title: Kunitz-type protease inhibitor bikunin disrupts phorbol ester-induced oligomerization of CD44 variant isoforms containing epitope v9 and subsequently suppresses expression of urokinase-type plasminogen activator in human chondrosarcoma cells.
abstract: We previously found that bikunin (bik), a Kunitz-type protease inhibitor, suppresses phorbol ester (PMA)-stimulated expression of urokinase-type plasminogen activator (uPA). In the present study, we tried to answer this mechanism using human chondrosarcoma HCS-2/8 cells. Our results showed the following novel findings: (a) the standard form of CD44 (CD44s; 85 kDa) is expressed in both unstimulated and PMA-stimulated cells, while CD44v isoforms containing epitope v9 (110 kDa) are strongly up-regulated in response to treatment with PMA; (b) CD44v isoforms containing epitope v9 present on the same cell exclusively form aggregates in stimulated cells; (c) induction of uPA mRNA expression could be achieved by using a second cross-linker antibody to cross-link Fab monomers of anti-CD44; (d) co-treatment of stimulated cells with anti-CD44 mAb alone or anti-CD44v9 mAb alone suppresses PMA-induced clustering of CD44, which results in inhibition of uPA overexpression; (e) bikunin efficiently disrupts PMA-induced clustering of CD44, but does not prevent PMA-induced up-regulation of CD44v isoforms containing epitope v9; and (f) after exposure to bik, approximately 150-kDa band is mainly detected with immunoprecipitation and this band is shown to be a heterodimer composed of the 110-kDa v9-containing CD44v isoforms and a 45-kDa bik receptor (bik-R). In conclusion, we provide, for the first time, evidence that the bik-R can physically interact with the CD44v isoforms containing epitope v9 and function as a repressor to down-regulate PMA-stimulated uPA expression, at least in part, by preventing clustering of CD44v isoforms containing epitope v9.
authors.affiliation: Department of Obstetrics and Gynecology, Hamamatsu University School of Medicine, Handacho 3600, Hamamatsu, Shizuoka, 431-3192, Japan.
authors.forename: Mika
authors.initials: M
authors.lastname: Suzuki
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kobayashi
authors.forename: Michio
authors.initials: M
authors.lastname: Fujie
authors.forename: Takashi
authors.initials: T
authors.lastname: Nishida
authors.forename: Masaharu
authors.initials: M
authors.lastname: Takigawa
authors.forename: Naohiro
authors.initials: N
authors.lastname: Kanayama
authors.forename: Toshihiko
authors.initials: T
authors.lastname: Terao
mesh_terms: D000911:Antibodies, Monoclonal / Q000378:metabolism; D019921:Biotinylation; D015152:Blotting, Northern; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000494:pharmacology; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000737:chemistry; D005434:Flow Cytometry; D006801:Humans; D018960:Hyaluronan Receptors / Q000096:biosynthesis / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000494:pharmacology*; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D010703:Phorbol Esters / Q000494:pharmacology*; D011233:Precipitin Tests; D011485:Protein Binding; D020033:Protein Isoforms; D012333:RNA, Messenger / Q000378:metabolism; D013388:Succinimides / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013997:Time Factors; D014360:Trypsin Inhibitor, Kunitz Soybean; D014407:Tumor Cells, Cultured; D015854:Up-Regulation; D014568:Urokinase-Type Plasminogen Activator / Q000096:biosynthesis*
chemical_list: D000911:Antibodies, Monoclonal; D003432:Cross-Linking Reagents; D000939:Epitopes; D018960:Hyaluronan Receptors; D008562:Membrane Glycoproteins; D010703:Phorbol Esters; D020033:Protein Isoforms; D012333:RNA, Messenger; C109148:SPINT2 protein, human; D013388:Succinimides; D014360:Trypsin Inhibitor, Kunitz Soybean; D014568:Urokinase-Type Plasminogen Activator; D013755:Tetradecanoylphorbol Acetate; C019358:disuccinimidyl suberate