title: Association of plasminogen activator inhibitor type 2 (PAI-2) with proteasome within endothelial cells activated with inflammatory stimuli.
abstract: Quiescent endothelial cells contain low concentrations of plasminogen activator inhibitor type 2 (PAI-2). However, its synthesis can be rapidly stimulated by a variety of inflammatory mediators. In this study, we provide evidence that PAI-2 interacts with proteasome and affects its activity in endothelial cells. To ensure that the PAI-2·proteasome complex is formed in vivo, both proteins were coimmunoprecipitated from endothelial cells and identified with specific antibodies. The specificity of this interaction was evidenced after (a) transfection of HeLa cells with pCMV-PAI-2 and coimmunoprecipitation of both proteins with anti-PAI-2 antibodies and (b) silencing of the PAI-2 gene using specific small interfering RNA (siRNA). Subsequently, cellular distribution of the PAI-2·proteasome complexes was established by immunogold staining and electron microscopy analyses. As judged by confocal microscopy, both proteins appeared in a diffuse cytosolic pattern, but they also could be found in a dense perinuclear and nuclear location. PAI-2 was not polyubiquitinated, suggesting that it bound to proteasome not as the substrate but rather as its inhibitor. Consistently, increased PAI-2 expression (a) abrogated degradation of degron analyzed after cotransfection of HeLa cells with pCMV-PAI-2 and pd2EGFP-N1, (b) prevented degradation of p53, as evidenced both by confocal microscopy and Western immunoblotting, and (c) inhibited proteasome cleavage of specific fluorogenic substrate. This suggests that PAI-2, in endothelial cells induced with inflammatory stimuli, can inhibit proteasome and thus tilt the balance favoring proapoptotic signaling.
authors: Institute of Medical Biology, Polish Academy of Sciences, Lodz, Poland.
authors: Joanna
authors: J
authors: Boncela
authors: Patrycja
authors: P
authors: Przygodzka
authors: Izabela
authors: I
authors: Papiewska-Pajak
authors: Elzbieta
authors: E
authors: Wyroba
authors: Czeslaw S
authors: CS
authors: Cierniewski
mesh_terms: D002460:Cell Line; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D008070:Lipopolysaccharides / Q000494:pharmacology; D018613:Microscopy, Confocal; D046529:Microscopy, Electron, Transmission; D017396:Plasminogen Activator Inhibitor 2 / Q000235:genetics / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism* / Q000648:ultrastructure; D011485:Protein Binding / Q000187:drug effects; D034622:RNA Interference; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D054875:Ubiquitination / Q000187:drug effects
chemical_list: D008070:Lipopolysaccharides; D017396:Plasminogen Activator Inhibitor 2; D014409:Tumor Necrosis Factor-alpha; D046988:Proteasome Endopeptidase Complex