title: Co-localization of urokinase and its receptor on established human umbilical vein endothelial cell.
abstract: Vascular endothelial cells possess antithrombotic properties, which are determined by the balance between plasminogen activators (PAs) and PA inhibitors (PAls). A cell line, TKM-33, has been established and cloned from human umbilical vein endothelial cells, was previously reported to produce a large amount of urokinase-type PA (u-PA) and small amounts of tissue-type plasminogen activator (t-PA) and PA inhibitor-1 (PAI-1). Moreover, TKM-33 expressed the u-PA receptor (u-PAR) which plays an important role in the localization of fibrinolytic activity on cell surface. In the present study, we investigated the localization of u-PA, t-PA, PAI-1 and u-PAR in TKM-33 by using immunofluorescence staining technique. The endothelial cells were strongly stained with anti-PAI-1, anti-u-PA and anti-u-PAR IgGs, and slightly with anti-t-PA IgG. The double immunofluorescence staining with mouse anti-u-PA IgG and rabbit anti-u-PAR IgG followed by rhodamine-conjugated anti-mouse IgG and FITC-conjugated anti-rabbit IgG showed the co-localization of u-PA and u-PAR on the same section of endothelial cells. Although u-PA antigen also existed in the cytoplasm of endothelial cells, u-PAR antigen did not. The treatment of endothelial cells with phorbol-myristate-acetate (PMA) upregulated the expression of u-PA and u-PAR antigens. In this stimulation, u-PAR antigen was detected not only on the surface of the cells but also in the cytoplasm. Thus, the binding of u-PA to u-PAR was confirmed by double immunofluorescence staining.
authors.affiliation: Department of Physiology, Kinki University School of Medicine, Osakasayama, Osaka, Japan. sueshima@med.kindai.ac.jp
authors.forename: S
authors.initials: S
authors.lastname: Ueshima
authors.forename: H
authors.initials: H
authors.lastname: Matsumoto
authors.forename: S
authors.initials: S
authors.lastname: Izaki
authors.forename: Y
authors.initials: Y
authors.lastname: Mitsui
authors.forename: H
authors.initials: H
authors.lastname: Fukao
authors.forename: K
authors.initials: K
authors.lastname: Okada
authors.forename: O
authors.initials: O
authors.lastname: Matsuo
mesh_terms: D000906:Antibodies; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry; D003593:Cytoplasm / Q000737:chemistry; D004730:Endothelium, Vascular / Q000737:chemistry* / Q000166:cytology / Q000187:drug effects; D005110:Extracellular Space / Q000737:chemistry; D005455:Fluorescent Antibody Technique; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology; D017395:Plasminogen Activator Inhibitor 1 / Q000032:analysis; D011956:Receptors, Cell Surface / Q000032:analysis*; D055293:Receptors, Urokinase Plasminogen Activator; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D010959:Tissue Plasminogen Activator / Q000032:analysis; D014471:Umbilical Veins; D015854:Up-Regulation / Q000187:drug effects; D014568:Urokinase-Type Plasminogen Activator / Q000032:analysis*
chemical_list: D000906:Antibodies; D007074:Immunoglobulin G; C524898:PLAUR protein, human; D017395:Plasminogen Activator Inhibitor 1; C524899:Plaur protein, mouse; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D010959:Tissue Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator; D013755:Tetradecanoylphorbol Acetate