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T1764 The Genetic Background Plays An Important Role in Mouse Hepatocyte Mallory-Denk Body Formation
Here, we applied confocal microscopy and RNA interference to investigate the copper induced translocation of the Wilson disease protein, testing current models considering an interaction of Murr1 and ATP7B during this intracellular trafficking step. Although we could document a partial colocalization between Murr1/ COMMD1 and rab7, the Wilson disease protein was not observed in rab7-positive endosomes, noteven after copper induced translocation to cytoplasmically dispersed vesicles. Independent from cellular copper levels we find no colocalization of ATP7B with Murr1/ COMMD1 or other rab marker proteins of the endolysosomal system. Consistent with this, the depletion of either Murr1/ COMMD1 or rab7 in Huh7 cells did not affect translocation of ATP7B. In conclusion, our data suggest that the translocation of ATP7B takes place independent of rab7 regulated endosomal traffic events. Murr1/ COMMD1 plays a role in a later step of the copper excretion pathway but is not involved in the translocation of the Wilson disease protein.
At 3 h after bile duct ligation, Notch2 was found in zone 1, while Numb and Msi1 were restricted to zone 3. Notch2 expression shifted to zone 3 after 7 days, yet rarely costained with Numb. PCNA expression increased in zone 2 and 3 after 3h, and found ubiquitously after 7 days. CONCLUSION: Zonal transition of Notch, Wnt and Hh expression precedes hepatocyte proliferation, whereas Numb and Msi1 expression remains in perivenular hepatocytes, irrespective of the injury site. Expression of Numb and the proteins related to Notch, Wnt and Hh pathways seem to be mutually exclusive, indicating the role for Numb as a negative regulator of these pathways. T1763
T1761
Background: Mallory-Denk bodies (MDBs) are cytoplasmic inclusions that develop primarily in hepatocytes of patients with alcoholic hepatitis, non-alcoholic steatohepatitis, and a few other liver diseases. MDBs are primarily composed of the cytoskeletal keratins 8 and 18 (K8/K18), ubiquitin, and the ubiquitin-binding protein p62. Mouse genetic studies showed that K8 and its transamidation by transglutaminase-2 (TG2) are essential for MDB formation. Aims: Identify the K8 glutamines and/or lysines that are involved in K8 transamidation in order to test their importance in inclusion formation, and as a first step towards ultimately testing in genetic models whether MDBs are beneficial or detrimental to the survival of hepatocytes. Methods: An In Vitro transamidation assay was developed and utilized using purified TG2 and detergent-solubilized cells that express endogenous or cell-transfected K8/ K18. A battery of K8 glutamine-to-asparagine and lysine-to-arginine mutants were generated and compared for their ability to serve as TG2 substrates after transfection into BHK cells. Results: The In Vitro transamidation assay showed that K8 is a preferential substrate to K18 for the formation of high molecular weight keratin and ubiquitin-containing complexes which are hallmarks of MDBs. Application of the assay to ten single or double K8 glutamineto-asparagine independent mutants showed that a single glutamine-to-asparagine mutation in the N-terminal exposed domain of K8 blocks more than 90% of the observed In Vitro crosslinking of wild-type K8 or other nine K8 glutamine mutants. In contrast, none of the 5 individual single or double lysine-to-arginine mutants interfered with K8 crosslinking. The importance of this unique K8 N-terminal domain glutamine in inclusion formation was verified using a cell culture transfection assay followed by immunofluorescence staining using antibodies to keratins, ubiquitin and p62. Conclusions: We have identified a unique K8 N-terminal domain glutamine that accounts for most of keratin crosslinking by transamidation after TG2 activation. Keratin crosslinking at this site is a likely trigger for large molecular mass complex formation during inclusion formation. Our findings suggest that this unique glutamine may be an important “crosslinking switch” for MDB formation in human and animal models of liver disease.
Hrp12, the Murine Member of the Highly Conserved But Enigmatic Yer057c/ Yjgf/Uk114 Family, Interacts with Mitochondrial and Secreted Liver Proteins in the Yeast-Based Two-Hybrid System Sam J. Samuel, Alia A. Alawneh, Michael D. Sitrin Hrp12 was isolated from mouse liver and characterized as a tissue-restricted, heat-responsive protein (S. J. Samuel et al. Hepatology (1997) 25: 1213-1222). We hypothesized that hrp12 may be a novel chaperone based on its heat-responsiveness and a limited similarity to hsp90. This was supported by the finding that the Drosophila homolog (DUK114) slowed down the heat-induced aggregation of citrate synthase and accelerated the recovery of active enzyme from heat-denatured and urea-denatured preparations In Vitro. Its efficacy in this function was comparable to hsp 90, the archetypal chaperone. Moreover, Schneider cells overexpressing a DUK114-GFP protein recovered from heat shock faster than control cells (A. Farkas et al. Biochem. J. (2004) 383:165-170). In yeast cells, the S. cerevisiae homolog that is targeted to mitochondria (Mmf1p) was necessary for the maintenance of the mitochondrial genome, possibly in the role of a chaperone (E. Lexmark et al. Mol. Cell Biol. (2000) 20: 7784-7797). We created a bait plasmid encoding the binding domain of Gal 4 fused inframe to the open reading frame (ORF) of hrp12 in the vector pGBKT7. Expression of hrp12 in the yeast strain AH109 transformed by this construct was confirmed by western blotting of yeast cell lysates. A mouse liver cDNA library created in the activation domain vector pACT2 was screened by sequential transformation into AH 109 stably pre-transformed with the bait plasmid. A small number of proteins, uniformly of mitochondrial or extra-cellular origin, were identified as putative interactors by moderate to high stringency screening. The mitochondrial protein, succinate dehydrogenase (subunit beta)(SDH beta), and the secreted protein, transthyretin, were repeatedly identified suggesting authentic interactions with hrp12 in the co-transformed yeast cell. The interactions of these proteins with hrp12 were confirmed by affinity pull-down assays with GST-hrp12 fusion proteins In Vitro. The results suggest that hrp12 does interact, both in the In Vivo environment of the yeast cell and In Vitro, with SDH beta and transthyretin and may be involved in the trafficking of proteins into mitochondria and across the ER membrane and/or the assembly of multimeric protein complexes within these organelles. Overview of results of two-hybrid screening.
T1764 The Genetic Background Plays An Important Role in Mouse Hepatocyte Mallory-Denk Body Formation Shinichiro Hanada, Pavel Strnad, Elizabeth M. Brunt, Bishr Omary Background: Mallory-Denk bodies (MDBs) (formerly known as Mallory bodies) are hepatocyte intracytoplasmic inclusions found in several specific liver diseases. They consist primarily of keratins 8 and 18 (K8/18), which are the cytoskeletal intermediate filament proteins of hepatocytes. Several genetic mouse studies have demonstrated that MDB formation requires a K8>K18 overexpression state and transamidation via transglutaminase-2 (TG2) in response to selected types of injury. Aims: Given that not all patients with an MDB-associated liver disease develop MDBs, we hypothesized that genetic variables likely contribute to the extent of MDB formation in a given patient. Methods: We tested our hypothesis using five divergent strains of mice (FVB/NJ, C3H/HeJ, Balb/cByJ, C57BL/6J, and 129X1/SvJ) fed for 3 months (8 mice/strain) the established MDB-inducing agent 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). MDB formation was assessed using routine histology staining and immunofluorescence staining with antibodies to K8/K18, ubiquitin, the ubiquitin-binding protein p62 and TG2. Livers were also scored for level of injury. Results: DDC feeding induced MDBs and keratin filament disruption in all strains, but there were dramatic strain differences between the lowest and highest MDB-forming strains (p<0.05) as determined using immunofluorescene and hematoxylin&eosin staining. Immunofluorescence was far more sensitive in detecting MDBs as compared with hematoxylin&eosin staining, and demonstrates some discordance between the two staining modalities. MDB formation correlated with hepatocyte ballooning in all strains except for one. Biochemical analysis demonstrated that MDB formation paralleled the generation of high molecular weight ubiquitinated keratin-containing complexes and induction of p62, and to a lesser extent with the induction of TG2. However, MDB formation does not correlate with changes in liver size or injury in response to DDC. Conclusions: Our findings support a role for genetic differences in mouse MDB formation. If extrapolated to humans, this may help explain why some patients develop MDBs while others with the same liver disease are less likely to do so. Among the genetic variables, induction of p62 and transamidation are evident. The genetic factors that contribute to MDB formation do not correlate with the extent of DDC-induced liver injury.
T1762 Numb Is Involved in Regulation of Hepatic Self-Repair By Antagonizing Notch, Wnt and Hedgehog Pathway Hiroshi Nagata, Miho Adachi, Toshifumi Hibi Notch pathway is implicated in binary cell-fate determination in progenitor cells and terminal differentiation in proliferating cells. Numb antagonizes Notch function by preventing nuclear translocation of activated Notch, whereas Musashi1 (Msi1) reactivates Notch pathway by repressing translation of Numb. Numb is a potent target of Wnt and Hedgehog (Hh) pathways in progenitor cells, while Numb is a suppressor of these pathways in tumorigenesis. We showed simultaneous activation of Notch, Wnt and Hh signaling during hepatic regeneration (DDW2007). In this study, we analyzed spatiotemporal changes of Numb and Msi1 expression after hepatic injury. METHODS: Time course changes in expression of Numb, Msi1 and proteins related to Notch, Wnt and Hh were analyzed using Western blot and immunostaining in the rat liver. Perivenular (Rappaport zone 3) injury was produced by carbon tetrachloride (CCl4) sc, and periportal (zone 1) injury was produced by bile duct ligation. RESULTS: Expression of Numb and Msi1 increased transiently 1 to 6 h after CCl4, and that of Notch2, Delta, Frizzled 7 and Patched increased after 1 to 24 h. Expression of Numb and Msi1 did not change initially, but declined 7 days after bile duct ligation. In the normal liver, Numb immunoreactivity was found in zone 3 hepatocytes, and a few Notch2 hepatocytes were in zone 2 (midzonal region). Hepatocytes labeled with PCNA were present in zone 2 and 3, and partly coexpressed Numb. Hepatocytes stained with Numb, Msi1 or Notch2 increased 1 h after CCl4 in zone 3, but these proteins were stained in different hepatocytes. Notch2 expression extended to zone 1 and 2 after 3h, while Numb and Msi1 expression was confined in zone 3 where Notch 2 hepatocytes decreased, and PCNA hepatocytes increased. PCNA hepatocytes increased in zone 1 after 24 h when Notch 2 expression began to decrease. Distribution of Notch2 hepatocytes returned to the basal level after 7 days. Notch4, Delta, Wnt3, Frizzled 7 and Patched were coexpressed with Notch2.
T1765 The Enhancement of Peroxisome Proliferator-Activated Receptor-Gamma Activity By Curcumin Blocks the Signaling Pathways for Platelet Derived Growth Factor and Epidermal Growth Factor in Activated Hepatic Satellite Cell In Vitro Jianguo Lin, Anping Chen BACKGROUND: During hepatic fibrogenesis, the reduction in the abundance of PPARγ is accompanied by activation of signaling for PDGF and for EGF in hepatic stellate cells, the major effector cells. We previously reported that curcumin, the yellow pigment in curry,
A-817
AASLD Abstracts
AASLD Abstracts
A Keratin 8 Single Glutamine “Switch” Is Essential for Its Crosslinking By Transglutaminase-2 and Inclusion Body Formation Raymond Kwan, Pavel Strnad, Shinichiro Hanada, Bishr Omary
At 3 h after bile duct ligation, Notch2 was found in zone 1, while Numb and Msi1 were restricted to zone 3. Notch2 expression shifted to zone 3 after 7 days, yet rarely costained with Numb. PCNA expression increased in zone 2 and 3 after 3h, and found ubiquitously after 7 days. CONCLUSION: Zonal transition of Notch, Wnt and Hh expression precedes hepatocyte proliferation, whereas Numb and Msi1 expression remains in perivenular hepatocytes, irrespective of the injury site. Expression of Numb and the proteins related to Notch, Wnt and Hh pathways seem to be mutually exclusive, indicating the role for Numb as a negative regulator of these pathways. T1763
T1761
Background: Mallory-Denk bodies (MDBs) are cytoplasmic inclusions that develop primarily in hepatocytes of patients with alcoholic hepatitis, non-alcoholic steatohepatitis, and a few other liver diseases. MDBs are primarily composed of the cytoskeletal keratins 8 and 18 (K8/K18), ubiquitin, and the ubiquitin-binding protein p62. Mouse genetic studies showed that K8 and its transamidation by transglutaminase-2 (TG2) are essential for MDB formation. Aims: Identify the K8 glutamines and/or lysines that are involved in K8 transamidation in order to test their importance in inclusion formation, and as a first step towards ultimately testing in genetic models whether MDBs are beneficial or detrimental to the survival of hepatocytes. Methods: An In Vitro transamidation assay was developed and utilized using purified TG2 and detergent-solubilized cells that express endogenous or cell-transfected K8/ K18. A battery of K8 glutamine-to-asparagine and lysine-to-arginine mutants were generated and compared for their ability to serve as TG2 substrates after transfection into BHK cells. Results: The In Vitro transamidation assay showed that K8 is a preferential substrate to K18 for the formation of high molecular weight keratin and ubiquitin-containing complexes which are hallmarks of MDBs. Application of the assay to ten single or double K8 glutamineto-asparagine independent mutants showed that a single glutamine-to-asparagine mutation in the N-terminal exposed domain of K8 blocks more than 90% of the observed In Vitro crosslinking of wild-type K8 or other nine K8 glutamine mutants. In contrast, none of the 5 individual single or double lysine-to-arginine mutants interfered with K8 crosslinking. The importance of this unique K8 N-terminal domain glutamine in inclusion formation was verified using a cell culture transfection assay followed by immunofluorescence staining using antibodies to keratins, ubiquitin and p62. Conclusions: We have identified a unique K8 N-terminal domain glutamine that accounts for most of keratin crosslinking by transamidation after TG2 activation. Keratin crosslinking at this site is a likely trigger for large molecular mass complex formation during inclusion formation. Our findings suggest that this unique glutamine may be an important “crosslinking switch” for MDB formation in human and animal models of liver disease.
Hrp12, the Murine Member of the Highly Conserved But Enigmatic Yer057c/ Yjgf/Uk114 Family, Interacts with Mitochondrial and Secreted Liver Proteins in the Yeast-Based Two-Hybrid System Sam J. Samuel, Alia A. Alawneh, Michael D. Sitrin Hrp12 was isolated from mouse liver and characterized as a tissue-restricted, heat-responsive protein (S. J. Samuel et al. Hepatology (1997) 25: 1213-1222). We hypothesized that hrp12 may be a novel chaperone based on its heat-responsiveness and a limited similarity to hsp90. This was supported by the finding that the Drosophila homolog (DUK114) slowed down the heat-induced aggregation of citrate synthase and accelerated the recovery of active enzyme from heat-denatured and urea-denatured preparations In Vitro. Its efficacy in this function was comparable to hsp 90, the archetypal chaperone. Moreover, Schneider cells overexpressing a DUK114-GFP protein recovered from heat shock faster than control cells (A. Farkas et al. Biochem. J. (2004) 383:165-170). In yeast cells, the S. cerevisiae homolog that is targeted to mitochondria (Mmf1p) was necessary for the maintenance of the mitochondrial genome, possibly in the role of a chaperone (E. Lexmark et al. Mol. Cell Biol. (2000) 20: 7784-7797). We created a bait plasmid encoding the binding domain of Gal 4 fused inframe to the open reading frame (ORF) of hrp12 in the vector pGBKT7. Expression of hrp12 in the yeast strain AH109 transformed by this construct was confirmed by western blotting of yeast cell lysates. A mouse liver cDNA library created in the activation domain vector pACT2 was screened by sequential transformation into AH 109 stably pre-transformed with the bait plasmid. A small number of proteins, uniformly of mitochondrial or extra-cellular origin, were identified as putative interactors by moderate to high stringency screening. The mitochondrial protein, succinate dehydrogenase (subunit beta)(SDH beta), and the secreted protein, transthyretin, were repeatedly identified suggesting authentic interactions with hrp12 in the co-transformed yeast cell. The interactions of these proteins with hrp12 were confirmed by affinity pull-down assays with GST-hrp12 fusion proteins In Vitro. The results suggest that hrp12 does interact, both in the In Vivo environment of the yeast cell and In Vitro, with SDH beta and transthyretin and may be involved in the trafficking of proteins into mitochondria and across the ER membrane and/or the assembly of multimeric protein complexes within these organelles. Overview of results of two-hybrid screening.
T1764 The Genetic Background Plays An Important Role in Mouse Hepatocyte Mallory-Denk Body Formation Shinichiro Hanada, Pavel Strnad, Elizabeth M. Brunt, Bishr Omary Background: Mallory-Denk bodies (MDBs) (formerly known as Mallory bodies) are hepatocyte intracytoplasmic inclusions found in several specific liver diseases. They consist primarily of keratins 8 and 18 (K8/18), which are the cytoskeletal intermediate filament proteins of hepatocytes. Several genetic mouse studies have demonstrated that MDB formation requires a K8>K18 overexpression state and transamidation via transglutaminase-2 (TG2) in response to selected types of injury. Aims: Given that not all patients with an MDB-associated liver disease develop MDBs, we hypothesized that genetic variables likely contribute to the extent of MDB formation in a given patient. Methods: We tested our hypothesis using five divergent strains of mice (FVB/NJ, C3H/HeJ, Balb/cByJ, C57BL/6J, and 129X1/SvJ) fed for 3 months (8 mice/strain) the established MDB-inducing agent 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). MDB formation was assessed using routine histology staining and immunofluorescence staining with antibodies to K8/K18, ubiquitin, the ubiquitin-binding protein p62 and TG2. Livers were also scored for level of injury. Results: DDC feeding induced MDBs and keratin filament disruption in all strains, but there were dramatic strain differences between the lowest and highest MDB-forming strains (p<0.05) as determined using immunofluorescene and hematoxylin&eosin staining. Immunofluorescence was far more sensitive in detecting MDBs as compared with hematoxylin&eosin staining, and demonstrates some discordance between the two staining modalities. MDB formation correlated with hepatocyte ballooning in all strains except for one. Biochemical analysis demonstrated that MDB formation paralleled the generation of high molecular weight ubiquitinated keratin-containing complexes and induction of p62, and to a lesser extent with the induction of TG2. However, MDB formation does not correlate with changes in liver size or injury in response to DDC. Conclusions: Our findings support a role for genetic differences in mouse MDB formation. If extrapolated to humans, this may help explain why some patients develop MDBs while others with the same liver disease are less likely to do so. Among the genetic variables, induction of p62 and transamidation are evident. The genetic factors that contribute to MDB formation do not correlate with the extent of DDC-induced liver injury.
T1762 Numb Is Involved in Regulation of Hepatic Self-Repair By Antagonizing Notch, Wnt and Hedgehog Pathway Hiroshi Nagata, Miho Adachi, Toshifumi Hibi Notch pathway is implicated in binary cell-fate determination in progenitor cells and terminal differentiation in proliferating cells. Numb antagonizes Notch function by preventing nuclear translocation of activated Notch, whereas Musashi1 (Msi1) reactivates Notch pathway by repressing translation of Numb. Numb is a potent target of Wnt and Hedgehog (Hh) pathways in progenitor cells, while Numb is a suppressor of these pathways in tumorigenesis. We showed simultaneous activation of Notch, Wnt and Hh signaling during hepatic regeneration (DDW2007). In this study, we analyzed spatiotemporal changes of Numb and Msi1 expression after hepatic injury. METHODS: Time course changes in expression of Numb, Msi1 and proteins related to Notch, Wnt and Hh were analyzed using Western blot and immunostaining in the rat liver. Perivenular (Rappaport zone 3) injury was produced by carbon tetrachloride (CCl4) sc, and periportal (zone 1) injury was produced by bile duct ligation. RESULTS: Expression of Numb and Msi1 increased transiently 1 to 6 h after CCl4, and that of Notch2, Delta, Frizzled 7 and Patched increased after 1 to 24 h. Expression of Numb and Msi1 did not change initially, but declined 7 days after bile duct ligation. In the normal liver, Numb immunoreactivity was found in zone 3 hepatocytes, and a few Notch2 hepatocytes were in zone 2 (midzonal region). Hepatocytes labeled with PCNA were present in zone 2 and 3, and partly coexpressed Numb. Hepatocytes stained with Numb, Msi1 or Notch2 increased 1 h after CCl4 in zone 3, but these proteins were stained in different hepatocytes. Notch2 expression extended to zone 1 and 2 after 3h, while Numb and Msi1 expression was confined in zone 3 where Notch 2 hepatocytes decreased, and PCNA hepatocytes increased. PCNA hepatocytes increased in zone 1 after 24 h when Notch 2 expression began to decrease. Distribution of Notch2 hepatocytes returned to the basal level after 7 days. Notch4, Delta, Wnt3, Frizzled 7 and Patched were coexpressed with Notch2.
T1765 The Enhancement of Peroxisome Proliferator-Activated Receptor-Gamma Activity By Curcumin Blocks the Signaling Pathways for Platelet Derived Growth Factor and Epidermal Growth Factor in Activated Hepatic Satellite Cell In Vitro Jianguo Lin, Anping Chen BACKGROUND: During hepatic fibrogenesis, the reduction in the abundance of PPARγ is accompanied by activation of signaling for PDGF and for EGF in hepatic stellate cells, the major effector cells. We previously reported that curcumin, the yellow pigment in curry,
A-817
AASLD Abstracts
AASLD Abstracts
A Keratin 8 Single Glutamine “Switch” Is Essential for Its Crosslinking By Transglutaminase-2 and Inclusion Body Formation Raymond Kwan, Pavel Strnad, Shinichiro Hanada, Bishr Omary