Nurdan Guldiken

Hepatic activation of IKK/NFκB signaling induces liver fibrosis via macrophage-mediated chronic inflammation.

Liver damage in humans is induced by various insults including alcohol abuse, hepatitis B/C virus infection, autoimmune or metabolic disorders and, when persistent, leads to development of liver fibrosis. Because the nuclear factor‐κB (NF‐κB) system is activated in response to several of these stresses, we hypothesized that NF‐κB activation in hepatocytes may contribute to fibrosis development. To activate the NF‐κB signaling pathway in a time‐ and cell‐type‐specific manner in the liver, we crossed transgenic mice carrying the tetracycline‐responsive transactivator under the control of the liver activator protein promotor with transgenic mice carrying a constitutively active form of the Ikbkb gene (IKK2 protein [CAIKK2]). Double‐transgenic mice displayed doxycycline‐regulated CAIKK2 expression in hepatocytes. Removal of doxycycline at birth led to activation of NF‐κB signaling, moderate liver damage, recruitment of inflammatory cells, hepatocyte proliferation, and ultimately to spontaneous liver fibrosis development. Microarray analysis revealed prominent up‐regulation of chemokines and chemokine receptors and this induction was rapidly reversed after switching off the CAIKK2 expression. Turning off the transgene expression for 3 weeks reversed stellate cell activation but did not diminish liver fibrosis. The elimination of macrophages by clodronate‐liposomes attenuated NF‐κB‐induced liver fibrosis in a liver‐injury‐independent manner. Conclusion: Our results revealed that hepatic activation of IKK/NF‐κB is sufficient to induce liver fibrosis by way of macrophage‐mediated chronic inflammation. Therefore, agents controlling the hepatic NF‐κB system represent attractive therapeutic tools to prevent fibrosis development in multiple chronic liver diseases. (HEPATOLOGY 2012;56:1117–1128)

High‐fat diet triggers Mallory‐Denk body formation through misfolding and crosslinking of excess keratin 8

Mallory‐Denk bodies (MDBs) are protein aggregates consisting of ubiquitinated keratins 8/18 (K8/K18). MDBs are characteristic of alcoholic and nonalcoholic steatohepatitis (NASH) and discriminate between the relatively benign simple steatosis and the more aggressive NASH. Given the emerging evidence for a genetic predisposition to MDB formation and NASH development in general, we studied whether high‐fat (HF) diet triggers MDB formation and liver injury in susceptible animals. Mice were fed a high‐fat (HF) or low‐fat (LF) diet plus a cofactor for MDB development, 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidine (DDC). Additionally, we fed nontransgenic and K8 overexpressing mice (K8tg) with the HF diet. The presence of MDB and extent of liver injury was evaluated using biochemical markers, histological staining, and immunofluorescence microscopy. In DDC‐fed animals, an HF diet resulted in greater liver injury and up‐regulation of inflammation‐related genes. As a potential mechanism, K8/K18 accumulation and increased ecto‐5′‐nucleotidase (CD73) levels were noted. In the genetically susceptible K8tg mice, HF diet triggered hepatocellular injury, ballooning, apoptosis, inflammation, and MDB development by way of 1) decreased expression of the major stress‐inducible chaperone Hsp72 with appearance of misfolded keratins; 2) elevated levels of the transglutaminase 2 (TG2); 3) increased K8 phosphorylation at S74 with subsequent TG2‐mediated crosslinking of phosphorylated K8; and 4) higher production of the MDB‐modifier gene CD73. Conclusion: Our data demonstrate that HF diet triggers aggregate formation and development of liver injury in susceptible individuals through misfolding and crosslinking of excess K8. (Hepatology 2014;60:169–178)

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.

Non-coding keratin variants associate with liver fibrosis progression in patients with hemochromatosis.

Background Keratins 8 and 18 (K8/K18) are intermediate filament proteins that protect the liver from various forms of injury. Exonic K8/K18 variants associate with adverse outcome in acute liver failure and with liver fibrosis progression in patients with chronic hepatitis C infection or primary biliary cirrhosis. Given the association of K8/K18 variants with end-stage liver disease and progression in several chronic liver disorders, we studied the importance of keratin variants in patients with hemochromatosis. Methods The entire K8/K18 exonic regions were analyzed in 162 hemochromatosis patients carrying homozygous C282Y HFE (hemochromatosis gene) mutations. 234 liver-healthy subjects were used as controls. Exonic regions were PCR-amplified and analyzed using denaturing high-performance liquid chromatography and DNA sequencing. Previously-generated transgenic mice overexpressing K8 G62C were studied for their susceptibility to iron overload. Susceptibility to iron toxicity of primary hepatocytes that express K8 wild-type and G62C was also assessed. Results We identified amino-acid-altering keratin heterozygous variants in 10 of 162 hemochromatosis patients (6.2%) and non-coding heterozygous variants in 6 additional patients (3.7%). Two novel K8 variants (Q169E/R275W) were found. K8 R341H was the most common amino-acid altering variant (4 patients), and exclusively associated with an intronic KRT8 IVS7+10delC deletion. Intronic, but not amino-acid-altering variants associated with the development of liver fibrosis. In mice, or ex vivo, the K8 G62C variant did not affect iron-accumulation in response to iron-rich diet or the extent of iron-induced hepatocellular injury. Conclusion In patients with hemochromatosis, intronic but not exonic K8/K18 variants associate with liver fibrosis development.

Keratin 23 is a stress-inducible marker of mouse and human ductular reaction in liver disease

BACKGROUND & AIMSnKeratins (K) constitute the epithelial intermediate filaments. Among them, K7/K19 are widely used markers of the regenerative liver response termed ductular reaction (DR) that consists of activated biliary epithelial cells (BECs) and hepatic progenitor cells (HPCs) and correlates with liver disease severity. In the present study we aimed to characterize K23 in the liver.nnnMETHODSnWe analyzed the expression and localization of K23 in the digestive system under basal conditions as well as in various human and mouse liver diseases/stress models. Cell culture studies were used to study factors regulating K23 expression.nnnRESULTSnIn untreated mice, K23 was restricted to biliary epithelia. It was (together with K7/K19) markedly upregulated in three different DR/cholestatic injury models, i.e., multidrug resistance protein 2 (Mdr2) knockouts, animals treated with 3,5-diethoxycarbonyl-1,4-dihydrocollidine or subjected to bile duct ligation. K23 levels correlated with the DR marker Fn14 and immunofluorescence staining showed a distinct co-localization with K7/K19. In chronic human liver disease, K23 expression increased in patients with a more prominent inflammation/fibrosis. A dramatic upregulation (>200times) was observed in patients with acute liver failure (ALF) and end-stage primary biliary cholangitis (PBC). Patients with alcoholic liver cirrhosis displayed increased K23 serum levels. In primary hepatocytes as well as hepatobiliary cell lines, treatment with TNF-related weak inducer of apoptosis (TWEAK), and the type I acute phase inducer interleukin (IL)-1β but not the type II inducer IL-6 elevated K23 expression.nnnCONCLUSIONSnK23 represents a specific, stress-inducible DR marker, whose levels correlate with liver disease severity. K23 may represent a useful non-invasive DR marker.nnnLAY SUMMARYnDuctular reaction represents a basic response to liver injury and correlates with liver disease severity. Our study identifies K23 as a novel ductular reaction marker in mice and humans.

399 HEPATIC ACTIVATION OF IKK/NF-KB SIGNALING INDUCES LIVER FIBROSIS VIA MACROPHAGE-MEDIATED CHRONIC INFLAMMATION

Liver damage in humans is induced by various insults including alcohol abuse, hepatitis B/C virus infection, autoimmune or metabolic disorders and, when persistent, leads to development of liver fibrosis. Because the nuclear factor-jB (NF-jB) system is activated in response to several of these stresses, we hypothesized that NF-jB activation in hepatocytes may contribute to fibrosis development. To activate the NF-jB signaling pathway in a timeand celltype-specific manner in the liver, we crossed transgenic mice carrying the tetracycline-responsive transactivator under the control of the liver activator protein promotor with transgenic mice carrying a constitutively active form of the Ikbkb gene (IKK2 protein [CAIKK2]). Double-transgenic mice displayed doxycycline-regulated CAIKK2 expression in hepatocytes. Removal of doxycycline at birth led to activation of NF-jB signaling, moderate liver damage, recruitment of inflammatory cells, hepatocyte proliferation, and ultimately to spontaneous liver fibrosis development. Microarray analysis revealed prominent up-regulation of chemokines and chemokine receptors and this induction was rapidly reversed after switching off the CAIKK2 expression. Turning off the transgene expression for 3 weeks reversed stellate cell activation but did not diminish liver fibrosis. The elimination of macrophages by clodronate-liposomes attenuated NF-jB-induced liver fibrosis in a liver-injury-independent manner. Conclusion: Our results revealed that hepatic activation of IKK/NF-jB is sufficient to induce liver fibrosis by way of macrophage-mediated chronic inflammation. Therefore, agents controlling the hepatic NF-jB system represent attractive therapeutic tools to prevent fibrosis development in multiple chronic liver diseases. (HEPATOLOGY 2012;56:1117-1128)

Loss of keratin 19 favours the development of cholestatic liver disease through decreased ductular reaction

Keratins (K) are cytoprotective proteins and keratin mutations predispose to the development of multiple human diseases. K19 represents the most widely used marker of biliary and hepatic progenitor cells as well as a marker of ductular reaction that constitutes the basic regenerative response to chronic liver injury. In the present study, we investigated the role of K19 in biliary and hepatic progenitor cells and its importance for ductular reaction. K19 wild‐type (WT) and knockout (KO) mice were fed: (a) 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidine (DDC); (b) cholic acid (CA); (c) a choline‐deficient, ethionine‐supplemented (CDE) diet; or (d) were subjected to common bile duct ligation (CBDL). The bile composition, liver damage, bile duct proliferation, oval cell content and biliary fibrosis were analysed. In untreated animals, loss of K19 led to redistribution of the K network in biliary epithelial cells (BECs) but to no obvious biliary phenotype. After DDC feeding, K19 KO mice exhibited (compared to WTs): (a) increased cholestasis; (b) less pronounced ductular reaction with reduced ductular proliferation and fewer oval cells; (c) impaired Notch 2 signalling in BECs; (d) lower biliary fibrosis score and biliary bicarbonate concentration. An attenuated oval cell proliferation in K19 KOs was also found after feeding with the CDE diet. K19 KOs subjected to CBDL displayed lower BEC proliferation, oval cell content and less prominent Notch 2 signal. K19 deficiency did not change the extent of CA‐ or CBDL‐induced liver injury and fibrosis. Our results demonstrate that K19 plays an important role in the ductular reaction and might be of importance in multiple chronic liver disorders that frequently display a ductular reaction. Copyright

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)

CHOP‐mediated hepcidin suppression modulates hepatic iron load

The liver is the central regulator of iron metabolism and accordingly, chronic liver diseases often lead to systemic iron overload due to diminished expression of the iron‐regulatory hormone hepcidin. To study the largely unknown regulation of iron metabolism in the context of hepatic disease, we used two established models of chronic liver injury, ie repeated carbon tetrachloride (CCl4) or thioacetamide (TAA) injections. To determine the impact of CCAAT/enhancer‐binding protein (C/EBP)‐homologous protein (CHOP) on hepcidin production, the effect of a single TAA injection was determined in wild‐type and CHOP knockout mice. Furthermore, CHOP and hepcidin expression was assessed in control subjects and patients with alcoholic liver disease. Both chronic injury models developed a distinct iron overload in macrophages. TAA‐, but not CCl4‐ injected mice displayed additional iron accumulation in hepatocytes, resulting in a significant hepatic and systemic iron overload which was due to suppressed hepcidin levels. C/EBPα signalling, a known hepcidin inducer, was markedly inhibited in TAA mice, due to lower C/EBPα levels and overexpression of CHOP, a C/EBPα inhibitor. A single TAA injection resulted in a long‐lasting (> 6 days) suppression of hepcidin levels and CHOP knockouts (compared to wild‐types) displayed significantly attenuated hepcidin down‐regulation in response to acute TAA administration. CHOP mRNA levels increased 5‐fold in alcoholic liver disease patients versus controls (p < 0.005) and negatively correlated with hepcidin expression. Our results establish CHOP as an important regulator of hepatic hepcidin expression in chronic liver disease. The differences in iron metabolism between the two widely used fibrosis models likely reflect the differential regulation of hepcidin expression in human liver disease. Copyright

Epiplakin attenuates experimental mouse liver injury by chaperoning keratin reorganization

Background & Aims Epiplakin is a member of the plakin protein family and exclusively expressed in epithelial tissues where it binds to keratins. Epiplakin-deficient (Eppk1−/−) mice displayed no obvious spontaneous phenotype, but their keratinocytes showed a faster keratin network breakdown in response to stress. The role of epiplakin in the stressed liver remained to be elucidated. Methods Wild-type (WT) and Eppk1−/− mice were subjected to common bile duct ligation (CBDL) or fed with a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-containing diet. The importance of epiplakin during keratin reorganization was assessed in primary hepatocytes. Results Our experiments revealed that epiplakin is expressed in hepatocytes and cholangiocytes, and binds to keratin 8 (K8) and K18 via multiple domains. In several liver stress models epiplakin and K8 genes displayed identical expression patterns and transgenic K8 overexpression resulted in elevated hepatic epiplakin levels. After CBDL and DDC treatment, Eppk1−/− mice developed a more pronounced liver injury and their livers contained larger amounts of hepatocellular keratin granules, indicating impaired disease-induced keratin network reorganization. In line with these findings, primary Eppk1−/− hepatocytes showed increased formation of keratin aggregates after treatment with the phosphatase inhibitor okadaic acid, a phenotype which was rescued by the chemical chaperone trimethylamine N-oxide (TMAO). Finally, transfection experiments revealed that Eppk1−/− primary hepatocytes were less able to tolerate forced K8 overexpression and that TMAO treatment rescued this phenotype. Conclusion Our data indicate that epiplakin plays a protective role during experimental liver injuries by chaperoning disease-induced keratin reorganization.