Signal Transduction and Cell Function


Sarcopenia in cirrhosis is caused by hyperammonemia induced skeletal muscle mitochondrial dysfunction

Samjhana Thapaliya2, Ciaran E. Fealy2, Anny Mulya2, Chris A. Flask3, John P. Kirwan2,1, Srinivasan Dasarathy1, 2

1Gastroenterology and hepatology, Cleveland Clinic Foundation, Cleveland, OH; 2PathobioIogy, Cleveland Clinic, Cleveland, OH; 3Imaging Research Core, Case Western Reserve University, Cleveland, OH

Background. Cirrhosis is a state of disordered energy metabolism that contributes to sarcopenia or loss of muscle mass. Hyperammonemia is a consistent abnormality in cirrhosis that we have previously shown as a critical mediator of sarcopenia due to impaired protein synthesis. Protein synthesis places a high energy demand on the cellular bioenergetic pathways. In the present study, we demonstrate that hyperammonemia results in abnormal skeletal muscle mitochondrial dynamics and bioenergetics. Methods. The portacaval anastamosis (PCA) rat, model of hyperammonemia and sarcopenia of liver disease and pair fed sham operated controls (n=6 each) were subjected to magnetic resonance spectroscopy to quantify in vivo, skeletal muscle ATP and phosphocreatinine (PC) content and ATP/PC ratio were determined. These data were complemented by in vitro studies in C2C12 murine myotubes exposed to 10mM hyperammonemia to study mitochondrial function. ATP production was quantified using a bioluminescence assay. Mitochondrial dynamics were evaluated by real time PCR for genes regulating mitochondrial fusion (Mfn1), & fission/mitophagy (DRP1, & DJ1). Reactive oxygen species (R〇S) was measured by a fluorometric method. Mitochondrial function was quantified using the Seahorse XF24® analyzer. Total cellular protein and ribosomal content were quantified for translational efficiency and cell size was quantified on phase contrast microscopy. Results. Gastrocnemius muscle PC content in PCA rats was 41.7±31.4% and of ATP content was 41.7±31.4% of controls rats. In C2C12 myotubes, ATP content was 20.3±3.4 μmol/mg protein lower in response to hyperammonemia compared to controls. The time course evaluation showed that this was an early abnormality beginning within 5 m and persisted to 24 h. This was accompanied by an increased generation of ROS (133.6±12.8% of controls). Mitochondrial stress test in C2C12 cells showed that hyperammonemia decreased basal respiration and increased the spare respiratory capacity at early time point. Mitochondrial dynamics were altered: specifically, relative expression of Mfn1 was 0.48±0.09, Drp1 was 8.4±2.3 fold and DJ1, 2.1 ±0.73 fold within 5 minutes and these changes persisted for to 24 h. of hyperammonemia suggesting an increase in net mitochondrial fragmentation. These changes were accompanied by reduced protein content, translational efficiency and myotubes size. Conclusions. Our observations suggest a novel hyperammonemia mediated mechanism of sarcopenia and decreased muscle protein synthesis in cirrhosis due to dysregulated mitochondrial dynamics and impaired mitochondrial function.


The following people have nothing to disclose: Samjhana Thapaliya, Ciaran E. Fealy, Anny Mulya, Chris A. Flask, John P. Kirwan, Srinivasan Dasarathy


Subcellular Trafficking of Ntcp is Regulated by the EGF Receptor

Xintao Wang1, Pijun Wang1, Wen-Jun Wang1 , 2 Yansen Xiao1, John W. Murray1 , 2 Allan W Wolkoff1 , 2

1Liver Research Center, Albert Einstein College of Medicine, Bronx, NY; 2Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY

Previous studies have shown that net cell surface expression of the hepatocyte specific bile acid transporter ntcp is determined by its trafficking between the cell surface and intracellular pools. This was found to result from bidirectional vesicular trafficking of ntcp along microtubules using kinesin-1 and dynein as plus and minus end directed motors, respectively. Motility required activity of PKCZ, an atypical kinase associated with ntcp-containing vesicles. The present study was designed to functionally elucidate the target of PKCځ activity. Methods: Immunopurified endocytic vesicles were prepared from rat liver and HuH7 cells stably expressing ntcp-sfGFP. Vesicles were incubated with −32P-ATP in the presence or absence of a PKCځspecific inhibitor. Vesicle lysates were subjected to SDS-PAGE, radioautography, and mass spectrometry (MS) analysis to identify the target of PKCځ activity. Results: A major 180 kD vesicleassociated phosphoglycoprotein was seen. Phosphorylation was abolished in the presence of the PKCځ inhibitor. MS analysis identified this protein as the EGF receptor (EGFR). Ntcp was not phosphorylated, although PKCځ immunoprecipitated individually with ntcp and EGFR. Motility of ntcp-containing vesicles on microtubules was quantified in an in vitro motility assay. Although 35% of ntcp-containing vesicles that colocalized with EGFR moved upon addition of 50 μM ATP, only 10% of those that were not colocalized with EGFR moved (p<0.01). The importance of colocalization of ntcp with EGFR on microtubulebased motility was validated in ntcp-sfGFP transfected HuH7 cells in which EGFR was knocked down with specific shRNA. Only 8% of ntcp-containing vesicles prepared from these cells moved on microtubules (p<0.03) and there was no change in motility with inhibition of PKCځ activity (p>0.45). EGFR knockdown was associated with a 45% reduction in cell surface ntcp content as revealed by a cell surface biotinylation assay (p<0.001) while total cell content of ntcp was unchanged (103±11.8%). Despite less ntcp accessible to cell surface biotinylation, there was no reduction in Na+-dependent uptake of taurocholate with EGFR knockdown. Conclusions: (1)Colocalization with EGFR is an early event in subcellular trafficking of ntcp. EGFR ultimately traffics to lysosomes while ntcp traffics between the cell surface and intracellular vesicles. (2) Knockdown of EGFR results in reduced trafficking of ntcp to the cell surface, although taurocholate transport activity is maintained. (3) Maintenance of ntcp-mediated taurocholate transport activity despite reduced cell surface content suggests a novel regulatory mechanism.


Allan W. Wolkoff - Grant/Research Support: Merck

The following people have nothing to disclose: Xintao Wang, Pijun Wang, WenJun Wang, Yansen Xiao, John W. Murray


MicroRNA-214 is involved in the early stage of liver development by regulating follistatin expression

Junichi Saito, Masao Hondo, Hikari Okado, Takayoshi Shirasaki, Taro Yamashita, Shuichi Koneko

Gastroenterology, Kanazawa University, Kanazawa, Japan

Background MicroRNAs (miRNAs) play important roles in many biological processes by regulating the expression of their targetes. Previously, we identified differentially expressed miRs in hepatocellular carcinoma; however, their roles in the liver in vivo are not clear. Here we performed loss-of-function experiments using Morpholino (Mo) antisense oligonucleotide injected into zebrafish embryos and examined the phenotype of the developing liver. Methods To knock down miRNAs, Mo antisense oligonucleotides against each miRNA were injected into 1-2 cell stage embryos. Liver and gut development was monitored by the expression of endogenous biotin, which was detected with in situ hybridization using a C3-labeled streptavidin (C3-SA) probe. The developmental stage of liver was evaluated using probes against the liver-specific marker fabp10a, early liver stage marker prox1, and endodermal marker foxa3. Gene expression profiling in 1-3 days post-fertilization (dpf) embryos and Mo antisense oligonucleotide-injected embryos was compared using an Affymetrix GeneChip. EGFP mRNA fused to the 3f-untranslated region (UTR) of the target genes was co-injected into the embryos with Mo antisense oligonucleotides or mimic miRNAs. Results Out of the 23 miRNAs repressed using Mo antisense oligonucleotides, knocking down some miRNAs (e. g., miR-92a, miR-125b, miR-199a, and miR-214) severely disrupted liver development; especially, injection of Mo antisense oligonucleotides to miR-214 (Mo214) showed remarkable impairment of liver development at 5 dpf using CY3-SA staining. In situ hybridization of 1-3 dpf embryos showed reduced expression of fabp10a and prox1, but no significant change in the expression of foxa3, indicating that miR214 is essential for the early development of the liver. Gene expression profiling demonstrated repressed signaling of, for example, angiogenesis, hedgehog, FGF, and Wnt in embryos. We found that miR-214 targets follistatin, which is antagonistic to important liver development genes, including, activin and BMP genes which facilitate liver budding from the endoderm, extracellular matrix production, and angiogenesis. The EGFP sensor assay verified that miR-214 targeted the 3しUTR of follistatin and modulated its expression. Conclusion We demonstrated that miR-2 14 regulates liver development in zebrafish via follistatin, which interferes with the function of activin and BMP genes, both of which are important for liver development. Therefore, miR-214 might play important roles in the pathogenesis of various liver diseases including regeneration, fibrosis, and cancer.


Shuichi Kaneko - Grant/Research Support: MDS, Co., Inc, Chugai Pharma., Co., Inc, Toray Co., Inc, Daiichi Sankyo., Co., Inc, Dainippon Sumitomo, Co., Inc, Ajinomoto Co., Inc, MDS, Co., Inc, Chugai Pharma., Co., Inc, Toray Co., Inc, Daiichi Sankyo., Co., Inc, Dainippon Sumitomo, Co., Inc, Ajinomoto Co., Inc, Bayer Japan

The following people have nothing to disclose: Junichi Saito, Masao Honda, Hikari Okada, Takayoshi Shirasaki, Taro Yamashita


DMT1 Deficient Hepatocytes have Diminished Lysosomal Iron Release and Hypoxia-Induced Death

Justin Schwartz1, Ray Deepe1, John J. Lemasters1,2

1Drug Discovery & Biomedical Sciences, Medical University of South Carolina, Charleston, SC; 2Biochemistry & Molecular Biology, Medical University of South Carolina, Charleston, SC

Background: During hypoxic and toxic stresses, lysosomes release chelatable iron that translocates through the cytosol into mitochondria to catalyze formation of reactive oxygen species (ROS) culminating in cell death. Previously, we demonstrated that pharmacological inhibitors of divalent metal transporter 1 (DMT1) inhibited lysosomal iron release, ROS production and cell death during hypoxic and ischemia/reperfusion injury. However, these inhibitors might prevent lysosomal iron release by off target pharmacological effects. Here, our Aim was to determine if hepatocytes lacking functional DMT1 have diminished lysosomal iron release and cell death during hypoxic injury. Methods: Hepatocyte-specific DMT1 knockout (K〇) mice were created by mating loxP-DMT1 mice with mice containing albumin-promoter cre recombinase. Livers from DMT1 KO mice were examined for damage, morphology, and fat content by serum ALT, hematoxylin and eosin and oil red O staining, respectively. Overnight-cultured DMT1 KO hepatocytes were loaded with calcein-AM (an iron indicator) into the cytosol and incubated with propidium iodide (an indicator of cell death) prior to bafilomycin-induced lysosomal alkalization or induction of chemical hypoxia with iodoacetic acid plus KCN. Fluorescence was measured with a multiwell plate reader. Results: Compared to wild type, DMT1 KO mice had indistinguishable serum ALT levels histology except for fewer hepatocellular lipid droplets. In DMT1 KO hepatocytes, calcein quenching after bafilomycin, signifying an increase of cytosolic chelatable iron, was inhibited by 99% compared to wild type hepatocytes. During 2 h of chemical hypoxia, calcein fluorescence progressively decreased in wild type hepatocytes, which was diminished by 78% in DMT1 KO hepatocytes. Similarly, loss of viability of wild type hepatocytes after 2 h of chemical hypoxia was 85% compared to 42% for DMT1 KO hepatocytes. Conclusion: ATP depletion during hypoxia inhibits the proton-pumping vacuolar ATPase of lysosomes, leading to lysosomal alkalization and iron release into the cytosol through DMT1. This cytosolic iron augments cell killing during hypoxia.


John J. Lemasters - Grant/Research Support: Proctor & Gamble

The following people have nothing to disclose: Justin Schwartz, Ray Deepe


Ca2+-depletion impairs transcytotic vesicle fusion with the apical membrane by altering SNARE docking complexes

Alfonso Lopez Coral, Julia F. Omotade, Pamela L. Tuma

Biology The Catholic University of Amercia, Washington, DC

Over 15 years ago, transcytosis in MDCK cells was reported to be Ca2+-dependent: thapsigargin enhanced transcytosis while BAPTA impaired it. To date, the Ca2+-dependence of apical to basolateral transcytosis has not been examined in polarized hepatocytes, cells that rely heavily on the transcytotic pathway for the delivery of newly-synthesized apical proteins. We determined that the transcytosis of three classes of newly synthesized apical proteins (GPI-anchored, single spanning and polymeric IgA receptor) was impaired by Ca2+ chelation with BAPTA in polarized WIF-B cells. We further noticed that the stalled transcytosing proteins accumulated in sub-apical structures suggesting vesicle fusion with the apical membrane is impaired. This observation prompted us to examine MAL2 (a known transcytotic regulator) and members of the SNARE docking and fusion machinery. Although it is not known what specific SNARE molecules mediate vesicle docking, some likely candidates in hepatocytes are SNAP23 and syntaxins 2 and 3 (QSNAREs), VAMP8 (R-SNARE), Munc18-2, rab17 and the synaptotagmin-like protein 5 (Slp5). Importantly, Slp5 contains two tandem C2 domains that mediate Ca2+-dependent binding to membrane lipids. We found that Ca2+-depletion led to the dramatic redistribution of MAl2 and syntaxins 2 and 3 (but not SNAP23) from the apical membrane to the sub-apcial puncta containing the stalled transcytosing proteins. To rule out that lost apical labeling was not due to a general disruption of apical protein retention, we examined the distributions of several apical ecto-enzymes and ABC transporters. No changes in distribution were observed for all proteins tested indicating the effect is selective. Furthermore, the distributions of basolateral residents and tight junction proteins were not changed in BAPTAtreated cells indicating tight junction integrity was maintained. Treatment with W7 (a calmodulin antagonist) and KN93 (a CaM kinase II inhibitor) also led to decreased apical labeling of MaL2 and the syntaxins which correlated with impaired transcytosis further suggesting CaM kinase II activation is required for fusion of apically-targeted vesicles. We are actively testing this hypothesis.


The following people have nothing to disclose: Alfonso Lopez Coral, Julia F. Omotade, Pamela L. Tuma


Sarcopenia in alcoholic liver disease and autophagy are mediated by a protein phosphatase dependent mechanism

Srinivasan Dasarathy1 , 2, Samjhana Thapaliya1, Laura E. Nagy2 , 1, Megan R. McMullen2, Sathyamangla V. Naga Prasad3

1Gastroenterology and hepatology, Cleveland Clinic Foundation, Cleveland, OH; 2PathobioIogy, Cleveland Clinic, Cleveland, OH; 3MoIecuIar cardiology Cleveland Clinic, Cleveland, OH

Background. Sarcopenia or loss of skeletal muscle mass is a major clinical consequence of alcohol use and alcoholic cirrhosis but the contribution of muscle autophagy is not known. Methods. Skeletal muscle biopsies from alcoholic cirrhotics and control subjects, gastrocnemius muscle from ethanol and pair fed mice and murine C2C12 myotubes exposed to 100 mM of ethanol were studied. Static autophagy readouts included LC3 lipidation, p62 degradation, Beclin 1 overexpression on immunoblots and RT PCR quantification of LC3 and Atg7 mRNA. Autophagic flux was measured in C2C12 myotubes treated with or without 3 methyladenine (autophagy blocker) in pulse chase experiments and in myotubes stably transfected with GFP-LC3 and GFP-mCherry-LC3 by quantifying the LC3 and mCherry positive punctae as well as LC3 lipidation. Myotubes were incubated with ethanol and methylpyrazone to block alcohol dehydrogenase or acetaldehyde and cyanamide, to inhibit acetaldehyde dehydrogenase. Phosphorylation of Akt, mT〇R and AMPK was determined by immunoblots and activity of protein phosphatase 2A (PP2A) was quantified by thin layer chromatography. Results. Skeletal muscle mass was lower in patients with alcoholic cirrhosis, and mice fed ethanol compared to controls. Myotubes size was significantly (p<0.01) lower (71.1 ±7.6% compared to controls) in response to ethanol. Static autophagy readouts including immunoblots for LC3 lipidation, p62 degradation and Beclin1 overexpression were increased in all 3 models. Autophagy flux quantified by LC3 lipidation, and LC3 and mCherry positive puncta quantification was increased in ethanol exposed myotubes. Consistent with previous reports, these were accompanied by unaltered or lower 20S Proteasome activity in these models compared to their respective controls. Increased autophagic proteolysis was also observed with pulse chase experiments. Interestingly, acetaldehyde rather than ethanol mediated increased autophagy in myotubes. mTOR phosphorylation was decreased without significant alteration in Akt. AMPK phosphorylation was variably altered in the different models. Increased PP2A suggested a novel mechanism of lower mTOR phosphorylation in response to ethanol. Conclusion. Muscle proteolysis by ethanol results from increased autophagy mediated by acetaldehyde. This was accompanied by impaired mTOR activity due to increased dephosphorylation mediated by PP2A. This novel pathway provides mechanistic insights and development of possible therapies to reverse sarcopenia in alcoholic liver disease.


The following people have nothing to disclose: Srinivasan Dasarathy, Samjhana Thapaliya, Laura E. Nagy, Megan R. McMullen, Sathyamangla V. Naga Prasad


Platelet internalization stimulates proliferation of HepG2 cells by a mechanism involving horizontal transfer of platelet RNA

Marc Kirschbaum1, Golnor Karimian1, Jelle Adelmeijer1, Ben N. Giepmons2, Robert J. Porte1,Ton Lisman1

1Surgical Research Laboratory, Department of Surgery, University of Groningen, University Medical Center Groningen, Groningen, Netherlands; 2Department of Cell Biology, University of Groningen, University Medical Center Groningen, Groningen, Netherlands

Background: Recent studies have demonstrated that platelets play a pivotal role in liver regeneration following partial hepatectomy. The molecular mechanisms of platelet-induced hepatocyte proliferation and liver regeneration are largely unexplored. Although platelets are anucleate, several studies have convincingly shown that platelets are capable of de novo protein synthesis from platelet mRNA. In addition, recent studies have demonstrated that platelets are capable of functional transfer of mRNA and microRNA to other cell types. This study aims to investigate the mechanisms involved in platelet-mediated hepatocyte proliferation, with a focus on the role of platelet mRNA. Methods: Platelets were fluorescently labeled and incubated under serum free conditions with HepG2 cells for up to 48 hours. Proliferation of the HepG2 cells was quantified by BrdU incorporation. Confocal fluorescence microscopy and transmission electron microscopy was performed to study platelet internalization by HepG2 cells. To investigate horizontal transfer of platelet mRNA, total mRNA of the megakaryocytic cell line Meg-01 was labeled by incorporation of 5-ethynyl-uridine (EU). In addition, these Meg-01 cells were transfected with a GFP-tagged actin mRNA construct. Subsequently, the MEG01 cells were stimulated to produce plateletlike particles (PLPs) which were isolated and added to HepG2 cells. Results: Platelets and PLPs stimulate HepG2 cell proliferation by ±70%, and were internalized within one hour by HepG2 cells. Internalized platelets and PLPs were predominantly located in the perinuclear region of the hepatocyte. Electron microscopy demonstrated direct contact between platelets and the HepG2 nucleus. Platelet-mediated stimulation of proliferation was decreased by inhibition of platelet uptake using AnnexinA5, and by treatment with RNA-degrading enzymes. Both approaches resulted in a ∼25% decrease in proliferation. Platelet mRNA was transferred to the HepG2 cell as EU-labeled mRNA was present not only within the internalized PLP, but also within the cytosol of the hepatocyte following incubation with EU-expressing PLPs. Incubation with PLPs containing GFPtagged actin mRNA, resulted in GFP expression throughout the hepatocyte cytoskeleton, suggesting translation of PLP-derived mRNA by the hepatocyte. Conclusions: Platelet mRNA is transferred to and translated by HepG2 cells following platelet internalization. This mechanism is, in part, responsible for platelet-mediated stimulation of proliferation of HepG2 cells. These findings broaden our understanding of the mechanisms by which platelets stimulate liver regeneration.


The following people have nothing to disclose: Marc Kirschbaum, Golnar Karimian, Jelle Adelmeijer, Ben N. Giepmans, Robert J. Porte, Ton Lisman


Rab17 and MAL2 interact to coordinate apical vesicle fusion

Anneliese C. Sfriz, Pamela L. Tuma

Biology, The Catholic University of America, Washington, DC

Unlike simple epithelial cells that directly target newly-synthesized proteins from the TGN to the apical membrane, hepatocytes use an indirect pathway: newly-synthesized apical proteins are first delivered to the basolateral domain, then selectively internalized and transcytosed to the apical surface. MAL2 has been identified as an important regulator of indirect apical delivery at two distinct steps: in transcytosis from the basolateral early endosome to the subapical compartment and in the basolateral delivery of polymeric IgA-receptor (pIgA-R) from the Golgi. Because vesicle budding, fusion and delivery require complex protein machineries, we predict MAL2 has multiple binding partners to specify its function at these and other as-yet-to-be-identified transport steps. To identify novel MAL2 binding proteins, we performed Split-ubiquitin yeast-2hybrid screens. One promising candidate we have chosen to further examine is rab17, a low molecular weight GTPase whose expression is restricted to polarized epithelial cells and enriched in liver. To initiate our studies on rab17, we generated three recombinant adenoviruses expressing wild type, constitutively active (GTP bound) or dominant negative (GDP bound) rab17. Like endogenous rab17 and as observed in other epithelial cell types, overexpressed wild type and the mutant rab17s were present at or near the apical membrane. Western blotting revealed immunoreactive species at 25 kDa (the predicted rab17 molecular weight) and 40 kDa. Mass spectrometry confirmed that both bands are rab17. When we expressed a prenylation deficient rab17 isoform, the 40 kDa band was lost suggesting the shift in molecular weight is due to acylation. Because many rabs participate in vesicle docking with members of the SNARE machinery, and because rab17 has been shown to bind syntaxin 3 in kidney, we began our analysis in WIF-B cells by analyzing rab17-syntaxin interactions. We limited our studies to syntaxins 2 and 3 (the apical isoforms) and for our negative control, syntaxin 4 (the basolateral isoform). As predicted, syntaxin 4 did not bind wild type or the mutant rab17s. However, unlike in kidney, wild type and GTP-bound rab17 bound syntaxin 2, not syntaxin 3. Interestingly, in both cases, only the 40 kDa rab17 species bound syntaxin 2 suggesting acylation is required for binding. We are currently examining interactions with other members of the SNARE machinery and their relationship to transcytotic vesicle docking and fusion.


The following people have nothing to disclose: Anneliese C. Striz, Pamela L. Tuma


Existence of 5-Hydroxytryptamine Autocrine in Hepatocytes

Xiaohui Liu1, Haiqing Guo1, Rui Liu1, Xinhuan Wei1, Lixia Ma1, Yujuan Gao1, Xiaoqing Li1, Xianshan Yang1, Feng Ren2, Jing Zhang1, Zhongping Duan1

1Artificial liver department, Beijing Youan Hosptitl, Capital Medical University, Beijing, China; 2Beijing Institute of Liver Diseases, Beijing, China

Background: Many tissues have autocrine system of 5-hydroxytryptamine (5-HT, serotonin). Previous investigations suggested that 5-HT has been implicated in liver regeneration. The source and molecular mechanism of 5-HT in the control of liver regeneration are not fully elucidated, although circulating 5-HT was assumed to come mainly from platelets. The present study was designed to explore whether hepatocytes are a source of 5-HT, and to investigate the regulation mechanism of 5-HT in process of liver regeneration. Methods and results: (1)70% partial hepatectomy(PH) model of Wistar rats were used to observe the 5HT synthesis during liver regeneration. Immunohistochemical staining showed that at 24 h and 36 h after 70% PH, the number of 5-HT immune-positive jejunum enterochromaffin cells was increased. The scanning electron microscopy also proved the results. WB tests demonstrated that expression of tryptophan hydroxylase type1 (TPH1), the key enzyme of 5-HT synthesis, in enterochromaffincells also up-regulated after PH. (2) Detected by ELISA, 5-HT levels deriving from platelets were also increased after PH from 12h to 72h. (3) After PH, the mRNA levels of TPH1, serotonin reuptake transporter(SERT), 5HT receptor 2A and 2B, as well as TPH1 protein, were increased in rat liver tissue, so did 5-HT level in liver tissue homogenate. (4) As the results of RT-PCR and Western blot, TPH1, SERT and 5-HT receptors were detected in cultured HepG2 cells.5-HT level in the culture supernatant was higher than those levels in serum free culture (ELISA). (5) 5-hydroxytryptophan (5-HTP) could be converted to 5-HT by aromatic L-amino acid decarboxylase. When HepG2 was cultured with 5-HTP, the levels of 5-HT in supernatant was increased together with PCNA positive^ells [(66.94±13.02)% vs (59.88±10.33)%, P=0.009]. (6) SERT was one of the targets of microRNA-16. Transfected by microRNA-16 lentivirus for 48h, HepG2 SERT and TPH1 expressions began to decrease, and decreased further at 72h. At the same time, the 5-HT levels in the supernatant decreased from 0.211 ±0.01 ng/ml to 0.151 ±0.008ng/ml(p<0.05). (7) The mRNA of TPH1, SERT, 5HT receptor 2B were also detected in human liver tissue. Conclusion: 5-HT may mediate hepatocytes regeneration by an autocrine or paracrine pattern of action. The synthesis of 5-HT may be partly inhibited by the mcrioRNA16 through targeting SERT.


The following people have nothing to disclose: Xiaohui Liu, Haiqing Guo, Rui Liu, Xinhuan Wei, Lixia Ma, Yujuan Gao, Xiaoqing Li, Xianshan Yang, Feng Ren, Jing Zhang, Zhongping Duan


Functional analysis of sodium taurocholate cotransporting polypeptide (NTCP) as a cellular receptor for entry of hepatits B virus

Sung Wook Yang, Eun Sun Jang, Sang Soo Lee, Jung Wha Chung, Sukho Hong, Seong Min Chung, Sook-Hyang Jeong, Jin-Wook Kim

Internal Medicine, Seoul National University Bundang Hospital, Seongnam, Republic of Korea

Background: Although many antiviral targets against hepatitis B virus (HBV) have been identified, the host receptor of HBV has not been fully documented. Recently, sodium taurocholate cotransporting polypeptide (NTCP) was reported to have affinity for HBsAg and may work as a receptor for HBV entry in HepG2 cells. This study aimed to validate whether NTCP could induce natural HBV infection in Huh7 cell line, and whether HBV infection affect the expression of NTCP. Methods: Huh-BAT cells, Huh7-derived cell line in which human NTCP under the control of CMV promoter was transfected, were used in this study. Supernatant of HepAD38 cells cultured in tetracyclinefree medium was concentrated by ultracentrifugation (x16, 000 g for 4 hours) for the generation of HBV concentrates. Huh7 / Huh-Bat cells were collected at 48 hours after addition of HBV concentrates. NTCP and HBV levels in cytosol of HBV treated cells were measured by real-time PCR. NTCP promoters were cloned in pGL3 plasmid and co-transfected with plasmids encoding HBV protein in Huh7 cells, and the promoter activity was assessed by luciferase assay Results: NTCP expression level was negligible in Huh7 cells, whereas the expression was readily detected in Huh-BAT cells. After 48 hours of treatment with HBV concentrates, cytosolic HBV RNA levels were significantly higher in Huh-BAT cells compared to Huh-7 cells in a dose-dependent manner. Knockdown of NTCP by siRNAs significantly suppressed the efficacy of HBV infection in Huh-Bat cells. Interestingly, NTCP levels were up-regulated by HBV infection in Huh-7 cells, but not in Huh-BAT cells. The promoter activity of NTCP was enhanced by HBV S, core, and X protein by 72, 49 and 68%, respectively. Conclusions: NTCP expression in a human HCC cell line allows permissive growth of HBV more effectively than cells without NTCP expression. HBV infection enhances NTCP expression via transcriptional activation, and this mechanism might facilitate further viral entry in the infected cell.


The following people have nothing to disclose: Sung Wook Yang, Eun Sun Jang, Sang Soo Lee, Jung Wha Chung, Sukho Hong, Seong Min Chung, Sook-Hyang Jeong, Jin-Wook Kim


Livin is associated with invasive and oncogenic phenotypes of human hepatocellular carcinoma cells

Sung Bum Cho, Young Eun Joo, Sung Kyu Choi

Internal Medicine, Chonnam National University Medical School, Gwang ju, Republic of Korea

Background: Livin, a member of the inhibitors of apoptosis proteins, has been found to be expressed in variable cancers, where its expression is considered to be a poor prognostic marker. The purposes of this study were to observe the effect of Livin on tumor cell behaviors of human hepatocellular carcinoma (HCC) cells and to evaluate its expression in human HCC tissues and its relation to prognosis. Methods: We investigated the biologic role of Livin on tumor cell behaviors by using the small interfering RNA (siRNA) in human HCC cell line, HepG2. The migration, invasion and proliferation assays were performed. To evaluate the impact of Livin on apoptosis and cell cycle, we performed flow cytometric analyses and Western blotting. The expression of Livin by western blotting and immunohistochemistry was investigated in human HCC tissues. Results: Knockdown of Livin suppressed tumor cell migration, invasion and proliferation in human HCC cells. The proportion of apoptotic cells induced by knockdown of Livin was greater than that induced by the scramble siRNA-transfected cells in human HCC cells. Knockdown of Livin activated cleaved caspase-3, −7, −9 and PARP, leading to induction of apoptosis in human HCC cells. Knockdown of Livin induced the G2/M phase arrest by decreasing cyclin D1, CDK4 and CDK6, and by increasing p16, p21, p27 and p57 expression in human HCC cells. The expression of Livin was significantly elevated in human HCC tissues compared to normal hepatic tissues. But Livin expression is not associated with various clinicopathological parameters including survival. Conclusions: These results suggest that Livin is associated with the alteration of invasive and oncogenic phenotypes such as tumor cell migration, invasion, proliferation, resistance to apoptosis and cell cycle arrest in human HCC cells.


The following people have nothing to disclose: Sung Bum Cho, Young Eun Joo, Sung Kyu Choi


Mitogen- and phosphorylation site-specific degradation mechanisms of tumor suppressor PDCD4 in human HCC cells

Iwata Ozaki1 , 2 Hiroshi Hamajima2 , 3, Jinghe Xia2, Jingyan Qi2, Toshihiko Mizuta2, Keizo Anzai2, Sachiko Matsuhashi2

1Health Adminsitration Cener, Saga Medical School, Saga, Japan; 2Internal Medicine, Saga Medical School, Saga, Japan; 3Agency of Liaison and Innovation in Saga, Saga University, Saga, Japan

Background: Programmed cell death 4(PDCD4) is a tumor suppressor and reported to be downregulated in many types of cancers including hepatocellular carcinoma(HCC). We have previously showed that TGF-1β induced the apoptosis of human HCC cells via an induction of PDCD4. Epidermal growth factor (EGF) is known to suppress the TGF-p1-induced apoptosis of hepatic cells and promote the degradation of PDCD4 by proteasome through the phosphorylation of serine residues by S6K. However, the molecular mechanisms of PDCD4 degradation by proteasome is not fully understood, we therefore investigated the phosphorylation-dependent control mechanisms of PDCD4 in human HCC cells. Materials and methods: Human HCC cell line Huh7 cells were treated with TGF-p1 with or without EGF or 12-〇-tetradecanoylphorbo-13-acetate (TPA) that activates protein kinase C(PKC). Apoptotic cells were calculated with Hoechst33342 staining under microscopy. Expressions of PDCD4, Bax, cytochrome c, capsase-3, −8, −9 and β-actin were determined by Western blotting. To identify the involved signaling pathways, cells were treated with protein kinase inhibitors, rapamycin, LY294002, wortmanin, Ro-318452, SB203580, PD98059, SP600125 and proteasome inhibitor MG132. To investigate the role of phosphorylation of PDCD4, Ser67, 71 and 76 of PDCD4 cDNA were mutated to Ala, then transfected and the PDCD4 protein levels were compared with wild type. Results: EGF and TPA inhibited TGF-β1 induced apoptosis by suppressing PDCD4 protein. EGF-mediated suppression of PDCD4 was strongly inhibited by rapamycin and slightly by PKC inhibitor but not by others, suggesting that mTOR/S6K and PKC are involved in the PDCD4 degradation by proteasome. Suppression of PDCD4 degradation by rapamycin and PKC inhibitor enhanced the TGF-β1induced apoptosis, and suppressed the EGF- and TPA-mediated inhibition of TGF-β1-induced apoptosis respectively. Furthermore PKC inhibitor did not affect the phosphorylation of S6K after EGF treatment, suggesting the independent role of PKC and S6K. Analysis using PDCD4 mutants at Ser67, 71 and 76 revealed that all three phosphorylation sites were required for the EGF-mediated PDCD4 degradation but Ser67 was not essential for the TPA-mediated degradation, indicating the differential role of S6K and PKC in the phosphorylation-dependent degradation of PDCD4. Conclusions: A tumor suppressor PDCD4 degradation is differentially regulated by S6K and PKC. Since the inhibition of S6K or PKC attenuated the EGFand TPA-mediated inhibition of TGF-β1-induced apoptosis, the inhibition of PDCD4 degradation might be a possible target for the treatment of HCC.


The following people have nothing to disclose: Iwata Ozaki, Hiroshi Hamajima, Jinghe Xia, Jingyan Qi, Toshihiko Mizuta, Keizo Anzai, Sachiko Matsuhashi


Utility of Fatty Acid Binding Protein 5 expression as a recurrence and prognostic factor in hepatocellular carcinoma

Takanari Ohata, Hideki Yokoo, Toshiya Kamiyama, Kenji Wakayama, Tatsuyo Orimo, Tatsuhiko Kakisaka, Yosuke Tsuruga, Hirofumi Kamachi, Akinobu Taketomi

Gastroenterological Surgery I, Hokkaido University Graduate School of Medicine, Sapporo, Japan

Background: Hepatocellular carcinoma (HCC) is one of the cancer types with poor prognosis. At present, the serum level of Alphafetoprotein (AFP), Prothrombin Induced by Vitamin K Absence II (PIVKA II) or Alphafetoprotein Lens culinaris agglutiin 3 (AFP-L3%) are used for tumor markers of HCC, but they are not adequate to predict survival or recurrence after curative hepatectomy. Fatty Acid Binding Protein 5 (FABP5) has been reported in several cancers such as pancreatic cancer, breast cancer and prostatic cancer, also involved with tumor growth or invasiveness by using several cancer cell lines. However, there are few reports mentioned about HCC. Purpose: To analyze the correlation between the expression of FABP5 and malignant behavior of HCC using HCC tissues and human HCC cell lines. Methods: A total of 243 HCC samples were obtained through our institute and subjected to immunohistochemical analysis. In vitro, protein expression of FABP5 in HCC cell lines (HLE, HLF, and HepG2) was assessed by Western blot analysis. To the higher FABP5 expression cell lines, we performed invasion assay and proliferation assay with or without genetic down-regulation of FABP5 by small interfering RNA (siRNA). Results: The intensity of immunostaining in HCC tissues was determined comparing to immunoreactivity of Kupffer cells, and the patients were divided into a strong staining group (n=132) and a weak staining group (n=111) according to the intensity of immunostaining. In the strong staining group, 5-year overall survival rate was 59.5%, compared with 89.9% in the weak staining group (p<0.0001). In addition, 5-year relapse free survival rate in the strong staining group was 23.7%, compared with 47.2% in the weak staining group (p<0.0001). Moreover, the strong staining group of FABP5 was associated with poor prognosis and early recurrence (P<0.05), and also showed positive correlation of AFP, AFP-L3%, PIVKA II, tumor size, intrahepatic metastasis, and micro/macro vascular invasion from univariate and multivariate analysis (P<0.05). In vitro, Western blot analysis showed that protein expression of FABP5 in HLE and HLF which were highly invasive cell lines was high, while that of HepG2 which was lowly invasive cell was low. The knockdown of FABP5 in HLE and HLF also inhibited cell proliferation and invasiveness significantly. Conclusions: FABP5 was closely related with invasiveness and proliferation, and also behaved as a significant prognostic and recurrence factor for HCC patients. Therefore, FABP5 may serve as a new biomarker of HCC and a potential molecular target for the development of HCC therapies.


The following people have nothing to disclose: Takanori Ohata, Hideki Yokoo, Toshiya Kamiyama, Kenji Wakayama, Īatsuya Orimo, Tatsuhiko Kakisaka, Yosuke Tsuruga, Hirofumi Kamachi, Akinobu Taketomi


Bile acid flux essential for normal liver regeneration

Willscott E. Naugler1, Branden Tarlow3, Markus Grompe2

1Medicine/Gastroenterology, Oregon Health & Sciences University, Portland, OR; 2Pediatrics, Oregon Health & Sciences University, Portland, OR; 3Cell & Developmental Biology, Oregon Health & Sciences University, Portland, OR

Background: While many signals governing liver regeneration (LR) following 2/3 partial hepatectomy (PH) are known, the first signals are elusive. It is unclear what function the remant 1/3 liver cannot perform, and it is postulated that this function may stimulate LR. Aims: To examine bile acid (BA) flux and signaling in liver and intestine after PH and determine if BA are required for normal LR. Methods: Mice and rats underwent PH and were sacrificed in a standard timecourse following PH. Liver, blood, intestinal contents and ileum were collected. BA flux in the enterohepatic circulation (EHC) was determined by measuring BA in the various compartments over time, and BA signaling (Fgf15) and synthesis (Cyp7a) determined by qPCR. In order to drain the BA pool, rats were used in a biliary fistula (BF) model, wherein bile was drained for > 24 hours, followed by PH. In rats with BF, BA could be replaced via duodenal catheter to determine if effects of bile drainage on PH were specific to BA rather than other substances in bile. Three groups received PH: control rats with sham BF, rats with BF > 24 hours, and rats with BF > 24 hours and duodenal BA replacement. Rats were sacrificed at 0, 4, 12, and 24 hours, and tissues collected for analysis. Gene expression via RNA sequencing was done on liver samples, and pathway analysis software used to investigate transcriptional networks. Standard IHC staining for BrdU was used to determine hepatocyte proliferation. Results: BA rise quickly in blood immediately following PH, indicating that the remant 1/3 liver cannot handle the BA pool returning via the EHC. BA move from the intestinal compartment to the systemic blood after PH, and gradually return to the intestine over several days as the liver regenerates. Liver Cyp7a and intestinal Fgf15 are initially suppressed following PH. When the BA pool is drained prior to PH in the BF model, hepatocyte proliferation is markedly attenuated at 24 hours, and can be restored with the duodenal infusion of BA. RNA sequencing data of rat liver after PH and in the conditions of BF or BF + BA replacement reveal differences in MAPK pathway transcriptional networks, notably c-jun and AP-1 pathways. Conclusions: BA flux after PH is highly dynamic, with changes occurring in minutes of PH. Inability of the remant liver to secrete the returning BA pool after PH suggests a compelling reason for the liver to regenerate. Drainage of the BA pool prior to PH markedly attenuates hepatocyte proliferation, and BA replacement restores proliferation. Transcriptional networks involving c-jun and AP-1 are involved in the BA-specific effects on hepatocyte proliferation.


Markus Grompe - Board Membership: Yecuris Corp.; Consulting: Yecuris Corp.; Stock Shareholder: Yecuris Corp.

The following people have nothing to disclose: Willscott E. Naugler, Branden Tarlow


The novel membrane estrogen receptor GPER positively regulates liver growth during development

Saireudee Chaturantabut1, Claire C. Cutting2, Wolfram Goessling2, Trista E. North3

1MCB, Harvard University, Cambridge, MA; 2Genetics, Brigham and Women's Hospital, Boston, MA; 3PathoIogy, PaBeth Israel Deaconess Medical Center, Boston, MA

Estrogen plays crucial roles in adult reproductive organ growth and size determination; however, the impact of estrogen on non-reproductive organs such as liver is not well understood. We have previously performed a chemical screen of biologically active compounds in zebrafish and identified estrogen activity as a regulator of liver development. Using estrogen receptor agonists/antagonists, we demonstrated that the effect of estrogen on liver formation is time-dependent. We observed a biphasic effect of estrogen regulation: at the hepatic progenitor stage (<72hpf) exogenous estrogen decreases liver size (93%, n= 48), whereas once differentiated hepatocytes are present (>96 hpf), estrogen increases liver size (75%, n= 60) as determined by in situ hybridization for the hepatocyte-specific marker liver fatty acid binding protein (Ifabp, (fabp10)). Estrogen elicits regulatory effects through classical estrogen receptors (ERs) and a novel membrane G-coupled estrogen receptor (GPER). Our data demonstrate that the later developmental effect of estrogen on liver size is mediated by GPER; gper expression was detected in the developing liver from 48hpf when hepatocytes start to differentiate. Exposure to the GPER agonist (G1) and antagonist (G15), but not ER-specific agonists/antagonists, during liver maturation (>96hpf) dramatically increased (57%, n= 21), and decreased (92%, n= 38), the liver size respectively. Additionally, G15 treatment blocked the positive growth effects of estrogen on the maturing liver suggesting that estrogen signals through GPER to increase liver size. Estrogen or G1 exposure increased liver size by enhancing cellular proliferation and reducing apoptosis as determined by BrdU and TUNEL analysis. These results were confirmed by genetic knockdown of GPER using antisense morpholinos, which resulted in decreased liver size (46%, n= 28). Co-treatment of estrogen with GPER antagonist or genetically knockdown of GPER with morpholino further blocked the effects of estrogen on liver size determined by Ifabp expression (52%, n= 41). GPER morphants exhibited normal primitive endoderm and hepatoblast formation, but demonstrated defects in hepatocyte and bile duct formation as determined by in situ hybridization for Ifabp and sox9b. Taken together, these data identify GPER as a novel regulator of liver formation and growth during zebrafish embryonic development, where GPER is required for the differentiation and expansion of mature hepatic tissue in vivo.


Wolfram Goessling - Consulting: Fate Therapeutics, Fate Therapeutics; Patent Held/Filed: Fate Therapeutics, Fate Therapeutics

Trista E. North - Consulting: Fate Therapeutics

The following people have nothing to disclose: Saireudee Chaturantabut, Claire C. Cutting


FH535 Inhibits WNT/β-catenin signaling pathway in Liver cancer Stem Cells and HCC cell lines

Roberto R. Galuppo1, Roberto Gedaly1, Paul Angulo2, Michael F. Daily1, Malay Shah1, Changguo Chen1, Xiping Zhang3, Karyn Esser3, Mark Evers4, Jieyun Jiang5, Brett Spear5

1Surgery, Univ of Kentucky, Lexington, KY; 2Department of Internal Medicine, University of Kentucky, Lexington, KY; SDepattment of Physiology, University of Kentucky, Lexington, KY; 4Markey Cancer Center University of Kentucky, Lexington, KY; 5Department of Microbiology and Immunology, University of Kentucky, Lexington, KY

Background: The purpose of this study is to determine how FH535, an inhibitor of p-catenin/TCF/LEF activity, inhibits proliferation of liver cancer stem cells (LCSC) and HCC cell lines. Methods: FH535 induced inhibition of LCSC and HCC cell lines proliferation was assayed by 3H-thymidine incorporation. FH535 on inhibition of β-catenin/TCF/LEF activity was assayed by dual luciferase reporter assay by sequential measurement of firefly and Renilla luciferase activities after co-transfection of the cells with TOPFIash and p-RL plasmids. TCF/LEF mutant plasmid (FOPFIash) was used as a negative control. Real-time RTPCR and western-blot was used to detect mRNA and protein expression of β-catenin targeted genes of c-Myc, survivin and cycIin-D1. Cell cycle was assayed by flow cytometry. Results: FH535 inhibited 3H-thymidine incorporation significantly in LCSC with 50% inhibition concentration (IC50) of 15.4 μM when the cells were cultured in DMEM+1 0%fBs. When the LCSC were cultured in DMEM +1% FBS, the FH535 IC50 was 5.1 μM. FH535 inhibited HCC cell line Huh7 and PLC proliferation with IC50 of 10.9 and 9.3 μM respectively when the cells were cultured in DMEM+10%FBS. FH535 at 1 μM and 5 M significantly inhibited β-catenin/TCF/LEF reporter gene of TOPFIash plasmid transfected in LCSC, Huh7 and PLC. In addition, FH535 inhibited mutant β-catenin signaling in dual luciferase reporter assay. In comparison, FH535 is much more potent inhibitor than a tankyrase inhibitor XAV939 in β-catenin signaling pathway. FH535 dose dependently inhibited mRNA and protein expression of c-Myc, cycIin-D1 and survivin. FH535 induce cell cycle arrest from G1 to s phase. Conclusion: Targeting β-catenin signaling pathway caused significant inhibition of LCSC and HCC cell lines in vitro. Cell cycle was arrested from G1 to S phase correlating with inhibition of Cyclin-D1. Wtn-β-catenin pathway inhibition may play a role in the future treatment of HCC.


Paul Angulo - Grant/Research Support: NIDDK, Mochida, Genfit

The following people have nothing to disclose: Roberto R. Galuppo, Roberto Gedaly, Michael F. Daily, Malay Shah, Changguo Chen, Xiping Zhang, Karyn Esser, Mark Evers, Jieyun Jiang, Brett Spear


Participation of NF-қB in cell survival or proliferation is regulated by interaction with β-catenin

Kari Nejak-Bowen, Satdarshan (Paul) S. Monga

Pathology, University of Pittsburgh, Pittsburgh, PA

Understanding the signaling pathways involved in liver regeneration (LR) is important for identifying new therapies for treating liver injury. Because p-catenin and NF-қB share redundant biological effects during LR, we hypothesize that -catenin interacts with NF-қB to enhance cell proliferation via transactivation of cycIin-D1 gene expression, whereas the absence of β-catenin permits NF-қB to regulate cell survival via distinct target gene expression. We have previously shown that p-catenin sequestration of p65 in cytosol prevents it from translocating to the nucleus, and that lack of p65/p-catenin complex in hepatocytespecific p-catenin knockout (K〇) mice enhances survival. To determine whether this association regulates cell proliferation, WT and KO mice were treated with GalN/LPS to induce liver injury. WT livers display proliferating cell nuclear antigen (PCNA)-positive cells during the early phase of injury, which are absent at time of morbidity. Conversely K〇 display scant proliferation, but an increase in NF-қB-regulated pro-survival target genes. We then characterized the association and expression of NF-қB and p-catenin after LR by immunoprecipitation and demonstrated translocation and interaction of these two proteins in the nucleus within hours after partial hepatectomy (PH), which preceded a 2.5-fold increase in cycIin-D1 mRNA. Finally, Hep3B hepatoma cells transfected with cycIin-D1 reporter demonstrated a significant decrease in reporter activity when treated with β-catenin siRNA but not with p65 siRNA. Thus, βcatenin is an important component of balancing the contextdependent pro-proliferation versus pro-survival functions of NF-қB and could be an attractive target for clinical therapies that enhance cell survival and/or proliferation in liver injury.


Satdarshan (Paul) S. Monga - Consulting: Bristol Myers Squibb, Phase Rx, Merck The following people have nothing to disclose: Kari Nejak-Bowen


Canonical TGF-β Signaling drives Tumor-Progression via JAK/STAT3-signaling in Advanced HCC

Andres Rojas1, Jessica Cromheecke1,Lianchun Xiao2, Mong-Hong Lee3, Mien-Chie Hung3, Gregory J. Gores4, Boris Blechacz1

1Gastroenterology, hepatology and nutrition, MD Anderson Cancer center, Houston, TX; 2Department of Biostatistics, MD Anderson Cancer Center, Houston, TX; 3MoIecuIar & Cellular Oncology MD Anderson Cancer Center, Houston, TX; 4Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN

Background: More than 80% of Hepatocellular carcinoma (HCC) patients present at advanced stages not amenable to curative therapies. Currently existing palliative therapies achieve minimal survival benefits of 3 months. Hence, there is an urgent need for novel therapeutic strategies. Tumor Growth Factor-β (TGF-β) is a potent tumor suppressor via its Smad2/3 signaling axis; its inactivation has been implicated in hepatocarcinogenesis. In patients with advanced HCC, TGF- v serum concentrations are elevated and correlated to disease stage, metastases and worse prognosis. Our goal is to evaluate the status and function of canonical TGF-β signaling in advanced HCC. Material and Methods: Immunohistochemical analysis for pSer465/467-Smad2 and STAT3 in human HCC samples was performed using Tissue Micro Arrays. In vitro studies were performed using human HCC cell lines (HepG2, Hep3B, PLC/PRF/5, HUH-7, SNU-398, SNU-449). Pathway activation was evaluated by immunoblot analysis and immunofluorescence microscopy. Gene silencing was achieved by lentiviral mediated transduction with shRNA. Cell proliferation was assessed by cell quantification and MTS-assay. Apoptosis was quantified morphologically by DAPI stain and fluorescence microscopy. Migration was assessed by wound assay, and invasion by Boyden Chamber Assay using Matrigel. Results: Intranuclear pSer465/467-Smad2 was identified in up to 65% of human HCC samples, and correlated with TNM-stage, 3year survival and intranuclear pTyr705STAT3. In 50% of human HCC cell lines, TGF-β treatment (10 ng/mL) resulted in Ser465/467-phosphorylation and nuclear translocation of Smad2. Exclusively in HCC cells with an intact canonical TGFp signaling, TGF-β stimulation induced Tyr705-phosporylation of STAT3. Smad2-directed gene silencing inhibited TGF-pinduced STAT3 Tyr705-phosporylation. Further, pyridine 6 mediated Janus Kinase-1/2 (JAK1/2) inhibition prevented TGFp induced Tyr705-phosporylation of STAT3. In human HCC cell lines with intact canonical TGF-β signaling, TGF-β treatment induced invasion and migration, which was completely abrogated by STAT3-inhibition. Physiologic TGF-β mediated apoptosis induction and antiproliferative effects were neutralized in these cells. However, STAT3 inhibition restored TGF-β antiproliferative effects and sensitized HCC cells to TRAIL-mediated apoptosis. Conclusion: In advanced HCC, canonical TGF-β signaling is constitutively activated and mediates tumor progression via JAK/STAT3 signaling. STAT3-inhibition restores TGF-β tumor suppressor function. Hence, restoration of the natural TGF-β tumor suppressor function might be a promising new therapeutic strategy in advanced HCC.


Gregory J. Gores - Advisory Committees or Review Panels: Bayer, Chugia, Daiichi, Generon, Conatus, IntegraGen

The following people have nothing to disclose: Andres Rojas, Jessica Cromheecke, Lianchun Xiao, Mong-Hong Lee, Mien-Chie Hung, Boris Blechacz


The SUMOyation enzymes UBC9 and PIAS1 Serve as Novel Coactivitors of FXR-Target Genes

Natarajan Balasubramaniyan, Mosaab Esseid, Frederick J. Suchy

Department of Pediatrics and Children's Hospital Colorado Research Institute, University of Colorado School of Medicine, Aurora, CO

FXR is a ligand-regulated nuclear receptor (NR) that has a central role in bile acid homeostasis including biosynthesis, biotransformation and transport, and more recently has been recognized to regulate lipid and carbohydrate metabolism. Transcription dynamics at target gene promoters require ligand-dependent interactions of NRs with a large number of coregulatory proteins that possess an array of chromatin-remodeling and histone-modifying enzymatic activities. Post- translational modifications such as acetylation, methylation, demethylation, phosphorylation, ubiquitination and SUMOylation of histones have been shown to significantly influence gene transcription. We recently reported (J Biol Chem 288: 1385062, 2013) that FXR is posttranslationally modified by small ubiquitin-related modifier-1 (SUMO-1), resulting in attenuation of transcription. Here, we investigated the role of the SUM〇ylation enzymes, ubiquitin-conjugating enzyme 9 (UBC9) and protein inhibitors of activated STAT1 (PIAS1), in fXr mediated transcription of FXR target genes, BSEP and SHP. Transient transfection assays in HEPG2 cells showed Ubc9 and PIAS1 each significantly potentiated FXR-mediated transactivation of reporter constructs containing human BSEP or SHP promoters. Wild type and SUMOylation inactive mutants of UBC9 (C93S) and PIAS1 (C351S) similarly enhanced the FXR-mediated transactivation of the BSEP and SHP promoters, indicating that the coactivation potency of UBC9 and PIAS1 is independent of SUMOylation activity. Reduction of endogenous UBC9 and PIAS1 by introducing corresponding small interfering RNAs significantly reduced endogenous BSEP and SHP mRNA levels, indicating that they normally function as coactivators of FXR. Sequential chromatin immunoprecipitation (ChIP-ReChIP) verified the concurrent binding of FXR and UBC9 and PIAS1 to the BSEP and SHP promoters. These results demonstrated a novel role of Ubc9 and PIAS1 as coactivators of FXR-target genes. Further studies are needed to define how these enzymes can function alternatively in either activating or repressing FXR-mediated transcription.


The following people have nothing to disclose: Natarajan Balasubramaniyan, Mosaab Esseid, Frederick J. Suchy


Cyclic AMP increases expression of the Type 2 Inositol trisphosphate receptor (IP3R2) in hepatocytes

Emma A. Krugalov, Meena Ananthanarayanan, Jittima Weerachayaphorn, Michael H. Nathanson

Dept Int. Med, Section Dig Dis, Yale University School of Medicine, New Haven, CT

Background: Inositol trisphosphate receptor, type 2 (IP3R2) -mediated Ca2+-signaling is essential for canalicular bile secretion mediated by Mrp2. While IP3R2 has been shown to be phosphorylated by PKA via cAMP, it is unknown whether cAMP can also regulate IP3R2 transcription. AIM: To investigate whether cAMP mediates transcriptional regulation of the IP3R2 gene and if so whether this is mediated via CREs in proximal promoter region. Methods: Rat hepatocytes on collagen were incubated with Forskolin+IBMX (FSK) or CPT-cAMP for 12 hrs following which RNA and protein extracts were prepared. Hepatocytes were also treated with Actinomycin D to block new RNA synthesis. qPCR and western blotting were carried out to quantitate mRNA and protein levels following standard methods. Rat and Human promoters (2.0 kb) were analyzed for CREs (cAMP-responsive elements) using MatInspector algorithm. Canalicular secretion was monitored by CMFDA fluorescence in hepatocytes on collagen sandwich cultures Results: Incubation of rat hepatocytes with FSK+ IBMX (50 μM+ 200 μM) or CPT-cAMP (100 μM) for 12 hrs led to a significant increase in IP3R2 message levels compared to solvent-treatment (4.3 and 3.0-fold over controls respectively, p<0.01). Incubation with Actinomycin D in the presence of these compounds resulted in significant abrogation of the increase in IP3R2 mRNA indicating that the increase was due to enhanced transcription. Analysis of the proximal 2.0 kb rat and human IP3R2 promoters revealed that both promoters contained multiple CREs (cyclicAMP response elements). There was also an increase in IP3R2 protein levels under the same conditions (2.1 and 1.5-fold over controls with FSK and CPT-cAMP treatment respectively. p<0.0001) suggesting that the increased mRNA resulted in increased IP3R2 protein synthesis. Further confirmation of the effect of cAMP on IP3R2 gene regulation was obtained by our observation that hepatic IP3R2 protein levels were increased 3.7-fold (p<0.05) in fasted (high serum glucagon) vs. fed rats. IP3R2 stimulates Mrp2 function (Hepatology 52: 327-37, 2010) and canalicular secretion of the Mrp2 substrateCMFDA was increased by either FSK+IBMX or CPTcAMP relative to controls (1.4 or 1.6-fold respectively over controls). Conclusion: cAMP increases transcription of the IP3R2 gene. This increase in IP3R2 is responsible for the enhanced capacity for bile flow, which may represent an important adaptive mechanism during fasting.


The following people have nothing to disclose: Emma A. Krugalov, Meena Ananthanarayanan, Jittima Weerachayaphorn, Michael H. Nathanson


A novel RARa/CAR-mediated mechanism for regulation of the human organic solute transporter beta (OSTβ) gene expression

Shuhua Xu, An-QIang Sun, Frederick J. Suchy

University of Colorodo School of Medicine Aurora, CO

The organic solute transporter alpha-beta (OSTa/p) is a heteromeric transporter that is essential for bile acid and sterol disposition and for the enterohepatic circulation. To understand the mechanism of OSTα gene regulation, the effects of retinoic acid (RA) on OSTa/p gene expression were investigated. The results show a dose-dependent induction of OSTβ, but not OST, expression in both Huh7 and HepG2 cells by RA treatment. A novel functional retinoic acid receptor response element (RARE, so-called DR5) in the promoter of OSTβ gene was identified. The interaction of RARa/RXRa with the RARE to activate hOSTβ promoter activation was verified by luciferase reporter assay, electrophoretic mobility shift assay, chromatin immunoprecipitation assay, and mutagenesis studies. The studies demonstrated that the RARE is also a CAR binding site for OSTβ gene regulation. These results suggest that OSTβ is a target of both FXR-mediated (by binding to IR-1 element) and RARα- and CARmediated (by binding to DR5 element) gene regulation pathways. In summary, this study has uncovered a novel RARE (DR5) element in the promoter of OSTβ which appears to function synergistically with the IR-1 element to provide maximal induction of OSTβ in response to retinoic acid (RA). RARα or CAR heterodimerized with RXRα interacts with this DR5 element to activate hOSTβ expression, and RA plays a critical role in the crosstalk between the IR-1 and RARE mediated mechanisms. This finding underlines the physiological importance of RARα and CAR in controlling 〇STp expression levels.


The following people have nothing to disclose: Shuhua Xu, An-QIang Sun, Frederick J. Suchy


Persistent Activation of Nrf2 Causes Delay in S Phase Entry and Subsequent Acceleration in DNA Synthesis of Replicating Hepatocytes during Liver Regeneration

Min Hu1,2 Yuhong Zou2, Shashank M. Nambiar2, Joonyong Lee2, Yan Yang1, Guoli Dai2

1Department of Pharmacology, Anhui Medical University, Hefei, China; 2Department of Biology, Indiana University-Purdue University Indianapolis' Indianapolis, IN

Actin-binding Kelch-like ECH-associated protein 1 (Keap1) controls the activity of transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) by tethering or releasing Nrf2 in or from cytosol. This Keap1/Nrf2 pathway plays critical roles in maintaining tissue homeostasis and combating oxidative stress and inflammation. Several reports indicate its involvement in regulating cell proliferation. The aim of our study was to determine whether and if so how the pathway modulates the cell cycle progression of replicating hepatocytes during liver regeneration. keap 1+/- mice were used because it represents a mouse model with Keap1 knockdown and therefore Nrf2 persistent activation. Wild-type and keap 1+/- mice were subjected to 2/3 partial hepatectomy (PH) and various assessments were performed at multiple time points after surgery. We found that persistent activation of Nrf2 enhanced liver regrowth transiently on the third day after PH. Ki-67 immunostaining, BrdU incorporation, and mitotic figure quantification were used to evaluate the total numbers of hepatocytes undergoing proliferation, S phase, and M phase, respectively. To our surprise, when Nrf2 was persistently activated, S phase entry of proliferating hepatocytes was delayed by at least 6 hours, which was associated with complete prevention of c-Met, Akt, p70S6K and Cyclin A2 activation. It was even more surprising that, in keap 1+/- mice, subsequent to the severe delay in S phase entry, DNA synthesis was markedly accelerated. This was accompanied by restoration of Akt, p70S6K, and Cyclin A2 activation and increased expression of several genes essential for DNA synthesis including DNA polymerase and ligase. Eventually, S phase was shortened by at least half of the normal time due to persistent activation of Nrf2. As the cell cycle progressed to M phase, we did not see a significant difference in the numbers of hepatocytes undergoing mitosis in the regenerating livers between wild-type and Keap1 +/- mice. Thus, persistent activation of Nrf2 exerted potent effects on S phase entry and DNA synthesis of replicating hepatocytes during liver regeneration. Our study demonstrates that Keap1/Nrf2 pathway is a critical molecular mechanism transducing cell signaling to the cell cycle.


The following people have nothing to disclose: Min Hu, Yuhong Zou, Shashank M. Nambiar, Joonyong Lee, Yan Yang, Guoli Dai


Hepatic circadian rhythm regulation in human CYP7A1transgenic mice

Jessica M. Francl1, Tiangang Li2, 1, Shannon M. Boehme1, John Chiang1

1 Integrative Medical Sciences, Northeast Ohio Medical University, Rootstown, OH; 2Pharmacology, KUMC, Kansas City, KS

Introduction: Circadian rhythms regulate central and peripheral physiology with a period of 24 hr. Bile acids facilitate the absorption of dietary nutrients and also activate several nuclear receptors and a G-protein coupled receptor TGR5 to regulate lipid, glucose and energy metabolism. CYP7A1, the rate-limiting enzyme that converts cholesterol to bile acids, exhibits a circadian rhythm of expression in rodents and humans. Recently, it has been shown that disruptions in circadian rhythms (e. g., shift work, sleep deprivation, chronic jet lag) can lead to disturbed hepatic regulation and symptoms of metabolic disease. The mechanisms involved in CYP7A1 circadian gene regulation are not fully understood, though the human CYP7A1 gene promoter lacks a liver orphan receptor (LXR) binding site compared to mouse. Therefore, we utilized a human CYP7A1 transgenic mouse model (hCYP7A1-tg) to investigate the circadian regulation of liver metabolism. Results: Under a 12 hr light/dark cycle (ZT 12=lights off), genes which compose the molecular biological clock exhibited altered amplitude, but similar timing, in peak gene expression between genotypes. Cyp7a 1 gene expression was rhythmic, peaking at ZT 14 in wild type mice and 4 hr later in hCYP7A1-tg mice. ChlP assay showed HNF4α binding to the human CYP7A1 promoter was significantly increased at ZT 14, while HNF4a binding to mouse Cyp7a1 was not significantly increased at any time over 24 hr. HNF4α mRNA was rhythmically expressed in hCYP7A1-tg mice but not in wild type counterparts. SHP, a negative regulator of nuclear receptors, was also differentially expressed between genotypes, with peak expression occurring in early day in hCYP7A1-tg mice but not in wild type mice. ChIP assay also revealed SHP binding to the CYP7A1 promoter at ZT 14 and 22 in hCYP7A1-tg mice, while CYP7A1 circadian expression did not appear to be regulated by LRH. Conclusion: Epigenetic regulation of gene expression can affect cellular physiology. The human and mouse Cyp7a1 gene promoter exhibit low sequence homology, and the data presented here suggest differential circadian regulation of the human and mouse Cyp7a1 gene, possibly via HNF4a. Furthermore, the SHP/LRH pathway may not be involved, as SHP binding is increased when CYP7A1 mRNA peaks, while LRH binding remains constant. Further study of the circadian and molecular regulation of the human CYP7A1 gene could lead to better understanding of the detrimental effects of circadian disruption and the development of metabolic disease.


The following people have nothing to disclose: Jessica M. Francl, Tiangang Li, Shannon M. Boehme, John Chiang


Inhibitory effects and the molecular mechanism of tenofovir disoproxil fumarate and adefovir dipivoxil on proliferation of hepatic stellate cells

Hyung Joon Yim1, Ji Won Hwang1, Hyungshin Yim2, Hae Rim Kim1, Seong Hee Kang1, Sang Jun Suh1, Ji Hoon Kim1, Yeon Seok Seo1, Jong Eun Yeon1, Soon Ho Um1, Kwan Soo Byun1

1Department of Internal Medicine, Korea University Medical College, Ansan, Republic of Korea; 2Department of Pharmacy, Hanyang university, Ansan, Republic of Korea

Background: It has been reported that long-term suppression of replication of hepatitis B virus (HBV) with nucleotide analogues such as tenofovir (TDF) or adefovir (ADV) is associated with regression of advanced hepatic fibrosis and reversal of cirrhosis. These effects are considered mainly due to decreased liver injury caused by HBV, but direct anti-fibrotic effects of these drugs might be associated. We planned to investigate the effect of these drugs on the hepatic stellate cells which are key driver cells causing hepatic fibrosis. Methods: The immortalized human hepatic stellate cell line, LX-2 was cultured. The cells were treated with control, TDF and ADV for 24 and 48 hours. Cellular DNA synthesis was evaluated by measuring BrdU incorporation. Distribution of cell cycle was determined by propidium iodide (PI) staining and flow cytometry. Expression of proteins including cell cycle regulators (p21, p27, cyclin D1), NF-қB, TGF-β, α-smooth muscle actin (a-SMA), Erk1/2 and pErk1/2 were measured in Western blot analysis. Results: In BrdU-proliferation assay, both of two drugs effectively inhibited the proliferation dependent on the concentration. All of two drugs markedly reduced p27, p21 and induced cyclin D1 compared to control. In addition, the cell cycle arrest at the G2/M phase was observed according to the concentration of two drugs by using flow cytometry. The amounts of NF-kB and TGFp were decreased by both of two drugs. No significant changes of two drugs on protein expression of a-SMA, Erk1/2 and pErk1/2 were observed. Conclusions: TDF and ADF are capable of reducing proliferation of activated HSCs. We believe that the mechanism of anti-proliferative effect seems to be mediated by G2/M cell cycle arrest. Furthermore, both of two drugs suppressed NF-қB, TGF-β transduction signaling. These data suggest that TDF and ADF effectively block the proliferation and growth of activated HSCs, therefore both of two drugs would be associated with regression the liver fibrosis.


Hyung Joon Yim - Grant/Research Support: GSK Korea, Handok Pharm, Gilead Korea; Speaking and Teaching: BMS Korea

The following people have nothing to disclose: Ji Won Hwang, Hyungshin Yim, Hae Rim Kim, Seong Hee Kang, Sang Jun Suh, Ji Hoon Kim, Yeon Seok Seo, Jong Eun Yeon, Soon Ho Um, Kwan Soo Byun


Emodin attenuates bile acid-induced and palmitateinduced hepatocyte apoptosis by suppressing JNK activation

Yun Bin Lee1, Jung-Hwan Yoon1, Eun Ju Cho2, Dong Hyeon Lee1, Yuri Cho1, Su Jong Yu1, Jeong-Hoon Lee1, Yoon Jun Kim1, Hyo-Suk Lee1, Chung Yong Kim1

1Department of internal Medicine and Liver Research Institute, Seoul National University College of Medicine, Seoul, Republic of Korea; 2Department of Internal Medicine, Kangwon National University Hospital, Chuncheon, Republic of Korea

Background/Aim: Emodin, 1, 3, 8-trihydroxy-6-methyIanthraquinone, is an anthraquinone derivative contained in the rhizome of Rheum palmatum L., which is traditionally used in Chinese medicine. It has been reported that Emodin possesses biological activities such as anti-inflammatory or hepatoprotective effects. Thus, we aimed to investigate if Emodin may attenuate hepatocyte apoptosis induced by bile acid or saturated fatty acid. Methods: We performed in vitro studies using human hepatocellular carcinoma cell line, SNU761, which contains bile acid transporter and thereby, susceptible to bile acidinduced apoptosis. Deoxycholate (DC) and palmitate were used to induce apoptosis. Endoplasmic reticulum (ER) stress was induced by tunicamycin or thapsigargin. Cell growth and apoptosis were assessed using MTS assay, and apoptotic and kinase signaling pathways were explored by immunoblot analysis. Results: Emodin diminished DC- and palmitate-induced JNK activation, which in turn lead to attenuation of caspase-dependent hepatocyte apoptosis. However, Emodin did not affect tumor necrosis factor-related apoptosis- inducing ligand (TRAIL)induced and ER stress-induced apoptosis, indicating that it had no effects on death receptor- and ER stress-mediated pro-apoptotic signalings. Conclusions: Emodin attenuates bile acidinduced hepatocyte apoptosis and palmitate-induced hepatocyte 丨ipoapoptosis by suppressing JNK activation. Therefore, Emodin may exhibit hepatoprotective activities against cholestatic liver injury and steatohepatitis.


The following people have nothing to disclose: Yun Bin Lee, Jung-Hwan Yoon, Eun Ju Cho, Dong Hyeon Lee, Yuri Cho, Su Jong Yu, Jeong-Hoon Lee, Yoon Jun Kim, Hyo-Suk Lee, Chung Yong Kim


Enhanced autophagy promotes liver regeneration and hepatocyte proliferation after partial hepatectomy in mouse liver

Chih-Wen Lin1 , 2, Lein-Ray Mo2, Yaw-Sen Chen3, Yun-Ju Chen4 , 5, PoLin Kuo6, Ming-Lung Yu6

1Division of Gastroenterology and Hepatology, Department of Medicine, E-DA Hospital/ I-SHOU University' Kaohsiung, Taiwan; 2Graduate Institute of Medicine, College of Medicine,, Kaohsiung Medical University, Kaohsiung, Taiwan; 3Department of Surgery,, E-Da Hospital, I-Shou University,, Kaohsiung, Taiwan; 4Department of Medical Research, E-Da Hospital, IShou University,, Kaohsiung, Taiwan; 5Department of Biological Science & Technology,, I-Shou University, Kaohsiung, Taiwan; 6HepatobiIiary Division, Department of Internal Medicine,, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan

Background and Aims: Pharmacological approaches can potentially improve liver regeneration and decrease hepatic failure after partial hepatiectomy (PHx). Autophagy is a cellular degradation process that can remove subcellular organelles and known to participate in physiology and pathogenesis. However, the role of autophagy in liver regeneration after PHx is never discussed in the literature. Thus, we investigated that autophagy may play an important role in liver regeneration and pharmacological modulation of autophagy could be an effective approach to promote liver regeneration after PHx. Methods: c57BL/6 mice that underwent sham-operation and partial hepatectomy (two-third of liver removed) were used as models of liver regeneration. An autophagy enhancer, carbamazepine, or a lysosome inhibitor, chloroquine, was given before sacrifice. Activation of autophagy, liver growth, level of hepatocyte proliferation, and blood levels of liver enzyme were measured. Results: We found that autophagy was decreased in early phase of liver regeneration after PHx in vivo. However, pharmacological enhancement of autophagy by carbamazepine significantly enhanced autophagy during liver regeneration after PHx. Furthermore, carbamazepine promoted liver growth, enhanced hepatocyte proliferation, and alleviated liver injury during liver regeneration after PHx. In contrast, chloroquine decreased liver growth and hepatocyte proliferation. Moreover, carbamazepine significantly increased the PCNA and cyclin D1 protein levels but decreased TGF-β 1 protein levels. Conclusion: These findings indicate that autophagy plays a critical protective role in liver regeneration and pharmacological enhancement of autophagy in the mouse liver can be an effective strategy for promoting liver regeneration and hepatocyte proliferation and alleviating liver injury after PHx.


The following people have nothing to disclose: Chih-Wen Lin, Lein-Ray Mo, YawSen Chen, Yun-Ju Chen, Po-Lin Kuo, Ming-Lung Yu


Hepatic Receptor Activation By Polychlorinated Biphenyls - Implications For Xenobiotic/Energy Metabolism And Nonalcoholic Fatty Liver Disease

Banrida Wahlang3, Keith C. Falkner1, Heather B. Clair1, Laila AlEryani1, John J. Guardiola1, Russell A. Prough4, Matthew C. Cave1, 2

1Department of Medicine/GI, University of Louisville, Louisville, KY; 2Robley Rex VAMC, Louisville, KY; 3Pharmacology, University of Louisville, Louisville, KY; 4Biochemistry, University of Louisville, Louisville, KY

Purpose: Polychlorinated Biphenyls (PCBs) are environmental pollutants, present in 100% of the US adult population and theoretically predicted to act as ligands for hepatic transcription factors involved in xenobiotic/endobiotic detoxification, obesity, inflammation, and steatosis. These human receptors include arylhydrocarbon receptor (AhR), pregnane xenobiotic receptor (PXR), constitutive androstane receptor (CAR), liver X receptor (LXRa), farnesoid X receptor (FXR) and peroxisomal-proliferator activated receptor (PPARa). This study evaluates a PCB mixture, Aroclor 1260, and selected individual PCB congeners, as potential ligands for these receptors. Methods: MTT assay was performed to determine acutely toxic concentrations for Aroclor 1260. HepG2 cells were transfected with plasmids expressing human PXR, CAR, LXR, FXR or PPARα; and receptor-responsive plasmids. Transfected cells were treated with ligands for AhR (Benzanthracene: 10μM), PXR (Rifampicin: 10μM), CAR (CITCO: 10μM), LXR (T0901317: 100nM), FXR (GW4064: 0.5 μM) and PPARα (Nafenopin: 50μM); varying concentrations of Aroclor 1260 and individual congeners. Results: The toxicity threshold of Aroclor 1260 in HepG2 cells was 26.0 ± 3.7μg/mL. Aroclor 1260 did not activate AhR but the individual congener 126 was a potent inducer. Aroclor 1260 induced the PXR reporter system in a simple monotonic fashion with significant inductions being observed at concentrations of 10μg/mL or greater. Amongst congeners tested, PCBs 1 49, 138, 187, 174, 151, 183 and 196 activated human PXR. Aroclor 1260 appeared to activate CAR at lower concentrations and antagonize CAR activation by the CAR agonist, CITCO, at higher concentrations (>20 μg/mL). Aroclor 1260 also suppressed PPARα activation induced by the agonist Nafenopin at 20 μg/mL, although none of the selected congeners tested showed any significant inhibition. Aroclor 1260 did not interact with LXR or FXR and had no effect on LXR or FXR-dependent induction by T0901317 or GW4064 respectively. Conclusions: These data suggest that Aroclor 1260 is a human PXR agonist, a mixed agonist/antagonist for human CAR and an antagonist for human PPARα. However, this mixture did not significantly activate AhR. PCB 126 activated both PXR and AhR and 196 activated PXR only. Thus, receptor activation by PCBs was both congener and dose-dependent. Although, PCBs did not directly activate non-xenobiotic receptors (e. g. LXR and FXR), these pollutants could potentially influence expression of target genes for these nuclear receptors via cross-talk. We postulate that PCB-receptor interactions modify human xenobiotic metabolism, and contribute to obesity/metabolic syndrome and NAFLD.


The following people have nothing to disclose: Banrida Wahlang, Keith C. Falkner, Heather B. Clair, Laila Al-Eryani, John J. Guardiola, Russell A. Prough, Matthew C. Cave


Disruption of TGF-β pathway is associated with alcoholassociated hepatocellular cell cancer

Jian Chen1, Vivek Shukla1, Jiun-Sheng Chen1, Lior H. Katz1, Avijit Majumdar1, Walter N Hittelman1, Xiaoping Su1, Junjie Chen1, Xifeng Wu1, Patrizia Farci2, Lopa Mishra1

1MD Andrson Concer Center, Houston, TX; 2MD Andrson Cancer Center, Houston, TX

Objective: A crucial factor in protection from liver injury, fibrosis and cancer by agents such as alcohol and viral hepatitis is the enforcement of genomic stability. However, sensors for genotoxicity leading to aberrant DNA repair remain elusive. Due to the inactivation of the TGF-p pathway resulting in DNA damage, alcohol toxicity and hepatocellular cancer (HCC), TGF-β has been implicated as a critical promoter of genomic stability and tumor suppression; however, the framework by which this occurs is not known. We performed Whole-genome (WGS) and Exome sequencing (WES) on 4 pairs human HCC samples and identified inactivation of TGF-β pathway members as a prominent characteristic of alcohol induced HCC, potentially through inactivation of a TGF-β adaptor, p-2spectrin (p2SP). Materials & Methods: WGS and WES were performed on 4 pairs human HCC samples. p2SP mutant mice were treated with alcohol to determine their susceptibility to aldehyde-induced developmental abnormalities. Primary MEFs from β2SP+/+, β2SP+/- and β2SP-/- mice were treated with multiple DNA damaging agents and -irradiation to evaluate ۷-H2AX, 53BP1, NBS1 and Rad51 foci formation. ChIP assays were performed to determine the recruitment of p2SP/Smad3 at FancD2 promoter. Results: (1)Through our WGS and WES analyses, we discovered a novel inactivating mutation of TGFp/p2SP pathway in alcohol-associated HCC. This p2SP mutation results in functional disruption of TGF-β signaling. (2) p2SP-/- mouse embryos display features of human fetal alcohol syndrome (FAS). Alcohol treatment in the absence of p2SP mice results in teratogenicity and spontaneous fetal alcohol-like phenotype. (3) Over-expression of p2SP somatic mutation in HCC results in hypersensitivity to DNA interstrand crosslinking agents. (4) Deficiency of p2SP results in compromised DNA repair. (5) DNA damage response induces nuclear localization of p2SP in a TGF-β-dependent manner. (6) p2SP/Smad3 complexes regulate FancD2 at the transcriptional level. (7) The expression of p2SP and FANCD2 is correlated in alcoholic hepatitis and HCCs. Conclusions: Through WGS and WES, we found a novel inactivating mutation in p2SP which results in loss of TGF-β signaling pathway. TGF-p/p2SP/Smad3 pathway is crucial for protection against aldehyde genotoxicity through enforcing genomic stability. Importantly, these results could potentially lead to new therapeutics targeting toxin-induced DNA damage and tumorigenesis.


The following people have nothing to disclose: Jian Chen, Vivek Shukla, JiunSheng Chen, Lior H. Katz, Avijit Majumdar, Walter N Hittelman, Xiaoping Su, Junjie Chen, Xifeng Wu, Patrizia Farci, Lopa Mishra


Identification And Validation Of Environmental Chemical Nuclear Receptor Agonists Which Could Contribute To Nonalcoholic Fatty Liver Disease

Laila Al-Eryani1, Banrida Wahlang1, Heather B. Clair1, John J. Guardiola1, Keith C. Falkner1, Russell A. Prough3, Mattnew C. Cave1, 2

1Department of Medicine/GI, University of Louisville, Louisville, KY; 2Robley Rex VA Medical Center, Louisville, KY; 3Biochemistry' University of Louisville, Louisville, KY

Introduction: Environmental chemical exposures including organochlorine pesticides (OCPs) have been linked to nonalcoholic fatty liver disease (NAFLD) in epidemiologic studies. Environmental chemicals may activate hepatic xenobiotic receptors including the pregnane-xenobiotic-receptor (PXR) and constitutive androstane receptor (CAR). While these receptors are classically known to regulate xenobiotic metabolism, more recently they have been found to regulate energy metabolism 一 and PXR activation has been shown to worsen obesity and NAFLD. The purpose of this study is to data mine for environmental chemicals which activate xenobiotic receptors and then validate this activation for selected chemicals. Methods: The EPA-ToxCast Phase I database (320 chemicals) was used to identify compounds associated with PXR activation. In vitro assays used for selection included NCGC nuclear receptor assays, Attagene transcription factor assays and CellzDirect transcription assays. Human (h) and murine (m) PXR and CAR activation were then assessed/validated for selected compounds identified from the EPA screening assays and for OCPs which were associated with NAFLD in our previous National Health and Nutritional Examination Survey study. HepG2 cells were transfected with plasmids expressing h or m PXR/CAR and receptor-responsive plasmids, pGL3-PXR/CAR-REluciferase. Transfected cells were exposed to h or m PXR/CAR ligands or varying concentrations of OCPs. Results: Nearly 2/3 of chemicals screened positive for potential effects on PXR.67 compounds including alachlor and chlorothalonil and 102 compounds including lindane were found to activate hPXR by NGCg and Attagene assays respectively. CellzDirect assay identified 202 compounds including alachlor and atrazine that were found to change CYP3A4 (PXR target gene) expression. Among identified chemicals initially selected for validation (n=9) in cell-based reporter assays, trans-nonachlor, chlordane, DDE, DDT, lindane and alachlor activated hPXR and mPXR. Dieldrin activated mPXR only. Dieldrin, trans-nonachlor, DDT, lindane and alachlor also activated hCAR but none of the compounds activated mCAR. Chlorothalonil may have been an antagonist for h/m PXR/CAR. Conclusion: Potential PXR effects were identified for nearly 2/3 of toxCast Phase I chemicals. PXR activation was confirmed for many relevant pesticides. Not all chemicals identified by the ToxCast screening assays proved to be xenobiotic receptor agonists. We postulate that the pesticides which activate PXR may contribute to NAFLD through the effect of this receptor on energy metabolism, and these effects may be different in humans and rodent models.


The following people have nothing to disclose: Laila Al-Eryani, Banrida Wahlang, Heather B. Clair, John J. Guardiola, Keith C. Falkner, Russell A. Prough, Matthew C. Cave


Liver X Receptor p controls Aquaporin-1 and Aquaporin-4 expression and a sex-specific, caveolin-dependent cellular localization of Aquaporin-4 in the hepatocytes

Chiara Gabbi1,2 Hyun-Jin Kim1, Nathan Sweed1, Margaret Warner1, Jan-Åke Gustafsson1, 2

1 University of Houston, Houston, TX; 2Karolinska Institutet, Stockholm, Sweden

Introduction: Aquaporins (AQPs) are a family of channel proteins that facilitate the osmotic movement of water and small solutes across biological membranes. In the hepatobiliary tract, AQPs participate in formation and flow of bile. We have previously shown that Liver X Receptor p (LXRp), a nuclear receptor activated by oxysterols, controls the expression of AQP-1 in pancreas and kidney. Aim and methods: In the present study we aimed to investigate the role of LXRβ in controlling water channel homeostasis in the hepatobiliary tract, in-vivo. Both male and female WT and LXRp-/- mice were studied at the age of 12 months. Results: In WT mice, LXRβ protein was strongly expressed in the nuclei of intrahepatic cholangiocytes while a weak expression was detected in hepatocytes. Male LXRβ-/mice demonstrated a significant increase of serum levels of both total bilirubin and alkaline phosphatase, indicating the presence of a mild cholestasis. In the whole liver, there was a significant reduction in the levels of both the mRNA of AQP-1 and AQP-4 in male LXRβ-/- mice. Interestingly, AQP-4 protein expression was detected on the plasma membrane of male WT hepatocytes while in LXRp-/- mice, the immunoreactivity was identified in the cytosol, suggesting a possible defect in its transport to the membrane. No AQP-4 was detected in female mice either WT or LXRβ-/-. In the kidney, AQP-2 trafficking is controlled by caveoIin-1, a major component of caveolae, that is involved in transcytosis, endocytosis and signal transduction. In prostate cancer cells, caveoIin-1 is upregulated by testosterone and acts as Androgen Receptor (AR) co-regulator being involved in non-genomic effects of AR. In LXRβ-/- male mice, strong immunoreactivity of caveoIin-1 was detected in the cytosol while in WT mice it was on the cell surface. Thus there is impaired translocation of caveoIin-1 in the mutant mice. Interestingly, in LXRp-/- male mice, serum testosterone levels were in normal range. Conclusion: LXRβ regulates the level of AQP-1 and AQP-4 mRNA as well as the cellular localization of AQP4, via caveoIin-1, in male mice.


Jan-Åke Gustafsson - Advisory Committees or Review Panels: BioNovo; Consulting: KaroBio AB

The following people have nothing to disclose: Chiara Gabbi, Hyun-Jin Kim, Nathan Sweed, Margaret Warner