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 Center
The University of Kansas Cancer Center, and
                Bohan Fund

            Beller Conference Center
              Hemenway Building
We dedicate 2010 Liver Symposium to the people who contributed to
                  The Liver Center Tissue Bank

                 Patients who donated their tissue

                         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 Cazarez

Liver Tissue Procurement and Liver Tissue Banking Protocol (#11378)
                      PI: Yu-Jui Yvonne Wan

                     The University of Kansas Liver Center Tissue Bank

Natali 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 Wan

The University of Kansas Medical Center, Kansas City, KS

The University of Kansas Liver Center Tissue Bank is a human liver tissue supplier for
biomedical research at the University of Kansas Medical Center. This liver depository was
established in November 2008 supported by the Institution. The KU Liver Center Tissue Bank
activities and services include coordinating patient consent, collecting and banking fresh frozen
livers from excess surgical materials in liver transplants, resections, and biopsies, processing
and banking blood and urine, maintaining a specimen database with extensive clinical and
pathological data, and distributing samples to researchers. Currently, the bank maintains an
incredible success rate of 95% patient participation due to the great interest not only from
physicians, but also from patients eager to help fight liver diseases. Since inception, 562 tissue
samples have been deposited. In addition, the bank has collected 236 blood samples, 103
serum samples, and 27 urine samples. The KU Liver Center Tissue Bank can provide a variety
of 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 collects
clinical data including demographic information, BMI, past medical history, alcohol and tobacco
use, medication intake, HCV treatment details, and past liver transplant information. The
comprehensive clinical database allows the KU Liver Center Tissue Bank to distribute
specimens with extensive specificity. So far, seven ongoing studies are using specimens
obtained from the KU Liver Center Tissue Bank. Five scientific abstracts are presented in this
year’s Liver Center Symposium. One scientific abstract has been presented in a national
meeting and two manuscripts have been submitted for publication. This liver depository serves
as a meeting point for physicians and basic researchers and thus enhances the progress of
translational research.

                               Thursday, May 27, 2010

                                 Beller Conference Center

7:30 - 8:00 am       Continental Breakfast

8:00 - 8:10 am       Welcome note by Barbara F. Atkinson, M.D., Executive Vice Chancellor,
                     KUMC, Executive Dean, School of Medicine

8:10 - 8:30 am       Yu-Jui Yvonne Wan, Ph.D., Professor, Director, Liver Center
                     Liver Center Progress Report

8:30 - 8:50 am       Steven Weinman, M.D., Ph.D., Professor, Director, Translational Research
                     Translational Research Report

Hepatocellular Carcinoma – Faris Farassati, Pharm.D., Ph.D. - Moderator

8:50 - 9:20 am       Udayan Apte, Ph.D., Assistant Professor
                     Ablation off Farnesoid X receptor promotes hepatocellular carcinoma
                     development via activation of Wnt/beta-catenin pathway

9:20 - 9:50 am       Partha Krishnamurthy, Ph.D., Assistant Professor
                     ATP binding cassette transporter ABCB6; Potential therapeutic target in

9:50 - 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 partner

10:05 – 10:20 am     Break

Liver Injury – James Luyendyk, Ph.D. - Moderator

10:20 - 10:50 am     Wen-Xing Ding, Ph.D., Assistant Professor
                     Role of autophagy in acute alcoholic liver injury

10:50 -11:20 am      Hartmut Jaeschke, Ph.D., Professor
                     Translational research in acetaminophen hepatotoxicity

11:20 - 11:35 am     Hao Zhu, Ph.D., Assistant Professor
                     Ncb5or, fatty acid metabolism and oxidative stress

11:35 - 11:50 am     Ann Wozniak, Graduate Student - Steven Weinman’s laboratory
                     Function of the hepatitis C virus (HCV) viroporin ion channel p7

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 cholestasis

12:05 - 2:10 pm      Lunch served in Beller 1005-1007
                     Poster viewing in foyer of Beller conference area

Transport & Biotransformations – Sitta Sittampalam, Ph.D. - Moderator

2:10 - 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 diseases

2:40 - 3:10 pm       Jeff Staudinger, Ph.D., Associate Professor, Department of
                     Pharmacology & Toxicology, KU Lawrence, KS
                     Inflammation, PXR modification, and drug disposition

3:10 - 3:25 pm       Pallavi Limaye, Ph.D., Postdoctoral Fellow – Curtis Klaassen’s laboratory
                     Assessment of modulation of intestinal microflora following alterations in
                     bile acid transport

3:25 – 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

Plenary lecture – Steven Weinman, M.D., Ph.D. – Moderator

3:40 – 4:40 pm     Keith Lindor, M.D., Professor of Medicine, Dean, Mayo Medical School
                   New developments in primary sclerosing cholangitis

                                       Symposium End


                    Liver Center External Advisory Committee Members

Anna Mae Diehl, M.D., Chief Division of Gastroenterology, Director of the Liver Center,
Duke University Medical Center
Dr. Diehl received her medical degree from Georgetown University and completed her internal
medicine training and fellowship in gastroenterology and hepatology at Johns Hopkins
University. She was a faculty member at Johns Hopkins University most of her professional
career, and was promoted to Professor of Medicine there in 1997. In 2004, Dr. Diehl was
recruited to Duke University to lead their gastroenterology and hepatology programs and was
awarded the Florence A. McAlister Endowed Professorship. Her research interests include liver
injury and repair. Her lab focuses on the mechanisms that regulate this process using cultured
cells, animal models of acute and chronic liver damage, and samples from patients with various
types of liver disease. Her group also conducts clinical trials in patients with chronic liver

Alex Lentsch, Ph.D., Professor of Surgery, Vice Chairman for Research, Department of
Surgery, University of Cincinnati College of Medicine
Dr. Lentsch received his Bachelors of Science degree in Biological Sciences from Northern
Kentucky University, Highland Heights, Kentucky and his Physiology and Biophysics
from the University of Louisville, Louisville, Kentucky. Dr. Lentsch’s research interests include
regulation of inflammatory responses by cytokines, chemokines and adhesion molecules. A
central theme of his research is aimed at determining how specific signal transduction pathways
utilized by cytokines and chemokines control the development of inflammatory disease.

Keith Lindor, M.D., Professor of Medicine, Dean, Mayo Medical School
Dr. Lindor received a Bachelor’s of Chemistry degree from the University of Minnesota and his
MD degree from Mayo Medical School. Following this, he was a resident in internal medicine at
Bowman Grey School of Medicine of Wake Forest University. Before starting his
Gastroenterology Fellowship at Mayo, he served a year in the Indian Health Service in Southern
Arizona. Dr. Lindor’s research interests include cholestatic liver diseases in adults, particularly
primary biliary cirrhosis and primary sclerosing cholangitis as well as nonalcoholic
steatohepatitis. The focus of his research work is primarily on clinical trials and means of
optimizing the medical management of patients with these disorders. Dr. Lindor serves as the
Senior Associate Editor for Clinical Gastroenterology and Hepatology. He is on the editorial
boards of Gastroenterology and Journal of Clinical Gastroenterology.

Shelly Lu, M.D., Professor of Medicine Keck School of Medicine, Associate Director of USC
Liver Center, University of Southern California
After graduating from Medical School of UCLA, Dr. Lu has held positions at Cedars-Sinai
Medical 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 hepatic
GSH synthesis, the regulation of hepatic methionine adenosyltransferases (MATs),
abnormalities in methionine metabolism in alcoholic liver injury, the role of SAMe in liver function
and injury, and MAT1A knockout mice as an animal model to study the effect of ethanol. She is
also associate director of the NIH-funded USC Research Center for Liver Diseases and
associate editor of the American Journal of Physiology: GI and Liver and the Encyclopedia of

George Michalopoulos, M.D., Ph.D., Professor and Chair of Pathology, School of Medicine,
University of Pittsburgh
Dr. George K. Michalopoulos received his medical doctoral degree at Athens University School
of Medicine in 1969. A residency in Anatomic Pathology and a Ph.D. in Oncology was obtained
at the Wisconsin Medical Center in Madison in 1977. Dr. Michalopoulos moved to Duke
University as Assistant Professor in 1977 and stayed at Duke University until 1991. He then
moved 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 of
Medicine from November, 1995, until November, 1998. His research interests include growth
factors and receptors in hepatocytes, growth regulation of human hepatocytes, and hepatic

             Liver Center Internal Advisory Committee Members

Bashar Abdulkarim, M.D., Ph.D., Assistant Professor, Department of Surgery

Richard Gilroy, M.D., Associate Professor, Department of Internal Medicine

Hartmut Jaeschke, Ph.D., Professor, Department of Pharmacology, Toxicology &

Mojtaba Olyaee, M.D., Professor and Director, Gastroenterology/Hepatology/Motility,
                     Department of Internal Medicine

Peter Smith, Ph.D., Director, Department of KS Intellectual & Developmental Disabilities
                      Research Center

Yu-Jui Yvonne Wan, Ph.D., Professor, Department of Pharmacology, Toxicology &
                    Therapeutics, Director of the Liver Center

Steven Weinman, M.D., Ph.D., Professor, Department of Gastroenterology/Hepatology,
                   Director of Translational Research


            HCC (C1-C9)

        Liver Injury (L1-L11)

Transport & Biotransformation (T1-T8)

                         HCC Abstracts (C1-C9)

The differential effect of N-(4-hydroxyphenyl) retinamide and all-trans retinoic acid in
inducing apoptosis and autophagy in Huh7 cells

Nathan Bushue and Yu-Jui Yvonne Wan
Department of Pharmacology, Toxicology & Therapeutics, KUMC

N-(4-hydroxyphenyl) retinamide (4HPR, fenretinide) is a synthetic analog of all-trans retinoic
acid (ATRA). In contrast to the differentiation effect of ATRA, 4HPR induces apoptosis of human
hepatocellular carcinoma Huh7 cells. While previous data shows that both differentiation and
apoptosis are mediated through a retinoic acid receptor β (RARβ)-dependent manner, the
mechanisms by which 4HPR and ATRA exert their different roles are not well understood. Our
data showed that both chemicals induced RARβ expression level and regulated the activation of
ERK, JNK and Akt in the same manner. Both chemicals also induced the expression level of
LC3. 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 for
activation of death receptors and caspase 8-mediated apoptosis, was significantly lower in
ATRA than 4HPR-treated Huh7 cells. Furthermore, ATRA induced the expression of beclin
suggesting ATRA induced autophagy in Huh7 cell. Induction of autophagy by rapamycin
prevented 4HPR-induced apoptosis, while blocking autophagy with wortmannin increases
ATRA-induced cell death. Taken together, our data suggest that lack of autophagy in 4HPR
treated 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 the
differentiation effect of ATRA.


ABCB6; potential therapeutic target in HCC

Hemantkumar Chavan, Mahitha Oruganti, and Partha Krishnamurthy
Department of Pharmacology, Toxicology & Therapeutics, KUMC

Hepatocellular carcinoma (HCC) is a major health problem being the fifth most common cancer
worldwide and the third leading cause of cancer death. Despite significant progress in cancer
medicine, curative treatment options for HCC are still limited with an unsatisfactory survival rate.
Evidence suggests that molecular changes in gene expression that compromise proliferation
and cell death represent a pro-tumorigenic principle in human HCC. Here we show that Abcb6 a
mitochondrial ATP binding cassette transporter is upregulated in HCC. Abcb6 expression
promotes hepatocellular carcinoma cell growth and proliferation both in vitro in cell culture
models and in a human to mouse xenograft tumor model. These results suggest that Abcb6
might play a role in the development or progression of HCC. Future studies will address whether
Abcb6 expression in HCC is the cause or the result of the disease and explore the therapeutic
relevance of regulating Abcb6 expression in HCC.
(Supported by the Liver Tissue Bank of the Liver Center)


Ral overactivation in hepatocellular carcinoma: biology and therapeutic ramifications

Mohammad Taha, Mohamad Ezzeldin, and Faris Farassati
Department of Medicine, KUMC

Ras signaling pathway and its down-stream effectors are involved in the molecular etiology of
Hepatocellular Carcinoma. Overactivation of wild-type Ras and Ras gene mutations as well as
other oncogenes in Ras pathway are shown to be involved in more than 40% of human
malignancies. Ras pathway has been found to be overactivated in 30% of hepatocellular
carcinoma (HCC) cases. We have identified, for the first time, the overactivation of RalA, a
novel effector pathway down-stream of Ras in a series of HCC cell lines as well as tissue
samples. We have also investigated other key players of Ral pathway such as Aurora kinase
and 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 of
HCC cell lines (by gene specific silencing as well as an inhibitory peptide) and introduce our
results here in terms of targeting this pathway for treatment of HCC. The results of this research
not only enhance our knowledge about the biology of HCC but also provide a new strategy for
targeting this malignancy. Ras signaling pathway and its down-stream effectors are involved in
the pathogenesis of Hepatocellular Carcinoma (HCC). Overactivation of wild-type Ras and Ras
gene mutations as well as other oncogenes in Ras pathway are shown to be involved in more
than 40% of human malignancies. Ras pathway has been found to be overactivated in 30% of
HCC cases. We have identified, for the first time, the overactivation of RalA, a novel effector
pathway down-stream of Ras in a series of HCC cell lines as well as tissue samples. We have
also investigated other key players of Ral pathway such as Aurora kinase (stimulator of RalA
activation) and protein phosphatase 2A (PP2A, a negative regulator of RalA). While Aurora
kinase is over-expressed in HCC cell lines (but not normal hepatocytes), PP2A is expressed at
equal levels (Figure 1). Therefore, in HCC, RalA is mainly over-activated via stimulation by
Aurora kinases while PP2A may not play a major role in this case. We have also investigated
the effects of inhibition of RalA on the viability and invasiveness of HCC cell lines (by gene
specific silencing as well as an inhibitory peptide) and introduce our results here in terms of
targeting this pathway for treatment of HCC. The results of this research not only enhance our
knowledge about the biology of HCC but also provide a new strategy for targeting this
(Supported by the Liver Tissue Bank of the Liver Center)


Farnesoid x receptor activation mediates head-to-tail chromatin looping in the Nr0b2
gene encoding small heterodimer partner

Guodong Li¹, Ann M. Thomas¹, Steve N. Hart¹, Xiaobo Zhong¹, Dequan Wu², and Grace L. Guo¹
¹Department of Pharmacology, Toxicology & Therapeutics, KUMC
²Department of General Surgery, Second Affiliated Hospital, Harbin Medical University, Harbin
150086, China

As a unique nuclear receptor with only ligand-binding but no DNA-binding domain, small
heterodimer 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 in
the liver and previous studies have shown FXR highly induces SHP by binding to a FXR
response 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. This
downstream IR1 is highly conserved throughout mammalian species. We hypothesized that this
downstream FXRRE is functional and may mediate a head-to-tail chromatin looping by
interacting with the proximal promoter FRXRE to increase SHP transcription efficiency. In the
current study, a ChIP-qPCR assay revealed FXR strongly bound to this downstream FXRRE in
mouse livers. The downstream FXRRE is important for FXR-mediated transcriptional activation
revealed by luciferase gene transcription activation, as well as by deletion and site-directed
mutagenesis. The chromatin conformation capture (3C) assay was used to detect chromatin
looping and the result confirmed the two FXRREs located in the Nr0b2 promoter and
downstream enhancer interacted to form a head-to-tail chromatin loop. To date the head-to-tail
chromatin looping has not been reported in the liver. In conclusion, our results suggest a
mechanism by which activation of FXR efficiently induces SHP transcription is through head-to-
tail chromatin looping.


Yes-associated protein expression is induced in hepatocellular carcinoma and is
responsive to cell density.

Hua Li, Andy Wolfe, Genea Edwards, Grace L. Guo, Yu-Jui Yvonne Wan, and Udayan Apte
Department of Pharmacology, Toxicology & Therapeutics, KUMC

Hepatocellular carcinoma (HCC) is the most common malignancy of the liver associated with
high mortality. We are interested in understanding the role of pathways involved in organ size
regulation in pathogenesis of HCC. Specifically, we are investigating the role of Hippo Kinase
pathway and yes-associated protein (Yap), the downstream effector of the pathway. Three HCC
cell lines investigated, Hep3B, HepG2 and Huh7, demonstrated increase in Yap as compared to
normal human hepatocytes. The expression levels and cellular localization of Yap was
responsive to cell density. High cell confluency resulted in decreased Yap expression and
nuclear export. siRNA and shRNA-mediated knockdown of Yap resulted in decreased cell
proliferation of Hep3B cells. Immunohistochemical analysis of Yap in
phenobarbital+diethylnitrosamine-induced liver tumors in C57BL/6 mice, spontaneous tumors in
Farnesoid X Receptor knockout mice and human HCC biopsies revealed extensive nuclear
staining on Yap in highly proliferative tumors. Detailed analysis of Yap and its target genes
including CTGF, Glypican3, and Survivin by mRNA and Western blot indicated extensive Yap
activation in human HCC samples. These data indicate that Yap plays a critical role in
pathogenesis of HCC, possibly due to increased cell-adhesion independent growth and
suppression of apoptosis.
(Supported by the Liver Tissue Bank of the Liver Center)


Hippo kinase pathway is involved in postnatal liver development

Seth Septer1, Genea Edwards2, Andy Wolfe2, Hua Li2, and Udayan Apte2
 Department of Gastroenterology, Children’s Mercy Hospital, Kansas City, MO; 2Department of
Pharmacology, Toxicology & Therapeutics, KUMC

Liver exhibits size expansion and differentiation during postnatal period. During postnatal period
cells in the liver undergo a period of well-controlled proliferation to achieve the adult liver to
body weight ratio followed by hepatic differentiation. However, the mechanisms of liver size
regulation and differentiation are not completely clear. Herein we report that Hippo Kinase
signaling plays a role in liver size regulation and differentiation during postnatal liver growth
period. 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 associated
protein (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. Western
blot analysis showed increase in phopho-Yap between PND 20 to 30 indicating increased
degradation of Yap at those time points. Consistent with this, we observed increase in
expression of other components of Hippo Kinase pathway including Mst, Lats and Sav, which
regulate Yap phosphorylation. PCNA analysis indicated peak cell proliferation in the livers
between PND15-20. To further investigate the role of Yap in postnatal liver development we
studied liver growth in Yap+/- mice, which revealed small but significant decrease in the liver to
body weight ratio in the Yap+/- mice as compared to Yap+/+ mice. Taken together these data
indicate that Yap plays a role in both cell proliferation and possibly in hepatic differentiation
during postnatal liver development.


Hepatocyte nuclear factor 4 α (HNF4) is involved in regulation of hepatocyte

Chad Walesky1, Aaron Bell2, Genea Edwards1, Andy Wolfe1, and Udayan Apte1
 Department of Pharmacology, Toxicology & Therapeutics, KUMC; 2Department of Pathology,
University of Pittsburgh School of Medicine, Pittsburgh, PA

HNF4 is the master regulator of hepatocyte differentiation and regulates a wide variety of
hepatocyte-specific genes. Here we report that HNF4 is involved in inhibiting hepatocyte
proliferation. Decreased HNF4 expression was observed in hepatocyte specific integrin linked
kinase knockout mice (ILK-KO), which have sustained cell proliferation resulting in
hepatomegaly. Further analysis revealed decrease in expression of HNF4 targets such as p21
and claudin1, which are involved in cell-cycle regulation in ILK-KO livers. Hepatocyte specific
knockdown of HNF4 in HNF4-floxed mice using Cre recombinase carried by an adeno-
associated virus 8 (AAV-8) vectors resulted in induction of cell proliferation accompanied by
decreased expression of p21 and claudin1. Over-expression of rat HNF4 in rat hepatoma JM1
cells resulted in decreased proliferation and increased cells in G0-G1 phase of cell cycle. Taken
together, these data indicate that HNF4 plays an anti-proliferative role in the hepatocytes.


Bevacizumab and erlotinib in previously untreated inoperable and metastatic
hepatocellular carcinoma

Rangaswamy Govindarajan¹, Eric Siegel¹, Issam Makhoul¹, and Stephen Williamson²
¹University of Arkansas for Medical Sciences, Little Rock, AR
²Department of Medicine, KUMC

Purpose: The treatment options for metastatic and inoperable hepatocellular carcinoma are
limited to sorafenib, a RAF kinase inhibitor. This study was designed to study the efficacy
vascular. Endothelial growth factor (VEGF) antibody bevacizumab and VEGF tyrosine kinase
inhibitor erlotinib

Patients and Methods: Twenty-one subjects with metastatic and inoperable hepatocellular
carcinoma who had not been treated with local or systemic therapy were treated with
bevacizumab 15mg/kg every three weeks and oral daily dose of Erlotinib 150 mg. The primary
efficacy 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 for
the 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% CI
10%-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 consent
and one subject did not have adequate base line scans.

Conclusion: The study results met the predetermined criterion of >20% PFS at 27 weeks. The
combination of bevacizumab and erlotinib provides is a combination therapy to be explored
further in randomized setting.


HDAC inhibitors potentiate fenretinide-induced apoptosis through mitochondrial
enrichment of Nur77 in HCC cells

Hui Yang¹, ², and Yu-Jui Yvonne Wan¹
¹Department of Pharmacology, Toxicology & Therapeutics, KUMC
²Department of Gastroenterology, Second Affiliated Hospital, Guangzhou Medical College,
Guangzhou, China

Fenretinide [N-(4-hydroxyphenyl] retinamide), a synthetic retinoid derivative, can cause
ceramide accumulation and reactive oxygen species (ROS) generation to induce cell death.
Fenretinide is also implicated in preventing high-fat diet-induced obesity, insulin resistance, and
hepatic steatosis in animal models. Furthermore, fenretinide inhibits angiogenesis and hepatic
fibrosis. Previously, we have shown that fenretinide induced apoptosis in Huh7 human liver
cancer cells, however had no effect on HepG2 cells. In our current study, we demonstrate that
histone deacetylase inhibitors (HDACi) trichostatin (TSA) and scriptaid, can either enhance
fenretinide-induced apoptosis in the fenretinide sensitive HCC cells (Huh7 and Hep3B) or
sensitize 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 with
HDACi increased caspase 3/7 activity as well as cell death in comparison to fenretinide or
HDACi treatment alone. We did not observe the same effect in primary human hepatocytes as
seen in the cancer cell lines suggesting the ability of fenretinide and HDACi to induce apoptosis
is cancer cell specific. The fenretinide plus HDACi enhanced cell death observed in cancer cells
was positively associated with increased retinoic acid receptor β (RARβ) expression, induction
of Nur77 level in the mitochondria, as well as reduced RXRα expression. However, we did not
observe 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 3
activation and apoptosis of Huh7 cell. In addition, fenretinide- and HDACi-induced cytosolic
Nur77 expression was not detected in Huh7 cells in which RARβ expression was inhibited by
siRNA. Furthermore, Nur77 and RARβ are co-localized in the cytosol of fenretinide or HDACi
treated Huh7 cells revealed by confocal microscopy. Immunoprecipitation followed by western
blot indicated that Nur77 and RARβ interacted with each other upon fenretinide or HDACi
treatment. Taken together, our data indicated that fenretinide and HDACi combination treatment
is 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 RARβ
and Nur77 induction by fenretinide/HDACi treatment favors the interaction between RARβ and
Nur77 therefore resulting in the nuclear export of Nur77 to target mitochondria and induction of
apoptosis. Thus, HDACi in combination with fenretinide modulate the expression and
intracellular localization of nuclear receptors, which in turns regulate the apoptosis of liver
cancer cell. (CA53596)

                   Liver Injury Abstracts (L1-L11)

Low oxygen prevents mitochondrial superoxide production and attenuates
acetaminophen-induced hepatocyte cell death in culture.

Anup Ramachandran¹, Hui-min Yan¹, John J Lemasters², and Hartmut Jaeschke¹
¹Department of Pharmacology, Toxicology & Therapeutics, KUMC
²Department of Pharmacological Sciences, Medical University of South Carolina, Charleston,

Oxidative stress and mitochondrial dysfunction play an important role in acetaminophen-induced
hepatocyte cell death. However, exact mechanisms involved in the process are controversial, in
part, due to the disparity in findings between in vitro & in vivo studies. A major difference in this
context is the oxygen tension, with cells in culture being exposed to 21% oxygen while those in
the liver experience a gradient from 5-11% oxygen. To determine if oxygen tension could
modulate hepatocyte responses to acetaminophen, this study examined cells under normoxia
(21% O2), 10% O2 & 5% O2. Isolated primary mouse hepatocytes were treated with 5mM
acetaminophen for 15 hours under various oxygen tensions and cell death assessed by LDH
release. Mitochondrial function and membrane potential were assessed at 6 & 15h using the
XTT assay and JC-1 fluorescence respectively. Mitochondrial generation of superoxide was
measured using Mitosox Red® fluorescence. Exposure of hepatocytes to 5mM acetaminophen
for 15 hours at 21% O2 resulted in significant cell death, accompanied by deterioration of
mitochondrial function and loss of mitochondrial membrane potential, which was evident at 6
hours 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. Mitochondrial
function and membrane potential were better preserved at 6 hours, though the protective effect
decreased by 15 hours. Culturing cells at 10% O2 blocked elevation in mitochondrial superoxide
production at 6 hours; an increase being seen only by 15 hours. These results suggest that
oxygen tension can modulate hepatocyte responses to acetaminophen, with low physiological
levels decreasing mitochondrial superoxide production and delaying hepatocyte cell death. This
may have wide-ranging implications in other cellular contexts where oxidative metabolism plays
a role.


Direct viral cytotoxicity of HCV requires cofilin translocation from cytosol to

Roosevelt V. Campbell1, 2, Batbayar Tumurbaatar1, and Steven A. Weinman1
 Department of Internal Medicine, KUMC; 2Department of Neuroscience and Cell Biology,
University of Texas Medical Branch, Galveston, Texas

The development of the JFH1 strain of cell culture competent HCV has clearly shown that the
virus can directly induce apoptosis in infected cells. One mechanism that has been identified is
enhanced sensitivity to TRAIL. Previous work from our lab and others has shown that HCV
sensitizes cells to oxidant-induced cell death as well, but whether these are independent effects
and the molecular details of how they are induced by HCV remain uncertain. The AIM of this
study was to determine whether TRAIL-induced and ROS-induced cell death are independent
processes 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 by
Caspase 3/7 Glo assay and cell death was determined by LDH release. Cofilin was down
regulated 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 JFH1
virus. Viral infection increased TRAIL and tBOOH induced cell death by 5-6 fold compared to
uninfected 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 PARP
cleavage but tBOOH induced cell death was not. TRAIL induced cell death was blocked by
zVAD, a pan-caspase inhibitor but tBOOH cell death was not. However, tBOOH induced cell
death was completely prevented by calcium chelation or antioxidant treatment while these had
no effect against TRAIL induced cell death. We next examined the effect of HCV on the
mitochondrial association of molecules known to cause apoptosis and necrosis. In response to
JFH1 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 prior
to 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 distinct
mechanisms of cell death, TRAIL hypersensitivity and cofilin-dependent oxidant induced cell
death, are induced by HCV. These may explain the clinical observations of HCV as a sensitizer
to alcoholic and other forms of liver disease, and contribute to the ATP binding cassette


Mitigation of ethanol-induced liver injury by autophagy

Wen-Xing Ding
Department of Pharmacology, Toxicology & Therapeutics, KUMC

Alcohol abuse is a major cause of liver injury. Whereas the pathology of alcoholic liver disease
develops over a prolonged period, the cellular defense mechanisms against the detrimental
effects of alcohol are not well understood. We found that macroautophagy, an evolutionarily
conserved lysosomal degradation pathway commonly activated in response to stress, plays a
significant role in protecting hepatocytes from ethanol toxicity. Using both an animal binge
model and cell cultures, we found that ethanol treatment strongly activated macroautophagy.
Suppression of macroautophagy led to a significant increase in hepatocyte apoptosis and liver
injury. Further analysis demonstrated that macroautophagy protected hepatocytes by removing
damaged mitochondria and accumulated lipid droplets. These findings established the
physiological significance of macroautophagy in acute ethanol-induced pathogenesis in the liver
and the potential therapeutic values of modulating macroautophagy in alcoholic liver disease.


Monocyte chemoattractant protein-1-deficiency does not affect steatosis or inflammation
in livers of mice fed a methionine-choline-deficient diet

Karen M. Kassel¹, Grace L. Guo¹, Ossama Tawfik², and James P. Luyendyk¹
¹Departments of Pharmacology, Toxicology & Therapeutics, KUMC
²Pathology and Laboratory Medicine, KUMC

Background: Monocyte chemoattractant protein-1 (MCP-1, Ccl2) expression is increased in
livers 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 feeding
a 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 tested
the 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 a
MCD diet or control diet for 4 weeks. Results: MCP-1-deficiency did not affect steatohepatitis as
indicated by liver histopathology, nor did it affect serum alanine aminotransferase activity,
hepatic triglyceride levels, hepatic inflammatory gene induction, or macrophage accumulation in
mice fed the MCD diet. Interestingly, MCP-1-deficiency significantly reduced collagen deposition
in the livers of mice fed the MCD diet. Moreover, MCP-1-deficiency reduced the expression of
profibrogenic genes including pro-collagen 1a1, connective tissue growth factor, and
transforming growth factor- as well as -smooth muscle actin protein levels in mice fed the
MCD diet. Conclusions: The results indicate that MCP-1 does not contribute to liver steatosis or
inflammation in the MCD diet model of steatohepatitis. Rather, the data suggest that MCP-1
contributes to fibrosis in mice fed the MCD diet independent of effects on steatosis and


Acetaminophen toxicity in HepaRG cells: a novel human model for studies of drug

Mitchell R. McGill, Huimin Yan, Anup Ramachandran, Hartmut Jaeschke
Department of Pharmacology, Toxicology & Therapeutics, KUMC

Acetaminophen (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 reactive
metabolite, glutathione (GSH) depletion, mitochondrial dysfunction, and oncotic necrosis. To
investigate these mechanisms in a human system, a metabolically competent cell line is
needed. In this study, we tested the value of a human hepatoma-derived cell line (HepaRG) for
APAP toxicity studies. Cells were treated with 20 mM APAP and the time course of cell
dysfunction and injury was evaluated. APAP caused a decline in cellular GSH levels to 60% of
control at 6 h and 30 % at 24 h. The mitochondrial membrane potential (JC-1) was unaffected at
6 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, a
clear dose response (5 -20 mM) was observed with each of these parameters. Experiments with
HepG2 cells, which are not metabolically competent, showed no GSH depletion, mitochondrial
dysfunction or cell injury. Conclusion: The mechanism of APAP toxicity in HepaRG cells mimics
closely the sequence of events observed in rodents. Importantly, the time course of injury
resembles what has been reported in the clinic. Thus, HepaRG cells may be a useful human
model for mechanistic studies of drug hepatotoxicity.


Impact of hepatitis C on survival in alcoholic hepatitis.

PK Pandya ¹, GA Rao, and Steven Weinman²
¹VA Medical Center, ² School of Medicine, KUMC

Background: Alcoholic hepatitis (AH) is an acute inflammatory process associated with an
overall 30 day mortality of 15% that approaches 50% among severe cases. The prevalence of
hepatitis C virus (HCV) infection among alcoholics can exceed 30% compared to 1.8% in the
general population. This infection reduces the antioxidant capacity of hepatic mitochondria that
can further sensitize hepatocytes to alcohol-induced injury. The additive impact on survival
among AH CHC patients is unknown. Methods: We performed a retrospective database review
of a representative sample of 10 Veterans affairs (VA) VISNs that represents half of all veterans
receiving 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, acute
acetaminophen toxicity (ICD9 965.4), Acute hepatitis B infection (070.2, 070.3), Budd Chiari
Syndrome (453.0), Autoimmune hepatitis (571.42) or Portal vein thrombosis (452). A patient
was defined to have CHC if they had a VA documented positive antibody test at any time prior
to discharge. The severity of AH was determined by using MELD score. Cox proportional
hazard survival analysis was performed to identify independent predictors of 90 days mortality
post 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%. The
90day 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). When
we 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 the
rate of use of either Pentoxyphylline or glucocorticoids were lower among HCV (11%) vs. non
HCV (15.7%), RR =0.95, C.I., 0.88 to 1.02. Conclusion: We found that HCV is an independent
predictor of mortality in AH. The impact of HCV infection on mortality appears to be more
important in non-cirrhotic subjects as compared to cirrhotic subjects.


Thrombin signaling enhances TGFβ induction of integrin β6 in bile duct epithelial cells

Bradley P. Sullivan and James P. Luyendyk
Department of Pharmacology, Toxicology & Therapeutics, KUMC

Damage to intrahepatic bile ducts causes cholestasis, inflammation, coagulation cascade
activation and liver fibrosis. Recent studies have shown that the Vβ6 integrin, which activates
the fibrogenic cytokine TGFβ, contributes to liver fibrosis. Expression of the β6 integrin (Itgβ6) is
restricted 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. We

BDECs. Treatment of transformed human BDECs (MMNK-1) with thrombin or protease
activated receptor-1 (PAR-1) agonist peptides (TFLLRN or SFLLRN) induced the expression of
interleukin 8 (IL-8), a chemokine induced by PAR-1 signaling. However, neither thrombin nor
agonist peptides increased Itgβ6 mRNA levels in MMNK-1 cells. In contrast, treatment of the
MMNK-1 cells with TGFβ increased Itgβ6 mRNA levels without affecting IL-8 expression. As
thrombin and TGF are likely generated concurrently during cholestatic liver injury, we
determined whether thrombin enhanced TGFβ induction of Itgβ6 mRNA. Co-treatment with
either 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 not
TGFβ, activated the p38 and JNK1/2 MAPK signaling pathways. Itgβ6 mRNA induction in cells
co-treated with thrombin and TGFβ was significantly reduced by a p38 inhibitor (SB203580) and
a JNK1/2 inhibitor (SP600125). Taken together, the results suggest that thrombin activation of
p38 and JNK1/2 signaling enhances TGFβ dependent induction of the Itgβ6. Further elucidation
of the mechanisms whereby these fibrogenic mediators synergistically induce the Itgβ6 and
contribute to fibrogenesis may reveal novel strategies for the treatment of cholestatic liver


Pharmacologic inhibition of caspase 1 and modulation of the inflammatory response
during acetaminophen hepatotoxicity

C. David Williams, Mary Lynn Bajt, and Hartmut Jaeschke
Department of Pharmacology, Toxicology & Therapeutics, KUMC

Background and aims: Acetaminophen (APAP) hepatotoxicity is the most frequent cause of
acute liver failure in the US and many European countries. In addition to intracellular signaling
mechanisms leading to cell necrosis, recent data indicate that activation of toll-like receptors on
macrophages 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 of
inflammatory 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 to
evaluate the effect of a pancaspase inhibitor (Z-VD-fmk) on IL-1β formation, inflammation and
liver injury during APAP overdose. Methods: C57Bl/6 mice were given 10 mg/kg Z-VD-fmk or
vehicle two hours after 300 mg/kg APAP and the inflammatory response (IL-1β, neutrophils) and
liver injury (ALT, necrosis) were evaluated. Results: During APAP overdose liver IL-1β mRNA
increased six-fold with or without Z-VD-fmk. Plasma IL-1β protein was reduced below baseline
levels by Z-VD-fmk indicating specific inhibition of IL-1β processing, but no difference in ALT or
necrosis was observed. Recombinant IL-1β caused systemic neutrophil activation in vivo
(CD11b expression, ROS priming); such activation was not observed after APAP. Accumulation
of neutrophils in the liver in response to APAP was not affected by Z-VD-fmk. Similar results
were 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 DAMPs
by necrotic hepatocytes recruit neutrophils into the liver. These leukocytes do not cause injury
but are involved in removal of necrotic cell debris. Thus, intracellular events including
mitochondrial dysfunction, oxidant stress, JNK activation and nuclear DNA fragmentation are
critical determinants of APAP-induced cell death independent of the inflammatory response.


Function of the hepatitis C virus (HCV) viroporin ion channel p7

A.L Wozniak1, 3, M Yi2, S.M Lemon2, and S.A Weinman3
 Department of Neuroscience and Cell Biology, KUMC
 Department of Microbiology and Immunology, University of Texas-Medical Branch, Galveston,
 Department of Internal Medicine, KUMC

HCV is a major cause of liver disease and current therapy is ineffective in half of infected
patients. The viral p7 protein is an ion channel critical for infectious particle production. It is an
attractive antiviral target but the role that it plays in the HCV lifecycle is unclear. Understanding
how p7 promotes viral particle formation will be of critical importance in the development of new
antivirals. This work seeks to evaluate the contribution of p7 proton channel activity to organelle
pH 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
this activity is inhibited by several viroporin inhibitors. HCV infected hepatoma cells and p7
containing 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 by
p7 inhibitors and were not present in subgenomic replicons that do not express p7. To further
confirm that p7 caused the loss of acidification, we compared a chimeric virus containing wt p7
vs. a mutant p7 lacking ion channel activity. Wt cells lost their acidic vesicle-associated
fluorescence while the mutant cells had similar acidic compartments as control cells. Preliminary
studies showed that the acidification inhibitor bafilomycin partially restored virus production to
mutant p7 cells demonstrating that the p7-induced loss of acidification is a key requirement for
virus production. In conclusion, p7 serves as a H+ channel preventing acidification in the most
acidic intracellular compartments. This loss of acidification is required for productive HCV


Ncb5or, fatty acid metabolism and oxidative stress

Ming Xu1, WenFang Wang1, and Hao Zhu2
 Department of Physical Therapy & Rehabilitation Science
 Department of Clinical Laboratory Sciences, KUMC

NADH cytochrome b5 oxidoreductase (Ncb5or) is a ubiquitous redox enzyme found in all
animals. Ncb5or knockout (KO) mice develop maturity-onset diabetes and lipoatrophy.
Microarray analysis of 5-week-old livers reveals higher expression levels of genes in oxidative
stress responses and de novo lipogenesis in prediabetic KO than wild-type samples. This gene
expression profile was confirmed by quantitative PCR, and the increased oxidative stress in KO
livers was further supported by their higher content of oxidized glutathione. To study the
potential function of Ncb5or in metabolizing exogenous saturated fatty acids, we isolated
primary hepatocytes from prediabetic KO mice and compared them to cells from age-matched
wild-type controls. After an 8-hour treatment with a medium dose of palmitates, KO hepatocytes
had significantly higher levels of cell-death, intracellular fatty acids, transcripts of oxidative
stress and ER stress genes, reactive oxygen species (through DCHF–staining), and oxidized
glutathione. The oxidative stress in the KO hepatocytes can be alleviated by co-incubation with
oleate, which is the major intracellular monounsaturated fatty acid and a preferred substrate for
triglyceride synthesis. Our preliminary data also show a faster β-oxidation rate and a lower
desaturation index (monounsaturated: saturated ratio) in KO hepatocytes. Taken together, our
studies suggest that Ncb5or is essential for cells to metabolize exogenous saturated fatty acids
and to maintain cellular redox homeostasis. (Supported by NIH RO1 DK067355 grant)


Enhanced expression of Nrf2 in mice attenuates the fatty liver produced by a methionine
and choline deficient Diet

Yu-Kun Jennifer Zhang, Ronnie L. Yeager, Yuji Tanaka, and Curtis D. Klaassen
Department of Pharmacology, Toxicology & Therapeutics, KUMC

Oxidative stress has been proposed as an important promoter of the progression of fatty liver
diseases. The current study investigates the potential functions of the Nrf2-Keap1 signaling
pathway, an important hepatic oxidative stress sensor, in a rodent fatty liver model. Mice with
no (Nrf2-null), normal (wild-type, WT), and enhanced (Keap1 knockdown, K1-kd) expression of
Nrf2 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-null
mice and less in the K1-kd mice. The Nrf2-null mice had lower hepatic glutathione and
exhibited more lipid peroxidation, whereas the K1-kd mice had the highest amount of
glutathione in the liver and developed the least lipid peroxidation among the three genotypes fed
the 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-kd
mice. In addition, Nrf2-null mice on both control and MCD diets exhibited altered expression
profiles of fatty acid metabolism genes, indicating Nrf2 may influence the lipid metabolism in
liver. For example, mRNA levels of long chain fatty acid translocase CD36 and the endocrine
hormone Fgf21 were higher in livers of Nrf2-null mice and lower in the K1-kd mice than WT
mice fed the MCD diet. Taken together, these observations indicate that Nrf2 could decelerate
the onset of fatty livers caused by the MCD diet by increasing hepatic antioxidant and
detoxification capabilities.

      Transport and Biotransformation Abstracts

Bile acids induce inflammatory genes in hepatocytes: a novel mechanism of
inflammation during obstructive cholestasis

Katryn Allen, Hartmut Jaeschke, and Bryan L. Copple
Department of Pharmacology, Toxicology & Therapeutics, KUMC

Background and Aims: Inflammation contributes to liver injury during cholestasis. The
mechanisms by which cholestasis initiates an inflammatory response in the liver, however, are
not known. Two hypotheses were investigated in the current studies. First, activation of toll-like
receptor 4 (TLR4) by either bacterial lipopolysaccharide (LPS) or damage-associated molecular
pattern molecules (DAMPs) released from dead hepatocytes triggers an inflammatory response.
Second, bile acids act as inflammagens, and directly activate signaling pathways in hepatocytes
that stimulate production of proinflammatory mediators. Methods: LPS-resistant C3H/HeJ mice
and LPS-sensitive C3Heb/FeJ mice were subjected to bile duct ligation (BDL) and hepatic
inflammation 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 of
hepatocytes with bile acids which are found to be elevated in serum during BDL did not directly
cause cell toxicity but increased expression of numerous proinflammatory mediators including
cytokines, chemokines, adhesion molecules, and other proteins that influence immune cell
levels and function. Upregulation of several of these genes in hepatocytes and in the liver after
BDL 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 demonstrate
that 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 that
promotes hepatic inflammation during cholestasis.
(Supported by the Liver Tissue Bank of the Liver Center)


The Effect of Aging on Bile Acid Homeostasis

Zidong Donna Fu, Iván L. Csanaky, Curtis D. Klaassen
Department of Pharmacology, Toxicology, & Therapeutics, KUMC

Aging 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 during
aging. Therefore, the present study was performed to determine the effect of aging on
concentrations of 8 conjugated and 10 unconjugated BAs (using UPLC-MS/MS) in serum and
liver and the mRNA level of genes related to BA homeostasis (using multiplex suspension
assay) 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 (3-4
fold) for both genders. The mRNA of Cyp7a1, the rate-limiting enzyme in the classic BA
synthesis pathway, decreased after 15 months of age. Cyp27a1 and Cyp7b1, two enzymes
critical 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 (bile
acid CoA: amino acid N-acyltransferases) remained consistent with age. The main hepatic BA
transporters Ntcp (uptake) and Bsep (efflux) shared a bell shape curve with the peak at around
12 months of age. Therefore, the decrease in BA transporters after 12 months of age might be
the reason for increased BA concentrations despite the decreased expression of synthesis
enzymes. The present study suggests that the increased BA concentrations in serum and liver
during aging might be due to decreased expression of hepatic BA uptake and efflux
transporters. (Supported by NIH grants ES-009716, ES-009649, ES-013714, ES-007079, RR-


Suppression of cyp7a1 gene transcription by FXR in mice is mediated through the
intestine-initiated FGF15/FGFR4 pathway

Bo Kong¹, Jeff Aube³, Li Wang², and Grace L. Guo¹
¹Department of Pharmacology, Toxicology & Therapeutics, KUMC
²Department of Oncological Sciences, University of Utah, Salt Lake City, UT
³Department of Medicinal Chemistry, KU Lawrence, KS

The 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 the
classic 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, FXR
floxed/floxed villin Cre(+) mice) were used to clarify the pathway of FXR/SHP/LRH1 and
FXR/FGF15/FGFR4 in the inhibition of Cyp7a1 gene expression. Oral administration with FXR
agonist 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 data
demonstrated that the negative feedback regulation of Cyp7a1 gene expression is dependent
on 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 FXR
knockout mice markedly reduced liver Cyp7a1 gene expression and activated the MAPK
signaling pathways including JNK and ERK1/2 MAP kinases. FGF15 induced JNK and ERK1/2
phosphorylation in a time-dependent and dose-dependent way, and their major downstream
targets, c-Jun and early growth responsive-1 (Egr-1), might involved in the inhibition of Cyp7a1
gene transcription. In summary, the intestinal FGF15 inhibits liver Cyp7A1 expression through
activation of JNK and ERK1/2 pathway is the major mechanism for the feedback regulation of
bile acid biosynthesis in mice.


Assessment of modulation of intestinal microflora following alterations in bile acid

Pallavi B. Limaye, Youcai Zhang, and Curtis D. Klaassen
Department of Pharmacology, Toxicology & Therapeutics, KUMC

Normal intestinal microflora is essential for various physiological functions such as innate
immunity, energy harvest, and bile acid metabolism. Nearly 90% of the bile acids secreted in the
duodenum in response to food ingestion are reabsorbed through terminal ileum and taken up by
the liver via the Oatp1a1 and other bile acid transporters. It is known that bile acids reaching the
large intestine are further metabolized by the gut bacteria to potentially toxic secondary bile
acids. Investigations from our laboratory have revealed significant increase in secondary bile
acids including deoxycholic acid (DCA) and lithocholic acid (LCA) in the small (SI) and large
intestine (LI) of Oatp1a1 knockout mice. Oatp1a1 knockout mice also exhibited an increase in
unconjugated bile acids in the SI. We hypothesized that the increase in secondary bile acids in
the Oatp1a1 knockout mice is due to change in the intestinal bacterial profile. We quantified the
gut bacterial population using 16s rDNA probe-based branched DNA assay. The data indicate
significant overgrowth of the majority of the bacterial species assessed in small intestine of
Oatp1a1 knockout mice. In the large intestine however, a striking increase in Bacteroides
bacteria, 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 knockout
mice may be involved in increased secondary bile acid production.


Mechanism of polybrominated diphenyl ether (PBDE) uptake into human and mouse liver

Erik K. Pacyniak¹, Megan Roth¹, Bruno Hagenbuch¹, ², and Grace L. Guo¹
¹ Department of Pharmacology, Toxicology & Therapeutics, KUMC
² The Cancer Center, KUMC

2,2`,4,4`-tetrabromodiphenyl ether (BDE47), 2,2',4,4',5-pentabromodiphenyl ether (BDE-99) and
2,2',4,4',5,5'-hexabromodiphenyl ether (BDE-153) are the predominant polybrominated diphenyl
ether (PBDE) congeners detected in human samples. While the hepatic metabolism of PBDEs
has been investigated in humans and mice, the mechanism by which PBDEs enter the liver has
not been elucidated. However, due to their large molecular weights (~485 to 1000 Da), they
cannot enter hepatocytes by simple diffusion. Organic anion transporting polypeptides (OATPs:
humans; Oatps: rodents) are responsible for hepatic uptake of a variety of amphipathic
compounds of molecular weights larger than 350 Da. Therefore, the objective of the current
study was to identify OATPs/Oatps responsible for uptake of BDE47, BDE99 and BDE153 in
human and mouse liver. To test this hypothesis we used Chinese Hamster Ovary (CHO) cell
lines expressing either OATP1B1, OATP1B3 or OATP2B1 as well as Human Embryonic Kidney
293 (HEK293) cells transiently expressing the murine orthologs of the OATP1B (Oatp1b2) and
OATP2B (Oatp2b1) family. Direct uptake studies confirmed that all three PBDE congeners are
substrates for the tested human and mouse hepatic OATPs/Oatps. Detailed kinetic analysis
revealed that OATP1B1 transported BDE47 with the highest affinity (Km= 0.31μM) followed by
BDE99 (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, Oatp1b2
transported 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 presented
clearly suggest that uptake of PBDEs via these OATPs/Oatps is a mechanism responsible for
liver specific accumulation of PBDEs.


Isolation 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.
  Department of Pharmacology, Toxicology & Therapeutics, KUMC
  Department of Medicinal Chemistry, KU Lawrence, KS

Organic anion transporting polypeptides 1B1 and 1B3 (OATP1B1 and OATP1B3) are liver
specific transporters that mediate the uptake of a broad range of drugs into hepatocytes,
including statins, antibiotics and many anticancer drugs. Compounds which alter transport by
one or both of these OATPs could potentially be used to target drugs to hepatocytes or to
improve bioavailability of drugs that are cleared by the liver. In this study, we applied a
bioassay guided isolation approach to identify such compounds from the organic extract of
Rollinia emarginata Schlecht (Annonaceae). Fractions of the plant extract were screened for
effects on OATP1B1- and OATP1B3-mediated transport of 0.1µM estrone-3-sulfate and
estradiol-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-mediated
transport of both model substrates was strongly inhibited, as was OATP1B3-mediated transport
of estradiol-17β-glucuronide. However, OATP1B3-mediated uptake of estrone-3-sulfate was
stimulated 4- to 5-fold. Kinetic analysis of this stimulation revealed that the apparent affinity for
substrate was increased (Km reduced from 100μM to 10μM), while the Vmax was significantly
reduced. These results demonstrate a mechanism through which the hepatic uptake of drug
OATP substrates could be stimulated.


The coagulation system contributes to αVβ6 integrin expression and liver fibrosis
induced by cholestasis

Bradley P. Sullivan¹, Paul H. Weinreb², Shelia M. Violette³, and James P. Luyendyk¹
¹Department of Pharmacology, Toxicology & Therapeutics, KUMC
² Biogen Idec, Inc., ³ Stromedix Inc.

Chronic injury to intrahepatic bile duct epithelial cells (BDECs) elicits expression of various
mediators that promote liver fibrosis, including the V6 integrin. We tested the hypothesis that
tissue factor (TF)-dependent thrombin generation and protease activated receptor-1 (PAR-1)
activation contribute to liver fibrosis induced by cholestasis via induction of V6 expression. To
test 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 cascade
activation and liver fibrosis were reduced in genetically modified mice with a 50% reduction in
liver TF activity fed the ANIT diet. Similarly, liver fibrosis was significantly reduced in PAR-1-/-
mice fed an ANIT diet. Hepatic integrin 6 mRNA induction and the expression of V6 by
intrahepatic BDECs were reduced by TF-deficiency and PAR-1-deficiency in mice fed the ANIT
diet. Treatment with either anti-V6 antibody or soluble TGF- receptor reduced liver fibrosis in
mice fed the ANIT diet. PAR-1 activation enhanced TGF-1-induced integrin 6 mRNA
expression in transformed human BDECs. Interestingly, TF and PAR-1 mRNA levels were
increased 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 increasing
expression of the V6 integrin, an important regulator of TGF-1 activation.
(Supported by the Liver Tissue Bank of the Liver Center)


Tissue-specific FXR-DNA binding: genome-wide liver and intestine binding analysis by

Ann Thomas, Steven Hart, Bo Kong, Jianwen Fang, Xiaobo Zhong, and Grace L. Guo
Department of Pharmacology, Toxicology & Therapeutics, KUMC

Farnesoid X receptor (FXR) is a ligand activated transcription factor belonging to the nuclear
receptor superfamily. FXR is highly expressed in the liver and intestines and bile acids are its
endogenous ligands. FXR is essential in maintaining bile acid homeostasis, but is also critical in
regulating lipid and glucose metabolism. However, direct FXR target genes in the liver and
intestine are not completely known. The current study was designed to determine genome-wide
FXR target genes in the liver and intestine by chromatin immunoprecipitation (ChIP) coupled to
massively parallel sequencing (ChIP-Seq) following treatment of mice with a synthetic FXR
ligand (GW4064). The results revealed novel genome-wide FXR-DNA binding. (1) FXR binding
shows a large degree of tissue-specificity, with the liver and intestines sharing only 11% of total
FXR binding sites. (2) Uncharacterized binding sites were identified in different regulatory
regions 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 of
gene expression. Novel binding sites were also found with in regulatory regions of known FXR
target genes, such as Nr0b2 and Fgf15. In addition, FXR binding sites were discovered
upstream of the Slc10a1 gene in the liver and Osta lacked FXR binding sites in the liver, which
contradicts previous reports. (3) Motif analysis of FXR binding sites showed a nuclear receptor
half binding site was commonly associated with the classical FXR response element (inverted
repeat separated by 1 nucleotide [IR1]), indicating the involvement of other nuclear receptors or
transcription factors for facilitating regulatory functions of FXR. (4) Gene Ontology pathway
analysis revealed FXR is significantly involved in important metabolic pathways, including
organic acid, lipid, and fatty acid metabolic processes, which has not been previously identified
or sufficiently studied. In conclusion, this study provides a tissue-specific genome-wide
assessment of FXR-DNA binding in vivo. The results from this study suggest tissue-specific
regulatory functions of FXR as well as reveal novel metabolic pathways potentially regulated by


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