The keratin cytoskeleton in liver diseases

Authors


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. Copyright © 2004 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The keratin cytoskeleton of hepatocytes and biliary epithelial cells

Differentiated hepatocytes are epithelial cells with the least complex keratin expression pattern. They normally only express the type I keratin 8 (K8) and the type II keratin 18 (K18) 1, 2. K8 and K18 are assembled in equimolar ratios into intermediate filaments (IFs), which form a filamentous network in the cytoplasm of hepatocytes (for reviews see refs 3 and 4). Bile duct epithelia reveal a more complex keratin expression pattern since they express the keratins K7 and K19 in addition to K8 and K18 2. In contrast to mature bile ducts, reactive ductules, which are considered to originate from activated progenitor cells that differentiate into bile ducts, express K7 and, to a lesser extent, K19 (Figure 1) 5. The formation of K7-positive IFs in the absence of K19 indicates that in these situations the partner of K7 has to be K18. Under certain conditions, also hepatocytes express other keratins in addition to K8 and K18. During fetal development, expression of K14 as well as K7 and K19 was found in hepatoblasts and in hepatoblasts committed to the biliary lineage, respectively 6, 7. Furthermore, in certain disease conditions, such as cholestasis and alcoholic liver disease, groups of hepatocytes express K7 5, 6–8. The biological significance of the more complex keratin cytoskeleton in bile duct epithelia and the expression of bile duct-type or epidermal keratins in hepatocytes are unclear. However, there is emerging evidence that different keratin family members are associated with distinct cellular functions. This is most clearly evident in the epidermis, where keratinocytes replace the keratin pairs K5/K14, which are expressed in the basal cell layer, by K1/K10 during their course of differentiation 4, 9. Furthermore, keratinocytes produce K16 in situations of wound healing and in neoplasia, where expression of K16 is associated with enhanced cell proliferation and migration 10, 11. A relationship between specific keratin proteins and cell function was established in transfection studies. In transfected keratinocytes, expression of K16 resulted in higher cell proliferation rates, whereas expression of K10 reduced proliferation 12, 13.

Figure 1.

Immunohistochemical detection of keratin IF in human liver. (a) Antibodies to K8 and to K18. (b) Antibody to K7. (c) Antibody to K19. Note that reactive bile ductules (indicated by arrow-heads in b and c) express less K19 than bile ducts, whereas K7 is strongly expressed in bile ducts and reactive ductules. Scale bar = 20 µm

Proper assembly of keratins requires the presence of at least one type I and one type II family member. In vitro, several type I keratins were able to partner with various type II keratins 14. This promiscuity, however, appears to be limited in vivo since gene knock-out mice showed clear preferences in pair formation. For example, in bile duct epithelial cells of K8 knock-out mice, the deficiency of K8 was not compensated by K7, although K7 is constitutively expressed 15, 16. If a certain keratin is expressed in the absence of a partner, the keratin protein cannot assemble into IF and is rapidly degraded. In a variety of disease conditions, such as alcoholic steatohepatitis and hepatocellular carcinoma, imbalanced expression of keratin pairs was observed. There is increasing evidence that keratin proteins not assembled into IF are able to interact with numerous cellular proteins involved in signal transduction and apoptosis. Therefore, keratins not only act as skeletal proteins providing mechanical stability, but also exert several non-skeletal functions 4, 16, 17.

Keratin alterations in chronic metabolic liver diseases

Alcoholic and non-alcoholic steatohepatitis

Alterations of the hepatocytic keratin IF network are constant findings in alcoholic (ASH) and non-alcoholic steatohepatitis (NASH). These diseases are characterized by steatosis, ballooning of hepatocytes, cytoplasmic inclusions (ie Mallory bodies, MBs), pericellular fibrosis, and inflammation 18–23. Ballooned hepatocytes, which are hallmark lesions in ASH and NASH, reveal a reduced density or even loss of the cytoplasmic keratin IF network (Figure 2). Some of the ballooned hepatocytes also contain MBs, which consist of misfolded and aggregated keratins as well as several stress proteins (for a review see ref 24). The occurrence of MBs in hepatocytes with reduced or lost cytoplasmic keratin IFs was originally misinterpreted as MBs originating from a collapse of the keratin IFs. However, recent studies in mice, as well as detailed analysis of the protein composition of MBs, revealed that in MBs keratins have an altered molecular structure and chemical modifications compared with regular IFs. These alterations comprise increased phosphorylation, partial proteolytic degradation, cross-linking, conformational changes leading to epitope masking, and an increase of β-sheet conformation 25–28.

Figure 2.

Morphological alterations in ASH and NASH. (a–e) Same liver sample from a patient with ASH: (a) haematoxylin and eosin-stained section; (b) chromotrope aniline blue-stained section; (c) antibodies to K8 and K18; (d) antibody to p62; (e) antibody to ubiquitin. (f) Liver section from a patient with NASH stained with antibodies to K8 and K18. The arrows in panels a–c and f indicate ballooned hepatocytes with Mallory bodies (MBs) and reduced or even lost cytoplasmic keratin IF network. MBs react with keratin (c), p62 (d), and ubiquitin (e) antibodies. Scale bar = 20 µm

MBs as well as ballooning of hepatocytes can also be reproduced in mouse liver by chronic intoxication with griseofulvin (GF) or 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) (Figure 3) 29, 30. DDC and GF are metabolized by cytochrome P450, which leads to the formation of methyl radicals 31. It is assumed that the oxidative injury induced by the methyl radical is the common pathogenetic principle in GF- or DCC-fed animals and human livers with ASH or NASH. In ASH, free radicals produced by cytochrome P450-mediated oxidation of ethanol, mitochondrial injury caused by acetaldehyde, as well as alterations in glutathione metabolism play central roles 32–34. In NASH, free fatty acid overload causing mitochondrial injury is considered to be a major source of oxidative stress 35. DDC treatment of wild-type as well as of K8 and K18 gene knock-out mice provided insights into the role of keratin in the pathogenesis of these diseases. Hepatocytes show within 3 days of DDC intoxication marked overexpression of keratin, which was regarded as an active response of hepatocytes to toxic (oxidative) stress 36. Heterozygous K8 knock-out mice (K8+/−) are more sensitive to toxic injury than wild-type mice, which could be explained by the absence of one keratin allele resulting in a reduced capacity to increase keratin expression 16, 37. This indicates that increased concentrations of cellular keratin are associated with better tolerance to toxic cell injury. In this context, the report of Bauman et al 38 on the induction of multi-drug resistance in cultured mouse fibroblasts by transfection with K8 and/or K18 cDNAs further underlines the potential biological relevance of keratin in toxic stress.

Figure 3.

Morphological alterations in a mouse liver after 2 months of DDC treatment (a–d). (a) Haematoxylin and eosin-stained liver section. (b) Antibodies to K8 and K18. (c) Antibody to p62. (d) Antibody to ubiquitin. The arrows in a and b indicate ballooned hepatocytes with Mallory bodies and reduced or even lost cytoplasmic keratin IF network similar to the situation in human livers with ASH and NASH. The immunostaining of Mallory bodies resembles that observed in human liver (see Figure 1). Scale bar = 15 µm

Although it is still unclear how keratin fulfils its protective role, there are several cellular processes in which keratin polypeptides participate and can therefore be modulated by keratin. For example, keratin is a substrate of a variety of protein kinases involved in mitosis, apoptosis, and stress (for a review on post-translational modifications of keratins see ref 39). Comparative studies in human ASH and DDC-fed mice showed that keratin becomes phosphorylated at many sites (Figure 4) 25. Some of these phosphorylation sites were shown to influence IF assembly/disassembly as well as interaction of keratin with other cellular proteins involved in the regulation of cell proliferation and apoptosis, such as 14-3-3 40, 41. Since keratin is a highly abundant protein in hepatocytes, it is likely that concentration as well as assembly status of keratin has a major impact on the action of protein kinases. This hypothesis is further substantiated by observations in transgenic mice which express a keratin 18 with a mutated phosphorylation site. These mice were more sensitive to treatment with griseofulvin or microcystin than wild-type mice or transgenic mice expressing non-mutated K18 42. Furthermore, keratin is able to modulate TNF signalling and execution of apoptosis. It was shown that keratin binds to tumour necrosis factor receptor 2 (TNFR2) and thereby influences TNF-α-induced activation of Jun NH2-terminal kinase (JNK) as well as NFκB 43. These signalling pathways play central roles in ASH and NASH, where enhanced production of TNF-α is a common event 33, 35, 44, 45. TNF-α is a potent inducer of the neutrophilic inflammatory response, which is typically pronounced in proximity of ballooned hepatocytes with MBs. Furthermore, TNF-α and oxidative stress lead to activation of JNK, which results in phosphorylation of IRS-1. IRS-1 phosphorylated at Ser 307 by JNK1 inhibits insulin signalling and causes insulin resistance, which plays a major role in the pathogenesis of NASH and the metabolic syndrome 46. Studies in K8 and K18 knock-out mice revealed that keratins are also modulators of apoptosis. For example, hepatocytes of K8-deficient mice were three- to four-fold more sensitive to Fas-mediated apoptosis than those of wild-type mice. This increased sensitivity was related to enhanced targeting of Fas to the plasma membrane 47. Mice expressing a mutated K18 were also significantly more sensitive to Fas-mediated apoptosis 48. In another study, up to 100-fold increased sensitivity to TNF-α-induced cell death was reported in K8- as well as in K18-deficient mice, which was explained by the interaction of K8 and K18 with TNFR2 43. However, a relationship between TNF-α-induced apoptosis and K8 or K18 was not observed in other studies 47, 48. Keratins are also involved in the execution of apotosis since keratins are targets of caspases. Caspase-mediated cleavage of keratins results in disruption of the keratin IF network and the formation of small spheroidal cytoplasmic inclusions containing cleaved and hyperphosphorylated keratins as well as activated caspases 49. The sequestration of activated caspases into spheroidal inclusions is expected to influence the execution of the apoptotic programme and is a characteristic feature of epithelial cell apoptosis. In apoptotic hepatocytes, cleavage of K18 by caspase 3 generates a neo-epitope of K18, which is specifically detected by the M30 antibody and can be used as a marker of apoptosis 50.

Figure 4.

Detection of keratin phosphorylation in human liver with ASH (a) and mouse liver after 3 days of DDC treatment (b) by double-label immunofluorescence microscopy. Antibodies recognizing K8 and K18 independent of their phosphorylation status (red) and antibody to K8 phosphorylated at Ser 73 (green). Note that phosphorylated keratins accumulate in Mallory bodies (a). At 3 days of DDC intoxication, several hepatocytes with still intact keratin IF cytoskeleton reveal marked hyperphosphorylation of keratin IFs, indicating that keratins are major targets for stress-related protein kinases. Scale bar = 25 µm

Another mechanism by which keratin could influence toxic injury is its involvement in situations of oxidative stress as the preferred target of the response to unfolded proteins. It is essential for proper protein function that proteins acquire a specific structure after undergoing a series of folding steps. In properly folded cytoplasmic proteins, hydrophobic amino acid residues are buried in the centre of proteins or protein complexes 51, 52. As a result of the action of free radicals in situations of oxidative stress, amino acid residues are modified, which results in conformational changes of the affected protein and the exposure of hydrophobic residues. Proteins with exposed hydrophobic residues are not functional and have a great tendency to aggregate via hydrophobic interactions 53. Several cellular proteins are involved in proper folding of newly synthesized proteins, or in refolding or degradation of misfolded proteins. These proteins, for example, bind to misfolded proteins and support refolding (eg heat shock protein 70, HSP70), prevent aggregation (eg HSP25/27, αB-crystallin), mediate their degradation by the proteasome (ubiquitin), or associate with ubiquitinated misfolded proteins as aggregates (p62) 37, 51–58.

Analysis of MBs isolated from DDC-fed mice by mass spectrometry showed that MBs can be considered as the product of the response to modified and unfolded keratin since MBs consist mainly of keratin, ubiquitinated keratin, the stress-induced and ubiquitin binding protein p62, HSPs 70 and 25, as well as the not yet further characterized MM120-1 antigen 37, 59. Furthermore, αB-crystallin, a member of the small HSP family, was detected in MBs 60. Comparative immunohistochemical studies demonstrated that the compositions of experimentally induced MBs in mice by DDC or GF and MBs in human livers are very similar, if not identical 24, 61–65.

In this context, it is noteworthy that in all disease conditions in which MBs are formed (ASH, NASH, Wilson disease, primary biliary cirrhosis, and certain drug toxicities), chronic oxidative stress is a common and major feature 24, 32–35, 66, 67. The formation of cytoplasmic aggregates of misfolded proteins also occurs in cell culture, particularly if cells are exposed to stress and the degradation of misfolded proteins by the proteasome is inhibited. These cytoplasmic aggregates, also designated aggresomes or sequestosomes, share, at least in part, features with MBs 56, 66, 68, 69.

The occurrence of cytoplasmic inclusions is also a feature of a variety of diseases in the central nervous system and other organs, such as neurofibrillary tangles (NFTs) in neurons of patients with Alzheimer's disease, Lewy bodies in neurons of patients with Parkinson's disease, Lewy body-like or skein-like inclusions in amyotrophic lateral sclerosis, Rosenthal fibres in glial cells in Alexander disease, and inclusions in skeletal muscle fibres in patients with inclusion body myopathies (for reviews see refs 70–72). For most of these inclusions, it is still a matter of debate whether the inclusions are the cause or a consequence of the disease. At least for MBs, clues to their biological significance were obtained in studies with K8 and K18 knock-out mice. DDC feeding of these mice resulted in a markedly higher toxicity in K8 knock-out than in K18 knock-out or wild-type mice. Furthermore, no MBs were formed in the K8 knock-out mice, whereas K18 knock-out mice revealed even spontaneous MB formation at high age 16, 73. These studies demonstrated that K8, but not K18, is essential for MB formation and that the occurrence of MBs was associated with better tolerance to toxic stress. MBs by themselves are therefore not deleterious to cells, but can rather be considered as the product of a cellular defence response to toxic stress 16.

Copper toxicosis

Various types of copper storage diseases including Wilson disease, Indian childhood cirrhosis, as well as idiopathic copper storage diseases lead to alterations in hepatocytes similar to ASH or NASH with ballooning, loss of cytoplasmic keratin IF network, MB formation, and pericellular fibrosis. In Wilson disease, the presence of MBs is not a constant feature but the occurrence of MBs is related to the severity of liver disease and poor prognosis 74. Immunohistochemical analysis of copper storage disease-associated MBs revealed identical protein composition as in ASH/NASH, with constant presence of keratin, ubiquitin, and p62 (Figure 5) 75. The similarity of copper toxicosis with ASH/NASH further underlines the central role of oxidative stress, since accumulation of copper results in marked generation of hydroxyl radicals via the Fenton reaction.

Figure 5.

Morphological alterations in a human liver with Indian childhood cirrhosis (a–d). (a) Haematoxylin and eosin-stained liver section. (b) Antibodies to K8 and K18. (c) Antibody to p62. (d) Antibody to ubiquitin. The arrows in a and b indicate ballooned hepatocytes with Mallory bodies and reduced or even lost cytoplasmic keratin IF network. Note that Mallory bodies are positive for keratin (b), p62 (c), and ubiquitin (d). Scale bar = 30 µm

Cholestasis

Chronic cholestasis is characterized by decreased bile flow and retention of toxic bile acids as well as other bile constituents. In cholestatic liver disease, profound alterations of all three cytoskeletal systems, namely microtubules, microfilaments, and keratin IF, are seen. These alterations were regarded as a consequence of mechanical and toxic stress. Primary biliary cirrhosis (PBC) leads to characteristic alterations of the keratin cytoskeleton. Hepatocytes with clear cytoplasm (‘cholatestasis’) are typically present at the periphery of cirrhotic nodules. These hepatocytes, which are cholestatic as indicated by the reduced expression of the bile acid transporter NTCP and up-regulation of the efflux pump MRP3, show, as in ASH/NASH, a reduction or even loss of the cytoplasmic keratin IF network and occasionally MBs (Figure 6) 76. Furthermore, in hepatocytes without MBs, the keratin content is increased and keratins are hyperphosphorylated 67. Experiments in bile duct ligated mice as well as mice fed bile acids with different hydrophobicities (ie cholic acid and lithocholic acid) demonstrated that keratins are targets of bile acid toxicity. There was striking overexpression of keratin in bile duct epithelia in response to bile duct ligation and in hepatocytes in the proximity of bile infarcts (Figure 6) 77. Furthermore, bile acids were able to induce MB formation in DDC pre-exposed (primed) mice, suggesting a causal relationship between bile acid toxicity and MB formation in cholestatic diseases 78.

Figure 6.

Morphological alterations in a human liver with primary biliary cirrhosis (a–d). (a) Haematoxylin and eosin-stained liver section. (b) Antibodies to K8 and K18. The arrows in a and b indicate ballooned hepatocytes with Mallory bodies and reduced or even lost cytoplasmic keratin IF network. (c) Antibody to K7 reacts with bile ducts and reactive ductules at the periphery of cirrhotic nodules. (d) Antibody to K7 demonstrates the presence of intermediate hepatocytes in cirrhotic nodules. (e, f) Demonstration of K8 overexpression in a mouse liver 3 days after bile duct ligation by in situ hybridization. Note that hepatocytes in proximity of the bile infarct (n) show marked overexpression of K8. bd = bile duct; n = necrotic hepatocytes in a bile infarct. Scale bar = 30 µm

Another feature of cholestatic liver diseases as well as ASH is the expression of bile duct epithelia-type keratins (K7 and to a lesser extent K19) in hepatocytes. Hepatocytes expressing K7 were also designated as intermediate hepatocytes and have to be distinguished from reactive bile ductules which typically express K7 and K19 5. There are two possible mechanisms leading to the appearance of intermediate hepatocytes. On the one hand, intermediate hepatocytes are considered to originate from hepatic progenitor cells and contribute to tissue regeneration in situations when differentiated hepatocytes cannot sufficiently compensate liver cell injury. On the other hand, the expression of K7 in hepatocytes was also regarded as a sign of incomplete transdifferentiation of hepatocytes into a biliary epithelial phenotype (ductular metaplasia), which is expected to be more resistant to bile acid toxicity 8. The latter mechanism, however, was not observed in experimental cholestasis in mice where neither bile duct ligation nor feeding of toxic bile acids led to expression of K7 in hepatocytes 77.

Keratin expression in liver cancer

The general keratin expression pattern of hepatocytes and bile duct epithelia is preserved in hepatocellular (HCC) and cholangiocellular (CCC) carcinoma. HCCs express K8 and K18, but in some cancers solitary as well as groups of cancer cells also express K7, K19 or K20. Expression of K19 in HCC was associated with increased risk of intrahepatic metastases and poor prognosis 79. In CCC, the expression of K7, K19, and K20 is much more pronounced than in HCC, facilitating the distinction of these two tumour types as well as the recognition of combined HCC and CCC (Figure 7) 80–82. We observed that in several HCCs, particularly in nodule-in-nodule situations, neither K8 nor K18 was detectable by immunohistochemistry (Figure 6). These tumours still expressed K8 and K18 mRNA but at different ratios (Lackner et al, unpublished observations). The imbalanced expression of K8 and K18 could explain why in these tumours no stable keratin IFs were formed. Furthermore, keratin phosphorylation was more pronounced in HCCs than in adjacent non-neoplastic liver tissue (Figure 7). Based on the findings of essential non-skeletal roles of keratin in cell proliferation, stress responses, and apoptosis, one must assume that hyperphosphorylation as well as imbalanced expression of keratin pairs could affect the biological behaviour of the tumour.

Figure 7.

Alterations of the keratin cytoskeleton in human hepatocellular carcinoma (HCC). (a) Immunohistochemical staining for K8 and K18 in a trabecular HCC. Note that the HCC is negative for K8 and K18, whereas the surrounding non-neoplastic liver tissue is positive. (b) Immunohistochemical detection of K8 and K18 in a combined HCC/cholangiolar carcinoma (CCC). K8 and K18 are present at the cellular periphery of the tumour part with HCC differentiation. (c) Immunohistochemical detection of K7 expression in the same tumour as shown in b showing CCC typical differentiation. Note strong positivity for K7 in a tumour area with tubular differentiation (CCC). (d) Double-label immunofluorescence microscopy with antibodies recognizing K8 and K18 independent of their phosphorylation status (red) and an antibody to K8 phosphorylated at Ser 73 (green). Note that single cells and groups of HCC cells show increased phosphorylation of K8 (yellow). (e) Antibody to p62 demonstrates numerous intracytoplasmic hyaline bodies (IHBs) in a HCC. (f) Antibody to p62 demonstrates numerous Mallory bodies (MBs) in a HCC (different case as shown in f). Note the globular appearance of IHBs (e) and the reticular structure of MBs (f). Scale bar = 30 µm

In some HCCs, cytoplasmic inclusions are also found. The most frequent inclusions are MBs, intracytoplasmic hyaline bodies (IHBs), and pale bodies (Figure 7). MBs in HCC reveal the same protein composition as MBs in ASH or NASH. IHBs consist mainly of p62, are positive for ubiquitin to a variable extent, and are negative for keratin 83. Pale bodies consist of fibrinogen. Some HCCs also show MBs and IHBs as well as inclusions with combined features of MBs and IHBs, suggesting that IHBs may develop into MBs if keratin is integrated into the inclusion (Stumptner et al, unpublished observations). Gene expression profiling of HCCs with various types of inclusions revealed several gene clusters which were specific for HCCs containing MBs or IHBs, indicating that these tumours are distinct types of HCC (Neumann et al, unpublished observations).

Mutations of keratin genes and liver diseases

Mutations in the K8 and K18 genes were found in 3.6% of explanted livers from patients with acute and chronic liver diseases, whereas mutations were present in only 0.6% of a control cohort 84–86. There is a clear preference of keratin mutations in patients with cryptogenic liver cirrhosis with a frequency of 8.8%. The mutations identified affected both K8 and K18 with hot spots at K8 Y53H, K8 G61C, and K18 H127L, which contribute to approximately 75% of all mutations. Mutations of K8 or K18 are more likely risk factors with variable penetrance than being direct causes of liver disease. Therefore, patients harbouring keratin mutations require a second ‘hit’ to develop liver disease. The relatively high frequency of mutations in cryptogenic liver cirrhosis raises the possibility that the presence of a keratin mutation predisposes to the development of liver cirrhosis, particularly in the context of NASH, which is regarded as the major cause of cryptogenic liver cirrhosis. The keratin mutations were shown to affect keratin assembly in transfected cells exposed to oxidative stress 85 and lead to reorganization and partial collapse of the keratin IF network in patient livers 86. The mechanism of how mutations in K8 or K18 contribute to the development of liver disease remains to be elucidated.

Ancillary