title: Rat Mrp2 gene expression is regulated by an interleukin-1β-stimulated biphasic response with enhanced transcription and subcellular shuttling of YB-1.
abstract: BACKGROUND
  Expression 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.
  
  
  METHODS
  Liver sections from LPS-injected rats (20 h) 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.
  
  
  RESULTS
  In 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.
  
  
  CONCLUSIONS
  YB-1 is activated during the hepatic acute phase response. IL-1β promotes a rapid nuclear YB-1 protein shuttling in hepatoma cells within 90 min and a transcriptional induction thereafter. This biphasic response may explain the IL-1β-mediated suppression of Mrp2 expression in endotoxemic rats.
authors.affiliation: Department of Nephrology and Hypertension, Diabetes and Endocrinology, Otto-von-Guericke-University, Magdeburg, Germany. peter.mertens@med.ovgu.de
authors.forename: Peter R
authors.initials: PR
authors.lastname: Mertens
authors.forename: Ina V
authors.initials: IV
authors.lastname: Martin
authors.forename: Björn C
authors.initials: BC
authors.lastname: Frye
authors.forename: Thomas
authors.initials: T
authors.lastname: Rauen
authors.forename: Sonja
authors.initials: S
authors.lastname: Strauch
authors.forename: Melanie
authors.initials: M
authors.lastname: Pabst
authors.forename: Andreas
authors.initials: A
authors.lastname: Geier
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000096:biosynthesis / Q000235:genetics*; D000818:Animals; D045744:Cell Line, Tumor; D004195:Disease Models, Animal; D015536:Down-Regulation / Q000187:drug effects; D005786:Gene Expression Regulation / Q000187:drug effects; D056945:Hep G2 Cells; D006801:Humans; D007150:Immunohistochemistry; D053583:Interleukin-1beta / Q000378:metabolism / Q000494:pharmacology*; D008099:Liver / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D008297:Male; D018613:Microscopy, Confocal; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D014162:Transfection; D051840:Y-Box-Binding Protein 1 / Q000235:genetics* / Q000378:metabolism
chemical_list: D018528:ATP-Binding Cassette Transporters; C423013:Abcc2 protein, rat; D053583:Interleukin-1beta; D012333:RNA, Messenger; D051840:Y-Box-Binding Protein 1