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-2⅐proteasome 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-2⅐proteasome 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. Plasminogen activator inhibitor type-2 (PAI-2)2 has both extracellular and intracellular functions and acts as a multifunctional protein (1, 2). It exists predominantly as a 47-kDa nonglycosylated intracellular form (3). Due to the lack of an N-terminal signal peptide, only a small percentage of PAI-2 is exported from cells, by the nonclassical secretory pathway, as a glycosylated 60-kDa form (4). PAI-2 contains a unique domain bridging helices C and D, called the C-D loop (5), which directly interacts with different proteins, including retinoblastoma protein (6), proteasome subunit member ␤1 (7), interferon response factor 3 (8), ZNF198/FGFR1 fusion kinase (9), annexins (10), pre-mRNA processing factor 8 (7), and vitronectin (11).The primary physiological function of PAI-2 is thought to be the regulation of plasminogen activators in the extravascular compartment (1, 12). However, such a role is not supported by studies using PAI-2 Ϫ/Ϫ mice that show no discernable defects in fibrinolysis (13). Covalent PAI-2⅐uPA complexes, readily formed in vitro, have also never been unequivocally demonstrated in vivo (14). On the other hand, PAI-2 has been proposed to play a role in different processes, including tumor metastasis, embryo implantation, and macrophage survival (1, 2, 15). Consistently, a broad range of activities were ascribed to PAI-2, such as protection of the retinoblastoma protein f...

Screening of differentially expressed genes between multiple trauma patients with and without sepsis

“…In contrast, secreted phosphoprotein 1 (SPP1), a cytokine that up-regulates expression of interferon-gamma and IL-12, is down-regulated in sepsis patients. Plasminogen activator inhibitor-2 (SerpinB2), which inhibits the proteasome (Boncela et al, 2011), is down-regulated in our study, favoring protein degradation in accordance with the second function term, proteolysis. However, Schroder et al (2010) reported that SerpinB2 could play a positive role in adaptive immunity.…”

PAI-2/SerpinB2 inhibits proteolytic activity in a P. gingivalis -dominated multispecies bacterial consortium

“…a function in inhibition of apoptosis has been explored (Dickinson, Norris, Jensen, & Antalis, 1998). However, PAI-2 has also been demonstrated to block the proteasome, thereby promoting apoptosis (Boncela, Przygodzka, Papiewska-Pajak, Wyroba, & Cierniewski, 2011). Also roles such as protection against viral infections (Antalis et al, 1998) and regulation of the adaptive immunity (Schroder et al, 2010) have been explored.…”

Plasminogen Activator Inhibitor-2 Polymorphism Associates with Recurrent Coronary Event Risk in Patients with High HDL and C-Reactive Protein Levels

“…Whereas association of CVD risk with PAI-1 has been well-established [14-16]; for PAI-2, there are few studies addressing this issue [17]. However, with particular regard to the issue of inflammation, potential association of PAI-2 with CVD risk in this setting is suggested by results from a recent study indicating that PAI-2 can induce apoptosis in endothelial cells in the setting of inflammation through inhibitory effects on proteosome function [18]. …”

“…Recent efforts directed toward characterization of intracellular functions of PAI-2 suggest potential roles in regulation of apoptosis, cell differentiation, the innate immune response, cell signaling, and neuroprotection [18,20]. Conversely, the predominantly intracellular location of PAI-2 could call into question functionality with regard to inhibition of uPA and tPA.…”

“…This seemed like a potentially informative approach especially given the previously cited study demonstrating endothelial cell apoptosis induced by PAI-2 under inflammatory conditions [18]. Further, given the role of the LDLR family of receptors as related to PAI action as noted above, we assessed a polymorphism of LRP-1 (LRP1, rs1800156) [26] for effects on PAI-2 polymorphism-associated recurrent risk.…”