- Email: [email protected]
Keratin 23 Represents a Novel Liver Injury Marker Reflecting the Severity of Ductular Reaction
POSTER PRESENTATIONS THU-470 CHARACTERIZATION OF BILE ACID HOMEOSTASIS DURING LIVER REGENERATION UNDER NORMAL AND PATHOLOGICAL CONDITIONS M. Mueller1, S. Schultze1, N. Auer1, F. Pauler2, M. Trauner1. 1Hans Popper Laboratory of Molecular Hepatology, Division of Gastroenterology and Hepatology, Department of Internal Medicine III, Medical University of Vienna; 2CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria E-mail: [email protected] Background and Aims: The liver has unique regenerative capacities. However, patients undergoing extended liver resection may suffer from hepatic failure, if the remnant liver is too small to sustain organ function. This is known as the Small-for-size syndrome (SFSS), which is characterized by impaired liver regeneration. Molecular mechanisms to improve treatment remain to be elucidated. Partial hepatectomy ( pHX) results in a transient increase of serum BAs promoting liver regeneration via BA receptor (FXR) signaling. However, little is known about BA homeostasis during impaired liver regeneration after extended hepatectomy (eHX). We performed 86% eHX in mice to characterize the exposure of the liver remnant to BAs. Methods: 12 weeks old male C57BL/6 mice were subjected to 68% pHX and 86% eHX. We determined serum BA concentrations and expression of genes involved in BA homoestasis in liver samples after 68% pHX, 86% eHX and control mice was analyzed by RNA deep sequencing and qRT-PCR. Results: Mice showed decreased body weight to liver weight ratio ( p > 0.01) and reduced number of hepatocytes undergoing mitosis ( p > 0.05) at 48 h upon 86% eHX compared to 68/ pHX. Serum analysis revealed a significant elevation of serum BAs and alkaline phosphatase levels after 86% eHX (e.g. serum BAs, 269.8 ± 184.0 μmol/L vs 75.2 ± 56.6 μmol/L at 48 h after 86% eHX vs. 68% pHX, respectively; p < 0.05). Moreover, we found increased levels of serum urea at 24 h after 86% eHX suggesting impaired renal function due to high serum BA concentrations. Genes regulating BA synthesis and uptake were significantly downregulated after 86% eHX (e.g. Cyp7a1, p > 0.001; Cyp8b1, p > 0.005; at 48 h after 86% eHX vs. 68% pHX). Additionally, genes involved in BA detoxification and active biliary secretion, such as Cyp2b10, Gstm2, Mdr2 and Mrp4 were upregulated after 86% eHX (e.g. Mdr2, p > 0.05; at 48 h after 86% eHX vs. 68% pHX). Conclusions: Our data suggest that the remnant liver is exposed to a toxic BA overload, resulting in an adaptive response of genes involved in BA metabolism and transport. The BA overlaod may contribute to the inhibition of liver regeneration after 86% eHX. Currently, we are validating the expression of genes in BA homeostasis by qRT-PCR and Western blotting. Our future plan is to evaluate BA signaling via FXR and TGR5 after 86% eHX. This study will contribute to a better understanding of BA signaling during liver regeneration and may open up new revenues for therapeutic treatment of impaired liver regeneration. THU-471 CDK1 KNOCK OUT GIANT HEPATOCYTES INDUCE LIVER PROGENITOR CELLS N. Van Hul1, M. Caldez1, P. Kaldis1. 1Cell Division and Cancer Laboratory, Institute of Molecular and Cellular Biology, A*STAR, Singapore, Singapore E-mail: [email protected] Background and Aims: In order for cells to divide, cyclin-dependent protein kinases (Cdks) and associated cyclins are expressed in a wellorganized sequence coordinating the progression through the cell cycle. Lack of Cdk1, the major cell cycle regulator and the only essential Cdk, is embryonic lethal at a very early stage and Cdk1 conditional knockout mice are not viable. Previously, we have shown that Cdk1 is dispensable for liver regeneration as Cdk1 KO
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hepatocytes would grow in size instead of undergoing cell division upon 70% partial hepatectomy (PH). Methods: Livers from Cdk1flox/flox Alb Cre mice (or Cdk1LKO) were collected for immunohistochemistry and western blotting. 70% PH was performed and livers were retrieved at multiple time points post surgery. Results: Cdk1LKO are viable and survive to adulthood. Livers collected from 8 week-old Cdk1LKO mice show disrupted liver architecture by the presence of large, multinucleated and dysmorphic hepatocytes. These giant hepatocytes exhibit concomitant enlargement of cytoplasm, nuclei and nucleoli, suggesting a lack of size homeostasis causing an imbalanced cell growth. The enlarged nuclei remain positive for HNF4a, a marker of hepatocyte maturity and functionality, and show one large ring-shaped nucleolus as revealed by nucleolin immunostaining. CK19+ liver progenitor cells (LPC) are found to accumulate in the vicinity of these giant hepatocytes, accompanied by the deposition of collagen fibers as seen by sirius red staining. The bile ducts are not obviously affected, but are at times found far in the parenchyma, dissociated from their portal vein. Some Cdk1LKO livers also show a ductular reaction. Nodules of smaller, seemingly healthy hepatocytes are found in Cdk1LKO livers, appearing to compete with the giant hepatocytes to repopulate the liver. The source of these smaller hepatocytes is being investigated by lineage tracing studies. Livers collected from Cdk1LKO livers 8h after 70% PH, show less giant hepatocytes, reduced fibrosis and a decreased number of LPC, suggesting that the liver condition is improved compared to before PH. However, 36h post PH, the giant hepatocytes, fibrosis and inflammation reappeared. Conclusions: Deletion of Cdk1 blocks the proliferation of hepatocytes and causes cellular hypertrophy in a resting liver. The presence of liver progenitor cells indicates that the giant hepatocytes activate this rescue mechanism even without an external injury to the liver. The source of the smaller hepatocytes in the Cdk1LKO needs to be identified. THU-472 KERATIN 23 REPRESENTS A NOVEL LIVER INJURY MARKER REFLECTING THE SEVERITY OF DUCTULAR REACTION N. Guldiken1, G.K. Ensari2, P. Lahiri3, G. Couchy4, C. Preisinger5, C. Liedtke2, H.W. Zimmermann2, M. Ziol6, J. Zucman-Rossi4, C. Trautwein2, P. Strnad1. 1Department of Internal Medicine III and Interdisciplinary Center for Clinical Research (IZKF); 2Department of Internal Medicine III, RWTH University Hospital Aachen, Aachen, Germany; 3Institute of Pathology, Medical University of Graz, Graz, Austria; 4Génomique fonctionnelle des Tumeurs solides, Equipe Labellisée Ligue Contre le Cancer, Inserm, UMR-1162, Paris, France; 5 Interdisciplinary Center for Clinical Research (IZKF), RWTH University Hospital Aachen, Aachen, Germany; 6Centre de Ressources Biologiques, Hôpital Jean Verdier, Bondy, France E-mail: [email protected] Background and Aims: >Keratins (K) are the intermediate filaments of epithelial cells and constitute established diagnostic tools. In the liver, K7/K19 expression is restricted to hepatic progenitor cells (HPCs), intermediate (i.e. not fully differentiated) hepatocytes and biliary epithelial cells (BECs). Consequently, K7/K19 represent a widely used marker of the regenerative liver response termed ductular reaction (DR) that consists of activated BECs and HPCs. Since K23 is a largely unknown keratin family member, we analysed its expression and localization in selected human liver disorders and mouse liver injury models using custom-made antibodies. Methods: For expression analysis, >200 liver biopsies/surgical specimens were studies. Serum K23 levels were measured via dot blot analysis. K23 regulation in response to TWEAK and IL1b was studied in hepatocellular and bile duct carcinoma cell lines.
Journal of Hepatology 2016 vol. 64 | S213–S424
POSTER PRESENTATIONS Results: In untreated mice, K23 was found in biliary epithelia but not hepatocytes. It was (together with K7/K19) markedly upregulated in three different DR/cholestatic injury models, i.e. four months old MDR2 knockouts, animals treated with -diethoxycarbonyl-1,4-dihydrocollidine for four weeks or subjected to bile duct ligation for five days. No changes in K23 levels were seen in hepatocellular injury models such as partial hepatectomy or carbon tetrachloride-induced fibrogenesis. K23 levels correlated with the DR marker Fn14 and immunofluorescence staining showed a distinct but not perfect co-localization with K7/K19. In primary hepatocytes as well as hepatobiliary cell lines, treatment with TWEAK, and the type I acute phase inducer IL-1β but not the type II inducer IL-6 elevated K23 expression. In humans, only moderate K23 up-regulation was seen in most chronic liver diseases. Among them, K23 expression was higher in patients with more prominent inflammation/fibrosis. A dramatic increase (>200 times) was observed in patients with ALF and end-stage PBC. In ALF/PBC, K7/K23 positive, K19 negative cells corresponding to intermediate hepatocytes were frequently found. K23 serum levels were significantly higher in patients with alcoholic liver cirrhosis compared to control subjects. Conclusions: K23 represents a novel, stress inducible DR marker and its levels correlate with the severity of the liver disease. Given its release into the serum and its rather specific expression pattern, it may represent an attractive non-invasive marker of liver injury/ regeneration. THU-473 OSTEOPONTIN: A KEY REGULATOR OF LIVER METABOLISM DURING REGENERATION AFTER PARTIAL HEPATECTOMY M. Nuñez-García1, B. Gomez-Santos1, X. Buqué1,2, J.L. GarcíaRodriguez1, M.R. Romero3,4, J.J.G. Marín3,4, B. Arteta5, C. GarcíaMonzón6,7, W. Syn8,9, O. Fresnedo1, P. Aspichueta1,2. 1Department of Physiology, University of Basque Country UPV/EHU, Leioa; 2Biocruces Research Institute, Barakaldo; 3Experimental Hepatology and Drug Targeting (HEVEFARM), IBSAL, University of Salamanca, Salamanca; 4 Centro de Investigación Biomédica en Red para el Estudio de Enfermedades Hepáticas y Digestivas, (CIBERehd); 5Department of Biology and Histology, University of Basque Country UPV/EHU, Leioa; 6 Liver Research Unit, University Hospital Santa Cristina, Instituto de investigación Sanitaria Princesa, Madrid; 7Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas, (CIBERehd), Spain; 8Regeneration and Repair, Institute of Hepatology, Foundation for Liver Research; 9Transplantation Immunology and Mucosal Biology, Kings College, London, United Kingdom E-mail: [email protected] Background and Aims: The liver plays a central role in whole body metabolic homeostasis and has capacity for regeneration after cellular damage or surgery resection. During regeneration different metabolic networks turn on. Osteopontin (OPN), a multifunctional cytokine, plays a role in liver inflammation and its deficiency delays initiation of liver regeneration. Here we have evaluated the involvement of OPN in the regulation of liver metabolic homeostasis and in the response to the increased metabolic demands of the liver after partial hepatectomy (PH). Methods: Metabolic pathways were analyzed by lipidomic and radiometric techniques in hepatocytes and total livers from OPN-KO mice and WT littermates and from WT mice that received recombinant (r)OPN. PH was performed in OPN-KO mice, OPN-KO mice treated with intragastric atorvastatin for two weeks and WT littermates. Liver biopsies taken during programmed laparoscopic cholecystectomy and matched serum samples from non-obese subjects with non-alcoholic fatty liver (NAFL) (n = 13) or with normal liver (NL) (n = 13) were analyzed. Results: OPN-KO mice exhibited reduced de novo lipogenesis in hepatocytes and increased phosphorylation of liver acetyl-CoA carboxylase which was decreased in WT mice treated with rOPN.
OPN deficiency decreased liver phosphatidylcholine (PC) content while rOPN increased it in WT mice without affecting triglyceride metabolism. rOPN reduced liver expression of CYP7A1 protein, which was increased in OPN-KO mice liver, in which mdr2 mRNA levels were also increased when compared to WT mice. The increased conversion of cholesterol into bile acids was associated with increased de novo cholesterol synthesis. All these metabolic changes in OPN-KO mice liver led to a complete altered liver lipidoma and a lower modulation of it during initiation of regeneration. Inhibition of hepatic de novo cholesterol synthesis by atorvastatin treatment reprogrammed hepatic lipidoma increasing PC concentration in the liver and accelerating the initiation of regeneration in OPN-KO mice. Finally, serum OPN increases in non-obese patients with NAFL, in which CYP7A1 is decreased when compared to those with NL. Serum OPN correlates with liver PC concentration and also with liver cholesterol concentration only in NAFL patients. Conclusions: OPN is a key liver metabolic regulator that controls metabolic response during initiation of regeneration. The results suggest that OPN could be a serum biomarker of liver metabolic deregulation in non-obese patients with NAFL. THU-475 EGR1 MEDIATES INFLAMMATION AND REGENERATION IN CHOLESTATIC LIVER DISEASES S. Ibrahim1, R. Dayoub1, M. Melter1, T. Weiss1. 1Children Hospital University regensburg, University Hospital Regensburg, Regensburg, Germany E-mail: [email protected] Background and Aims: Cholestatic liver diseases result mainly when the excretion of bile from the liver is blocked. It has been shown that inflammation contributes to liver injury in cholestasis and previous studies indicated the role of bile acids in the up-regulation of early growth response fator-1 (EGR1) through activating MAPK signaling pathway. EGR1 is responsible for the initiation of the inflammation through up-regulation of several pro-inflammatory genes. Augmenter of liver regeneration (ALR), a hepatotrophic factor with anti-oxidative and anti-apoptotic properties, has been found to support the process of liver regeneration in human liver. Nevertheless, little is known about the impact of ALR in cholestasis but interestingly EGR1 has several binding sites within ALR promoter. Therefore, the aim of our study is to investigate the potential role of bile acids (GCDCA, GCA, DCA, CDCA) and EGR1 in regulating ALR expression, and to determine a dual role of EGR1 for initiating inflammation and regenerative process mediated by ALR. Methods: Promoter studies of ALR gene to determine potential binding sites for transcription factors and reporter-gene assays, using two different constructs of ALR promoter cloned into pGL2 vector, were performed. Electrophoretic mobility shift assay (EMSA) was performed to investigate the responsiveness of ALR promoter to bile acids and/or EGR1. Later, hepatoma cell lines (HepG2 and Huh7) were treated with different bile acids (100 μM) for 24 hours to evaluate ALR expression using Western blot technique. Results: Promoter studies revealed potential response elements for EGR1 and luciferase assays showed that treatment of HepG2 and Huh7 with different bile acids or transfection with EGR1 is able to enhance the activity of ALR promoter compared to control. Furthermore, we found increased levels of ALR protein in the bile acid-treated hepatoma cells and EGR1-overexpressing HepG2 cells compared to the control. In addition, EMSA was performed using nuclear extracts from hepatoma cells treated with bile acids or transfected with EGR1. We later analyzed the binding of nuclear extract to the EGR1 binding site and demonstrated specificity of EGR1 binding. Conclusions: Our data indicate that EGR1 induced by bile acids not only initiates inflammation but also regulates ALR expression as a marker of activated liver regeneration. More work is required to explore the impact of bile acids on the regulation of ALR expression.
Journal of Hepatology 2016 vol. 64 | S213–S424
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hepatocytes would grow in size instead of undergoing cell division upon 70% partial hepatectomy (PH). Methods: Livers from Cdk1flox/flox Alb Cre mice (or Cdk1LKO) were collected for immunohistochemistry and western blotting. 70% PH was performed and livers were retrieved at multiple time points post surgery. Results: Cdk1LKO are viable and survive to adulthood. Livers collected from 8 week-old Cdk1LKO mice show disrupted liver architecture by the presence of large, multinucleated and dysmorphic hepatocytes. These giant hepatocytes exhibit concomitant enlargement of cytoplasm, nuclei and nucleoli, suggesting a lack of size homeostasis causing an imbalanced cell growth. The enlarged nuclei remain positive for HNF4a, a marker of hepatocyte maturity and functionality, and show one large ring-shaped nucleolus as revealed by nucleolin immunostaining. CK19+ liver progenitor cells (LPC) are found to accumulate in the vicinity of these giant hepatocytes, accompanied by the deposition of collagen fibers as seen by sirius red staining. The bile ducts are not obviously affected, but are at times found far in the parenchyma, dissociated from their portal vein. Some Cdk1LKO livers also show a ductular reaction. Nodules of smaller, seemingly healthy hepatocytes are found in Cdk1LKO livers, appearing to compete with the giant hepatocytes to repopulate the liver. The source of these smaller hepatocytes is being investigated by lineage tracing studies. Livers collected from Cdk1LKO livers 8h after 70% PH, show less giant hepatocytes, reduced fibrosis and a decreased number of LPC, suggesting that the liver condition is improved compared to before PH. However, 36h post PH, the giant hepatocytes, fibrosis and inflammation reappeared. Conclusions: Deletion of Cdk1 blocks the proliferation of hepatocytes and causes cellular hypertrophy in a resting liver. The presence of liver progenitor cells indicates that the giant hepatocytes activate this rescue mechanism even without an external injury to the liver. The source of the smaller hepatocytes in the Cdk1LKO needs to be identified. THU-472 KERATIN 23 REPRESENTS A NOVEL LIVER INJURY MARKER REFLECTING THE SEVERITY OF DUCTULAR REACTION N. Guldiken1, G.K. Ensari2, P. Lahiri3, G. Couchy4, C. Preisinger5, C. Liedtke2, H.W. Zimmermann2, M. Ziol6, J. Zucman-Rossi4, C. Trautwein2, P. Strnad1. 1Department of Internal Medicine III and Interdisciplinary Center for Clinical Research (IZKF); 2Department of Internal Medicine III, RWTH University Hospital Aachen, Aachen, Germany; 3Institute of Pathology, Medical University of Graz, Graz, Austria; 4Génomique fonctionnelle des Tumeurs solides, Equipe Labellisée Ligue Contre le Cancer, Inserm, UMR-1162, Paris, France; 5 Interdisciplinary Center for Clinical Research (IZKF), RWTH University Hospital Aachen, Aachen, Germany; 6Centre de Ressources Biologiques, Hôpital Jean Verdier, Bondy, France E-mail: [email protected] Background and Aims: >Keratins (K) are the intermediate filaments of epithelial cells and constitute established diagnostic tools. In the liver, K7/K19 expression is restricted to hepatic progenitor cells (HPCs), intermediate (i.e. not fully differentiated) hepatocytes and biliary epithelial cells (BECs). Consequently, K7/K19 represent a widely used marker of the regenerative liver response termed ductular reaction (DR) that consists of activated BECs and HPCs. Since K23 is a largely unknown keratin family member, we analysed its expression and localization in selected human liver disorders and mouse liver injury models using custom-made antibodies. Methods: For expression analysis, >200 liver biopsies/surgical specimens were studies. Serum K23 levels were measured via dot blot analysis. K23 regulation in response to TWEAK and IL1b was studied in hepatocellular and bile duct carcinoma cell lines.
Journal of Hepatology 2016 vol. 64 | S213–S424
POSTER PRESENTATIONS Results: In untreated mice, K23 was found in biliary epithelia but not hepatocytes. It was (together with K7/K19) markedly upregulated in three different DR/cholestatic injury models, i.e. four months old MDR2 knockouts, animals treated with -diethoxycarbonyl-1,4-dihydrocollidine for four weeks or subjected to bile duct ligation for five days. No changes in K23 levels were seen in hepatocellular injury models such as partial hepatectomy or carbon tetrachloride-induced fibrogenesis. K23 levels correlated with the DR marker Fn14 and immunofluorescence staining showed a distinct but not perfect co-localization with K7/K19. In primary hepatocytes as well as hepatobiliary cell lines, treatment with TWEAK, and the type I acute phase inducer IL-1β but not the type II inducer IL-6 elevated K23 expression. In humans, only moderate K23 up-regulation was seen in most chronic liver diseases. Among them, K23 expression was higher in patients with more prominent inflammation/fibrosis. A dramatic increase (>200 times) was observed in patients with ALF and end-stage PBC. In ALF/PBC, K7/K23 positive, K19 negative cells corresponding to intermediate hepatocytes were frequently found. K23 serum levels were significantly higher in patients with alcoholic liver cirrhosis compared to control subjects. Conclusions: K23 represents a novel, stress inducible DR marker and its levels correlate with the severity of the liver disease. Given its release into the serum and its rather specific expression pattern, it may represent an attractive non-invasive marker of liver injury/ regeneration. THU-473 OSTEOPONTIN: A KEY REGULATOR OF LIVER METABOLISM DURING REGENERATION AFTER PARTIAL HEPATECTOMY M. Nuñez-García1, B. Gomez-Santos1, X. Buqué1,2, J.L. GarcíaRodriguez1, M.R. Romero3,4, J.J.G. Marín3,4, B. Arteta5, C. GarcíaMonzón6,7, W. Syn8,9, O. Fresnedo1, P. Aspichueta1,2. 1Department of Physiology, University of Basque Country UPV/EHU, Leioa; 2Biocruces Research Institute, Barakaldo; 3Experimental Hepatology and Drug Targeting (HEVEFARM), IBSAL, University of Salamanca, Salamanca; 4 Centro de Investigación Biomédica en Red para el Estudio de Enfermedades Hepáticas y Digestivas, (CIBERehd); 5Department of Biology and Histology, University of Basque Country UPV/EHU, Leioa; 6 Liver Research Unit, University Hospital Santa Cristina, Instituto de investigación Sanitaria Princesa, Madrid; 7Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas, (CIBERehd), Spain; 8Regeneration and Repair, Institute of Hepatology, Foundation for Liver Research; 9Transplantation Immunology and Mucosal Biology, Kings College, London, United Kingdom E-mail: [email protected] Background and Aims: The liver plays a central role in whole body metabolic homeostasis and has capacity for regeneration after cellular damage or surgery resection. During regeneration different metabolic networks turn on. Osteopontin (OPN), a multifunctional cytokine, plays a role in liver inflammation and its deficiency delays initiation of liver regeneration. Here we have evaluated the involvement of OPN in the regulation of liver metabolic homeostasis and in the response to the increased metabolic demands of the liver after partial hepatectomy (PH). Methods: Metabolic pathways were analyzed by lipidomic and radiometric techniques in hepatocytes and total livers from OPN-KO mice and WT littermates and from WT mice that received recombinant (r)OPN. PH was performed in OPN-KO mice, OPN-KO mice treated with intragastric atorvastatin for two weeks and WT littermates. Liver biopsies taken during programmed laparoscopic cholecystectomy and matched serum samples from non-obese subjects with non-alcoholic fatty liver (NAFL) (n = 13) or with normal liver (NL) (n = 13) were analyzed. Results: OPN-KO mice exhibited reduced de novo lipogenesis in hepatocytes and increased phosphorylation of liver acetyl-CoA carboxylase which was decreased in WT mice treated with rOPN.
OPN deficiency decreased liver phosphatidylcholine (PC) content while rOPN increased it in WT mice without affecting triglyceride metabolism. rOPN reduced liver expression of CYP7A1 protein, which was increased in OPN-KO mice liver, in which mdr2 mRNA levels were also increased when compared to WT mice. The increased conversion of cholesterol into bile acids was associated with increased de novo cholesterol synthesis. All these metabolic changes in OPN-KO mice liver led to a complete altered liver lipidoma and a lower modulation of it during initiation of regeneration. Inhibition of hepatic de novo cholesterol synthesis by atorvastatin treatment reprogrammed hepatic lipidoma increasing PC concentration in the liver and accelerating the initiation of regeneration in OPN-KO mice. Finally, serum OPN increases in non-obese patients with NAFL, in which CYP7A1 is decreased when compared to those with NL. Serum OPN correlates with liver PC concentration and also with liver cholesterol concentration only in NAFL patients. Conclusions: OPN is a key liver metabolic regulator that controls metabolic response during initiation of regeneration. The results suggest that OPN could be a serum biomarker of liver metabolic deregulation in non-obese patients with NAFL. THU-475 EGR1 MEDIATES INFLAMMATION AND REGENERATION IN CHOLESTATIC LIVER DISEASES S. Ibrahim1, R. Dayoub1, M. Melter1, T. Weiss1. 1Children Hospital University regensburg, University Hospital Regensburg, Regensburg, Germany E-mail: [email protected] Background and Aims: Cholestatic liver diseases result mainly when the excretion of bile from the liver is blocked. It has been shown that inflammation contributes to liver injury in cholestasis and previous studies indicated the role of bile acids in the up-regulation of early growth response fator-1 (EGR1) through activating MAPK signaling pathway. EGR1 is responsible for the initiation of the inflammation through up-regulation of several pro-inflammatory genes. Augmenter of liver regeneration (ALR), a hepatotrophic factor with anti-oxidative and anti-apoptotic properties, has been found to support the process of liver regeneration in human liver. Nevertheless, little is known about the impact of ALR in cholestasis but interestingly EGR1 has several binding sites within ALR promoter. Therefore, the aim of our study is to investigate the potential role of bile acids (GCDCA, GCA, DCA, CDCA) and EGR1 in regulating ALR expression, and to determine a dual role of EGR1 for initiating inflammation and regenerative process mediated by ALR. Methods: Promoter studies of ALR gene to determine potential binding sites for transcription factors and reporter-gene assays, using two different constructs of ALR promoter cloned into pGL2 vector, were performed. Electrophoretic mobility shift assay (EMSA) was performed to investigate the responsiveness of ALR promoter to bile acids and/or EGR1. Later, hepatoma cell lines (HepG2 and Huh7) were treated with different bile acids (100 μM) for 24 hours to evaluate ALR expression using Western blot technique. Results: Promoter studies revealed potential response elements for EGR1 and luciferase assays showed that treatment of HepG2 and Huh7 with different bile acids or transfection with EGR1 is able to enhance the activity of ALR promoter compared to control. Furthermore, we found increased levels of ALR protein in the bile acid-treated hepatoma cells and EGR1-overexpressing HepG2 cells compared to the control. In addition, EMSA was performed using nuclear extracts from hepatoma cells treated with bile acids or transfected with EGR1. We later analyzed the binding of nuclear extract to the EGR1 binding site and demonstrated specificity of EGR1 binding. Conclusions: Our data indicate that EGR1 induced by bile acids not only initiates inflammation but also regulates ALR expression as a marker of activated liver regeneration. More work is required to explore the impact of bile acids on the regulation of ALR expression.
Journal of Hepatology 2016 vol. 64 | S213–S424
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