Kateryna Levada

Keratins 8 and 18 are type II acute‐phase responsive genes overexpressed in human liver disease

Keratins (Ks) 7, 8, 18 and 19 constitute important markers and modifiers of liver disease. In mice, K8 and K18 are stress inducible and a dysregulated K8 > K18 stoichiometry predisposes to formation of Mallory–Denk bodies (MDBs), i.e. aggregates characteristic of chronic liver disorders such as alcoholic liver disease (ALD). In our study, we analyse the expression and the regulation of keratins in context of human liver disease.

Human keratin 8 variants promote mouse acetaminophen hepatotoxicity coupled with c‐jun amino‐terminal kinase activation and protein adduct formation

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. (Hepatology 2015) Hepatology 2015;62:876–886)

Hsp72 protects against liver injury via attenuation of hepatocellular death, oxidative stress, and JNK signaling

BACKGROUND & AIMS Heat shock protein (Hsp) 72 is a molecular chaperone that has broad cytoprotective functions and is upregulated in response to stress. To determine its hepatic functions, we studied its expression in human liver disorders and its biological significance in newly generated transgenic animals. METHODS Double transgenic mice overexpressing Hsp72 (gene Hspa1a) under the control of a tissue-specific tetracycline-inducible system (Hsp72-LAP mice) were produced. Acute liver injury was induced by a single injection of acetaminophen (APAP). Feeding with either a methionine choline-deficient (MCD; 8 weeks) or a 3,5-diethoxycarbonyl-1,4-dihydrocollidine-supplemented diet (DDC; 12 weeks) was used to induce lipotoxic injury and Mallory-Denk body (MDB) formation, respectively. Primary hepatocytes were treated with palmitic acid. RESULTS Patients with non-alcoholic steatohepatitis and chronic hepatitis C infection displayed elevated HSP72 levels. These levels increased with the extent of hepatic inflammation and HSP72 expression was induced after treatment with either interleukin (IL)-1β or IL-6. Hsp72-LAP mice exhibited robust, hepatocyte-specific Hsp72 overexpression. Primary hepatocytes from these animals were more resistant to isolation-induced stress and Hsp72-LAP mice displayed lower levels of hepatic injury in vivo. Mice overexpressing Hsp72 had fewer APAP protein adducts and were protected from oxidative stress and APAP-/MCD-induced cell death. Hsp72-LAP mice and/or hepatocytes displayed significantly attenuated Jnk activation. Overexpression of Hsp72 did not affect steatosis or the extent of MDB formation. CONCLUSIONS Our results demonstrate that HSP72 induction occurs in human liver disease, thus, HSP72 represents an attractive therapeutic target owing to its broad hepatoprotective functions. LAY SUMMARY HSP72 constitutes a stress-inducible, protective protein. Our data demonstrate that it is upregulated in patients with chronic hepatitis C and non-alcoholic steatohepatitis. Moreover, Hsp72-overexpressing mice are protected from various forms of liver stress.

Hepcidin knockout mice spontaneously develop chronic pancreatitis owing to cytoplasmic iron overload in acinar cells

Iron is both an essential and a potentially toxic element, and its systemic homeostasis is controlled by the iron hormone hepcidin. Hepcidin binds to the cellular iron exporter ferroportin, causes its degradation, and thereby diminishes iron uptake from the intestine and the release of iron from macrophages. Given that hepcidin‐resistant ferroportin mutant mice show exocrine pancreas dysfunction, we analysed pancreata of aging hepcidin knockout (KO) mice. Hepcidin and Hfe KO mice were compared with wild‐type (WT) mice kept on standard or iron‐rich diets. Twelve‐month‐old hepcidin KO mice were subjected to daily minihepcidin PR73 treatment for 1 week. Six‐month‐old hepcidin KO mice showed cytoplasmic acinar iron overload and mild pancreatitis, together with elevated expression of the iron uptake mediators DMT1 and Zip14. Acinar atrophy, massive macrophage infiltration, fatty changes and pancreas fibrosis were noted in 1‐year‐old hepcidin KO mice. As an underlying mechanism, 6‐month‐old hepcidin KO mice showed increased pancreatic oxidative stress, with elevated DNA damage, apoptosis and activated nuclear factor‐κB (NF‐κB) signalling. Neither iron overload nor pancreatic damage was observed in WT mice fed iron‐rich diet or in Hfe KO mice. Minihepcidin application to hepcidin KO mice led to an improvement in general health status and to iron redistribution from acinar cells to macrophages. It also resulted in decreased NF‐κB activation and reduced DNA damage. In conclusion, loss of hepcidin signalling in mice leads to iron overload‐induced chronic pancreatitis that is not seen in situations with less severe iron accumulation. The observed tissue injury can be reversed by hepcidin supplementation. Copyright

288 Hsp72 OVEREXPRESSION PROTECTS FROM ACETAMINOPHEN-, BUT EXACERBATES Fas-INDUCED LIVER INJURY

Background: Heat shock protein (Hsp) 72 is a molecular chaperone which is upregulated in response to a variety of stress situations and possesses broad cytoprotective functions. However its hepatic function remains largely unknown. Aims and Methods: To study the importance of Hsp72 in the liver, we generated transgenic mice overexpressing Hsp72 under the control of a tissue-specific tetracycline-inducible system and crossed them with animals carrying the tetracycline-responsive transactivator under the control of the liver activator protein promotor (Hsp72-LAP mice). Acute liver injury was induced by a single intraperitoneal injection of acetaminophen (800mg/kg) or the anti-CD95 antibody Jo2 (0.30mg/g). The severity of experimental liver injury was determined via serological and histological analyses. Results: Hsp72-LAP mice displayed doxycycline-regulated, robust Hsp72 overexpression in hepatocytes, but not in the other tissues tested. 18 hours after acetaminophen injection, a significantly lower liver injury was noted in Hsp72-LAP mice compared to single transgenes (ALT: 933 vs. 1977, p < 0.05). No differences in acetaminophen metabolism were seen. On the other hand, injection of anti-CD95 antibody resulted in a more profound injury in Hsp72-LAP vs. single transgenic animals (ALT level: 6435 vs. 2574, p = 0.004). Conclusions: Hsp72-LAP mice represent a unique tool to study the role of Hsp72 in the liver in a timeand cell-type-specific manner. Our results suggest that Hsp72, dependent on context, both ameliorates and promotes liver injury.