IL-1 and IL-6 induce hepatocyte plasminogen activator inhibitor-1 expression through independent signaling pathways converging on C/EBPδ

To elucidate signaling pathways activated by IL-1 and IL-6 that contribute to increased expression of plasminogen activator inhibitor-1 (PAI-1), we studied human hepatoma (HepG2) cells and primary mouse hepatocytes. HepG2 cell PAI-1 mRNA increased in response to IL-1 beta, IL-6, and IL-1 beta plus IL-6 as shown by real- time PCR. Activity of the transiently transfected PAI-1 promoter (-829 to +36 bp) increased as well. Systematic promoter deletion assays showed that the region from -239 to -210 bp containing a putative CCAAT-enhancer binding protein (C/EBP) binding site was critical. Point mutations in this region abolished the IL-1 beta and IL-6 responses. Antibody interference electrophoretic mobility shift assays showed that C/EBP delta (but not C/EBP alpha or C/EBP beta) binding and protein were increased by IL-1 beta, IL-6, and IL-1 beta plus IL-6 in HepG2 cells. IL-1 beta and IL-6 increased expression of both PAI-1 mRNA and C/EBP delta mRNA in mouse primary hepatocytes as well. Downregulation of C/EBP delta induced with small interfering RNA (siRNA) decreased secretion of PAI-1. As judged from results obtained with inhibitors, signal transduction in all three of the mitogen-activated protein kinase pathways was involved in IL-1-inducible PAI-1 expression. By contrast, JAK signaling was responsible for the IL-6-induced inducible expression. Thus IL-1 and IL-6 exert directionally similar effects on PAI-1 expression, but the induction involves distinct signaling pathways with a final common mediator, C/EBP delta.