- Email: [email protected]
Autosomal dominant reticulo-endothelial iron overload associated with a three base pair deletion in the ferroportin 1 gene
182
Tuesday, April 16, 2002
used all-trans-retinoic acid (ATRA) to examine the expression of MICA on human hepatoma cells as well as their susceptibility to NK cells. Results: The expression of MICA were responsible for NK-mediated cytotoxicity of hepatoma cells. ATRA-treated hepatoma cells conferred increased production of IFN gamma as well as enhanced ability of cytolysis upon NK cells, which was dependent on up-regulation of MICA. Conclusion: These findings suggested that MICA on human hepatoma cells may play an pivotal role in NK cell activity, and raise the possibility that administration of retinoid may be useful for augmenting an innate immunity against HCC.
Parallel Session 4: Molecular Hepatology, Regeneration and Cancer ~-]
AUTOSOMAL DOMINANT RETICULO-ENDOTHELIAL IRON OVERLOAD ASSOCIATEO WITH A THREE BASE PAIR DELETION IN THE FERROPORTIN 1 GENE
reactive oxygen species (ROS) in vascular smooth muscle cell (VSMC). Here, we report a novel mechanism by which NAC blocks PDGF-induced signaling pathways in stellate cell (HSC). Methods: HSC and VSMC were isolated from male Wistar rats. Liver fibrosis was induced in rats by thioacetoamide (TAA) or bile duct ligation. NAC was administered at 100 mg/body every day orally and i.p. Results: NAC inhibited the PDGF-BB-induced proliferation of HSC, and the activation of Akt/PKB and MAP kinase. Interestingly this was caused by the downregulation of PDGFR itself. Thiol protease inhibitors, such as E64, attenuated the NAC effect. In vitro degradation assay showed that cathepsin B mainly contributes to degradation of PDGFR-b and reducing agent plays a role for activation of cathepsin B. Moreover, E64-c, membrane-impermeable analogue, had a similar effect to E64. HSC secreted both pre- and mature-cathepsin B, suggesting that PDGFR proteolysis happened extracellularly. In liver fibrosis models, NAC dramatically attenuated the progress of liver fibrosis. Conclusion: In culture, NAC blocked PDGF-dependent proliferation and signal transduction of HSC through the degradation of PDGFR protein assisted by secreted cathepsin B from HSC. NAC inhibited the progress of TAA-induced hepatic fibrosis.
~-]
V. Devalia I , K. Carter 2, A.P. Walker 3, S.J. Perkins 4, M. Worwood 2, A. May 2, J.S. Dooley 3. 1Departmentof Haematology, Princess of Wales
Hospital, Bridgend; 2Departmentof Haematology, University of Wales College of Medicine, Cardiff; 3Centrefor Hepatology, Department of Medicine, Royal Free and University CollegeMedical School, University College London, Royal Free Campus, London; 4Departmentof Biochemistry and Molecular Biology, Royal Free and University College Medical School, University CollegeLondon, Royal Free Campus, London, UK The majority of UK patients with genetic haemochromatosis have autosomal recessive inheritance, parenchymal iron overload and are homozygous for the C282Y mutation of the HFE gene. We have studied a family with autosomal dominant inheritance of reticuloendothelial (RE) cell iron overload. Affected individuals had an elevated serum ferritin concentration but normal transferrin saturation. Liver biopsy in affected adults showed increased iron deposition predominantly in Kupffer cells, without fibrosis. Patients did not have any of the clinical features associated with classical HFE-related haemochromatosis. Treatment by venesection was not well tolerated in the two patients treated, with anemia occurring early. None of the family had C282Y or H63D mutations in the HFE gene. In one affected adult, studies showed normal coding sequence and splice sites for both HFE and beta-2-microglobulin genes. Analysis of the ferroportin 1 gene showed a 3 base pair deletion in exon 5. This was present in affected but not in 100 control subjects. The predicted consequence of this mutation is the loss of one of three valine residues in the extracellular loop between the third and fourth transmembrane helices. This site lies within a spatial cluster of the other known ferroportin 1 mutations. In summary, mutation of the ferroportin 1 gene was associated with RE cell iron accumulation in this family. Structural predictions suggest that this extracellular cluster is functionally important for the transport of iron from RE cells.
~-~
REGULATION OF STELLATE CELL GROWTH BY REDOX-MEDIATED EXTRACELLULAR PROTEOLYSIS OF PLATELET-DERIVED GROWTH FACTOR RECEPTOR-B
Hiroaki Okuyama 1, Yasuyuki Shimahara 1, Norifumi Kawada 2, Yoshio Yamaoka I . 1Departmentof Gastroenterological Surgery, Kyoto
University, kyoto; 2Departmentof Hepatology, Osaka City University, Osaka, Japan Introduction: Reducing agents, such as N-acetylcysteine (NAC), are known to regulate growth-related signal transduction through scavenging
INHIBITION MECHANISMS OF CYP 3A4, 2B6 AND 2C9 EXPRESSION BY INTERLEUKINE 1 AND 6 IN HUMAN HEPATOCYTES
Eric Assenat I , Sabine Gerbal 2, Made Jose Vilarem 2, Dominique Larrey ~.
1CHUMontpellier; 2INSERM U128 Montpellier, France During the acute phase of inflammation, a marked lowering of CYP450 expression, induced by cytokines (IL 1 and IL6) has previously been shown; But the mechanisms leading to gene repression are still unknown. Two nuclear receptors, the Constitutively Activated Receptor (CAR) and the Pregnane X Receptor (PXR) have been found to play a key role in the induction of the CYP P450. We have shown that the expression of these receptors is under the control of the glucocorticoid pathway. We have, then, studied the effects of cytokines (ILl, IL6) on the glucocorticoid pathway and consequences on CAR, PXR and CYPs expression. We have found that ILl and IL6 are responsible for the inhibition of the expression of PXR and CAR mRNAs. Also, GR mRNA level was not affected while its activation (measured as Tyrosine Amino Transferase TAT-mRNA) was dramatically reduced by ILl and IL6 treatment. This inhibition was found for both the basal and induced levels of the CYPs. Kinetic studies showing a rapid but reversible inhibition of TAT, CAR and PXR mRNA levels. Lastly, in human hepatocytes, ILl induced a rapid and transient activation of the NFkB pathway measured by the nuclear translocation of the p65 sub-unit, leading to a physical interaction with the GR as assessed by immuno-precipitation experiment. These results suggest that the inhibition of the CYP450 in response to ILl treatment could be due to an activation of the NFkB pathway leading to a direct repression of the GR transcriptional activity on its target genes.
[6~
ANALYSES OF JAGGED1 AND NOTCH1 PROTEINS IN HEPATOCYTE CULTURES AND LIVER REGENERATION
Christoph Koehler, William C. Bowen, Peter Pediaditakis, Satdarshan P.S. Monga, Jens Walldorf, Steven C. Strom, George K. Michalopoulos.
Department of Pathology, University of Pittsburgh, Medical School, Pittsburgh, PA, USA The Jagged/Notch signaling pathway controls cell fate determination and differentiation, and its dysfunction is associated with human pathologies, including Alagille syndrome. To determine the putative role of the transmembrane receptor Notch and its ligand Jagged in cell-cell interaction and proliferation of hepatocytes, we analyzed Jaggedl and Notchl proteins in the primary cultures of rat and human hepatocytes and after partial hepatectomy of rat liver. Human hepatocytes and rat hepatocytes were cultured in
Tuesday, April 16, 2002
used all-trans-retinoic acid (ATRA) to examine the expression of MICA on human hepatoma cells as well as their susceptibility to NK cells. Results: The expression of MICA were responsible for NK-mediated cytotoxicity of hepatoma cells. ATRA-treated hepatoma cells conferred increased production of IFN gamma as well as enhanced ability of cytolysis upon NK cells, which was dependent on up-regulation of MICA. Conclusion: These findings suggested that MICA on human hepatoma cells may play an pivotal role in NK cell activity, and raise the possibility that administration of retinoid may be useful for augmenting an innate immunity against HCC.
Parallel Session 4: Molecular Hepatology, Regeneration and Cancer ~-]
AUTOSOMAL DOMINANT RETICULO-ENDOTHELIAL IRON OVERLOAD ASSOCIATEO WITH A THREE BASE PAIR DELETION IN THE FERROPORTIN 1 GENE
reactive oxygen species (ROS) in vascular smooth muscle cell (VSMC). Here, we report a novel mechanism by which NAC blocks PDGF-induced signaling pathways in stellate cell (HSC). Methods: HSC and VSMC were isolated from male Wistar rats. Liver fibrosis was induced in rats by thioacetoamide (TAA) or bile duct ligation. NAC was administered at 100 mg/body every day orally and i.p. Results: NAC inhibited the PDGF-BB-induced proliferation of HSC, and the activation of Akt/PKB and MAP kinase. Interestingly this was caused by the downregulation of PDGFR itself. Thiol protease inhibitors, such as E64, attenuated the NAC effect. In vitro degradation assay showed that cathepsin B mainly contributes to degradation of PDGFR-b and reducing agent plays a role for activation of cathepsin B. Moreover, E64-c, membrane-impermeable analogue, had a similar effect to E64. HSC secreted both pre- and mature-cathepsin B, suggesting that PDGFR proteolysis happened extracellularly. In liver fibrosis models, NAC dramatically attenuated the progress of liver fibrosis. Conclusion: In culture, NAC blocked PDGF-dependent proliferation and signal transduction of HSC through the degradation of PDGFR protein assisted by secreted cathepsin B from HSC. NAC inhibited the progress of TAA-induced hepatic fibrosis.
~-]
V. Devalia I , K. Carter 2, A.P. Walker 3, S.J. Perkins 4, M. Worwood 2, A. May 2, J.S. Dooley 3. 1Departmentof Haematology, Princess of Wales
Hospital, Bridgend; 2Departmentof Haematology, University of Wales College of Medicine, Cardiff; 3Centrefor Hepatology, Department of Medicine, Royal Free and University CollegeMedical School, University College London, Royal Free Campus, London; 4Departmentof Biochemistry and Molecular Biology, Royal Free and University College Medical School, University CollegeLondon, Royal Free Campus, London, UK The majority of UK patients with genetic haemochromatosis have autosomal recessive inheritance, parenchymal iron overload and are homozygous for the C282Y mutation of the HFE gene. We have studied a family with autosomal dominant inheritance of reticuloendothelial (RE) cell iron overload. Affected individuals had an elevated serum ferritin concentration but normal transferrin saturation. Liver biopsy in affected adults showed increased iron deposition predominantly in Kupffer cells, without fibrosis. Patients did not have any of the clinical features associated with classical HFE-related haemochromatosis. Treatment by venesection was not well tolerated in the two patients treated, with anemia occurring early. None of the family had C282Y or H63D mutations in the HFE gene. In one affected adult, studies showed normal coding sequence and splice sites for both HFE and beta-2-microglobulin genes. Analysis of the ferroportin 1 gene showed a 3 base pair deletion in exon 5. This was present in affected but not in 100 control subjects. The predicted consequence of this mutation is the loss of one of three valine residues in the extracellular loop between the third and fourth transmembrane helices. This site lies within a spatial cluster of the other known ferroportin 1 mutations. In summary, mutation of the ferroportin 1 gene was associated with RE cell iron accumulation in this family. Structural predictions suggest that this extracellular cluster is functionally important for the transport of iron from RE cells.
~-~
REGULATION OF STELLATE CELL GROWTH BY REDOX-MEDIATED EXTRACELLULAR PROTEOLYSIS OF PLATELET-DERIVED GROWTH FACTOR RECEPTOR-B
Hiroaki Okuyama 1, Yasuyuki Shimahara 1, Norifumi Kawada 2, Yoshio Yamaoka I . 1Departmentof Gastroenterological Surgery, Kyoto
University, kyoto; 2Departmentof Hepatology, Osaka City University, Osaka, Japan Introduction: Reducing agents, such as N-acetylcysteine (NAC), are known to regulate growth-related signal transduction through scavenging
INHIBITION MECHANISMS OF CYP 3A4, 2B6 AND 2C9 EXPRESSION BY INTERLEUKINE 1 AND 6 IN HUMAN HEPATOCYTES
Eric Assenat I , Sabine Gerbal 2, Made Jose Vilarem 2, Dominique Larrey ~.
1CHUMontpellier; 2INSERM U128 Montpellier, France During the acute phase of inflammation, a marked lowering of CYP450 expression, induced by cytokines (IL 1 and IL6) has previously been shown; But the mechanisms leading to gene repression are still unknown. Two nuclear receptors, the Constitutively Activated Receptor (CAR) and the Pregnane X Receptor (PXR) have been found to play a key role in the induction of the CYP P450. We have shown that the expression of these receptors is under the control of the glucocorticoid pathway. We have, then, studied the effects of cytokines (ILl, IL6) on the glucocorticoid pathway and consequences on CAR, PXR and CYPs expression. We have found that ILl and IL6 are responsible for the inhibition of the expression of PXR and CAR mRNAs. Also, GR mRNA level was not affected while its activation (measured as Tyrosine Amino Transferase TAT-mRNA) was dramatically reduced by ILl and IL6 treatment. This inhibition was found for both the basal and induced levels of the CYPs. Kinetic studies showing a rapid but reversible inhibition of TAT, CAR and PXR mRNA levels. Lastly, in human hepatocytes, ILl induced a rapid and transient activation of the NFkB pathway measured by the nuclear translocation of the p65 sub-unit, leading to a physical interaction with the GR as assessed by immuno-precipitation experiment. These results suggest that the inhibition of the CYP450 in response to ILl treatment could be due to an activation of the NFkB pathway leading to a direct repression of the GR transcriptional activity on its target genes.
[6~
ANALYSES OF JAGGED1 AND NOTCH1 PROTEINS IN HEPATOCYTE CULTURES AND LIVER REGENERATION
Christoph Koehler, William C. Bowen, Peter Pediaditakis, Satdarshan P.S. Monga, Jens Walldorf, Steven C. Strom, George K. Michalopoulos.
Department of Pathology, University of Pittsburgh, Medical School, Pittsburgh, PA, USA The Jagged/Notch signaling pathway controls cell fate determination and differentiation, and its dysfunction is associated with human pathologies, including Alagille syndrome. To determine the putative role of the transmembrane receptor Notch and its ligand Jagged in cell-cell interaction and proliferation of hepatocytes, we analyzed Jaggedl and Notchl proteins in the primary cultures of rat and human hepatocytes and after partial hepatectomy of rat liver. Human hepatocytes and rat hepatocytes were cultured in