Genesis and consequences of inborn and acquired alterations of hepatocellular keratin architecture
Final Report Abstract
Background: Keratins (K) 7, 8 and 18-20/23 are the intermediate filaments of digestive epithelia displaying an important cytoprotective function. In the liver, hepatocytes express only K8/K18 while biliary and progenitor cells also harbour K7/K19. K8/K18 variants predispose humans to acute acetaminophen toxicity and chronic liver disease development/progression. Under stress conditions, keratins become overexpressed, posttranslationally modified or redistributed into Mallory-Denk bodies (MDBs) that represent hallmarks of alcoholic and non-alcoholic steatohepatitis. Results: Our in silico analysis indicated that keratins of digestive epithelia differ from hair and epidermal keratins in that they lack cysteine residues and harbor abundant acidid/basic amino acids within their head/tail domains. Inherited K8/K18 variants are overrepresented in subjects with acute liver failure, drug-induced liver injury or chronic hepatitis C infection and predispose them to an adverse disease outcome. We also identified the first epidermal-like K18 mutation leading to a massive disruption of keratin architecture in a patient who died due to isoniacide hepatotoxicity. Of note, these variants were previously thought to be embryolethal. In transgenic animals, overexpression of K8 G62, K8 R341H or K8 R341C resulted in an increased acetaminophen hepatotoxicity accompanied by higher JNK activation and formation of acetaminophen-cystein adducts. Loss of K19 impaired oval cell proliferation and resulted in a stronger liver injury in mice fed with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) or choline-deficient, ethionine-supplemented diet, an effect that seems to be due to impaired Notch signalling. K7/8 and K18-K20 were found to be upregulated in several liver disorders and their elevation associated with liver disease severity. In cell culture, K8/K18 overexpression was induced via IL-6, but not TNF-α or IL1-β suggesting that K8/K18 represent type II acute-phase proteins. K23 is expressed at low levels in the unstressed liver, but is more abundant in cholangiocytes and patients with chronic liver disease of multiple etiologies. A combination of murine and human data suggests that K23 is a novel marker of ductal reaction. High fat diet induced robust MDB formation in K8 mice and aggregate appearance was accompanied by liver injury with stellate cell activation, induction of transglutaminase 2 with higher K8/p62 crosslinking and autophagic dysfunction leading to accumulation of p62 and ubiquitinated proteins. To study the impact of chaperones on MDB formation, we generated a new mouse line overexpressing Hsp72 in a tissue-specific and inducible manner. When fed with DDC, Hsp72 displayed lower weight loss and significantly lower ALT levels, but, surprisingly, a more prominent MDB formation which was paralleled by increased p62 and stronger accumulation of insoluble, ubiquitinated protein. As a potential mechanism, these animals seemed to have decreased autophagic activity. Glutamine 70 of K8 was detected to represent the major K8 crosslinking residue and K8 crosslinking was enhanced through phosphorylation at the adjacent K8 S74 residue. Plectin was found to be crosslinked during MDB formation and this modification was confirmed in several independent MDB models. To further characterize the process of MDB formation as well as to define the protein dynamics within aggregates, we analyze primary hepatocytes obtained from transgenic mice expressing a fusion protein consisting of K8 and enhanced yellow fluorescent protein. Last but not least, microarray of human samples identified a change in K8/K18 expression ratio, overexpression of metallothioneins and diminished Hsp72 production as the major events associated with MDB formation.
Publications
- (2009) Keratins modulate the shape and function of hepatocyte mitochondria: a mechanism for protection from apoptosis. J Cell Sci 122:3851-3855
Tao GZ, Looi KS, Toivola DM, Strnad P, Zhou Q, Liao J, Omary MB
- (2010) Cytoskeletal keratin glycosylation protects from epithelial tissue injury. Nat Cel Biol 12:876-885
Ku NO, Toivola DM, Strnad P, Omary MB
- (2010) Intermediate filaments take the heat as stress proteins. Trends Cell Biol 20:79-91
Toivola DM, Strnad P, Habtezion A, Omary MB
- (2010) Keratin variants predispose to acute liver failure and adverse outcome: race and ethnic associations. Gastroenterology 139:828-35
Strnad P, Zhou Q, Hanada S, Lazzeroni LC, Zhong B, So P, Davern TJ, Lee WM, the Acute Liver Failure Study Group (ALFSG), Omary MB
- (2011) Cytoskeleton in non-alcoholic steatohepatitis-100 years old but still youthful. Expert Rev Gastroenterol Hepatol 5:167-177
Molnar A, Haybaeck J, Lackner C, Strnad P
- (2011) Unique amino acid signatures that are evolutionarily conserved distinguish simple-type, epidermal and hair keratins. J Cell Sci 124:4221-32
Strnad P, Usachov V, Debes C, Gräter F, Parry DAD, Omary MB
- (2012) Keratin 8 phosphorylation regulates its transamidation and hepatocyte Mallory-Denk body formation. FASEB J 26:2318-26
Kwan R, Hanada S, Harada M, Strnad P, Li DH, Omary MB
(See online at https://doi.org/10.1096/fj.11-198580) - (2012) Keratin 8 variants are infrequent in patients with alcohol-related liver cirrhosis and do not associate with development of hepatocellular carcinoma. BMC Gastroenterol 12:147
Usachov V, Nahon P, Lunova M, Ziol M, Rufat P, Sutton A, Beaugrand M, Strnad P
(See online at https://doi.org/10.1186/1471-230X-12-147) - (2012) Keratins - markers and modulators of liver disease. Curr Opin Gastroenterol 28:209-16
Strnad P, Paschke S, Jang KH, Ku NO
(See online at https://doi.org/10.1097/MOG.0b013e3283525cb8) - (2012) Noncoding keratin variants associate with liver fibrosis progression in patients with hemochromatosis. PLoS One 7: e32669
Strnad P, Kucukoglu O, Lunova M, Güldiken N, Lienau TC, Stickel F, Omary MB
(See online at https://doi.org/10.1371/journal.pone.0032669) - (2013) Broad Spectrum of Hepatocyte Inclusions in Humans, Animals, and Experimental Models. Compr Physiol 3:1393-1436
Strnad P, Nuraldeen R, Guldiken N, Hartmann D, Mahajan V, Denk H, Haybaeck J
(See online at https://doi.org/10.1002/cphy.c120032) - High-fat diet triggers Mallory-Denk body formation through misfolding and crosslinking of excess keratin 8. Hepatology, Vol 60 Issue 1, July 2014, Pages 169-178
Kucukoglu O, Guldiken N, Chen Y, Usachov V, El-Heliebi A, Haybaeck J, Denk H, Trautwein C, Strnad P
(See online at https://doi.org/10.1002/hep.27068) - Keratins 8 and 18 are type II acute-phase responsive genes overexpressed in human liver disease. Liver International, Vol 35 Issue 4, April 2015, Pages 1203-1212
Nurdan Guldiken, Valentyn Usachov, Kateryna Levada, Christian Trautwein, Marianne Ziol, Pierre Nahon, Pavel Strnad
(See online at https://doi.org/10.1111/liv.12608)
