Mallory body (cytokeratin aggresomes) formation is prevented in vitro by p38 inhibitor

Microarray analysis of livers from mice fed diethyl-1,4-dihydro-2,4,6-trimethyl-3,5-pyridinedicarboxylate (DDC) to induce Mallory body (MB) cytokeratin aggresome formation showed that gene expression for cellular adhesion molecules, cytokeratins, kinases and aggresome forming proteins were upregulated, when MBs were formed in vivo. This response was enhanced when the DDC was refed (mice fed DDC for 10 weeks followed by DDC withdrawal for 1 month, then refed DDC for 7 days). Immunofluorescent antibody staining of the MBs that formed showed that MAPK p38 was colocalized with ubiquitin and p62 in the MBs. To investigate further the mechanisms of MB formation, primary cultures derived from DDC primed mice and their controls were incubated for 6 days. Liver cells cultured for 3 h and 6 days were used for microarray analysis. At 3 h, there were no MBs formed, but MBs were numerous after 6 days of culture. At 3 h, the expression of a large number of genes was different when the control, and the DDC primed hepatocytes were compared, which indicates that the primed hepatocytes were phenotypically changed. The gene expression of many kinases including p38 was upregulated after 6 days where the gene expression of cytokeratins, adhesion molecules and aggresome forming proteins were upregulated when MBs formed. An inhibitor of p38 phosphorylation (SB202190) completely prevented MB formation. Western blot showed that phosphorylated p38 MAPK and total p38 were absent in vitro after the p38 inhibitor treatment. Immunostaining of 6-day DDC-primed hepatocyte cultures stained with antibodies to p62 and phospho-p38 MAPK showed that phosphorylated p38 MAPK was concentrated within the MBs. Antibodies to specific serine phosphorylated sites 73 and 431, located in cytokeratin 8, localized to Mallory bodies in vivo, indicating that cytokeratin 8 was hyperphosphorylated. The data supported the concept that MBs form as the result of hyperphosphorylation of cytokeratin 8 by p38.     

Mallory body forming cells express the preneoplastic hepatocyte phenotype

The livers of mice fed diethyl 1,4-dihydro-2,4,6,-trimethyl-3,5-pyridinedicarboxylate (DDC) for 10 weeks formed Mallory bodies (MBs) in clusters of hepatocytes. Mice withdrawn from DDC for 9 months developed liver tumors. In the present study, the phenotype of the hepatocytes that formed MBs and tumors was characterized. Immunoperoxidase and immunofluorescent stains were done on the DDC-treated mouse livers, as well as mouse liver tumors and a human hepatocellular carcinoma that formed MBs. Antibodies to markers of hepatocellular neoplasms such as alpha-fetoprotein (AFP), ubiquitin B (UbB) fatty acid synthase (FAS) and alpha2 macroglobulin (A2m) stained the MB forming cells positive. Quantitative real-time RT-PCR assay was used to measure AFP, UbB, FAS and GCP-3 A2m mRNA levels in the livers of DDC fed mice and the DDC-induced mouse liver tumors. The FAS, UbB, GPC-3 and AFP mRNA levels were significantly increased in the MB forming liver cells. The in vitro model of MB formation was used to correlate MB formation with gene and protein expression. Primary cultures of DDC-primed hepatocytes were compared with the controls. A2m and UbB expression increased in the primary cultures of DDC-primed hepatocytes when MBs formed. Thus, the tumor markers used to identify hepatocellular carcinoma were upregulated in cells forming MBs in vivo and in vitro, suggesting that MB forming cells express preneoplastic phenotypic features.     

The role of laminin-integrin signaling in triggering MB formation. An in vivo and in vitro study

It is still unclear as to how hepatocytes perceive external factors and transduce the signals which initiate MB formation. To investigate this phenomenon, the model of MB formation in liver in vivo and in primary culture of hepatocytes derived from drug-primed mice was used. Control mice were fed the control diet (group 1). MBs were induced in the livers of mice fed diethyl-1, 4-dihydro-2, 4, 6-trimethyl-3, 5-pyridinedicarboxylate (DDC) for 10 weeks (group 2). The induced MBs completely disappeared after the withdrawal of DDC for 4 weeks (group 3). Newly formed MBs were numerous after DDC was refed for 1 week (group 4). Relative mRNA abundance was determined by quantitative real-time RT-PCR in the liver from the mice. The expression of integrin alpha(6) and beta(2) was significantly increased in the livers of DDC-treated (group 2) and drug refed mice (group 4), when compared with the livers from controls (group 1) and DDC-withdrawn (group 3) mice. The increased mRNA of these two integrin genes was associated with the increased expression of laminin (a ligand for integrin alpha(6)beta(1) and alpha(6)beta(4)), Icam1 (a ligand of alphaLbeta2), Src, MEKK1, and ERK1. Primary cultures of isolated DDC-primed hepatocytes (group 4 mice were withdrawn from DDC-CMZ for 4-6 weeks) produced significantly more MBs on laminin-coated coverslips compared with plastic uncoated, fibronectin-, collagen-, or fibrinogen-coated coverslips. U0126, an inhibitor of MEK1 protein, significantly reduced the phosphorylated forms of ERK1/2 and MB formation in vitro. In conclusion, the current study revealed an association between MB formation and integrin-mediated signaling in vivo. The data indicate that laminin-integrin signaling which activates ERK, triggered MB formation in vitro, and an inhibitor of the signaling cascade reduced MB formation.     

Mallory bodies formed in proteasome-depleted hepatocytes: an immunohistochemical study

Mallory bodies (MBs) are aggregates of proteins, principally cytokeratin proteins found in liver cells. They are also found in a few other cell types such as type II pneumocytes and trophoblasts. Studies on the liver thus far indicate that MBs are derived from hyperphosphorylated, heavily ubiquitinated proteins which have undergone conformational change. The aggregated protein may accumulate because of the failure of the proteasome to remove the altered proteins from the cytoplasm of liver cells. To investigate this possibility, the proteasomes were assessed immunohistochemically in individual liver cells of mice fed a drug which induced MB formation. To accelerate and enhance MB formation, cytochrome P450 2EI knockout mice were used. Proteasomes in individual cells were visualized by immunofluorescence using an antibody to a subunit of the proteasome (P25). The results showed that the groups of liver cells that had formed MBs were often partially depleted of proteasomes. These findings support the possibility that MBs formed as a result of the loss of the proteasome to remove misfolded cytokeratin proteins. Thus MBs may share their pathogenesis with other types of cellular inclusions seen where proteins aggregate in the cytoplasm due to mutation, misfolding, or loss of proteasomes.     

Mallory body formation and amyloid deposition in the liver of aged mice fed a vitamin A deficient diet for a prolonged period

When twenty Swiss albino mice (male) were fed a vitamin A deficient diet for 18-24 months, Mallory bodies (MB(s] were observed. MBs were found by light microscopy and electron microscopy in the hepatocytes of six mice fed the vitamin A deficient diet and in one of the 15 control mice. Coincidentally, amyloid deposits were found in the space of Disse in the livers of 8 mice fed the deficient diet (three of the six mice with MBs). Amyloid was seen in two control mice suggesting that vitamin A enhanced amyloid deposition and MB formation in aging mice which spontaneously form amyloid and MBs. The possible role of vitamin A deficiency in the pathogenesis of MB formation is briefly discussed.     

Heat shock proteins are present in mallory bodies (cytokeratin aggresomes) in human liver biopsy specimens

Mallory bodies (MBs) are aggresomes, composed of cytokeratin and various other proteins, which form in diseased liver because of disruption in the ubiquitin-proteasome protein degradation pathway. Heat shock proteins (hsp's) are thought to be involved in this process because it was discovered that MB formation is induced by heat shock in drug-primed mice. It has been reported that ubiquitin and a mutant form of ubiquitin (UBB(+1)) are found in aggresomes formed in the neurons in Alzheimer's disease and in the liver MBs in various liver diseases. In addition, hsp 70 has been found in aggresomes in Alzheimer's and in MBs in drug-primed mice. Therefore, we hypothesized that hsp's might be involved in MB formation in human liver diseases. Liver biopsy sections were double-stained using ubiquitin and hsp 70 or 90b antibodies. Both hsps 70 and 90b were found in MBs in all liver diseases investigated including primary billiary cirrhosis, nonalcoholic steatohepatitis, hepatitis B and C, idiopathic cirrhosis, alcoholic hepatitis, and hepatocellular carcinoma. Ubiquitin and the hsp's colocalized in all MBs in the diseased liver sections. These results indicate that hsp involvement in MB formation is similar to that seen in aggresome formation in other conformational diseases.     

Hepatocyte cytokeratins are hyperphosphorylated at multiple sites in human alcoholic hepatitis and in a mallory body mouse model

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.     

Epigenetic mechanisms regulate Mallory Denk body formation in the livers of drug-primed mice

The mechanism of Mallory Denk body formation is still not fully understood, but growing evidence implicates epigenetic mechanisms in MDB formation. In a previous study the epigenetic memory of MDB formation remained intact for at least 4 months after withdrawal from the DDC diet. In the present study, mice were fed a diet containing DDC or a diet containing DDC and S-adenosylmethionine (SAMe) to investigate the epigenetic memory of MDB formation. DDC feeding caused an increase in histone 3 acetylation, a decrease in histone 3 trimethylation, and an increase in histone ubiquitinylation. The addition of SAMe to the DDC diet prevented the DDC induced decrease of H3K4 and H3K9 trimethylation and the increase in histone ubiquitinylation. Changes in histone modifying enzymes (HATs and HDACs), were also found in the liver nuclear extracts of the DDC/SAMe fed mice. Data mining of microarray analysis confirmed that gene expression changed with DDC refeeding, particularly the SAMe metabolizing enzymes, Mat2a, AMD, AHCY and Mthfr. SAMe supplementation prevented the decrease of AHCY and GNMT, and prevented the increase in Mthfr, which provides a mechanism to explain how DDC inhibits methylation of histones. The results indicate that SAMe prevented the epigenetic cellular memory involved in the MDB formation.     

High molecular weight component of Mallory bodies detected by a monoclonal antibody.

To identify Mallory body (MB) constituents, monoclonal antibodies to murine MBs induced by long-term griseofulvin (GF) feeding were produced. One of these, antibody MM 120-1, specifically reacted in immunofluorescence microscopy with MBs in all developmental stages but not with other cell structures of human and mouse liver and other organs. The MM 120-1 antigen was present in murine MBs induced by griseofulvin or 3,5-diethoxycarbonyl-1,4-dihydrocollidine feeding and also in human MBs in livers of patients with alcoholic hepatitis. In immunoblots, the MM 120-1 antigen was detectable in the high molecular weight fraction of MB preparations, most of which remained in the well and at the interphase between stacking and resolving gel. No reactivity with cytokeratin polypeptides of different conformational states (i.e., isolated cytoker atin components A and D, heterotetramers A2D2, reconstituted intermediate filaments) was found. It is concluded that the antibody MM 120-1 is a highly specific and sensitive marker for murine and human MBs recognizing a high molecular weight nonkeratin component. This component could play a central role in the pathogenesis of MBs.     

High molecular weight components are main constituents of Mallory bodies isolated with a fluorescence activated cell sorter.

Mallory bodies (MBs) are cytoplasmic filamentous aggregates containing cytokeratin (CK) material. They occur in hepatocytes of patients with alcoholic liver disease (i.e., alcoholic hepatitis) and can also be induced experimentally in mice by chronic griseofulvin intoxication. To further investigate components and mechanisms involved in MB formation, a new method for MB purification was established. MBs present in a liver homogenate of griseofulvin-fed mice were labeled with a murine monoclonal antibody specific for MBs and a second fluorescein isothiocyanate-conjugated (anti-mouse IgG and IgM) antibody and subsequently isolated by two sequential sorting procedures using a fluorescence activated cell sorter (FACS). Purity of MB isolates was over 90% as revealed by computer analysis of sorting signals and fluorescence and electron microscopy. Electrophoretic separation on sodium dodecyl sulfate-polyacrylamide gels revealed three MB-related polypeptides with apparent molecular masses of 48, 55, and 65 kilodaltons but most of the highly purified MB material did not enter the gel or remained at the interphase between stacking and resolving gels. Western blotting with CK-specific antibodies showed the presence of CK epitopes in the high molecular weight MB material, which has a similar amino acid composition as normal liver CKs. These results establish that very high molecular weight material is the main constituent of MBs and suggest that a post-translational modification of CKs by covalent crosslinks is a principal mechanism of MB pathogenesis.     

Aggresome formation in liver cells in response to different toxic mechanisms: role of the ubiquitin-proteasome pathway and the frameshift mutant of ubiquitin.

Aggresomes form in cells when intracellular proteins undergo conformational changes, as in so-called conformational diseases. This phenomenon has been observed in the liver and brain and in cell culture in response to abnormal protein formation, such as mutant proteins. In the case of the brain the frameshift mutant ubiquitin (UBB+1) is involved. Mallory body formation in the liver is one example of this phenomenon in vivo. Mallory body formation is common in a variety of liver diseases of diverse pathogenesis. The study of the Mallory body forming model indicated that drug-conditioned hepatocytes form Mallory bodies when mice are given colchicine, ethanol, okadaic acid, or exposure to heat shock. These findings suggest that aggresome formation is a common pathway of liver injury due to diverse mechanisms. To further characterize the role of this common pathway, drug-primed mice were exposed to different types of liver injury, i.e., using such drugs as thioacetamide, galactosamine, tautomycin, and the proteasome inhibitor PS341. Mallory body formation was induced by treatment with all the toxins tested, giving credence to the proposal that aggresome formation in the liver is a common pathway in response to different primary mechanisms of liver injury. The frameshift mutant UBB+1 was invariably found to colocalize with ubiquitin in the Mallory body, indicating its essential involvement in the mechanism of MB formation.     

The proteasome inhibitor, PS-341, causes cytokeratin aggresome formation

Mallory body (MB) experimental induction takes 10 weeks of drug ingestion. Therefore, it is difficult to study the dynamics and mechanisms involved in vivo. Consequently, an in vitro study was done using primary tissue culture of hepatocytes from drug-primed mice livers in which MBs had already formed. The hypothesis to be tested was that MBs are cytokeratin aggresomes, which form when hepatocytes have a defective ubiquitin-proteasome pathway by which turnover of cytokeratin proteins is prevented. To test this hypothesis, primary tissue cultures of the hepatocytes from normal and MB-forming livers were incubated with the proteasome inhibitor PS-341 and then the cytokeratin filaments and the filament connecting proteins, that is, β-actin, and ZO1, were visualized by immunofluorescence microscopy. PS-341 caused detachment of the cytokeratins from the cell surface plasma membrane. The cytokeratin filaments retracted toward the nucleus and cytokeratin aggresomes formed. In human livers, MBs showed colocalization of cytokeratin-8 (CK-8) with ubiquitin but not with β-actin or ZO1. Mouse hepatoma cell lines were studied using PS-341 to induce cytokeratin aggresome formation. In these cell lines, the cytokeratin filaments first retracted toward the nucleus then formed cytokeratin-ubiquitin aggresomes polarized at one side of the nucleus. At the same time, the cells became dissociated from each other, however. The results simulated MB formation. MBs differ from cytokeratin aggresomes both morphologically and in ultrastructure.     

High amount of epsilon-(gamma-glutamyl)lysine cross-links in Mallory bodies.

Alcoholic hepatitis, the most severe form of alcoholic liver disease, is associated with inflammation, liver cell necrosis, and the appearance of Mallory bodies (MBs) in hepatocytes. Identical MBs can be experimentally induced in mouse livers by chronic griseofulvin or 3,5-diethoxycarbonyl-1,4-dihydrocollidine treatment. MBs are filamentous cytoplasmic inclusions containing insoluble high molecular weight protein material. Covalent polymerization of intracellular proteins may occur through formation of epsilon-(gamma-glutamyl)lysine cross-links catalyzed by Ca(2+)-dependent transglutaminases. Therefore, isolated experimentally-induced MBs were analyzed for the presence of epsilon-(gamma-glutamyl)lysine bonds. Highly purified MBs contained 19.7 nmole (griseofulvin-induced) and 14.4 nmoles (3,5-diethoxycarbonyl-1,4-dihydrocollidine induced) of isodipeptide linkage, respectively, per mg of protein. These results suggest that transglutaminase-induced cross-linking of proteins plays a major role in MB formation.     

Ubiquitin is present on the cytokeratin intermediate filaments and Mallory bodies of hepatocytes

To investigate the relationship of cytokeratin intermediate filaments (IFs) and Mallory bodies (MBs) to the regulatory protein ubiquitin, the griseofulvin-fed mouse was examined by double-label immunocytochemistry. In controls, immunofluorescence of hepatocytes showed that an antiserum specific to ubiquitin stained the cell border and the cytoplasm as well as the nuclear rim. In griseofulvin-fed liver cells, the MBs induced by this treatment were stained in an identical pattern by the antiserum to ubiquitin and a monoclonal antibody specific to cytokeratin (TROMA 1). Upon examination of the immunoreaction at the ultrastructural level, the ubiquitin antiserum decorated the cytokeratin filaments as well as MB filaments. Particularly striking was the coincidence of localization of TROMA 1 and ubiquitin epitopes, many IF surrounding MBs being either intensely decorated or alternatively nonimmunoreactive. These results suggest that normal cytokeratin IFs are lightly ubiquitinated, whereas MBs are heavily ubiquitinated. Immunoblot analysis of extracted cytoskeletal proteins separated by gel electrophoresis showed that extensive ubiquitination of peptides was present in the livers of the griseofulvin-fed mice. Further, the lack of ubiquitin and TROMA 1 epitopes in some liver IF suggest that loss of the TROMA 1 epitope may lead to concomitant loss of the ability to bind ubiquitin. Although the role ubiquitin plays in Mallory body formation remains to be elucidated, we suggest that its significance here may be related to its normal association with cytokeratin.     

Common epitopes of human and murine Mallory bodies and Lewy bodies as revealed by a neurofilament antibody.

The antibody SMI 31, which is directed against a phosphorylated epitope, associated with neurofilaments and recognizes Lewy bodies in brains of patients with Parkinson's disease (Bancher C, Lassmann H, Budka H, Jellinger K, Grundgke-Iqbal I, Iqbal K, Wiche G, Seitelberger F, Wisniewski H: J Neuropathol Exp Neurol 1:81, 1989), decorated in immunofluorescence microscopy Mallory bodies (MBs) present in livers of mice chronically treated with griseofulvin and 3,5-diethoxycarbonyl-1,4-dihydrocollidine. In immunoblots it recognized very acidic MB components in a molecular weight range between 55 and 69.5 kilodaltons in addition to poorly soluble high molecular weight material. Moreover, an antibody to tau protein showed similar reactivities in immunofluorescence microscopy and immunoblotting experiments. Both antibodies also stained MBs in human liver with alcoholic hepatitis. These observations support and extend earlier findings which indicate that several intermediate filament-related cellular inclusion bodies, including MBs, share a variety of morphologic, structural and antigenic features. They also suggest the involvement of tau or tau-like proteins in MB formation.     

Mallory body formation is associated with epigenetic phenotypic change in hepatocytes in vivo

Microarrays were done on the livers of mice fed DDC for 10 weeks, withdrawn 1 month (DDC primed livers) and refed 6 days, and compared with mice fed the control diet. The expression of a large number of genes changed when DDC was fed or refed. A Venn diagram analysis identified 649 genes where gene expression was changed in the same direction. The epigenetic memory of the DDC primed liver involved an increase in the expression of ubiquitin D, alpha fetoprotein, connective tissue growth factor, integrin beta 2, DNA methyl transferase 3a and DNA damage-inducible 45 gamma. DNA methyl transferase 3b was down-regulated as was Cbp/p300. When DDC was refed, DNA methyltransferase and histone deacetylase were up-regulated as shown by microarray analysis. Histone3 lysine9 acetylation was increased by DDC and DDC refeeding and DNA methyltransferases were not changed as shown by Western blot analysis. The data suggest the concept that the epigenetic memory that explains why DDC primed hepatocytes form MBs in 7 days of DDC refeeding is primarily the result of epigenetic modifications of gene expression through changes in histone acetylation and methylation, as well as DNA methylation.     

Cytokeratin 8 protects from hepatotoxicity, and its ratio to cytokeratin 18 determines the ability of hepatocytes to form Mallory bodies

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.     

Experimental induction of hepatocellular hyalin (Mallory bodies) in mice by griseofulvin treatment. 1. Light microscopic observation.

Griseofulvin (GF) feeding of mice resulted in protoporphyria, liver cell damage, bile duct alterations, and finally hepatoma formation. In addition, hepatocellular hyalin developed, resembling in its morphology classic Mallory bodies (MB) as seen in alcoholic and nonalcoholic liver disorders in man. Liver cells containing MB often displayed features of severe cell damage and MB were finally released into the sinusoids and degraded by macrophages. The rapid disappearance of MB following GF discontinuation and the reappearance after resumption of GF feeding suggest an intimate relationship between metabolic alterations in the hepatocytes exerted by the drug and MB formation. This assumption is further supported by the fact that MB change their tinctoreal properties in chromotrope aniline blue-stained sections after GF discontinuation, possibly relfecting degeneration. Long term GF treatment apparently primed the liver for MB formation since the cells were able to respond almost instantly with MB to a GF challenge after a 1-month GF-free period.