Hepatitis: Zatloukal K

Strnad P, Stumptner C, Zatloukal K, Denk H. · Department of Internal Medicine I, University of Ulm, Robert-Koch-Strabe 8, 89081, Ulm, Germany. · Histochem Cell Biol. · Pubmed #18443813 links to  free full text

Abstract: Intermediate filaments (IFs) represent the largest cytoskeletal gene family comprising approximately 70 genes expressed in tissue specific manner. In addition to scaffolding function, they form complex signaling platforms and interact with various kinases, adaptor, and apoptotic proteins. IFs are established cytoprotectants and IF variants are associated with >30 human diseases. Furthermore, IF-containing inclusion bodies are characteristic features of several neurodegenerative, muscular, and other disorders. Acidic (type I) and basic keratins (type II) build obligatory type I and type II heteropolymers and are expressed in epithelial cells. Adult hepatocytes contain K8 and K18 as their only cytoplasmic IF pair, whereas cholangiocytes express K7 and K19 in addition. K8/K18-deficient animals exhibit a marked susceptibility to various toxic agents and Fas-induced apoptosis. In humans, K8/K18 variants predispose to development of end-stage liver disease and acute liver failure (ALF). K8/K18 variants also associate with development of liver fibrosis in patients with chronic hepatitis C. Mallory-Denk bodies (MDBs) are protein aggregates consisting of ubiquitinated K8/K18, chaperones and sequestosome1/p62 (p62) as their major constituents. MDBs are found in various liver diseases including alcoholic and non-alcoholic steatohepatitis and can be formed in mice by feeding hepatotoxic substances griseofulvin and 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). MDBs also arise in cell culture after transfection with K8/K18, ubiquitin, and p62. Major factors that determine MDB formation in vivo are the type of stress (with oxidative stress as a major player), the extent of stress-induced protein misfolding and resulting chaperone, proteasome and autophagy overload, keratin 8 excess, transglutaminase activation with transamidation of keratin 8 and p62 upregulation.

Zatloukal K, French SW, Stumptner C, Strnad P, Harada M, Toivola DM, Cadrin M, Omary MB. · Institute of Pathology, Medical University of Graz, Auenbruggerplatz 25, A-8036 Graz, Austria. · Exp Cell Res. · Pubmed #17531973 No free full text.

Abstract: Frank B. Mallory described cytoplasmic hyaline inclusions in hepatocytes of patients with alcoholic hepatitis in 1911. These inclusions became known as Mallory bodies (MBs) and have since been associated with a variety of other liver diseases including non-alcoholic fatty liver disease. Helmut Denk and colleagues described the first animal model of MBs in 1975 that involves feeding mice griseofulvin. Since then, mouse models have been instrumental in helping understand the pathogenesis of MBs. Given the tremendous contributions made by Denk to the field, we propose renaming MBs as Mallory-Denk bodies (MDBs). The major constituents of MDBs include keratins 8 and 18 (K8/18), ubiquitin, and p62. The relevant proteins and cellular processes that contribute to MDB formation and accumulation include the type of chronic stress, the extent of stress-induced protein misfolding and consequent proteasome overload, a K8-greater-than-K18 ratio, transamidation of K8 and other proteins, presence of p62 and autophagy. Although it remains unclear whether MDBs serve a bystander, protective or injury promoting function, they do serve an important role as histological and potential progression markers in several liver diseases.

Zatloukal K, Stumptner C, Fuchsbichler A, Fickert P, Lackner C, Trauner M, Denk H. · Institute of Pathology, Medical University of Graz, A-8036 Graz, Austria. · J Pathol. · Pubmed #15495250 No free full text.

Abstract: The keratin intermediate filament (IF) cytoskeleton of hepatocytes has continuously gained medical relevance over the last two decades. Originally it was mainly recognized as a differentiation marker for diagnostic purposes in pathology. However, keratin IFs were soon identified as major cellular structures to be affected in a variety of chronic liver diseases, such as alcoholic and non-alcoholic steatohepatitis (ASH, NASH), copper toxicosis, and cholestasis. Based on observations in keratin gene knock-out mice, the insight into the functional role of keratins was extended from a mere structural role providing mechanical stability to hepatocytes, to an additional role as target and modulator of toxic stress and apoptosis. The functional relevance of keratins in human diseases has recently been underlined by the identification of mutations in keratin genes in patients with liver cirrhosis.

Lackner C, Gogg-Kamerer M, Zatloukal K, Stumptner C, Brunt EM, Denk H. · Institute of Pathology, Medical University of Graz, Auenbruggerplatz 25, Graz, Austria. · J Hepatol. · Pubmed #18329127 No free full text.

Abstract: BACKGROUND/AIMS: Hepatocyte "ballooning" is an often used but ill defined term in liver pathology to designate a special form of liver cell degeneration associated with cell swelling and enlargement found particularly in steatohepatitis. Alterations of the intermediate filament cytoskeleton of the hepatocyte may contribute to the pathogenesis of this microscopic change. Ballooning degeneration is considered a hallmark of steatohepatitis, but enlarged hepatocytes may also be observed in a variety of other acute and chronic liver diseases. METHODS: The intermediate filament cytoskeleton was investigated using keratin 8 and 18 immunohistochemistry in liver diseases associated with enlarged or ballooned hepatocytes. RESULTS: Keratin 8/18 immunostaining was drastically reduced or lost in the cytoplasm of ballooned hepatocytes in alcoholic and non-alcoholic steatohepatitis, chronic cholestatic conditions, ischemia/reperfusion injury and in ballooned hepatocytes in chronic hepatitis C cases with concurrent steatohepatitis. In contrast, substantial decrease or loss of keratin 8/18 immunostaining was not noted in cases of acute hepatitis, giant cell hepatitis, chronic hepatitis B, or autoimmune hepatitis. CONCLUSIONS: Loss of keratin 8/18 immunostaining can serve as an objective marker of a specific type of ballooning degeneration of hepatocytes. Oxidative stress may be a common denominator in the pathogenesis of keratin filament alterations in these conditions.

Hasselblatt P, Rath M, Komnenovic V, Zatloukal K, Wagner EF. · Research Institute of Molecular Pathology, Doktor Bohr Gasse 7, A-1030 Vienna, Austria. · Proc Natl Acad Sci U S A. · Pubmed #17940019 links to  free full text

Abstract: Analysis of the molecular factors determining hepatocyte survival or death in response to inflammatory stimuli is essential for understanding the pathogenesis of inflammatory liver disease and for identifying novel therapeutic approaches. c-Jun N-terminal kinase (JNK) is a major mediator of cytokine-induced cell death during hepatitis, but the signaling pathways downstream of JNK remain less well defined. Here we show that the transcription factor c-Jun/AP-1, a prototypic target of JNK, is strongly expressed in the liver of patients with acute liver injury. The molecular function of c-Jun in inflammatory liver disease was analyzed in mice by using the Con A model of T cell-mediated hepatitis. Mice lacking c-Jun in hepatocytes display increased liver cell death and mortality upon Con A injection. This phenotype is caused by impaired expression of inducible nitric oxide synthase (nos2), a direct transcriptional target of c-Jun, and reduced production of hepatoprotective nitric oxide (NO). Moreover, increased hepatotoxicity in mutant mice is likely caused by hypoxia and oxidative stress and can be rescued pharmacologically by liver-specific NO delivery. These findings demonstrate that c-Jun/AP-1 is hepatoprotective during acute hepatitis by regulating nos2/NO expression and thus functionally antagonizes the cell death-promoting functions of JNK.

Fickert P, Trauner M, Fuchsbichler A, Stumptner C, Zatloukal K, Denk H. · Department of Medicine, Karl-Franzens University Graz, Auenbruggerplatz 25, A-8036 Graz, Austria. · J Hepatol. · Pubmed #12663227 No free full text.

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.

Zollner G, Fickert P, Zenz R, Fuchsbichler A, Stumptner C, Kenner L, Ferenci P, Stauber RE, Krejs GJ, Denk H, Zatloukal K, Trauner M. · Division of Gastroenterology and Hepatology, Department of Internal Medicine, Karl-Franzens University Graz, Austria. · Hepatology. · Pubmed #11230744 No free full text.

Abstract: Reduced hepatobiliary transporter expression could explain impaired hepatic uptake and excretion of bile salts and other biliary constituents resulting in cholestasis and jaundice. Because little is known about alterations of hepatobiliary transport systems in human cholestatic liver diseases, it was the aim of this study to investigate such potential changes. Hepatic mRNA levels in hepatobiliary transport systems for bile salts (NTCP, BSEP), organic anions (OATP2, MRP2, MRP3), organic cations (MDR1), phospholipids (MDR3), and aminophospholipids (FIC1) were determined in 37 human liver biopsies and control livers by competitive reverse-transcription polymerase chain reaction (RT-PCR). Transporter tissue distribution was investigated by immunofluorescence microscopy. In patients with inflammation-induced icteric cholestasis (mainly cholestatic alcoholic hepatitis), mRNA levels of NTCP, OATP2, and BSEP were reduced by 41% (P <.001), 49% (P <.005), and 34% (P <.05) compared with controls, respectively. In addition, NTCP and BSEP immunostaining was reduced. MRP2 mRNA levels remained unchanged, but canalicular immunolabeling for MRP2 was also decreased. mRNA expression of MRP3, MDR1, MDR3, and FIC1 remained unchanged. In contrast to the alterations of transporter expression in inflammation-induced icteric cholestasis, transporter expression did not change in anicteric cholestasis caused by primary biliary cirrhosis (PBC) stages I and II. In conclusion, reduced expression of hepatobiliary transport systems for bile salts and other organic anions may contribute to inflammation-induced cholestasis in humans. Reduction of transporter gene expression can occur at the mRNA level as observed for NTCP, OATP2, and BSEP. However, reduced MRP2 immunostaining in the presence of conserved MRP2 mRNA levels suggests an additional role for posttranscriptional/posttranslational mechanisms.

Zatloukal K, Stumptner C, Lehner M, Denk H, Baribault H, Eshkind LG, Franke WW. · Department of Pathology, University of Graz, Graz, Austria. · Am J Pathol. · Pubmed #10751352 links to  free full text

Abstract: In alcoholic hepatitis, a severe form of alcohol-induced toxic liver injury, as well as in experimental intoxication of mice with the porphyrinogenic drugs griseofulvin and 3,5-diethoxycarbonyl-1, 4-dihydrocollidine, hepatocytes form cytoplasmic protein aggregates (Mallory bodies; MBs) containing cytokeratins (CKs) and non-CK components. Here we report that mice lacking the CK8 gene and hence CK intermediate filaments in hepatocytes, but still expressing the type I partner, ie, the CK18 gene, do not form MBs but suffer from extensive porphyria and progressive toxic liver damage, leading to the death of a considerable number of animals (7 of 12 during 12 weeks of intoxication). Our observations show that 1) in the absence of CK8 as well as in the situation of a relative excess of CK18 over CK8 no MBs are formed; 2) the loss of CK8 is not compensated by other type II CKs; and 3) porphyria and toxic liver damage are drastically enhanced in the absence of CK8. Our results point to a protective role of CKs in certain types of toxic liver injury and suggest that MBs by themselves are not harmful to hepatocytes but may be considered as a product of a novel defense mechanism in hepatocytes.

Stumptner C, Omary MB, Fickert P, Denk H, Zatloukal K. · Departments of Pathology and Medicine, University of Graz, Graz, Austria. · Am J Pathol. · Pubmed #10623656 links to  free full text

Abstract: Alcoholic hepatitis (AH) is associated with cytokeratin 8 and 18 (CK8/18) accumulation as cytoplasmic inclusion bodies, termed Mallory bodies (MBs). Studies with MB mouse models and cultured hepatocytes suggested that CK8/18 hyperphosphorylation might be involved in MB formation. However, no data exist on phosphorylation of CK8/18 in human AH. In this study, antibodies that selectively recognize phosphorylated epitopes of CK8 or CK18 were used to analyze CK8/18 phosphorylation states in normal human and murine livers, human AH biopsies, and livers of 3,5-diethoxycarbonyl-1, 4-dihydrocollidine (DDC)-intoxicated mice, the last serving as model for MB induction. Hepatocyte cytokeratins become hyperphosphorylated at multiple sites in AH and in DDC-intoxicated mice. Hyperphosphorylation of CK8/18 occurred rapidly, after 1 day of DDC intoxication and preceded architectural changes of the cytoskeleton. In long-term DDC-intoxicated mice as well as in human AH, MBs preferentially contain hyperphosphorylated CK8/18 as compared with the cytoplasmic cytokeratin intermediate filament network suggesting that CK8/18 hyperphosphorylation may play a contributing role in MB pathogenesis. Furthermore, the site-specific phosphorylation of cytokeratin in different stages of MB induction provides indirect evidence for the involvement of a variety of protein kinases known to be activated in stress responses, mitosis, and apoptosis.