Successful reactivation of hepatic Mrp transporters in endotoxininduced cholestasis in mice by the CAR activator 6,7-Dimethylesculetin

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1 Successful reactivation of hepatic Mrp transporters in endotoxininduced cholestasis in mice by the activator 6,7-Dimethylesculetin Ina V. Martin *, Sebastian Voigt*, Michael Trauner, Christian Trautwein and Andreas Geier Dept. of Gastroenterology & Hepatology, University Hospital Aachen (UKA), Germany Division of Gastroenterology & Hepatology, Laboratory for Experimental Hepatology, University Hospital Zurich (USZ), Switzerland Falk Bile Acid Meeting Amsterdam, June 14th 2008

2 Hepatic organic anion transporters in obstructive cholestasis reduced maintained / increased Oatps MDR3/Mdr2 Mdr1 Mrp3 Mrp4 Pro-cholestatic (causal) Anti-cholestatic (adaptive)

3 Hepatic organic anion transporters in inflammatory cholestasis reduced maintained / increased LPS Oatps MDR3/Mdr2 Mdr1 Mrp3 Mrp4 Pro-cholestatic (causal) Anti-cholestatic (adaptive)

4 Hepatic organic anion transporters in inflammatory cholestasis LPS Shuttling of nuclear receptors IL-6 TNFα Il-1β IL-1β Oatp1 Mdr2 Mdr1 Mrp3 Mrp4 α nuclear protein CO 4h 8h 16h kd TNFα IL-1β 60 Siewert BBRC 2004 Geier Am J Physiol 2005 Ghose Nuclear Receptors 2004

5 Regulation of hepatic transport systems by nuclear receptors Mrp4 Mrp3 Activators as targets for intervention Oatp1 Oatp2 Oatp4 HNF4 HNF1 - RAR Stat5 HNF1 HNF1 HNF1 RAR SHP - FXR RAR FXR RAR FXR LRH-1 FXR TCPOBOP Phenobarbital 6,7-Dimethylesculetin PCN Atorvastatin Rifampicin GW4064 UDCA/TUDCA Geier Biochim Biophys Acta 2007 Karpen J Hepatology 2005

6 Induction of hepatic transport systems in healthy liver and CBDL Mrp4 Mrp3 Bili-Gluc -SO 2 Ugt1a1 TCPOBOP PB Sult2a1 PCN Atorvastatin Oatp1 Oatp2 Oatp4 Mrp3 Mrp4 Wagner Hepatology 2005

7 Induction of hepatic transport systems in inflammatory cholestasis Mrp3 30% RAR? Rosiglitazone? FXR 31% IR-1 DR-2 Ghose J Hepatology 2007

8 Background & Aims Inflammatory cholestasis has been previously characterized as a result of reduced organic anion transporter expression mediated by a decreased expression and altered subcellular localization of class II nuclear hormone receptors (NHR). We hypothesized that treatment with the activator 6,7-dimethylesculetin (DE) in endotoxemic mice may induce expression and putatively a nuclear redistribution of NHR leading to a maintained transporter gene expression. 6,7-dimethylesculetin as the active substance of Yin Chin has been used in Asian traditional medicine to treat neonatal jaundice for centuries.

9 Yin Chin (6,7-Dimethylesculetin) enhances bilirubin clearance by activating humanized transgenic mice YinChin/ 6,7-DE Artemisia capillaris Huang J Clin Invest 2004

10 Methods 6,7-DE or corn oil (Sham) C57/BL6 (25g; n=5) LPS Animal model - Inflammatory liver injury was induced in male C57/BL6 mice by intraperitoneal injection of endotoxin (0,5 mg/kg). - Intervention: Simultaneous application of 6,7- Dimethylesculetin (Sigma; 100 mg/kg) or corn oil only by i.p. injection. Livers were harvested after 4h and 16h h mrna Expression (RT-PCR, Northern) - hepatic transporters (,, -4) - nuclear receptors (,, FXR, RAR,, Lrh-1) -cytokines(tnfα, IL-1β) immunohistochemistry in fixed liver sections

11 Results & Conclusions (Abstract) Treatment with the activator 6,7-DE in endotoxemia induces NHR gene expression with maximum effects on and Lrh-1 RNA at 16 hours compared to an extensive suppression in control mice. Immunohistochemistry staining provided no definite DE-specific effect on localization, since sham controls were also characterized by a partial nuclear redistribution of the receptor protein. At 16h transcriptional and Lrh-1 activation was associated with a prominent induction of Mrp3 and a maintained expression of (and ) compared to a substantial decrease in controls. This study provides a potential protective interventional approach to selectively modify an otherwise unselective negative hepatic acute phase response during endotoxemia at the molecular level.

12 Hypothesis: targeting in inflammatory cholestasis Mrp4 Mrp3?? LRH-1 LRH-1 6,7-DE Mrp3 TNFα Il-1β IL-1β?

13 Acknowledgements Christoph Dietrich, Aschaffenburg Carsten Gartung, Minden Ina V. Martin Sebastian Voigt Hella Scotland Anne Porn Ann-Kathrin Vehr Alexander Minkenberg Joba Arikkat Sonja Strauch Michael Trauner, Graz Peter Mertens, Aachen Frank Lammert, Bonn Steven Dooley, Mannheim Elke Roeb, Giessen Bruno Stieger, Zürich Gerd Kullak-Ublick, Zürich M. Ananthanarayanan, New York