Binding of PAI-1 to endothelial cells stimulated by thymosin beta4 and modulation of their fibrinolytic potential.
Our previous studies showed that thymosin beta4 (Tbeta4) induced the synthesis of plasminogen activator inhibitor-1 (PAI-1) in cultured human umbilical vein endothelial cells (HUVECs) via the AP-1 dependent mechanism and its enhanced secretion. In this work we provide evidence that the released PAI-1 is accumulated on the surface of HUVECs, exclusively in its active form, in a complex with alpha1-acid glycoprotein (AGP) that is also up-regulated and released from the cells. This mechanism is supported by several lines of experiments, in which expression of both proteins was analyzed by flow cytometry and their colocalization supported by confocal microscopy. PAI-1 did not bind to quiescent cells but only to the Tbeta4-activated endothelial cells. In contrast, significant amounts of AGP were found to be associated with the cells overexpressing enhanced green fluorescent protein (EGFP)-alpha1-acid glycoprotein (AGP) without Tbeta4 treatment. The AGP.PAI-1 complex was accumulated essentially at the basal surface of endothelial cells, and such cells showed (a) morphology characteristic for strongly adhered and spread cells and (b) significantly reduced plasmin formation. Taken together, these results provide the evidence supporting a novel mechanism by which active PAI-1 can be bound to the Tbeta4-activated endothelial cells, thus influencing their adhesive properties as well as their ability to generate plasmin.
Boncela
Joanna
J
Center of Medical Biology, Polish Academy of Sciences, Lodz.
Smolarczyk
Katarzyna
K
Wyroba
Elzbieta
E
Cierniewski
Czeslaw S
CS
D002448:Cell Adhesion; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000166:cytology; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D005341:Fibrinolysin / Q000378:metabolism; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D007249:Inflammation; D018613:Microscopy, Confocal; D009961:Orosomucoid / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D010958:Plasminogen / Q000737:chemistry; D017395:Plasminogen Activator Inhibitor 1 / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013947:Thymosin / Q000737:chemistry* / Q000378:metabolism; D013997:Time Factors; D020298:U937 Cells; D014471:Umbilical Veins / Q000166:cytology; D015854:Up-Regulation; D014746:Vimentin / Q000737:chemistry; D000099201:Thymosin beta(4)
D005341:Fibrinolysin; D049452:Green Fluorescent Proteins; D009961:Orosomucoid; D010958:Plasminogen; D017395:Plasminogen Activator Inhibitor 1; D013947:Thymosin; D014746:Vimentin; D000099201:Thymosin beta(4)