Hyaluronan Oligosaccharides Induce CD44 Cleavage and Promote Cell Migration in CD44-expressing Tumor Cells
2003; Elsevier BV; Volume: 278; Issue: 34 Linguagem: Inglês
10.1074/jbc.m300347200
ISSN1083-351X
AutoresKazuki N. Sugahara, Toshiyuki Murai, Hitomi Nishinakamura, Hiroto Kawashima, Hideyuki Saya, Masayuki Miyasaka,
Tópico(s)Fibroblast Growth Factor Research
ResumoCD44 is an adhesion molecule that serves as a cell surface receptor for several extracellular matrix components, including hyaluronan (HA). The proteolytic cleavage of CD44 from the cell surface plays a critical role in the migration of tumor cells. Although this cleavage can be induced by certain stimuli such as phorbol ester and anti-CD44 antibodies in vitro, the physiological inducer of CD44 cleavage in vivo is unknown. Here, we demonstrate that HA oligosaccharides of a specific size range induce CD44 cleavage from tumor cells. Fragmented HA containing 6-mers to 14-mers enhanced CD44 cleavage dose-dependently by interacting with CD44, whereas a large polymer HA failed to enhance CD44 cleavage, although it bound to CD44. Examination using uniformly sized HA oligosaccharides revealed that HAs smaller than 36 kDa significantly enhanced CD44 cleavage. In particular, the 6.9-kDa HA (36-mers) not only enhanced CD44 cleavage but also promoted tumor cell motility, which was completely inhibited by an anti-CD44 monoclonal antibody. These results raise the possibility that small HA oligosaccharides, which are known to occur in various tumor tissues, promote tumor invasion by enhancing the tumor cell motility that may be driven by CD44 cleavage. CD44 is an adhesion molecule that serves as a cell surface receptor for several extracellular matrix components, including hyaluronan (HA). The proteolytic cleavage of CD44 from the cell surface plays a critical role in the migration of tumor cells. Although this cleavage can be induced by certain stimuli such as phorbol ester and anti-CD44 antibodies in vitro, the physiological inducer of CD44 cleavage in vivo is unknown. Here, we demonstrate that HA oligosaccharides of a specific size range induce CD44 cleavage from tumor cells. Fragmented HA containing 6-mers to 14-mers enhanced CD44 cleavage dose-dependently by interacting with CD44, whereas a large polymer HA failed to enhance CD44 cleavage, although it bound to CD44. Examination using uniformly sized HA oligosaccharides revealed that HAs smaller than 36 kDa significantly enhanced CD44 cleavage. In particular, the 6.9-kDa HA (36-mers) not only enhanced CD44 cleavage but also promoted tumor cell motility, which was completely inhibited by an anti-CD44 monoclonal antibody. These results raise the possibility that small HA oligosaccharides, which are known to occur in various tumor tissues, promote tumor invasion by enhancing the tumor cell motility that may be driven by CD44 cleavage. CD44 is a widely distributed cell adhesion protein that serves as a cell surface receptor for several extracellular matrix components, including hyaluronan (HA) 1The abbreviations used are: HA, hyaluronan; PMA, phorbol myristate acetate; mAb, monoclonal antibody; pAb, polyclonal antibody; HA-n, hyaluronan n-mers; FL-HA, fluorescein-conjugated hyaluronan; HPLC, high performance liquid chromatography; ELISA, enzyme-linked immunosorbent assay; frHA, fragmented hyaluronan; PBS, phosphate-buffered saline; BSA, bovine serum albumin.1The abbreviations used are: HA, hyaluronan; PMA, phorbol myristate acetate; mAb, monoclonal antibody; pAb, polyclonal antibody; HA-n, hyaluronan n-mers; FL-HA, fluorescein-conjugated hyaluronan; HPLC, high performance liquid chromatography; ELISA, enzyme-linked immunosorbent assay; frHA, fragmented hyaluronan; PBS, phosphate-buffered saline; BSA, bovine serum albumin. (1Aruffo A. Stamenkovic I. Melnick M. Underhill C.B. Seed B. Cell. 1990; 61: 1303-1313Abstract Full Text PDF PubMed Scopus (2134) Google Scholar). CD44 participates in various biological processes, such as lymphocyte rolling, tumor cell migration, and invasion (2Naor D. Sionov R.V. Ish-Shalom D. Adv. Cancer Res. 1997; 71: 241-319Crossref PubMed Google Scholar). The cell surface CD44 is proteolytically cleaved at the extracellular domain by membrane-bound metalloproteases such as MT1-MMP (3Kajita M. Itoh Y. Chiba T. Mori H. Okada A. Kinoh H. Seiki M. J. Cell Biol. 2001; 153: 893-904Crossref PubMed Scopus (610) Google Scholar); this cleavage has been suggested to play an important role in tumor cell migration along extracellular matrix components (4Okamoto I. Kawano Y. Tsuiki H. Sasaki J. Nakao M. Matsumoto M. Suga M. Ando M. Nakajima M. Saya H. Oncogene. 1999; 18: 1435-1446Crossref PubMed Scopus (214) Google Scholar, 5Kawano Y. Okamoto I. Murakami D. Itoh H. Yoshida M. Ueda S. Saya H. J. Biol. Chem. 2000; 275: 29628-29635Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Indeed, enhanced CD44 cleavage has been reported in invasive tumors such as gliomas, breast carcinomas, non-small cell lung carcinomas, colon carcinomas, and ovarian carcinomas (6Okamoto I. Tsuiki H. Kenyon L.C. Godwin A.K. Emlet D.R. Holgado-Madruga M. Lanham I.S. Joynes C.J. Vo K.T. Guha A. Matsumoto M. Ushio Y. Saya H. Wong A.J. Am. J. Pathol. 2002; 160: 441-447Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Although several reagents such as phorbol myristate acetate (PMA) (7Okamoto I. Kawano Y. Matsumoto M. Suga M. Kaibuchi K. Ando M. Saya H. J. Biol. Chem. 1999; 274: 25525-25534Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar) and an anti-CD44 monoclonal antibody (mAb), IM7 (8Shi M. Dennis K. Peschon J.J. Chandrasekaran R. Mikecz K. J. Immunol. 2001; 167: 123-131Crossref PubMed Scopus (55) Google Scholar), can potently induce CD44 cleavage in vitro, the physiological inducer of CD44 cleavage in vivo remains uncharacterized.HA is a nonsulfated linear glycosaminoglycan that consists of repeating disaccharide units of d-glucuronic acid and N-acetyl-d-glucosamine (9Laurent T.C. Fraser J.R. FASEB J. 1992; 6: 2397-2404Crossref PubMed Scopus (2052) Google Scholar). Accumulating evidence indicates that HA not only acts as an extracellular matrix component but also participates in a number of physiological events such as cell adhesion, migration, and proliferation (10Toole B.P. J. Intern. Med. 1997; 242: 35-40Crossref PubMed Scopus (278) Google Scholar). HA also plays a role in pathological conditions, including cancer. An increased synthesis of HA has been reported in various malignant tumors (11Ropponen K. Tammi M. Parkkinen J. Eskelinen M. Tammi R. Lipponen P. Ågren U. Alhava E. Kosma V.M. Cancer Res. 1998; 58: 342-347PubMed Google Scholar, 12Auvinen P. Tammi R. Parkkinen J. Tammi M. Ågren U. Johansson R. Hirvikoski P. Eskelinen M. Kosma V.M. Am. J. Pathol. 2000; 156: 529-536Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar). HA enhances tumor cell adhesion and migration (13Itano N. Atsumi F. Sawai T. Yamada Y. Miyaishi O. Senga T. Hamaguchi M. Kimata K. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 3609-3614Crossref PubMed Scopus (263) Google Scholar) and activates the Ras-mitogen-activated protein kinase pathway as well as the phosphoinositide 3-kinase pathway (14Sohara Y. Ishiguro N. Machida K. Kurata H. Thant A.A. Senga T. Matsuda S. Kimata K. Iwata H. Hamaguchi M. Mol. Biol. Cell. 2001; 12: 1859-1868Crossref PubMed Scopus (89) Google Scholar). HA forms a protective barrier around tumor cells, which may help the tumor cells to evade immune surveillance (15Toole B.P. Glycobiology. 2002; 12: 37R-42RCrossref PubMed Scopus (180) Google Scholar). Although HA usually exists as a high molecular mass polymer (in excess of 1000 kDa) as a component of the extracellular matrix under physiological conditions (9Laurent T.C. Fraser J.R. FASEB J. 1992; 6: 2397-2404Crossref PubMed Scopus (2052) Google Scholar), HA of a much lower molecular mass is detected in association with certain pathological conditions, such as inflammation (16Balazs E.A. Watson D. Duff I.F. Roseman S. Arthritis Rheum. 1967; 10: 357-376Crossref PubMed Scopus (465) Google Scholar) and tumors (17Dahl I.M. Laurent T.C. Cancer. 1988; 62: 326-330Crossref PubMed Scopus (66) Google Scholar, 18Kumar S. West D.C. Ponting J.M. Gattamaneni H.R. Int. J. Cancer. 1989; 44: 445-448Crossref PubMed Scopus (66) Google Scholar, 19Lokeshwar V.B. Öbek C. Soloway M.S. Block N.L. Cancer Res. 1997; 57: 773-777PubMed Google Scholar, 20Lokeshwar V.B. Rubinowicz D. Schroeder G.L. Forgacs E. Minna J.D. Block N.L. Nadji M. Lokeshwar B.L. J. Biol. Chem. 2001; 276: 11922-11932Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar). It is becoming increasingly clear that the low molecular mass HA fragments can induce a variety of biological events, such as chemokine gene expression (21McKee C.M. Penno M.B. Cowman M. Burdick M.D. Strieter R.M. Bao C. Noble P.W. J. Clin. Invest. 1996; 98: 2403-2413Crossref PubMed Scopus (687) Google Scholar, 22Horton M.R. McKee C.M. Bao C. Liao F. Farber J.M. Hodge-DuFour J. Pure E. Oliver B.L. Wright T.M. Noble P.W. J. Biol. Chem. 1998; 273: 35088-35094Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 23Horton M.R. Burdick M.D. Strieter R.M. Bao C. Noble P.W. J. Immunol. 1998; 160: 3023-3030PubMed Google Scholar), activation of transcription factors such as NF-κB (24Noble P.W. McKee C.M. Cowman M. Shin H.S. J. Exp. Med. 1996; 183: 2373-2378Crossref PubMed Scopus (277) Google Scholar), cell proliferation (19Lokeshwar V.B. Öbek C. Soloway M.S. Block N.L. Cancer Res. 1997; 57: 773-777PubMed Google Scholar, 25Slevin M. Kumar S. Gaffney J. J. Biol. Chem. 2002; 277: 41046-41059Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar), and angiogenesis (26West D.C. Hampson I.N. Arnold F. Kumar S. Science. 1985; 228: 1324-1326Crossref PubMed Scopus (961) Google Scholar, 27Rooney P. Wang M. Kumar P. Kumar S. J. Cell Sci. 1993; 105: 213-218Crossref PubMed Google Scholar, 28Sattar A. Rooney P. Kumar S. Pye D. West D.C. Scott I. Ledger P. J. Invest. Dermatol. 1994; 103: 576-579Abstract Full Text PDF PubMed Scopus (155) Google Scholar, 29Montesano R. Kumar S. Orci L. Pepper M.S. Lab. Invest. 1996; 75: 249-262PubMed Google Scholar) and that they are more potent than the high molecular mass HA in mediating these biological processes. High levels of angiogenic HA fragments are detected in several human tumors, such as bladder cancers (19Lokeshwar V.B. Öbek C. Soloway M.S. Block N.L. Cancer Res. 1997; 57: 773-777PubMed Google Scholar), prostate cancers (20Lokeshwar V.B. Rubinowicz D. Schroeder G.L. Forgacs E. Minna J.D. Block N.L. Nadji M. Lokeshwar B.L. J. Biol. Chem. 2001; 276: 11922-11932Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar), Wilms' tumors (18Kumar S. West D.C. Ponting J.M. Gattamaneni H.R. Int. J. Cancer. 1989; 44: 445-448Crossref PubMed Scopus (66) Google Scholar), and mesothelioma (17Dahl I.M. Laurent T.C. Cancer. 1988; 62: 326-330Crossref PubMed Scopus (66) Google Scholar), and interestingly, high levels of hyaluronidase activity are also found in bladder and prostate cancer (20Lokeshwar V.B. Rubinowicz D. Schroeder G.L. Forgacs E. Minna J.D. Block N.L. Nadji M. Lokeshwar B.L. J. Biol. Chem. 2001; 276: 11922-11932Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar, 30Pham H.T. Block N.L. Lokeshwar V.B. Cancer Res. 1997; 57: 778-783PubMed Google Scholar), raising the interesting possibility that the autoregulatory degradation of HA in tumor tissues may enhance tumor invasion and metastasis.These reports have led us to speculate that HA and/or its degradation products may be involved in the CD44 cleavage that enhances tumor motility. We herein demonstrate that small HA oligosaccharides but not large HA polymers efficiently induce CD44 cleavage and also promote tumor cell migration in a CD44-dependent fashion. Our results suggest that HA fragments may serve as a physiological inducer of CD44 cleavage in vivo and reinforce the notion that HA degradation products play an important role in tumor invasion.EXPERIMENTAL PROCEDURESReagents—A rabbit polyclonal antibody (pAb), anti-CD44cyto pAb, which is directed against the cytoplasmic domain of CD44, was raised as described previously (4Okamoto I. Kawano Y. Tsuiki H. Sasaki J. Nakao M. Matsumoto M. Suga M. Ando M. Nakajima M. Saya H. Oncogene. 1999; 18: 1435-1446Crossref PubMed Scopus (214) Google Scholar). An anti-human CD44 mAb, BRIC235, was purchased from the International Blood Group Reference Laboratory (Bristol, UK). An anti-β-tubulin mAb was purchased from Calbiochem (Cambridge, MA). A mouse IgG was purchased from Sigma-Aldrich. Horseradish peroxidase-conjugated anti-rabbit IgG and horseradish peroxidase-conjugated anti-mouse IgG were purchased from American Qualex (San Clemente, CA). HA oligosaccharides (HA-2, HA-4, HA-6, HA-8, HA-10, HA-12, 6.9-kDa HA, and 36-kDa HA) and fluorescein-conjugated HA (FL-HA) were generously provided by Seikagaku Kogyo Co. (Tokyo, Japan) (31Tawada A. Masa T. Oonuki Y. Watanabe A. Matsuzaki Y. Asari A. Glycobiology. 2002; 12: 421-426Crossref PubMed Scopus (82) Google Scholar). Human umbilical cord HA that mainly consists of 200-kDa HA and human umbilical cord HA that mainly consists of 1000-kDa HA were purchased from ICN Biomedicals (Costa Mesa, CA) and Sigma-Aldrich, respectively. Sheep testicular hyaluronidase and hyaluronidase from Streptococcus dysgalactiae (hyaluronidase SD) were purchased from Sigma-Aldrich and Seikagaku Kogyo Co., respectively. Carbobenzoxyl-leucinyl-leucinyl-leucinal (MG132) was from the Peptide Institute (Osaka, Japan), and PMA was from Sigma-Aldrich.Cell Culture—The human pancreatic carcinoma cell line MIA PaCa-2 was obtained from the Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University (Sendai, Japan). The cells were grown in RPMI 1640 medium (Sigma-Aldrich) supplemented with 10% fetal calf serum, 1% (v/v) 100× nonessential amino acids, 1 mm sodium pyruvate, 2 mml-glutamine, 50 μm 2-mercaptoethanol, 100 units/ml penicillin, and 100 μg/ml streptomycin at 37 °C in an atmosphere containing 5% CO2.Fluorescein Isothiocyanate Conjugation to BRIC235—The BRIC235 mAb solution was first dialyzed against a 0.1 m sodium carbonate buffer, pH 9.2. A 400-μl aliquot of 1.4 mg/ml BRIC235 was incubated with 28 μg of fluorescein isothiocyanate (Dojindo, Kumamoto, Japan) for 4 h at room temperature. The solution was applied to a PD-10 column (Amersham Biosciences) equilibrated with PBS containing 0.05% sodium azide. The eluates were monitored by absorbance at 280 nm.BRIC235 Fab Generation and Purification—BRIC235 Fab fragments were generated and purified using the ImmunoPure Fab preparation kit (Pierce) according to the manufacturer's instructions. Briefly, the BRIC235 antibody solution was extensively dialyzed against a 20 mm sodium phosphate, 10 mm EDTA buffer at pH 7.0. After concentration to ∼20 mg/ml, the antibody was digested by incubation with immobilized papain for 5 h at 37 °C. The Fab fragments were then separated using a protein A column and dialyzed against PBS overnight at 4 °C.Preparation of HA Fragments—The 200-kDa HA (200 mg) was digested with 3,600 units of sheep testicular hyaluronidase at 37 °C for 19 h, and the mixture of HA fragments was separated on a Bio-Gel P-10 column (1.5 cm × 100 cm; Bio-Rad) that had been equilibrated with 1 m NaCl containing 10% ethanol. The column fractions were applied to a Sephadex G-25 column (1 cm × 50 cm; Bio-Rad) equilibrated with distilled water for desalting, and the eluates were analyzed by high performance liquid chromatography (HPLC). HPLC analysis was performed with the GULLIVER system (Nippon Bunko Engineering, Tokyo, Japan) on a YMC-Pack PA-03 column (YMC, Wilmington, NC; 4.6 mm × 250 mm) using a programmed linear gradient elution from 16 mm to 1 m NaH2PO4 over 70 min at a flow rate of 1.0 ml/min. The eluates were monitored by absorbance at 210 nm. The HA fragments (10 mg) ranging from HA 6-mers to 14-mers were digested with 0.1 unit of hyaluronidase SD at 37 °C for 3 h and then applied to the Sephadex G-25 column equilibrated with distilled water for desalting. The eluates were analyzed by HPLC as described above. In some experiments, hyaluronidase SD was heat-inactivated by boiling before use.CD44 Cleavage Assay—MIA PaCa-2 cells were seeded into 24-well plates at 5 × 104 cells/well, cultured overnight at 37 °C, and then incubated with 10 μm MG132 for 30 min at 37 °C to inhibit the secondary cleavage of the CD44 intracellular domain (32Okamoto I. Kawano Y. Murakami D. Sasayama T. Araki N. Miki T. Wong A.J. Saya H. J. Cell Biol. 2001; 155: 755-762Crossref PubMed Scopus (303) Google Scholar). The cells were then incubated with HA for 1 h or with 100 ng/ml PMA for 30 min at 37 °C in the presence of 10 μm MG132, and the culture supernatants were collected for analysis by enzyme-linked immunosorbent assay (ELISA). The cells were lysed with SDS sample buffer (2% SDS, 10% glycerol, 0.1 m dithiothreitol, 120 mm Tris-HCl, pH 6.8, 0.02% bromphenol blue) and boiled for 5 min. Samples extracted from equal numbers of cells were separated by electrophoresis on an SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride filter. The filter was blocked in PBS containing 3% BSA and then incubated with anti-CD44cyto pAb or with anti-β-tubulin mAb. The filters were then incubated with horseradish peroxidase-conjugated anti-rabbit IgG to detect the anti-CD44cyto pAb or with horseradish peroxidase-conjugated anti-mouse IgG to detect the anti-β-tubulin mAb. The secondary Abs were detected using ECL Western blotting detection reagents (Amersham Biosciences). The densitometric analysis of the bands was performed on an image analysis program (NIH Image; National Institutes of Health, Bethesda, MD). For ELISA, the culture supernatants from the stimulated cells were filtered using a 0.22-μm Millipore filter (Millipore Co., Bedford, MA) before analysis. Soluble human CD44 in the samples was quantified using a soluble CD44H ELISA kit (Bender MedSystems, Vienna, Austria) according to the manufacturer's instructions.Flow Cytometry—The binding of FL-HA to the cell surface was determined as reported previously using an EPICS XL flow cytometer (Coulter, Hialeah, FL) (33Ishiwatari-Hayasaka H. Fujimoto T. Osawa T. Hirama T. Toyama-Sorimachi N. Miyasaka M. J. Immunol. 1999; 163: 1258-1264PubMed Google Scholar). The binding of FL-HA to CD44 was verified by its inhibition with the anti-CD44 mAb BRIC235. The binding of the HAs to the cell surface was analyzed by observing the inhibition of FL-HA binding upon the addition of the HAs as follows. One million cells were incubated with serial dilutions of the HAs for 20 min at 4 °C, then 1.0 μg/ml of FL-HA was added, and the cells were incubated for another 30 min at 4 °C, followed by two washes with cold PBS containing 0.1% BSA. The cells were then analyzed by flow cytometry.Immunofluorescence Microscopy—MIA PaCa-2 cells were seeded at a concentration of 3 × 105 cells/well on 1000-kDa HA-coated cover glasses placed in 6-well plates and incubated overnight at 37 °C. The medium was changed to serum-free RPMI medium containing the same supplements as above, and then the cells were incubated with or without 100 ng/ml PMA for 30 min or 6.9-kDa HA for 1 h. In this assay, the cells were not pretreated with a proteasome inhibitor MG132. The cells were then fixed with 4% paraformaldehyde/PBS for 10 min followed by treatment with 0.2% Triton X-100/PBS for 5 min and blocked in PBS containing 1% BSA for 30 min at room temperature. After being washed with PBS, the cells were incubated with the anti-CD44cyto pAb for 1 h at room temperature and then with fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) and rhodamine-conjugated phalloidin (Molecular Probes, Eugene, OR) for 1 h at room temperature. After being washed with PBS, the samples were mounted in Prolong Antifade (Molecular Probes) and visualized with an LSM 410 confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany).Migration Assay—Cell migration was analyzed using 24-well Costar Transwell chambers (Corning Inc., Corning, NY) containing polycarbonate filters with an 8-μm pore size. Both sides of the filter were coated with 100 μg/ml of 1000-kDa HA. The lower compartment of the chamber was filled with 600 μl of RPMI medium containing 0.1% BSA, and the filters were placed into the chamber. MIA PaCa-2 cells at a logarithmic phase of growth were detached by brief exposure to trypsin-EDTA and resuspended at 2 × 105 cells/ml in RPMI medium containing 0.1% BSA. A 100-μl aliquot of the cell suspension was added to the upper compartment. After incubation at 37 °C for 3 h so that the cells become attached to the filter, the cells were incubated with or without 10 μg/ml BRIC235 or mouse IgG in RPMI medium containing 0.1% BSA 20 min prior to and during the migration assay. Finally, HAs or PBS were added to the upper compartment of the wells at the final concentration of 50 μg/ml. The chambers were subsequently incubated at 37 °C in a 5% CO2 atmosphere for 24 h. As in the immunofluorescence microscopy, the cells were not treated with MG132. After the cells on the upper side of the filters were gently wiped off, the filters were fixed in methanol, stained with hematoxylin and eosin, and mounted on glass slides. The cells that had migrated to the lower side of the filters were counted under a light microscope. The number of cells in five defined high power fields (×200) was counted, and the average was determined. Each assay was performed five times.RESULTSHA Fragments Induce CD44 Cleavage in a Human Pancreatic Carcinoma Cell Line, MIA PaCa-2—The degradation products of HA have been shown to have a variety of biological activities in CD44-expressing cells (21McKee C.M. Penno M.B. Cowman M. Burdick M.D. Strieter R.M. Bao C. Noble P.W. J. Clin. Invest. 1996; 98: 2403-2413Crossref PubMed Scopus (687) Google Scholar, 22Horton M.R. McKee C.M. Bao C. Liao F. Farber J.M. Hodge-DuFour J. Pure E. Oliver B.L. Wright T.M. Noble P.W. J. Biol. Chem. 1998; 273: 35088-35094Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 24Noble P.W. McKee C.M. Cowman M. Shin H.S. J. Exp. Med. 1996; 183: 2373-2378Crossref PubMed Scopus (277) Google Scholar, 25Slevin M. Kumar S. Gaffney J. J. Biol. Chem. 2002; 277: 41046-41059Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar, 26West D.C. Hampson I.N. Arnold F. Kumar S. Science. 1985; 228: 1324-1326Crossref PubMed Scopus (961) Google Scholar, 27Rooney P. Wang M. Kumar P. Kumar S. J. Cell Sci. 1993; 105: 213-218Crossref PubMed Google Scholar, 28Sattar A. Rooney P. Kumar S. Pye D. West D.C. Scott I. Ledger P. J. Invest. Dermatol. 1994; 103: 576-579Abstract Full Text PDF PubMed Scopus (155) Google Scholar, 29Montesano R. Kumar S. Orci L. Pepper M.S. Lab. Invest. 1996; 75: 249-262PubMed Google Scholar, 34Xu H. Ito T. Tawada A. Maeda H. Yamanokuchi H. Isahara K. Yoshida K. Uchiyama Y. Asari A. J. Biol. Chem. 2002; 277: 17308-17314Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). We therefore examined their ability to induce CD44 cleavage using a human pancreatic carcinoma cell line, MIA PaCa-2, which has been reported to show substantial CD44 cleavage (3Kajita M. Itoh Y. Chiba T. Mori H. Okada A. Kinoh H. Seiki M. J. Cell Biol. 2001; 153: 893-904Crossref PubMed Scopus (610) Google Scholar). As shown in Fig. 1, MIA PaCa-2 cells expressed CD44 abundantly (Fig. 1A) and bound FL-HA; this binding was completely blocked by an anti-CD44 mAb, BRIC235, indicating that these cells require CD44 for HA binding. In agreement with previous reports (4Okamoto I. Kawano Y. Tsuiki H. Sasaki J. Nakao M. Matsumoto M. Suga M. Ando M. Nakajima M. Saya H. Oncogene. 1999; 18: 1435-1446Crossref PubMed Scopus (214) Google Scholar, 6Okamoto I. Tsuiki H. Kenyon L.C. Godwin A.K. Emlet D.R. Holgado-Madruga M. Lanham I.S. Joynes C.J. Vo K.T. Guha A. Matsumoto M. Ushio Y. Saya H. Wong A.J. Am. J. Pathol. 2002; 160: 441-447Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 7Okamoto I. Kawano Y. Matsumoto M. Suga M. Kaibuchi K. Ando M. Saya H. J. Biol. Chem. 1999; 274: 25525-25534Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar), Western blotting with anti-CD44cyto pAb showed that MIA PaCa-2 cells expressed CD44H, the 90-kDa standard form of CD44 (Fig. 1B), and that treatment of these cells with PMA significantly up-regulated the extracellular domain cleavage of CD44, as evidenced by an increase in the membrane-bound 25-kDa cleavage product (Fig. 1B). The enhancement of CD44 cleavage upon stimulation with PMA was confirmed by ELISA (Fig. 1C).When MIA PaCa-2 cells were treated with HA fragments that mainly consisted of 6–14-mers (frHA) (Fig. 2A), they showed enhanced CD44 cleavage in an HA concentration-dependent manner (Fig. 2B). In contrast, when the cells were treated with the undigested 200-kDa HA, which mainly consisted of polymers of 1000-mers or larger, no increase in CD44 cleavage was observed (Fig. 2C). Basically identical results were obtained using the human glioblastoma cell line U251MG and the human pancreatic carcinoma cell line Panc-1 (data not shown), indicating that small HA fragments, but not large HA polymers, have the ability to enhance CD44 cleavage in various tumor cell types.Fig. 2CD44 cleavage is enhanced by fragmented HA but not by 200-kDa HA. A, HPLC profile of frHA. 200-kDa HA was digested with sheep testicular hyaluronidase. The degraded HA fragments were then analyzed by HPLC as described under “Experimental Procedures.” The number of monosaccharide units in each oligosaccharide peak is indicated above each peak. B, MIA PaCa-2 cells were cultured overnight as described in the legend to Fig. 1 and then treated with MG132, followed by incubation with (lane 1) or without (lane 2) 100 ng/ml PMA for 30 min, or with frHA at the indicated concentration for 1 h (lanes 3–7). The cells were lysed, and the samples were subjected to immunoblotting as described in Fig. 1. C, cells were treated as above except that 200-kDa HA was used instead of frHA.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Digestion of HA Fragments to Disaccharides Abolishes the HA Fragment-induced CD44 Cleavage—Various HA fragment-dependent phenomena hitherto reported, such as inflammatory cytokine gene expression (21McKee C.M. Penno M.B. Cowman M. Burdick M.D. Strieter R.M. Bao C. Noble P.W. J. Clin. Invest. 1996; 98: 2403-2413Crossref PubMed Scopus (687) Google Scholar) and inducible nitric-oxide synthase gene induction (35McKee C.M. Lowenstein C.J. Horton M.R. Wu J. Bao C. Chin B.Y. Choi A.M. Noble P.W. J. Biol. Chem. 1997; 272: 8013-8018Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar, 36Yang T. Witham T.F. Villa L. Erff M. Attanucci J. Watkins S. Kondziolka D. Okada H. Pollack I.F. Chambers W.H. Cancer Res. 2002; 62: 2583-2591PubMed Google Scholar), cannot be seen when the HA fragments are digested into disaccharides. This was also the case with the HA fragment-induced CD44 cleavage, as shown in Fig. 3. When completely digested to disaccharides by hyaluronidase derived from S. dysgalactiae (Fig. 3A), the HA fragments up-regulated CD44 cleavage only marginally, if at all (Fig. 3C), whereas the HA preparation that had been treated with heat-inactivated hyaluronidase and hence still contained undigested HA fragments (Fig. 3B) up-regulated the CD44 cleavage to a level comparable with that induced by untreated HA fragments (Fig. 3C). These results clearly indicate that CD44 cleavage is induced by HA fragments but not by HA disaccharides and also exclude the possibility that non-HA molecules that might have been present in the frHA sample induced the CD44 cleavage.Fig. 3Fragmented HA loses its ability to induce CD44 cleavage upon digestion with intact hyaluronidase but not with boiled hyaluronidase. A and B, HPLC profiles of hyaluronidase-treated HA preparations. The frHA was treated with intact hyaluronidase SD (A) or boiled hyaluronidase SD (B). The resulting HA fragments, frHA-HAase and frHA-HAase/boiled, were analyzed by HPLC. Note that frHA was completely digested to disaccharides by intact hyaluronidase but remained undigested with heat-inactivated hyaluronidase. C, MIA PaCa-2 cells were cultured overnight as described in the legend to Fig. 1 and then treated with MG132, followed by incubation with (lane 2) or without (lane 1) 100 ng/ml PMA for 30 min or with 25 μg/ml of frHA-HAase (lane 3), frHA-HAase/boiled (lane 4), or frHA (lane 5) for 1 h. CD44 cleavage was examined as described in the legend to Fig. 2.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Only HA Fragments of a Certain Size Range Can Induce CD44 Cleavage—To further examine the role of HA size in mediating CD44 cleavage, we used uniformly sized HA fragments ranging from 2- to 12-mers and also the 6.9-kDa HA preparation that contained mainly 36-mers. As shown in Fig. 4 (A and C), HA 2- and 4-mers had only marginal CD44 cleavage up-regulating activity, whereas HA 6-mers and larger fragments induced CD44 cleavage in a manner that was apparently dependent on the size of the fragment. In contrast, HA preparations of much larger sizes, i.e. 36, 200, and 1000 kDa, uniformly failed to up-regulate the CD44 cleavage (Fig. 4, B and C). These results show that only HA fragments of a certain size range can up-regulate the CD44 cleavage.Fig. 4CD44 cleavage is induced by uniformly sized small HA saccharides but not by large polymers. MIA PaCa-2 cells were cultured overnight as described in the legends to Figs. 2 and 3 and treated with 10 mm MG132 for 30 min. Subsequently, in A, the cells were incubated with culture medium alone (lane 1), with 100 ng/ml PMA (lane 2) f
Referência(s)