The keratin cytoskeleton of hepatocytes is affected in a variety of chronic liver diseases, such as alcoholic and nonalcoholic steatohepatitis (ASH, NASH), copper toxicosis, cholestasis and hepatocellular carcinoma. In these diseases hepatocytes reveal a derangement or even loss of the cytoplasmic keratin intermediate filament cytoskeleton and formation of cytoplasmic inclusions (Mallory bodies) consisting of misfolded and aggregated keratin as well as a variety of stress proteins. Keratin gene knock-out mice demonstrated that keratins fulfil besides a structural role providing mechanical stability to hepatocytes a role as target and modulator of toxic stress responses in that keratins interact with a variety of stress-related signaling pathways, are preferred targets of stress-induced protein misfolding and are substrates for caspases. Furthermore, the identification of mutations in keratin genes in patients with liver cirrhosis suggests that keratins act as genetic modifiers in liver diseases.

The Keratin Cytoskeleton of Hepatocytes and Bile Duct Epithelial Cells

The keratin intermediate filament (IF) cytoskeleton of hepatocytes is composed of the type I keratin 18 (K18) and type II keratin 8 (K8) in equimolar ratios.^1-3 Bile duct epithelia have a more complex keratin composition and express in addition to K8 and K18 also K7 and K19. Since K7 is present in higher concentrations than K19 the major partner for K7 in bile duct epithelia is K18. This preferred partnership of K7 and K18 was demonstrated in K18 knock-out mice where in the absence of K18 there was also a loss of K7 in bile duct epithelia.⁴ In certain liver diseases, such as alcoholic steatohepatitis (ASH) and chronic cholestasis also single hepatocytes as well as small groups of hepatocytes express in addition to K8 and K18, K7 and, to a lesser extent, K19. This phenomenon was originally regarded as evidence for ductular metaplasia (i.e., hepatocytes acquire features of bile duct epithelial).^5,6 A metaplastic transition of hepatocytes to bile duct epithelia, however, was so far not observed in mouse models for ASH or cholestasis.⁷ Another explanation for the occurrence of K7-positive hepatocytes is that in certain liver diseases regeneration of hepatocytes involves proliferation and differentiation of hepatocytic precursor cells (oval cells), which normally express K8, K18 as well as K7 and K19.^8,9 Therefore, the presence of hepatocytes expressing K7 indicates that these hepatocytes originated from precursor cells and not by proliferation of differentiated hepatocytes, the latter of which is the classical mode of hepatocyte regeneration.⁹

The biological significance of the expression of different keratin pairs in bile duct epithelia and the expression of bile duct-type keratins in hepatocytes is unclear. Studies in keratin gene knock-out mice demonstrated that a regular liver can be formed in the absence either of K8, K18 or K19.^4,10-12 However mice lacking K8 or K18 or expressing a mutated K18 gene were more sensitive to a variety of toxins and stress conditions.¹³ Furthermore, studies performed with epidermal keratins provided evidence that different keratin family members are associated with distinct cellular functions. In the epidermis keratinocytes replace the keratin pairs K5 / K14 by K1 / K10 during their course of differentiation.^14,15 In situations of wound healing and in neoplasia keratinocytes start expressing different keratins, such as K16, which is associated with enhanced cell proliferation and migration.^16,17 A relationship between specific keratin proteins and cell function was recently demonstrated in transfected keratinocytes, where expression of K16 resulted in higher cell proliferation rates whereas expression of K10 reduced proliferation.^18,19

Alterations of the Keratin Cytoskeleton in Human Liver Diseases

Alterations of the hepatocytic keratin IFs is a characteristic feature of ASH, which develops in approximately 20% of heavy drinkers and rapidly leads to the development of liver cirrhosis the major cause of death of alcoholics. In ASH hepatocytes on the one hand accumulate fat in the cytoplasm (steatosis), because of a general disturbance of lipid metabolism and, on the other hand, there are non steatotic hepatocytes, which are enlarged (ballooned) and may contain cytoplasmic protein aggregates termed Mallory bodies (MBs). Further morphologic alterations in ASH are a chicken wirelike fibrosis, an inflammatory reaction with predominantly polymorphonuclear granulocytes, and cholestasis.^20-22 It has been demonstrated more than 20 years ago that the major cellar structure that is affected in ballooned hepatocytes is the keratin cytoskeleton. Ballooned hepatocytes reveal a derangement or even loss of the cytoplasmic keratin IFs and aggregation of keratin proteins as MBs²³ (fig. 1). These alterations of the keratin cytoskeleton are characteristic but not specific of ASH since they are also seen in a variety of other liver diseases such as (i) nonalcoholic steatohepatitis (NASH), which occurs in patients with type II diabetes and obesity, after intestinal bypass surgery, and can be induce by certain drugs, (ii) chronic cholestasis, particularly primary biliary cirrhosis, (iii) copper intoxication as it occurs in patients with Wilson disease and Indian childhood cirrhosis, (iv) hepatocellular carcinoma^24,25 (fig. 1).

Figure 1

Alterations of the keratin cytoskeleton in human liver diseases. A) Hematoxylin eosin-stained human liver section with alcoholic hepatitis. B) Same liver as shown in (A) stained with antibodies to K8 and 18, C) stained with an antibody to p62, D) stained (more...)

The common occurrence of alterations of the hepatocytic keratin cytoskeleton in such a variety of diseases poses the question whether there is a unique pathogenetic principle leading to this phenotype. Although the above mentioned diseases originate from different aetiologies, increased oxidative stress is a constant and common feature. For example, in ASH oxidation of ethanol results in the formation acetaldehyde, which depletes glutathione and disturbs mitochondrial function, both of which result in increased oxidative stress. Furthermore metabolism of alcohol by cytochrome P450 2E1 leads to generation of reactive oxygen species (ROS) as by-product.²⁶ In NASH oxidative stress is an indirect consequence of mitochondrial overload with free fatty acids, and the increased release of TNF-α from adipose tissue.²⁷ In chronic cholestasis there is accumulation of bile acids in hepatocytes, which also results in oxidative stress either directly by affecting signaling and mitochondrial function or indirectly by activation of polymorphonuclear granulocytes.²⁸ In copper toxicosis copper leads to oxidative stress by mediating the formation of hydroxyl radicals by the Fenton reaction. The biological role of oxidative stress in copper storage-associated liver diseases was recently underlined by the common presence of MBs containing the stress-induced, ubiquitin binding protein p62 in livers with Wilson disease or Indian childhood cirrhosis.²⁹ In liver cancer ballooned tumor cells containing MBs or cytoplasmic hyaline bodies, which share features with MBs and can be considered as precursors of MBs, are found in 10-20% of cancer cases³⁰ (Denk et al, manuscript submitted). Comparative gene expression profiling of HCC with and without inclusions demonstrated increased expression of heat shock proteins, p62 and keratin in HCC containing inclusions, which supports the hypothesis that formation of cytoplasmic inclusions is associated with increased stress (Neumann et al, unpublished observations). This observation is in line with the emerging general evidence that cancer cells experience increased oxidative stress and reveal altered responses to reactive oxygen species.³¹

Animal Models to Study Hepatocytic Keratin Alterations

The alterations of the keratin cytoskeleton observed in human liver diseases can be reproduced in mice by feeding a diet containing griseofulvin (GF) or 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)^24,32,33 (fig. 2). Time course studies in GF- or DDC-fed mice, immunohistochemical and biochemical analyses of MBs as well as studies in K8 and K18 knock-out mice provided insights into the functional relevance of the keratin alterations in liver disease. DDC intoxication rapidly (within 2 days) leads to phosphorylation of keratin IFs at various phosphorylation sites.³⁴ Some of these phosphorylation sites are targets for protein kinases typically activated in stress conditions indicating that DDC induces a stress response in hepatocytes.35 Furthermore mice respond to intoxication with marked overexpression of K8 and K18 resulting in a denser keratin IF network in enlarged hepatocytes^34,36 (fig. 3). This overexpression of keratin apparently is an active (defence)response of hepatocytes to toxic injury since mice with one deleted K8 allele (K8+/-), which had limited capacities to increase keratin expression, were more sensitive to toxic injury than wild-type mice.³⁷ The concept that keratin overexpression confers resistance to drug toxicity is not restricted to DCC intoxication since in a previous report marked induction of multidrug resistance in cultured mouse fibroblasts was achieved by transfection with K8 and K18 cDNAs.³⁸ Interestingly, the overexpression of keratin in hepatocytes is a specific reaction to certain types of injury, such as ethanol, DDC, GF or cholestasis and not seen after intoxication with a series of cytostatic drugs^7,34,36,39 (Lackner et al, unpublished observation). The mode of transcriptional control, which leads to these rather specific alterations in keratin expression in liver diseases, is unknown. Continuation of DDC or GF intoxication for up to two months leads to a reduction of the density to a loss of cytoplasmic keratin IFs in hepatocytes (fig. 3). The loosening of the keratin network is accompanied by the appearance small MBs at intersections of the keratin IF network at around 4-6 weeks of DDC intoxication. After 8 weeks larger MBs are found typically in the perinuclear region without association with keratin IFs (fig. 3). These alterations are reversible upon cessation of intoxication. After 4 weeks of recovery only granular remnants of MBs are found at the cell periphery preferentially in association with desmosomes of hepatocytes most of which are still devoid of a cytoplasmic keratin IF network (fig. 3). At this stage of recovery mice rapidly respond with MB formation upon reexposure to DDC.³⁴ This enhanced reappearance of MBs is also seen in humans with ASH if patients are exposed to alcohol even after prolonged periods of abstinence and is reminiscent of a “toxic memory response”. Recent comparative studies in naïve mice and mice recovered from DDC intoxication, which were exposed to DDC for 3 days revealed marked differences in cytochrome P450 contents in these two groups of mice (Stumptner et al, unpublished observation). These findings indicate a relationship between DDC metabolism and cytoskeletal alterations. DDC is known to be metabolized by cytochrome P450 by N-demethylation, which leads to the generation of a methyl radical.⁴⁰ This indicates that chronic radical injury is a major consequence of DDC intoxication and the putative common pathogenetic principle in human ASH, NASH and other diseases associated with MB formation.

Figure 2

Double-label immunofluorescence microscopy of human liver with alcoholic steatohepatitis (ASH) (A,C) and mouse liver treated with DDC for 2 months (B,D) using antibodies to K8 and 18 (A,B) and to p62 (C,D). Electron microscopy of a Mallory body in human (more...)

Figure 3

Time course of keratin alterations in DDC-treated mice. Immunofluorescence microscopy using antibodies to K8 and 18 of normal mouse liver (A), after 7 days DDC feeding (B), after 2 months DDC feeding (C), after 2 months DDC feeding and 1 month of recovery (more...)

Mallory Bodies As a Product of the Cellular Response to Misfolded Keratin

Occurrence of misfolded proteins is a major consequence of radical injury. Oxidation of amino acid residues results in conformational changes and exposure of hydrophobic residues at the protein surface. Such misfolded proteins are inactive, can disturb cellular functions and have the tendency to aggregate via hydrophobic interactions.⁴¹ Several cellular defence mechanisms against misfolded proteins exist. Misfolded proteins can either be refolded under consumption of ATP by heat shock protein 70 (HSP), preserved in a folding competent state by HSP 25/27, tagged by ubiquitin for degradation by the proteasome or aggregate in association with ubiquitin binding protein p62 as cytoplasmic inclusions recently designated in cultured cells as aggresomes or sequestosomes.^41-50 Analysis of the protein composition of MBs by mass spectrometry revealed that MBs consist predominantly of keratin, ubiquitinated keratin, HSP 70, HSP 25 and p62.⁵⁰ Immunohistochemical studies which confirmed the mass spectrometry data revealed in addition the presence α-B crystallin, another member of the small HSP family, and proteasomal subunits^23,24,51-55 (Table 1). The protein composition of MBs indicates hat in these diseases all major cellular defence mechanism against misfolded proteins (i.e., misfolded keratin) are involved. Abnormal conformation of keratins in human ASH and experimentally induced MBs has been demonstrated by monoclonal antibodies recognizing conformation-dependent epitopes, the excess of keratin 8 over keratin 18 in MBs, which is not compatible with proper assembly of IFs, and the reduction in α-helically structured domains.^{34,56- 58} These data imply that keratin is the primary target for modification by radicals and misfolding, whereas all other MB components reflect the cellular response to misfolded proteins. The central role of keratins in MB formation has been demonstrated with keratin 8 and keratin 18 null mice. Keratin 18 null mice responded to DDC-intoxication with enhanced MB formation and formed MBs spontaneously at high age⁴ (Stumptner et al, manuscript submitted). In contrast no MBs were formed in the absence of keratin 8³⁷ (fig. 4). Surprisingly, keratin 8 null mice, which did not form MBs revealed markedly increased toxicity, whereas keratin 18 null mice tolerated DDC intoxication like wild type mice. This discrepancy in the phenotypes of DDC-treated keratin 8 null and keratin 18 null mice allows drawing several important conclusions: (i) Keratin 8 is the core component of MBs and all other MB components bind to or coassemble with keratin, (ii) MBs by themselves are not toxic to cells but MB occurrence is associated with better tolerance to toxic stress, (iii) since in both types of knock-out mice hepatocytes are devoid of a keratin IF cytoskeleton, the increased sensitivity of keratin 8 null mice cannot by related to the loss of keratin IFs but indicates that keratins fulfil in addition nonstructural roles in toxic stress situations.^37,59

Table 1

Summary of major Mallory body components.

Figure 4

Requirement of K8 for Mallory body formation. Immunofluorescence microscopy of mouse livers stained with antibodies to K8 and K18. A) K8 null mouse liver treated for 2 months with DDC. B) K18 null mouse liver at 18 month of age. Note that in the absence (more...)

How Can Keratins Influence Toxic Cell Injury?

Keratins were shown to undergo extensive modifications in various cell culture and in vivo conditions. They include phosphorylation, ubiquitination, proteolytic cleavage, and cross linking, which modulate the status of the keratin IF cytoskeleton and the interaction of keratins with several cellular proteins, particularly signaling molecules.^35,54,60,61 K8 and 18 are substrates of a variety of protein kinases involved in mitosis, apoptosis and stress.³⁵ Antibodies directed against specific phosphoepitopes on keratin revealed that K8 and 18 become phosphorylated at many sites in human ASH as well as in DDC-fed mice.⁶² Some of these phosphorylation sites were targets of stress-induced protein kinases and were phosphorylated already within one day of intoxication. This indicates that phosphorylation of keratin might be one of the initial mechanisms of involving keratins in toxic stress responses. A direct role of keratin phosphorylation as modulator of cell toxicity was demonstrated in transgenic mice expressing a K18 with a mutated phosphorylation site. These mice were more sensitive to treatment with griseofulvin or microcystin than wild-type mice or transgenic mice expressing nonmutated K18.⁶³ The functional relationship between keratin phosphorylation and toxicity could be based on the fact that phosphorylation status of keratins regulates IF assembly/disassembly as well as interaction of keratin with various signaling proteins. For example, a phosphorylation-dependent interaction of K18 with 14-3-3 was demonstrated recently.⁶⁴ 14-3-3 occurs in various isoforms and is an important integrator of major signaling pathways regulating cell proliferation and apoptosis.⁶⁵ The biological significance of the interaction of keratin with 14-3-3 was show in mice with an impaired keratin cytoskeleton, in which an association between alterations of keratin, 14-3-3 and cell proliferation was observed.⁶⁶ Furthermore, keratin is able to modulateTNF-α signaling. Association of keratin to tumor necrosis factor receptor 2 (TNFR2) influences TNF-α-induced activation of Jun NH₂-terminal kinase (JNK) as well as NFκB.⁶⁷ The interaction of keratin with TNF-α signaling could play a central role in ASH and NASH where enhanced production of TNF-α and activation of JNK and NFκB are constant findings.^27,68-70 Furthermore keratins were shown to modulate induction and execution of apoptosis by interfering at various levels with the apoptotic program. In K8-deficient mice there is enhanced targeting of Fas to the plasma membrane, which results in three- to four-fold increased sensitivity to Fas-mediated apoptosis.⁷¹ A similar observation of increased Fas-mediated apoptosis was made in mice expressing mutated K18.⁷² Furthermore K8- as well as in K18- deficient mice were up to 100-fold more sensitive to TNF-α-induced cell death, which was explained by the interaction of K8 and K18 with TNFR2.⁶⁷ Other studies, however, did only demonstrate a relationship between K8 or K18 and Fas but not TNF-α- induced apoptosis.^71,72 A further mechanism by which keratin modulates Fas or TNF-α mediated apoptosis was recently demonstrated in keratin 8 null mice. These mice showed marked reduction of c-Flip and did not respond with Erk1/2 activation upon stimulation of Fas, TNF-α receptor or TNF-α-related apoptosis-inducing ligand receptor.⁷³ A direct role of keratin in this pathway was suggested by the fact that keratin was found in association with a complex consisting of c-Flip, Raf, and Erk1/2.

Keratins are also targets in and modulators of the execution of apoptosis since keratins are cleaved by and associate with caspases. Cleavage of keratins by caspases leads to a disruption of the keratin IF network and the formation of small globular cytoplasmic inclusions containing cleaved and hyperphosphorylated keratins as well as activated caspases.⁷⁴ The association of activated caspases with keratin inclusions is a characteristic feature of epithelial cell apoptosis and is expected to influence the activity of caspases in the execution of the apoptotic program.

Mutations of Keratin Genes and Liver Diseases

Heterozygous mutations in the K8 and K18 genes were found in 17 out of 467 (3.6%) explanted livers from patients with end-stages of various acute and chronic liver diseases.^75-77 75% of all mutations identified were at K8 Y53H, K8 G61C and K18 H127L, suggesting existence of mutation hot spots. The keratin mutations were shown to lead to reorganization and partial collapse of the keratin IF network in patient livers.⁷⁷ Furthermore the mutations affected keratin assembly in transfected cells if they were exposed to oxidative stress.⁷⁶ The preference of keratin mutations in patients with cryptogenic liver cirrhosis indicates that the presence of a keratin mutation might predispose to the development of liver cirrhosis, in particular in patients with NASH since NASH is regarded as the major cause of cryptogenic liver cirrhosis.

Since keratin mutations were found also in a healthy cohort in a frequency of 0.6%, heterozygous keratin mutations per se do not lead to liver disease but could rather act as a genetic modifier. Interestingly, keratin mutations were not found in patients with earlier stages of liver diseases.⁷⁸ This discrepancy to the studies performed by B. Omary in end-stage liver disease^75-77 underlines that heterozygous mutations of K8 and K18 are not the cause of liver disease in humans but could characterize patients with risk for enhanced progression of liver disease to cirrhosis requiring liver transplantation.

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