Protective Effect of Nerium oleander Distillate and Tarantula cubensis Alcoholic Extract on Cancer Biomarkers in Colon and Liver Tissues of Rats with Experimental Colon Cancer.

BACKGROUND: Colon cancers are among the top three causes of cancer-related deaths. This study is a continuation of previous research aiming to identify effective treatments. OBJECTIVE: This study investigated the effects of Tarantula cubensis alcoholic extract (TCAE) and Nerium oleander (NO) distillate on the levels of midkine, transforming growth factor (TGF)-ß, vascular endothelial growth factor (VEGF), alpha-fetoprotein (AFP), cyclooxygenase (COX)-2, insulin-like growth factor (IGF) and caspase-3 in the liver and colon tissues of rats with experimentally induced colon cancer. METHODS: The liver and colon tissues of rats were homogeneously divided into control, colon cancer (azoxymethane, AZM), AZM + TCAE, and AZM + NO distillate groups. The levels of midkine, TGF-ß, VEGF, AFP, COX-2, IGF, and caspase-3 in the colon and liver tissues were measured by ELISA. RESULTS: The levels of all parameters in colon and liver tissues in the AZM group were higher (p<0.05) than those in the control group. TCAE and NO distillate prevented (p < 0.05) increases in midkine, TGF-ß, VEGF, AFP, COX-2, IGF, and caspase-3 levels in the colon. NO distillate prevented the increase in all parameters except IGF, whereas TCAE prevented the increase in all values apart from COX-2 and IGF levels in the liver (p<0.05). CONCLUSION: NO distillate and TCAE may prevent the studied markers from reaching specified levels observed in the colon in AZM-induced colon cancer. The increases in the levels of the parameters in the liver were not as severe as those in the colon; however, an 18-week study period may not be sufficient for liver metastasis formation. Future molecular studies should investigate the mechanisms and pathways of these treatments in greater detail.

H. sinensis mycelium inhibits epithelial-mesenchymal transition by inactivating the midkine pathway in pulmonary fibrosis.

The medical fungus Hirsutella sinensis has been used as a Chinese folk health supplement because of its immunomodulatory properties. Our previous studies established the antifibrotic action of Hirsutella sinensis mycelium (HSM) in the lung. The epithelial-mesenchymal transition (EMT) is involved in the pathogenesis of idiopathic pulmonary fibrosis. The present study investigates the role of HSM in mediating EMT during the development of pulmonary fibrosis. HSM significantly inhibits bleomycin (BLM)-induced pulmonary fibrosis by blocking the EMT. In addition, the expression levels of midkine are increased in the lungs of the BLM-induced group. Further analysis of the results indicates that the mRNA level of midkine correlated positively with EMT. HSM markedly abrogates the transforming growth factor ß-induced EMT-like phenotype and behavior in vitro. The activation of midkine related signaling pathway is ameliorated following HSM treatment, whereas this extract also caused an effective attenuation of the induction of EMT (caused by midkine overexpression) in vitro. Results further confirm that oral medication of HSM disrupted the midkine pathway in vivo. Overall, findings suggest that the midkine pathway and the regulation of the EMT may be considered novel candidate therapeutic targets for the antifibrotic effects caused by HSM.

H. sinensis mycelium inhibits epithelial-mesenchymal transition by inactivating the midkine pathway in pulmonary fibrosis / 医学前沿

The medical fungus Hirsutella sinensis has been used as a Chinese folk health supplement because of its immunomodulatory properties. Our previous studies established the antifibrotic action of Hirsutella sinensis mycelium (HSM) in the lung. The epithelial-mesenchymal transition (EMT) is involved in the pathogenesis of idiopathic pulmonary fibrosis. The present study investigates the role of HSM in mediating EMT during the development of pulmonary fibrosis. HSM significantly inhibits bleomycin (BLM)-induced pulmonary fibrosis by blocking the EMT. In addition, the expression levels of midkine are increased in the lungs of the BLM-induced group. Further analysis of the results indicates that the mRNA level of midkine correlated positively with EMT. HSM markedly abrogates the transforming growth factor β-induced EMT-like phenotype and behavior in vitro. The activation of midkine related signaling pathway is ameliorated following HSM treatment, whereas this extract also caused an effective attenuation of the induction of EMT (caused by midkine overexpression) in vitro. Results further confirm that oral medication of HSM disrupted the midkine pathway in vivo. Overall, findings suggest that the midkine pathway and the regulation of the EMT may be considered novel candidate therapeutic targets for the antifibrotic effects caused by HSM.

Electroacupuncture Alleviated Neuronal Apoptosis Following Ischemic Stroke in Rats via Midkine and ERK/JNK/p38 Signaling Pathway.

This study aimed to evaluate the effects of electroacupuncture (EA) intervention administered at rats of middle cerebral artery occlusion (MCAO)/reperfusion. Fifty-four male Sprague-Dawley rats were divided into three groups, consisting of sham group, MCAO/R group, and EA group. EA treatment at Quchi and Zusanli acupoints was applied in rats of EA group at 24 h after MCAO once per day for 3 days. Our results indicated that EA treatment reduced infarct volumes and neurological deficits, as well alleviated the apoptotic cells in peri-infarct cortex, indicating that EA exerted neuroprotective effect in cerebral ischemic rats. Moreover, EA treatment may effectively reverse the upregulation of caspase-3 and Bim and alleviate the inhibition of Bcl-2 following 72-h ischemic stroke. EA may significantly reverse the promoted relative density level of p-ERK1/2, p-JNK, and p-p38 in the EA group compared with the MCAO/R group. In addition, the growth factor midkine (MK) was upregulated at 72 h after MCAO/R, and EA treatment may significantly prompt expression of MK. Our study demonstrated that EA exerted neuroprotective effect against neuronal apoptosis and the mechanism might involve in upregulation of MK and mediation of ERK/JNK/p38 signal pathway.

The Protein Tyrosine Phosphatase Rptpζ Suppresses Osteosarcoma Development in Trp53-Heterozygous Mice.

Osteosarcoma (OS), a highly aggressive primary bone tumor, belongs to the most common solid tumors in growing children. Since specific molecular targets for OS treatment remain to be identified, surgical resection combined with multimodal (neo-)adjuvant chemotherapy is still the only way to help respective individuals. We have previously identified the protein tyrosine phosphatase Rptpζ as a marker of terminally differentiated osteoblasts, which negatively regulates their proliferation in vitro. Here we have addressed the question if Rptpζ can function as a tumor suppressor protein inhibiting OS development in vivo. We therefore analyzed the skeletal phenotype of mice lacking Ptprz1, the gene encoding Rptpζ on a tumor-prone genetic background, i.e. Trp53-heterozygosity. By screening a large number of 52 week old Trp53-heterozygous mice by contact radiography we found that Ptprz1-deficiency significantly enhanced OS development with 19% of the mice being affected. The tumors in Ptprz1-deficient Trp53-heterozygous mice were present in different locations (spine, long bones, ribs), and their OS nature was confirmed by undecalcified histology. Likewise, cell lines derived from the tumors were able to undergo osteogenic differentiation ex vivo. A comparison between Ptprz1-heterozygous and Ptprz1-deficient cultures further revealed that the latter ones displayed increased proliferation, a higher abundance of tyrosine-phosphorylated proteins and resistance towards the influence of the growth factor Midkine. Our findings underscore the relevance of Rptpζ as an attenuator of proliferation in differentiated osteoblasts and raise the possibility that activating Rptpζ-dependent signaling could specifically target osteoblastic tumor cells.

The Circulating Levels of Selenium, Zinc, Midkine, Some Inflammatory Cytokines, and Angiogenic Factors in Mitral Chordae Tendineae Rupture.

Chordae tendineae rupture process is associated with increased production of inflammatory and angiogenesis mediators in connective tissues, which contributes to chronic inflammation and pathogenesis of degenerative chordae. A few trace elements are known to possess antioxidant, anti-inflammatory, and antiangiogenic properties. Therefore, the aim of this study was to determine whether zinc, selenium, midkine (MK), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor-alpha (TNF-α), vascular endothelial growth factor-A (VEGF-A), platelet-derived growth factor-BB (PDGF-BB), and reduced glutathione (GSH) levels are associated with inflammation and angiogenesis processes in the context of a potential etiology causing aggravation of mitral regurgitation and/or ruptured chordae tendineae. Seventy-one subjects comprising 34 patients with mitral chordae tendineae rupture (MCTR) and 37 healthy controls diagnosed on the basis of their clinical profile and transthoracic echocardiography were included in this study. The levels of GSH, MK, selenium, and zinc were found to be lower in the patients group when compared to control group. There were no significant difference in plasma TNF-α, IL-1ß, IL-6, IL-8, VEGF-A, and PDGF-BB levels between two groups. There were positive significant correlations between MK and GSH, MK, and selenium levels in patients with MCTR. According to our data in which selenium, zinc, MK, and GSH decreased in MCTR patients, inflammatory response, oxidative stress, and trace element levels may contribute to etiopathogenesis of mitral regurgitation and/or ruptured chordae tendineae.

Sorafenib and lithium chloride combination treatment shows promising synergistic effects in human glioblastoma multiforme cells in vitro but midkine is not implicated.

OBJECTIVES: The objectives of this study were to test the effects of the new combination treatment modality, sorafenib (SOR) and lithium chloride (LiCl) and to assess whether midkine (MK) protein has a role in any potential effects. METHODS: Monolayer and spheroid cultures of T98G human glioblastoma multiforme (GBM) cells were treated with LiCl and SOR (inhibition concentration 50 value  =  100 µM), or their combination, or were left untreated (control). Cell proliferation and apoptotic indices, the mechanism of action, and the levels of apoptotic and anti-apoptotic proteins were evaluated in monolayer cultures and ultrastructure was evaluated by transmission electron microscopy (TEM) in spheroid cultures after for 72 hours. RESULTS: All drug applications decreased cell numbers and increased the apoptotic index. The combination shows a synergistic effect. In the combination group, the decrease in cell numbers and the increase in the apoptotic index were significantly greater than with the individual drugs (P < 0.01). The combination treatment led to the greatest decreases in MRP-1 and p170 levels; but the greatest decreases in p-STAT-3, p-ERK (P < 0.05), p-AKT, p-GSK-3-beta (P < 0.01), EGFR (P < 0.01), NF-kappa-ß levels were with SOR alone, followed by the combination. The decreases in MK levels in the SOR and combination groups were similar (P  =  0.06). Severe ultrastructural damage was more frequently observed in the combination group compared with the other groups. CONCLUSIONS: These results suggest the possibility that the addition of LiCl to SOR could improve the prognosis in at least some patients who need both cancer and psychotherapy and indicate the need for further studies.

Involvement of midkine expression in the inhibitory effects of low-frequency magnetic fields on cancer cells.

Effects of magnetic fields (MFs) on cancer cells may depend on cell type and exposure conditions. Gene expression levels are different among cancer cells. However, the effect of MFs on cancer cells with different gene expressions is still unclear. In this study, the cancer cell lines BGC-823, MKN-45, MKN-28, A549, SPC-A1, and LOVO were exposed to a low-frequency MF. Specific parameters of MFs were determined. Furthermore, the potential of the MF to influence cancer cell growth with midkine (MK) expression was evaluated. Cell proliferation and cell cycle were detected using the CCK-8 assay and flow cytometry. Cell ultrastructure was observed by transmission electron microscopy. BGC-823 cells with over-expression of MK (BGC-MK cells) and stanniocalcin-1 were generated by plasmid construction and transfection. Results showed that exposure to a 0.4-T, 7.5 Hz MF inhibited the proliferation of BGC-823, MKN-28, A549, and LOVO cells, but not MKN-45 and SPC-A1 cells. Moreover, the inhibitory effect of the MF on BGC-MK cells was lower (12.3%) than that of BGC-823 cells (20.3%). Analysis of the cell cycle showed that exposure to the MF led to a significant increase in the S phase in BGC-823 cells, but not in BGC-MK cells. In addition, organelle morphology was modified in BGC-823 cells exposed to the MF. These results suggest that exposure to a 0.4-T, 7.5 Hz MF could inhibit tumor cell proliferation and disturb the cell cycle. The alteration of MK expression in cancer cells may be related to the inhibitory effect of the MF on these cells.

Midkine regulation of the renin-angiotensin system.

Hypertension and chronic kidney disease are often associated. The pathogenesis of these diseases involves the renin-angiotensin system. We have recently reported that the growth factor midkine is a novel regulator of the renin-angiotensin system. Midkine is a heparin-binding growth factor so far implicated in neuronal differentiation, neuroprotection, cardioprotection, inflammation, and cancer development. In a mouse model of chronic kidney disease induced by 5/6 nephrectomy, midkine is produced in the lung and in turn upregulates angiotensin-converting enzyme expression. Hypertension associated with 5/6 nephrectomy is not observed in midkine-deficient mice. Conversely, supplemental administration of midkine to the deficient mice induces hypertension. This review describes the molecular characteristics of midkine and its significance in the renin-angiotensin system and the kidney-lung interaction.

Dopaminergic hypofunctions and prepulse inhibition deficits in mice lacking midkine.

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.

Abilities of cumulus and granulosa cells to enhance the developmental competence of bovine oocytes during in vitro maturation period are promoted by midkine; a possible implication of its apoptosis suppressing effects.

We previously reported that when midkine (MK), a heparin-binding growth differentiation factor was used in in vitro maturation (IVM) culture of bovine cumulus-enclosed oocytes (CEOs), their developmental competence to the blastocyst stage after in vitro fertilization (IVF) was enhanced and the effect of MK might be mediated by its action upon mural granulosa cells and cumulus cells that closely surround the oocyte. In the present study, when denuded oocytes (DOs) were matured in IVM medium with or without MK (200 ng/ml) in the presence or absence of isolated cumulus cell masses and subjected to IVF, the enhancing effects of MK on the developmental competence of DOs to the blastocyst stage after IVF were exerted only in the presence of cumulus cells. In addition, we prepared the conditioned media of granulosa cells cultured with or without 200 ng MK/ml (CMMK+ or CMMK- respectively) and examined their effects on the IVM of DOs in terms of their developmental competence to the blastocyst stage after IVF. The supplementation of CMMK+ into IVM medium at 40% (v/v) significantly enhanced the blastocyst development compared with the no additive control and the CMMK- supplemented groups. Furthermore, the effects of MK during IVM of bovine CEOs on the cumulus cell apoptosis were investigated. CEOs were cultured up to 24 h in IVM medium without (control) or with 200 ng MK/ml. The genomic DNA was extracted from CEOs at 0, 6, 12, 18 and 24 h of IVM and subjected to ligation-mediated PCR (LM-PCR) to detect the apoptotic internucleosomal DNA fragmentation. DNA fragmentation was scarcely detected at the start of IVM, whereas it increased time-dependently as the IVM culture progressed. The degree of the fragmentation was significantly lower in the MK-treatment group compared with the control group at 18 and 24 h of IVM. The apoptosis-suppressing effect of MK on cumulus cells was further confirmed in situ by using TUNEL on CEOs. In conclusion, data from the present study further confirmed that MK enhances the developmental competence of bovine oocytes via cumulus and granulosa cells. It was also demonstrated that MK suppresses the apoptosis that occurs in cumulus cells during the period of IVM of bovine CEOs. The putative soluble factor(s) from cumulus cells was suggested from the experiment using CMMK+ . MK may promote the production of such factors in part by its anti-apoptotic effects on cumulus cells.

Introduction of midkine gene into human bladder cancer cells enhances their malignant phenotype but increases their sensitivity to antiangiogenic therapy.

PURPOSE: Midkine (MK) is a member of a family of heparin-binding growth factors, which was reported to have an important role in angiogenesis. Although MK was reported to be associated with bladder cancer progression, the functional significance of MK expression in bladder cancer progression has not been elucidated. The objectives of this study were to determine whether overexpression of MK in bladder cancer cells enhances their malignant potential and to evaluate the inhibitory effect of the antiangiogenic agent TNP-470 on the growth of MK-overexpressing bladder cancer cells in vivo. EXPERIMENTAL DESIGN: We introduced the MK gene into human bladder cancer UM-UC-3 cells that do not secrete a detectable level of MK protein and generated the MK-overexpressing cell line UM-UC-3/MK. The biological activity of secreted MK was evaluated using a human umbilical vein endothelial cell proliferation assay. To investigate the in vivo effects of MK overexpression on tumor growth, each cell line was injected s.c. and orthotopically into nude mice. To evaluate the therapeutic effects of the antiangiogenic agent, mice were given TNP-470 after s.c. injection of each cell line. The microvessel density of tumors was quantitated by immunohistochemistry of CD31. RESULTS: The heparin affinity-purified conditioned media of UM-UC-3/MK cells significantly enhanced human umbilical vein endothelial cell proliferation. MK expression had no effect on in vitro growth but conferred a growth advantage on both s.c. and orthotopic tumors in vivo. Furthermore, enhanced tumor growth was closely associated with increased microvessel density. Significant inhibition of tumor growth by TNP-470 treatment was observed only in UM-UC-3/MK tumors and not in control tumors. CONCLUSIONS: We demonstrated that overexpression of the MK gene causes an increase in the angiogenic activity of cells through vascular endothelial cell growth, resulting in enhanced malignant potential of human bladder cancer cells. Moreover, the present findings suggest that TNP-470 could be used as a novel therapeutic adjunct to conventional agents for patients with advanced bladder cancer overexpressing MK.

HB-GAM/Pleiotrophin and Midkine are differently expressed and distributed during retinoic acid-induced neural differentiation of P19 cells.

HB-GAM/Pleiotrophin and Midkine (MK) are developmentally-regulated proteins with putative functions during cell growth and differentiation. Using the P19 cell which is a model to study the events associated with early development, we examined the expression and cellular localization of HB-GAM and MK during neural differentiation of P19 cells induced by retinoic acid (RA). The temporal expressions of HB-GAM and MK transcripts and both the levels and cellular localizations of the corresponding proteins appeared dramatically different. MK mRNA, already expressed in untreated P19 cells, was transiently increased by exposure to RA and then largely down regulated. More interestingly, HB-GAM which was not detected in untreated P19 cells was strongly expressed after 2 days of RA treatment and this expression persists throughout the duration of the culture suggesting that it could be involved in different aspects of this differentiation process.

The growth factor midkine is modulated by both glucocorticoid and retinoid in fetal lung development.

The glucocorticoids (GC) and retinoids (RA) modulate branching morphogenesis and cytodifferentiation in the developing lung. We investigated downstream target genes that link glucocorticoid stimulation to the achievement of a mature lung in glucocorticoid receptor (GR) knockout mice. All GR(null) mice and approximately 80% of mice homozygous for a hypomorphic allele (GR(hypo)) die shortly after birth of respiratory failure. cDNA microarray analysis showed organ-specific upregulation of the retinoic acid responsive gene midkine (MK) and its chondroitin-sulfate binding partner PG-M/versican at fetal day 18 and at neonatal day 1 in lungs of GR(hypo) mice, and at neonatal day 1 in lungs of GR(null) mice. By contrast, lung MK and PG-M/versican were downregulated in these mice at fetal day 16.5. In situ hybridization studies showed a dramatic decrease in MK and PG-M/versican RNA between days 16.5 and 17.5 in GR(WT) but not in GR(null) mice. Continued diffuse and robust expression of MK protein was observed in GR(null) mice at neonatal day 1. These findings suggest that MK may contribute to the dysmature lung phenotype in GR-deficient mice. Exposure of cultured day 21 fetal rat lung cells to GC downregulated MK, whereas RA enhanced MK expression. Our findings demonstrate the coincident modulation of expression of MK at the same developmental time point by both GC and RA, providing a potential mechanism for the integration of GC and RA effects on fetal lung development.

Midkine binds to anaplastic lymphoma kinase (ALK) and acts as a growth factor for different cell types.

Midkine (MK) is a developmentally regulated, secreted growth factor homologous to pleiotrophin (PTN). To investigate the potential role of MK in tumor growth, we expressed MK in human SW-13 cells and studied receptor binding, signal transduction, and activity of MK. The MK protein stimulates soft agar colony formation in vitro and tumor growth of SW-13 cells in athymic nude mice, as well as proliferation of human endothelial cells from brain microvasculature and umbilical vein (HUVEC) in the low ng/ml range. MK binds to anaplastic lymphoma kinase (ALK), the receptor for PTN, with an apparent K(d) of 170 pm in intact cells, and this receptor binding of MK is competed by PTN with an apparent K(d) of approximately 20 pm. Monoclonal antibodies raised against the extracellular ligand-binding domain of ALK inhibit ALK receptor binding of MK as well as MK-stimulated colony formation of SW-13 cells. Furthermore, MK stimulates ALK phosphorylation in WI-38 human fibroblasts and activates PI3-kinase and MAP kinase signal transduction in WI-38, HUVEC, neuroblastoma (SH SY-5Y) and glioblastoma (U87MG) cells that express the ALK protein. We conclude that MK can act as a growth, survival, and angiogenic factor during tumorigenesis and signals through the ALK receptor.

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.

A role of midkine in the development of the neuromuscular junction.

Midkine (MK) is a member of a family of developmentally regulated neurotrophic and heparin-binding growth factors. It is expressed during the midgestation period in a retinoid-acid dependent manner during embryogenesis in the mouse. In vitro, it promotes neurite outgrowth from spinal cord neurons and cell migration. It expression is strongest in the central nervous system, thus suggesting a function for this protein in neural development. In this study, the role of MK in synaptogenesis was examined in the Xenopus system. A Xenopus MK cDNA was cloned from an embryonic library encompassing neurulation and synaptogenesis stages. By Northern blot analysis, MK mRNA was detected from the onset of neurulation and throughout the stages of synaptogenesis in the Xenopus embryo. This suggests that MK is also an important growth regulator in Xenopus embryogenesis. To study the function of MK in the development of the neuromuscular junction (NMJ), fusion proteins were made and their ability to induce the formation of acetylcholine receptor (AChR) clusters in cultured muscle cells was studied. Beads coated with MK strongly induce AChR clustering. When nerve-muscle cocultures were labeled with antibodies made against the MK fusion protein, MK immunoreactivity was detected at the NMJ. Unlike heparin-binding growth-associated molecule (HB-GAM), another member of this growth factor family, MK expression cannot be detected in the muscle but is present in spinal cord neurites. Consistent with these in vitro data is the observation that MK mRNA is only localized in the central nervous system but the protein is deposited at the intersomitic junction where the NMJ is located in vivo. Exogenously applied MK does bind to the heparan sulfate proteoglycan on the surface of Xenopus muscle cells. Agrin, a heparan-sulfate proteoglycan that induces the formation of AChR clusters in cultured muscle cells, binds strongly to MK. Bath application of MK in conjunction with agrin results in a change in the pattern of AChR clustering induced by agrin alone. These data suggest that MK is a neuron-derived factor that participates in the signal transduction process during NMJ development.

Developmental and differential regulations in gene expression of Xenopus pleiotrophic factors-alpha and -beta.

Using conserved nucleotide sequences in mammalian osteoblast specific factor-1 (OSF-1) coding regions, we isolated two kinds of cDNA clones from the Xenopus brain library. The encoded proteins, named Xenopus pleiotrophic factors (X-PTFs)-alpha and -beta, were 65 and 87% homologous to human midkine and OSF-1, respectively. In the adult frog, X-ptf-alpha was expressed in the ovary, brain, eye, bone, heart and lung, whereas X-ptf-beta was expressed in the brain, eye and bone. By in situ hybridization of the tailbud embryo, X-ptf-alpha mRNA was detected rather broadly in the head/tail regions including the central nervous system (CNS), whereas X-ptf-beta mRNA was restricted to the CNS, particularly in the hind-brain. During embryogenesis, X-ptf-alpha mRNA was detected in the one-cell stage embryo, whereas only zygotic expression was observed in X-ptf-beta. X-ptf-beta mRNAs contained approximately 79 bp tandem repeats in the 3'-untranslated region, complementary to those found in retinoic acid cellular receptor mRNA and in the sense strand of short interspersed repeat transcripts in X. laevis.

The midkine (MK) family of growth/differentiation factors: structure of an MK-related sequence in a pseudogene and evolutionary relationships among members of the MK family.

Midkine (MK) is a novel heparin-binding growth/differentiation factor coded by a retinoic acid-responsive gene. MK cDNA probe reacts with two bands, a 4 kb one and a 3 kb one, upon Southern blot analysis of Hin dIII fragments of mouse genomic DNA: the midkine gene (Mdk) is on the 4 kb fragment. Sequence analysis of the 3 kb fragment revealed that it has an Mdk-related sequence (Mdk-rs) highly homologous to MK cDNA, three mouse Aluequivalent repeats and seven A+T-rich segments. The Mdk-rs carried an inserted microsatellite DNA, is flanked by imperfect direct repeats observed in many retroposons, and lacks introns. Interspecific hybrid analysis revealed that Mdk-rs is located on chromosome 11, while Mdk is known to be on chromosome 2. The evolutional velocity of Mdk-rs was calculated to be 11 times higher than that of mouse Mdk. These features suggest that Mdk-rs is a processed pseudogene generated in the mouse genome. The 3 kb fragment with Mdk-rs, which is rich in inserted DNA sequences probably due to the presence of A+T-rich segments, may be a hot spot for amplification and evolution of genomic DNA. Mdk-rs was estimated to have been generated about 19.1 million years ago. A chicken protein retinoic acid-induced, heparin-binding protein (RIHB), is highly homologous to MK, and its divergence from human MK was estimated to have occurred about 250 million years ago, suggesting that RIHB is the chicken homology of MK. Thus, so far there are only two established protein members, MK and heparin-binding, growth-associated molecule (HB-GAM)/pleiotrophin (PTN) in the MK family.