Suppression of urokinase receptor expression by bikunin is associated with inhibition of upstream targets of extracellular signal-regulated kinase-dependent cascade.
Our laboratory showed that bikunin, a Kunitz-type protease inhibitor, suppresses 4beta-phorbol 12-myristate 13-acetate (PMA)- or tumor necrosis factor-alpha (TNFalpha)-induced urokinase-type plasminogen activator (uPA) expression in different cell types. In addition to its effects on protease inhibition, bikunin could be modulating other cellular events associated with the metastatic cascade. To test this hypothesis, we examined whether bikunin was able to suppress the expression of uPA receptor (uPAR) mRNA and protein in a human chondrosarcoma cell line, HCS-2/8, and two human ovarian cancer cell lines, HOC-I and HRA. The present study showed that (a) bikunin suppresses the expression of constitutive and PMA-induced uPAR mRNA and protein in a variety of cell types; (b) an extracellular signal-regulated kinase (ERK) activation system is necessary for the PMA-induced increase in uPAR expression, as PD098059 and U0126, which prevent the activation of MEK1, reduce the uPAR expression; (c) bikunin markedly suppresses PMA-induced phosphorylation of ERK1/2 at the concentration that prevents uPAR expression, but does not reduce total ERK1/2 antigen level; (d) bikunin has no ability to inhibit overexpression of uPAR in cells treated with sodium vanadate; and (e) we further studied the inhibition of uPAR expression by stable transfection of HRA cells with bikunin gene, demonstrating that bikunin secretion is necessary for inhibition of uPAR expression. We conclude that bikunin downregulates constitutive and PMA-stimulated uPAR mRNA and protein possibly through suppression of upstream targets of the ERK-dependent cascade, independent of whether cells were treated with exogenous bikunin or transfected with bikunin gene.
Department of Obstetrics and Gynecology, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka, Japan. hirokoba@haa-med.ac.jp
Hiroshi
H
Kobayashi
Mika
M
Suzuki
Naohiro
N
Kanayama
Takashi
T
Nishida
Masaharu
M
Takigawa
Toshihiko
T
Terao
D001859:Bone Neoplasms / Q000473:pathology; D002070:Butadienes / Q000494:pharmacology; D002277:Carcinoma / Q000473:pathology; D002813:Chondrosarcoma / Q000473:pathology; D003864:Depression, Chemical; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D005419:Flavonoids / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D048369:MAP Kinase Kinase 1; D020935:MAP Kinase Signaling System / Q000187:drug effects*; D008562:Membrane Glycoproteins / Q000235:genetics / Q000494:pharmacology* / Q000502:physiology; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020929:Mitogen-Activated Protein Kinase Kinases / Q000037:antagonists & inhibitors; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000235:genetics; D009570:Nitriles / Q000494:pharmacology; D010051:Ovarian Neoplasms; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011956:Receptors, Cell Surface / Q000096:biosynthesis* / Q000235:genetics; D055293:Receptors, Urokinase Plasminogen Activator; D011993:Recombinant Fusion Proteins / Q000502:physiology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection; D014360:Trypsin Inhibitor, Kunitz Soybean; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism
D002070:Butadienes; D004791:Enzyme Inhibitors; D005419:Flavonoids; D008562:Membrane Glycoproteins; D009363:Neoplasm Proteins; D009570:Nitriles; C524898:PLAUR protein, human; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D011993:Recombinant Fusion Proteins; C109148:SPINT2 protein, human; C113580:U 0126; D014360:Trypsin Inhibitor, Kunitz Soybean; D017346:Protein Serine-Threonine Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D048369:MAP Kinase Kinase 1; C482125:MAP2K1 protein, human; D020929:Mitogen-Activated Protein Kinase Kinases; D013755:Tetradecanoylphorbol Acetate; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one