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Samuel W. French - One of the best experts on this subject based on the ideXlab platform.
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Mallory Body formation is associated with epigenetic phenotypic change in hepatocytes in vivo
Experimental and Molecular Pathology, 2007Co-Authors: Fawzia Bardaggorce, Barbara A. French, Jennifer Dedes, Joan Oliva, Jun Li, Samuel W. FrenchAbstract:Microarrays were done on the livers of mice fed DDC for 10 weeks, withdrawn 1 month (DDC primed livers) and refed 6 days, and compared with mice fed the control diet. The expression of a large number of genes changed when DDC was fed or refed. A Venn diagram analysis identified 649 genes where gene expression was changed in the same direction. The epigenetic memory of the DDC primed liver involved an increase in the expression of ubiquitin D, alpha fetoprotein, connective tissue growth factor, integrin beta 2, DNA methyl transferase 3a and DNA damage-inducible 45 gamma. DNA methyl transferase 3b was down-regulated as was Cbp/p300. When DDC was refed, DNA methyltransferase and histone deacetylase were up-regulated as shown by microarray analysis. Histone3 lysine9 acetylation was increased by DDC and DDC refeeding and DNA methyltransferases were not changed as shown by Western blot analysis. The data suggest the concept that the epigenetic memory that explains why DDC primed hepatocytes form MBs in 7 days of DDC refeeding is primarily the result of epigenetic modifications of gene expression through changes in histone acetylation and methylation, as well as DNA methylation.
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Mallory Body formation runs parallel to γ glutamyl transferase induction in hepatocytes of griseofulvin fed mice
Hepatology, 2007Co-Authors: Junichi Tazawa, Tetsuya Irie, Samuel W. FrenchAbstract:To evaluate whether Mallory bodies (MBs) are linked to the induction of the enzyme gamma-glutamyl transferase (GGT), mice were fed 2.5% griseofulvin (GF). The experimental and control mice livers were examined at four time periods, i.e., after 4 months' of GF feeding, 1 month after GF withdrawal and 13 days after GF refeeding, and at sacrifice after 4 months of GF withdrawal. The livers from mice continuously fed GF or control diet for 10 months were also examined. Tumors and nontumorous livers were examined histologically, histochemically, electron microscopically, and immunocytochemically. The tumors consisted of hepatomas and hyperplastic nodules. To localize MBs inside GGT-positive cells, a double-staining method was employed; GGT-positive cells were identified histochemically followed by staining for MBs using the unlabeled immunoperoxidase technique. The per cent area of the GGT-positive foci was closely correlated with the frequency of MBs observed in the course of a GF feeding and withdrawal. Almost all of the MBs were located in GGT-positive cells in both tumors and nontumor liver tissue. MBs and GGT positivity involved the same liver cells. They both were found in high frequency in tumors induced by GF. These results indicate that MB formation, like GGT induction, is a phenotypic change induced by GF. The coexistence of the two phenomenon in the same cell throughout all phases of tumor formation suggests that MBs may be related to the neoplastic process in the GF-fed mouse model.
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cyp2e1 induced by ethanol causes oxidative stress proteasome inhibition and cytokeratin aggresome Mallory Body like formation
Experimental and Molecular Pathology, 2006Co-Authors: Fawzia Bardaggorce, Li Nan, Barbara A. French, Helen Song, Sheila Khanh Nguyen, Holly Yong, Jennifer Dede, Samuel W. FrenchAbstract:The role of oxidative stress in alcoholic liver disease and cytokeratin aggresome formation is the focus of this in vitro study. HepG2 cells transduced to over express CYP2E1 (E47) and control HepG2 cells (C34) were first treated with arachidonic acid, then Fe-NAT, and finally with ethanol. In the E47 ethanol-treated cells, CYP2E1 was induced and a higher level of reactive oxygen species and carbonyl proteins were generated. The proteasome activity decreased significantly in the E47 ethanol-treated cells. This inhibition was prevented when CYP2E1 was inhibited by DAS. Microarray analysis showed gene expression down regulation of the proteasome subunit, as well as ubiquitin pathway proteins in the E47 ethanol-treated cells. 4-Hydroxynonenal (4-HNE) adducts were increased in the E47 cells treated with ethanol. Furthermore, the immunoprecipitated 4-HNE modified proteins from these cells stained positive with antibodies to the proteasome subunit alpha 6. These results indicate that the ethanol induced CYP2E1 generates oxidative stress that is responsible for the decrease in proteasome activity. Cytokeratin 8 and 18 were induced by ethanol treatment of E47 cells and polyubiquitinated forms of these proteins were found in the polyubiquitin smear upon Western blots analysis. Cytokeratin aggresomes and Mallory Body-like inclusions formed in the ethanol-treated E47 cells, indicating that the ubiquitinated cytokeratins accumulated as a result of the inhibition of the proteasome by ethanol treatment when oxidation of ethanol induced oxidative stress. This is the first report where ethanol caused Mallory Body-like cytokeratin inclusions in transformed human liver cells in vitro.
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Mallory Body cytokeratin aggresomes formation is prevented in vitro by p38 inhibitor
Experimental and Molecular Pathology, 2006Co-Authors: Li Nan, Barbara A. French, Fawzia Bardaggorce, Jennifer Dedes, Samuel W. FrenchAbstract:Microarray analysis of livers from mice fed diethyl-1,4-dihydro-2,4,6-trimethyl-3,5-pyridinedicarboxylate (DDC) to induce Mallory Body (MB) cytokeratin aggresome formation showed that gene expression for cellular adhesion molecules, cytokeratins, kinases and aggresome forming proteins were upregulated, when MBs were formed in vivo. This response was enhanced when the DDC was refed (mice fed DDC for 10 weeks followed by DDC withdrawal for 1 month, then refed DDC for 7 days). Immunofluorescent antiBody staining of the MBs that formed showed that MAPK p38 was colocalized with ubiquitin and p62 in the MBs. To investigate further the mechanisms of MB formation, primary cultures derived from DDC primed mice and their controls were incubated for 6 days. Liver cells cultured for 3 h and 6 days were used for microarray analysis. At 3 h, there were no MBs formed, but MBs were numerous after 6 days of culture. At 3 h, the expression of a large number of genes was different when the control, and the DDC primed hepatocytes were compared, which indicates that the primed hepatocytes were phenotypically changed. The gene expression of many kinases including p38 was upregulated after 6 days where the gene expression of cytokeratins, adhesion molecules and aggresome forming proteins were upregulated when MBs formed. An inhibitor of p38 phosphorylation (SB202190) completely prevented MB formation. Western blot showed that phosphorylated p38 MAPK and total p38 were absent in vitro after the p38 inhibitor treatment. Immunostaining of 6-day DDC-primed hepatocyte cultures stained with antibodies to p62 and phospho-p38 MAPK showed that phosphorylated p38 MAPK was concentrated within the MBs. Antibodies to specific serine phosphorylated sites 73 and 431, located in cytokeratin 8, localized to Mallory bodies in vivo, indicating that cytokeratin 8 was hyperphosphorylated. The data supported the concept that MBs form as the result of hyperphosphorylation of cytokeratin 8 by p38.
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Mallory Body forming cells express the preneoplastic hepatocyte phenotype.
Experimental and molecular pathology, 2006Co-Authors: Li Nan, Fawzia Bardag-gorce, Barbara A. French, Samuel W. FrenchAbstract:The livers of mice fed diethyl 1,4-dihydro-2,4,6,-trimethyl-3,5-pyridinedicarboxylate (DDC) for 10 weeks formed Mallory bodies (MBs) in clusters of hepatocytes. Mice withdrawn from DDC for 9 months developed liver tumors. In the present study, the phenotype of the hepatocytes that formed MBs and tumors was characterized. Immunoperoxidase and immunofluorescent stains were done on the DDC-treated mouse livers, as well as mouse liver tumors and a human hepatocellular carcinoma that formed MBs. Antibodies to markers of hepatocellular neoplasms such as α-fetoprotein (AFP), ubiquitin B (UbB) fatty acid synthase (FAS) and α2 macroglobulin (A2m) stained the MB forming cells positive. Quantitative real-time RT-PCR assay was used to measure AFP, UbB, FAS and GCP-3 A2m mRNA levels in the livers of DDC fed mice and the DDC-induced mouse liver tumors. The FAS, UbB, GPC-3 and AFP mRNA levels were significantly increased in the MB forming liver cells. The in vitro model of MB formation was used to correlate MB formation with gene and protein expression. Primary cultures of DDC-primed hepatocytes were compared with the controls. A2m and UbB expression increased in the primary cultures of DDC-primed hepatocytes when MBs formed. Thus, the tumor markers used to identify hepatocellular carcinoma were upregulated in cells forming MBs in vivo and in vitro, suggesting that MB forming cells express preneoplastic phenotypic features.
Barbara A. French - One of the best experts on this subject based on the ideXlab platform.
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Mallory Body formation is associated with epigenetic phenotypic change in hepatocytes in vivo
Experimental and Molecular Pathology, 2007Co-Authors: Fawzia Bardaggorce, Barbara A. French, Jennifer Dedes, Joan Oliva, Jun Li, Samuel W. FrenchAbstract:Microarrays were done on the livers of mice fed DDC for 10 weeks, withdrawn 1 month (DDC primed livers) and refed 6 days, and compared with mice fed the control diet. The expression of a large number of genes changed when DDC was fed or refed. A Venn diagram analysis identified 649 genes where gene expression was changed in the same direction. The epigenetic memory of the DDC primed liver involved an increase in the expression of ubiquitin D, alpha fetoprotein, connective tissue growth factor, integrin beta 2, DNA methyl transferase 3a and DNA damage-inducible 45 gamma. DNA methyl transferase 3b was down-regulated as was Cbp/p300. When DDC was refed, DNA methyltransferase and histone deacetylase were up-regulated as shown by microarray analysis. Histone3 lysine9 acetylation was increased by DDC and DDC refeeding and DNA methyltransferases were not changed as shown by Western blot analysis. The data suggest the concept that the epigenetic memory that explains why DDC primed hepatocytes form MBs in 7 days of DDC refeeding is primarily the result of epigenetic modifications of gene expression through changes in histone acetylation and methylation, as well as DNA methylation.
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cyp2e1 induced by ethanol causes oxidative stress proteasome inhibition and cytokeratin aggresome Mallory Body like formation
Experimental and Molecular Pathology, 2006Co-Authors: Fawzia Bardaggorce, Li Nan, Barbara A. French, Helen Song, Sheila Khanh Nguyen, Holly Yong, Jennifer Dede, Samuel W. FrenchAbstract:The role of oxidative stress in alcoholic liver disease and cytokeratin aggresome formation is the focus of this in vitro study. HepG2 cells transduced to over express CYP2E1 (E47) and control HepG2 cells (C34) were first treated with arachidonic acid, then Fe-NAT, and finally with ethanol. In the E47 ethanol-treated cells, CYP2E1 was induced and a higher level of reactive oxygen species and carbonyl proteins were generated. The proteasome activity decreased significantly in the E47 ethanol-treated cells. This inhibition was prevented when CYP2E1 was inhibited by DAS. Microarray analysis showed gene expression down regulation of the proteasome subunit, as well as ubiquitin pathway proteins in the E47 ethanol-treated cells. 4-Hydroxynonenal (4-HNE) adducts were increased in the E47 cells treated with ethanol. Furthermore, the immunoprecipitated 4-HNE modified proteins from these cells stained positive with antibodies to the proteasome subunit alpha 6. These results indicate that the ethanol induced CYP2E1 generates oxidative stress that is responsible for the decrease in proteasome activity. Cytokeratin 8 and 18 were induced by ethanol treatment of E47 cells and polyubiquitinated forms of these proteins were found in the polyubiquitin smear upon Western blots analysis. Cytokeratin aggresomes and Mallory Body-like inclusions formed in the ethanol-treated E47 cells, indicating that the ubiquitinated cytokeratins accumulated as a result of the inhibition of the proteasome by ethanol treatment when oxidation of ethanol induced oxidative stress. This is the first report where ethanol caused Mallory Body-like cytokeratin inclusions in transformed human liver cells in vitro.
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Mallory Body cytokeratin aggresomes formation is prevented in vitro by p38 inhibitor
Experimental and Molecular Pathology, 2006Co-Authors: Li Nan, Barbara A. French, Fawzia Bardaggorce, Jennifer Dedes, Samuel W. FrenchAbstract:Microarray analysis of livers from mice fed diethyl-1,4-dihydro-2,4,6-trimethyl-3,5-pyridinedicarboxylate (DDC) to induce Mallory Body (MB) cytokeratin aggresome formation showed that gene expression for cellular adhesion molecules, cytokeratins, kinases and aggresome forming proteins were upregulated, when MBs were formed in vivo. This response was enhanced when the DDC was refed (mice fed DDC for 10 weeks followed by DDC withdrawal for 1 month, then refed DDC for 7 days). Immunofluorescent antiBody staining of the MBs that formed showed that MAPK p38 was colocalized with ubiquitin and p62 in the MBs. To investigate further the mechanisms of MB formation, primary cultures derived from DDC primed mice and their controls were incubated for 6 days. Liver cells cultured for 3 h and 6 days were used for microarray analysis. At 3 h, there were no MBs formed, but MBs were numerous after 6 days of culture. At 3 h, the expression of a large number of genes was different when the control, and the DDC primed hepatocytes were compared, which indicates that the primed hepatocytes were phenotypically changed. The gene expression of many kinases including p38 was upregulated after 6 days where the gene expression of cytokeratins, adhesion molecules and aggresome forming proteins were upregulated when MBs formed. An inhibitor of p38 phosphorylation (SB202190) completely prevented MB formation. Western blot showed that phosphorylated p38 MAPK and total p38 were absent in vitro after the p38 inhibitor treatment. Immunostaining of 6-day DDC-primed hepatocyte cultures stained with antibodies to p62 and phospho-p38 MAPK showed that phosphorylated p38 MAPK was concentrated within the MBs. Antibodies to specific serine phosphorylated sites 73 and 431, located in cytokeratin 8, localized to Mallory bodies in vivo, indicating that cytokeratin 8 was hyperphosphorylated. The data supported the concept that MBs form as the result of hyperphosphorylation of cytokeratin 8 by p38.
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Mallory Body forming cells express the preneoplastic hepatocyte phenotype.
Experimental and molecular pathology, 2006Co-Authors: Li Nan, Fawzia Bardag-gorce, Barbara A. French, Samuel W. FrenchAbstract:The livers of mice fed diethyl 1,4-dihydro-2,4,6,-trimethyl-3,5-pyridinedicarboxylate (DDC) for 10 weeks formed Mallory bodies (MBs) in clusters of hepatocytes. Mice withdrawn from DDC for 9 months developed liver tumors. In the present study, the phenotype of the hepatocytes that formed MBs and tumors was characterized. Immunoperoxidase and immunofluorescent stains were done on the DDC-treated mouse livers, as well as mouse liver tumors and a human hepatocellular carcinoma that formed MBs. Antibodies to markers of hepatocellular neoplasms such as α-fetoprotein (AFP), ubiquitin B (UbB) fatty acid synthase (FAS) and α2 macroglobulin (A2m) stained the MB forming cells positive. Quantitative real-time RT-PCR assay was used to measure AFP, UbB, FAS and GCP-3 A2m mRNA levels in the livers of DDC fed mice and the DDC-induced mouse liver tumors. The FAS, UbB, GPC-3 and AFP mRNA levels were significantly increased in the MB forming liver cells. The in vitro model of MB formation was used to correlate MB formation with gene and protein expression. Primary cultures of DDC-primed hepatocytes were compared with the controls. A2m and UbB expression increased in the primary cultures of DDC-primed hepatocytes when MBs formed. Thus, the tumor markers used to identify hepatocellular carcinoma were upregulated in cells forming MBs in vivo and in vitro, suggesting that MB forming cells express preneoplastic phenotypic features.
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CYP2E1 inhibition enhances Mallory Body formation
Experimental and molecular pathology, 2005Co-Authors: Fawzia Bardag-gorce, Li Nan, Barbara A. French, Latoyia Wilson, Timothy R. Morgan, Kengathevy Morgan, Samuel W. FrenchAbstract:Mallory Body (MB) formation is a complex phenomenon seen in chronic liver disease. CYP2E1 may play a role in preventing MB formation since it is involved in the elimination of toxic drugs and chemicals. When mice were fed with diethyl-1,4-dihydro-2,4,6-trimethyl-3,5-pyridinedicarboxylate (DDC) for 10 weeks, Mallory bodies (MBs) developed in the liver at the end of this period. When DDC feeding was combined with CMZ (an efficient in vivo CYP2E1 inhibitor), more MBs formed compared to DDC feeding alone. DDC was shown to be a suicide inhibitor of CYP2E1. The level of CYP2E1 protein in the liver was further reduced by the DDC and CMZ treatment when measured by Western blot. To test whether CYP2E1 reduced MB formation, CYP2E1 knockout mice and CYP2E1 overexpressed mice were fed with DDC or DDC and CMZ for 10 weeks. MB formation increased markedly in the liver of CYP2E1 knockout mice when fed with DDC only. CYP2E1 overexpressed mice showed an increase in MB formation when the mice were fed with the combination of DDC and CMZ where the amount of CYP2E1 was reduced to levels seen in wild type mice. It was concluded that CYP2E1 inhibits MB formation by increasing the rate of elimination of DDC and/or its toxic intermediates.
Helmut Denk - One of the best experts on this subject based on the ideXlab platform.
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changes of cytokeratin filament organization in human and murine Mallory Body containing livers as revealed by a panel of monoclonal antibodies
Liver, 2008Co-Authors: Karlheinz Preisegger, Gerlinde Spurej, Kurt Zatloukal, Helmut DenkAbstract:— Mallory bodies (MBs) are characteristic morphologic features of alcoholic hepatitis and can be produced in mouse hepatocytes by chronic griseofulvin (GF) intoxication. The formation of MBs, which share some immunological, biochemical, and ultrastructural features with cytokeratin (CK) filaments of normal liver, is accompanied by derangement and even loss of the CK cytoskeleton of hepatocytes (“empty cells”) as revealed by immunofluorescence microscopy. To clarify whether this diminution or lack of CK-related staining of MB-containing hepatocytes was due to loss of CK filaments or changes in antigenicity or accessibility of antigenic determinants immunohistochemical studies using a battery of monoclonal and polyclonal CK antibodies were performed. It could be shown that all these antibodies directed against different CK polypeptide components and antigenic determinants of CKs revealed a highly reduced or even undetectable cytoplasmic CK meshwork in most cells with fully developed large MBs. In the light of our present knowledge of the organization of CK intermediate filaments, these results indicate that the phenomenon of the “empty cells” reflects a diminution of CK meshwork rather than altered antigenic determinants.
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Mallory Body formation in primary biliary cirrhosis is associated with increased amounts and abnormal phosphorylation and ubiquitination of cytokeratins
Journal of Hepatology, 2003Co-Authors: Peter Fickert, Michael Trauner, Andrea Fuchsbichler, Conny Stumptner, Kurt Zatloukal, Helmut DenkAbstract:Abstract Background/Aims : Animal studies revealed a key role of toxic bile acids in the regulation of hepatocytic cytokeratin (CK) expression and Mallory Body (MB) formation. In this study, we compared CK expression, phosphorylation, and ubiquitination in primary biliary cirrhosis (PBC), chronic hepatitis C (CHC) and control livers to determine whether bile acid-induced CK alterations are associated with cytoskeletal alterations and MB formation in a prototypic chronic cholestatic liver disease. Methods : CK 8 and CK 18 mRNA and protein levels were investigated by reverse transcriptase-polymerase chain reaction and Western blotting. Intermediate filament (IF) cytoskeletal alterations were assessed by immunofluorescence microscopy using antibodies against CKs, CK phosphoepitopes, MBs, and ubiquitin. Results : Despite unchanged mRNA levels, CK 8 and CK 18 protein levels were significantly elevated in PBC suggesting stabilization of CKs, possibly due to decreased degradation. CK-IF alterations in PBC comprised increased density with abnormal phosphorylation of the IF network of hepatocytes in acinar zone 1 and in the periphery of cirrhotic nodules. In addition, in these areas hepatocytes with diminished IF network containing MBs consisting of abnormally phosphorylated and ubiquitinated CK were observed. Conclusions : These findings support our concept that IF cytoskeletal alterations and MB formation in cholestatic liver diseases are related to bile acid-induced cell stress.
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bile acid induced Mallory Body formation in drug primed mouse liver
American Journal of Pathology, 2002Co-Authors: Peter Fickert, Michael Trauner, Andrea Fuchsbichler, Conny Stumptner, Kurt Zatloukal, Helmut DenkAbstract:Chronic cholestasis is associated with retention of bile acids and profound cytoskeletal alterations in hepatocytes including Mallory Body (MB) formation. The mechanisms responsible for MB formation in cholestatic liver diseases are unclear. The aim of our study was to determine the relevance of cholestasis and bile acids for MB formation. For this purpose mice received a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-supplemented diet for 2.5 months to induce MB formation. After recovery from DDC intoxication for 4 weeks followed by disappearance of MBs, these drug-primed mice were subjected to DDC refeeding, common bile duct ligation (CBDL), and feeding of a cholic acid (CA)-supplemented diet for 7 days, respectively. Cytokeratin (CK) 8 and CK 18 expression was studied by competitive reverse transcriptase-polymerase chain reaction and Western blot analysis. Cytoskeletal alterations of hepatocytes and MB formation were monitored by immunofluorescence microscopy and immunohistochemistry using CK-, ubiquitin-, and MB-specific antibodies. Like DDC refeeding, both CBDL and CA feeding of drug-primed mice significantly increased CK 8 and CK 18 mRNA and protein levels (with excess of CK 8) and resulted in ubiquitination and abnormal phosphorylation of CKs. Furthermore, CBDL and CA feeding resulted in rapid neoformation of MBs in drug-primed mice. It is concluded that MB formation in cholestatic liver diseases may be triggered by the action of potentially toxic bile acids.
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Mallory Body a disease associated type of sequestosome
Hepatology, 2002Co-Authors: C Stumptner, Kurt Zatloukal, Andrea Fuchsbichler, Hans Heid, Helmut DenkAbstract:Mallory bodies (MBs) consist of abnormal keratins, ubiquitin, heat shock proteins, and the protein p62. p62 is encoded by an immediate-early response gene that rapidly responds to a variety of extracellular signals involved in cell proliferation, differentiation, and particularly oxidative stress. It acts as an adapter in signal transduction and binds noncovalently to ubiquitin, possibly being involved in the regulation of the fate of ubiquitinated proteins by segregation (i.e., sequestosome or aggresome formation). The presence of p62 together with ubiquitinated abnormal keratins in the MB characterizes MBs as a disease-associated type of sequestosome. A detailed study on the expression of p62 and its relationship to MB formation in the 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-treated mouse liver is reported based on immunohistochemical, immunoblot, and Northern blot analyses. The results indicate that p62 is rapidly induced in hepatocytes of intoxicated animals preceding MB formation. As suggested by experiments with short-term DDC-treated naive mice and mice refed DDC after recovery from long-term DDC treatment (primed mice), p62 does not exert an initiating effect on MB formation but the appearance of MBs requires the presence of abnormal keratins, which associate with p62 after ubiquitination. The rapid induction of p62 and its association with MBs further support the role of oxidative stress in MB formation. In conclusion, the constant presence of p62 in MBs suggests that binding of p62 to abnormal keratins may allow hepatocytes to dispose potentially harmful proteins in a biologically inert manner.
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sequence of events in the assembly of Mallory Body components in mouse liver clues to the pathogenesis and significance of Mallory Body formation
Journal of Hepatology, 2001Co-Authors: C Stumptner, Kurt Zatloukal, Andrea Fuchsbichler, Manfred Lehner, Helmut DenkAbstract:Abstract Background/Aims : Chronic intoxication of mice with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) or griseofulvin (GF) results in appearance of Mallory bodies (MBs) and alterations of the keratin cytoskeleton, which are reversible upon drug withdrawal but recur after readministration within 2–3 days. Methods : DDC- or GF-treated and recovered mice were reintoxicated with the original drugs but also colchicine and lumicolchicine. Cytoskeletal alterations of hepatocytes and MB formation were monitored by immunofluorescence microscopy using keratin, MB-specific antibodies, antibodies to phosphoepitopes and to HSP70. Keratin 8/18 mRNA expression and protein levels were determined by competitive reverse transcription-polymerase chain reaction, in situ-hybridization and western blotting. Results : Duration of pretreatment was important for the efficiency of MB triggering. Rapid increase of keratin 8/18 mRNA and proteins was found in all reintoxicated mice concomitant with MB formation, whereby keratin 8 prevailed over keratin 18. Keratins and a protein with heat shock characteristics (M M 120-1 antigen) were the earliest detectable MB components, whereas ubiquitination and phosphorylation followed later. Conclusions : Overproduction of keratins is a major but not the only step responsible for MB formation. Additional components (e.g. M M 120-1 antigen) and excess of keratin 8 over keratin 18 are essential.
Qi X Yuan - One of the best experts on this subject based on the ideXlab platform.
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dexamethasone enhances Mallory Body formation in drug primed mouse liver
Experimental and Molecular Pathology, 2000Co-Authors: Qi X Yuan, Barbara A. French, Yasuyuki Nagao, Yujui Yvonne Wan, Samuel W. FrenchAbstract:Abstract In a clinical study in which patients with alcoholic hepatitis were treated with prednisone for 1 month, posttreatment liver biopsies showed diminished inflammation, but Mallory bodies were not diminished. This suggested that steroid treatment may reduce inflammation by inhibiting NFκB activation. Sparing of Mallory bodies suggests that NFκB activation may not be involved mechanistically in Mallory Body formation. To test this idea, we induced Mallory Body formation in drug-primed mice with or without dexamethasone treatment. As predicted, dexamethasone decreased NFκB activation; however, Mallory Body formation was increased. Surprisingly, TNFα and iNOS, which normally increase as a result of NFκB activation, were upregulated by the dexamethasone treatment. It was concluded that NFκB activation is not involved in Mallory Body formation. Despite this, induced increases in TNFα, iNOS, c- jun /API and c- myc expression indicate that oxidative stress is likely involved in Mallory Body formation.
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pathogenesis of Mallory Body formation studies using the drug primed mouse model
Hepatology Research, 1998Co-Authors: Yasuyuki Nagao, Barbara A. French, Qi X Yuan, Yujui Yvonne Wan, Samuel W. FrenchAbstract:Abstract Mallory bodies (MB) are formed in preneoplastic foci in the drug primed mouse model. These foci expand when the drug is refed. To better understand this phenotypic change in hepatocytes, the livers response to refeeding the drug was studied for transcriptional and post-translational changes. The post-translational change, hyperphosphorylation of MBs, was associated with changes in gene expression and activation of transcription regulation factors. Hyperphosphorylation of MBs appeared on the second day of refeeding the drug, the same time interval when MB formation began. Prior studies using this model showed an increase in the mitotic index and an increase in cytokeratin proteins at this time interval. In the present study, cytokeratin mRNA increased significantly, compared to the 0 day base line. Likewise, c-jun mRNA increased significantly at the same time interval. Previous studies on mice fed the drug for 5 months showed a significant increase in c-fos mRNA. The binding activity of AP-1 consensus oligonucleotide was significantly increased on the 5th day of refeeding. Taken together, the data support the conclusion that the MB phenotype change expands as the result of a response to stimulation of liver cell proliferation.
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mechanisms of Mallory Body formation induced by okadaic acid in drug primed mice
Experimental and Molecular Pathology, 1998Co-Authors: Qi X Yuan, Yasuyuki Nagao, Karl Gaal, Samuel W. FrenchAbstract:Drug-primed mice form Mallory bodies in their liver after various types of liver injury such as heat shock, drug refeeding, or ethanol ingestion. However, the mechanisms involved that lead to Mallory Body formation after these different treatments are unknown. There may be a common pathway of Mallory Body formation that is initiated by these different types of injuries. Recently it was shown that the phosphatase 1/2A inhibitor okadaic acid induced Mallory Body formation, suggesting that the mechanism of formation involves hyperphosphorylation or oxidative stress-induced NFkappaB activation. To test this hypothesis we exposed drug-primed mice to okadaic acid and measured phosphorylation of Mallory Body proteins immunohistochemically and by immunoblot chemiluminescence using an antiBody specific for phosphothreonine. NFkappaB activation was measured by a gel shift retardation assay of nuclear lysates. Beginning 15 min after okadaic acid injection, complex changes were progressively seen in the liver cells focally including aggregation of cytokeratins 8 and 18 in hepatocytes which otherwise failed to stain normally with cytokeratin antiBody. The aggregates stained positive with ubiquitin and phosphothreonine antibodies. Immunoblots showed a progressive increase in positive staining of the Mallory Body band with the antiBody to phosphothreonine. NFkappaB activation was progressive up to 2 h after okadaic acid treatment but was downregulated 7 days later. In summary we show for the first time the effect of okadaic acid on the liver cytokeratins in vivo. We conclude that hyperphosphorylation and NFkappaB activation may play a role in the early phases of Mallory Body formation.
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Mallory Body formation by ethanol feeding in drug primed mice
Hepatology, 1998Co-Authors: Zhiqi Zhanggouillon, Barbara A. French, Qi X Yuan, Yujui Yvonne Wan, Karl Gaal, Norman Marceau, Yasuki Nagao, Samuel W. FrenchAbstract:Drug-primed mice, created by a 5-month feeding of diethyl-1,4-dihydro-2,4,6-trimethyl-3,5-pyridinedicarboxylate (DDC), followed by a 1-month withdrawal, were refed ethanol or isocaloric dextrose (control) diets intragastrically for 7 days. The formation of Mallory bodies (MBs) was monitored by immunofluorescence and immunoperoxidase microscopy using antibodies to cytokeratin and ubiquitin, and also by electron microscopy. The changes in cytokeratin 55 (CK55), ubiquitin conjugate, nuclear factor kappaB (NFkappaB) p65, NFkappaB p50, inhibitor kappaB alpha, c-myc, tumor necrosis factor alpha, and cytochrome P450 2E1 (CYP2E1) contents were determined by Western blotting using appropriate antibodies. The messenger RNA (mRNA) for CYP2E1, cytokeratin, ubiquitin, hepatocyte growth factor activator, and tissue transglutaminase was quantitated. MBs were present at 5 to 7 days' postfeeding with ethanol, but not with dextrose. They developed in clusters of "empty hepatocytes," where the cytokeratin antiBody failed to recognize the typical filament structures seen in normal hepatocytes. MBs were larger and more numerous in the subcapsular region. Northern blots showed that CK55 mRNA was decreased by the ethanol treatment, but protein levels were increased, suggesting a decreased turnover of the cytokeratin. Likewise, the increase in CYP2E1 protein in the face of a lack of an increase in mRNA for CYP2E1 could be explained by a decreased turnover of this cytochrome. This is the first report of MB formation induced by ethanol ingestion in an experimental model.
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Mallory Body induction in drug primed mouse liver
Hepatology, 1996Co-Authors: Qi X Yuan, Barbara A. French, N Marceau, Samuel W. FrenchAbstract:The aim of this study was to determine the various factors that are involved in the induction of Mallory Body (MB) formation. A model was developed where MB formation was induced by refeeding either of the drugs griseofulvin or diethyl 1,4-dihydro-1,4,6-trimethyl-3,5-pyridinedicarboxylate (DDC). Mice were fed the drugs for 5 months, followed by withdrawal of the drugs for 1 month (drug-primed livers). The drugs were refed for 1,3,5,7, or 11 days. Early MBs first appeared as small, enlarged aggregates of filaments in the perinuclear or pericanalicular location on the third day of refeeding. Mature MBs appeared on the fifth day of refeeding. MBs reached maximum concentration on day 5 of refeeding. Western blots showed a progressive increase in the cytokeratin proteins (CK49 and CK55) and actin while refeeding the drugs. Liver cell regeneration, as indicated by the percent of proliferating cell nuclear antigen (PCNA)-positive nuclei, increased on the third day of refeeding. However, there was no correlation between the frequency of MBs and the percent of PCNA-positive nuclei. It is concluded that MB formation is not related to the liver cell regeneration response to injury but rather involves a separate regulation pathway. The MBs were heavily ubiquitinated and were associated with increased ubiquitin-protein conjugates as indicated by Western blotting, suggesting that ubiquitinization of cytokeratin protein are involved in the formation of MB aggregation.
Kurt Zatloukal - One of the best experts on this subject based on the ideXlab platform.
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changes of cytokeratin filament organization in human and murine Mallory Body containing livers as revealed by a panel of monoclonal antibodies
Liver, 2008Co-Authors: Karlheinz Preisegger, Gerlinde Spurej, Kurt Zatloukal, Helmut DenkAbstract:— Mallory bodies (MBs) are characteristic morphologic features of alcoholic hepatitis and can be produced in mouse hepatocytes by chronic griseofulvin (GF) intoxication. The formation of MBs, which share some immunological, biochemical, and ultrastructural features with cytokeratin (CK) filaments of normal liver, is accompanied by derangement and even loss of the CK cytoskeleton of hepatocytes (“empty cells”) as revealed by immunofluorescence microscopy. To clarify whether this diminution or lack of CK-related staining of MB-containing hepatocytes was due to loss of CK filaments or changes in antigenicity or accessibility of antigenic determinants immunohistochemical studies using a battery of monoclonal and polyclonal CK antibodies were performed. It could be shown that all these antibodies directed against different CK polypeptide components and antigenic determinants of CKs revealed a highly reduced or even undetectable cytoplasmic CK meshwork in most cells with fully developed large MBs. In the light of our present knowledge of the organization of CK intermediate filaments, these results indicate that the phenomenon of the “empty cells” reflects a diminution of CK meshwork rather than altered antigenic determinants.
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Mallory Body formation in primary biliary cirrhosis is associated with increased amounts and abnormal phosphorylation and ubiquitination of cytokeratins
Journal of Hepatology, 2003Co-Authors: Peter Fickert, Michael Trauner, Andrea Fuchsbichler, Conny Stumptner, Kurt Zatloukal, Helmut DenkAbstract:Abstract Background/Aims : Animal studies revealed a key role of toxic bile acids in the regulation of hepatocytic cytokeratin (CK) expression and Mallory Body (MB) formation. In this study, we compared CK expression, phosphorylation, and ubiquitination in primary biliary cirrhosis (PBC), chronic hepatitis C (CHC) and control livers to determine whether bile acid-induced CK alterations are associated with cytoskeletal alterations and MB formation in a prototypic chronic cholestatic liver disease. Methods : CK 8 and CK 18 mRNA and protein levels were investigated by reverse transcriptase-polymerase chain reaction and Western blotting. Intermediate filament (IF) cytoskeletal alterations were assessed by immunofluorescence microscopy using antibodies against CKs, CK phosphoepitopes, MBs, and ubiquitin. Results : Despite unchanged mRNA levels, CK 8 and CK 18 protein levels were significantly elevated in PBC suggesting stabilization of CKs, possibly due to decreased degradation. CK-IF alterations in PBC comprised increased density with abnormal phosphorylation of the IF network of hepatocytes in acinar zone 1 and in the periphery of cirrhotic nodules. In addition, in these areas hepatocytes with diminished IF network containing MBs consisting of abnormally phosphorylated and ubiquitinated CK were observed. Conclusions : These findings support our concept that IF cytoskeletal alterations and MB formation in cholestatic liver diseases are related to bile acid-induced cell stress.
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bile acid induced Mallory Body formation in drug primed mouse liver
American Journal of Pathology, 2002Co-Authors: Peter Fickert, Michael Trauner, Andrea Fuchsbichler, Conny Stumptner, Kurt Zatloukal, Helmut DenkAbstract:Chronic cholestasis is associated with retention of bile acids and profound cytoskeletal alterations in hepatocytes including Mallory Body (MB) formation. The mechanisms responsible for MB formation in cholestatic liver diseases are unclear. The aim of our study was to determine the relevance of cholestasis and bile acids for MB formation. For this purpose mice received a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-supplemented diet for 2.5 months to induce MB formation. After recovery from DDC intoxication for 4 weeks followed by disappearance of MBs, these drug-primed mice were subjected to DDC refeeding, common bile duct ligation (CBDL), and feeding of a cholic acid (CA)-supplemented diet for 7 days, respectively. Cytokeratin (CK) 8 and CK 18 expression was studied by competitive reverse transcriptase-polymerase chain reaction and Western blot analysis. Cytoskeletal alterations of hepatocytes and MB formation were monitored by immunofluorescence microscopy and immunohistochemistry using CK-, ubiquitin-, and MB-specific antibodies. Like DDC refeeding, both CBDL and CA feeding of drug-primed mice significantly increased CK 8 and CK 18 mRNA and protein levels (with excess of CK 8) and resulted in ubiquitination and abnormal phosphorylation of CKs. Furthermore, CBDL and CA feeding resulted in rapid neoformation of MBs in drug-primed mice. It is concluded that MB formation in cholestatic liver diseases may be triggered by the action of potentially toxic bile acids.
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Mallory Body a disease associated type of sequestosome
Hepatology, 2002Co-Authors: C Stumptner, Kurt Zatloukal, Andrea Fuchsbichler, Hans Heid, Helmut DenkAbstract:Mallory bodies (MBs) consist of abnormal keratins, ubiquitin, heat shock proteins, and the protein p62. p62 is encoded by an immediate-early response gene that rapidly responds to a variety of extracellular signals involved in cell proliferation, differentiation, and particularly oxidative stress. It acts as an adapter in signal transduction and binds noncovalently to ubiquitin, possibly being involved in the regulation of the fate of ubiquitinated proteins by segregation (i.e., sequestosome or aggresome formation). The presence of p62 together with ubiquitinated abnormal keratins in the MB characterizes MBs as a disease-associated type of sequestosome. A detailed study on the expression of p62 and its relationship to MB formation in the 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-treated mouse liver is reported based on immunohistochemical, immunoblot, and Northern blot analyses. The results indicate that p62 is rapidly induced in hepatocytes of intoxicated animals preceding MB formation. As suggested by experiments with short-term DDC-treated naive mice and mice refed DDC after recovery from long-term DDC treatment (primed mice), p62 does not exert an initiating effect on MB formation but the appearance of MBs requires the presence of abnormal keratins, which associate with p62 after ubiquitination. The rapid induction of p62 and its association with MBs further support the role of oxidative stress in MB formation. In conclusion, the constant presence of p62 in MBs suggests that binding of p62 to abnormal keratins may allow hepatocytes to dispose potentially harmful proteins in a biologically inert manner.
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sequence of events in the assembly of Mallory Body components in mouse liver clues to the pathogenesis and significance of Mallory Body formation
Journal of Hepatology, 2001Co-Authors: C Stumptner, Kurt Zatloukal, Andrea Fuchsbichler, Manfred Lehner, Helmut DenkAbstract:Abstract Background/Aims : Chronic intoxication of mice with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) or griseofulvin (GF) results in appearance of Mallory bodies (MBs) and alterations of the keratin cytoskeleton, which are reversible upon drug withdrawal but recur after readministration within 2–3 days. Methods : DDC- or GF-treated and recovered mice were reintoxicated with the original drugs but also colchicine and lumicolchicine. Cytoskeletal alterations of hepatocytes and MB formation were monitored by immunofluorescence microscopy using keratin, MB-specific antibodies, antibodies to phosphoepitopes and to HSP70. Keratin 8/18 mRNA expression and protein levels were determined by competitive reverse transcription-polymerase chain reaction, in situ-hybridization and western blotting. Results : Duration of pretreatment was important for the efficiency of MB triggering. Rapid increase of keratin 8/18 mRNA and proteins was found in all reintoxicated mice concomitant with MB formation, whereby keratin 8 prevailed over keratin 18. Keratins and a protein with heat shock characteristics (M M 120-1 antigen) were the earliest detectable MB components, whereas ubiquitination and phosphorylation followed later. Conclusions : Overproduction of keratins is a major but not the only step responsible for MB formation. Additional components (e.g. M M 120-1 antigen) and excess of keratin 8 over keratin 18 are essential.