- Email: [email protected]
280 MICRORNA-29: A NOVEL ANTIFIBROGENIC MEDIATOR IN LIVER FIBROGENESIS
S110
Poster Session − Thursday, April 23
278 HEPATITIS B REPLICATION INDUCES FIBROGENIC ACTIVATION OF HEPATIC STELLATE CELLS M. Demir, S. Schulte, G. Suckau, B. Jakob, T. Goeser, H.-M. Steffen, U. T¨ox. Klinik f¨ur Gastroenterologie und Hepatologie, Universit¨at zu K¨oln, K¨oln, Germany E-mail: [email protected] Background and Aims: The mechanisms by which hepatitis B virus (HBV) induces liver fibrosis are unknown. Our aim was to characterize the profibrogenic potential of HBV replication on hepatic stellate cells (HSCs) as fibrogenic effector cells. Methods: HBV-replicating HepG2.2.15 cells and Mock transfected Hep G2 cells (control) were cultivated for 7 and 14 days. HBV-Replication was controlled by measurement of HBV-DNA in the supernatants. Thereafter LX-2 cells (human HSC cell line) were incubated with conditioned media from HepG2 2.15 and HepG2 cells for 7 and 14 days. Expression of fibrosis-related genes in LX-2 cells was quantified by real-time polymerase chain reaction. Secretion of bioactive transforming growth factor-b1 (TGF-b1), the major fibrogenic cytokine, was assessed by ELISA. Results: LX-2 cells exposed to conditioned media from HepG2 2.15 cells vs. Mock transfected HepG2 cells showed an increased expression of key genes involved in liver fibrosis in a profibrogenic manner: there was an up to 25-fold up-regulation of mRNA levels of a-smooth muscle actin, angiotensin II receptor (type1), collagen I a 1, connective tissue growth factor, endothelin-1, matrix metalloproteinase-13, tissue inhibitor of metalloproteinase-1 and TGF-b1 compared to control (Mock), with rising mRNA levels from day 7 to day 14. TGF-b1 bioactivity was markedly increased in supernatants of HepG2 2.15 cells vs. Mock transfected HepG2 cells, also with increasing tendency from day 7 to day 14. Conclusions: HBV replication induces fibrogenic activation of hepatic stellate cells in vitro. HBV-induced liver fibrosis may be caused by a direct interaction between HBV proteins or virus particles and hepatic stellate cells. 279 BONE MORPHOGENETIC PROTEIN (BMP)-9: A NEW MEMBER OF THE TGF-b SUPERFAMILY WHICH IS SECRETED BY ACTIVATED HEPATIC STELLATE CELLS K. Breitkopf1 , A. M¨uller1 , L. Ciuclan1 , E. Wiercinska2 , P. Ten Dijke2 , S. Dooley1 . 1 Molecular Alcohol Research in Gastroenterology, Medical Clinic II, University Hospital at Mannheim, University of Heidelberg, Mannheim, Germany; 2 Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands E-mail: [email protected] Not much is known yet about BMP-9 and its potential relevance in liver pathologies. We therefore analyzed which liver cell types express BMP-9 and if they respond to it. The aim of these studies is to find hints about its function in the liver. As determined by real-time PCR, primary hepatic stellate cells (HSC) expressed increasing amounts of BMP-9 mRNA during early stages of in vitro transdifferentiation and conditioned medium from these cells mimicked effects of recombinant BMP-9, indicating that HSC secrete functional protein. Primary cultured hepatocytes (HC), in contrast, expressed if any only very low amounts of BMP-9 mRNA, but were strongly responsive to stimulation with rec. BMP-9 as determined by rapid and sustained Smad1 phosphorylation. Overexpression of the TGF-b type I receptor ALK-1 but not ALK-2 or −5 increased BMP-9 dependent Smad1 phosphorylation in HC, implying that ALK-1 represents its signalling receptor in HC. Unexpectedly, HSC did not show such responsiveness, although these cells express ALK1. To learn about possible functions of BMP-9 in the liver, micro-array analyses were performed with samples from HC ±BMP-9 stimulation. Target genes of BMP-9 in HC were, among others, strongly related to proliferation (diverse cyclins, Id1 and −3, Cdc20) and indeed BMP-9 induced proliferation of AML-12 hepatocytes. These findings and the down regulation of certain pro-apoptotic proteins (e.g. c-Src and NRADD) point to a role for BMP-9 during liver regeneration or
carcinoma development, where proliferation of HC is induced. In addition, BMP-9 induced expression of matrix proteins from the basal membrane (coll IV, laminin and fibronectin) and down-regulated certain proteins involved in coagulation (fibrinogens and factors II, X and XII). In summary, our results show that upon activation HSC rapidly secrete increasing amounts of BMP-9, which then induces Smad1-mediated signaling in HC thus affecting expression of genes mediating proliferation and/or fibrogenesis.
280 MICRORNA-29: A NOVEL ANTIFIBROGENIC MEDIATOR IN LIVER FIBROGENESIS M. Kwiecinski, I. Strack, A. Noetel, S. Schievenbusch, M. Scheffler, N. Elfimova, H.P. Dienes, M. Odenthal. Institute for Pathology, University Hospital of Cologne, Cologne, Germany E-mail: [email protected] Background: MicroRNAs (miRNA) are small noncoding RNAs that regulate gene expression by targeting mRNAs through translational repression or RNA degradation. Many fundamental biological processes, such as embryogenesis, carcinogenesis and cell differentiation, are modulated by miRNAs. In the present study, we investigated the role of the miRNA-29 family members in regulation of extracellular matrix production during liver fibrogenesis. Methods: In order to identify miRNA species involved in repression of collagen synthesis, the databases of Miranda, Targetscan and Pictar were screened. MiR-29a and miR-29b were analysed during in vitro induced myofibroblastic differentiation of primary hepatic stellate cells (HSC), in human biopsies of chronically HCV infected liver representing different fibrotic stages (S0-S4), and in experimental rat fibrosis after bile-duct ligation by real time PCR of total RNA extracted by the Trizol method. The 3 -untranslated regions (UTR) of collagen subunits 1A2, 4A1, and 4A5 or miR-29a/b complementary sequences were fused downstream to the luciferase gene of the psiCheck vector providing endogenous normalisation of the luciferase reporter activity. Ago-miR-29, mimicking miR-29, was transfected into HSC and collagen synthesis was determined by Western blot. Results: Searching different databases revealed putative miR-29 target sequences in transcripts of various collagen subunits but also e.g. in fibrillin and identified the members of the miR-29 family as probable regulators of extracellular matrix depositon. Analysis of miR-29a and miR-29b expression in chronically diseased liver demonstrated prominent downregulation of miR-29a and moderate downregulation of miR-29b upon experimental and human liver fibrosis. MiR-29 as repressors of collagen synthesis were proven by reporter assays of chimeric collagen 3 -UTR luciferase fusion constructs in HSC overexpressing miR-29 by ago-miR treatment. Additionally, in these miR-29 overexpressing cells, downregulation of collagen synthesis could also be shown by real-time PCR and Western blot analyses. Conclusion: Members of miR-29 family, repressed during liver fibrogenesis, are suggested to act as antifibrogenic mediators.
281 THE NEURONAL GUIDANCE CUE AND SURVIVAL FACTOR, NETRIN-1, IN LIVER FIBROGENESIS D. Lopez1 , M. Scheffler1 , F. Schulze1 , K. Wendland1 , S. Mesghenna2 , U. T¨ox2 , U. Drebber1 , D. Nierhoff2 , H.P. Dienes1 , M. Odenthal1 . 1 Institute for Pathology, University Hospital Cologne, 2 Department of Gastroenterology and Hepatology, University Cologne, Koeln, Germany E-mail: [email protected] Background: Netrin-1 has been discovered as a guidance cue for commissural axons via its receptor DCC (deleted in colorectal cancer) und the UNC5H receptor family. The netrin-1 receptors including UNC5H have been shown to be dependence receptors which induce apoptosis when devoid of their ligand. Accordingly, netrin-1 serves as a survival factor for different cell types. In the present study we investigated the expression of
Poster Session − Thursday, April 23
278 HEPATITIS B REPLICATION INDUCES FIBROGENIC ACTIVATION OF HEPATIC STELLATE CELLS M. Demir, S. Schulte, G. Suckau, B. Jakob, T. Goeser, H.-M. Steffen, U. T¨ox. Klinik f¨ur Gastroenterologie und Hepatologie, Universit¨at zu K¨oln, K¨oln, Germany E-mail: [email protected] Background and Aims: The mechanisms by which hepatitis B virus (HBV) induces liver fibrosis are unknown. Our aim was to characterize the profibrogenic potential of HBV replication on hepatic stellate cells (HSCs) as fibrogenic effector cells. Methods: HBV-replicating HepG2.2.15 cells and Mock transfected Hep G2 cells (control) were cultivated for 7 and 14 days. HBV-Replication was controlled by measurement of HBV-DNA in the supernatants. Thereafter LX-2 cells (human HSC cell line) were incubated with conditioned media from HepG2 2.15 and HepG2 cells for 7 and 14 days. Expression of fibrosis-related genes in LX-2 cells was quantified by real-time polymerase chain reaction. Secretion of bioactive transforming growth factor-b1 (TGF-b1), the major fibrogenic cytokine, was assessed by ELISA. Results: LX-2 cells exposed to conditioned media from HepG2 2.15 cells vs. Mock transfected HepG2 cells showed an increased expression of key genes involved in liver fibrosis in a profibrogenic manner: there was an up to 25-fold up-regulation of mRNA levels of a-smooth muscle actin, angiotensin II receptor (type1), collagen I a 1, connective tissue growth factor, endothelin-1, matrix metalloproteinase-13, tissue inhibitor of metalloproteinase-1 and TGF-b1 compared to control (Mock), with rising mRNA levels from day 7 to day 14. TGF-b1 bioactivity was markedly increased in supernatants of HepG2 2.15 cells vs. Mock transfected HepG2 cells, also with increasing tendency from day 7 to day 14. Conclusions: HBV replication induces fibrogenic activation of hepatic stellate cells in vitro. HBV-induced liver fibrosis may be caused by a direct interaction between HBV proteins or virus particles and hepatic stellate cells. 279 BONE MORPHOGENETIC PROTEIN (BMP)-9: A NEW MEMBER OF THE TGF-b SUPERFAMILY WHICH IS SECRETED BY ACTIVATED HEPATIC STELLATE CELLS K. Breitkopf1 , A. M¨uller1 , L. Ciuclan1 , E. Wiercinska2 , P. Ten Dijke2 , S. Dooley1 . 1 Molecular Alcohol Research in Gastroenterology, Medical Clinic II, University Hospital at Mannheim, University of Heidelberg, Mannheim, Germany; 2 Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands E-mail: [email protected] Not much is known yet about BMP-9 and its potential relevance in liver pathologies. We therefore analyzed which liver cell types express BMP-9 and if they respond to it. The aim of these studies is to find hints about its function in the liver. As determined by real-time PCR, primary hepatic stellate cells (HSC) expressed increasing amounts of BMP-9 mRNA during early stages of in vitro transdifferentiation and conditioned medium from these cells mimicked effects of recombinant BMP-9, indicating that HSC secrete functional protein. Primary cultured hepatocytes (HC), in contrast, expressed if any only very low amounts of BMP-9 mRNA, but were strongly responsive to stimulation with rec. BMP-9 as determined by rapid and sustained Smad1 phosphorylation. Overexpression of the TGF-b type I receptor ALK-1 but not ALK-2 or −5 increased BMP-9 dependent Smad1 phosphorylation in HC, implying that ALK-1 represents its signalling receptor in HC. Unexpectedly, HSC did not show such responsiveness, although these cells express ALK1. To learn about possible functions of BMP-9 in the liver, micro-array analyses were performed with samples from HC ±BMP-9 stimulation. Target genes of BMP-9 in HC were, among others, strongly related to proliferation (diverse cyclins, Id1 and −3, Cdc20) and indeed BMP-9 induced proliferation of AML-12 hepatocytes. These findings and the down regulation of certain pro-apoptotic proteins (e.g. c-Src and NRADD) point to a role for BMP-9 during liver regeneration or
carcinoma development, where proliferation of HC is induced. In addition, BMP-9 induced expression of matrix proteins from the basal membrane (coll IV, laminin and fibronectin) and down-regulated certain proteins involved in coagulation (fibrinogens and factors II, X and XII). In summary, our results show that upon activation HSC rapidly secrete increasing amounts of BMP-9, which then induces Smad1-mediated signaling in HC thus affecting expression of genes mediating proliferation and/or fibrogenesis.
280 MICRORNA-29: A NOVEL ANTIFIBROGENIC MEDIATOR IN LIVER FIBROGENESIS M. Kwiecinski, I. Strack, A. Noetel, S. Schievenbusch, M. Scheffler, N. Elfimova, H.P. Dienes, M. Odenthal. Institute for Pathology, University Hospital of Cologne, Cologne, Germany E-mail: [email protected] Background: MicroRNAs (miRNA) are small noncoding RNAs that regulate gene expression by targeting mRNAs through translational repression or RNA degradation. Many fundamental biological processes, such as embryogenesis, carcinogenesis and cell differentiation, are modulated by miRNAs. In the present study, we investigated the role of the miRNA-29 family members in regulation of extracellular matrix production during liver fibrogenesis. Methods: In order to identify miRNA species involved in repression of collagen synthesis, the databases of Miranda, Targetscan and Pictar were screened. MiR-29a and miR-29b were analysed during in vitro induced myofibroblastic differentiation of primary hepatic stellate cells (HSC), in human biopsies of chronically HCV infected liver representing different fibrotic stages (S0-S4), and in experimental rat fibrosis after bile-duct ligation by real time PCR of total RNA extracted by the Trizol method. The 3 -untranslated regions (UTR) of collagen subunits 1A2, 4A1, and 4A5 or miR-29a/b complementary sequences were fused downstream to the luciferase gene of the psiCheck vector providing endogenous normalisation of the luciferase reporter activity. Ago-miR-29, mimicking miR-29, was transfected into HSC and collagen synthesis was determined by Western blot. Results: Searching different databases revealed putative miR-29 target sequences in transcripts of various collagen subunits but also e.g. in fibrillin and identified the members of the miR-29 family as probable regulators of extracellular matrix depositon. Analysis of miR-29a and miR-29b expression in chronically diseased liver demonstrated prominent downregulation of miR-29a and moderate downregulation of miR-29b upon experimental and human liver fibrosis. MiR-29 as repressors of collagen synthesis were proven by reporter assays of chimeric collagen 3 -UTR luciferase fusion constructs in HSC overexpressing miR-29 by ago-miR treatment. Additionally, in these miR-29 overexpressing cells, downregulation of collagen synthesis could also be shown by real-time PCR and Western blot analyses. Conclusion: Members of miR-29 family, repressed during liver fibrogenesis, are suggested to act as antifibrogenic mediators.
281 THE NEURONAL GUIDANCE CUE AND SURVIVAL FACTOR, NETRIN-1, IN LIVER FIBROGENESIS D. Lopez1 , M. Scheffler1 , F. Schulze1 , K. Wendland1 , S. Mesghenna2 , U. T¨ox2 , U. Drebber1 , D. Nierhoff2 , H.P. Dienes1 , M. Odenthal1 . 1 Institute for Pathology, University Hospital Cologne, 2 Department of Gastroenterology and Hepatology, University Cologne, Koeln, Germany E-mail: [email protected] Background: Netrin-1 has been discovered as a guidance cue for commissural axons via its receptor DCC (deleted in colorectal cancer) und the UNC5H receptor family. The netrin-1 receptors including UNC5H have been shown to be dependence receptors which induce apoptosis when devoid of their ligand. Accordingly, netrin-1 serves as a survival factor for different cell types. In the present study we investigated the expression of