Abstract 686: IL-1 and IL-6 Induce Hepatocyte Plasminogen Activator Inhibitor-1 Expression Through Independent Signaling Pathways Converging on C/EBPδ
Atherosclerosis is accelerated by inflammatory processes and increased expression of plasminogen activator inhibitor (PAI)-1, the major physiologic inhibitor of fibrinolysis and an acute-phase reactant produced in liver. We have previously shown that prototypical pro-inflammatory cytokines, interleukin (IL)-1β and IL-6, synergistically increase PAI-1 mRNA and protein in a well-differentiated human liver cell line (HepG2). To elucidate signaling pathways activated by IL-1β and IL-6 that contribute to increased expression of PAI-1, we studied HepG2 cells and primary mouse hepatocytes. PAI-1mRNA was increased by IL-1β (10.8 ± 0.4 (SD) fold over control at 1 ng/ml, real-time PCR), IL-6 (2.1 ± 0.4 fold at 1 ng/ml) and IL-1β + IL-6 (12.5 ± 0.8 fold). Transient transfection and luciferase assay of the PAI-1 promoter (−829bp to + 39bp) demonstrated that the PAI-1 promoter activity was increased by IL-1β (4.5 ± 0.3 fold), IL-6 (2.4 ± 0.2 fold) and IL-1β + IL-6 (8.8 ± 0.8 fold). Systematic deletion assay of the PAI-1promoter demonstrated that the region from −239bp to −210bp containing a putative CCAAT-enhancer binding protein (C/EBP) binding site was critical. Point mutation in C/EBP site abolished both IL-1β and IL-6 responses. Antibody interference electrophoretic mobility-shift assays showed that among C/EBP isoforms C/EBPδ (but not C/EBPα or C/EBPβ) protein level and its binding to PAI-1 promoter were increased by IL-1β, IL-6 and IL-1β + IL-6 in both HepG2 cells and mouse hepatocytes. Down-regulation of C/EBPδ induced with specific small interfering RNA (siRNA, 30 nM) decreased PAI-1 production. IL-1β inducible C/EBPδ and PAI-1 expressions were inhibited by mitogen activated protein kinase (MAPK) homolog-p38 inhibitor (SB 203580), c-Jun N-terminal kinase/MAPK inhibitor (SP600125) and ERK/MAPK pathway inhibitor (U0126). By contrast, IL-6 inducible C/EBPδ and PAI-1 expressions were inhibited by Janus kinase inhibitor I. Thus, IL-1β and IL-6 exert directionally similar effects on PAI-1 expression, but the induction involves distinct signaling pathways with a final common mediator, C/EBPδ. siRNAs aiming at C/EBP family of transcription factors may diminish atherothrombosis and provide vascular protection by regulating downstream PAI-1 expression.