Early perturbations in keratin and actin gene expression and fibrillar organisation in griseofulvin-fed mouse liver.

BACKGROUND/AIMS: Long-term feeding of mice with a diet containing griseofulvin results in the formation of Mallory bodies, keratin K8 and K18 containing aggregates in hepatocytes. These bodies are biochemically and morphologically identical to the Mallory bodies that emerge in several human liver disorders. The aim of this study was to examine the contribution of K8 and K18 and actin to Mallory body formation. METHODS: Mice were fed griseofulvin over a period ranging from 1 day to 20 months. Hepatocyte morphology was monitored by immunocytochemistry, gene expression by Northern and run-off transcription assays, and protein level by Western blotting. RESULTS: Griseofulvin feeding induced a series of morphological alterations in hepatocytes that could be grouped into 3 phases: appearance of cholestasis during the first week (phase I), partial hepatocyte recovery at 3 months (phase II), and development of typical Mallory bodies after 3 to 5 months (phase III). All these cellular alterations were associated with perturbations in keratin and actin fibrillar status, coupled with increases in K8, K18 and actin mRNA steady-state level and, in K8 and K18 protein content. The transcriptional activity of the genes was not affected. CONCLUSIONS: Perturbations in keratin and actin gene expression and fibrillar organisation constitute early events in the griseofulvin-induced pathological process that in the long-term leads to Mallory body formation. The higher keratin and actin mRNA levels reflect significant increases in mRNA stability taking place at the early phase of griseofulvin intoxication in hepatocytes.

Reduction of cytochemical ecto-ATPase activities in keratin 8-deficient FVB/N mouse livers.

Keratin 8 (K8) and keratin 18 are the intermediate filament proteins that are expressed in hepatocytes. A K8-deficient FVB/N mouse is a unique animal model for assessing the contribution of keratin intermediate filaments (IFs) to the structural and functional integrity of hepatocytes. Hepatocytes from homozygous (-/-) K8-deficient mice manifest a reduced bile acid secretion and an increased fragility to mechanical stress and hepatotoxic drugs. Hepatocytes from heterozygous (+/-) mice are more susceptible to drug-induced injury. Immunofluorescent microscopy revealed that hepatocytes from (+/-) mice maintained K8 IFs and F-actin that are similar to those in wild-type (+/+) mouse hepatocytes. In (-/-) mouse hepatocytes, K8 protein was negative and F-actin presented a coarse and irregular pattern. Ecto-ATPase, detected by enzyme histochemistry and observed by electron microscopy, was reduced in the bile canaliculi of both (+/-) and (-/-) mouse livers, in comparison with that of (+/+) mouse livers. These results reveal for the first time different microscopical findings regarding the livers of these three genotypes. They also suggest that the reduction of ecto-ATPase plays a role in the increased fragility of (+/-) and (-/-) mouse livers.

Subcellular localization of G-proteins in primary-cultured mouse preadipocytes and adipocytes.

The subcellular localization of G5 alpha, Gi alpha 1&2, Gi alpha 3, and G beta was studied in primary-cultured undifferentiated and differentiated, lipid replete, adipose cells. The results show a distinct distribution for each of these G-proteins and differences between differentiated and undifferentiated cells. All the G-proteins examined had a cytoplasmic localization; only Gi alpha 1 and 2 showed a significant colocalization with the plasma membrane and this only in differentiated cells. Most studies using cells in culture have reported an intracellular localization for G-proteins, whereas in tissue sections the localization has been reported to be largely with the plasma membrane, with some intracellular localization. The results suggest that the cell-cell interactions or the specific geometry imposed by culture conditions favor the intracellular compared to peripheral localization of G-proteins. Alternately, the posttranslational modifications necessary for G-protein insertion in the plasma membrane may be deficient in cultured cells.

Comparison of the subcellular distribution of G-proteins in hepatocytes in situ and in primary cultures.

The subcellular localization of the heterotrimeric G-proteins in hepatocytes in situ was compared to that in hepatocytes in primary culture. The ability of various ligands to activate adenylyl cyclase (AC) in membrane preparations was also investigated. In hepatocytes in situ the G proteins were mainly localized at the plasma membrane while in hepatocytes in culture they were predominantly cytoplasmic. The localization of the G-proteins in hepatocytes in situ correlates with their role in signal transduction. In homogenates prepared from the cultured cells, ligands which stimulate AC via Gs alpha were without effect, which was consistent with the localization of Gs alpha in the cytoplasmic and nuclear compartments. The "relocalization" of the G proteins to the cytoplasm when cells are cultured suggests that transmembrane signalling may be regulated by cell differentiation and cell-cell and cell-extracellular matrix interactions.

Alterations of intermediate filaments in various histopathological conditions.

Intermediate filament proteins belong to a multigene family and constitute an important cytoskeletal component of most vertebrate cells. Their pattern of expression is tissue specific and is highly controlled during embryonic development. Numerous pathologies are known to be associated with modifications of intermediate filament organisation, although their precise role has not yet been elucidated. The present review focuses on the most recent data concerning the possible causes of intermediate filaments disorganization in specific pathologic conditions affecting the epidermis, the liver, and the nervous system. We discuss the formation of abnormal intermediate filament networks that arise as a consequence of mutations that directly affect intermediate filament structure or are induced by multifactorial causes such as modifications of post-translational processes and changes in the levels of expression.

Modifications in cytokeratin and actin in cultured liver cells derived from griseofulvin-fed mice.

BACKGROUND: Hepatocytes from mice fed griseofulvin (GF) for 8 months form Mallory bodies (MBs), which represent a pathologic state of intermediate filaments (IFs). The cellular mechanisms that lead to MB formation are not known. EXPERIMENTAL DESIGN: This study was aimed to investigate if MB formation could be related to modification in cytokeratin (CK) metabolism. Primary cultures of hepatocytes from control and GF livers were studied. Immunofluorescence microscopy was used to study the organization of the cytoskeleton in these cells. The hepatocytes were labeled with [35S]methionine or [32P]orthophosphate to study, respectively, the level of amino acid incorporation into IF proteins (CK 8 and CK 18) and their phosphorylation levels. The response to the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate stimulation of the phosphorylation of CK 8 and CK 18 was also elicited in contrast to control hepatocytes. RESULTS: We found that there was a change in the organization of actin and the IF network in the hepatocytes from GF-treated animals. This was associated with an increase in labeled amino acid incorporation into CK 8 and CK 18 as well as in actin. Although there was no significant difference in the absolute level of CK phosphorylation, we found modifications in the phosphorylated isomers of CK 8, the more phosphorylated isomers becoming more prominent. The treatment of the hepatocytes with 12-O-tetradecanoyl-phorbol-13-acetate did not induce changes in the level of CK phosphorylation in GF-pretreated hepatocytes. CONCLUSIONS: These results suggest that the modification of the IF network and MB formation are the consequences of increased CK synthesis and the modification of phosphorylation. They could alter the normal interaction of the IFs with different cellular components, which results in conformational changes of CKs and the reorganization of the IF network to the form of MBs.

Identification and localization of G(i) alpha 3 in the clonal adipocyte cell lines HGFu and Ob17.

The HGFu and Ob17 cell lines, derived from adipose tissue of lean (+/?) and ob/ob mice, respectively, express several G-protein peptides. Investigation of the expression and subcellular localization of the G(i) alpha 3 subunit showed that this peptide is associated with the Golgi apparatus. These findings indicate a role for this subunit in vesicular traffic and are in agreement with the view of the adipocyte as a secretory cell.

Identification and localization of G-proteins in the clonal adipocyte cell lines HGFu and Ob17.

HGFu and Ob17 are cell lines derived from adipose tissue of lean (+/?) and ob/ob mice, respectively. Neither adenylyl cyclase activity nor G protein abundance and subcellular distribution have been assessed previously in these cells. Cyclase activity was low and resistant to catecholamine stimulation in both cell lines. However, the enzyme could be stimulated to high levels by forskolin and Mn2+. Gs alpha (largely the long isoform), Gi alpha 2, and G beta were the major G protein subunits identified. The levels of G protein mRNA expression were similar in both cell lines and, unlike actin expression, did not change as a result of differentiation. Immunoblotting and ADP-ribosylation of the G peptides corroborated these results. Assessment of the subcellular localization of the subunits by indirect epifluorescence and scanning confocal microscopy showed that each of the subunits had a characteristic subcellular pattern. Gs alpha showed vesicular cytoplasmic and nuclear staining; Gi alpha 2 colocalized with actin stress fibers and disruption of these structures altered the distribution of Gi alpha 2; beta subunits showed some colocalization with the stress fibers as well as a cytoplasmic vesicular and nuclear pattern. As a result of differentiation, there was reorganization of the actin, together with the Gi alpha 2 and beta fibrous patterns. Both cell lines showed similar modifications. The induction of differentiation in these cells is therefore not associated with changes in adenylyl cyclase activity nor of the abundance of G-protein subunits, although reorganization of some of these subunits does accompany actin reorganization.

Synthesis of Mallory body, intermediate filament, and microfilament proteins in liver cell primary cultures. An electron microscopic autoradiography assay.

BACKGROUND: The synthesis of Mallory body (MB) and cytoskeletal proteins in hepatocytes have not been studied in vitro. To visualize the incorporation of radiolabeled amino acid into MBs and cytoskeletal filaments separately in the same cell, we developed a new technique using high resolution electron microscopic autoradiography. EXPERIMENTAL DESIGN: Primary cultures of hepatocytes isolated from griseofulvin (GF)-fed mice and control mice were used. The hepatocytes labeled with [35S]methionine were extracted with detergent. The detergent-resistant residual cytoskeleton was characterized by polyacrylamide gel electrophoresis and autoradiography. For light microscopic and electron microscopic autoradiography, the hepatocytes were labeled with [3H]methionine or [3H]leucine, then extracted with detergent and then embedded in plastic. To obtain high resolution of the labeled cytoskeleton, exposed ultrathin sections were treated by the gold latensification and physical developing methods and then examined by electron microscopy. RESULTS: Autoradiography of polyacrylamide gel electrophoresis showed [35S]methionine was incorporated into three major bands: cytokeratin 55, cytokeratin 49, and actin in control and GF hepatocytes. In GF hepatocytes, high molecular weight proteins that remained near the top of the gel (MB-derived proteins) were also labeled. The electron microscopic autoradiography revealed that the intermediate filaments, microfilaments, MBs, and nuclear matrix were labeled and the resolution of the labeling was high enough to localize the labeled amino acids incorporated into the filaments. The background was very low. The labeling using [3H]methionine was greater than [3H]leucine, but the pattern of labeling was the same. In control mice, hepatocytes intermediate filaments and microfilaments were heavily labeled at the cell border. In GF-fed mice, the MBs were heavily labeled compared with the intermediate filaments at the cell borders within the same cells. The intensity of labeling varied from cell to cell. CONCLUSIONS: These results suggested that the newly synthesized cytokeratin monomers were incorporated into the already polymerized filamentous network as well as the MB filaments in cell culture.

Reversible disassembly of transcription domains in lymphocyte nuclei during inhibition of RNA synthesis by DRB.

We are investigating the roles of RNA synthesis, chromatin structure and nuclear matrix organization in establishing and maintaining transcription domains, using mitogen stimulated lymphocytes as a model system. In a continuing study, the effects of the RNA polymerase inhibitor DRB and of its removal on nuclear organization have been examined by EM cytochemistry and by immunofluorescence labelling of the nuclear matrix PI1, Sm and nucleolar fibrillarin antigens. Chromatin, interchromatin granules and nucleoli were extensively restructured after DRB, as were matrix antigens. According to cytochemical staining properties, the conformation of DRB-induced condensed chromatin resembled that in partially stimulated lymphocytes. The nucleoplasmic fibrogranular RNP network appeared little altered, but the fibrillar proteinaceous interchromatinic regions, interpreted as representing the nuclear matrix in situ, were more affected. After removal of DRB, nuclei recovered the organization and transcriptional activity of controls within 8 h. These results suggest that the matrix subtending transcription domains remains stable when transcription is arrested, even though the chromatin and individual RNP components of the domains are disorganized. The data further indicate that absence of transcription is not solely accountable for the highly aggregated state of the chromatin in resting lymphocytes.

Differential phosphorylation of CK8 and CK18 by 12-O-tetradecanoyl-phorbol-13-acetate in primary cultures of mouse hepatocytes.

The phosphorylation of cytokeratin was investigated in primary cultures of hepatocytes. The two hepatocyte cytokeratins CK8 and CK18 (55,000 and 49,000 M(r) respectively) were phosphorylated, CK8 being more phosphorylated than CK18. Treatment of the hepatocytes with 150 nM 12-O-tetradecanoyl-phorbol-13-acetate (TPA) an activator of protein kinase C induced a transient increase in the level of phosphorylation of CK8 but not CK18. This effect was maximal after 15 min of TPA treatment and was maintained for up to 3 h. After 22 h of treatment with TPA, which down-regulates protein kinase C, CK8 phosphorylation was returned to the basal level. Further addition of TPA to the 22-h treated cells did not cause an increase in CK8 phosphorylation. Indirect immunofluorescence microscopy with a monoclonal antibody to CK8 indicated that while the addition of TPA induced the formation of granular cytokeratin aggregates in some hepatocytes, in most hepatocytes no major changes in the intermediate filament network were observed. Staining for actin showed that actin microfilaments were rapidly reorganized after the treatment and a loss of stress fibres were observed. We propose that CK8 is an in vivo substrate for protein kinase C and that the specific phosphorylation of CK8 plays a role in protein kinase C signal transduction.

Cytokeratin of apparent high molecular weight in livers from griseofulvin-fed mice.

Livers from mice fed griseofulvin (GF) for 12 months contain abnormal cytokeratin (CK) intermediate filaments (IFs) that aggregate to form Mallory bodies (MBs). Sections from control and GF-treated livers were extracted with Triton X-100. IF-enriched fractions were analyzed by gel electrophoresis and IF proteins were identified by Western blotting using anti-CK 55 (TROMA 1) and CK 49 (TROMA 2), and the anti-'universal' IF known as aIF. Western blotting on control and GF-treated livers with TROMA 1 and TROMA 2 obtained with gels loaded with 2 micrograms of protein revealed the presence of CK 55 and CK 49. In contrast, when a larger amount of protein was loaded on the gel (10 or 12 micrograms) the results were different. In the control we detected bands corresponding to CK 55 and CK 49 plus a faint band at approx. 97 kDa. In the GF-treated animals, we detected a more pronounced reaction with the 97 kDa band and in addition we now detected several higher molecular weight bands which reacted with anti-CK antibodies. These results demonstrated that the induced changes in CKs in the GF liver are associated with the increase of apparent high molecular weight CK. These apparent high molecular weight CKs could be obtained by a pathological cross-linking of the preexisting liver CK monomers in different combinations such as CK 55-55, CK 49-49 and/or with other proteins.

Distinctive infrared spectral features in liver tumor tissues of mice: evidence of structural modifications at the molecular level.

Mice were treated with griseofulvin (GF) containing diet or control diet for 12 months. The livers from mice fed griseofulvin showed large tumors that were excised and used for analysis. The infrared spectra from control liver tissue and tumor tissue from GF livers were measured and compared as a function of pressure up to 27 kbar. Many changes in the infrared spectral features of the tumor tissue were observed. Results showed that neoplasm formation involved structural modifications of nucleic acids, lipids, carbohydrates, and proteins in the liver cells, which were detected from the abnormal vibrations of the functional groups in these biomolecules. The amount of glycogen was dramatically decreased in the tumor tissue compared to the control tissue. Important changes in the strength of hydrogen-bondings in the phosphodiester backbone of the nucleic acids and in the C-O groups of tissue proteins and carbohydrates were observed. Stronger interchain interactions and thus close interchain packing among the lipids in the GF liver were evident. These results showed very close similarities with those obtained with other types of tumors such as human colon cancer, suggesting that a common pattern of molecular changes has been identified in neoplastic transformation.

Alteration in molecular structure of cytoskeleton proteins in griseofulvin-treated mouse liver: a pressure tuning infrared spectroscopy study.

Different liver diseases are associated with modifications in hepatocyte cytoskeletal organization and formation of Mallory bodies (MBs). Since the structure of a protein is critical for its function, we studied the changes in the molecular structure of the cytoskeletal protein in the liver from mice fed griseofulvin (GF), which is a good animal model for studying liver disease. Using pressure-tuning infrared spectroscopy we compared the infrared spectra of the cytoskeletal proteins from control liver and griseofulvin treated liver. The results show that the overall structure of the cytoskeletal protein was modified by the griseofulvin treatment. A relative increase in the amount of alpha-helices to beta-sheets was observed in the liver cytoskeleton from the GF-treated mice. Moreover, the random coil and the turn segments were dramatically decreased compared to controls. Pressure-induced modifications including denaturation were irreversible in the control samples whereas they were reversible in the griseofulvin-treated samples. These changes reflect important fundamental modifications in the molecular structure of the cytoskeletal proteins in the griseofulvin-treated hepatocytes. We suggest that these changes are related to the modification of the organization of intermediate filaments and the formation of MBs that occur in the GF-treated liver.

Role of cytokeratin intermediate filaments in transhepatic transport and canalicular secretion.

The role of cytokeratin filaments in the function of hepatocytes was investigated using a nickel-treated hepatocyte in vitro model. Cytokeratin intermediate filaments were selectively dissociated from the cell cortex by nickel treatment. Cytokeratins and ubiquitin were observed using immunofluorescence and immunoelectron microscopy. Hepatocytic function was assessed by visualizing uptake, transhepatic transport and secretion of fluorescein diacetate and horseradish peroxidase into the bile canaliculi. In control primary cultures, most of the bile canaliculi were surrounded by an inner layer of actin filaments and an outer pericanalicular sheath of cytokeratin filaments and microtubules. The cytoplasmic distribution of ubiquitin was diffuse and particulate. After treatment with NiCl2 (150 micrograms/ml) for 24 hr, the cytokeratin filaments and desmoplakin became focally detached from the cell cortex and retracted to form an aggregate around the nucleus. These aggregates were associated with intense ubiquitin immunoreactivity. Only a few attachments of the cytokeratin filaments to the cell cortex remained. F-actin remained attached to the cell cortex in the areas where the cytokeratin filaments had become detached. The pericanalicular sheath of cytokeratin filaments and the bile canaliculi disappeared and actin was dispersed over the entire cell periphery. Fluorescein diacetate secretion and horseradish peroxidase uptake were almost completely absent in the hepatocytes treated with nickel. The effects of nickel persisted 24 hr after its removal from the medium. It is concluded that cytokeratin intermediate filaments play a critical role in the formation of the bile canaliculus, secretion of fluorescein diacetate and uptake of horseradish peroxidase. Further, our study indicates that cytokeratin ubiquitination occurs during collapse and aggregation of the cytokeratin filaments. The formation of cytokeratin-ubiquitin conjugates during aggregation suggests a role of ubiquitin in the control of cytokeratin organization in hepatocytes in the response to cell stress.

Ethanol-induced phosphorylation of cytokeratin in cultured hepatocytes.

We studied the effect of ethanol on the phosphorylation of cytokeratins (CKs) in cultured hepatocytes since CK filaments are regulated by phosphorylation and they are abnormal in alcoholic liver disease. Hepatocytes were obtained from 14-day-old rats and cultured for 48 hrs. The hepatocytes were exposed to ethanol (300 mM) for 30 min. The cells were extracted with the buffer containing Triton X-100. The residual insoluble cytoskeletons were analyzed by two dimensional (2D) gel electrophoresis and autoradiography. 2D gel electrophoresis showed CK 55 and CK 49 or 8 and 18 and actin. The CKs had several isoelectric variants. The most basic spot was the dominant protein which was not phosphorylated. The more acidic spots were phosphorylated. After ethanol treatment, the phosphorylation of CK 55 and CK 49 were markedly increased over controls. We compared these results, with the effect of vasopressin (10 nM), TPA (150 nM) and db-cAMP (1 mM) on the phosphorylation of CKs. Vasopressin and TPA caused the phosphorylation of CK 55 and 49 but db-cAMP did not. The results suggest that CKs are phosphorylated by protein kinase C through the phosphoinositide-linked transduction system activated by ethanol.

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.

The effects of methylmercury on the cytoskeleton of murine embryonal carcinoma cells.

Immunofluorescence staining with antibodies to tubulin and vimentin and staining with phalloidin have been used to examine the effects of methylmercury on the cytoskeleton of embryonal carcinoma cells in culture. Exposure of embryonal carcinoma cells to methylmercury (0.01 to 10 microns) resulted in concentration- and time-dependent disassembly of microtubules in interphase and mitotic cells. These effects were reversible when cultures were washed free of methylmercury. Spindle microtubules were more sensitive than those of interphase cells. Spindle damage resulted in an accumulation of cells in prometaphase/metaphase, which correlated with a temporary delay in the resumption of normal proliferation rate upon removal of methylmercury. Of the interphase cytoskeletal components, microtubules were the first affected by methylmercury. Vimentin intermediate filaments appeared relatively insensitive to methylmercury, but showed a reorganization secondary to the microtubule disassembly. Actin microfilaments appeared unchanged in cells showing complete absence of microtubules. Our results 1) support previous reports suggesting that microtubules are a primary target of methylmercury, 2) document a differential sensitivity of mitotic and interphase microtubule systems and 3) demonstrate the relative insensitivities of other cytoskeletal components.

Effects of methylmercury on retinoic acid-induced neuroectodermal derivatives of embryonal carcinoma cells.

Immunofluorescence staining with antibodies to tubulin, neurofilaments and glial filaments was used to study the effects of methylmercury on the differentiation of retinoic acid-induced embryonal carcinoma cells into neurons and astroglia and on the cytoskeleton of these neuroectodermal derivatives. Methylmercury did not prevent undifferentiated embryonal carcinoma cells from developing into neurons and glia. Treatment of committed embryonal carcinoma cells with methylmercury doses exceeding 1 microM resulted in the formation of neurons with abnormal morphologies. In differentiated cultures, microtubules were the first cytoskeletal element to be affected. Their disassembly was time- and concentration-dependent. Microtubules in glial cells and in neuronal perikarya were more sensitive than those in neuronal processes. Neurofilaments and glial filaments appeared relatively insensitive to methylmercury treatment but showed reorganization after complete disassembly of the microtubules. The data demonstrate 1) the sensitivity of microtubules of both neurons and glia to methylmercury-induced depolymerization, and 2) the heterogeneous response of neuronal microtubules to methylmercury, presumably reflecting posttranslational modifications of different subpopulations of microtubules in the perikarya and neurite.