A Novel Function of Syndecan-2, Suppression of Matrix Metalloproteinase-2 Activation, Which Causes Suppression of Metastasis
2007; Elsevier BV; Volume: 282; Issue: 38 Linguagem: Inglês
10.1074/jbc.m609812200
ISSN1083-351X
AutoresSeiichi Munesue, Yasuo Yoshitomi, Yuri Kusano, Yoshie Koyama, Akiko Nishiyama, Hayao Nakanishi, Kaoru Miyazaki, Takeshi Ishimaru, Shuichi Miyaura, Minoru Okayama, Kayoko Oguri,
Tópico(s)Peptidase Inhibition and Analysis
ResumoThe syndecans comprise a family of cell surface heparan sulfate proteoglycans exhibiting complex biological functions involving the interaction of heparan sulfate side chains with a variety of soluble and insoluble heparin-binding extracellular ligands. Here we demonstrate an inverse correlation between the expression level of syndecan-2 and the metastatic potential of three clones derived from Lewis lung carcinoma 3LL. This correlation was proved to be a causal relationship, because transfection of syndecan-2 into the higher metastatic clone resulted in the suppression of both spontaneous and experimental metastases to the lung. Although the expression levels of matrix metalloproteinase-2 (MMP-2) and its cell surface activators, such as membrane-type 1 matrix metalloproteinase and tissue inhibitor of metalloproteinase-2, were similar regardless of the metastatic potentials of the clones, elevated activation of MMP-2 was observed in the higher metastatic clone. Removal of heparan sulfate from the cell surface of low metastatic cells by treatment with heparitinase-I promoted MMP-2 activation, and transfection of syndecan-2 into highly metastatic cells suppressed MMP-2 activation. Furthermore, transfection of mutated syndecan-2 lacking glycosaminoglycan attachment sites into highly metastatic cells did not have any suppressive effect on MMP-2 activation, suggesting that this suppression was mediated by the heparan sulfate side chains of syndecan-2. Actually, MMP-2 was found to exhibit a strong binding ability to heparin, the dissociation constant value being 62 nm. These results indicate a novel function of syndecan-2, which acts as a suppressor for MMP-2 activation, causing suppression of metastasis in at least the metastatic system used in the present study. The syndecans comprise a family of cell surface heparan sulfate proteoglycans exhibiting complex biological functions involving the interaction of heparan sulfate side chains with a variety of soluble and insoluble heparin-binding extracellular ligands. Here we demonstrate an inverse correlation between the expression level of syndecan-2 and the metastatic potential of three clones derived from Lewis lung carcinoma 3LL. This correlation was proved to be a causal relationship, because transfection of syndecan-2 into the higher metastatic clone resulted in the suppression of both spontaneous and experimental metastases to the lung. Although the expression levels of matrix metalloproteinase-2 (MMP-2) and its cell surface activators, such as membrane-type 1 matrix metalloproteinase and tissue inhibitor of metalloproteinase-2, were similar regardless of the metastatic potentials of the clones, elevated activation of MMP-2 was observed in the higher metastatic clone. Removal of heparan sulfate from the cell surface of low metastatic cells by treatment with heparitinase-I promoted MMP-2 activation, and transfection of syndecan-2 into highly metastatic cells suppressed MMP-2 activation. Furthermore, transfection of mutated syndecan-2 lacking glycosaminoglycan attachment sites into highly metastatic cells did not have any suppressive effect on MMP-2 activation, suggesting that this suppression was mediated by the heparan sulfate side chains of syndecan-2. Actually, MMP-2 was found to exhibit a strong binding ability to heparin, the dissociation constant value being 62 nm. These results indicate a novel function of syndecan-2, which acts as a suppressor for MMP-2 activation, causing suppression of metastasis in at least the metastatic system used in the present study. Tumor metastasis is accomplished through a multistep process in which individual tumor cells disseminate from a primary tumor to distant secondary sites. In the process of metastasis, tumor cells are involved in numerous interactions with the extracellular matrix (ECM) 2The abbreviations used are: ECMextracellular matrixMMPmatrix metalloproteinaseTIMPtissue inhibitor of metalloproteinaseMT1-MMPmembrane-type 1 MMPPBSphosphate-buffered salineH11LM66-H11GAPDHglyceraldehyde-3-phosphate dehydrogenase.2The abbreviations used are: ECMextracellular matrixMMPmatrix metalloproteinaseTIMPtissue inhibitor of metalloproteinaseMT1-MMPmembrane-type 1 MMPPBSphosphate-buffered salineH11LM66-H11GAPDHglyceraldehyde-3-phosphate dehydrogenase. providing information that controls the behavior of tumor cells. The information inscribed in the ECM is transmitted to tumor cells through interaction between individual ECM ligands and the respective cell surface receptors. extracellular matrix matrix metalloproteinase tissue inhibitor of metalloproteinase membrane-type 1 MMP phosphate-buffered saline LM66-H11 glyceraldehyde-3-phosphate dehydrogenase. extracellular matrix matrix metalloproteinase tissue inhibitor of metalloproteinase membrane-type 1 MMP phosphate-buffered saline LM66-H11 glyceraldehyde-3-phosphate dehydrogenase. One class of cell surface receptors with such functions is cell surface heparan sulfate proteoglycans, including the transmembrane-type syndecan family and the glycosylphosphatidylinositol-anchored-type glypican family (1Bernfield M. Kokenyesi R. Kato M. Hinkes M.T. Spring J. Gallo R.L. Lose E.J. Annu. Rev. Cell Biol. 1992; 8: 365-393Crossref PubMed Scopus (964) Google Scholar, 2David G. FASEB J. 1993; 7: 1023-1030Crossref PubMed Scopus (373) Google Scholar, 3Bernfield M. Götte M. Park P.W. Reizes O. Fitzgerald M.L. Lincecum J. Zako M. Annu. Rev. Biochem. 1999; 68: 729-777Crossref PubMed Scopus (2317) Google Scholar). However, cell surface heparan sulfate proteoglycans are unique and are different from proteinous cell surface receptors in terms of the binding redundancy for ligands. This is because the many ligand-binding sites reside in the polysaccharide moiety (i.e. heparan sulfate side chains). Therefore, theoretically, they can be receptors for all heparin-binding molecules. Actually, a large number of reports have demonstrated that cell surface heparan sulfate proteoglycans function as receptors for soluble heparin-binding ligands, such as cell growth factors and insoluble molecules such as ECM constituents. In many cases, it seems that they cooperate with intrinsic high affinity receptors for heparin-binding molecules as low affinity receptors and thereby regulate their signal transduction. For example, they act as co-receptors for heparin-binding growth factors, such as fibroblast growth factor, hepatocyte growth factor, and vascular endothelial growth factor, and control the strength of the signals (4Yayon A. Klagsbrun M. Esko J.D. Leder P. Ornitz D.M. Cell. 1991; 64: 841-848Abstract Full Text PDF PubMed Scopus (2081) Google Scholar, 5Mizuno K. Inoue H. Hagiya M. Shimizu S. Nose T. Shimohigashi Y. Nakamura T. J. Biol. Chem. 1994; 269: 1131-1136Abstract Full Text PDF PubMed Google Scholar, 6Tessler S. Rockwell P. Hicklin D. Cohen T. Levi B.Z. Witte L. Lemischka I.R. Neufeld G. J. Biol. Chem. 1994; 269: 12456-12461Abstract Full Text PDF PubMed Google Scholar, 7Gengrinovitch S. Berman B. David G. Witte L. Neufeld G. Ron D. J. Biol. Chem. 1999; 274: 10816-10822Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 8Horowitz A. Tkachenko E. Simons M. J. Cell Biol. 2002; 157: 715-725Crossref PubMed Scopus (150) Google Scholar, 9Derksen P.W. Keehnen R.M. 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Nagayasu Y. Munesue S. Ishihara M. Saiki I. Yonekura H. Yamamoto H. Okayama M. Exp. Cell Res. 2000; 256: 434-444Crossref PubMed Scopus (87) Google Scholar, 15Munesue S. Kusano Y. Oguri K. Itano N. Yoshitomi Y. Nakanishi H. Yamashina I. Okayama M. Biochem. J. 2002; 363: 201-209Crossref PubMed Scopus (63) Google Scholar, 16Kusano Y. Yoshitomi Y. Munesue S. Okayama M. Oguri K. J. Biochem. (Tokyo). 2004; 135: 129-137Crossref PubMed Scopus (32) Google Scholar). Furthermore, besides the function regarding this signal transduction, cell surface proteoglycans provide initial docking sites for the binding to various viruses (17Gallay P. Microbes Infect. 2004; 6: 617-622Crossref PubMed Scopus (43) Google Scholar, 18Spear P.G. Cell Microbiol. 2004; 6: 401-410Crossref PubMed Scopus (453) Google Scholar). In such cases, viruses also recognize other proteinous receptors expressed simultaneously on the cell surface. Overall, it can be considered that the common function of cell surface heparan sulfate proteoglycans is regulation of the functions of other proteinous molecules on the cell surface. Therefore, heparan sulfate proteoglycans on tumor cells have various functions in the process of metastasis that force the tumor cells to always interact in non-self circumstances. During metastasis, tumor cells must respond to and adapt to the host ECM, which in general, acts defensively against the tumor cells. First of all, tumor cells have to degrade the host ECM in order to disseminate. A family of ECM degradation enzymes called matrix metalloproteinases (MMPs) has been implicated in not only physiological processes of tissue remodeling but also pathological conditions, such as cancer (19Visse R. Nagase H. Circ. Res. 2003; 92: 827-839Crossref PubMed Scopus (3654) Google Scholar). Although various types of MMPs are involved in metastasis, a great deal of emphasis has been placed on type IV collagenases (20Chambers A.F. Matrisian L.M. J. Natl. Cancer Inst. 1997; 89: 1260-1270Crossref PubMed Scopus (1433) Google Scholar, 21John A. Tuszynski G. Pathol. Oncol. Res. 2001; 7: 14-23Crossref PubMed Scopus (546) Google Scholar), and, in particular, activation of MMP-2 appears to be closely correlated with tumor metastasis (22Sato H. Seiki M. J. Biochem. (Tokyo). 1996; 119: 209-215Crossref PubMed Scopus (195) Google Scholar). In common with all MMPs, MMP-2 is synthesized as a latent form, requiring proteolytic removal of the propeptide for activation. Therefore, regulation of MMP-2 occurs through three steps (i.e. alteration of gene expression, activation of latent zymogens, and inhibition by tissue inhibitors of metalloproteinases). The mechanism of activation of MMP-2 on the cell surface has been well documented. This event is triggered by the formation of a tertiary complex comprising (or including) secreted pro-MMP-2, tissue inhibitor of metalloproteinase-2 (TIMP-2), and membrane-type 1 MMP (MT1-MMP) on the cell surface and is followed by the cleavage of the peptide of the pro-MMP-2 by TIMP-2-free MT1-MMP (see Fig. 10A) (23Sato H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. Nature. 1994; 370: 61-65Crossref PubMed Scopus (2368) Google Scholar, 24Strongin A.Y. Collier I. Bannikov G. Marmer B.L. Grant G.A. Goldberg G.I. J. Biol. Chem. 1995; 270: 5331-5338Abstract Full Text Full Text PDF PubMed Scopus (1436) Google Scholar, 25Butler G.S. Butler M.J. Atkinson S.J. Will H. Tamura T. Schade van Westrum S. Crabbe T. Clements J. d'Ortho M.-P. Murphy G. J. Biol. Chem. 1998; 273: 871-880Abstract Full Text Full Text PDF PubMed Scopus (540) Google Scholar, 26Kinoshita T. Sato H. Okada A. Ohuchi E. Imai K. Okada Y. Seiki M. J. Biol. 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Previously, we found an inverse correlation between the expression level of syndecan-2 and the metastatic potential of clones established from Lewis lung carcinoma 3LL (14Kusano Y. Oguri K. Nagayasu Y. Munesue S. Ishihara M. Saiki I. Yonekura H. Yamamoto H. Okayama M. Exp. Cell Res. 2000; 256: 434-444Crossref PubMed Scopus (87) Google Scholar, 15Munesue S. Kusano Y. Oguri K. Itano N. Yoshitomi Y. Nakanishi H. Yamashina I. Okayama M. Biochem. J. 2002; 363: 201-209Crossref PubMed Scopus (63) Google Scholar, 16Kusano Y. Yoshitomi Y. Munesue S. Okayama M. Oguri K. J. Biochem. (Tokyo). 2004; 135: 129-137Crossref PubMed Scopus (32) Google Scholar). In the present study, we further demonstrated the causal relationship in this inverse correlation and the possible mechanism underlying this relation. The results obtained indicate a novel function of syndecan-2, of which the heparan sulfate side chains suppress the activation of MMP-2 on the cell surface. Cell Culture and Transfection—P29, LM12-3, and LM66-H11 cells exhibiting low, intermediate, and high metastatic potential, respectively, were cloned from Lewis lung carcinoma 3LL on the basis of spontaneous metastatic potential (28Nakanishi H. Takenaga K. Oguri K. Yoshida A. Okayama M. Virchows Arch. A. 1992; 420: 163-170Crossref Scopus (22) Google Scholar, 29Itano N. Oguri K. Nakanishi H. Okayama M. J. Biochem. (Tokyo). 1993; 114: 862-873Crossref PubMed Scopus (14) Google Scholar) (where "P" represents "parent" and LM represents "lung metastasis"; i.e. the LM12 and LM66 series were cloned from the populations undergoing 12 and 66 cycles of spontaneous metastasis from a subcutaneous primary tumor to the lung). Cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% (v/v) fetal bovine serum (Invitrogen), streptomycin (100 μg/ml), and penicillin (100 units/ml) at 37 °C under a humidified 5% CO2 atmosphere. The cells were harvested after incubation with 2 mm EDTA in phosphate-buffered saline (EDTA/PBS) for 10 min at 37 °C, followed by gentle flushing with a pipette, and subcultured twice a week. After transfection of the expression plasmid for each protein using Tfx-50 reagent (Promega Corp.) according to the manufacturer's instructions, cells were cultured in the presence of G418 (400 μg/ml), and the G418-resistant colonies were picked up 2 weeks later. Construction of Syndecan-2 Core Protein and a Mutant of It—cDNA of mouse syndecan-2 core protein containing the entire coding region was generated as described previously (15Munesue S. Kusano Y. Oguri K. Itano N. Yoshitomi Y. Nakanishi H. Yamashina I. Okayama M. Biochem. J. 2002; 363: 201-209Crossref PubMed Scopus (63) Google Scholar). The mutant core protein of syndecan-2, of which the glycosaminoglycan-attachable serine residues were changed to alanine residues (S41A, S53A, S55A, and S57A), was generated using a TaKaRa Mutan-Super Express Km Kit (TaKaRa BIO Inc.). The four mutagenic primers that changed thymine to guanine were primer A (5′-GAG GAA GCT CA GGA GTA TA-3′), primer B (5′-TGA CTA TTC TC TGC CTC AG-3′), primer C (5′-TAT TCT CT GCC GCA GGC T C-3′), and primer D (5′-AGG CC AGG AGC TGA TGA AT-3′). The double underlining in the sequences indicates the altered residues. First, primer A was used to mutate codon 41 of syndecan-2 cDNA cloned into the pkF19k vector (TaKaRa BIO Inc.) by the oligonucleotide-directed dual amber-long and accurate (ODA-LA) PCR method. After confirmation of the sequence of the mutant cDNA, primers B, C, and D were used similarly to mutate codons 53, 55, and 57 of the cDNA in that order. Finally, the mutant syndecan-2/pkF19k was inserted into pcDNA3 (Promega Corp.) at a site downstream of the cytomegalovirus promoter. The nucleotide sequence of the cloned mutant syndecan-2 cDNA was verified with an ABI PRISM 310 sequencer (Applied Biosystems). Treatment of Cells with Antisense Oligonucleotides of Syndecan-2—Treatments of P29 cells with antisense or sense oligonucleotides of syndecan-2 were performed as described previously (14Kusano Y. Oguri K. Nagayasu Y. Munesue S. Ishihara M. Saiki I. Yonekura H. Yamamoto H. Okayama M. Exp. Cell Res. 2000; 256: 434-444Crossref PubMed Scopus (87) Google Scholar). Briefly, antisense phosphorothioate oligonucleotides complementary to the region around the initiation codon of mouse syndecan-2 mRNA (5′-CAC GCG CGC TGC ATA TT-3′) and the corresponding sense (5′-AAT ATG CAG CGC GCG TG-3′) and scrambled antisense (5′-ACGT CCC TGA GCG ATC T-3′) phosphorothioate oligonucleotides were synthesized using a model 392 DNA synthesizer (Applied Biosystems Inc.). The oligodeoxynucleotides were purified by two cycles of reverse-phase high pressure liquid chromatography. P29 cells were cultured in 10% fetal bovine serum/Dulbecco's modified Eagle's medium containing 10 μm oligonucleotides for 4 days and then cultured for another 1 day in the medium without fetal bovine serum. The conditioned media were concentrated for gelatin zymography. Antibodies—Rabbit polyclonal antibodies, SN1Ab, SN2Ab, SN3Ab, and SN4Ab, specific to ectodomain recombinants of the mouse syndecan-1, -2, -3, and -4 core proteins, respectively, were prepared as described previously (14Kusano Y. Oguri K. Nagayasu Y. Munesue S. Ishihara M. Saiki I. Yonekura H. Yamamoto H. Okayama M. Exp. Cell Res. 2000; 256: 434-444Crossref PubMed Scopus (87) Google Scholar, 16Kusano Y. Yoshitomi Y. Munesue S. Okayama M. Oguri K. J. Biochem. (Tokyo). 2004; 135: 129-137Crossref PubMed Scopus (32) Google Scholar). The commercially available antibodies used were as follows: goat anti-human integrin α5β1 antibodies (Chemicon International); mouse monoclonal antibodies F58-10E4, specific to heparan sulfate, and F69-3G10, specific to the unsaturated nonreducing end of heparan sulfate chain, generated upon heparitinase digestion (Seikagaku Corp.); and mouse monoclonal antibody F-68 anti-human MMP-2 reactive to mouse MMP-2 (DAIICHI Fine Chemical Co., Ltd.). Flow Cytometric Assay—Subconfluent cell layers were washed with Dulbecco's modified Eagle's medium and then digested with or without 0.1 unit/ml of heparitinase-I (EC 4.2.2.8; Seikagaku) for 24 h at 37 °C. Cells were harvested with EDTA/PBS, suspended in 0.2% bovine serum albumin/Dulbecco's modified Eagle's medium (3 × 105 cells/50 μl), and then incubated with antibodies or the respective nonimmune serum or Ig for 1 h at 4 °C with gentle agitation. After washing three times with PBS, they were exposed to an fluorescein isothiocyanate-conjugated second antibody for 30 min. The labeled cells were washed, and the intensity of fluorescence was measured with a flow cytometer, FACSort (BD Biosciences). Metastasis Assay—The metastasis assay was performed as described previously (28Nakanishi H. Takenaga K. Oguri K. Yoshida A. Okayama M. Virchows Arch. A. 1992; 420: 163-170Crossref Scopus (22) Google Scholar). Briefly, cells (2 × 105 cells) suspended in 0.2 ml of PBS were injected into the tail veins of 6-week-old male C57BL/6 mice for experimental metastasis or subcutaneously into the right abdominal flank for spontaneous metastasis. The animals were sacrificed 4 weeks later, and the numbers of visible nodules in lungs fixed in Bouin's solution were determined. Northern Blot Analysis—Poly(A)+ RNA was isolated from 1 × 107 cells of each clone, using a QuickPrep mRNA Purification Kit (GE Healthcare Bio-Sciences Corp.). Northern blot analysis was performed as described previously (15Munesue S. Kusano Y. Oguri K. Itano N. Yoshitomi Y. Nakanishi H. Yamashina I. Okayama M. Biochem. J. 2002; 363: 201-209Crossref PubMed Scopus (63) Google Scholar). In brief, 2 μg of each poly(A)+ RNA from cells was electrophoresed on a 1.0% agarose gel containing 1.1 m formaldehyde, transferred to a Hybond N+ nylon membrane (GE Healthcare Bio-Sciences Corp.), and then hybridized to a cDNA fragment of mouse syndecan-1 (nucleotides 409-967), syndecan-2 (nucleotides 487-1175), syndecan-3 (nucleotides 370-931), syndecan-4 (nucleotides 92-534), MMP-2 (nucleotides 683-1484), MT1-MMP (nucleotides 1115-1696), TIMP-2 (nucleotides 227-691), β-actin (nucleotides 183-722), or glyceraldehyde-3-phosphate dehydrogenase (nucleotides 319-1047), which had been labeled with [α-32P]dCTP by the random labeling method. After hybridization for 14 h at 42 °C, the membranes were exposed to x-ray films, and the films were scanned with a CanoScan 600 (Canon). Quantification of the bands was performed with the public domain NIH Image program in the 256-grayscale mode. Western Blot Analysis—Cell surface proteoglycans were extracted from cell layers with 2% Triton X-100, 25 mm KCl, 50 mm Tris-HCl, pH 7.3, containing 10 mm EDTA, 10 mm N-ethylmaleimide, 1 mm phenylmethylsulfonyl fluoride, 0.036 mm pepstatin A as proteinase inhibitors for 12 h on ice and then purified as described previously (14Kusano Y. Oguri K. Nagayasu Y. Munesue S. Ishihara M. Saiki I. Yonekura H. Yamamoto H. Okayama M. Exp. Cell Res. 2000; 256: 434-444Crossref PubMed Scopus (87) Google Scholar). Samples were digested with heparitinase-I plus chondroitinase ABC to remove glycosaminoglycan side chains (29Itano N. Oguri K. Nakanishi H. Okayama M. J. Biochem. (Tokyo). 1993; 114: 862-873Crossref PubMed Scopus (14) Google Scholar) and then subjected to SDS-PAGE, followed by transfer to Hybond-P membranes (GE Healthcare Bio-Sciences Corp.). The membranes were blocked with 3% bovine serum albumin in PBST for 1 h and then reacted with antibodies for 1 h. After washing with PBST, the membranes were reacted with horseradish peroxidase-conjugated second antibodies for 1 h and then stained with Immunostain (Konica) or ECL detection reagent (GE Healthcare Bio-Sciences Corp.). Gelatin Zymography—Gelatin zymography was conducted on an SDS-polyacrylamide gel containing gelatin (1 mg/ml) as described previously (23Sato H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. Nature. 1994; 370: 61-65Crossref PubMed Scopus (2368) Google Scholar). Briefly, conditioned media in which cells were cultured for 15 h without fetal bovine serum were concentrated with a Microcon YM-30 centrifugal filter device (Millipore), adjusted to the SDS-PAGE loading buffer without the reducing agent, and then subjected to electrophoresis through a gel containing gelatin at room temperature. After electrophoresis, the gels were washed twice for 30 min at 37 °C with 2.5% Triton X-100, 10 mm Tris-HCl, pH 8.0, and then incubated in 1 μm ZnCl2, 10 mm CaCl2, 0.2 m NaCl, 50 mm Tris-HCl, pH 8.0, for 18 h at 37 °C. Enzyme activity was visualized as negative staining with Coomassie Brilliant Blue R250. The band density ratios, active form to latent form, were calculated according to Isnard et al. (30Isnard N. Robert L. Renard G. Cell Biol. Int. 2003; 27: 779-784Crossref PubMed Scopus (47) Google Scholar). If necessary, the following polypeptides were used as a substratum; human plasma fibronectin (Iwaki Glass) and its recombinant polypeptides (i.e. the RGD-containing cell-binding domain C-274, the C-terminal heparin-binding domain H-271, and CH-271, a fusion peptide of C-274 and H-271) (TaKaRa Bio Inc.) (14Kusano Y. Oguri K. Nagayasu Y. Munesue S. Ishihara M. Saiki I. Yonekura H. Yamamoto H. Okayama M. Exp. Cell Res. 2000; 256: 434-444Crossref PubMed Scopus (87) Google Scholar). Affinity Chromatography of MMP-2 on a Heparin-Sepharose CL-6B Column—The conditioned medium of H11 cells (1 × 108 cells) was applied to a column (1 × 9 cm) of Heparin-Sepharose CL-6B (GE Healthcare Bio-Sciences Corp.) equilibrated with 10 mm phosphate buffer, pH 7.3. The column was then washed extensively with the equilibrating buffer. The bound material was eluted with a linear gradient of 0-1.0 m NaCl in the same buffer, 1-ml fractions being collected. The conductivity of each fraction was measured by CD-35MII (M & S Instruments Inc.). Each fraction was concentrated and then subjected to gelatin zymography as described above. Surface Plasmon Resonance Binding Analysis—Surface plasmon resonance measurements were carried out with a Biacore X system (Biacore AB). A C1 biosensor chip was activated according to the manufacturer's recommendation (Biacore AB). 1340 response units of streptavidin (Sigma) was coupled to the surface of the C1 biosensor chip. Then 150 response units of biotinylated heparin (Seikagaku), produced as described previously (31Yang B. Yang B.L. Goetinck P.F. Anal. Biochem. 1995; 228: 299-306Crossref PubMed Scopus (44) Google Scholar), was captured by streptavidin on the chip. The amount of biotinylated heparin coupled to the sensor surface was limited to avoid mass transfer limitation. Kinetic binding analysis was performed in 10 mm HEPES buffer containing 150 mm NaCl and 5 mm CaCl2, pH 7.4, at a constant flow rate of 5 μl/min, the instrument being equilibrated at 25 °C. Mouse calvaria-derived pro-MMP-2 (Calbiochem) was perfused at various concentrations. At least two different replicated experiments were performed. Response curves were generated by subtraction of the background signal generated simultaneously in the control flow cell. Kinetic parameters were obtained by fitting of the sensorgrams to a 1:1 (Langmuir) binding model using the BIAevaluation 3.1 software (Biacore AB). Inverse Correlation between Metastatic Potential and Syndecan-2 Expression Level in Clones Derived from Lewis Lung Carcinoma 3LL—Our previous paper (16Kusano Y. Yoshitomi Y. Munesue S. Okayama M. Oguri K. J. Biochem. (Tokyo). 2004; 135: 129-137Crossref PubMed Scopus (32) Google Scholar) showed that Lewis lung carcinoma 3LL-derived low metastatic P29 and highly metastatic LM66-H11 (abbreviated as H11 hereafter) clones expressed three members of the syndecan family (i.e. syndecan-1, -2, and -4). Among them the expression level of only syndecan-2, a key molecule for regulation of actin cytoskeletal organization in cooperation with integrin α5β1, was different between the clones, although the expression levels of the other two syndecans were very similar in the two clones (14Kusano Y. Oguri K. Nagayasu Y. Munesue S. Ishihara M. Saiki I. Yonekura H. Yamamoto H. Okayama M. Exp. Cell Res. 2000; 256: 434-444Crossref PubMed Scopus (87) Google Scholar, 15Munesue S. Kusano Y. Oguri K. Itano N. Yoshitomi Y. Nakanishi H. Yamashina I. Okayama M. Biochem. J. 2002; 363: 201-209Crossref PubMed Scopus (63) Google Scholar). In this study, to verify the correlation between the metastatic potential and the expression level of syndecan-2, another clone, LM12-3, with intermediate metastatic potential (28Nakanishi H. Takenaga K. Oguri K. Yoshida A. Okayama M. Virchows Arch. A. 1992; 420: 163-170Crossref Scopus (22) Google Scholar), was used. As shown in Table 1, both the experimental and spontaneous metastatic potentials of the three clones used gradually increased in the order of P29 < LM12-3 < H11. Northern blot analysis clearly showed that the expression levels of mRNA of syndecan-1 (2.6 kbp) and -4 (5.6 kbp) were similar among the three clones, but that of only syndecan-2 was different (Fig. 1A, left). Quantification of the densities of the bands followed by normalization as to the respective β-actin bands revealed that syndecan-2 mRNA linearly decreased as the metastatic potential of the clones increased (Fig. 1A, right). These results were very consistent with the results of flow cytometric analysis showing cell surface expression of the syndecan family in the clones (Fig. 1B). Syndecan-3 expression was hardly detected on any of these clones, and the expression levels of syndecan-1 and -4 were not significantly different among the clones. In contrast, syndecan-2 expression decreased inversely with the metastatic potentials of the clones. Since it became clear that there is an inverse correlation between the syndecan-2 expression level and the metastatic potential in these clones, next the causality underlying this inverse correlation was investigated.TABLE 1Experimental and spontaneous metastatic potentials of the three clones derived from Lewis lung carcinoma 3LLInjection and cloneIncidenceNumber of lung fociRangeMeanIntravenousP293/70-10.4LM12-37/719-9247.9LM66-H117/7467-744516.2SubcutaneousP290/700LM12-36/70-144.5LM66-H117/723-5635.2 Open table in a new tab Causal Relationship of the Inverse Correlation between the Metastatic Potential and Syndecan-2 Expression—In order to examine the causality, a stable transfectant, the H11-SN2 clone, which was obtained upon transfection of syndecan-2 core protein cDNA into H11 cells, and a mock transfectant, H11-Vec (15Munesue S. Kusano Y. Oguri K. Itano N. Yoshitomi Y. Nakanishi H. Yamashina I. Okayama M. Biochem. J. 2002; 363: 201-209Crossref PubMed Scopus (63) Google Scholar), were used. Northern blot analysis confirmed that the expression of syndecan-2 mRNA in the H11-SN2 clone was elevated, although the level of H11-Vec was similar to that in the parent H11 cells (Fig. 2A). Western blot analysis of the core proteins obtained upon digestion of samples with heparitinase-I plus chond
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