Mallory-Denk-bodies: lessons from keratin-containing hepatic inclusion bodies.

Inclusion bodies are characteristic morphological features of various neuronal, muscular and other human disorders. They share common molecular constituents such as p62, chaperones and proteasome subunits. The proteins within aggregates are misfolded with increased beta-sheet structure, they are heavily phosphorylated, ubiquitinylated and partially degraded. Furthermore, involvement of proteasomal system represents a common feature of virtually all inclusions. Multiple aggregates contain intermediate filament proteins as their major constituents. Among them, Mallory-Denk bodies (MDBs) are the best studied. MDBs represent hepatic inclusions observed in diverse chronic liver diseases such as alcoholic and non-alcoholic steatohepatitis, chronic cholestasis, metabolic disorders and hepatocellular neoplasms. MDBs are induced in mice fed griseofulvin or 3,5-diethoxycarbonyl-1,4-dihydrocollidine and resolve after discontinuation of toxin administration. The availability of a drug-induced model makes MDBs a unique tool for studying inclusion formation. Our review summarizes the recent advances gained from this model and shows how they relate to observations in other aggregates. The MDB formation-underlying mechanisms include protein misfolding, chaperone alterations, disproportional protein expression with keratin 8>keratin 18 levels and subsequent keratin 8 crosslinking via transglutaminase. p62 presence is crucial for MDB formation. Proteasome inhibitors precipitate MDB formation, whereas stimulation of autophagy with rapamycin attenuates their formation.

Intermediate filament cytoskeleton of the liver in health and disease.

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.

How a cell deals with abnormal proteins. Pathogenetic mechanisms in protein aggregation diseases.

Defective protein folding is responsible for many diseases. Although these diseases seem to be quite diverse at the first glance, there is evidence for common pathogenetic principles. The basis of the pathological changes is the cell's inability to prevent protein misfolding, to revert misfolded proteins to normal or to eliminate misfolded proteins by degradation. This could result in deposition of potentially cytotoxic protein aggregates (protein aggregation diseases). Chronic degenerative diseases of the central nervous system (e.g. Alzheimer's and Parkinson's disease), the amyloidoses, but also chronic liver diseases, for example alcoholic and nonalcoholic steatohepatitis, belong to this category of disorders. This review highlights general pathogenic principles of protein aggregation diseases based on immunohistochemical and biochemical studies as well as observations in a mouse model for protein aggregation in the context of alcoholic and nonalcoholic steatohepatitis. The cellular defense mechanisms involved in protein quality control as well as the pathogenesis of protein aggregation diseases will be discussed.

Are the Mallory bodies and intracellular hyaline bodies in neoplastic and non-neoplastic hepatocytes related?

Mallory bodies (MBs) and intracellular hyaline bodies (IHBs) are cytoplasmic hepatocellular inclusions that consist of aggregated proteins. MBs are characteristically associated with alcoholic and non-alcoholic steatohepatitis, but may also be found in chronic cholestatic and metabolic (eg copper intoxication) diseases and hepatocellular neoplasms, particularly hepatocellular carcinomas. IHBs have hitherto only been described in hepatocellular carcinoma cells. In the present study hepatocellular carcinomas (HCCs) and a case of idiopathic copper toxicosis were evaluated with respect to the presence and mutual relationship of MBs and IHBs. IHBs alone were present in 8.6%, MBs alone in 16.1% and both types of inclusion in 7.5% of HCCs. It is shown that IHBs may also occur in non-neoplastic hepatocytes in association with idiopathic copper toxicosis, together with MBs. In HCCs and idiopathic copper toxicosis, MBs and IHBs may be present within the same cell. Moreover, hybrid inclusions holding an intermediate position between MBs and IHBs regarding light microscopy, ultrastructure and composition exist. MBs and IHBs contain p62, a stress-inducible adapter protein, as the major constituent. In MBs p62 is associated with keratins, whereas classical IHBs lack keratins. Light microscopic, electron microscopic and immunohistochemical data suggest a close pathogenetic relationship between MBs and IHBs. Both types of inclusion are the result of over-expression and accumulation of the stress protein p62. If p62 is induced alone, or at least prevails, IHBs may arise by aggregation. However, if abnormal keratins are present in addition to p62, p62 associates and co-aggregates with keratins, finally leading to classical MBs.

[Alcoholic and non-alcoholic steatohepatitis]. / Alkoholische und nicht-alkoholische Steatohepatitis.

Morphologic criteria of steatohepatitis are steatosis, ballooning of hepatocytes, often but not constantly associated with Mallory bodies, pericellular fibrosis and inflammation. Liver cirrhosis follows in about 20-50%. With respect to etiology an alcoholic and non-alcoholic type can be distinguished, the latter being a characteristic hepatic lesion associated with the metabolic syndrome (type II diabetes, insulin resistance, obesity, dyslipidemia). Ballooning of hepatocytes as well as Mallory body formation are associated with a disturbance of the keratin intermediate filament cytoskeleton. Mallory bodies are protein aggregates consisting of keratin (particularly keratin 8), p62, a stress-induced adapter protein involved in signal transduction pathways, heat shock proteins, and ubiquitin. Oxidative stress is involved in Mallory body formation. Major sources of oxidative stress in alcoholic and non-alcoholic steatohepatitis are the microsomal biotransformation system (cytochrome P-450) and the mitochondria, together with an impaired antioxidant defense system. Oxidative stress leads to misfolding/unfolding, abnormal phosphorylation of keratins and disturbance of keratin 8: keratin 18 ratio, and thus interferes with intermediate filament assembly. Moreover, impairment of cellular defense against abnormal proteins, i. e. chaperone action and proteasomal degradation, leads to the accumulation of abnormal aggregation--prone keratins (particularly keratin 8) which after ubiquitination associate with the stress-induced ubiquitin-binding protein p62 to form Mallory bodies. Thus, Mallory body formation resembles an "off-folding" protein response of the amyloid type. These pathogenetic principles of the human disease are supported by immunohistochemical and gene expression studies in experimental animals and by transfection experiments in tissue culture cells.

Sequence of events in the assembly of Mallory body components in mouse liver: clues to the pathogenesis and significance of Mallory body formation.

BACKGROUND/AIMS: Chronic intoxication of mice with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) or griseofulvin (GF) results in appearance of Mallory bodies (MBs) and alterations of the keratin cytoskeleton, which are reversible upon drug withdrawal but recur after readministration within 2-3 days. METHODS: DDC- or GF-treated and recovered mice were reintoxicated with the original drugs but also colchicine and lumicolchicine. Cytoskeletal alterations of hepatocytes and MB formation were monitored by immunofluorescence microscopy using keratin, MB-specific antibodies, antibodies to phosphoepitopes and to HSP70. Keratin 8/18 mRNA expression and protein levels were determined by competitive reverse transcription-polymerase chain reaction, in situ-hybridization and western blotting. RESULTS: Duration of pretreatment was important for the efficiency of MB triggering. Rapid increase of keratin 8/18 mRNA and proteins was found in all reintoxicated mice concomitant with MB formation, whereby keratin 8 prevailed over keratin 18. Keratins and a protein with heat shock characteristics (M(M) 120-1 antigen) were the earliest detectable MB components, whereas ubiquitination and phosphorylation followed later. CONCLUSIONS: Overproduction of keratins is a major but not the only step responsible for MB formation. Additional components (e.g. M(M) 120-1 antigen) and excess of keratin 8 over keratin 18 are essential.

Effects of ursodeoxycholic and cholic acid feeding on hepatocellular transporter expression in mouse liver.

BACKGROUND AND AIMS: Cholestasis is associated with retention of potentially toxic bile acids and alterations in hepatocellular transporter expression. Conversely, nontoxic ursodeoxycholic acid (UDCA) stimulates bile secretion and counteracts cholestasis. This study aimed to determine the effects of UDCA and cholic acid (CA) on the expression of hepatocellular transporters for bile acids (Ntcp, Bsep), organic anions (Oatp1, Mrp2), organic cations (Mdr1a/b), and phospholipids (Mdr2) in mouse liver. METHODS: Bile flow/composition was analyzed in UDCA- or CA-fed mice. Transporter expression was studied by reverse-transcription polymerase chain reaction, Western blotting, and immunofluorescence microscopy. RESULTS: UDCA had no effect on basolateral Ntcp and down-regulated Oatp1, whereas canalicular Bsep and Mrp2 were up-regulated. CA down-regulated basolateral Ntcp and Oatp1, whereas canalicular Bsep, Mrp2, and Mdr1a/b were up-regulated. Neither UDCA nor CA affected Mdr2 expression. Both UDCA and CA stimulated biliary bile acid and glutathione excretion, although only CA increased phospholipid and cholesterol excretion. CONCLUSIONS: Down-regulation of basolateral and up-regulation of canalicular transporters in response to CA may represent a defense mechanism, in an attempt to prevent hepatocellular accumulation of potentially toxic bile acids. The therapeutic effects of UDCA may be caused in part by stimulation of canalicular transporter expression in the absence of hepatocellular toxicity.

Hepatobiliary transporter expression in percutaneous liver biopsies of patients with cholestatic liver diseases.

Reduced hepatobiliary transporter expression could explain impaired hepatic uptake and excretion of bile salts and other biliary constituents resulting in cholestasis and jaundice. Because little is known about alterations of hepatobiliary transport systems in human cholestatic liver diseases, it was the aim of this study to investigate such potential changes. Hepatic mRNA levels in hepatobiliary transport systems for bile salts (NTCP, BSEP), organic anions (OATP2, MRP2, MRP3), organic cations (MDR1), phospholipids (MDR3), and aminophospholipids (FIC1) were determined in 37 human liver biopsies and control livers by competitive reverse-transcription polymerase chain reaction (RT-PCR). Transporter tissue distribution was investigated by immunofluorescence microscopy. In patients with inflammation-induced icteric cholestasis (mainly cholestatic alcoholic hepatitis), mRNA levels of NTCP, OATP2, and BSEP were reduced by 41% (P <.001), 49% (P <.005), and 34% (P <.05) compared with controls, respectively. In addition, NTCP and BSEP immunostaining was reduced. MRP2 mRNA levels remained unchanged, but canalicular immunolabeling for MRP2 was also decreased. mRNA expression of MRP3, MDR1, MDR3, and FIC1 remained unchanged. In contrast to the alterations of transporter expression in inflammation-induced icteric cholestasis, transporter expression did not change in anicteric cholestasis caused by primary biliary cirrhosis (PBC) stages I and II. In conclusion, reduced expression of hepatobiliary transport systems for bile salts and other organic anions may contribute to inflammation-induced cholestasis in humans. Reduction of transporter gene expression can occur at the mRNA level as observed for NTCP, OATP2, and BSEP. However, reduced MRP2 immunostaining in the presence of conserved MRP2 mRNA levels suggests an additional role for posttranscriptional/posttranslational mechanisms.

[Alcoholic and nonalcoholic steatohepatitis. Histopathologic and pathogenetic considerations]. / Alkoholische und nichtalkoholische Steatohepatitis. Histopathologie und pathogenetische Uberlegungen.

Alcoholic (ASH) and nonalcoholic (NASH) steatohepatitis show an almost identical morphology. Since the clinical picture is not characteristic, liver biopsy is still the diagnostic gold standard. ASH and NASH are morphologically characterized by a combination of steatosis, hepatocellular injury (ballooning degeneration, apoptosis, necrosis), perivenular and pericellular fibrosis, and inflammation (mostly neutrophils). A definitive differentiation of ASH and NASH is only possible by exclusion of alcohol abuse. Although NASH comprises a syndrome with a multifactorial etiology, adipositas seems to be the most constant associated causal factor. The pathogenesis of both diseases is still unclear. Clinical evidence and experimental studies suggest an important toxic role of reactive oxygen species (oxidative stress). According to our experience, ballooning of hepatocytes is a constant morphologic feature of ASH and NASH and already present in the early stages of disease. Ballooned cells often (but not always) contain Mallory bodies (alcoholic hyalin), which are irregular cytoplasmic inclusions consisting of keratins and nonkeratin components, including ubiquitin. Ballooning is associated with a disturbance and finally almost disappearance of the keratin-intermediate filament cytoskeleton. In our studies on the pathogenesis of ASH and NASH, we concentrated on these cytoskeletal alterations and Mallory body formation. It could be shown that in the early stages overexpression and hyperphosphorylation of keratins take place. Moreover, the 1:1 ratio of keratin type I (keratin 18) and type II (keratin 8) necessary for the assembly of intermediate filaments is disturbed and the equilibrium shifted toward keratin 8. Thus, the pool of soluble keratin 8 increases. The resulting keratin monomers are sensitive to misfolding and either degraded or aggregated as inclusion bodies. If the proteolytic capacity is impaired (e.g., by inhibition of the proteasomal system) in the chronically stressed cell aggregation prevails,finally leading to Mallory body formation. Convincing evidence exists on the basis of clinical and experimental studies that keratins exert a nonskeletal protective function in simple epithelia (e.g., liver cells). Disturbance of the keratin system may thus significantly contribute to cell damage.

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.

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.

Analysis of intracytoplasmic hyaline bodies in a hepatocellular carcinoma. Demonstration of p62 as major constituent.

Intracytoplasmic hyaline bodies (IHBs) resemble inclusions in hepatocellular carcinoma cells, which so far have escaped further characterization. A relationship to Mallory bodies was suggested on the basis of light microscopy and filamentous ultrastructure. A hepatocellular carcinoma containing numerous IHBs was studied. Our studies revealed immunoreactivity of IHBs with the monoclonal antibodies SMI 31 and MPM-2, which recognize hyperphosphorylated epitopes present on paired helical filaments in Alzheimer's disease brains (SMI 31) or on diverse proteins hyperphosphorylated by mitotic kinases in the M-phase of the cell cycle (MPM-2). One- and two-dimensional gel electrophoresis of tumor extracts followed by immunoblotting with SMI 31 and MPM-2 antibodies revealed a major immunoreactive protein with an apparent molecular weight between 62 and 65 kd, which was resolved into several highly acidic (pH 4.5) protein components in two-dimensional gels. This protein was undetectable in non-neoplastic liver tissue. Sequence analysis identified the SMI 31 and MPM-2 immunoreactive material as p62, indicating that p62 is a major constituent of IHBs. p62 is an only recently discovered protein that is a phosphotyrosine-independent ligand of the SH2 domain of p56(lck), a member of the c-src family of cytoplasmic kinases. Moreover, p62 binds ubiquitin and may act as an adapter linking ubiquitinated species to other proteins. These features suggest a role of p62 in signal transduction and possibly also carcinogenesis. IHBs observed in the hepatocellular carcinoma cells presented are the first indications of a role of p62 in disease.

Atypical ductular proliferation and its inhibition by transforming growth factor beta1 in the 3,5-diethoxycarbonyl-1,4-dihydrocollidine mouse model for chronic alcoholic liver disease.

Many acute and chronic liver diseases are often associated with atypical ductular proliferation (ADP). These ADPs have gained increasing interest since a number of recent observations suggest that ADPs may represent progenies of the putative liver stem cell compartment. In this study, we show that feeding mice with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) results in persistent proliferation of primitive ductules with poorly defined lumens. Similar to oval cell proliferation in other rodent models as well as in various human liver diseases, DDC-induced ADP originated from the portal tract, spread into the hepatic lobule, and was associated closely with appearance of hepatocytes harboring an antigen (A6), which normally is expressed in biliary epithelium. Furthermore, DDC treatment severely inhibited the regenerative capacity of mice after partial hepatectomy. The development of ADP was selectively blocked in DDC-fed TGF-beta1 transgenic mice producing active TGF-beta1 in the liver and no accumulation of new hepatocytes expressing the A6 antigen was observed. Moreover, the transforming growth factor beta1 (TGF-beta1) transgenic mice did not survive beyond 3 weeks from starting the DDC-containing diet. The results suggest that persistent activation of the hepatic stem cell compartment is essential for liver regeneration in the DDC model and that active TGF-beta1 may negatively control activation of stem cells in the liver. These data further emphasize the relevance of the DDC model as an experimental tool for studying chronic liver diseases.

Altered microtubule-associated tau messenger RNA isoform expression in livers of griseofulvin- and 3,5-diethoxycarbonyl-1, 4-dihydrocollidine-treated mice.

Tau proteins belong to the family of microtubule-associated proteins (MAPs), which so far have been mostly detected in neuronal cells. Different domains on the protein serve different functions. By alternative splicing, several mRNAs and tau isoforms are created from one gene, which contain these functionally important domains to various degrees, and thus differ in their microtubule-related properties. In the present article, several novel observations are reported. Tau mRNA and proteins have been identified and further characterized in mouse liver. It is shown on the basis of mRNA determinations that at least three tau isoforms differing particularly with respect to their amino-terminal domains are present in mouse liver. The major and predominant isoform (isoform 1) lacks portions encoded by exons 2 and 3, which are responsible for cross-talk of microtubules with their environment ("projection domain"). Moreover, mRNA encoding tau protein with four repeats of the microtubule binding domain predominate in embryonal as well as adult mouse liver in contrast to brain, in which a shift from the predominant three-repeat isoform to the four-repeat isoform characterizes the transition from the embryonic to the adult stage. Intoxication with griseofulvin (GF) or 3,5-diethoxycarbonyl-1, 4-dihydrocollidine (DDC) significantly affects in a reversible manner the levels of tau mRNA as well as isoform ratios in mouse liver, but not in mouse brain. Tau mRNAs are significantly increased in intoxicated mouse livers. Moreover, a shift to isoform 1 lacking exons 2 and 3 occurs. However, the increase in liver tau protein was less than expected from increased mRNA levels, which could be the result of translational or posttranslational regulation. The consequences on microtubular function are as yet unclear, but impairment can be expected because the overexpressed tau mRNA isoform lacks the domain that mediates interaction of microtubules with their environment. On the other hand, the ratio of polymerized (microtubules) to nonpolymerized tubulin remained unaffected.

Identification of p62, a phosphotyrosine independent ligand of p56lck kinase, as a major component of intracytoplasmic hyaline bodies in hepatocellular carcinoma.

Intracytoplasmic hyaline bodies (IHBs) resemble a peculiar type of cytoplasmic inclusions in cells of hepatocellular carcinoma (HCC) which so far have escaped further characterization. In order to determine protein composition of IHBs we investigated tissue of a HCC containing numerous IHBs by immunohistochemistry and Western blot analysis using a large panel of different antibodies. Our studies revealed immunoreactivity of IHBs with the monoclonal antibodies SMI 31 and MPM-2 which recognize hyperphosphorylated epitopes present on paired helical filaments in Alzheimer's disease brains (SMI 31) and on proteins hyperphosphorylated by mitotic kinases (MPM-2), respectively. In two-dimensional Western blots of HCC extracts SMI 31 and MPM-2 antibodies detected a 62 to 65 kD protein with an isoelectric point around 4.5. Microsequencing identified this protein as p62, a recently identified phosphotyrosine-independent ligand of the SH2 domain of tyrosine kinase p56lck. Immunoreactivity of p62 protein spots with antibodies to phosphorylated epitopes (i.e. SMI 31 and MPM-2) suggest that p62 is highly phosphorylated in IHBs. This is the first report on accumulation of p62 as cellular inclusions and its association with human disease.

Experimental Mallory body formation is accompanied by modulation of the expression of multidrug-resistance genes and their products.

Mallory bodies (MBs) are characteristic morphological features of alcoholic hepatitis and are also found in other chronic liver disorders and hepatocellular neoplasms. MBs can be produced in mouse liver by chronic administration of the porphyrinogenic drugs griseofulvin (GF) and 3,5-diethoxy-carbonyl-1,4-dihydrocollidine (DDC). The mechanisms causing the formation of MBs are poorly understood, and the significance of MB formation during the course of liver disease remains unclear. We investigated the relationship between the mechanisms underlying the formation of MBs and the regulation of multidrug resistance (mdr) genes and their products, the P-glycoproteins (Pgp). Immunofluorescence microscopy using the monoclonal antibody C219 revealed an increase of Pgp expression in almost all hepatocytes after 3 to 8 days of feeding mice DDC- and GF-containing diets. However, after approximately 4 weeks of DDC and approximately 8 weeks of GF feeding, when the first small MBs appeared and loosening and diminution of keratin intermediate filament (KIF) cytoskeleton occurred in some hepatocytes, a decrease or loss of Pgp staining in affected hepatocytes was observed. After feeding mice DDC for 6 weeks and GF for 12 weeks, many hepatocytes contained MBs and displayed a disruption of the immunohistochemically demonstrable KIF meshwork. Double immunofluorescence microscopy with the keratin polyclonal antibody and the mab C219 at this time point revealed a complete loss of Pgp staining in affected cells, although remaining hepatocytes with unaltered KIF meshwork showed a strong reaction with the C219 antibody. Northern blot analyses revealed a significant increase of mdr2 mRNA and, to a lesser extent, of mdr1a mRNA in the livers of DDC- and GF-fed animals.

Disturbance of keratin homeostasis in griseofulvin-intoxicated mouse liver.

BACKGROUND: Alterations of the hepatocytic intermediate filament (IF) cytoskeleton, i.e., derangement and diminution of the keratin network and appearance of cytoplasmic aggregates of keratin-containing material, termed Mallory bodies, are characteristic features of human alcoholic hepatitis. Mallory bodies can be experimentally produced in mouse liver by chronic griseofulvin (GF) administration. GF intoxication of mice is, therefore, a suitable model to study the mechanisms of Mallory body formation and related cytoskeletal changes. EXPERIMENTAL DESIGN: To investigate the correlation between morphologic alterations of the keratin cytoskeletal network and the mRNA levels for liver keratins A (8) and D (18) in this pathologic situation immunohistochemical studies and northern blot analyses were performed. The amount of mRNA for both keratins was also analyzed by nuclease S1 protection assay. RESULTS: In GF-treated livers (4 months of treatment) an increase of mRNA for both liver keratins was found. This increase of mRNA was unexpected under these conditions, since in longterm GF-fed animals, the amount of keratin IFs was reduced as revealed by immunofluorescence and electron microscopy and by biochemical analysis of keratin proteins. In livers treated for 2 months with GF the IF meshwork seemed to be still intact, but the increase of RNA was already detectable indicating that alterations of keratin mRNA precede detectable morphologic alterations. When using this mRNA for in vitro translation experiments, strong keratin polypeptide spots could be detected by autoradiography of 2-dimensional gels. CONCLUSIONS: These results strongly suggest that in vivo under the conditions of GF intoxication posttranslational modifications, like phosphorylation, proteolysis and covalent cross-linking, could influence IF homeostasis and interfere with IF assembly. Increase of mRNA for liver keratins despite IF protein reduction might be due to negative feedback regulation.