Nurdan Guldiken1,2, Qin Zhou3, Ozlem Kucukoglu2, Melanie Rehm2, Kateryna Levada1, Annika Gross1, Raymond Kwan4, Laura P James5, Christian Trautwein1, M Bishr Omary4, Pavel Strnad1,2. 1. IZKF and Department of Internal Medicine III, University Hospital Aachen, Aachen, Germany. 2. Department of Internal Medicine I, University Hospital Ulm, Ulm, Germany. 3. Department of Medicine, Palo Alto VA Medical Center, Palo Alto, CA. 4. Department of Molecular & Integrative Physiology, University of Michigan Medical School, and the VA Ann Arbor Health Care System, Ann Arbor, MI. 5. Arkansas Children's Hospital Research Institute and Department of Pediatrics, University of Arkansas for Medical Sciences, Little Rock, AR.
Abstract
UNLABELLED: Keratins 8 and 18 (K8/K18) are the intermediate filaments proteins of simple-type digestive epithelia and provide important cytoprotective function. K8/K18 variants predispose humans to chronic liver disease progression and poor outcomes in acute acetaminophen (APAP)-related liver failure. Given that K8 G62C and R341H/R341C are common K8 variants in European and North American populations, we studied their biological significance using transgenic mice. Mice that overexpress the human K8 variants, R341H or R341C, were generated and used together with previously described mice that overexpress wild-type K8 or K8 G62C. Mice were injected with 600 mg/kg of APAP or underwent bile duct ligation (BDL). Livers were evaluated by microarray analysis, quantitative real-time polymerase chain reaction, immunoblotting, histological and immunological staining, and biochemical assays. Under basal conditions, the K8 G62C/R341H/R341C variant-expressing mice did not show an obvious liver phenotype or altered keratin filament distribution, whereas K8 G62C/R341C animals had aberrant disulphide cross-linked keratins. Animals carrying the K8 variants displayed limited gene expression changes, but had lower nicotinamide N-methyl transferase (NNMT) levels and were predisposed to APAP-induced hepatotoxicity. NNMT represents a novel K8/K18-associated protein that becomes up-regulated after K8/K18 transfection. The more pronounced liver damage was accompanied by increased and prolonged JNK activation; elevated APAP protein adducts; K8 hyperphosphorylation at S74/S432 with enhanced keratin solubility; and prominent pericentral keratin network disruption. No differences in APAP serum levels, glutathione, or adenosine triphosphate levels were noted. BDL resulted in similar liver injury and biliary fibrosis in all mouse genotypes. CONCLUSION: Expression of human K8 variants G62C, R341H, or R341C in mice predisposes to acute APAP hepatotoxicity, thereby providing direct evidence for the importance of these variants in human acute liver failure.
UNLABELLED: Keratins 8 and 18 (K8/K18) are the intermediate filaments proteins of simple-type digestive epithelia and provide important cytoprotective function. K8/K18 variants predispose humans to chronic liver disease progression and poor outcomes in acute acetaminophen (APAP)-related liver failure. Given that K8G62C and R341H/R341C are common K8 variants in European and North American populations, we studied their biological significance using transgenic mice. Mice that overexpress the humanK8 variants, R341H or R341C, were generated and used together with previously described mice that overexpress wild-type K8 or K8G62C. Mice were injected with 600 mg/kg of APAP or underwent bile duct ligation (BDL). Livers were evaluated by microarray analysis, quantitative real-time polymerase chain reaction, immunoblotting, histological and immunological staining, and biochemical assays. Under basal conditions, the K8G62C/R341H/R341C variant-expressing mice did not show an obvious liver phenotype or altered keratin filament distribution, whereas K8G62C/R341C animals had aberrant disulphide cross-linked keratins. Animals carrying the K8 variants displayed limited gene expression changes, but had lower nicotinamide N-methyl transferase (NNMT) levels and were predisposed to APAP-induced hepatotoxicity. NNMT represents a novel K8/K18-associated protein that becomes up-regulated after K8/K18 transfection. The more pronounced liver damage was accompanied by increased and prolonged JNK activation; elevated APAP protein adducts; K8 hyperphosphorylation at S74/S432 with enhanced keratin solubility; and prominent pericentral keratin network disruption. No differences in APAP serum levels, glutathione, or adenosine triphosphate levels were noted. BDL resulted in similar liver injury and biliary fibrosis in all mouse genotypes. CONCLUSION: Expression of humanK8 variants G62C, R341H, or R341C in mice predisposes to acute APAP hepatotoxicity, thereby providing direct evidence for the importance of these variants in humanacute liver failure.
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