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Unlabelled: Midkine (MK) is a heparin-binding growth factor or cytokine and forms a small protein family, the other member of which is pleiotrophin. MK enhances survival, migration, cytokine expression, differentiation and other activities of target cells. MK is involved in various physiological processes, such as development, reproduction and repair, and also plays important roles in the pathogenesis of inflammatory and malignant diseases. MK is largely composed of two domains, namely a more N-terminally located N-domain and a more C-terminally located C-domain. Both domains are basically composed of three antiparallel β-sheets. In addition, there are short tails in the N-terminal and C-terminal sides and a hinge connecting the two domains. Several membrane proteins have been identified as MK receptors: receptor protein tyrosine phosphatase Z1 (PTPζ), low-density lipoprotein receptor-related protein, integrins, neuroglycan C, anaplastic lymphoma kinase and Notch-2. Among them, the most established one is PTPζ. It is a transmembrane tyrosine phophatase with chondroitin sulfate, which is essential for high-affinity binding with MK. PI3K and MAPK play important roles in the downstream signalling system of MK, while transcription factors affected by MK signalling include NF-κB, Hes-1 and STATs. Because of the involvement of MK in various physiological and pathological processes, MK itself as well as pharmaceuticals targeting MK and its signalling system are expected to be valuable for the treatment of numerous diseases., Linked Articles: This article is part of a themed section on Midkine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-4., (© 2013 The British Pharmacological Society.)

The cytokine midkine (MK) promotes tumor growth mainly by inducing angiogenesis. Here, we identified the source of MK in the vascular system under hypoxic conditions and demonstrated the relevance of MK during ischemia of normal tissue. Hypoxia increased MK protein expression in human polymorphonuclear neutrophils (PMN), monocytes, and human umbilical vein endothelial cells (HUVEC) compared with normoxia. Immunoelectron microscopy showed elevated cell surface expression of MK in PMN and monocytes during hypoxia. However, only HUVEC released significant amounts of soluble MK during hypoxia compared with normoxia (301 ± 81 pg/ml vs. 158 ± 45 pg/ml; P < 0.05). Exogenous MK induced neovascularization in a chorioallantoic membrane (CAM) assay compared with negative control as measured by counting the number of branching points per visual field (1,074 ± 54 vs. 211 ± 70; P < 0.05). In a hind limb ischemia model, the angiogenic response was almost completely absent in MK-deficient mice, whereas control animals showed a profound angiogenic response measured as proliferating endothelial cells per visual field (45 ± 30 vs. 169 ± 34; P < 0.01). These unanticipated results identified endothelial cells as the source of soluble MK in the vascular system during hypoxia and defined MK as a pivotal player of angiogenesis during ischemia in nonmalignant tissue.

Purpose: The aim of this study was the identification of novel tumor suppressor genes (TSG) silenced by DNA hypermethylation in hepatocellular carcinoma (HCC)., Experimental Design: We conducted integrative array-based approach for genome-wide screening of methylation targets using a methylated DNA immunoprecipitation-CpG island microarray and expression array in three universal hepatoma cell lines and normal liver tissue. Through detailed expression and functional analyses using hepatoma cell lines and primary HCC samples, we isolated novel TSGs for HCC., Results: A total of 642 genes were identified as methylated in three hepatoma cell lines but unmethylated in normal liver tissue, whereas 204 genes on autosomes were identified as genes unexpressed but restored after treatment with 5-aza-2'-deoxycytidine in these cell lines and expressed in normal tissue. Through the integration of results of the two-array analyses and further validation analyses of expression and methylation status in 17 cell lines and 30 primary tumors of hepatoma, we identified MZB1, marginal zone B and B1 cell-specific protein, encoding an endoplasmic reticulum protein, as a putative TSG frequently methylated within its CpG island in hepatoma. Among 162 patients with primary HCC, silencing of MZB1 protein was significantly and independently associated with a worse outcome. Restoration of MZB1 expression in hepatoma cells reduced cell proliferation in vitro and in vivo through G(1)-arrest., Conclusions: These results suggest that methylation-mediated silencing of MZB1 expression leads to loss of its tumor-suppressive activity, which may be a factor in the hepatocarcinogenesis, and is a useful prognosticator in HCC., (©2012 AACR.)

Long-term peritoneal dialysis induces peritoneal fibrosis with submesothelial fibrotic tissue. Although angiogenesis and inflammatory mediators are involved in peritoneal fibrosis, precise molecular mechanisms are undefined. To study this, we used microarray analysis and compared gene expression profiles of the peritoneum in control and chlorhexidine gluconate (CG)-induced peritoneal fibrosis mice. One of the 43 highly upregulated genes was pleiotrophin, a midkine family member, the expression of which was also upregulated by the solution used to treat mice by peritoneal dialysis. This growth factor was found in fibroblasts and mesothelial cells within the underlying submesothelial compact zones of mice, and in human peritoneal biopsy samples and peritoneal dialysate effluent. Recombinant pleiotrophin stimulated mitogenesis and migration of mouse mesothelial cells in culture. We found that in wild-type mice, CG treatment increased peritoneal permeability (measured by equilibration), increased mRNA expression of TGF-β1, connective tissue growth factor and fibronectin, TNF-α and IL-1β expression, and resulted in infiltration of CD3-positive T cells, and caused a high number of Ki-67-positive proliferating cells. All of these parameters were decreased in peritoneal tissues of CG-treated pleiotrophin-knockout mice. Thus, an upregulation of pleiotrophin appears to play a role in fibrosis and inflammation during peritoneal injury.

Protein production within the secretory pathway is accomplished by complex but organized processes. Here, we demonstrate that the growth factor midkine interacts with LDL receptor-related protein 1 (LRP1) at high affinity (K(d) value, 2.7 nm) not only at the cell surface but also within the secretory pathway during biosynthesis. The latter premature ligand-receptor interaction resulted in aggregate formation and consequently suppressed midkine secretion and LRP1 maturation. We utilized an endoplasmic reticulum (ER) retrieval signal and an LRP1 fragment, which strongly bound to midkine and the LRP1-specialized chaperone receptor-associated protein (RAP), to construct an ER trapper. The ER trapper efficiently trapped midkine and RAP and mimicked the premature ligand-receptor interaction, i.e. suppressed maturation of the ligand and receptor. The ER trapper also diminished the inhibitory function of LRP1 on platelet-derived growth factor-mediated cell migration. Complementary to these results, an increased expression of RAP was closely associated with midkine expression in human colorectal carcinomas (33 of 39 cases examined). Our results suggest that the premature ligand-receptor interaction plays a role in protein production within the secretory pathway.

Objectives: Midkine and Pleiotrophin are low molecular weight basic proteins with closely related structures and serve as growth/differentiation factors. They have been reported to be expressed in the cochlea during the embryonic and perinatal periods. In the present study, we focused on the roles of midkine and pleiotrophin in the stria vascularis and investigated morphological changes using mice deficient in these genes., Methods: Midkine knockout, pleiotrophin knockout, and double knockout mice were used and compared to wild-type mice. Auditory brain stem responses (ABRs) and cochlear blood flows were measured in each type of mice. Pathological changes in the stria vascularis were examined by light microscopy, including immunohistochemical staining with anti-Kir4.1 antibody, and electron microscopy., Results: Hearing thresholds examined by ABRs were significantly higher in midkine knockout and pleiotrophin knockout mice than in wild-type mice. Double knockout mice showed higher thresholds compared to midkine knockout and pleiotrophin knockout mice. Blood flow in the lateral walls did not significantly differ and light microscopy examination showed an almost normal appearance of the stria vascularis in these knockout mice. However, the expression of Kir4.1 was weak in the knockout mice and severe vacuolar degeneration was observed by electron microscopy in the intermediate cells of the double knockout mice., Conclusion: The present study demonstrates that midkine and pleiotrophin play some roles for the morphological maintenance of intermediate cell in the stria vascularis., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

The 13 kDa heparin-binding growth factor midkine (MK) was originally identified as a molecule involved in the orchestration of embryonic development. Recent studies provided evidence for a new role of MK in acute and chronic inflammatory processes. Accordingly, several inflammatory diseases including nephritis, arthritis, atherosclerosis, colitis, and autoimmune encephalitis have been shown to be alleviated in the absence of MK in animal models. Reduced leukocyte recruitment to the sites of inflammation was found to be one important mechanism attenuating chronic inflammation when MK was absent. Furthermore, MK was found to modulate expression of proinflammatory cytokines and the expansion of regulatory T-cells. Here, we review the current understanding of the role of MK in different inflammatory disorders and summarize the knowledge of MK biology.

Midkine (MK) is a heparin-binding cytokine, and promotes growth, survival, migration and other activities of target cells. After describing the general properties of MK, this review focuses on MK and MK inhibitors as therapeutics for diseases in the central nervous system. MK is strongly expressed during embryogenesis especially at the midgestation period, but is expressed only at restricted sites in adults. MK expression is induced upon tissue injury such as ischemic brain damage. Since exogenously administered MK or the gene transfer of MK suppresses neuronal cell death in experimental systems, MK has the potential to treat cerebral infarction. MK might become important also in the treatment of neurodegenerative diseases such as Alzheimer's disease. MK is involved in inflammatory diseases by enhancing migration of leukocytes, inducing chemokine production and suppressing regulatory T cells. Since an aptamer to MK suppresses experimental autoimmune encephalitis, MK inhibitors are promising for the treatment of multiple sclerosis. MK is overexpressed in most malignant tumors including glioblastoma, and is involved in tumor invasion. MK inhibitors may be of value in the treatment of glioblastoma. Furthermore, an oncolytic adenovirus, whose replication is under the control of the MK promoter, inhibits the growth of glioblastoma xenografts. MK inhibitors under development include antibodies, aptamers, glycosaminoglycans, peptides and low molecular weight compounds. siRNA and antisense oligoDNA have proved effective against malignant tumors and inflammatory diseases in experimental systems. Practical information concerning the development of MK and MK inhibitors as therapeutics is described in the final part of the review.

In this study, we examined the effects of switching from acarbose or voglibose to miglitol in type 2 diabetes mellitus patients for 3 months on gene expression of inflammatory cytokines/cytokine-like factors in peripheral leukocytes and on glucose fluctuations. We enrolled 47 Japanese patients with type 2 diabetes mellitus, aged 26 to 81 years, with hemoglobin A(₁c) levels ranging from 6.5% to 7.9% and who were treated with the highest approved dose of acarbose (100 mg per meal) or voglibose (0.3 mg per meal) in combination with insulin or sulfonylurea. Their prior α-glucosidase inhibitors were switched to a medium dose of miglitol (50 mg per meal), and the new treatments were maintained for 3 months. Forty-three patients completed the 3-month study and were analyzed. The switch to miglitol for 3 months did not affect hemoglobin A(₁c), fasting glucose, triglycerides, total cholesterol, or C-reactive protein levels, or adverse events other than hypoglycemia symptoms. Hypoglycemia symptoms and glucose fluctuations were significantly improved by the switch. The expression of interleukin-1β, tumor necrosis factor-α, and S100a4/6/9/10/11/12 genes in peripheral leukocytes, and the serum tumor necrosis factor-α protein levels were suppressed by switching to miglitol. Miglitol reduces glucose fluctuations and gene expression of inflammatory cytokines/cytokine-like factors in peripheral leukocytes of type 2 diabetes mellitus patients more than other α-glucosidase inhibitors and with fewer adverse effects., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Midkine (Mdk) and pleiotrophin (Ptn) comprise a family of heparin-binding growth factors known primarily for their effects on neuronal cells. Since transgenic mice overexpressing Ptn have been reported to display increased bone density, we have previously analyzed Ptn-deficient mice but failed to detect any abnormality of skeletal development and remodeling. Together with the finding that Mdk expression increases in the course of primary osteoblast differentiation, we reasoned that Mdk, rather than Ptn, could play a physiologic role in bone formation. Here, we show that Mdk-deficient mice display an increased trabecular bone volume at 12 and 18 months of age, accompanied by cortical porosity. Histomorphometric quantification demonstrated an increased bone-formation rate compared with wild-type littermates, whereas bone resorption was differentially affected in trabecular and cortical bone of Mdk-deficient mice. To understand the effect of Mdk on bone formation at the molecular level, we performed a genome-wide expression analysis of primary osteoblasts and identified Ank and Enpp1 as Mdk-induced genes whose decreased expression in Mdk-deficient osteoblasts may explain, at least in part, the observed skeletal phenotype. Finally, we performed ovariectomy and observed bone loss only in wild-type but not in Mdk-deficient animals. Taken together, our data demonstrate that Mdk deficiency, at least in mice, results in an increased trabecular bone formation, thereby raising the possibility that Mdk-specific antagonists might prove beneficial in osteoporosis therapy.

Midkine is a heparin-binding cytokine or a growth factor with a molecular weight of 13 kDa. Midkine binds to oversulfated structures in heparan sulfate and chondroitin sulfate. The midkine receptor is a molecular complex containing proteoglycans. Midkine promotes migration, survival and other activities of target cells. Midkine has about 50% sequence identity with pleiotrophin. Mice deficient in both factors exhibit severe abnormalities including female infertility. In adults, midkine is expressed in damaged tissues and involved in the reparative process. It is also involved in inflammatory reactions by promoting the migration of leukocytes, induction of chemokines and suppression of regulatory T cells. Midkine is expressed in a variety of malignant tumors and promotes their growth and invasion. Midkine appears to be helpful for the treatment of injuries in the heart, brain, spinal cord and retina. Midkine inhibitors are expected to be effective in the treatment of malignancies, rheumatoid arthritis, multiple sclerosis, renal diseases, restenosis, hypertension and adhesion after surgery.

Midkine (MK) is a growth factor implicated in the development and repair of various tissues, especially neural tissues. MK acts as a reparative neurotrophic factor in damaged peripheral nerves. A postulated role of MK in the degeneration and regeneration of sciatic nerves was explored by comparing wild-type (Mdk(+/+)) mice with MK-deficient (Mdk(-/-)) mice after freezing injury. In the Mdk(-/-) mice, a regenerative delay was observed, preceded by a decelerated Wallerian degeneration (WD). The relative wet weight of the soleus muscle slowly declined, and recovery was delayed compared with that in the Mdk(+/+) mice. In the regenerating nerve, unmyelinated axons were unevenly distributed, and some axons contained myelin-like, concentrically lamellated bodies. In the endplates of soleus muscles, nerve terminals containing synaptic vesicles disappeared in both mice. In Mdk(-/-) mice, the appearance of nerve terminals was delayed in synaptic vesicles of terminal buttons after injury. The recovery of evoked electromyogram was delayed in Mdk(-/-) mice compared with Mdk(+/+) mice. Our results suggested a delay in axonal degeneration and regeneration in Mdk(-/-) mice compared with Mdk(+/+) mice, and the delayed regeneration was associated with a delayed recovery of motor function. These findings show that a lack of MK following peripheral nerve injury is a critical factor in degeneration and regeneration, and manipulation of the supply of MK may offer interesting therapeutic options for the treatment of peripheral nerve damage., (2009 Wiley-Liss, Inc.)

Midkine is a 13-kDa retinoic acid-induced heparin-binding growth factor involved in various biological phenomena such as cell migration, neurogenesis, and tissue repair. We previously demonstrated that midkine-deficient (Mdk(-/-)) mice exhibited a delayed hippocampal development with impaired working memory and increased anxiety only at the age of 4 weeks. To assess whether midkine gene could play important roles in development and maintenance of central nervous system, we investigated biochemical and behavioral parameters in dopamine and glutamate neurotransmission of Mdk(-/-) mice. The Mdk(-/-) mice exhibited a hypodopaminergic state (i.e., decreased levels of dopamine and its receptors in the striatum) with no alterations of glutamatergic system (i.e., normal level of glutamate, glutamine, glycine, d-serine, l-serine, and NMDA receptors in the frontal cortex and hippocampus). We also found prepulse inhibition deficits reversed by clozapine and haloperidol in the Mdk(-/-) mice. Our results suggested that midkine deficiency may be related to neurochemical and behavioral dysfunctions in dopaminergic system.

A significant proportion of cytokines bind to glycosaminoglycans such as heparin. Glycosaminoglycans are involved in signaling, stabilization and/or storage of these cytokines. Typical examples of glycosaminoglycan-binding cytokines are basic fibroblast growth factor (bFGF), interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1), vascular endothelial growth factor (VEGF), VEGF-C, hepatocyte growth factor (HGF), granulocyte colony-stimulating factor (G-CSF), midkine, and pleiotrophin. All are present in the tumor microenvironment and promote tumor growth, tumor invasion and/or tumor angiogenesis. Serum or plasma levels of glycosaminoglycan-binding cytokines are frequently elevated in patients with various malignant tumors. High levels of these cytokines are usually correlated with the occurrence of metastasis and a poor prognosis. The mode of elevation of individual glycosaminoglycan-binding cytokines in patients with malignant tumors is summarized here. Further studies, especially with multiple cytokines, are expected to make assays clinically useful for both early detection and prognostic prediction.

CD4(+)CD25(+) regulatory T (Treg) cells are crucial mediators of autoimmune tolerance. The factors that regulate Treg cells, however, are largely unknown. Here, we show that deficiency in midkine (MK), a heparin-binding growth factor involved in oncogenesis, inflammation, and tissue repair, attenuated experimental autoimmune encephalomyelitis (EAE) because of an expansion of the Treg cell population in peripheral lymph nodes and decreased numbers of autoreactive T-helper type 1 (T(H)1) and T(H)17 cells. MK decreased the Treg cell population ex vivo in a dose-dependent manner by suppression of STAT5 phosphorylation that is essential for Foxp3 expression. Moreover, administration of anti-MK RNA aptamers significantly expanded the Treg cell population and alleviated EAE symptoms. These observations indicate that MK serves as a critical suppressor of Treg cell expansion, and inhibition of MK using RNA aptamers may provide an effective therapeutic strategy against autoimmune diseases, including multiple sclerosis.

Objective: This study was to clarify the roles of midkine (MK) in the brain., Methods: We determined cerebrospinal fluid MK levels in patients with neurological disorders by enzyme-linked immunoassay and immunostained autopsied brain samples in patients with meningitis., Results: MK levels were 0.37+/-0.21 ng/ml in controls (n=46, mean +/- S.D.), 0.67+/-0.19 ng/ml in patients with cerebral infarction (n=8), 1.78+/-1.32 ng/ml in patients with meningitis (n=25; ANOVA and post-hoc Fisher's PLSD test, p<0.0001), 0.31+/-0.25 ng/ml in patients with human T-lymphotrophic virus type I-associated myelopathy/tropical spastic paraparesis (n=29), and 0.42+/-0.17 ng/ml in patients with amyotrophic lateral sclerosis (n=8). The regression equations were Y=0.005X+0.498 (Y, CSF MK level; X, cell number) and Y=0.007X+0.326 (Y, MK level; X, protein level) for all CSF samples. Autopsy brain samples from patients with meningitis expressed MK weakly in mononuclear cells on immunohistochemical examination. Western blot and polymerase chain reaction analyses showed that leukocytes were MK positive. CSF MK levels were not high in patients with cerebral infarction but were increased in patients with meningitis. CSF MK levels were high in normal controls, compared to those of other cytokines. MK was expressed in choroid plexus of normal brain and released there., Conclusion: Our findings suggested that MK may maintain normal adult brain as a neurotrophic factor, and that MK may be released from leucocytes in brain of patients with meningitis as an immunological mediator.

In order to find informative salivary biomarkers specific to oral cancer we examined expression of 4 kinds of cytokine in saliva. Levels of interleukins (IL-1beta, -6, -8) and osteopontin were measured by ELISA using whole saliva samples collected from 19 patients with oral cancer (9 men, 10 women; mean age, 60.9 years) and 20 healthy persons (15 men, 5 women; mean age, 32 years). Expression of the 4 cytokines was higher in patients with oral cancer than in healthy controls. The difference was significant in IL-6, in particular. The results suggest that saliva offers a potential target for a screening test aimed at detection of precancerous lesions.

Problem: Ovarian follicles undergo a dynamic change to provide a mature ovum, and the process involves angiogenesis, follicular cell proliferation and leukocyte recruitment. Midkine (MK) is a heparin-binding growth factor that has angiogenic, mitogenic, and chemotactic activities. In the present study, we investigated the presence of MK and its possible role in human ovarian follicles., Method of Study: Follicular fluid (FF) and luteinized granulosa cells (LGC) were collected from women undergoing in vitro fertilization and embryo transfer. Expression of MK protein in FF was examined by Western blotting. Concentrations of MK, estradiol and oxygen in FF were measured. 5-bromo-2'-deoxyuridine (BrdU) incorporation assay was performed in LGC. Normal ovarian tissues were obtained surgically and used in in-situ hybridization of MK mRNA., Results: The presence of MK protein was verified in FF. MK mRNA was expressed in both granulosa cells and theca cells of large follicles. There is a significant negative correlation between the concentrations of MK and oxygen in FF, and a significant positive correlation between the concentrations of MK and estradiol. MK promoted BrdU uptake in LGC., Conclusion: The present findings imply that hypoxic condition, a characteristic of growing follicles, associates with the production of MK. Given that MK is involved in granulosa cell proliferation and estradiol production in developing follicles, MK may play a role as a local regulator in the human ovary.

Purpose: Midkine (MK), a heparin-binding growth factor, has an important role in cancer progression. The outcome of patients with gastrointestinal stromal tumors (GISTs) is correlated with tumor size and mitotic count. The aim of this study was to determine MK expression in GISTs., Methods: Midkine was detected in 31 (55%) of 57 surgically resected GISTs by immunohistochemistry with a rabbit antibody against MK and peroxidase method., Results: A significant worse outcome of MK-positive patients was found (P < 0.05; log rank test). Multivariate Cox regression analysis showed an independent prognostic impact (relative risk for overall survival 3.64; P < 0.05). Interestingly, MK expression was significantly associated with mitotic rate (P < 0.05; Chi-squared test), but not with tumor size (P = 0.97)., Conclusions: Taken together, MK is a prognostic marker for GIST patients. MK might also be a useful peripheral tumor marker since it can be detected in peripheral serum. Future studies should involve higher GIST patient numbers including tumor and serum samples for detection of MK.

Intraperitoneally administered chondroitin sulfate E inhibited the development of antibody-induced arthritis, a model of rheumatoid arthritis, while chondroitin 4-sulfate showed no effects. Chondroitin sulfate E inhibited in vitro differentiation of osteoclasts, which play key roles in the etiology of rheumatoid arthritis. One of the targets of chondroitin sulfate E is midkine, a heparin-binding growth factor or cytokine. Indeed, a chimeric-type siRNA for midkine inhibited the development of antibody-induced arthritis and adhesion of the omentum to the injured abdominal wall. These results indicate the significance of midkine as a molecular target to treat or prevent rheumatoid arthritis and adhesion after surgery, and the utility of chondroitin sulfate E to inhibit midkine in vivo.

Midkine, a heparin-binding growth factor, was found to be expressed in neural precursor cells, which consist of neural stem cells and the progenitor cells. When embryonic brain cells were allowed to form neurospheres enriched in neural precursor cells, numbers were significantly smaller from the midkine-deficient brain than from the wild-type brain. Dissociated neurosphere cells yielded nestin-positive neural precursor cells and differentiated neuronal cells upon culture on a substratum. Neural precursor cells from the midkine-deficient brain spread poorly and grew less effectively on a substratum coated with poly-l-lysine than the cells on midkine-coated substratum. Neural precursor cells from the wild-type brain spread and grew well on both the substrata. Differentiation to neurons and glia cells was not affected by the absence of midkine. Heparitinase digestion of dissociated neurosphere cells resulted in poor growth of neural precursor cells, while chondroitinase digestion had no effect. These results indicate that midkine is involved in the growth of neural precursor cells and suggest that the interaction with heparan sulfate proteoglycans is important in midkine action to these cells.

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.

Background: Midkine (MK) is a heparin-binding growth factor involved in diverse biological phenomena, eg, neural survival, carcinogenesis, and tissue repair. MK could have a protective action against ischemia/reperfusion (I/R) injury in the heart, because MK was shown to have cytoprotective activity in cultured neurons and tumor cells. We investigated this hypothesis in mice with and without genetic MK deletion., Methods and Results: Myocardial injury after I/R was produced by transient occlusion of coronary arteries. In wild-type (Mdk+/+) mice, MK expression was increased after I/R in the periinfarct area. Infarct size/area at risk 24 hours after I/R in MK-deficient (Mdk-/-) mice was larger than in Mdk+/+ mice (55.4+/-9.1% versus 32.1+/-5.3%, P<0.05). Terminal dUTP nick end-labeling-positive myocyte population in the periinfarct area in Mdk-/- mice was higher than in Mdk+/+ mice (6.8+/-0.9% versus 3.2+/-0.6%, P<0.05). Left ventricular fractional shortening 24 hours after I/R in Mdk-/- mice was significantly less than that in Mdk+/+ mice (34.3+/-4.4% versus 50.8+/-2.1%, P<0.05). Supplemental application of MK protein to left ventricle of Mdk-/- mice at the time of I/R resulted in reduction of the infarct size. Application of exogenous MK to cultured cardiomyocytes resulted in increased Bcl-2 expression and decreased apoptosis after hypoxia/reoxygenation., Conclusions: These results suggest that MK plays a protective role against I/R injury, most likely through a prevention of apoptotic reaction. MK is a potentially important new molecular target for treatment of ischemic heart disease.

Objective: Intimal hyperplasia is a major obstacle to patency after vein grafting. Despite of a diverse array of trials to prevent it, a satisfactory therapeutic strategy for clinical use has not been established. However, sufficient inhibition of early stages of intimal hyperplasia may prevent this long-term progressive disease. Midkine (MK) is a heparin-binding growth factor that was originally discovered as the product of a retinoic acid-responsive gene. We previously demonstrated that MK-deficient mice exhibit a striking reduction of neointima formation in a restenosis model, which is reversed on systemic MK administration. In this study, we evaluated a strategy of using small interfering RNA (siRNA) targeting MK as a therapy for vein graft failure., Methods: We first made a highly effective siRNA to rabbit MK. Jugular vein-to-carotid artery interposition vein grafts, which are applied to a low flow condition, were made in Japanese white rabbits. Small interfering RNA mixed with atelocollagen was administrated to the external wall of grafted veins. Cy3-conjugated stabilized siRNA was used to confirm its stability and successful transfer into the vein graft wall. Neointimal hyperplasia was evaluated 4 weeks after the operation. The proliferation index and leukocyte infiltration were determined., Results: MK expression was induced and reached the maximum level 7 days after operation. Fluorescence of Cy3-labeled siRNA could be detected in the graft wall even 7 days after operation. Knockdown of the gradually increasing expression was achieved by perivascular application of siRNA using atelocollagen. The intima-media ratio and the intima thickness at 28 days after grafting were both reduced >90% by this treatment compared with controls. This phenomenon was preceded by significant reductions of inflammatory cell recruitment to the vessel walls and subsequent cell proliferation in MK siRNA-treated grafts., Conclusions: These results suggest that midkine is a candidate molecular target for preventing vein graft failure. Furthermore, for clinical applications of siRNA, a single intraoperative atelocollagen-based nonviral delivery method could be a reliable approach to achieve maximal function of siRNA in vivo. This strategy may be a useful and practical form of gene therapy against human vein graft failure.

Background: Midkine (MK) is a heparin-binding growth factor that promotes the growth, migration, and survival of various cells. Its overexpression has been observed in many human malignancies, making it an attractive therapeutic target., Methods: We established a human midkine blockade system involving RNA interference., Results: The synthetic small interfering RNA (siRNA) targeting human midkine almost completely inhibited the secretion of midkine by a human prostate cancer cell line, PC-3, and consequently suppressed the proliferation and anchorage-independent growth of the transfected cells. The midkine siRNA, together with atelocollagen, significantly suppressed the growth of PC-3 tumors, which had been subcutaneously xenografted. These inhibitory effects of midkine siRNA were augmented by combinational treatment with a chemotherapeutic, paclitaxel (PTX), both in vitro and in vivo. The dose of PTX (12 mg/kg) used for the combinational treatment did not cause leukopenia or liver damage, which are often reported as serious adverse effects of PTX. The effect of the combination on tumor growth was comparable with that of PTX alone at higher doses that have exhibited severe adverse effects. Midkine siRNA suppressed mainly cell proliferation and slightly angiogenesis, whereas PTX enhanced apoptosis and slightly suppressed angiogenesis. The combination treatment was particularly effective for suppression of angiogenesis; consequently, it had striking effects on all aspects (proliferation, apoptosis, and angiogenesis)., Conclusions: We established a novel and safe cancer therapy strategy involving midkine siRNA combined with PTX, with less adverse effects.

alpha-Tectorin and beta-tectorin are major noncollagenous proteins of the tectorial membrane, which plays a crucial role in the reception of sonic signals in the cochlea. Midkine and pleiotrophin are closely related proteins that serve as growth factors and cytokines. In mice doubly deficient in the midkine gene and pleiotrophin gene, expression of beta-tectorin mRNA was nearly abolished in the cochlea on day 1 and 7 after birth. Expression of alpha-tectorin mRNA was unaffected in the double knockout mice, and expression of beta-tectorin mRNA was not altered in mice deficient in only the midkine or pleiotrophin gene. In newborn wild-type mice, both midkine and pleiotrophin were expressed in the greater epithelial ridge of the cochlea, in which beta-tectorin mRNA was strongly expressed. These results indicate that either midkine or pleiotrophin is required for significant expression of beta-tectorin. In agreement with the view that beta-tectorin is essential for normal auditory function, mice doubly deficient in both midkine and pleiotrophin genes exhibited very severe auditory deficits. We observed that mice deficient in either midkine or pleiotrophin gene were also impaired in their auditory response, but the level of the deficit was generally low or moderate. The present finding illustrates the importance of growth factor expression in the cochlea for auditory function.

Background: Midkine is a heparin-binding growth factor preferentially expressed in tumor cells. The present study was performed to utilize anti-midkine antibody for tumor therapy., Methods: A monoclonal antibody to midkine was raised by immunizing mice deficient in the midkine gene. The binding site of the antibody was studied by using N-terminal half and C-terminal half of midkine, both of which were chemically synthesized. Doxorubicin (DOX)-conjugate of the antibody was produced by chemical conjugation. The effects of the antibody and the conjugate on cell growth were examined using a midkine-secreting tumor cell, i.e. human hepatocellular carcinoma cell (HepG2)., Results: The monoclonal antibody bound to the N-terminal half of midkine. The antibody did not inhibit the growth of HepG2 cells probably because the active domain of midkine is in the C-terminal half. We produced the antibody conjugated with DOX with the hope that the conjugate would be internalized accompanied with midkine. Indeed, the antibody-DOX conjugate significantly inhibited the growth of HepG2 cells compared with DOX-conjugated control IgG., Conclusion: The result raises the possibility of using anti-midkine antibody conjugated with DOX for cancer therapy.

Midkine is a heparin-binding growth factor and is expressed by a number of tumor cells, contributing to their growth both in vitro and in vivo. Spontaneous lung metastasis of Lewis lung carcinoma cells, which did not significantly express MK, was significantly less extensive in mice deficient in the midkine gene than in wild-type mice, when the tumor was subcutaneously grown above the thigh. Midkine strongly enhanced migration of Lewis lung carcinoma cells in vitro. Therefore, midkine is also a host factor enhancing tumor metastasis. Anti-midkine therapy for malignancy may act on midkine produced by both the tumor and host.

Diabetic nephropathy is a life-threatening disease associated with diabetes mellitus. Longstanding hyperglycemia induces pathological reactions of glomerular mesangial cells, such as overproduction of extracellular matrix, which finally lead to nephropathy. However, the mechanisms underlying its pathogenesis have not been completely elucidated. Using the Streptozotocin-induced model of diabetes, we report that mice deficient in the growth factor midkine (Mdk-/-) exhibited strikingly milder nephropathy than Mdk+/+ mice, even though both mice showed similar extents of hyperglycemia after Streptozotocin injection. Midkine expression was induced in the glomerular mesangium of Mdk+/+ mice with diabetic nephropathy and in primary cultured mesangial cells exposed to high glucose. Mdk-/- mesangial cells exhibited reduced phosphorylation of protein kinase C and extracellular signal-regulated kinase as well as reduced production of transforming growth factor-beta(1) on high glucose loading. Addition of exogenous midkine restored extracellular signal-regulated kinase phosphorylation in Mdk-/- cells under high glucose conditions, whereas a midkine antisense oligodeoxynucleotide suppressed midkine in Mdk+/+ cells. Therefore, this study identifies midkine as a key molecule in diabetic nephropathy and suggests that midkine accelerates the intracellular signaling network evoked by hyperglycemia in nephropathy.

Mammalian brains contain significant amounts of chondroitin sulfate (CS), dermatan sulfate (DS), and CS/DS hybrid chains. CS/DS chains isolated from embryonic pig brains (E-CS/DS) promote the outgrowth of neurites in embryonic mouse hippocampal neurons in culture by interacting with pleiotrophin (PTN), a heparin-binding growth factor. Here, we analyzed oligosaccharides isolated from E-CS/DS, which showed that octasaccharides were the minimal size capable of interacting with PTN at a physiological salt concentration. Five and eight sequences were purified from fluorescently labeled PTN-bound and -unbound octasaccharide fractions, respectively, by enzymatic digestion followed by PTN-affinity chromatography. Their sequences were determined by enzymatic digestion in conjunction with high performance liquid chromatography, revealing a critical role for oversulfated D and/or iD disaccharides in the low yet significant affinity for PTN, which is required for neuritogenesis. The critical D and iD units are GlcUA(2-O-sulfate)beta1-3GalNAc(6-O-sulfate) and IdoUA(2-O-sulfate)alpha1-3GalNAc(6-O-sulfate), respectively, where IdoUA represents L-iduronic acid. In contrast, high affinity interactions with PTN required decasaccharides with E units (GlcUAbeta1-3GalNAc(4, 6-O-disulfate)), B units (GlcUA(2-O-sulfate)beta1-3GalNAc(4-O-sulfate)), and/or their IdoUA-containing counterparts (iE and iB) in addition to D/iD units, although the biological significance of such strong interactions remains to be investigated. Thus, chain size and composition are crucial to the interaction with PTN, and PTN binds to multiple sequences in E-CS/DS chains with distinct affinity. Notably, not only heparan sulfate but also CS/DS hybrid chain structures of mammalian brains contain a high degree of microheterogeneity with a cluster of oversulfated disaccharides and appear to play roles in regulating the functions of PTN.

Guluronate and mannuronate oligomers with various degree of polymerization were prepared from polyguluronate (PG) and polymannuronate (PM) with an alginate lyase from a Pseudoalteromonas sp., and their activities to induce cytokine secretion from mouse macrophage cell line RAW264.7 cells were examined. Enzymatically depolymerized unsaturated alginate oligomers induced tumor necrosis factor (TNF)-alpha secretion from RAW264.7 cells in a structure-depending manner, while the activities of saturated alginate oligomers prepared by acid hydrolysis were fairly low or only trace levels. These results suggest that unsaturated end-structure of alginate oligomers was important for the TNF-alpha-inducing activity. Among the unsaturated guluronate (G3-G9) and mannuronate (M3-M9) oligomers, G8 and M7 showed the most potent activity, respectively. Bio-Plex assay revealed that interleukin (IL)-1alpha, IL-1beta, and IL-6 secretion from RAW264.7 cells were also induced by unsaturated alginate oligomers with similar structure-activity relationship profiles as seen in TNF-alpha, and the most potent activities were observed with G8 and M7. These results suggest that G8 and M7 may have the most suitable molecular size or entire structural conformation as stimulant for cytokine secretion. Since antibodies to Toll-like receptor (TLR)2 and TLR4 effectively inhibited the G8- and M7-induced production of TNF-alpha, these alginate oligomers may stimulate innate immunity through the pattern recognition receptors on macrophages similar to microbial products.

Restenosis is the major clinical problem of angioplasty. We have previously shown that neointima formation is strikingly suppressed in midkine (MK)-deficient mice. Neointima formation is restored if MK protein is administrated to the deficient mice. MK is a heparin-binding growth factor and implicated in the migration of inflammatory cells and vascular smooth muscle cells. Consistently, the suppression of neointima formation in the deficient mice is accompanied by suppression of recruitment of inflammatory cells into the vascular wall. Here, we evaluated the potential of MK antisense oligodeoxyribonucleotide (ODN) for the prevention of restenosis. We cloned the cDNA of rabbit MK, which showed a strongly conserved sequence in mammals. The balloon injury induced MK expression, with the maximum level occurring 7-14 days after angioplasty, in the rabbit carotid artery. Two antisense ODNs suppressed the production of MK in a rabbit kidney cell line, RK13 cells, one of which was then transfected into the arterial wall by means of lipofection immediately after balloon treatment. The antisense ODN suppressed MK induction in vivo and consequently suppressed neointima formation to 60% of the control level. These results suggest that MK is a candidate molecular target for the therapy for vascular restenosis.

Midkine (MK) is a heparin binding growth factor and promotes growth, survival and migration of various cells including neurons. It is also known to accumulate in senile plaques of patients with Alzheimer's disease (AD). To investigate the involvement of serum MK in the pathophysiology of AD, serum MK levels were determined in patients with AD (n=36) and age- and sex-matched healthy controls (n=32), using an enzyme-linked immunosorbent assay (ELISA). The serum MK values of the patients with AD (median 560 and interquartile range (500-755) pg/ml) were significantly (U=278.5, P=0.0003, Mann-Whitney U-test) higher than those of the controls (median 500 and interquartile range (385-520) pg/ml). Moreover, 17 patients (47.2%) had abnormally high values of more than 600 pg/ml, but no controls (0%) did. There was no correlation between serum MK level and the mini mental state examination (MMSE) score in the patients. The demonstration of elevated MK levels in sera of patients with AD may contribute toward an understanding the pathophysiology of this disease, and provide a novel potential therapeutic strategy for decreasing neuronal damages in patients with AD. We found that serum MK levels in patients with AD were increased in comparison with those of normal controls.

Overexpression of a heparin-binding growth factor, midkine (MK), has been observed in many malignancies, making it an attractive therapeutic target. We used morpholino antisense oligomers to downregulate human MK expression in human prostate (PC-3) and colon carcinoma (SW620) cells, and determined the practical advantages of this anticancer therapeutic. Morpholino antisense oligomers directed against MK caused a dramatic and sequence-specific decrease of the target protein level, resulting in the inhibition of growth and anchorage-independent growth of the transfected cells. Furthermore, MK morpholino antisense oligomers exhibited a significant anticancer effect in the PC-3- and SW620-xenograft models. In comparison with phosphorothioate-modified oligodeoxynucleotide, morpholino oligomers showed 2 major advantages, stability and non-toxicity. MALDI-TOF mass spectrometric analysis showed that morpholino antisense oligomers were completely stable in the presence of serum nuclease(s). Serological examinations demonstrated no toxicity of MK morpholino antisense oligomers. Our study indicates that inhibition of MK expression by morpholino antisense oligomer is a promising novel and safe therapeutic strategy for cancers., ((c) 2004 Wiley-Liss, Inc.)

Background: Midkine, a heparin-binding growth factor, is involved in the migration of inflammatory cells. The inflammatory cell migration to the tubulointerstitium of the kidney after ischemia/reperfusion (I/R) injury is attenuated in midkine gene-deficient mice, resulting in better preservation of the tubulointerstitium compared with wild-type mice. In the present investigation, we planned to evaluate the usefulness of antisense midkine for the therapy of ischemic renal failure., Methods: Midkine antisense phosphorothioate oligodeoxyribonucleotide (ODN) at a dose of 1 mg/kg in saline was intravenously administered to mice 1 day before or after I/R. The kidneys were removed for examination 1, 2, 3, and 7 days after I/R., Results: It was rapidly incorporated into proximal tubular epithelial cells, and inhibited midkine synthesis, leading to reduced migration of inflammatory cells to the injured epithelial layer. Consequently, the midkine antisense ODN-treated animals exhibited less severe renal damage than untreated or midkine sense ODN-treated animals 2 days after I/R as assessed by morphologic criteria and blood urea nitrogen (BUN) and serum creatinine levels. Midkine expression, BUN, and serum creatinine levels were not significantly different between injection of midkine antisense ODN before and after ischemic injury., Conclusion: These results indicate that intravenous injection of midkine antisense ODN is a candidate for a novel therapeutic strategy against acute tubulointerstitial injury induced by I/R injury.

Background: The present study was conducted to assess whether midkine (MK), a multifunctional molecule known to stimulate tumor growth, may be involved in the development of endometriosis., Methods: The mitogenic activity of MK on cultured endometriotic stromal cells was examined by measuring 5-bromo-2'-deoxyuridine (BrdU) incorporation. Concentrations of MK in the peritoneal fluid (PF) of women without or with endometriosis and those under GnRH agonist treatment were measured using a specific enzyme immunoassay. The expression of MK mRNA in peritoneal bone marrow-derived cells, peritoneum and endometriotic tissues was evaluated by RT-PCR., Results: MK significantly increased BrdU incorporation into the DNA of cultured endometriotic stromal cells. The MK concentrations in the PF of the women with advanced endometriosis (stages II, III and IV) Median: 1.21 ng/ml; interquartile range 0.80-2.27 were significantly higher than those of the women without endometriosis and with stage I endometriosis (0.06 ng/ml, 0.67-1.26, P < 0.05). As for the menstrual phase, the MK concentration in PF in the inteal phase (1.32 ng/ml. 0.72-2.21) were significantly higher than those in the follicular phase (0.95 ng/ml, 0.68-1.24, P < 0.05). In addition, women with adnexal adhesions had higher concentrations of MK in PF than those without adhesions (P < 0.05). The MK concentrations of the women under GnRH agonist treatment were significantly lower than those of the other groups (P < 0.001). The expression of MK mRNA was detected in peritoneal bone marrow-derived cells, peritoneum and endometriotic tissues., Conclusions: The present findings suggest that MK may play roles, such as stimulation of endometriotic cell proliferation, in the development of endometriosis.

Chondroitin sulfate (CS) and dermatan sulfate (DS) chains play roles in the central nervous system. Most notably, CS/DS hybrid chains (E-CS/DS) purified from embryonic pig brains bind growth factors and promote neurite outgrowth toward embryonic mouse hippocampal neurons in culture. However, the neuritogenic mechanism is not well understood. Here we showed that pleiotrophin (PTN), a heparin-binding growth factor, produced mainly by glia cells, was the predominant binding partner for E-CS/DS in the membrane-associated protein fraction of neonatal rat brain. The CS/DS chains were separated on a PTN column into unbound, low affinity, and high affinity fractions. The latter two fractions promoted outgrowth of dendrite- and axon-like neurites, respectively, whereas the unbound fraction showed no such activity. The activity of the low affinity fraction was abolished by an anti-PTN antibody or when glia cells were removed from the culture. In contrast, the high affinity fraction displayed activity under both these conditions. Hence, PTN mainly from glia cells mediated the activity of the low affinity but not the high affinity fraction. The anti-CS antibody 473HD neutralized the neuritogenic activities of both fractions. Interaction analysis indicated that the 473HD epitope and PTN-binding domains in the E-CS/DS chains largely overlap. The three affinity subfractions differed in disaccharide composition and the distribution of l-iduronic acid-containing disaccharides along the chains. Oversulfated disaccharides and nonconsecutive iduronic acid-containing units were the requirements for the E-CS/DS chains to bind PTN and to exhibit the neuritogenic activities. Thus, CS subpopulations with distinct structures in the mammalian brain play different roles in neuritogenesis through distinct molecular mechanisms, at least in part by regulating the functions of growth factors.

Gene therapy may be a promising approach for treatment of brain ischemia. In this study, we examined the effect of postischemic gene transfer of midkine, a heparin-binding neurotrophic factor, using a focal brain ischemia model with the photothrombotic occlusion method. At 90 min after induction of brain ischemia in spontaneously hypertensive rats, a replication-deficient recombinant adenovirus encoding mouse midkine (AdMK, n=7) or a control vector encoding beta-galactosidase (Adbetagal, n=7) was injected into the lateral ventricle ipsilateral to ischemia. At 2 days after ischemia, we determined infarct volume by 2,3,5-triphenyltetrazolium chloride staining. There were no significant differences in cerebral blood flow 1 h after ischemia between AdMK and Adbetagal groups. Infarct volume of AdMK group was 51+/-27 mm3, which was significantly smaller than that of Adbetagal group (86+/-27 mm3, P<0.05). TUNEL-positive and cleaved caspase-3-positive cells in the periischemic area of AdMK-treated rats were significantly fewer than those in Adbetagal-treated rats, suggesting that the reduction of infarct volume by midkine was partly mediated by its antiapoptotic action. Thus, gene transfer of midkine to the ischemic brain may be effective in the treatment of brain ischemia.

Midkine (MK) is one of a family of heparin-binding growth factors, and increased MK expression is reported in various types of human carcinomas. To clarify the association between serum MK (S-MK) concentrations and gastric cancer, we examined S-MK concentrations of gastric cancer patients (n=275) and healthy controls (n=275). S-MK concentrations of all subjects were measured by enzyme-linked immunosorbent assay (elisa). The medians (25th and 75th percentiles) of S-MK were 192 (123 and 314) pg/mL in the cases and 170 (81 and 273) pg/mL in the controls (P <0.01). We also compared S-MK concentrations in each group divided by the progression stage or histological type of cancer. A difference was observed in the median S-MK concentrations between early and advanced cancers [182 (105 and 301) pg/mL vs 203 (139 and 331) pg/mL, P=0.07], but not between intestinal and diffuse type cancers [185 (121 and 306) pg/mL vs 198 (127 and 323) pg/mL, P=0.51]. We found that those progression stages affect S-MK concentration more strongly than the histological types in gastric cancer patients. Because S-MK seems to reflect the progression stage of gastric cancer, it may serve as a useful marker in the clinical follow-up of gastric cancer patients.

Midkine (MK), a heparin-binding growth factor, is produced in the developing and damaged nervous system. However, the role of MK in peripheral nerve injury has not been clarified. Here, we investigated MK expression in lumbar spinal motor neurons after rat sciatic nerve injury by immunohistochemical, in situ hybridization, and Western blot analyses. The rat sciatic nerve showed complete degeneration after local freezing. Numerous regenerated myelinated and thin nerve fibers were observed 3 weeks after injury. Intense MK immunoreactivity was detected in the ipsilateral spinal motor neurons of the anterior horn of the lumbar spinal cord after 1 day and in ipsilateral and contralateral spinal motor neurons from 4 days to 1 week after injury. It decreased after 2 weeks and again transiently increased in spinal motor neurons after 3 weeks. MK was found in the motor neurons and axon of the sciatic nerve. However, it was not detected in normal neurons and axon. In situ hybridization showed the expression of MK mRNA in lumbar spinal motor neurons of the anterior horn, but it was not present in Schwann cells or non-neuronal cells. Low-density lipoprotein receptor-related protein (LRP) immunoreactivity, a cell membrane receptor of MK, was observed in anterior horn motor neurons, but receptor-type protein tyrosine phosphatase zeta (PTPzeta) immunoreactivity as a signaling receptor complex of MK was not observed. LRP and PTPzeta immunoreactivities were observed in Schwann cells of the injured and uninjured sciatic nerve. Our findings suggest that MK is synthesized, released, and taken up in anterior horn motor neurons in an autocrine fashion with LRP. MK may have a role in degeneration and regeneration after peripheral nerve injury.

Although cisplatin acts directly on proximal tubule epithelial cells and causes cell death, little is known regarding the biological significance of its secondary effects, such as inflammation. The growth factor midkine is highly expressed in the proximal tubule and exerts ambivalent activities as to cisplatin nephrotoxicity, ie, anti-apoptotic and chemotactic ones. Here we report that midkine-deficient mice show a significantly higher survival rate than wild-type mice. The levels of blood urea nitrogen and tubular degeneration and apoptosis were higher in wild-type mice despite the anti-apoptotic activity of midkine. We found that recruitment of neutrophils was more enhanced in wild-type mice, this being consistent with the chemotactic activity of midkine. Midkine expression in wild-type mice persisted for 24 hours, and then dramatically decreased. Preadministration of midkine anti-sense oligodeoxyribonucleotide to wild-type mice suppressed midkine expression, and consequently neutrophil infiltration. It is of note that neutrophil infiltration, apoptosis, and elevation of blood urea nitrogen became conspicuous sequentially, namely 1, 2, and 3 days after cisplatin administration, respectively. These findings suggest that early molecular events involving midkine induce inflammatory response and their circuits eventually enhance the death of the proximal tubule epithelial cells. The results indicate the crucial role of inflammation in cisplatin-induced renal damage, and provide a candidate molecular target for its prevention.

Midkine is a heparin-binding growth factor that promotes the growth, survival, migration and differentiation of various target cells. So far, receptor-type protein tyrosine phosphatase zeta, low-density-lipoprotein-receptor-related protein and anaplastic lymphoma kinase have been identified as receptors for midkine. We found beta1 integrin in midkine-binding proteins from 13-day-old mouse embryos. beta1-Integrin bound to a midkine-agarose column and was eluted mostly with EDTA. Further study revealed that the alpha-subunits capable of binding to midkine were alpha4 and alpha6. Purified alpha4beta1- and alpha6beta1-integrins bound midkine. Anti-alpha4 antibody inhibited the midkine-dependent migration of osteoblastic cells, and anti-alpha6 antibody inhibited the midkine-dependent neurite outgrowth of embryonic neurons. After midkine treatment, tyrosine phosphorylation of paxillin, an integrin-associated molecule, was transiently increased in osteoblastic cells. Therefore, we concluded that alpha4beta1- and alpha6beta1-integrins are functional receptors for midkine. We observed that the low-density-lipoprotein-receptor-related-protein-6 ectodomain was immunoprecipitated with alpha6beta1-integrin and alpha4beta1-integrin. The low-density-lipoprotein-receptor-related-protein-6 ectodomain was also immunoprecipitated with receptor-type protein tyrosine phosphatase zeta. alpha4beta1- and alpha6beta1-Integrins are expected to co-operate with other midkine receptors, possibly in a multimolecular complex that contains other midkine receptors.

Background/purpose: We studied roles of two closely related heparin-binding growth factors, midkine (MK) and plieotrophin (PTN) in the processes of liver regeneration after partial hepatectomy., Methods: Liver regeneration was comparatively analyzed using mice deficient in the MK gene (Mdk), mice deficient in the pleiotrophin gene (Ptn) and wild-type mice., Results: Migration of neutrophils and macrophages to the liver 1-5 days after partial hepatectomy was suppressed in Mdk (-/-) mice. Liver damage as assessed by serum aspartate aminotransferase was also less severe in the deficient mice. On the other hand, cell growth in the liver was generally suppressed in the deficient mice 3-14 days after partial hepatectomy. The weight of the liver 7 and 14 days after partial hepatectomy was significantly less in Mdk (-/-) mice than wild-type mice. Very similar results were obtained using Ptn (-/-) mice except that a significant difference in the repaired liver weight was not observed between Ptn (-/-) mice and wild-type mice., Conclusions: MK and PTN are involved both in the inflammatory and reparative processes after partial hepatectomy, and as a whole are beneficial for liver regeneration.

We examined possible application of a regulatory region of midkine (MK) gene, which is frequently upregulated in a number of human tumours but not in normal cells, to cancer gene therapy. We examined transcriptional activity of the MK genomic fragments in paired cell lines, immortalized cells and their parental normal fibroblasts, and found that the MK fragments activated a fused reporter or a suicide gene preferentially in the immortalized cells. Recombinant adenoviruses (Ad), in which the MK fragment was inserted upstream to the E1A gene (AdMK), replicated preferentially in the immortalized cells and were cytotoxie to them. Human hepatocellular carcinoma cells were significantly susceptible to AdMK compared with human normal fibroblasts in vitro and the replication of AdMK was less than that of wild-type Ad in the infected fibroblasts. Hepatocellular carcinoma cells infected with AdMK did not form tumours in immunocompromised mice and intratumoural injection of AdMK into the hepatocellular carcinoma developed in mice retarded the subsequent tumour growth. Expression of E1A and necrosis of tumours were detected in AdMK-injected but not control Ad-injected cases. The MK promoter-driven suicide gene therapy and -mediated replicative Ad can thereby produce cytotoxic effects to immortalized and tumour cells with minimal damage to normal cells.

Objective: Midkine (MK), a heparin-binding growth factor, promotes growth, survival, and migration of various cells. The essential role of MK in migration of inflammatory cells has been shown using mice deficient in the MK gene (Mdk(-/-) mice). We undertook this study to investigate the role of MK in the pathogenesis of rheumatoid arthritis (RA)., Methods: MK levels in specimens from patients were determined by enzyme-linked immunosorbent assay, and localization of MK was revealed by immunohistochemical analysis. Susceptibility to antibody-induced arthritis was compared between Mdk(-/-) and wild-type (WT) mice. Osteoclast differentiation was monitored using macrophage-like cells isolated from human synovial tissue and macrophages from mouse bone marrow., Results: MK levels in sera and synovial fluid were increased in most RA patients, indicating a strong correlation between MK expression and RA. MK was expressed in macrophage-like cells and fibroblast-like cells in synovial membranes from the patients. In antibody-induced arthritis, Mdk(-/-) mice seldom developed the disease, while most of the WT mice did. Administration of MK to the Mdk(-/-) mice increased the frequency of antibody-induced arthritis. Migration of inflammatory leukocytes to the synovial membranes in the disease model was suppressed in the Mdk(-/-) mice. Furthermore, MK was found to promote the differentiation of osteoclasts from macrophages., Conclusion: MK participates in each of the two distinct phases of RA development, namely, migration of inflammatory leukocytes and osteoclast differentiation, and is a key molecule in the pathogenesis of RA.

It is widely held that growth factor signaling is terminated by lysosomal degradation of its activated receptor and the endocytosed growth factor is transported to lysosomes. Nuclear targeting is another important pathway through which signals of growth factors are mediated. However, mechanisms underlying desensitization of nuclear targeting growth factors are poorly understood. Here we report that the nuclear targeting pathway is down-regulated by the proteasome system. Degradation of endocytosed midkine, a heparin-binding growth factor, was suppressed by both proteasome and lysosome inhibitors to similar extents. By contrast, a proteasome inhibitor, but not lysosome ones, accelerated the nuclear accumulation of midkine. An expression vector of signal sequence-less midkine, which is produced in the cytosol, was constructed because endocytosed midkine may be translocated to the cytosol from cellular compartments before entering the nucleus. The cytosol-produced midkine underwent proteasomal degradation and accumulated in the nucleus as did the endocytosed midkine. It was polyubiquitinated, and its nuclear accumulation was enhanced by a proteasome inhibitor. We further dissected the midkine molecule to investigate roles in degradation and trafficking. The N-terminal half-domain of midkine was significantly more susceptible to proteasomal degradation, whereas the C-terminal half-domain was sufficient for nuclear localization. Together, these data highlight the desensitization of nuclear targeting by growth factors and indicate a critical role of the proteasome system in it.

Spinal cord injury is a debilitating condition. Midkine (MK) is involved in the generation of the central nervous system during development; however, the role of MK in the mature spinal cord has not been clarified. We examined the expression of MK, which has neurotrophic activity, before and after traumatic injury to the adult rat spinal cord. Following laminectomy, the rat spinal cord was injured at the T-9 level by applying extradural static weight-compression, in which a cylindrical compressor was used to induce complete and irreversible transverse spinal cord injury with paralysis of the lower extremities. The expression of MK was examined up to 14 days after the injury by immunohistochemical and Western blot analyses. Intense MK immunoreactivity was observed in the gray matter around the injury site but not in the necrotic lesion 1-7 days postinjury, although it was slightly positive 14 days after the injury. MK immunoreactivity was not detected in the normal spinal cord. The expression of MK was an early event, and its expression was compared to the increased production of glial fibrillary acidic protein (GFAP), a marker of reactive astrocytes, that was elevated at 2 days postinjury and continued over a 14 day period following the injury. Double immunostaining with anti-MK and anti-GFAP showed the existence of MK in the astrocytic cytoplasm. These findings suggest that MK was produced in astrocytes approximating the damaged region and may represent a reparative neurotrophic factor during the early phase of traumatic injury of the spinal cord.

The midkine (MK) family consists of only two members, namely heparin-binding growth factors MK and pleiotrophin (PTN). During embryogenesis, MK is highly expressed in the mid-gestational period, whereas PTN expression reaches the maximum level around birth. Both proteins are localized in the radial glial processes of the embryonic brain, along which neural stem cells migrate and differentiate. Zebrafish and Xenopus MK can induce neural tissues. In addition, deposits of MK and/or PTN are found in neurodegenerative diseases, such as Alzheimer's disease and multiple system atrophy. Both molecules are induced in reactive astrocytes by ischemic insults. In this context, it is interesting that LDL receptor-related protein is a receptor for MK and PTN, and this receptor has been implicated in the pathogenesis of Alzheimer's disease. MK and PTN share receptors, and show similar biological activities that include fibrinolytic, anti-apoptotic, mitogenic, transforming, angiogenic, and chemotactic ones. These activities explain how these molecules are involved in carcinogenesis. MK is detected in human carcinoma specimens from pre-cancerous stages to advanced stages. Strong expression of PTN is also detected in several carcinomas, although, in general, MK is expressed more intensely and in a wide range of carcinomas than PTN. The blood MK level is frequently elevated in advanced human carcinomas, decreases after surgical removal of the tumors, and is correlated with prognostic factors. Thus, it is a good market for evaluating the progress of carcinomas. Furthermore, antisense oligonucleotides for MK and ribozymes for PTN show anti-tumor activity. Therefore, MK and PTN are candidate molecular targets for therapy for human carcinomas.

We examined the expression level of the midkine (MK) and c-erbB-2 genes in both tumorous and matched nontumorous specimens from 18 patients with breast cancer. Expression of the MK and c-erbB-2 genes in nontumorous regions was relatively low, and the expression levels of both genes were not markedly different among the nontumorous samples. In contrast, the expression of the MK and c-erbB-2 genes in tumorous specimens was upregulated in 18 and 6 specimens, respectively. Regulatory regions of the MK gene were able to activate a reporter gene to a similar degree as those of the c-erbB-2 gene in the human breast cancer cell lines tested. Transfection of breast cancer cells with either the MK promoter- or the c-erbB-2 promoter-linked herpes simplex virus thymidine kinase gene increased their sensitivity to the prodrug ganciclovir. These data showed that the MK promoter activated a therapeutic gene in a wider range of human breast cancer than the c-erbB-2 promoter and suggest that MK promoter-mediated gene therapy is potentially more favorable in clinical settings., (Copyright 2004 S. Karger AG, Basel)