Abstract
IntroductionExpression of hepatobiliary transporters is decreased during endotoxemia. Reduction of Mrp2 is mediated by IL-1β-dependent signals but underlying mechanisms are still unclear. YB-1 is a predominantly cytoplasmic protein that translocates to the nucleus in response to various stimuli. Previously we have shown that YB-1 down-regulates Mrp2 expression in vitro. Therefore we investigated the potential role of YB-1 as regulator of hepatic acute phase genes. MethodsLiver sections from LPS-injected rats (20h) were stained with YB-1-specific antibodies. Real-time RT-PCR quantification was performed for Mrp2, MMP-2 and YB-1. YB-1 protein was quantified from IL-1β- or TNFα-stimulated rat hepatoma cells (FaO) and the localization of a YFP-YB-1-CFP fusion protein was visualized by confocal microscopy in HepG2 human hepatocellular carcinoma cells. ChIP-assays and EMSA were performed to analyze YB-1 binding to DNA promoter elements. ResultsIn endotoxemic livers Mrp2 mRNA was down-regulated by 80%, while YB-1 mRNA expression increased 2.5-fold. Immunohistochemical staining showed a marked up-regulation and predominant nuclear localization of YB-1 protein in LPS challenged rats. In FAO cells IL-1β incubation increased cytoplasmic YB-1 protein content up to 16h. IL-1β stimulation resulted in a 6-fold up-regulation of endogenous YB-1 in the nuclear compartment, which occurred within 90min. In accord with these findings nuclear fluorescence was detected with a YFP-YB-1-CFP fusion protein introduced in HepG2 cells. In addition to DNA binding studies with endotoxemic rat liver tissue, ChIP assays revealed an IL-1β-dependent increase of YB-1 binding to the Mrp2-promoter in FAO cells. ConclusionYB-1 is activated during the hepatic acute phase response. IL-1β promotes a rapid nuclear YB-1 protein shuttling in hepatoma cells within 90min and a transcriptional induction thereafter. This biphasic response may explain the IL-1β-mediated suppression of Mrp2 expression in endotoxemic rats.
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