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	Mika Suzuki, Hiroshi Kobayashi, Michio Fujie, Takashi Nishida, Masaharu Takigawa, Naohiro Kanayama, Toshihiko Terao
Journal	The Journal of biological chemistry (J Biol Chem) Vol. 277 Issue 10 Pg. 8022-32 (Mar 08 2002) ISSN: 0021-9258 [Print] United States
PMID	11777908 (Publication Type: Journal Article)
Chemical References	Antibodies, Monoclonal Cross-Linking Reagents Epitopes Hyaluronan Receptors Membrane Glycoproteins Phorbol Esters Protein Isoforms RNA, Messenger SPINT2 protein, human Succinimides Trypsin Inhibitor, Kunitz Soybean Urokinase-Type Plasminogen Activator Tetradecanoylphorbol Acetate disuccinimidyl suberate
Topics	Antibodies, Monoclonal (metabolism) Biotinylation Blotting, Northern Blotting, Western Cell Membrane (metabolism) Cross-Linking Reagents (pharmacology) Dimerization Dose-Response Relationship, Drug Down-Regulation Enzyme-Linked Immunosorbent Assay Epitopes (chemistry) Flow Cytometry Humans Hyaluronan Receptors (biosynthesis, metabolism) Membrane Glycoproteins (chemistry, pharmacology) Microscopy, Fluorescence Models, Biological Phorbol Esters (pharmacology) Precipitin Tests Protein Binding Protein Isoforms RNA, Messenger (metabolism) Succinimides (pharmacology) Tetradecanoylphorbol Acetate (pharmacology) Time Factors Trypsin Inhibitor, Kunitz Soybean Tumor Cells, Cultured Up-Regulation Urokinase-Type Plasminogen Activator (biosynthesis)

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