title: A kunitz-type protease inhibitor bikunin disrupts ligand-induced oligomerization of receptors for transforming growth factor (TGF)-beta and subsequently suppresses TGF-beta signalings.
abstract: We previously found that bikunin (bik), a Kunitz-type protease inhibitor, suppresses transforming growth factor-beta1 (TGF-beta1)-stimulated expression of urokinase-type plasminogen activator (uPA) in human ovarian cancer cells that lack endogenous bik. In the present study, we tried to elucidate the mechanism by which bik also inhibits plasminogen activator inhibitor type-1 (PAI-1) and collagen synthesis using human ovarian cancer cells. Here, we show that (a) there was an enhanced production of both uPA and PAI-1 in HRA cells in response to TGF-beta1; (b) the overexpression of bik in the cells or exogenous bik results in the inhibition of TGF-beta1 signaling as measured by phosphorylation of the downstream signaling effector Smad2, nuclear translocation of Smad3, and production of PAI-1 and collagen; (c) bik neither decreased expression of TGF-beta receptors (TbetaRI and TbetaRII) in either cell types nor altered the specific binding of 125I TGF-beta1 to the cells, indicating that the effects of bik in these cells are not mediated by ligand sequestration; (d) TbetaRI and TbetaRII present on the same cells exclusively form aggregates in TGF-beta1-stimulated cells; (e) co-treatment of TGF-beta1-stimulated cells with bik suppresses TGF-beta1-induced complex formation of TbetaRI and TbetaRII; and (f) a chondroitin-4-sulfate side chain-deleted bik (deglycosylated bik) does not inhibit TGF-beta1 signaling or association of type I/type II receptor. We conclude that glycosylated bik attenuates TGF-beta1-elicited signaling cascades in cells possibly by abrogating the coupling between TbetaRI and TbetaRII and that this probably provides the mechanism for the suppression of uPA and PAI-1 expression.
authors.affiliation: NetForce Co. Ltd., Taiko 3-1-18, Nakamura, Nagoya, Aichi 453-0801, Japan.
authors.forename: Tatsuo
authors.initials: T
authors.lastname: Yagyu
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kobayashi
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Wakahara
authors.forename: Hidenori
authors.initials: H
authors.lastname: Matsuzaki
authors.forename: Toshiharu
authors.initials: T
authors.lastname: Kondo
authors.forename: Noriyuki
authors.initials: N
authors.lastname: Kurita
authors.forename: Hideo
authors.initials: H
authors.lastname: Sekino
authors.forename: Kiyokazu
authors.initials: K
authors.lastname: Inagaki
authors.forename: Mika
authors.initials: M
authors.lastname: Suzuki
authors.forename: Naohiro
authors.initials: N
authors.lastname: Kanayama
authors.forename: Toshihiko
authors.initials: T
authors.lastname: Terao
mesh_terms: D002455:Cell Division / Q000187:drug effects; D045744:Cell Line, Tumor; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D018076:DNA, Complementary / Q000235:genetics; D005260:Female; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology* / Q000652:urine; D010051:Ovarian Neoplasms; D011347:Procollagen / Q000378:metabolism; D018125:Receptors, Transforming Growth Factor beta / Q000187:drug effects / Q000502:physiology*; D011994:Recombinant Proteins / Q000187:drug effects / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013936:Thymidine / Q000378:metabolism; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000187:drug effects / Q000502:physiology*; D014360:Trypsin Inhibitor, Kunitz Soybean / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology* / Q000652:urine
chemical_list: D015853:Cysteine Proteinase Inhibitors; D018076:DNA, Complementary; D008562:Membrane Glycoproteins; D011347:Procollagen; D018125:Receptors, Transforming Growth Factor beta; D011994:Recombinant Proteins; C109148:SPINT2 protein, human; D016212:Transforming Growth Factor beta; D014360:Trypsin Inhibitor, Kunitz Soybean; D013936:Thymidine