Intermediate filament cytoskeleton of the liver in health and disease

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.

Fig. 1

MDBs are readily detected by different methods. In clinical routine, MDBs are usually detected as eosinophilic aggregates in standard hematoxylin and eosin stained sections (a). After chromotrope aniline blue staining, MDBs appear as blue structures, often with red center (b). Immunofluorescence or immunohistochemical staining represents a more sensitive method for MDB detection than conventional histological stainings, but is strongly dependent on the antibody used as well as the staining protocol. MDBs can be reliably detected with antibodies against K8/K18 [green and redchannel in (c) and (d), respectively] or p62 [red channel in (c)], whereas only some MDBs stain with antibodies to phosphorylated keratins such as K8 pS431 antibody [green channel in (d)]. In both immunofluorescence pictures, MDBs are seen as yellow structure due to co-localization of both visualized epitopes

Fig. 2

MDBs are seen in various human liver diseases. Immunohistochemical staining with p62 antibody visualizes the presence of multiple irregularly shaped aggregates in patients with alcoholic steatohepatitis (a), non-alcoholic steatohepatitis (b), Indian childhood cirrhosis and (c), idiopathic copper toxicosis (d)

Fig. 3

MDBs formed in DDC-fed animals resemble inclusion bodies observed in human diseases. Liver sections were double labeled with antibodies to K8/K18 (green) and p62 (red). Samples from mouse fed DDC for 12 weeks (d) and from a patient with alcoholic steatohepatitis (b) exhibit multiple irregularly shaped inclusion bodies, which appear yellow due to presence of both epitopes. In contrast, control human (a) and mouse liver (c) display an unaffected keratin network with no apparent p62 staining. Note that DDC feeding leads to deposition of protoporphyrin, which can be seen as occasional blue pigment in (d)

Fig. 4

MDBs formation results from a complex interplay of several contributing factors. Since the cytoplasm represents a hydrophilic milieu, all exposed hydrophobic molecules (depicted by red stretches within the protein) are predisposed to aggregation. Properly folded proteins usually hide their hydrophobic stretches inside, but these get exposed in nascent protein chains or after proteins become misfolded as a consequence of oxidative stress. Chaperones bind to these hydrophobic residues and facilitate protein refolding. Alternatively, damaged proteins become polyubiquitinated and degraded either by the proteasomal or autophagic system. MDB-causing agents typically generate extensive amount of oxidative stress with increased protein misfolding. In addition, chaperone levels are downregulated and/or chaperone function is compromised. Dysbalanced K8/K18 expression precedes MDB formation, likely increases keratin misfolding and predisposes to posttranslational modifications, which may interfere with keratin refolding and/or repair. Accumulated misfolded proteins are sequestered as inclusions through the action of p62. p62 also shuttles polyubiquitinated proteins to degradative machineries. However, proteasomal degradation might be impaired by oxidative stress, may not be able to digest highly cross-linked protein species or might simply be overwhelmed by the excessive supply. On the other hand, autophagy is upregulated during MDB formation in mice and additional stimulation of autophagy attenuates MDB formation in certain conditions. Of note, supplementation of S-adenosylmethionine or mitochondrially targeted antioxidants effectively diminishes MDB formation, thereby pointing to a central role of oxidative stress in MDB generation