RESUMEN
INTRODUCTION: Expression of hepatobiliary transporters is decreased during endotoxemia. Reduction of Mrp2 is mediated by IL-1ß-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-1ß- 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-1ß incubation increased cytoplasmic YB-1 protein content up to 16h. IL-1ß 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-1ß-dependent increase of YB-1 binding to the Mrp2-promoter in FAO cells. CONCLUSION: YB-1 is activated during the hepatic acute phase response. IL-1ß 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-1ß-mediated suppression of Mrp2 expression in endotoxemic rats.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Interleucina-1beta/farmacología , Proteína 1 de Unión a la Caja Y/genética , Transportadoras de Casetes de Unión a ATP/biosíntesis , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Regulación hacia Abajo/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Células Hep G2 , Humanos , Inmunohistoquímica , Interleucina-1beta/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Masculino , Microscopía Confocal , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Transfección , Proteína 1 de Unión a la Caja Y/metabolismoRESUMEN
The cold shock protein Y-box (YB) binding-1 is an example of a highly regulated protein with pleiotropic functions. Besides activities as a transcription factor in the nucleus or regulator of translation in the cytoplasm, recent findings indicate extracellular effects and secretion via a non-classical secretion pathway. This review summarizes regulatory pathways in which YB-1 participates, all iterating auto-regulatory loops. Schematics are developed that elucidate the cold shock protein activities in (i) fine-tuning its own expression level following platelet-derived growth factor-B-, thrombin- or interferon-γ-dependent signaling, (ii) as a component of the messenger ribonucleoprotein (mRNP) complex for interleukin-2 synthesis in T-cell commitment/activation, (iii) pro-fibrogenic cell phenotypic changes mediated by transforming growth factor-ß, and (iv) receptor Notch-3 cleavage and signal transduction. Emphasis is put forward on subcellular protein translocation mechanisms and underlying signaling pathways. These have mostly been analysed in cell culture systems and rarely in experimental models. In sum, YB-1 seems to fulfill a pacemaker role in diverse diseases, both inflammatory/pro-fibrogenic as well as tumorigenic. A clue towards potential intervention strategies may reside in the understanding of the outlined auto-regulatory loops and means to interfere with cycling pathways.