A kunitz-type protease inhibitor bikunin disrupts ligand-induced oligomerization of receptors for transforming growth factor (TGF)-β and subsequently suppresses TGF-β signalings

Abstract 

We previously found that bikunin (bik), a Kunitz-type protease inhibitor, suppresses transforming growth factor-β1 (TGF-β1)–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-β1; (b) the overexpression of bik in the cells or exogenous bik results in the inhibition of TGF-β1 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-β receptors (TβRI and TβRII) in either cell types nor altered the specific binding of ¹²⁵I TGF-β1 to the cells, indicating that the effects of bik in these cells are not mediated by ligand sequestration; (d) TβRI and TβRII present on the same cells exclusively form aggregates in TGF-β1-stimulated cells; (e) co-treatment of TGF-β1–stimulated cells with bik suppresses TGF-β1–induced complex formation of TβRI and TβRII; and (f) a chondroitin-4-sulfate side chain-deleted bik (deglycosylated bik) does not inhibit TGF-β1 signaling or association of type I/type II receptor. We conclude that glycosylated bik attenuates TGF-β1–elicited signaling cascades in cells possibly by abrogating the coupling between TβRI and TβRII and that this probably provides the mechanism for the suppression of uPA and PAI-1 expression.

Keywords:  Bikunin, CD44, Dimerization, Ovarian cancer, TGF-β receptor, Signal transduction

Abbreviations:  bik, bikunin, Bik⁺, bikunin transfection, BSA, bovine serum albumin, FBS, fetal bovine serum, HA, hyaluronan, LP, Link protein, Luc⁺, luciferase transfection, PAI-1, plasminogen activator inhibitor type-1, PBS, phosphate-buffered saline, PVDF, polyvinylidene difluoride, uPA, urokinase-type plasminogen activator, TGF-β, transforming growth factor-β, TβRI, TGF-β receptor type-I, TβRII, TGF-β receptor type-II