Midkine Induces Tumor Cell Proliferation and Binds to a High Affinity Signaling Receptor Associated with JAK Tyrosine Kinases*

Edward A. RatovitskiDagger §, Paul T. Kotzbauer, Jeffrey Milbrandt, Charles J. LowensteinDagger , and Christopher R. Burrowpar

From the Dagger  Departments of Pathology and Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, the  Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110, and the par  Department of Medicine, Division of Nephrology, Mount Sinai School of Medicine, New York, New York 10029

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The G401 cell line derived from a rhabdoid tumor of the kidney secretes the heparin-binding growth factors midkine and pleiotrophin. Both proteins act as mitogens for diverse cells, but only midkine serves as an autocrine mitogen for G401 tumor cells. We show that midkine specifically binds a protein or complex of molecular mass greater than 200 kDa with high affinity (Kd = 0.07 ± 0.01 nM). Midkine, but not pleiotrophin, stimulates tyrosine phosphorylation of several cellular proteins with molecular mass of 100, 130, and 200+ kDa. Upon midkine binding, the midkine-receptor complex associates with the Janus tyrosine kinases, JAK1 and JAK2. MK stimulates tyrosine phosphorylation of JAK1, JAK2, and STAT1alpha . Our initial characterization of the midkine receptor suggests that midkine autocrine stimulation of tumor cell proliferation is mediated by a cell-surface receptor which in turn might activate the JAK/STAT pathway.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Mesenchymal-epithelial interactions during development involve reciprocal inductive stimuli that are critically important in the regulation of cellular proliferation, differentiation, and tissue morphogenesis. The elucidation of the molecular basis for these events is a major goal relevant to advancing our understanding of basic developmental processes. Kidney organogenesis depends upon a set of molecular signaling events that form the basis for the induction of nephron formation in the metanephric mesenchyme by the ureteric bud (1, 2). Midkine (MK),1 a recently identified growth factor, may play an important regulatory role at sites of mesenchymal-epithelial interaction during tooth development and during organogenesis of the kidney (3, 4).

MK and pleiotrophin (PTN) are developmentally regulated heparin-binding proteins that regulate cell growth, survival, and differentiation (5-8). Both MK and PTN are products of retinoic acid-responsive genes (8). Expression of PTN is also regulated by platelet-derived growth factor (9). Mature MK and PTN are basic, cysteine-rich polypeptides of 123 and 136 amino acids, respectively, with approximately 50% homology to each other (5, 10, 11). MK and PTN are conserved between mammalian species, and both are distinct from other heparin binding growth factors such as basic and acidic fibroblast growth factors (6, 10, 12).

MK is mitogenic to a number of cell lines and induces neurite outgrowth of embryonic brain cells, PC 12 cells, and dorsal root ganglion cells (12-15). MK also promotes survival of retinal cells in vivo, astrocytes and mesencephalic neurons in culture (17-16). MK stimulates differentiation of P19 embryonic carcinoma cells into nerve cells, and this stimulation is inhibited by anti-MK antibodies (17). In addition, MK enhances plasminogen activator and plasmin activity in a dose- and time-dependent manner, implying a role for MK in tissue repair and angiogenesis (18-20).

MK is expressed in a characteristic pattern in the developing embryo and may play a role in neurogenesis, kidney organogenesis, and in mesodermal-epithelial interactions (3, 4, 19-22). MK is absent in the mouse embryo until day 5 of gestation. MK expression then increases until it is widely expressed at 7-9 days of gestation; its expression then decreases. In the mid-gestation period (days 11-13) MK is expressed in the brain and kidney and in epithelial cells of the small intestine, pancreas, lung, and stomach. At day 15 of gestation MK expression is limited to the kidney (22-26). The expression of MK decreases in later embryogenesis but then increases again postnatally in certain organs and tissues. In adult mice and humans, MK is expressed in the kidney, testis, stomach, and small intestine (24-28).

MK is expressed in various human cancers, including neuroblastomas, hepatocellular carcinomas, gastric, colorectal, pancreatic, esophageal, lung, and breast carcinomas, and kidney cancers (27-31). MK is thought to be an autocrine tumor growth factor, since the G401 cell line derived from a rhabdoid kidney tumor expresses MK and since anti-MK antibodies partially inhibit the in vitro growth of these cells (27, 31). However, definitive evidence for a direct role of MK as an autocrine mitogenic factor in tumor cells has not yet been established.

Tyrosine phosphorylation of growth factor receptors upon ligand binding is an important signaling mechanism for cellular activation, proliferation, and differentiation (32). MK and PTN might exert their functions through interaction with specific cell-surface signaling receptors and induction of tyrosine phosphorylation of cellular proteins. Previous studies have shown that PTN induces tyrosine phosphorylation of cellular proteins (33). However, the cellular signaling receptors for MK and PTN have not yet been identified and characterized. Study of the molecular basis for signal transduction pathways of MK and PTN would further enhance the understanding of their roles in development and cancer. We report that MK, but not PTN, stimulates tumor (G401) cell proliferation in a dose-dependent and time-dependent manner. MK specifically binds to a high affinity cell-surface receptor, which is tyrosine-phosphorylated after MK binding and stably associates with the Janus non-receptor tyrosine kinases JAK1 and JAK2. MK also stimulates tyrosine phosphorylation of JAK1, JAK2, and STAT1alpha . Our studies support the hypothesis that this newly identified MK receptor is the signal transduction receptor that mediates the MK-dependent autocrine stimulation of this tumor cell line in vitro.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Cultures, Media, and Antibodies-- Human pediatric rhabdoid tumor kidney-derived G401 cell line (CRL 1441), normal rat kidney cells (CRL 6509), and NIH 3T3 mouse embryo fibroblasts (CRL 1658) were obtained from the American Tissue and Cell Culture Association. G401 cells (passages 15-45) were cultured in McCoy's 5A medium (Sigma) containing heat-inactivated 1% fetal bovine serum (Lot A2934J; Gemini, CA), penicillin (50 units/ml), and streptomycin (50 µg/ml). Normal rat kidney cells (passages 10-17) and NIH 3T3 fibroblasts (passages 6-14) were cultured in bicarbonate-buffered Dulbecco's modified Eagle's media with 5% fetal calf serum. Human adult kidneys were obtained under sterile conditions by the National Disease Research Interchange (Philadelphia, PA) within 24 h of harvest in Collins solution (98 mM Na+, 107 mM K+, 14 mM Cl-, 9.3 mM HCO3, 93 mM PO4, 182 mM glucose, pH 7.0) at 4 °C. Monoclonal antibody to phosphotyrosine (PT-66) was purchased from Sigma. Polyclonal antibodies to focal adhesion kinase, JAK1, JAK2, STAT2, gp130, and antibodies to MK and to PTN (without cross-reactivity) and monoclonal antibodies to STAT1alpha were purchased from Santa Cruz Biotechnology. Streptavidin-Sepharose was purchased from Pierce.

Expression of MK and PTN in COS1 Cells-- Total cellular RNA was extracted from human adult kidney (34). cDNA was synthesized from total RNA (5-10 µg) in 50 µl of 20 mM Tris-HCl, pH 8.4, 50 mM KCl, 2.5 mM MgCl2, and 1 mg/ml nuclease-free bovine serum albumin with Moloney murine leukemia virus reverse transcriptase (200 units) in the presence of RNasin (1 unit/µl), 1 mM each dNTP, 100 pmol of random hexamer primers (Boehringer Mannheim). For first strand synthesis, the mixture was incubated 10 min at 23 °C and 60 min at 42 °C, followed by denaturation at 95 °C for 10 min, and then quick-chilled on ice. One-µl aliquot of the 10-fold diluted first strand cDNA solution was used as the template in a 100-µl polymerase chain reaction with Taq DNA polymerase (Boehringer Mannheim) performed as follows: denaturation at 92 °C for 4 min; 35 cycles of 92 °C for 1 min, 60 °C for 1 min, 72 °C for 1 min; with a final extension 72 °C for 10 min; MK, sense: 5'-TCAGATCTGATGCAGCACCGAGGCTTCG-3' and antisense: 5'-TACAAGCTTCTGGTGGGTCACATCTCGG-3'; and PTN, sense: 5'-TCAGATCTAATGCAGGCTCAACAGTACC-3' and antisense: 5'-TACAAGCTTTTTAATCCAGCATCTTCTCCTG-3' with sites for BglII and XbaI for cloning in pCB6 vector. Polymerase chain reaction fragments were first subcloned into pCR 2.1 vector (InVitrogen). The resulting constructs were digested with BglII and XbaI; fragments corresponding to MK or PTN were isolated from a gel and inserted in the pCB6 (InVitrogen). COS1 cells were transiently transfected with pCB6-MK, pCB6-PTN, and pCB6 alone (control) in the following manner. COS1 cell cultures were grown in RPMI 1640 with 5% fetal calf serum; 5-10 µg of plasmid DNA was introduced into cells using DEAE-dextran (35). After 60 h, 200 ml of conditioned media (8.8-9.5 µg/ml total protein) was harvested from COS1 cells transfected with pCB6-MK or pCB6-PTN or pCB6 alone, and the conditioned media were concentrated 50-fold on an ultrafiltration stirrer device (YM-5, Amicon) followed by the purification of heparin-binding proteins by heparin affinity chromatography (Poros HE-1, PerSeptive Biosystems, Cambridge, MA) equilibrated with 0.2 M NaCl in 20 mM Tris-HCl buffer, pH 7.5. Columns were washed with 20 ml of 0.5 M NaCl in the same buffer, and proteins were eluted with 5 ml of the same buffer containing 1.5 M NaCl. The yield of MK or PTN secreted by transfected COS1 cells purified on heparin column was 0.3-0.4 µg/ml starting conditioned media. Purity of MK and PTN used in the experiments was near 90-95% based on SDS-PAGE analysis of iodinated MK and PTN.

Proliferation Assay on Different Cell Lines-- G401, normal rat kidney, and NIH 3T3 cells (2,000 cells/well) were plated onto Corning 96-well plates and were cultured at 37 °C in the presence of MK or PTN purified from conditioned media of transfected COS1 cells. (MK and PTN purified to near-homogeneity from media collected from either mouse MK or rat PTN stably transfected Chinese hamster ovary cell lines were also used in pilot proliferation and binding experiments, and produced equivalent results.) NIH 3T3 and normal rat kidney cells were treated for 24 h with 0-5 nM MK or PTN or control conditioned media in McCoy's 5A medium, 5% fetal calf serum, 10 µg/ml transferrin, 10 mM HEPES, pH 7.4. For dose-response experiments G401 cells were treated for 24 h with 0-5 nM MK or PTN or control conditioned media in serum-free McCoy's 5A medium, 10 µg/ml transferrin, 10 mM HEPES, pH 7.4. For time course experiments G401 cells were treated with 1 nM MK or 1 nM PTN or control for 0-120 h in McCoy's media, 1% fetal bovine serum, 10 µg/ml transferrin, 10 mM HEPES, pH 7.4. In both cases, a CellTiter 96TM AQueous non-radioactive colorimetric proliferative assay was used (Promega). 20 µl of combined MTS/PMS (20:1) reagent was added to 96-well cell-free plates and plates containing cells in the presence of necessary supplements, and assay was performed for 4 h at 37 °C. A cell-free duplicate plate with identical protein fractions and media was assayed as a control. Both cell-free plates and cell containing plates were analyzed on plate reader Labsystems Multiscan MCC/340 at A490-A690. Then data obtained from cell-free plate were subtracted from experimental results obtained from plate containing cells, individually. All subtracted data were in quadruplicate, and average results with error bars were plotted as graphs using Cricket-Graph computer program.

Iodination and Ligand-Receptor Cross-linking-- Labeling of ligands (MK and PTN purified from transfected COS1 cells) was performed by the incubation of proteins (50 µg) with Na125I (1.3-1.5 mCi, 16.8 mCi/mg, Amersham Corp.) during 1 min at 4 °C in the presence of chloramine T (1 mg/ml) and PBS, then stopping the reaction with sodium metabisulfite, followed by desalting chromatography on a PD-10 column (Pharmacia Biotech Inc.) in PBS (36). Specific activity of iodinated proteins was 1.1-1.3 × 105 cpm/µg. 125I-MK and 125I-PTN were stored at 4 °C and used within 2 weeks of preparation. For detection of cell-surface receptor, 0.5 nM 125I-MK or 0.5 nM 125I-PTN was incubated with intact G401 cells (2-5 × 107 cells) in 1 ml of Ca2+/Mg2+-free PBS, pH 7.4, for 60 min at 4 °C. Cells were washed 2-3 times with PBS containing 0.2 mM Na3VO4, 1 mM EDTA, and protease inhibitors, and ligand-receptor complexes were stabilized chemically with 0.5 mM disuccinimidyl suberate (DSS, Sigma), the homo-bifunctional cross-linking agent, for 20 min at 4 °C. The reaction was stopped with 100 mM Tris-HCl, pH 7.0, and 150 mM NaCl to inactivate DSS (36). Ligand-receptor binding experiments were done in the absence or presence of a 100-fold excess of unlabeled MK, PTN, or control protein fraction. Cell pellets were treated with liquid nitrogen and extracted with 1 ml/107 cells of lysis buffer A (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 0.1% of Nonidet P-40, 1 mM PMSF, 4 µg/ml aprotinin, 1 µg/ml pepstatin A, 1 µg/ml leupeptin, 1 mM Na3VO4, 50 mM NaF) for 30 min on ice. Postnuclear supernatants were incubated with 1.0% Nonidet P-40 for the next 30 min and cleared by centrifugation for 15 min at 15,000 × g. Supernatants 40-60 µl were boiled with SDS and beta -mercaptoethanol and resolved by denaturing 6% SDS-PAGE; dried gels were examined by autoradiography (36).

Saturation and Scatchard Analysis of the MK Cell-surface Receptor-- Confluent intact G401 cells (3 × 107) were incubated for 1 h at 4 °C with 125I-MK (0.005-1.0 nM), cross-linked, and treated as above; the samples were resolved by denaturing 6% SDS-PAGE; dried gels were autoradiographed and sliced into 1-cm2 pieces in regions of radioactive bands and background. The slices were counted in a Beckman 5500 gamma counter for 1 min. Scatchard analysis of saturation isotherms were performed using a linear least-squares regression LIGAND program (37). Scatchard analysis of MK ligand-receptor binding was also performed on intact G401 cells in Eppendorf tubes (without subsequent SDS-PAGE and excision of the radioactive receptor band). G401 cells (5 × 107) were incubated in 100 µl of PBS for 60 min at 4 °C with increasing amounts (0.01 to 1 nM) of 125I-MK cross-linked for 20 min at 4 °C with DSS (as above), spun down, and washed 5 times with 1 ml of PBS, and the resultant pellets were dried and counted in a gamma counter for 1 min. The binding experiments were repeated 4 times, normalized for cell number, and averaged.

Tyrosine Phosphorylation Analysis-- Confluent G401 cells (2 × 107) were incubated overnight in serum-free McCoy's 5A medium, washed with the same media containing 1 mM Na3VO4 and 50 mM NaF, and treated with MK or PTN or control proteins at 37 °C. Stimulation was stopped with ice-cold PBS containing 1 mM Na3VO4 and 50 mM NaF. Cells were scraped off the flasks with 1 ml of ice-cold lysis buffer (50 mM Tris-HCl, pH 7.5, 2 mM EDTA, 0.1% Nonidet P-40 containing 1 mM Na3VO4, 50 mM NaF, and protease inhibitors mixture, as above). The postnuclear supernatants were treated with 1.0% Nonidet P-40 on ice for 30 min and spun at 15,000 × g for 15 min (38). Supernatants containing 20-100 µg per lane were boiled with 1% SDS and beta -mercaptoethanol, resolved by SDS-PAGE, and transferred onto Immobilon P membrane sheets. Sheets were blocked with 10% non-fat milk, PBS, 0.05% Tween 20, probed with antibody to phosphotyrosine (PT-66) (1: 5,000), followed by washing in PBS, 0.05% Tween 20. Then sheets were incubated with goat anti-mouse antibody conjugated with horseradish peroxidase (1:10,000), washed with PBS, 0.05% Tween 20 and visualized with ECL (Amersham Corp.). Quantitative densitometry of phosphotyrosine signals was performed as follows; slides of films with immunoblot data were made, scanned, and plotted as diagrams using the Macros (NIH Image 1.58b 39f) computer program.

Streptavidin Precipitation of Biotinylated Ligand-Receptor Complexes and Western Blot Analysis-- PTN and MK were prepared from conditioned media of transfected COS1 cells as above. For biotinylation, 10 µg of MK or 10 µg of PTN or 10 µg of control proteins were incubated with 1 mg/ml sulfosuccinimidyl-2-(biotinamido) ethyl-1,3-dithiopropionate (Immunopure Sulfo-NHS-SS-Biotin, Pierce) for 10 min at 4 °C in 0.1 M HEPES buffer, pH 7.5, with gentle shaking (36). The reaction was stopped by 0.01 M glycine and diafiltrated against 10 mM PBS through 3K Centriprep concentrator (Amicon).

Intact G401 cells (5-8 × 107 cells) were cross-linked with 1 nM biotinylated MK or 1 nM biotinylated PTN or biotinylated control proteins for 30 min at 4 °C in serum-free McCoy's 5A media (1 ml/107 cells). Cells were centrifuged and resuspended in 1 ml/107 cells of ice-cold PBS with 0.5 mM DSS for 20 min at 4 °C, then washed with ice-cold PBS. The cells were solubilized (1 ml/107 cells) in 100 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 0.5% Nonidet P-40, 0.5% Brij-50, 1 mM PMSF, 4 µg/ml aprotinin, 1 µg/ml pepstatin A, 1 µg/ml leupeptin, 1 mM Na3VO4, 50 mM NaF for 30 min on ice, and supernatants were recovered by centrifugation at 15,000 × g for 15 min. Then 500-µl supernatants were pre-cleared with 10 µl of normal rabbit serum for 30 min and incubated with 50 µl of protein A-Sepharose 4B (Pharmacia) for 30 min (36). Supernatants were recovered by centrifugation and then used for subsequent precipitation. 40 µl of a 50% suspension of streptavidin-Sepharose (Pierce) was added to 500 µl of supernatant and incubated for 4 h at 4 °C with rotation, washed three times with 1 ml of cold 20 mM Tris-HCl, pH 7.4, 125 mM NaCl, 1 mM Na3VO4, 50 mM NaF, 1 mM EDTA, 0.5% Nonidet P-40, 0.2 mM PMSF (34). Samples were boiled with SDS and beta -mercaptoethanol, resolved by denaturing 6% SDS-PAGE, and transferred onto Immobilon P sheets. Blocked sheets were incubated for 2 h at room temperature with antibodies directed against phosphotyrosine (PT-66) (1:5,000), MK, JAK1, JAK2, focal adhesion kinase, gp130 (each in dilution 1:1,000), washed, incubated for 1 h with goat anti-mouse antibodies (1:10,000) or protein A (1:10,000) coupled to horseradish peroxidase (Sigma), and visualized with ECL.

Immunoprecipitation-- For immunoprecipitation experiments, G401 cells were starved for 24 h in serum-free media, incubated for 20 min with MK (0.5 nM), PTN (0.5 nM), or control (concentrated conditioned media from COS1 cells transfected with pCB6 vector alone). Cell extracts were prepared as above, including pre-clearing with protein A-agarose (Sigma) beads or goat anti-mouse immunoglobulin-bound agarose beads. Supernatants were precipitated with primary antibodies directed to JAK1, JAK2, STAT2, and STAT1alpha for 16-18 h at 4 °C and then with secondary antibodies (protein A or goat anti-mouse immunoglobulin) coupled to Sepharose/agarose beads. Antigen-antibody complexes were washed three times with 0.05% PBS, Tween 20, boiled in sample buffer containing SDS and beta -mercaptoethanol, and separated by SDS-PAGE followed by transfer onto Immobilon P membrane, and probed with antibody to phosphotyrosine (PT-66) and antibody to JAK1, JAK2, STAT2, or STAT1alpha . Immunoreactive bands were visualized by ECL as above.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

MK, but Not PTN, Stimulates G401 Cell Proliferation-- To analyze the mitogenic effect of MK and PTN on G401 cells, we cloned human MK and PTN using reverse transcription-polymerase chain reactions with human kidney RNA as a template. The MK (522 base pairs) and PTN (542 base pairs) cDNA fragments were inserted into the pCB6 vector, and COS1 cells were transfected with the pCB6-MK or pCB6-PTN expression constructs. COS1 cells transfected with pCB6-MK secrete MK, and COS1 cells transfected with pCB6-PTN secrete PTN, in contrast to COS1 cells transfected with pCB6 vector alone (Fig. 1A). MK, PTN, and control proteins were prepared from concentrated conditioned media of COS1 cells transfected with pCB6-MK, pCB6-PTN, or pCB6 alone, respectively, and purified by heparin affinity column chromatography (Fig. 1B).


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Fig. 1.   Expression of human MK and PTN. Western blot assay of media from G401 cells (lanes 1 and 2) and from COS-1 cells transfected with pCB6-PTN (lane 3) or pCB6-MK (lane 4) or pCB6 vector alone (lane 5), using antibody to PTN (lanes 1, 3, and 5) or to MK (lanes 2, 4, and 5). B, electrophoresis of 125I-PTN (lane 1) and 125I-MK (lane 2) purified on heparin affinity chromatography column were separated by 15% SDS-PAGE.

We then studied the mitogenic properties of MK and PTN in the pediatric tumor kidney-derived G401 cell line, using the non-radioactive colorimetric MTS/PMS proliferation assay. Since NIH 3T3 fibroblasts proliferate in response to both MK and PTN, and normal rat kidney cells proliferate in response to PTN only (6, 13, 39), we used these cell lines as positive controls in our experiments (Fig. 2, A and B). MK stimulates G401 cell proliferation in a time-dependent and dose-dependent fashion (Fig. 3, A and B); PTN and control protein have no effect. These data (Fig. 1 and Fig. 3, A and B) demonstrate that MK might regulate G401 cell proliferation in an autocrine manner. Titration of MK from 0.05 to 5 nM indicates that half-maximal stimulation of G401 cell proliferation occurs at approximately 0.3-0.5 nM.


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Fig. 2.   Mitogenic activity of MK and PTN on NIH 3T3 and NRK cells. Cells (2000 cells/well) were stimulated with 0.05-5 nM MK or PTN for 24 h prior to addition of MTS/PMS reagent. A, dose dependence of MK and PTN on NIH 3T3. B, dose dependence of MK and PTN on NRK cells. Units of mitogenic stimulation defined as corrected A490-A690 obtained from MTS/PMS assay performed for 4 h after subtraction of data from cell-free plate (see "Experimental Procedures"). Data shown are means ± S.D. (n = 4) from each experiment and are representative of three similar experiments. Some error bars are too small to be visible within the symbols.


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Fig. 3.   Mitogenic activity of MK and PTN on G401 cells. A, dose dependence of MK and PTN on G401 cells (0.05-5 nM MK or PTN for 24 h. B, time dependence of MK and PTN on G401 cells (1 nM MK or PTN, 0-120 h). Units of mitogenic stimulation defined as corrected A490-690 obtained from MTS/PMS assay performed for 4 h after subtraction of data from cell-free plate (see "Experimental Procedures"). Data shown are means ± S.D. (n = 4) from each experiment and are representative of three similar experiments. Some error bars are too small to be visible within the symbols.

MK, but Not PTN, Binds a G401 Cell-surface Receptor-- To test our hypothesis that MK mitogenic activity is mediated by its binding to a high affinity cell-surface receptor, we performed chemical cross-linking assays. Intact G401 cells were incubated with 125I-MK or 125I-PTN in the absence or presence of a 100-fold excess unlabeled MK or PTN, followed by cross-linking with DSS. Cell lysates were resolved by denaturing SDS-PAGE and examined by autoradiography. MK but not PTN is cross-linked to a protein (Fig. 4); the total mass of the MK-protein complex is between 200 and 250 kDa (200+ kDa). A 100-fold excess of unlabeled MK prevents 125I-MK from binding to this molecule, but the same amount of the unlabeled PTN does not (Fig. 4). Taken together, the data of cross-linking and cross-competition experiments suggest that G401 cells have a specific cell-surface receptor for MK, but not for PTN.


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Fig. 4.   Interaction of MK and PTN with G401 MK receptor. Intact G401 cells (2-5 × 107) were incubated with 0.5 nM 125I-PTN or 0.5 nM 125I-MK in PBS for 60 min at 4 °C and cross-linked. Cross-linking was performed with 0.5 nM 125I-PTN (lane 1), 0.5 nM 125I-MK (lane 2), or in the presence of the 100 × excess unlabeled MK (lane 3), unlabeled G401 conditioned media (lane 4), unlabeled PTN (lane 5), or unlabeled control (lane 6). Cells were extracted as described (see "Experimental Procedures") and resolved by 6% SDS-PAGE; dried gels were examined by autoradiography. Arrows indicate positions of cross-linked complexes.

To quantitate the binding of MK to its receptor, we cross-linked intact G401 cells with increasing amounts of 125I-MK and resolved ligand-receptor complexes by SDS-PAGE. Saturation and Scatchard analysis showed that G401 cells displayed receptor molecules for MK with high ligand binding affinity (200+ kDa; average data from four experiments; Kd = 0.072 nM, 7,700 receptors/cell) (Fig. 5, A and B). Similar data (average data from four experiments; Kd = 0.056 nM, 6,600 receptors/cell) were obtained for the G401 cell-surface MK receptor using a complementary approach of cross-linking of 125I-MK with intact G401 cells without subsequent lysis and SDS-PAGE (see "Experimental Procedures") (Fig. 6, A and B).


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Fig. 5.   Quantitative analysis of MK·MK receptor complex formation (gel assay). A, saturation plot analysis. B, Scatchard plot analysis. Intact G401 cells were cross-linked with increasing amount of 125I-MK (0.005-1.0 nM) at 4 °C for 1 h, and then cell extracts were resolved by SDS-PAGE followed by autoradiography. 1 cm2 gel pieces were counted in a Beckman gamma counter. Radioactive data were plotted using LIGAND computer program (see "Experimental Procedures").


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Fig. 6.   Quantitative analysis of MK·MK receptor complex formation (tube assay). A, saturation plot analysis. B, Scatchard plot analysis. Intact G401 cells were cross-linked with increasing amounts of 125I-MK (0.01-1.0 nM) at 4 °C for 1 h in Eppendorf test tubes, and then cells were washed extensively with PBS and counted in a gamma counter. Radioactive data were plotted using LIGAND computer program (see "Experimental Procedures").

MK Induces Tyrosine Phosphorylation of Its Cell-surface Receptor That Is Associated with JAK1 and JAK2 Tyrosine Kinases-- We next analyzed the ligand-induced tyrosine phosphorylation pattern of G401 cell proteins using immunoblotting with anti-phosphotyrosine antibodies (see "Experimental Procedures"). Stimulation with MK, but not PTN or control proteins, resulted in tyrosine phosphorylation of several cellular proteins, including 100-, 130-, and 200+-kDa proteins (Fig. 7A). Immunoblotting with antibody to phosphotyrosine shows that the MK-induced tyrosine phosphorylation was time-dependent (Fig. 7B). Immunoblotting and quantitative densitometry show that the MK-induced tyrosine phosphorylation is dose-dependent (Fig. 7C and Fig. 8).


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Fig. 7.   MK-induced tyrosine phosphorylation of G401 cell proteins. G401 cells were plated on T-25 flasks and serum-starved for 18-20 h. The cells were stimulated with 0.33 nM MK or 0.25 nM PTN or 5 ng/ml control (Con) for 15 min (A); 0.33 nM MK for various times (B); and increasing doses of MK (0.002-0.8 nM) for 15 min (C). G401 cell extracts were probed with antibody to phosphotyrosine (PT-66) (see "Experimental Procedures"). Samples were normalized by protein amount loaded on the lane. Arrows indicate positions of tyrosine-phosphorylated proteins upon MK stimulation.


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Fig. 8.   Quantitative analysis of tyrosine phosphorylation. The immunoblot data from Fig. 7 were scanned, and the intensity of the bands were quantified.

To determine the identity of proteins binding MK and tyrosine phosphorylated upon MK treatment, we used streptavidin-Sepharose to precipitate complexes of biotinylated MK and MK-binding proteins. Intact G401 cells (1 ml/107 cells) were incubated with 1.0 nM biotinylated-MK or 1.0 nM biotinylated-PTN or biotinylated-control proteins for 15 min at 4 °C, followed by DSS cross-linking. Cells were lysed as above, and cross-linked complexes were precipitated with streptavidin-Sepharose beads followed by blotting with antibodies to phosphotyrosine (lanes 1-3), MK (lanes 4-6), JAK1 (lanes 7-9), or JAK2 (lanes 10-12) (Fig. 9). The antibody to phosphotyrosine recognizes three streptavidin-precipitated, biotinylated MK·MK receptor complex components of 100, 130, and 200+ kDa. These data also demonstrate that MK binds to the 200+-kDa receptor that becomes tyrosine-phosphorylated (Fig. 9, lanes 1 and 4).


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Fig. 9.   Tyrosine phosphorylation of MK receptor and its association with JAK kinases. Intact G401 cells were cross-linked at 4 °C for 15 min with 1.0 nM biotinylated MK (lanes 1, 4, 7, and 10), 1.0 nM biotinylated PTN (lanes 2, 5, 8, and 11), or biotinylated control (lanes 3, 6, 9, and 12). Cell extracts were pre-cleared with protein A-Sepharose. Biotinylated ligand-receptor complexes were precipitated with 40 µl of streptavidin-Sepharose, washed, resolved by 6% SDS-PAGE, and blotted in parallel experiments with antibodies directed against phosphotyrosine (P(Tyr)) (lanes 1-3), MK (lanes 4-6), JAK1 (lanes 7-9), and JAK2 (lanes 10-12). IP, immunoprecipitated.

Moreover, antibodies to JAK1 and JAK2 detected JAK1 (126 kDa) and JAK2 (130 kDa) tyrosine kinases associated with the protein complex bound to biotinylated MK (but not to biotinylated PTN or biotinylated control proteins) (Fig. 9, lanes 7 and 10). Probing the same blots with antibody to JAK3 or anti-gp130 or focal adhesion kinase failed to detect any proteins associated with MK·MK·receptor complex (data not shown).

By comparing data presented on lanes 1, 7, and 10 (Fig. 9), we show that the apparent molecular mass of JAK1 and JAK2 are identical to the 130-kDa phosphoproteins that co-precipitate with biotinylated MK·MK receptor complexes. Taken together, these findings suggest that MK but not PTN stimulates G401 cell proliferation by binding to a high affinity cell-surface receptor that undergoes tyrosine phosphorylation and that appears to activate and recruit the downstream signaling molecules JAK1 and JAK2.

MK Activates the JAK/STAT Pathway-- We next analyzed the phosphorylation of various members of the JAK and the STAT family. G401 cells were stimulated with MK, PTN, and control as above for 20 min. Cell extracts were precipitated separately with antibodies against JAK1, JAK2, STAT1alpha , or STAT2. Precipitated complexes were electrophoresed, transferred to a membrane, and then hybridized with antibodies against JAK1, JAK2, STAT1alpha , or STAT2, as well as antibodies against phosphotyrosine as above. Stimulation of G401 cells with MK, but not PTN or control proteins, resulted in tyrosine phosphorylation of JAK1, JAK2, and STAT1alpha (Fig. 10) but not STAT 2 (not shown).


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Fig. 10.   MK induces tyrosine phosphorylation of JAK and STAT members. Intact G401 cells were stimulated with 0.5 nM MK (lanes 1 and 4), 0.5 nM PTN (lanes 2 and 5), or control (lanes 3 and 6) for 20 min. Cell extracts were pre-cleared with protein A-Sepharose or goat anti-mouse immunoglobulin coupled to agarose. Proteins were precipitated with 5 µl of antibody, electrophoresed, transferred to a membrane, and probed with antibodies. A, immunoprecipitation (IP) with antibody to JAK1, and immunoblot with antibody to JAK1 (left) and to phosphotyrosine (right). B, immunoprecipitation (IP) with antibody to JAK2, and immunoblot with antibody to JAK2 (left) and to phosphotyrosine (right). C, immunoprecipitation (IP) with antibody to STAT1alpha , and immunoblot with antibody to STAT1alpha (left) and to phosphotyrosine (right).

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

The pediatric tumor kidney-derived G401 cell line was chosen to elucidate molecular mechanisms underlying MK signal transduction because these cells produce high amounts of both MK and these cells also proliferate in response to MK. Garvin et al. (40) suggest the G401 cell line is of rhabdoid tumor, rather than Wilms' tumor origin. Regardless of its origin, the G401 cell line is a useful tool to study MK signal transduction.

Our results demonstrate that although G401 tumor cells secrete both MK and PTN, MK but not PTN stimulates G401 tumor cell proliferation in a time-dependent and dose-dependent fashion. These data also show that MK, but not PTN, interacts with a G401 cell-surface receptor and induces tyrosine phosphorylation of several cellular proteins. A quantitative analysis of MK-induced G401 cell proliferation (Fig. 3), MK/MK receptor binding affinity (Figs. 5 and 6), and MK-stimulated tyrosine phosphorylation of G401 cellular proteins (Fig. 8) strongly suggests that MK acts as an autocrine mitogen through its interaction with its cell-surface high affinity receptor identified in our studies.

Ligand-binding assay of MK cell-surface receptor was assessed by two independent techniques and demonstrates that this receptor binds MK with high affinity (Kd = 0.056-0.072 nM). This Kd value fits nicely within the range of values for other cytokine/growth factor receptors as follows: interleukin-3 and granulocyte-monocyte colony-stimulating factor (0.14 nM); interleukin-4 (0.16 nM); interleukin-5 (0.03 nM); interferon-alpha (0.02 nM); interferon-gamma (0.1 nM); insulin-like growth factor (0.06 nM); and transforming growth factor-beta (0.07 nM) (41-47).

The precise identity of the 200+-kDa high affinity MK receptor is unknown. Our experiments demonstrate that a 200+-kDa protein binds MK and becomes tyrosine-phosphorylated upon MK binding; cross-linking of MK pulls down a 200+-kDa protein (Fig. 4), and anti-phosphotyrosine immunoblot of total proteins from MK-stimulated cells also identifies a 200+-kDa protein (Fig. 7). Although the species identified by cross-linking could represent multiple polypeptides cross-linked to MK, this anti-phosphotyrosine immunoblot demonstrates a single 200+-kDa polypeptide chain. However, we cannot exclude the possibility of a multi-subunit composition of the high affinity MK cell-surface receptor, as has been demonstrated for number of cytokine receptors.

To identify the proteins phosphorylated upon MK exposure, we precipitated biotinylated MK cross-linked to an MK receptor, followed by Western analysis with various antibodies. This set of experiments demonstrates that MK binds to a 200+-kDa G401 cell-surface receptor complex that is associated with the non-receptor tyrosine kinases JAK1 and JAK2 (Fig. 9). Furthermore, protein species of the same molecular mobility as JAK1 and JAK2 are tyrosine-phosphorylated upon MK stimulation. To prove that MK induces tyrosine phosphorylation of specific members of the JAK/STAT pathway, we performed further immunoprecipitation experiments, with antibodies against JAK/STAT family members, and probing these complexes with anti-phosphotyrosine antibodies (Fig. 10). These experiments show that MK stimulates phosphorylation of specific members of the JAK/STAT pathway, namely JAK1, JAK2, and STAT1alpha . These data suggest that the mitogenic effect of MK on G401 cells is mediated by binding MK to a G401 cell-surface receptor; the MK receptor then becomes tyrosine-phosphorylated upon ligand-receptor interaction.

Previous studies have not yet identified a high affinity signaling receptor for either MK or PTN. Cross-linking studies with PTN performed on NIH 3T3 cells detected 155- and 127-kDa PTN binding proteins (Kd = 0.6 nM, 5000 molecules/cell) that are not yet purified or completely characterized (48). It was reported that MK and PTN in vitro also bind to the proteoglycans N-syndecan (syndecan-3) and ryudocan (syndecan-4), respectively (49, 50). MK was also shown to bind nucleolin (100 kDa), a major nucleolar protein that acts as a shuttle between the nucleus and the cytoplasm (51, 52). Perhaps the MK·nucleolin complex can be translocated to the nucleus where it might function in MK signal transduction, as recently suggested for platelet-derived growth factor and fibroblast growth factor (52, 53). However, our results suggest that MK signal transduction at the cell surface appears to involve a cell-surface receptor distinct from nucleolin with activation of the JAK/STAT pathway.

Growth factors and cytokines bind to cell-surface receptors with different affinities. For example, fibroblast growth factor and transforming growth factor-beta both bind to signaling receptor tyrosine kinases with high affinity (54). However, the same growth factors bind with low affinity to cell-surface proteoglycans that cannot transmit signals alone but modulate the ability of these growth factors to generate a biological response through other high affinity signaling receptors (57). Both ryudocan and N-syndecan, which interact with PTN and MK, are members of a type I integral membrane heparan sulfate proteoglycan family and were also reported to bind the basic fibroblast growth factor and tissue factor pathway inhibitor (49, 50, 55-58). Our studies have not yet identified a type I proteoglycan as an MK receptor in G401 cells. However, given the previous work suggesting the potential importance of proteoglycans in signal transduction of heparin binding growth factors, we cannot exclude that the high affinity cell-surface MK receptor identified here may interact with as yet unidentified proteoglycans.

Autocrine control mechanisms that regulate cell proliferation have been found to be important in tumorigenesis in many model systems (59). Our studies have demonstrated that the G401 cell line both secretes MK and expresses a high affinity cell-surface receptor for MK that appears to be directly involved in the regulation of cell proliferation in vitro. We have now demonstrated that the candidate MK signal transduction receptor is a 200+-kDa plasma membrane protein that is tyrosine-phosphorylated promptly after MK binding. In addition, we have shown that MK stimulation results in association of JAK1 and JAK2 with the MK ligand-receptor complex in the plasma membrane, followed by tyrosine phosphorylation of JAK1 and JAK2 and STAT1alpha . However, the architecture of the MK receptor complex as well as the specific molecular mechanisms underlying MK-induced signal transduction remain to be identified.

Janus non-receptor tyrosine kinases can be recruited by both growth factor receptors with intrinsic kinase activity and also by cytokine receptors that are associated with separate tyrosine kinase molecules only upon ligand binding (60, 61). Cross-talk between signaling pathways of receptor tyrosine kinases and cytokine receptors that both involve JAK kinases might be an important molecular mechanism mediating regulation of cell proliferation, differentiation, and development (60, 61). We cannot yet ascertain whether the MK high affinity receptor belongs to the receptor tyrosine kinase superfamily or cytokine receptor superfamily, but our data support the possible involvement of the JAK/STAT pathway in MK signal transduction. Additional studies, including the cloning of the 200+-kDa MK receptor to assess its functional role in signal transduction, are needed to elucidate the molecular events associated with MK regulation of cell proliferation.

    ACKNOWLEDGEMENTS

We gratefully acknowledge Dr. Richard Vandlen (Genentech) and Dr. Patricia Wilson (Mt. Sinai School of Medicine, NY) for their reviews of the manuscript. We also thank Dr. Eudora Eng (The Johns Hopkins Medical Institutions, Baltimore) for contribution to the development of the G401 cell proliferation assay.

    FOOTNOTES

* This work was supported in part by Genentech (to C. R. B.), American Cancer Society Grant JFRA-529 (to C. R. B.), National Institutes of Health Grant P50 HL52315 (to E. A. R. and C. J. L.), National Institutes of Health Grant R01 HL5361 (to C. J. L.), the Cora and John H. Davis Foundation (to E. A. R. and C. J. L.), and the Bernard Bernard Foundation (to C. J. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom reprint requests and correspondence should be addressed: Dept. of Pathology, The Johns Hopkins University School of Medicine, 950 Ross Bldg., 720 Rutland Ave., Baltimore, MD 21205. Tel.: 410-614-0071; Fax: 410-955-0485; E-mail: erat{at}welchlink.welch.jhu.edu.

1 The abbreviations used are: MK, midkine; PTN, pleiotrophin; PAGE, polyacrylamide gel electrophoresis; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxyl-methoxyphenyl)-2-4-sulfophenyl-2H-tetrazoli-um; PMS, phenazine methosulfate; DSS, disuccinimidyl suberate; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride; ECL, enhanced chemiluminescence; STAT, signal transducers and activators of transcription.

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