protein-tyrosine phosphatase inhibitor. These findings suggested that P...

Midkine and pleiotrophin form a family of growth factors. Mice deficient in one of the genes show few abnormalities on reproduction and development. To understand their roles in these processes, we produced mice deficient in both genes; the double deficient mice were born in only one third the number expected by Mendelian segregation and 4 weeks after birth weighed about half as much as wild-type mice. Most of the female double deficient mice were infertile. In these mice, the numbers of mature follicles and of ova at ovulation were reduced compared to numbers in wild-type mice. Both midkine and pleiotrophin were expressed in the follicular epithelium and granulosa cells of the ovary. The expression of these factors in the uterus was dramatically altered during the estrous cycle. The diestrus and proestrus periods were long and the estrus period was short in the double deficient mice, indicating the role of the factors in the estrous cycle. Furthermore, vaginal abnormality was found in about half of the double deficient mice. These abnormalities in combination resulted in female infertility. Therefore, midkine and pleiotrophin, together with their signaling receptors, play important roles in the female reproductive system.

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.

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 midkineinduced 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 PTP, 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 PTP and PI3-kinase. These results indicate that PTP 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.

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