Identification of Galectin-3-binding Protein as a Factor Secreted by Tumor Cells That Stimulates Interleukin-6 Expression in the Bone Marrow Stroma
2008; Elsevier BV; Volume: 283; Issue: 27 Linguagem: Inglês
10.1074/jbc.m803115200
ISSN1083-351X
AutoresYasushi Fukaya, Hiroyuki Shimada, Ling-Chi Wang, Ebrahim Zandi, Yves A. DeClerck,
Tópico(s)Neuroblastoma Research and Treatments
ResumoWe have previously demonstrated that neuroblastoma cells increase the expression of interleukin-6 by bone marrow stromal cells and that stimulation does not require cell-cell contact. In this study we report the purification and identification of a protein secreted by neuroblastoma cells that stimulates interleukin-6 production by stromal cells. Using a series of chromatographic purification steps including heparin-affinity, ion exchange, and molecular sieve chromatography followed by trypsin digestion and liquid chromatography tandem mass spectrometry, we identified in serum-free conditioned medium of neuroblastoma cells several secreted peptides including galectin-3-binding protein, also known as 90-kDa Mac-2-binding protein. We demonstrated the presence of the galectin-3-binding protein in the conditioned medium of several neuroblastoma cell lines and in chromatographic fractions with interleukin-6 stimulatory activity. Consistently, bone marrow stromal cells express galectin-3, the receptor for galectin-3-binding protein. Supporting a role for galectin-3-binding protein in stimulating interleukin-6 expression in bone marrow stromal cells, we observed that recombinant galectin-3-binding protein stimulated interleukin-6 expression in these cells and that interleukin-6 stimulation by neuroblastoma-conditioned medium was inhibited in the presence of lactose or a neutralizing anti-galectin-3 antibody. Down-regulation of galectin-3-binding protein expression in neuroblastoma cells also decreased the interleukin-6 stimulatory activity of the conditioned medium on bone marrow stromal cells. We also provide evidence that stimulation of interleukin-6 by galectin-3-binding protein involves activation of the Erk1/2 pathway. The data, thus, identifies galectin-3-binding protein as a factor secreted by neuroblastoma cells that stimulates the expression of interleukin-6 in bone marrow stromal cells and provides a novel function for this protein in cancer progression. We have previously demonstrated that neuroblastoma cells increase the expression of interleukin-6 by bone marrow stromal cells and that stimulation does not require cell-cell contact. In this study we report the purification and identification of a protein secreted by neuroblastoma cells that stimulates interleukin-6 production by stromal cells. Using a series of chromatographic purification steps including heparin-affinity, ion exchange, and molecular sieve chromatography followed by trypsin digestion and liquid chromatography tandem mass spectrometry, we identified in serum-free conditioned medium of neuroblastoma cells several secreted peptides including galectin-3-binding protein, also known as 90-kDa Mac-2-binding protein. We demonstrated the presence of the galectin-3-binding protein in the conditioned medium of several neuroblastoma cell lines and in chromatographic fractions with interleukin-6 stimulatory activity. Consistently, bone marrow stromal cells express galectin-3, the receptor for galectin-3-binding protein. Supporting a role for galectin-3-binding protein in stimulating interleukin-6 expression in bone marrow stromal cells, we observed that recombinant galectin-3-binding protein stimulated interleukin-6 expression in these cells and that interleukin-6 stimulation by neuroblastoma-conditioned medium was inhibited in the presence of lactose or a neutralizing anti-galectin-3 antibody. Down-regulation of galectin-3-binding protein expression in neuroblastoma cells also decreased the interleukin-6 stimulatory activity of the conditioned medium on bone marrow stromal cells. We also provide evidence that stimulation of interleukin-6 by galectin-3-binding protein involves activation of the Erk1/2 pathway. The data, thus, identifies galectin-3-binding protein as a factor secreted by neuroblastoma cells that stimulates the expression of interleukin-6 in bone marrow stromal cells and provides a novel function for this protein in cancer progression. It has become increasingly evident that the tumor microenvironment plays an important contributory role to cancer progression (1Fidler I.J. Nat. Rev. Cancer. 2003; 3: 453-458Crossref PubMed Scopus (3552) Google Scholar). Tumor cells closely interact with non-malignant cells present in the tumor stoma through a series of adhesion-dependent and adhesion-independent mechanisms. Among the adhesion-independent interactions, cytokines, chemokines, and growth factors play a critical role (2Singh S. Sadanandam A. Singh R.K. Cancer Metastasis Rev. 2007; 26: 453-467Crossref PubMed Scopus (150) Google Scholar, 3Lin W.W. Karin M. J. Clin. Investig. 2007; 117: 1175-1183Crossref PubMed Scopus (1528) Google Scholar). In this regard, the bone marrow, which is a frequent site of metastasis, provides a microenvironment that is particularly favorable to cancer progression because it is a source of hematopoietic and mesenchymal cells that produce a large number of cytokines and chemokines (4Bonfil R.D. Chinni S. Fridman R. Kim H.R. Cher M.L. Urol. Oncol. 2007; 25: 407-411Crossref PubMed Scopus (33) Google Scholar, 5Mitsiades C.S. McMillin D.W. Klippel S. Hideshima T. Chauhan D. Richardson P.G. Munshi N.C. Anderson K.C. Hematol. Oncol. Clin. North Am. 2007; 21: 1007-1034Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 6Roodman G.D. N. Engl. J. Med. 2004; 350: 1655-1664Crossref PubMed Scopus (1822) Google Scholar). Neuroblastoma is a neural crest-derived cancer that is the second most common solid tumor in children (7Brodeur G.M. Nat. Rev. Cancer. 2003; 3: 203-216Crossref PubMed Scopus (1696) Google Scholar). At the time it is diagnosed, it has metastasized in 56% of the cases, primarily to the bone marrow, the bone, and the liver (8Dubois S.G. Kalika Y. Lukens J.N. Brodeur G.M. Seeger R.C. Atkinson J.B. Haase G.M. Black C.T. Perez C. Shimada H. Gerbing R. Stram D.O. Matthay K.K. J. Pediatr. Hematol. Oncol. 1999; 21: 181-189Crossref PubMed Scopus (310) Google Scholar). The mechanisms by which neuroblastoma cells invade the bone have been recently elucidated by several laboratories, including our group (9van Golen C.M. Schwab T.S. Kim B. Soules M.E. Su O.S. Fung K. van Golen K.L. Feldman E.L. Cancer Res. 2006; 66: 6570-6578Crossref PubMed Scopus (62) Google Scholar, 10Michigami T. Ihara-Watanabe M. Yamazaki M. Ozono K. Cancer Res. 2001; 61: 1637-1644PubMed Google Scholar, 11Ara T. DeClerck Y.A. Cancer Metastasis Rev. 2006; 25: 645-657Crossref PubMed Scopus (70) Google Scholar). We have previously shown that bone metastasis in human neuroblastoma is associated with activation of osteoclasts and an increase in bone resorption (12Sohara Y. Shimada H. Scadeng M. Pollack H. Yamada S. Ye W. Reynolds C.P. DeClerck Y.A. Cancer Res. 2003; 63: 3026-3031PubMed Google Scholar). We more recently reported (13Sohara Y. Shimada H. Minkin C. Erdreich-Epstein A. Nolta J.A. DeClerck Y.A. Cancer Res. 2005; 65: 1129-1135Crossref PubMed Scopus (68) Google Scholar) that activation of osteoclasts by neuroblastoma cells involves the stimulation of interleukin-6 (IL-6) 2The abbreviations used are: IL-6, interleukin-6; rh, recombinant human; TNF, tumor necrosis factor-α; BMSC, bone marrow stromal cells; CM, conditioned medium; Gal-3BP, galectin-3-binding protein; Erk, extracellular signal-regulated kinase; MS, mass spectroscopy; siRNA, small interfering RNA. production by bone marrow stromal cells (BMSC), IL-6 being a potent activator of osteoclasts. A similar interaction between tumor cells and BMSC has been previously demonstrated to play a critical role in multiple myeloma, a cancer also characterized by an increase in bone destruction (14Teoh G. Anderson K.C. Hematol. Oncol. Clin. North Am. 1997; 11: 27-42Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 15Urashima M. Teoh G. Chauhan D. Hoshi Y. Ogata A. Treon S.P. Schlossman R.L. Anderson K.C. Blood. 1997; 90: 279-289Crossref PubMed Google Scholar). However, in contrast to myeloma, where the production of IL-6 by BMSC is dependent on an adhesion contact between BMSC and tumor cells, in neuroblastoma, stimulation of IL-6 does not involve cell-cell contact and occurs in the presence of serum-free conditioned medium (CM) from neuroblastoma cells. In this manuscript we report the purification and characterization of a protein secreted by human neuroblastoma cells and present in their culture medium that stimulates the expression of IL-6 by human BMSC. This protein was identified as galectin-3-binding protein (Gal-3BP), also known as 90-kDa Mac-2 binding protein, a secreted glycoprotein with a molecular mass of ∼90 kDa initially identified and characterized in breast cancer cells and in human milk (16Koths K. Taylor E. Halenbeck R. Casipit C. Wang A. J. Biol. Chem. 1993; 268: 14245-14249Abstract Full Text PDF PubMed Google Scholar). Cell Lines and Cell Culture—The human neuroblastoma cell lines CHLA-255 and SK-N-BE(2) were kindly provided by Dr. C. P. Reynolds (Childrens Hospital Los Angeles), and the NB-19 cell line was purchased from RIKEN BioResource Center (Tsukuba, Japan). CHLA-255 cells were cultured in Iscove's modified Dulbecco's medium containing HEPES (25 mm), penicillin (100 IU/ml), and streptomycin (100 μg/ml) (Cellgro). The medium was supplemented with 10% (v/v) fetal bovine serum, insulin (0.25 μg/ml), transferrin (0.25 μg/ml), selenium (0.25 ng/ml) (Lonza Inc., Basel, Switzerland) and l-glutamine (2 mm). NB-19 cells and SK-N-BE(2) cells were cultured in RPMI1640 (Invitrogen) with penicillin-streptomycin, 10% (v/v) fetal bovine serum, insulin (0.25 μg/ml), transferrin (0.25 μg/ml), selenium (0.25 ng/ml), and l-glutamine. Human BMSC were obtained from ALLCELLS (Emeryville, CA) and maintained in basal medium with stimulatory supplements containing basic fibroblast growth factor (10 ng/ml). Reagents and Antibodies—Trypsin was purchased from Sigma-Aldrich Corp., dissolved in 1 mm HCl, and stored at a concentration of 1 mg/ml at –20 °C. Soybean trypsin inhibitor was purchased from EMD Bioscience, Inc. (San Diego, CA) and stored at –20 °C at a concentration of 10 mg/ml in potassium phosphate (67 mm), pH 7.6. PD98059 was obtained from Calbiochem. Lactose and sucrose were purchased from Sigma-Aldrich. A goat anti-human Gal-3BP antibody was purchased from R&D Systems (Minneapolis, MN). Antibodies against Erk1/2 and phospho-Erk1/2 were purchased from Cell Signaling Technology, Inc. (Danvers, MA). A mouse monoclonal antibody against human actin was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and recombinant human (rh) Gal-3BP was purchased from R&D Systems. A mouse monoclonal antibody against human Gal-3 used for Western blot was purchased from Santa Cruz, and a mouse neutralizing monoclonal antibody against Gal-3 (TIB-166) was kindly provided by Dr. Avraham Raz (Karmanos Cancer Institute, Wayne State University, Detroit, MI). Trypsin Digestion—CM obtained from human neuroblastoma cell lines was incubated with 100 μg/ml trypsin at 37 °C for 1 h. The reaction was stopped by adding 200 μg/ml soybean trypsin inhibitor for 30 min. Preparation of CM from Neuroblastoma Cells—Neuroblastoma cells were plated at 5 × 106 in 150 cm2 tissue culture flasks for 72 h in culture medium containing 10% (v/v) fetal bovine serum. The tissue culture flasks were then washed twice with phosphate-buffered saline (4 mm KCl, 0.5 mm Na2HPO4, 140 mm NaCl, and 0.15 mm KH2PO4), and serum-free medium was added. After 24 h, the CM was collected and centrifuged at 1500 rpm for 10 min at 4 °C before being filtered through a 0.2-μm filter (Corning Inc., Corning, NY) and stored at –20 °C until used for chromatographic separation. Before chromatography, the CM was concentrated 10- and 50-fold by pressure dialysis using a 10,000 nominal molecular weight limit polyethersulfone membrane in a stirred ultra-filtration cell (Millipore, Billerica, MA) at 4 °C. Protein Separation by Column Chromatography—All purification procedures were carried out at 4 °C using an AKTA-prime plus chromatography system (GE Healthcare). Briefly, the concentrated CM was first applied to either a 1- or 5-ml heparin-Sepharose column (HiTrap Heparin, GE Healthcare) equilibrated with 10 mm sodium phosphate buffer, pH 7.0, at a flow rate of 1–2 ml/min. The bound proteins were eluted in one step with 1 m NaCl. The active fractions (see below) were pooled, desalted by passing through a YM-10 membrane, and resuspended in 20 mm Tris-HCl, pH 7.4. The pool was then applied to a 1-ml diethylaminoethyl-Sepharose column (HiTrap DEAE Fast Flow, GE Healthcare) equilibrated with the same buffer at a flow rate of 1 ml/min. Bound proteins were eluted using a linear gradient of NaCl from 0 to 0.5 m. The active fractions were pooled and concentrated by passing through a YM-10 membrane, equilibrated in 20 mm Tris-HCl, pH 7.4, and applied to a molecular sieve column (Superdex 200, 5 mm × 150 mm, GE Healthcare) at a flow rate of 0.2 ml/min. Each fraction eluting from the chromatography was examined for protein concentration using the BCA protein assay reagent kit (Pierce) for IL-6 stimulatory activity (see below) and for protein content by SDS-PAGE (see below). Measurement of IL-6 Stimulatory Activity—Individual fractions separated by chromatography were tested for their ability to simulate the expression of IL-6 in BMSC in culture. In brief, BMSC were seeded in 24-well tissue culture plates at 104 cells/well (1.9 cm2, Corning, Inc.) and maintained for 48 h in serum-containing medium. Each well was then washed twice with phosphate-buffered saline and incubated for 24 h with 1 ml of serum-free medium supplemented with an aliquot (50 or 100 μl) of individual chromatographic fractions. The supernatant of the culture medium was then collected, clarified by centrifugation at 12,000 rpm for 5 min at 4 °C, and examined for IL-6 concentration by enzyme-linked immunosorbent assay (ELISA) using a human IL-6 ELISA kit purchased from R&D Systems. The specific activity of each fraction was expressed in pg of IL-6/mg of protein in the fraction tested. SDS-PAGE and Immunoblotting—SDS-PAGE was performed using 4–12% gradient acrylamide gels (Invitrogen). Proteins were visualized by silver staining or transferred onto nitrocellulose membranes by semi-dry blotting (Bio-Rad). After being blocked in 10 mm Tris-HCl, pH 8, 150 mm NaCl, 0.1% Tween 20 containing 5% nonfat dried milk, the membranes were incubated overnight at 4 °C with the primary antibody in blocking buffer. After washing, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (dilution 1:10,000 v/v) for 1 h. The presence of immune complexes was identified by enhanced chemiluminescence (Amersham Biosciences). Gels were scanned, and quantitative analysis was done using the NIH J image software. Mass Spectrometry; Trypsin Digestion and Liquid Chromatography Tandem Mass Spectrometry (MS/MS)—The protein mixture was dissolved in 20 μl of 50 mm ammonium bicarbonate and 4 m urea and reduced by adding dithiothreitol to a final concentration of 10 mm at 95 °C for 10 min. The sample was immediately alkylated by adding 2 μl of 50 mm iodoacetamide at 37 °C for 30 min. The urea was diluted to 1 m with 72 μl of 100 mm ammonium bicarbonate, and the proteins were digested with 1 μg of sequencing grade modified trypsin (Promega, Madison, WI) at 37 °C overnight. The digestion was continued for an additional 24 h by adding 1 μg of trypsin. For MS analysis, 13 μl of 10% (v/v) formic acid was added, and the sample was desalted and concentrated using C18 reverse phase. One-fourth of the sample was loaded on a 10-cm reversed phase chromatography capillary column connected to a spray tip column through a MicroTee with in-union high voltage contact as previously described (17Wang L.C. Okitsu C.Y. Kochounian H. Rodriguez A. Hsieh C.L. Zandi E. Proteomics. 2008; 8: 1758-1761Crossref PubMed Scopus (19) Google Scholar). Peptides were eluted into the mass spectrometer using the gradients 5–50% acetonitrile + 0.1% formic acid over 75 min and 50–90% acetonitrile + 0.1% formic acid over 40 min. A split tee with a fused silica capillary (25-μm internal diameter) on one end was used to split the high pressure liquid chromatography flow from 50 to 500 μl/min. The other end of the tee was connected to a six-port divert/inject valve, which was used to introduce peptide samples into the column. A linear ion trap LTQ (Thermo Electron, Inc.) was used to acquire tandem MS/MS spectra with Xcalibur 2.1 software using the following method; a full MS scan was followed by seven consecutive MS2 scans of the top 7 ion peaks from the preceding full scan using dynamic exclusion (4 repetitions in 1.5 min were excluded for 15 min). Data were analyzed using Bioworks 3.2, utilizing the SEQUEST algorithm and Sage-N Sorcerer to determine cross-correlation scores between acquired spectra and NCBI protein FASTA databases. The following parameters were used for the TurboSEQUEST search: molecular weight range, 0–5000; threshold, 1000; monoisotopic; precursor mass, 1.4; group scan, 10; minimum ion count, 20; charge state, auto; peptide, 1.5; fragment ions, 0; static amino acid modifications, Cys 57.05 and Met 15.99. Results were filtered using SEQUEST cross-correlation scores greater than 2.0 for +1 ions, 3.0 for +2 ions, and 3.5 for +3 ions. Gal-3BP-blocking Experiment—Lactose and a blocking antibody against Gal-3 (TIB-166) were used in blocking experiments with BMSC. Lactose (50 to 150 mm) or TIB-166 (dilutions of 1/1000 to 1/100 v/v) were added for 2 h to the culture medium of BMSC before the addition of neuroblastoma CM or active pooled chromatographic fractions. Sucrose and mouse nonspecific IgG were used as a negative control for lactose and TIB-166, respectively. Fluorescence-activated Cell Sorter Analysis—Cells were washed in phosphate-buffered saline, detached in cell dissociation buffer, and recovered by centrifugation at 4500 rpm for 5 min before being resuspended in phosphate-buffered saline containing 1% (v/v) bovine serum albumin. Aliquots containing 1 × 106 cells were incubated with antibodies at a final concentration of 1 μg/106 cells. After incubation for 15 min, cells were washed and reincubated with a secondary fluorescein isothiocyanate-conjugated anti-mouse IgG antibody (1:1000 v/v). Cells were then analyzed using a FACScan flow cytometer, and the data were analyzed using CellQuest software (BD Bioscience). Nonspecific fluorescence was determined in the presence of an unlabeled anti-mouse IgG antibody. Transfection of siRNA—CHLA-255 neuroblastoma cells were seeded onto 24-well plates and cultured in medium without antibiotics for 24 h before transfection. Two different siRNAs for Gal-3BP were purchased from Qiagen, Inc. (Valencia, CA): Hs_LGALS3BP#1, 5′-CCG UCA UCC UGA CUG CCA A-3′, and Hs_LGLS3BP#5, sense 5′-CCA UGA GUG UGG AUG CUG A-3′. A scrambled control sequence was used as the control. siRNA transfection was done in the presence of Lipofectamine 2000 (Invitrogen), and a fluorescein isothiocyanate-labeled siRNA was used to assess the transfection efficiency. Statistical Analysis—Student's t test was used for the comparison between two sets of data. The analysis of variance was used for the analysis of trends. A p value of <0.05 was considered statistically significant. CM of Neuroblastoma Cells Contains a Protein(s) That Stimulates IL-6 Production in BMSC—In support of the concept that the stimulation of IL-6 by neuroblastoma CM was mediated by a protein(s) produced by neuroblastoma cells, we had previously demonstrated that treatment of neuroblastoma cells with cycloheximide resulted in a loss of IL-6 stimulatory activity in the CM (13Sohara Y. Shimada H. Minkin C. Erdreich-Epstein A. Nolta J.A. DeClerck Y.A. Cancer Res. 2005; 65: 1129-1135Crossref PubMed Scopus (68) Google Scholar). To obtain additional evidence in support of the stimulatory factor being a protein, we demonstrated that treatment of CM from two neuroblastoma cell lines (NB-19 and CHLA-255) with trypsin significantly decreased the amount of IL-6 produced by BMSC cultured in the presence of these CM (Fig. 1A). Treatment of CHLA-255 CM with heat (50 and 90 °C) also resulted in a substantial loss of IL-6 stimulatory activity. The data are, thus, consistent with the IL-6 stimulatory factor present in neuroblastoma CM being a (or possibly several) portein. Interestingly, we also observed an increase in Erk1/2 activation (phosphorylation) when BMSC were cultured in the presence of neuroblastoma CM, raising the possibility that the stimulatory protein could be a ligand signaling via Erk1/2 (Fig. 1B). Chromatographic Purification of a Protein with IL-6 Stimulatory Activity—We, therefore, initiated a series of chromatographic separations in an attempt to isolate and identify the active protein(s). A typical example of the purification protocol used is shown in Fig. 2. Serum-free CM from CHLA-255 cells was concentrated 50-fold by pressure dialysis on a 10,000 nominal molecular weight limit polyethersulfone membrane, applied to a heparin affinity column, and eluted with 1 m NaCl (Fig. 2A). This initial purification step resulted in the identification of 2 sets of fractions (heparin-unbound and heparin-bound) with IL-6 stimulatory activity. The heparin-bound fractions (13Sohara Y. Shimada H. Minkin C. Erdreich-Epstein A. Nolta J.A. DeClerck Y.A. Cancer Res. 2005; 65: 1129-1135Crossref PubMed Scopus (68) Google Scholar and 14Teoh G. Anderson K.C. Hematol. Oncol. Clin. North Am. 1997; 11: 27-42Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar), which had a higher specific activity than the heparin-unbound fractions, were then pooled, desalted, applied to a DEAE anion exchange column, and eluted with a 0–0.5 m NaCl gradient (Fig. 2B). This resulted in the identification of a broad peak of active fractions (20Sasaki T. Brakebusch C. Engel J. Timpl R. EMBO J. 1998; 17: 1606-1613Crossref PubMed Scopus (213) Google Scholar, 21Raz A. Lotan R. Cancer Metastasis Rev. 1987; 6: 433-452Crossref PubMed Scopus (294) Google Scholar, 22Inohara H. Akahani S. Koths K. Raz A. Cancer Res. 1996; 56: 4530-4534PubMed Google Scholar, 23Rao S.P. Wang Z. Zuberi R.I. Sikora L. Bahaie N.S. Zuraw B.L. Liu F.T. Sriramarao P. J. Immunol. 2007; 179: 7800-7807Crossref PubMed Scopus (88) Google Scholar, 24Sugiura Y. Ma L.Q. Sun B. Shimada H. Laug W.E. Seeger R.C. DeClerck Y.A. Cancer Res. 1999; 59: 1327-1336PubMed Google Scholar, 25Marchetti A. Tinari N. 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This chromatographic purification step resulted in the identification of 2 peaks of activity that, however, remained poorly separated. The activity and specific activity recovered during a typical purification procedure of CM from CHLA-255 cells is shown in Table 1. From 6 liters of CM containing 45.26 mg of protein, we obtained at the end 4.6 μg of active material with a specific activity of 203,977 pg of IL-6/mg of protein in the first peak eluting from the molecular sieve column and 7.6 μg of protein with a specific activity of 124,466 pg of IL-6/mg of protein in the second peak. Aliquots of the active fractions identified during these chromatographic steps were examined by SDS-PAGE and silver stain for protein content (Fig. 3). This analysis indicated after chromatographic separation by heparin, ion exchange, and molecular sieve chromatography, the presence of several proteins with Mr between 45 and 250 in the last pool of active fractions obtained by molecular sieve chromatography (fractions 7–9 in peak 1) (Fig. 3, lane 6). These pooled fractions were digested in solution with trypsin, and peptides were identified by liquid chromatography-MS/MS. This analysis identified a total of 65 proteins, most of them corresponding to intracellular and membrane-associated proteins (data not shown). However, among these, six proteins corresponded to secreted and extracellular matrix proteins (Table 2). Three peptides were from extracellular matrix proteins including laminin (β and γ chains) and tenascin. One peptide corresponded to the λ heavy chain of immunoglobulin (a likely contaminant from residual fetal bovine serum) and one to a hypothetical protein T08772. The protein with the highest score (4Bonfil R.D. Chinni S. Fridman R. Kim H.R. Cher M.L. Urol. Oncol. 2007; 25: 407-411Crossref PubMed Scopus (33) Google Scholar) was Gal-3BP or Mac-2BP, a 587-amino acid-secreted glycosylated protein with a molecular mass of 65.3 and 90 kDa when glycosylated (16Koths K. Taylor E. Halenbeck R. Casipit C. Wang A. J. Biol. Chem. 1993; 268: 14245-14249Abstract Full Text PDF PubMed Google Scholar). This latter protein particularly raised our interest as a likely candidate because a literature search revealed that it was the only protein among the six secreted peptides that had been previously reported to stimulate IL-6 expression (18Kalayci O. Birben E. Tinari N. Oguma T. Iacobelli S. Lilly C.M. Ann. Allergy Asthma Immunol. 2004; 93: 485-492Abstract Full Text PDF PubMed Scopus (22) Google Scholar, 19Powell T.J. Schreck R. McCall M. Hui T. Rice A. App H. Azam M. Ullrich A. Shawver L.K. J. Immunother. Emphasis Tumor Immunol. 1995; 17: 209-221Crossref PubMed Scopus (49) Google Scholar).TABLE 1Chromatographic purification of conditioned medium from CHLA-255 cellsVolumeConcentrationProteinActivitySpecific activityRecovery-Fold purificationmlmg/mlmgpg/mlpg/ml/mg%%50× CM1200.37745.261,545,92434,155100.00100Heparin-bound500.1256.24621,56899,57040.21291DEAE-bound200.0480.96177,249184,71511.47540Molecular sieve peak 10.80.0060.0046935203,9770.06597Molecular sieve peak 20.80.0100.0076947124,4660.06364 Open table in a new tab FIGURE 3SDS-PAGE of active fractions separated by chromatography. Aliquots of active fractions pooled after each purification steps shown in Fig. 2 were electrophoresed on a 4–12% gradient acrylamide gel under reducing conditions and visualized by silver staining.View Large Image Figure ViewerDownload Hi-res image Download (PPT)TABLE 2Soluble proteins separated by molecular sieve chromatography and identified by liquid chromatography-MS/MSReference scan(s)P (pro) P (pep)Score XCMr SpGI Accession RSpPeptide (hits) ionsGalectin 3-binding protein2.02E—0620.1865,312.65,031,8634T08772 hypothetical protein1.18E—1020.20104,220.57,512,9262LMG1_human laminin γ1 chain3.89E—0920.29177,588.0126,3692Tenascin C (hexabrachion)4.25E—0620.22240,847.14,504,5492Human laminin, β1 chain1.20E—0710.24198,047.64,504,9511Immunoglobulin λ heavy chain8.22E—022.1952,709.42,765,4251 Open table in a new tab Gal-3BP Is Expressed by Neuroblastoma Cells and Is Present in Active Chromatographic Fractions—We first confirmed that Gal-3BP was produced by neuroblastoma cells by Western blot analysis using a commercially available anti-human Gal-3BP antibody (Fig. 4). This analysis revealed the presence of 3 broad peptide bands with Mr values in the range of 93, 70, and 26 in the CM of 3 neuroblastoma cell lines that stimulated IL-6 expression in BMSC (Fig. 4A). The diffuseness of these bands was consistent with their being glycoproteins, and their Mr values were consistent with previously published data. Gal-3BP has been reported to be cleaved by plasmin and another endogenous protease into a 70-kDa N-terminal fragment with activity and a 26-kDa C-terminal domain (16Koths K. Taylor E. Halenbeck R. Casipit C. Wang A. J. Biol. Chem. 1993; 268: 14245-14249Abstract Full Text PDF PubMed Google Scholar, 20Sasaki T. Brakebusch C. Engel J. Timpl R. EMBO J. 1998; 17: 1606-1613Crossref PubMed Scopus (213) Google Scholar). A similar Western blot analysis performed on aliquots of pooled active fractions obtained by chromatography (Fig. 4B) revealed the presence of the intact 93-kDa protein in the active heparin-unbound fractions and of the N-terminal 70-kDa proteolytic fragment and 26-kDa C-terminal fragment in the active heparin-bound fractions and all other active fractions obtained by ion exchange and molecular sieve chromatography. In contrast, Gal-3BP was not identified by Western blot analysis in chromatographic fractions that had no IL-6 stimulatory activity (data not shown). BMSC Express Galectin-3—Gal-3BP binds to Gal-3, a member of the galectin family of lectins that specifically recognizes β-galactosidic carbohydrate structures and is localized not only at the cell membrane but also in the cytoplasm and the nucleus of cells (21Raz A. Lotan R. Cancer Metastasis Rev. 1987; 6: 433-452Crossref PubMed Scopus (294) Google Scholar, 22Inohara H. Akahani S. Koths K. Raz A. Cancer Res. 1996; 56: 4530-4534PubMed Google Scholar). We, therefore, determined whether Gal-3 was expressed by BMSC (Fig. 5). A Western blot analysis of whole cell lysates of BMSC indicated the presence of Gal-3 when cells were cultured in serum-free medium o
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