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  1. 1. The University of Kansas Medical Center The Liver Center 2010 Liver Symposium May 27th & 28th, 2010 Program and Abstracts This symposium is supported by The University of Kansas Medical CenterThe University of Kansas Cancer Center, and Bohan Fund Beller Conference Center Hemenway Building
  2. 2. We dedicate 2010 Liver Symposium to the people who contributed to The Liver Center Tissue Bank Patients who donated their tissue Physicians: Bashar Abdulkarim Ivan Damjanov Jameson Forster Richard Gilroy Tamer Malik Atta Nawabi Mojtaba Olyaee Ossam Tawfik Ryan Taylor Steven Weinman Stephen Williamson (in alphabetical order) GI & OR Staff Liver Transplant Coordinators Liver Center Tissue Bank Coordinator: Natali Navarro CazarezLiver Tissue Procurement and Liver Tissue Banking Protocol (#11378) PI: Yu-Jui Yvonne Wan 2
  3. 3. The University of Kansas Liver Center Tissue BankNatali Navarro Cazarez, Bashar Abdulkarim, Ivan Damjanov, Jameson Forster, Richard Gilroy,Tamer Malik, Atta Nawabi, Mojtaba Olyaee, Ossama Tawfik, Ryan Taylor, Steven Weinman,Stephen Williamson (in alphabetical order), and Yu-Jui Yvonne WanThe University of Kansas Medical Center, Kansas City, KSThe University of Kansas Liver Center Tissue Bank is a human liver tissue supplier forbiomedical research at the University of Kansas Medical Center. This liver depository wasestablished in November 2008 supported by the Institution. The KU Liver Center Tissue Bankactivities and services include coordinating patient consent, collecting and banking fresh frozenlivers from excess surgical materials in liver transplants, resections, and biopsies, processingand banking blood and urine, maintaining a specimen database with extensive clinical andpathological data, and distributing samples to researchers. Currently, the bank maintains anincredible success rate of 95% patient participation due to the great interest not only fromphysicians, but also from patients eager to help fight liver diseases. Since inception, 562 tissuesamples have been deposited. In addition, the bank has collected 236 blood samples, 103serum samples, and 27 urine samples. The KU Liver Center Tissue Bank can provide a varietyof tissue types ranging from normal livers to livers obtained from patients who have liver cancer,cirrhosis, hepatitis, steatosis, fibrosis, and bile duct diseases. Along with samples, it collectsclinical data including demographic information, BMI, past medical history, alcohol and tobaccouse, medication intake, HCV treatment details, and past liver transplant information. Thecomprehensive clinical database allows the KU Liver Center Tissue Bank to distributespecimens with extensive specificity. So far, seven ongoing studies are using specimensobtained from the KU Liver Center Tissue Bank. Five scientific abstracts are presented in thisyear’s Liver Center Symposium. One scientific abstract has been presented in a nationalmeeting and two manuscripts have been submitted for publication. This liver depository servesas a meeting point for physicians and basic researchers and thus enhances the progress oftranslational research. 3
  4. 4. Program Thursday, May 27, 2010 Beller Conference Center7:30 - 8:00 am Continental Breakfast8:00 - 8:10 am Welcome note by Barbara F. Atkinson, M.D., Executive Vice Chancellor, KUMC, Executive Dean, School of Medicine8:10 - 8:30 am Yu-Jui Yvonne Wan, Ph.D., Professor, Director, Liver Center Liver Center Progress Report8:30 - 8:50 am Steven Weinman, M.D., Ph.D., Professor, Director, Translational Research Translational Research ReportHepatocellular Carcinoma – Faris Farassati, Pharm.D., Ph.D. - Moderator8:50 - 9:20 am Udayan Apte, Ph.D., Assistant Professor Ablation off Farnesoid X receptor promotes hepatocellular carcinoma development via activation of Wnt/beta-catenin pathway9:20 - 9:50 am Partha Krishnamurthy, Ph.D., Assistant Professor ATP binding cassette transporter ABCB6; Potential therapeutic target in HCC9:50 - 10:05 am Grace Guo, Ph.D., Assistant Professor Farnesoid x receptor activation mediates head-to-tail chromatin looping in the Nr0b2 gene encoding small heterodimer partner10:05 – 10:20 am BreakLiver Injury – James Luyendyk, Ph.D. - Moderator10:20 - 10:50 am Wen-Xing Ding, Ph.D., Assistant Professor Role of autophagy in acute alcoholic liver injury10:50 -11:20 am Hartmut Jaeschke, Ph.D., Professor Translational research in acetaminophen hepatotoxicity11:20 - 11:35 am Hao Zhu, Ph.D., Assistant Professor Ncb5or, fatty acid metabolism and oxidative stress11:35 - 11:50 am Ann Wozniak, Graduate Student - Steven Weinman’s laboratory Function of the hepatitis C virus (HCV) viroporin ion channel p7 4
  5. 5. 11:50 - 12:05 pm Brad Sullivan, Graduate Student – James Luyendyk’s laboratory The coagulation system contributes to alphaVbeta6 integrin expression and liver fibrosis induced by cholestasis12:05 - 2:10 pm Lunch served in Beller 1005-1007 Poster viewing in foyer of Beller conference areaTransport & Biotransformations – Sitta Sittampalam, Ph.D. - Moderator2:10 - 2:40 pm Curtis Klaassen, Ph.D., University Distinguished Professor & Chair The possibility of adapting a Chinese herbal drug to a prescription drug for liver diseases2:40 - 3:10 pm Jeff Staudinger, Ph.D., Associate Professor, Department of Pharmacology & Toxicology, KU Lawrence, KS Inflammation, PXR modification, and drug disposition3:10 - 3:25 pm Pallavi Limaye, Ph.D., Postdoctoral Fellow – Curtis Klaassen’s laboratory Assessment of modulation of intestinal microflora following alterations in bile acid transport3:25 – 3:40 pm Megan Roth, Graduate Student – Bruno Hagenbuch’s laboratory Isolation of a modulator of the liver specific organic anion transporting polypeptides (OATPs) 1B1 and 1B3 from Rollinia emarginata Schlecht (Annonaceae)Plenary lecture – Steven Weinman, M.D., Ph.D. – Moderator3:40 – 4:40 pm Keith Lindor, M.D., Professor of Medicine, Dean, Mayo Medical School New developments in primary sclerosing cholangitis Symposium End ~~~~~ 5
  6. 6. Liver Center External Advisory Committee MembersAnna Mae Diehl, M.D., Chief Division of Gastroenterology, Director of the Liver Center,Duke University Medical CenterDr. Diehl received her medical degree from Georgetown University and completed her internalmedicine training and fellowship in gastroenterology and hepatology at Johns HopkinsUniversity. She was a faculty member at Johns Hopkins University most of her professionalcareer, and was promoted to Professor of Medicine there in 1997. In 2004, Dr. Diehl wasrecruited to Duke University to lead their gastroenterology and hepatology programs and wasawarded the Florence A. McAlister Endowed Professorship. Her research interests include liverinjury and repair. Her lab focuses on the mechanisms that regulate this process using culturedcells, animal models of acute and chronic liver damage, and samples from patients with varioustypes of liver disease. Her group also conducts clinical trials in patients with chronic liverdisease.Alex Lentsch, Ph.D., Professor of Surgery, Vice Chairman for Research, Department ofSurgery, University of Cincinnati College of MedicineDr. Lentsch received his Bachelors of Science degree in Biological Sciences from NorthernKentucky University, Highland Heights, Kentucky and his Physiology and Biophysicsfrom the University of Louisville, Louisville, Kentucky. Dr. Lentsch’s research interests includeregulation of inflammatory responses by cytokines, chemokines and adhesion molecules. Acentral theme of his research is aimed at determining how specific signal transduction pathwaysutilized by cytokines and chemokines control the development of inflammatory disease.Keith Lindor, M.D., Professor of Medicine, Dean, Mayo Medical SchoolDr. Lindor received a Bachelor’s of Chemistry degree from the University of Minnesota and hisMD degree from Mayo Medical School. Following this, he was a resident in internal medicine atBowman Grey School of Medicine of Wake Forest University. Before starting hisGastroenterology Fellowship at Mayo, he served a year in the Indian Health Service in SouthernArizona. Dr. Lindor’s research interests include cholestatic liver diseases in adults, particularlyprimary biliary cirrhosis and primary sclerosing cholangitis as well as nonalcoholicsteatohepatitis. The focus of his research work is primarily on clinical trials and means ofoptimizing the medical management of patients with these disorders. Dr. Lindor serves as theSenior Associate Editor for Clinical Gastroenterology and Hepatology. He is on the editorialboards of Gastroenterology and Journal of Clinical Gastroenterology.Shelly Lu, M.D., Professor of Medicine Keck School of Medicine, Associate Director of USCLiver Center, University of Southern CaliforniaAfter graduating from Medical School of UCLA, Dr. Lu has held positions at Cedars-SinaiMedical Center and numerous academic appointments at the USC Keck School of Medicine.Dr. Lu is a physician-scientist. Her research interests include the study of regulation of hepaticGSH synthesis, the regulation of hepatic methionine adenosyltransferases (MATs),abnormalities in methionine metabolism in alcoholic liver injury, the role of SAMe in liver functionand injury, and MAT1A knockout mice as an animal model to study the effect of ethanol. She isalso associate director of the NIH-funded USC Research Center for Liver Diseases andassociate editor of the American Journal of Physiology: GI and Liver and the Encyclopedia ofGastroenterology. 6
  7. 7. George Michalopoulos, M.D., Ph.D., Professor and Chair of Pathology, School of Medicine,University of PittsburghDr. George K. Michalopoulos received his medical doctoral degree at Athens University Schoolof Medicine in 1969. A residency in Anatomic Pathology and a Ph.D. in Oncology was obtainedat the Wisconsin Medical Center in Madison in 1977. Dr. Michalopoulos moved to DukeUniversity as Assistant Professor in 1977 and stayed at Duke University until 1991. He thenmoved to Pittsburgh as Professor and Chairman of the Department of Pathology in April, 1991.He served as Associate Vice Chancellor for Health Sciences and Interim Dean of the School ofMedicine from November, 1995, until November, 1998. His research interests include growthfactors and receptors in hepatocytes, growth regulation of human hepatocytes, and hepaticcarcinogenesis. 7
  8. 8. Liver Center Internal Advisory Committee MembersBashar Abdulkarim, M.D., Ph.D., Assistant Professor, Department of SurgeryRichard Gilroy, M.D., Associate Professor, Department of Internal MedicineHartmut Jaeschke, Ph.D., Professor, Department of Pharmacology, Toxicology & TherapeuticsMojtaba Olyaee, M.D., Professor and Director, Gastroenterology/Hepatology/Motility, Department of Internal MedicinePeter Smith, Ph.D., Director, Department of KS Intellectual & Developmental Disabilities Research CenterYu-Jui Yvonne Wan, Ph.D., Professor, Department of Pharmacology, Toxicology & Therapeutics, Director of the Liver CenterSteven Weinman, M.D., Ph.D., Professor, Department of Gastroenterology/Hepatology, Director of Translational Research 8
  9. 9. Abstracts HCC (C1-C9) Liver Injury (L1-L11)Transport & Biotransformation (T1-T8) 9
  10. 10. HCC Abstracts (C1-C9)#C1The differential effect of N-(4-hydroxyphenyl) retinamide and all-trans retinoic acid ininducing apoptosis and autophagy in Huh7 cellsNathan Bushue and Yu-Jui Yvonne WanDepartment of Pharmacology, Toxicology & Therapeutics, KUMCN-(4-hydroxyphenyl) retinamide (4HPR, fenretinide) is a synthetic analog of all-trans retinoicacid (ATRA). In contrast to the differentiation effect of ATRA, 4HPR induces apoptosis of humanhepatocellular carcinoma Huh7 cells. While previous data shows that both differentiation andapoptosis are mediated through a retinoic acid receptor β (RARβ)-dependent manner, themechanisms by which 4HPR and ATRA exert their different roles are not well understood. Ourdata showed that both chemicals induced RARβ expression level and regulated the activation ofERK, JNK and Akt in the same manner. Both chemicals also induced the expression level ofLC3. However, the conversion of LC3-I to LC3-II, a hallmark of autophagy, was higher in ATRA-than 4HPR-treated cells. In addition, the level of signaling adaptor p62, which is crucial foractivation of death receptors and caspase 8-mediated apoptosis, was significantly lower inATRA than 4HPR-treated Huh7 cells. Furthermore, ATRA induced the expression of beclinsuggesting ATRA induced autophagy in Huh7 cell. Induction of autophagy by rapamycinprevented 4HPR-induced apoptosis, while blocking autophagy with wortmannin increasesATRA-induced cell death. Taken together, our data suggest that lack of autophagy in 4HPRtreated cells leads to apoptosis of Huh7 cells. In contrast to 4HPR, ATRA induces autophagy,which may protect the cells from apoptosis and allow the survived cells to respond to thedifferentiation effect of ATRA. 10
  11. 11. #C2ABCB6; potential therapeutic target in HCCHemantkumar Chavan, Mahitha Oruganti, and Partha KrishnamurthyDepartment of Pharmacology, Toxicology & Therapeutics, KUMCHepatocellular carcinoma (HCC) is a major health problem being the fifth most common cancerworldwide and the third leading cause of cancer death. Despite significant progress in cancermedicine, curative treatment options for HCC are still limited with an unsatisfactory survival rate.Evidence suggests that molecular changes in gene expression that compromise proliferationand cell death represent a pro-tumorigenic principle in human HCC. Here we show that Abcb6 amitochondrial ATP binding cassette transporter is upregulated in HCC. Abcb6 expressionpromotes hepatocellular carcinoma cell growth and proliferation both in vitro in cell culturemodels and in a human to mouse xenograft tumor model. These results suggest that Abcb6might play a role in the development or progression of HCC. Future studies will address whetherAbcb6 expression in HCC is the cause or the result of the disease and explore the therapeuticrelevance of regulating Abcb6 expression in HCC.(Supported by the Liver Tissue Bank of the Liver Center) 11
  12. 12. #C3Ral overactivation in hepatocellular carcinoma: biology and therapeutic ramificationsMohammad Taha, Mohamad Ezzeldin, and Faris FarassatiDepartment of Medicine, KUMCRas signaling pathway and its down-stream effectors are involved in the molecular etiology ofHepatocellular Carcinoma. Overactivation of wild-type Ras and Ras gene mutations as well asother oncogenes in Ras pathway are shown to be involved in more than 40% of humanmalignancies. Ras pathway has been found to be overactivated in 30% of hepatocellularcarcinoma (HCC) cases. We have identified, for the first time, the overactivation of RalA, anovel effector pathway down-stream of Ras in a series of HCC cell lines as well as tissuesamples. We have also investigated other key players of Ral pathway such as Aurora kinaseand protein phosphatase 2A (PP2A). While Aurora kinase (an activator of Ral) is over-expressed in HCC cell lines, PP2A (a negative regulator of Ral) is expressed at equal levels.We have also investigated the effects of inhibition of RalA on the viability and invasiveness ofHCC cell lines (by gene specific silencing as well as an inhibitory peptide) and introduce ourresults here in terms of targeting this pathway for treatment of HCC. The results of this researchnot only enhance our knowledge about the biology of HCC but also provide a new strategy fortargeting this malignancy. Ras signaling pathway and its down-stream effectors are involved inthe pathogenesis of Hepatocellular Carcinoma (HCC). Overactivation of wild-type Ras and Rasgene mutations as well as other oncogenes in Ras pathway are shown to be involved in morethan 40% of human malignancies. Ras pathway has been found to be overactivated in 30% ofHCC cases. We have identified, for the first time, the overactivation of RalA, a novel effectorpathway down-stream of Ras in a series of HCC cell lines as well as tissue samples. We havealso investigated other key players of Ral pathway such as Aurora kinase (stimulator of RalAactivation) and protein phosphatase 2A (PP2A, a negative regulator of RalA). While Aurorakinase is over-expressed in HCC cell lines (but not normal hepatocytes), PP2A is expressed atequal levels (Figure 1). Therefore, in HCC, RalA is mainly over-activated via stimulation byAurora kinases while PP2A may not play a major role in this case. We have also investigatedthe effects of inhibition of RalA on the viability and invasiveness of HCC cell lines (by genespecific silencing as well as an inhibitory peptide) and introduce our results here in terms oftargeting this pathway for treatment of HCC. The results of this research not only enhance ourknowledge about the biology of HCC but also provide a new strategy for targeting thismalignancy.(Supported by the Liver Tissue Bank of the Liver Center) 12
  13. 13. #C4Farnesoid x receptor activation mediates head-to-tail chromatin looping in the Nr0b2gene encoding small heterodimer partnerGuodong Li¹, Ann M. Thomas¹, Steve N. Hart¹, Xiaobo Zhong¹, Dequan Wu², and Grace L. Guo¹¹Department of Pharmacology, Toxicology & Therapeutics, KUMC²Department of General Surgery, Second Affiliated Hospital, Harbin Medical University, Harbin150086, ChinaAs a unique nuclear receptor with only ligand-binding but no DNA-binding domain, smallheterodimer partner (SHP) interacts with many transcription factors to inhibit their function.However, the regulation of SHP expression is not well understood. SHP is highly expressed inthe liver and previous studies have shown FXR highly induces SHP by binding to a FXRresponse element (FXRRE) in the promoter of the Nr0b2 gene, which encodes SHP. The FXR-SHP pathway is critical in maintaining bile-acid and fatty-acid homeostasis. Following genome-wide FXR binding by chromatin immunoprecipitation coupled to massively parallel sequencing(ChIP-seq), a novel FXRRE was found in the 3’ enhancer region of the Nr0b2 gene. Thisdownstream IR1 is highly conserved throughout mammalian species. We hypothesized that thisdownstream FXRRE is functional and may mediate a head-to-tail chromatin looping byinteracting with the proximal promoter FRXRE to increase SHP transcription efficiency. In thecurrent study, a ChIP-qPCR assay revealed FXR strongly bound to this downstream FXRRE inmouse livers. The downstream FXRRE is important for FXR-mediated transcriptional activationrevealed by luciferase gene transcription activation, as well as by deletion and site-directedmutagenesis. The chromatin conformation capture (3C) assay was used to detect chromatinlooping and the result confirmed the two FXRREs located in the Nr0b2 promoter anddownstream enhancer interacted to form a head-to-tail chromatin loop. To date the head-to-tailchromatin looping has not been reported in the liver. In conclusion, our results suggest amechanism by which activation of FXR efficiently induces SHP transcription is through head-to-tail chromatin looping. 13
  14. 14. #C5Yes-associated protein expression is induced in hepatocellular carcinoma and isresponsive to cell density.Hua Li, Andy Wolfe, Genea Edwards, Grace L. Guo, Yu-Jui Yvonne Wan, and Udayan ApteDepartment of Pharmacology, Toxicology & Therapeutics, KUMCHepatocellular carcinoma (HCC) is the most common malignancy of the liver associated withhigh mortality. We are interested in understanding the role of pathways involved in organ sizeregulation in pathogenesis of HCC. Specifically, we are investigating the role of Hippo Kinasepathway and yes-associated protein (Yap), the downstream effector of the pathway. Three HCCcell lines investigated, Hep3B, HepG2 and Huh7, demonstrated increase in Yap as compared tonormal human hepatocytes. The expression levels and cellular localization of Yap wasresponsive to cell density. High cell confluency resulted in decreased Yap expression andnuclear export. siRNA and shRNA-mediated knockdown of Yap resulted in decreased cellproliferation of Hep3B cells. Immunohistochemical analysis of Yap inphenobarbital+diethylnitrosamine-induced liver tumors in C57BL/6 mice, spontaneous tumors inFarnesoid X Receptor knockout mice and human HCC biopsies revealed extensive nuclearstaining on Yap in highly proliferative tumors. Detailed analysis of Yap and its target genesincluding CTGF, Glypican3, and Survivin by mRNA and Western blot indicated extensive Yapactivation in human HCC samples. These data indicate that Yap plays a critical role inpathogenesis of HCC, possibly due to increased cell-adhesion independent growth andsuppression of apoptosis.(Supported by the Liver Tissue Bank of the Liver Center) 14
  15. 15. #C6Hippo kinase pathway is involved in postnatal liver developmentSeth Septer1, Genea Edwards2, Andy Wolfe2, Hua Li2, and Udayan Apte21 Department of Gastroenterology, Children’s Mercy Hospital, Kansas City, MO; 2Department ofPharmacology, Toxicology & Therapeutics, KUMCLiver exhibits size expansion and differentiation during postnatal period. During postnatal periodcells in the liver undergo a period of well-controlled proliferation to achieve the adult liver tobody weight ratio followed by hepatic differentiation. However, the mechanisms of liver sizeregulation and differentiation are not completely clear. Herein we report that Hippo Kinasesignaling plays a role in liver size regulation and differentiation during postnatal liver growthperiod. Postnatal liver growth was studied in C57BL/6 mice over a time course of 0 to 30 days.Western blot and immunofluorescence studies revealed increased expression of yes associatedprotein (Yap), the downstream effector of the Hippo Kinase pathway, starting postnatal day(PND) 5. Yap expression was biphasic with first peak at PND15 and second at PND30. Westernblot analysis showed increase in phopho-Yap between PND 20 to 30 indicating increaseddegradation of Yap at those time points. Consistent with this, we observed increase inexpression of other components of Hippo Kinase pathway including Mst, Lats and Sav, whichregulate Yap phosphorylation. PCNA analysis indicated peak cell proliferation in the liversbetween PND15-20. To further investigate the role of Yap in postnatal liver development westudied liver growth in Yap+/- mice, which revealed small but significant decrease in the liver tobody weight ratio in the Yap+/- mice as compared to Yap+/+ mice. Taken together these dataindicate that Yap plays a role in both cell proliferation and possibly in hepatic differentiationduring postnatal liver development. 15
  16. 16. #C7Hepatocyte nuclear factor 4 α (HNF4α) is involved in regulation of hepatocyteproliferation.Chad Walesky1, Aaron Bell2, Genea Edwards1, Andy Wolfe1, and Udayan Apte11 Department of Pharmacology, Toxicology & Therapeutics, KUMC; 2Department of Pathology,University of Pittsburgh School of Medicine, Pittsburgh, PAHNF4α is the master regulator of hepatocyte differentiation and regulates a wide variety ofhepatocyte-specific genes. Here we report that HNF4α is involved in inhibiting hepatocyteproliferation. Decreased HNF4α expression was observed in hepatocyte specific integrin linkedkinase knockout mice (ILK-KO), which have sustained cell proliferation resulting inhepatomegaly. Further analysis revealed decrease in expression of HNF4α targets such as p21and claudin1, which are involved in cell-cycle regulation in ILK-KO livers. Hepatocyte specificknockdown of HNF4α in HNF4α-floxed mice using Cre recombinase carried by an adeno-associated virus 8 (AAV-8) vectors resulted in induction of cell proliferation accompanied bydecreased expression of p21 and claudin1. Over-expression of rat HNF4α in rat hepatoma JM1cells resulted in decreased proliferation and increased cells in G0-G1 phase of cell cycle. Takentogether, these data indicate that HNF4α plays an anti-proliferative role in the hepatocytes. 16
  17. 17. #C8Bevacizumab and erlotinib in previously untreated inoperable and metastatichepatocellular carcinomaRangaswamy Govindarajan¹, Eric Siegel¹, Issam Makhoul¹, and Stephen Williamson²¹University of Arkansas for Medical Sciences, Little Rock, AR²Department of Medicine, KUMCPurpose: The treatment options for metastatic and inoperable hepatocellular carcinoma arelimited to sorafenib, a RAF kinase inhibitor. This study was designed to study the efficacyvascular. Endothelial growth factor (VEGF) antibody bevacizumab and VEGF tyrosine kinaseinhibitor erlotinibPatients and Methods: Twenty-one subjects with metastatic and inoperable hepatocellularcarcinoma who had not been treated with local or systemic therapy were treated withbevacizumab 15mg/kg every three weeks and oral daily dose of Erlotinib 150 mg. The primaryefficacy end point was the percentage of subjects who are progression free at 27 weeks.Results: 21 subjects were enrolled (16 Child- Pugh class A, 5 class B); 18 were evaluable forthe primary endpoint; all subjects were evaluable for toxicity. The median age was 60 yrs.(range 33-81). Five subjects (28%) were progression-free at 27 weeks of enrollment (95% CI10%-53%). Median (quartiles) time to progression was 11.0 weeks (9.0-28.0). The median(quartiles) overall survival (OS) was 35.7 weeks (23.6-74.9). Two subjects withdrew consentand one subject did not have adequate base line scans.Conclusion: The study results met the predetermined criterion of >20% PFS at 27 weeks. Thecombination of bevacizumab and erlotinib provides is a combination therapy to be exploredfurther in randomized setting. 17
  18. 18. #C9HDAC inhibitors potentiate fenretinide-induced apoptosis through mitochondrialenrichment of Nur77 in HCC cellsHui Yang¹, ², and Yu-Jui Yvonne Wan¹¹Department of Pharmacology, Toxicology & Therapeutics, KUMC²Department of Gastroenterology, Second Affiliated Hospital, Guangzhou Medical College,Guangzhou, ChinaFenretinide [N-(4-hydroxyphenyl] retinamide), a synthetic retinoid derivative, can causeceramide accumulation and reactive oxygen species (ROS) generation to induce cell death.Fenretinide is also implicated in preventing high-fat diet-induced obesity, insulin resistance, andhepatic steatosis in animal models. Furthermore, fenretinide inhibits angiogenesis and hepaticfibrosis. Previously, we have shown that fenretinide induced apoptosis in Huh7 human livercancer cells, however had no effect on HepG2 cells. In our current study, we demonstrate thathistone deacetylase inhibitors (HDACi) trichostatin (TSA) and scriptaid, can either enhancefenretinide-induced apoptosis in the fenretinide sensitive HCC cells (Huh7 and Hep3B) orsensitize the fenretinide resistant cells (HepG2) to the apoptotic effect of fenretinide. Huh7,Hep3B, and HepG2 cells were treated with fenretinide and/or HDACi. Fenretinide treatment withHDACi increased caspase 3/7 activity as well as cell death in comparison to fenretinide orHDACi treatment alone. We did not observe the same effect in primary human hepatocytes asseen in the cancer cell lines suggesting the ability of fenretinide and HDACi to induce apoptosisis cancer cell specific. The fenretinide plus HDACi enhanced cell death observed in cancer cellswas positively associated with increased retinoic acid receptor β (RARβ) expression, inductionof Nur77 level in the mitochondria, as well as reduced RXRα expression. However, we did notobserve any correlation between the enhanced cell death and the level of ROS generation.Inhibiting the expression of Nur77 by siRNA blocked fenretinide- and HDCAi-induced caspase 3activation and apoptosis of Huh7 cell. In addition, fenretinide- and HDACi-induced cytosolicNur77 expression was not detected in Huh7 cells in which RARβ expression was inhibited bysiRNA. Furthermore, Nur77 and RARβ are co-localized in the cytosol of fenretinide or HDACitreated Huh7 cells revealed by confocal microscopy. Immunoprecipitation followed by westernblot indicated that Nur77 and RARβ interacted with each other upon fenretinide or HDACitreatment. Taken together, our data indicated that fenretinide and HDACi combination treatmentis effective in inducing apoptosis of liver cancer cells in a Nur77 and RARβ-dependent manner.Nur77 may compete with RXRα in dimerization with RARβ. RXRα reduction as well as RARβand Nur77 induction by fenretinide/HDACi treatment favors the interaction between RARβ andNur77 therefore resulting in the nuclear export of Nur77 to target mitochondria and induction ofapoptosis. Thus, HDACi in combination with fenretinide modulate the expression andintracellular localization of nuclear receptors, which in turns regulate the apoptosis of livercancer cell. (CA53596) 18
  19. 19. Liver Injury Abstracts (L1-L11)#L1Low oxygen prevents mitochondrial superoxide production and attenuatesacetaminophen-induced hepatocyte cell death in culture.Anup Ramachandran¹, Hui-min Yan¹, John J Lemasters², and Hartmut Jaeschke¹¹Department of Pharmacology, Toxicology & Therapeutics, KUMC²Department of Pharmacological Sciences, Medical University of South Carolina, Charleston,SCOxidative stress and mitochondrial dysfunction play an important role in acetaminophen-inducedhepatocyte cell death. However, exact mechanisms involved in the process are controversial, inpart, due to the disparity in findings between in vitro & in vivo studies. A major difference in thiscontext is the oxygen tension, with cells in culture being exposed to 21% oxygen while those inthe liver experience a gradient from 5-11% oxygen. To determine if oxygen tension couldmodulate hepatocyte responses to acetaminophen, this study examined cells under normoxia(21% O2), 10% O2 & 5% O2. Isolated primary mouse hepatocytes were treated with 5mMacetaminophen for 15 hours under various oxygen tensions and cell death assessed by LDHrelease. Mitochondrial function and membrane potential were assessed at 6 & 15h using theXTT assay and JC-1 fluorescence respectively. Mitochondrial generation of superoxide wasmeasured using Mitosox Red® fluorescence. Exposure of hepatocytes to 5mM acetaminophenfor 15 hours at 21% O2 resulted in significant cell death, accompanied by deterioration ofmitochondrial function and loss of mitochondrial membrane potential, which was evident at 6hours of treatment. Mitochondrial generation of superoxide was also elevated at 6 hours.Culture of cells at 5 & 10% O2 resulted in significant protection against cell death. Mitochondrialfunction and membrane potential were better preserved at 6 hours, though the protective effectdecreased by 15 hours. Culturing cells at 10% O2 blocked elevation in mitochondrial superoxideproduction at 6 hours; an increase being seen only by 15 hours. These results suggest thatoxygen tension can modulate hepatocyte responses to acetaminophen, with low physiologicallevels decreasing mitochondrial superoxide production and delaying hepatocyte cell death. Thismay have wide-ranging implications in other cellular contexts where oxidative metabolism playsa role. 19
  20. 20. #L2Direct viral cytotoxicity of HCV requires cofilin translocation from cytosol tomitochondriaRoosevelt V. Campbell1, 2, Batbayar Tumurbaatar1, and Steven A. Weinman11 Department of Internal Medicine, KUMC; 2Department of Neuroscience and Cell Biology,University of Texas Medical Branch, Galveston, TexasThe development of the JFH1 strain of cell culture competent HCV has clearly shown that thevirus can directly induce apoptosis in infected cells. One mechanism that has been identified isenhanced sensitivity to TRAIL. Previous work from our lab and others has shown that HCVsensitizes cells to oxidant-induced cell death as well, but whether these are independent effectsand the molecular details of how they are induced by HCV remain uncertain. The AIM of thisstudy was to determine whether TRAIL-induced and ROS-induced cell death are independentprocesses and to elucidate the molecular basis responsible. METHODS: Human Hepatoma(Huh7.5) cells were infected with the JFH1 strain of HCV. Caspase activity was measured byCaspase 3/7 Glo assay and cell death was determined by LDH release. Cofilin was downregulated using cofilin siRNA from Dharmacon following the manufacturer’s instruction.Mitochondrial isolations and Western blots were done following standard protocols. RESULTS:Huh7.5 cells were treated with either tBOOH or TRAIL either before or after infection with JFH1virus. Viral infection increased TRAIL and tBOOH induced cell death by 5-6 fold compared touninfected cells but the characteristics of these 2 cell death processes were markedly different.TRAIL induced cell death was preceded by high caspase 3/7 activity and prominent PARPcleavage but tBOOH induced cell death was not. TRAIL induced cell death was blocked byzVAD, a pan-caspase inhibitor but tBOOH cell death was not. However, tBOOH induced celldeath was completely prevented by calcium chelation or antioxidant treatment while these hadno effect against TRAIL induced cell death. We next examined the effect of HCV on themitochondrial association of molecules known to cause apoptosis and necrosis. In response toJFH1 infection there was a marked mitochondrial translocation and dephosphorylation of cofilin,a cytoskeletal associated protein that has been shown to function as a mediator of oxidant-induced mitochondrial permeability transition. Treatment of Huh7.5 cells with cofilin siRNA priorto viral infection decreased tBOOH-induced cell death by 50% but had not effect on TRAIL-induced cell death. CONCLUSION: Our results demonstrate that at least two distinctmechanisms of cell death, TRAIL hypersensitivity and cofilin-dependent oxidant induced celldeath, are induced by HCV. These may explain the clinical observations of HCV as a sensitizerto alcoholic and other forms of liver disease, and contribute to the ATP binding cassettetransporter 20
  21. 21. #L3Mitigation of ethanol-induced liver injury by autophagyWen-Xing DingDepartment of Pharmacology, Toxicology & Therapeutics, KUMCAlcohol abuse is a major cause of liver injury. Whereas the pathology of alcoholic liver diseasedevelops over a prolonged period, the cellular defense mechanisms against the detrimentaleffects of alcohol are not well understood. We found that macroautophagy, an evolutionarilyconserved lysosomal degradation pathway commonly activated in response to stress, plays asignificant role in protecting hepatocytes from ethanol toxicity. Using both an animal bingemodel and cell cultures, we found that ethanol treatment strongly activated macroautophagy.Suppression of macroautophagy led to a significant increase in hepatocyte apoptosis and liverinjury. Further analysis demonstrated that macroautophagy protected hepatocytes by removingdamaged mitochondria and accumulated lipid droplets. These findings established thephysiological significance of macroautophagy in acute ethanol-induced pathogenesis in the liverand the potential therapeutic values of modulating macroautophagy in alcoholic liver disease. 21
  22. 22. #L4Monocyte chemoattractant protein-1-deficiency does not affect steatosis or inflammationin livers of mice fed a methionine-choline-deficient dietKaren M. Kassel¹, Grace L. Guo¹, Ossama Tawfik², and James P. Luyendyk¹¹Departments of Pharmacology, Toxicology & Therapeutics, KUMC²Pathology and Laboratory Medicine, KUMCBackground: Monocyte chemoattractant protein-1 (MCP-1, Ccl2) expression is increased inlivers of patients with non-alcoholic steatohepatitis and in murine models of steatohepatitis.Several studies in rodents indicate that MCP-1 contributes to liver steatosis induced by feedinga high fat diet. However, the extent of MCP-1 involvement in the widely utilized methionine-choline-deficient (MCD) diet model of steatohepatitis has not been determined. Aims: We testedthe hypothesis that MCP-1 contributes to steatohepatitis in mice fed an MCD diet. Methods:MCP-1-deficient mice on a C57Bl/6J background and age-matched C57Bl/6J mice were fed aMCD diet or control diet for 4 weeks. Results: MCP-1-deficiency did not affect steatohepatitis asindicated by liver histopathology, nor did it affect serum alanine aminotransferase activity,hepatic triglyceride levels, hepatic inflammatory gene induction, or macrophage accumulation inmice fed the MCD diet. Interestingly, MCP-1-deficiency significantly reduced collagen depositionin the livers of mice fed the MCD diet. Moreover, MCP-1-deficiency reduced the expression ofprofibrogenic genes including pro-collagen 1a1, connective tissue growth factor, andtransforming growth factor-β as well as α-smooth muscle actin protein levels in mice fed theMCD diet. Conclusions: The results indicate that MCP-1 does not contribute to liver steatosis orinflammation in the MCD diet model of steatohepatitis. Rather, the data suggest that MCP-1contributes to fibrosis in mice fed the MCD diet independent of effects on steatosis andinflammation. 22
  23. 23. #L5Acetaminophen toxicity in HepaRG cells: a novel human model for studies of drughepatotoxicityMitchell R. McGill, Huimin Yan, Anup Ramachandran, Hartmut JaeschkeDepartment of Pharmacology, Toxicology & Therapeutics, KUMCAcetaminophen (APAP) overdose is the most common cause of acute liver failure in the U.S.and U.K. The mechanism of APAP hepatotoxicity in rodents involves formation of a reactivemetabolite, glutathione (GSH) depletion, mitochondrial dysfunction, and oncotic necrosis. Toinvestigate these mechanisms in a human system, a metabolically competent cell line isneeded. In this study, we tested the value of a human hepatoma-derived cell line (HepaRG) forAPAP toxicity studies. Cells were treated with 20 mM APAP and the time course of celldysfunction and injury was evaluated. APAP caused a decline in cellular GSH levels to 60% ofcontrol at 6 h and 30 % at 24 h. The mitochondrial membrane potential (JC-1) was unaffected at6 h but was reduced to 50% of control at 12 h and 40% at 24 h. Lactate dehydrogenase (LDH)release was not observed until 24 h (30%) and 48 h (64%) indicating cell necrosis. In addition, aclear dose response (5 -20 mM) was observed with each of these parameters. Experiments withHepG2 cells, which are not metabolically competent, showed no GSH depletion, mitochondrialdysfunction or cell injury. Conclusion: The mechanism of APAP toxicity in HepaRG cells mimicsclosely the sequence of events observed in rodents. Importantly, the time course of injuryresembles what has been reported in the clinic. Thus, HepaRG cells may be a useful humanmodel for mechanistic studies of drug hepatotoxicity. 23
  24. 24. #L6Impact of hepatitis C on survival in alcoholic hepatitis.PK Pandya ¹, GA Rao, and Steven Weinman²¹VA Medical Center, ² School of Medicine, KUMCBackground: Alcoholic hepatitis (AH) is an acute inflammatory process associated with anoverall 30 day mortality of 15% that approaches 50% among severe cases. The prevalence ofhepatitis C virus (HCV) infection among alcoholics can exceed 30% compared to 1.8% in thegeneral population. This infection reduces the antioxidant capacity of hepatic mitochondria thatcan further sensitize hepatocytes to alcohol-induced injury. The additive impact on survivalamong AH CHC patients is unknown. Methods: We performed a retrospective database reviewof a representative sample of 10 Veterans affairs (VA) VISNs that represents half of all veteransreceiving care at the VA. All patients with an admission diagnosis of AH (ICD9 code 571.1)during the years 2004 to 2009 and without concomitant cancer, HIV/AIDS, acuteacetaminophen toxicity (ICD9 965.4), Acute hepatitis B infection (070.2, 070.3), Budd ChiariSyndrome (453.0), Autoimmune hepatitis (571.42) or Portal vein thrombosis (452). A patientwas defined to have CHC if they had a VA documented positive antibody test at any time priorto discharge. The severity of AH was determined by using MELD score. Cox proportionalhazard survival analysis was performed to identify independent predictors of 90 days mortalitypost discharge. Results: Of the 4129 patients admitted with alcoholic hepatitis, 1028 (24.9%)had HCV. The MELD score for all patients, HCV and non HCV patients were 13.9(±8.9),14.2(±9.6), 13.8(±8.7) and prevalence of cirrhosis prevalence were 17.7%, 23.6%, 15.7%. The90day survival rates for HCV versus non HCV patients were % and % for all patients(p=0.0007), % and % for cirrhotics (p=0.0705) and % and % for non cirrhotic (p=0.0373). Whenwe stratified the analysis by cirrhosis there was no effect of MELD score or HCV on mortality.Among non cirrhotics the impact of HCV on survival was most among patients with MELD > 11.The final predictors were age (HR 1.05, p 0.0002), albumin (0.41, <0.0001), total bilirubin (1.07,< 0.0001), creatinine (1.16, 0.0461) and HCV (1.78, 0.0121). For patients with MELD > 11 therate of use of either Pentoxyphylline or glucocorticoids were lower among HCV (11%) vs. nonHCV (15.7%), RR =0.95, C.I., 0.88 to 1.02. Conclusion: We found that HCV is an independentpredictor of mortality in AH. The impact of HCV infection on mortality appears to be moreimportant in non-cirrhotic subjects as compared to cirrhotic subjects. 24
  25. 25. #L7Thrombin signaling enhances TGFβ induction of integrin β6 in bile duct epithelial cellsBradley P. Sullivan and James P. LuyendykDepartment of Pharmacology, Toxicology & Therapeutics, KUMCDamage to intrahepatic bile ducts causes cholestasis, inflammation, coagulation cascadeactivation and liver fibrosis. Recent studies have shown that the αVβ6 integrin, which activatesthe fibrogenic cytokine TGFβ, contributes to liver fibrosis. Expression of the β6 integrin (Itgβ6) isrestricted to bile duct epithelial cells (BDECs) in the liver after common bile duct ligation.However, the mechanisms of Itgβ6 mRNA regulation in BDECs have not been identified. Wetested the hypothesis that coagulation protease signaling induces Itge 6 mRNA expression inBDECs. Treatment of transformed human BDECs (MMNK-1) with thrombin or proteaseactivated receptor-1 (PAR-1) agonist peptides (TFLLRN or SFLLRN) induced the expression ofinterleukin 8 (IL-8), a chemokine induced by PAR-1 signaling. However, neither thrombin noragonist peptides increased Itgβ6 mRNA levels in MMNK-1 cells. In contrast, treatment of theMMNK-1 cells with TGFβ increased Itgβ6 mRNA levels without affecting IL-8 expression. Asthrombin and TGFβ are likely generated concurrently during cholestatic liver injury, wedetermined whether thrombin enhanced TGFβ induction of Itgβ6 mRNA. Co-treatment witheither thrombin or the agonist peptides significantly enhanced TGFβ induction of Itgβ6 mRNA,whereas co-treatment with a scrambled peptide (FSLLRN) was without effect. Thrombin, but notTGFβ, activated the p38 and JNK1/2 MAPK signaling pathways. Itgβ6 mRNA induction in cellsco-treated with thrombin and TGFβ was significantly reduced by a p38 inhibitor (SB203580) anda JNK1/2 inhibitor (SP600125). Taken together, the results suggest that thrombin activation ofp38 and JNK1/2 signaling enhances TGFβ dependent induction of the Itgβ6. Further elucidationof the mechanisms whereby these fibrogenic mediators synergistically induce the Itgβ6 andcontribute to fibrogenesis may reveal novel strategies for the treatment of cholestatic liverdisease. 25
  26. 26. #L8Pharmacologic inhibition of caspase 1 and modulation of the inflammatory responseduring acetaminophen hepatotoxicityC. David Williams, Mary Lynn Bajt, and Hartmut JaeschkeDepartment of Pharmacology, Toxicology & Therapeutics, KUMCBackground and aims: Acetaminophen (APAP) hepatotoxicity is the most frequent cause ofacute liver failure in the US and many European countries. In addition to intracellular signalingmechanisms leading to cell necrosis, recent data indicate that activation of toll-like receptors onmacrophages by damage-associated molecular patterns (DAMPs) leads to cytokine formation,especially interleukin-1β (IL-1β). It was suggested that IL-1β is a critical mediator ofinflammatory injury during APAP overdose (Nat Med 13:851, 2007; J Clin Invest 119:305,2009). Since caspase-1 processes pro-IL-1β to its active, soluble form, the objective was toevaluate the effect of a pancaspase inhibitor (Z-VD-fmk) on IL-1β formation, inflammation andliver injury during APAP overdose. Methods: C57Bl/6 mice were given 10 mg/kg Z-VD-fmk orvehicle two hours after 300 mg/kg APAP and the inflammatory response (IL-1β, neutrophils) andliver injury (ALT, necrosis) were evaluated. Results: During APAP overdose liver IL-1β mRNAincreased six-fold with or without Z-VD-fmk. Plasma IL-1β protein was reduced below baselinelevels by Z-VD-fmk indicating specific inhibition of IL-1β processing, but no difference in ALT ornecrosis was observed. Recombinant IL-1β caused systemic neutrophil activation in vivo(CD11b expression, ROS priming); such activation was not observed after APAP. Accumulationof neutrophils in the liver in response to APAP was not affected by Z-VD-fmk. Similar resultswere obtained with mice deficient in the IL-1 receptor or beta2 integrin (CD18). Conclusions:During APAP hepatotoxicity, IL-1β is formed through a caspase-dependent mechanism(inflammasome). Multiple cytokines, not just IL-1β, generated in response to release of DAMPsby necrotic hepatocytes recruit neutrophils into the liver. These leukocytes do not cause injurybut are involved in removal of necrotic cell debris. Thus, intracellular events includingmitochondrial dysfunction, oxidant stress, JNK activation and nuclear DNA fragmentation arecritical determinants of APAP-induced cell death independent of the inflammatory response.(DK070195) 26
  27. 27. #L9Function of the hepatitis C virus (HCV) viroporin ion channel p7A.L Wozniak1, 3, M Yi2, S.M Lemon2, and S.A Weinman31 Department of Neuroscience and Cell Biology, KUMC2 Department of Microbiology and Immunology, University of Texas-Medical Branch, Galveston,Texas3 Department of Internal Medicine, KUMCHCV is a major cause of liver disease and current therapy is ineffective in half of infectedpatients. The viral p7 protein is an ion channel critical for infectious particle production. It is anattractive antiviral target but the role that it plays in the HCV lifecycle is unclear. Understandinghow p7 promotes viral particle formation will be of critical importance in the development of newantivirals. This work seeks to evaluate the contribution of p7 proton channel activity to organellepH regulation and virus production. Incorporating a fluorescent pH sensor within native,intracellular vesicles demonstrated that p7 is able to rapidly equilibrate H+ gradients and that thisactivity is inhibited by several viroporin inhibitors. HCV infected hepatoma cells and p7containing replicon cells displayed a 25% decrease in the number of acidic compartments(pH<5) and an alkaline shift in pH in the remaining vesicles. These effects could be reversed byp7 inhibitors and were not present in subgenomic replicons that do not express p7. To furtherconfirm that p7 caused the loss of acidification, we compared a chimeric virus containing wt p7vs. a mutant p7 lacking ion channel activity. Wt cells lost their acidic vesicle-associatedfluorescence while the mutant cells had similar acidic compartments as control cells. Preliminarystudies showed that the acidification inhibitor bafilomycin partially restored virus production tomutant p7 cells demonstrating that the p7-induced loss of acidification is a key requirement forvirus production. In conclusion, p7 serves as a H+ channel preventing acidification in the mostacidic intracellular compartments. This loss of acidification is required for productive HCVinfection. 27
  28. 28. #L10Ncb5or, fatty acid metabolism and oxidative stressMing Xu1, WenFang Wang1, and Hao Zhu21 Department of Physical Therapy & Rehabilitation Science2 Department of Clinical Laboratory Sciences, KUMCNADH cytochrome b5 oxidoreductase (Ncb5or) is a ubiquitous redox enzyme found in allanimals. Ncb5or knockout (KO) mice develop maturity-onset diabetes and lipoatrophy.Microarray analysis of 5-week-old livers reveals higher expression levels of genes in oxidativestress responses and de novo lipogenesis in prediabetic KO than wild-type samples. This geneexpression profile was confirmed by quantitative PCR, and the increased oxidative stress in KOlivers was further supported by their higher content of oxidized glutathione. To study thepotential function of Ncb5or in metabolizing exogenous saturated fatty acids, we isolatedprimary hepatocytes from prediabetic KO mice and compared them to cells from age-matchedwild-type controls. After an 8-hour treatment with a medium dose of palmitates, KO hepatocyteshad significantly higher levels of cell-death, intracellular fatty acids, transcripts of oxidativestress and ER stress genes, reactive oxygen species (through DCHF–staining), and oxidizedglutathione. The oxidative stress in the KO hepatocytes can be alleviated by co-incubation witholeate, which is the major intracellular monounsaturated fatty acid and a preferred substrate fortriglyceride synthesis. Our preliminary data also show a faster β-oxidation rate and a lowerdesaturation index (monounsaturated: saturated ratio) in KO hepatocytes. Taken together, ourstudies suggest that Ncb5or is essential for cells to metabolize exogenous saturated fatty acidsand to maintain cellular redox homeostasis. (Supported by NIH RO1 DK067355 grant) 28
  29. 29. #L11Enhanced expression of Nrf2 in mice attenuates the fatty liver produced by a methionineand choline deficient DietYu-Kun Jennifer Zhang, Ronnie L. Yeager, Yuji Tanaka, and Curtis D. KlaassenDepartment of Pharmacology, Toxicology & Therapeutics, KUMCOxidative stress has been proposed as an important promoter of the progression of fatty liverdiseases. The current study investigates the potential functions of the Nrf2-Keap1 signalingpathway, an important hepatic oxidative stress sensor, in a rodent fatty liver model. Mice withno (Nrf2-null), normal (wild-type, WT), and enhanced (Keap1 knockdown, K1-kd) expression ofNrf2 were fed a methionine and choline deficient (MCD) diet or control diet for 5 days.Compared to WT mice, the MCD diet-caused hepatosteatosis was more severe in the Nrf2-nullmice and less in the K1-kd mice. The Nrf2-null mice had lower hepatic glutathione andexhibited more lipid peroxidation, whereas the K1-kd mice had the highest amount ofglutathione in the liver and developed the least lipid peroxidation among the three genotypes fedthe MCD diet. The Nrf2 signaling pathway was activated by the MCD diet, and the Nrf2-targeted cytoprotective genes Nqo1 and Gstα1/2 were induced in WT, and even more K1-kdmice. In addition, Nrf2-null mice on both control and MCD diets exhibited altered expressionprofiles of fatty acid metabolism genes, indicating Nrf2 may influence the lipid metabolism inliver. For example, mRNA levels of long chain fatty acid translocase CD36 and the endocrinehormone Fgf21 were higher in livers of Nrf2-null mice and lower in the K1-kd mice than WTmice fed the MCD diet. Taken together, these observations indicate that Nrf2 could deceleratethe onset of fatty livers caused by the MCD diet by increasing hepatic antioxidant anddetoxification capabilities. 29
  30. 30. Transport and Biotransformation Abstracts (T1-T8)#T1Bile acids induce inflammatory genes in hepatocytes: a novel mechanism ofinflammation during obstructive cholestasisKatryn Allen, Hartmut Jaeschke, and Bryan L. CoppleDepartment of Pharmacology, Toxicology & Therapeutics, KUMCBackground and Aims: Inflammation contributes to liver injury during cholestasis. Themechanisms by which cholestasis initiates an inflammatory response in the liver, however, arenot known. Two hypotheses were investigated in the current studies. First, activation of toll-likereceptor 4 (TLR4) by either bacterial lipopolysaccharide (LPS) or damage-associated molecularpattern molecules (DAMPs) released from dead hepatocytes triggers an inflammatory response.Second, bile acids act as inflammagens, and directly activate signaling pathways in hepatocytesthat stimulate production of proinflammatory mediators. Methods: LPS-resistant C3H/HeJ miceand LPS-sensitive C3Heb/FeJ mice were subjected to bile duct ligation (BDL) and hepaticinflammation was quantified. Hepatocytes, isolated from wild-type and Egr-1 knockout mice,were treated with bile acids, and expression of proinflammatory genes was evaluated Results:Liver inflammation was not affected in TLR4-deficient mice after BDL. In contrast, treatment ofhepatocytes with bile acids which are found to be elevated in serum during BDL did not directlycause cell toxicity but increased expression of numerous proinflammatory mediators includingcytokines, chemokines, adhesion molecules, and other proteins that influence immune celllevels and function. Upregulation of several of these genes in hepatocytes and in the liver afterBDL required Egr-1. In addition, Egr-1 was upregulated in the livers of patients with cholestasis,and correlated with levels of inflammatory mediators. Conclusions: These data demonstratethat TLR4 is not required for the initiation of acute inflammation during cholestasis. In contrast,bile acids such as taurocholic acid directly activate a signaling network in hepatocytes thatpromotes hepatic inflammation during cholestasis.(Supported by the Liver Tissue Bank of the Liver Center) 30
  31. 31. #T2The Effect of Aging on Bile Acid HomeostasisZidong Donna Fu, Iván L. Csanaky, Curtis D. KlaassenDepartment of Pharmacology, Toxicology, & Therapeutics, KUMCAging is a physiological process accompanied with declining body function over time. Bile acids(BAs) are signaling molecules whose homeostasis is crucial for normal physiological functions.The alteration in BA homeostasis is likely related to several pathophysiological changes duringaging. Therefore, the present study was performed to determine the effect of aging onconcentrations of 8 conjugated and 10 unconjugated BAs (using UPLC-MS/MS) in serum andliver and the mRNA level of genes related to BA homeostasis (using multiplex suspensionassay) in both male and female C57BL/6 mice, from 3 to 29 months of age. Total, conjugated,and unconjugated BA concentrations increased with age in both liver (2-3 fold) and serum (3-4fold) for both genders. The mRNA of Cyp7a1, the rate-limiting enzyme in the classic BAsynthesis pathway, decreased after 15 months of age. Cyp27a1 and Cyp7b1, two enzymescritical for the alternative synthesis pathway, exhibited a gradual decrease in mRNA with age.However, the expression of BA conjugation enzymes BAL (bile acid-CoA ligase) and BAT (bileacid CoA: amino acid N-acyltransferases) remained consistent with age. The main hepatic BAtransporters Ntcp (uptake) and Bsep (efflux) shared a bell shape curve with the peak at around12 months of age. Therefore, the decrease in BA transporters after 12 months of age might bethe reason for increased BA concentrations despite the decreased expression of synthesisenzymes. The present study suggests that the increased BA concentrations in serum and liverduring aging might be due to decreased expression of hepatic BA uptake and effluxtransporters. (Supported by NIH grants ES-009716, ES-009649, ES-013714, ES-007079,RR-021940) 31
  32. 32. #T3Suppression of cyp7a1 gene transcription by FXR in mice is mediated through theintestine-initiated FGF15/FGFR4 pathwayBo Kong¹, Jeff Aube³, Li Wang², and Grace L. Guo¹¹Department of Pharmacology, Toxicology & Therapeutics, KUMC²Department of Oncological Sciences, University of Utah, Salt Lake City, UT³Department of Medicinal Chemistry, KU Lawrence, KSThe farnesoid X receptor (FXR), cellular bile acids sensor, involves in bile acids homeostasis,and represses the transcription of Cyp7a1 gene, which encodes the rate-limiting enzyme in theclassic pathway of bile acid synthesis. In this study, the mice with hepatocyte- or enterocyte-specific deletion of the FXR gene (AFXR, FXR floxed/floxed albumin Cre(+) mice; VFXR, FXRfloxed/floxed villin Cre(+) mice) were used to clarify the pathway of FXR/SHP/LRH1 andFXR/FGF15/FGFR4 in the inhibition of Cyp7a1 gene expression. Oral administration with FXRagonist GW4064 significantly repressed Cyp7a1 expression in AFXR mice but not VFXR mice,and intestine FGF15 mRNA levels are inversely correlated to Cyp7a1 mRNA levels. These datademonstrated that the negative feedback regulation of Cyp7a1 gene expression is dependenton intestine FXR/FGF15/FGFR4 pathway than on liver FXR/SHP/LRH pathway in mice.Furthermore, intravenous infusion of recombinant FGF15 in wild type and tissue specific FXRknockout mice markedly reduced liver Cyp7a1 gene expression and activated the MAPKsignaling pathways including JNK and ERK1/2 MAP kinases. FGF15 induced JNK and ERK1/2phosphorylation in a time-dependent and dose-dependent way, and their major downstreamtargets, c-Jun and early growth responsive-1 (Egr-1), might involved in the inhibition of Cyp7a1gene transcription. In summary, the intestinal FGF15 inhibits liver Cyp7A1 expression throughactivation of JNK and ERK1/2 pathway is the major mechanism for the feedback regulation ofbile acid biosynthesis in mice. 32
  33. 33. #T4Assessment of modulation of intestinal microflora following alterations in bile acidtransportPallavi B. Limaye, Youcai Zhang, and Curtis D. KlaassenDepartment of Pharmacology, Toxicology & Therapeutics, KUMCNormal intestinal microflora is essential for various physiological functions such as innateimmunity, energy harvest, and bile acid metabolism. Nearly 90% of the bile acids secreted in theduodenum in response to food ingestion are reabsorbed through terminal ileum and taken up bythe liver via the Oatp1a1 and other bile acid transporters. It is known that bile acids reaching thelarge intestine are further metabolized by the gut bacteria to potentially toxic secondary bileacids. Investigations from our laboratory have revealed significant increase in secondary bileacids including deoxycholic acid (DCA) and lithocholic acid (LCA) in the small (SI) and largeintestine (LI) of Oatp1a1 knockout mice. Oatp1a1 knockout mice also exhibited an increase inunconjugated bile acids in the SI. We hypothesized that the increase in secondary bile acids inthe Oatp1a1 knockout mice is due to change in the intestinal bacterial profile. We quantified thegut bacterial population using 16s rDNA probe-based branched DNA assay. The data indicatesignificant overgrowth of the majority of the bacterial species assessed in small intestine ofOatp1a1 knockout mice. In the large intestine however, a striking increase in Bacteroidesbacteria, while a decrease in Firmicutes bacteria was observed in the Oatp1a1 knockout mice.These findings indicate that the change in the intestinal bacterial profile in the Oatp1a1 knockoutmice may be involved in increased secondary bile acid production. 33
  34. 34. #T5Mechanism of polybrominated diphenyl ether (PBDE) uptake into human and mouse liverErik K. Pacyniak¹, Megan Roth¹, Bruno Hagenbuch¹, ², and Grace L. Guo¹¹ Department of Pharmacology, Toxicology & Therapeutics, KUMC² The Cancer Center, KUMC2,2`,4,4`-tetrabromodiphenyl ether (BDE47), 2,2,4,4,5-pentabromodiphenyl ether (BDE-99) and2,2,4,4,5,5-hexabromodiphenyl ether (BDE-153) are the predominant polybrominated diphenylether (PBDE) congeners detected in human samples. While the hepatic metabolism of PBDEshas been investigated in humans and mice, the mechanism by which PBDEs enter the liver hasnot been elucidated. However, due to their large molecular weights (~485 to 1000 Da), theycannot enter hepatocytes by simple diffusion. Organic anion transporting polypeptides (OATPs:humans; Oatps: rodents) are responsible for hepatic uptake of a variety of amphipathiccompounds of molecular weights larger than 350 Da. Therefore, the objective of the currentstudy was to identify OATPs/Oatps responsible for uptake of BDE47, BDE99 and BDE153 inhuman and mouse liver. To test this hypothesis we used Chinese Hamster Ovary (CHO) celllines expressing either OATP1B1, OATP1B3 or OATP2B1 as well as Human Embryonic Kidney293 (HEK293) cells transiently expressing the murine orthologs of the OATP1B (Oatp1b2) andOATP2B (Oatp2b1) family. Direct uptake studies confirmed that all three PBDE congeners aresubstrates for the tested human and mouse hepatic OATPs/Oatps. Detailed kinetic analysisrevealed that OATP1B1 transported BDE47 with the highest affinity (Km= 0.31μM) followed byBDE99 (Km= 0.91μM) and BDE153 (Km = 1.9 μM). For OATP1B3 the order was the same(BDE47: Km = 0.41 μM; BDE99: Km = 0.70 μM; BDE153: Km = 1.7 μM). Similarly, Oatp1b2transported BDE47 (Km= 0.46μM) with the greatest affinity followed by BDE99 (Km= 0.71μM)and BDE153 (Km= 1.4μM). However, both OATP2B1 (BDE47: Km = 0.81 μM; BDE99: Km =0.87 μM; BDE153: Km = 0.65 μM) and Oatp2b1 (BDE47: Km = 0.95μM; BDE99: Km = 1.1μM;BDE153: Km = 1.0) transported all three congeners with similar affinities. The results presentedclearly suggest that uptake of PBDEs via these OATPs/Oatps is a mechanism responsible forliver specific accumulation of PBDEs. 34
  35. 35. #T6Isolation of a modulator of the liver specific organic anion transporting polypeptides(OATPs) 1B1 and 1B3 from rollinia emarginata schlecht (annonaceae)Megan Roth1, Juan Araya2, Barbara Timmermann2, and Bruno Hagenbuch1.1 Department of Pharmacology, Toxicology & Therapeutics, KUMC2 Department of Medicinal Chemistry, KU Lawrence, KSOrganic anion transporting polypeptides 1B1 and 1B3 (OATP1B1 and OATP1B3) are liverspecific transporters that mediate the uptake of a broad range of drugs into hepatocytes,including statins, antibiotics and many anticancer drugs. Compounds which alter transport byone or both of these OATPs could potentially be used to target drugs to hepatocytes or toimprove bioavailability of drugs that are cleared by the liver. In this study, we applied abioassay guided isolation approach to identify such compounds from the organic extract ofRollinia emarginata Schlecht (Annonaceae). Fractions of the plant extract were screened foreffects on OATP1B1- and OATP1B3-mediated transport of 0.1µM estrone-3-sulfate andestradiol-17β-glucuronide. A rare compound, quercetin 3-O-α-l-arabinopyranosyl (1→2) α-l-rhamnopyranoside, was isolated that had distinct effects on each OATP. OATP1B1-mediatedtransport of both model substrates was strongly inhibited, as was OATP1B3-mediated transportof estradiol-17β-glucuronide. However, OATP1B3-mediated uptake of estrone-3-sulfate wasstimulated 4- to 5-fold. Kinetic analysis of this stimulation revealed that the apparent affinity forsubstrate was increased (Km reduced from 100μM to 10μM), while the Vmax was significantlyreduced. These results demonstrate a mechanism through which the hepatic uptake of drugOATP substrates could be stimulated. 35
  36. 36. #T7The coagulation system contributes to αVβ6 integrin expression and liver fibrosisinduced by cholestasisBradley P. Sullivan¹, Paul H. Weinreb², Shelia M. Violette³, and James P. Luyendyk¹¹Department of Pharmacology, Toxicology & Therapeutics, KUMC² Biogen Idec, Inc., ³ Stromedix Inc.Chronic injury to intrahepatic bile duct epithelial cells (BDECs) elicits expression of variousmediators that promote liver fibrosis, including the αVβ6 integrin. We tested the hypothesis thattissue factor (TF)-dependent thrombin generation and protease activated receptor-1 (PAR-1)activation contribute to liver fibrosis induced by cholestasis via induction of αVβ6 expression. Totest this hypothesis, mice deficient in either TF or PAR-1 were fed a diet containing 0.025%alpha-naphthylisothiocyanate (ANIT), a BDEC selective xenobiotic. Coagulation cascadeactivation and liver fibrosis were reduced in genetically modified mice with a 50% reduction inliver TF activity fed the ANIT diet. Similarly, liver fibrosis was significantly reduced in PAR-1-/-mice fed an ANIT diet. Hepatic integrin β6 mRNA induction and the expression of αVβ6 byintrahepatic BDECs were reduced by TF-deficiency and PAR-1-deficiency in mice fed the ANITdiet. Treatment with either anti-αVβ6 antibody or soluble TGF-β receptor reduced liver fibrosis inmice fed the ANIT diet. PAR-1 activation enhanced TGF-β1-induced integrin β6 mRNAexpression in transformed human BDECs. Interestingly, TF and PAR-1 mRNA levels wereincreased in livers from patients with cholestatic liver disease. The results indicate that a TF-PAR-1 pathway contributes to liver fibrosis induced by chronic cholestasis by increasingexpression of the αVβ6 integrin, an important regulator of TGF-β1 activation.(Supported by the Liver Tissue Bank of the Liver Center) 36
  37. 37. #T8Tissue-specific FXR-DNA binding: genome-wide liver and intestine binding analysis byChIP-SeqAnn Thomas, Steven Hart, Bo Kong, Jianwen Fang, Xiaobo Zhong, and Grace L. GuoDepartment of Pharmacology, Toxicology & Therapeutics, KUMCFarnesoid X receptor (FXR) is a ligand activated transcription factor belonging to the nuclearreceptor superfamily. FXR is highly expressed in the liver and intestines and bile acids are itsendogenous ligands. FXR is essential in maintaining bile acid homeostasis, but is also critical inregulating lipid and glucose metabolism. However, direct FXR target genes in the liver andintestine are not completely known. The current study was designed to determine genome-wideFXR target genes in the liver and intestine by chromatin immunoprecipitation (ChIP) coupled tomassively parallel sequencing (ChIP-Seq) following treatment of mice with a synthetic FXRligand (GW4064). The results revealed novel genome-wide FXR-DNA binding. (1) FXR bindingshows a large degree of tissue-specificity, with the liver and intestines sharing only 11% of totalFXR binding sites. (2) Uncharacterized binding sites were identified in different regulatoryregions of putative FXR target genes, including upstream, intragenic, or downstream of genes.Nearly 35-40% of total FXR binding sites were located more than 10 kb from a refseq gene,indicating a long-distance chromatin interaction as a potential mechanism for FXR regulation ofgene expression. Novel binding sites were also found with in regulatory regions of known FXRtarget genes, such as Nr0b2 and Fgf15. In addition, FXR binding sites were discoveredupstream of the Slc10a1 gene in the liver and Osta lacked FXR binding sites in the liver, whichcontradicts previous reports. (3) Motif analysis of FXR binding sites showed a nuclear receptorhalf binding site was commonly associated with the classical FXR response element (invertedrepeat separated by 1 nucleotide [IR1]), indicating the involvement of other nuclear receptors ortranscription factors for facilitating regulatory functions of FXR. (4) Gene Ontology pathwayanalysis revealed FXR is significantly involved in important metabolic pathways, includingorganic acid, lipid, and fatty acid metabolic processes, which has not been previously identifiedor sufficiently studied. In conclusion, this study provides a tissue-specific genome-wideassessment of FXR-DNA binding in vivo. The results from this study suggest tissue-specificregulatory functions of FXR as well as reveal novel metabolic pathways potentially regulated byFXR. 37