Binding of PAI-1 to Endothelial Cells Stimulated by Thymosin β4 and Modulation of Their Fibrinolytic Potential

Abstract

Our previous studies showed that thymosin ␤4 (T␤4) 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 ␣1-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 T␤4-activated endothelial cells. In contrast, significant amounts of AGP were found to be associated with the cells overexpressing enhanced green fluorescent protein (EGFP)-␣1-acid glycoprotein (AGP) without T␤4 treatment. The AGP⅐PAI-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 T␤4-activated endothelial cells, thus influencing their adhesive properties as well as their ability to generate plasmin.Thymosin ␤4 (T␤4) 2 promotes skin and corneal wound healing through its effects on cell migration, angiogenesis and possibly cell survival (1-3). However, the precise molecular mechanism through which it functions and its potential role in solid organ wound healing remains unknown. In our studies of the biological activity of T␤4 we found that this 43-amino acid peptide strongly activates endothelial cells to express and release plasminogen activator inhibitor type 1 (PAI-1). It occurs via the mechanism involving the signaling pathway leading to activation of the MAPK cascade and enhanced c-Fos/c-Jun DNA binding activity (4). Such a role of AP-1 in activation of transcriptional events by T␤4 was confirmed by independent studies (5). Recently, T␤4 was reported to stimulate migration of cardiomyocytes and endothelial cells and promote survival of cardiomyocytes by formation of a functional complex with PINCH and integrin-linked kinase, resulting in activation of the survival kinase Akt (6). These observations revealed that T␤4 affects cellular functions by activation of several signaling pathways and induces changes in the cell properties necessary for both migration and survival. Here, we show that T␤4 up-regulates expression and induces the surface accumulation of ␣1-acid glycoprotein (AGP) known to bind PAI-1 and prolong its inhibitory activity (7). AGP is expressed by activated endothelial cells and exerts its effect by interacting with components of the endothelial glycocalyx (8). In this study, we identify a previously unrecognized function of AGP, a direct and primary role in plasminogen activation. We provide evidence that t...

Adipocytokine Orosomucoid Integrates Inflammatory and Metabolic Signals to Preserve Energy Homeostasis by Resolving Immoderate Inflammation

“…Additionally, we cannot exclude the possibility that a chronic increase in Orm expression in circulation might play unwanted roles in affecting metabolic complications in obese subjects. Indeed, it has been recently reported that ORM interacts with PAI-1 to recruit prothrombotic activity to atherosclerotic lesions (44). Thus, further studies are required to explain the persistence of elevated inflammatory responses despite the induced expression of Orm in obese mice and to address the role of increased levels of Orm expression in obesity-related metabolic complications and cardiovascular diseases, emphasizing the importance of analyzing conditional Orm gene knock-out mice.…”

Ku80 as a Novel Receptor for Thymosin β4 That Mediates Its Intracellular Activity Different from G-actin Sequestering

“…Proteins released from the resin were separated by electrophoresis by 7% SDS-PAGE, electrotransfered onto nitrocellulose membranes, and immunodetected by HA tag mouse monoclonal antibody. Immunoprecipitation of the Ku80⅐T␤4 complex from endothelial cell extracts was performed as described previously (26).…”

“…Thus, T␤4 externally added to endothelial cells: (a) induces expression and release of plasminogen activator inhibitor type 1 (PAI-1) by the mechanism involving activation of the mitogen-activated protein kinase cascade leading to enhanced c-Fos/c-Jun binding to the AP-1-like element present in the PAI-1 promoter (25,26); (b) binds to PINCH and integrinlinked kinase, resulting in activation of the survival kinase Akt, and thus promotes myocardial and endothelial cell migration in the embryonic heart (27) and (c) promotes skin and corneal wound healing through its effects on cell migration, angiogenesis, and possibly cell survival (4,6,11).…”

Interleukin-18-mediated interferon-gamma secretion is regulated by thymosin beta 4 in human NK cells

“…T␤4 could also induce NK cell activation via direct stimulation of the signal machinery or the phosphorylation of transcription factors. It is well known that T␤4 stimulates integrin-linked kinase (ILK) and AKT/Protein Kinase B in cardiomyocyte survival and ERK phosphorylation in paclitaxelinduced cell death and activates plasminogen activator inhibitor-1 (PAI-1) and then induces JAK/STAT cascade (Srivastava et al 2007;Boncela et al 2006;Oh et al 2006). Furthermore, IFN-␥ induction is mediated by the JAK/STAT pathway, which is also involved in actin polymerization (Jacobson et al 1995).…”