Protein phosphatase inhibition in normal and ... - Wiley Online Library

Protein Phosphatase Inhibition in Normal and Keratin 8/18 Assembly–Incompetent Mouse Strains Supports a Functional Role of Keratin Intermediate Filaments in Preserving Hepatocyte Integrity DIANA M. TOIVOLA,1,2 M. BISHR OMARY,3 NAM-ON KU,3 OLLI PELTOLA,4 HE´ LE` NE BARIBAULT,5

The function and regulation of keratin 8 (K8) and 18 (K18), intermediate filament (IF) proteins of the liver, are not fully understood. We employed the liver damage induced by microcystin-LR (MC-LR), a liver-specific inhibitor of type-1 and type-2A protein phosphatases, in normal and in keratin assembly–incompetent mouse strains as a model to elucidate the roles of IF phosphorylation in situ. The mouse strains used were wild-type (wt) mice and mice with abnormal filament assembly, caused by a targeted null mutation of the K8 gene or caused by expression of a point-mutated dominant negative human K18. In vivo 32P-labeled wt mice, subsequently injected with a lethal dose of MC-LR, showed hyperphosphorylation, disassembly, and reorganization of K8/K18, in particular K18, indicating high phosphate turnover on liver keratins in situ. At lethal doses, the keratin assembly–incompetent mice displayed liver lesions faster than wt mice, as indicated histopathologically and by liver-specific plasma enzyme elevations. The histological changes included centrilobular hemorrhage in all mouse strains. The assembly-incompetent mice showed a marked vacuolization of periportal hepatocytes. Indistinguishable MC-LR–induced reorganization of microfilaments was observed in all mice, indicating that this effect on microfilaments is not dependent on the presence of functional K8/K18 networks. At sublethal doses of MC-LR, all animals had the same potential to recover from the liver damage. Our study shows that K8/K18

Abbreviations: IF, intermediate filament; K8, keratin 8; K18, keratin 18; MF, microfilament; MT, microtubule; hK18, human K18; ser/thr, serine/threonine; MC-LR, microcystin-LR; mK8, mouse keratin 8; TG2, mouse strain with hK18; F22, mouse strain with mutated hK18 (arg89=cys); wt, wild-type; LD50, half-maximal lethal dose; PBS, phosphate-buffered saline; TEM, transmission electron microscopy; AST, aspartate transaminase; ALT, alanine transaminase; SDS, sodium dodecyl sulfate. From the 1Department of Biology, Åbo Akademi University, BioCity, Turku, Finland; 2Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, BioCity, Turku, Finland; 3Division of Gastroenterology, Veterans Administration Palo Alto Health Care System and Stanford University School of Medicine, Palo Alto, CA; 4Department of Clinical Chemistry, Turku University Hospital, Turku, Finland; and 5The Burnham Institute, La Jolla, CA. Received November 20, 1997; accepted March 17, 1998. Supported by the Academy of Finland, Åbo Akademi University, the foundations ¨ flunds stiftelse (J.E.E./D.M.T.), Department of Veterans Viktoriastiftelsen and Oscar O Affairs merit and career development awards (M.B.O.), and the NIAMSD from the NIH (H.B.). Address reprint requests to: John E. Eriksson, Turku Centre for Biotechnology, P.O. B. 123, BioCity, FIN-20521 Turku, Finland. Fax: 358-2-333-8000. Copyright r 1998 by the American Association for the Study of Liver Diseases. 0270-9139/98/2801-0017$3.00/0

AND JOHN

E. ERIKSSON2

filament assembly is regulated in vivo by serine phosphorylation. The absence or occurrence of defective K8/K18 filaments render animals more prone to liver damage, which supports the previously suggested roles of keratin IFs in maintenance of structural integrity. (HEPATOLOGY 1998;28: 116-128.) The intermediate filament (IF) proteins of hepatocytes consist of the simple epithelial IFs, keratin 8 (K8; type II) and keratin 18 (K18; type I). K8 and K18 form obligate heteropolymers assembling into complex filamentous networks spanning the hepatocyte cytoplasm.1,2 There is significant evidence for the structural importance of epidermal keratins,3 a functional role that is well illustrated by a number of severe skin and corneal diseases associated with keratin mutations.4-6 Keratins participate in attachment to basement membranes as well as in intercellular contacts, by interacting with specific desmosomal and hemidesmosomal proteins.3 Furthermore, there are indications that they could be linked to microfilaments (MFs) and microtubules (MTs),3,7-9 and may be of importance for intracellular organization.10-12 The nonepidermal IF proteins are also likely to be important for mechanical and structural strength as well as integrity on tissue level (reviewed in Omary13), as indicated by the observed defects in K8-deficient mice14 and mice expressing a dominant negative mutation of human K18 (hK18).15 A liver-specific organizational role of K8/K18 has been implicated, because they form a specialized pericanalicular sheet enclosing the actin filaments surrounding the bile canaliculus.16 Phosphorylation is regarded as a major regulatory mechanism of all IF proteins. The state of phosphorylation has been shown to play an important role in IF function and assembly dynamics (for reviews, see Eriksson17 and Ku18). In addition to changes in IF protein phosphorylation during mitosis (e.g., Chou19 and Tsujimura20), there appears to be a continuous phosphate turnover on IFs in interphase.17,21,22 This is also exemplified by results obtained from primary hepatocyte cultures in which inhibition of the K8 and K18 dephosphorylation leads to a rapid hyperphosphorylation of these proteins, accompanied with filament dissociation and reorganization.8 Recently, several IF transgenic mouse models have been developed in attempts to address IF protein function (for review, see Omary13). In the present work, we focused on two of the mouse models with genetically manipulated K8 and

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K18 genes: 1) the K8 knockout mouse (mK82),23 which is unable to form K8/K18 filaments; and 2) the mouse model expressing human K18 (hK18) that is mutated at a highly conserved arg89=cys, a mutation that leads to assembly incompetence.15,24 These genetic manipulations lead to phenotypes described in previous reports.13-15,23 Microcystins are cyclic liver-targeted peptides that have been established as strong hepatotoxins.25 Their action is caused by specific and potent inhibition of type 1 and type 2A serine/threonine (ser/thr) protein phosphatases.26,27 Apart from their acute effects, microcystins are also tumor promoters,26 because chronic sublethal administration induces neoplastic nodules in the liver.28 The hepatotrophism of microcystins is caused by uptake through the multispecific bile acid transport system of hepatocytes.29,30 In mice, microcystins cause extensive hepatic hemorrhage with disruption of the lobular and sinusoidal liver architecture, leading to rapid death by hemodynamic shock.31-33 Studies with liver cells indicate disruption of cell morphology as a consequence of a remarkable reorganization of IFs, MFs,8,34-37 and MTs.36,38 However, the cytoskeletal effects in intact mouse liver have not yet been sufficiently assessed. Hence, we used microcystin-LR (MC-LR) to alter phosphatase/kinase equilibria in the liver and examined the resultant effects on cytoskeletal structure and tissue integrity. The aims of our study were to assess the roles of reversible K8/K18 phosphorylation in the intact liver and to study the microcystin-induced effects when two of the major phosphorylation targets, K8 and K18, are absent or malfunctional by using the transgenic keratin mouse models. We show that liver K8/K18 assembly is dynamically regulated by ser/thr protein phosphorylation in vivo. The lack of proper K8/K18 filament networks renders the keratin assembly–incompetent mouse strains more susceptible for liver damage by MC-LR. MC-LR induces in the keratin assembly–incompetent mice a marked vacuolization in the hepatocytes of the portal tract. Interestingly, the characteristic MC-LR–induced MF reorganization seems to be largely independent of the presence of K8/K18 filaments. Our results illustrate some of the structural roles of keratins in the liver. MATERIALS AND METHODS Chemicals and Antibodies. MC-LR was isolated and purified from the blue-green algae Microcystis aeruginosa as described.39 Tetramethylrhodamine-conjugated phalloidin (P-5157, Sigma Chemical Company, St. Louis, MO) was used to localize F-actin. Antibodies used were the polyclonal rabbit antibodies against rat K8 and K188 that recognize both human and mouse K8/K18 and a rabbit polyclonal anti-desmoplakin antibody to bovine desmosomal proteins.40 Secondary antibodies used for fluorescence microscopy were fluorescein isothiocyanate–conjugated swine anti-rabbit immunoglobulins (F205, Dakopatt, Glostrup, Denmark) and tetramethylrhodamineconjugated goat anti-rabbit immunoglobulins (Molecular Probes, Eugene, OR). Secondary antibodies used for immunoblotting were peroxidase-labeled donkey or goat anti-rabbit immunoglobulins (NIF 824, Amersham, Buckinghamshire, England; and W401B, Promega, Madison, WI, respectively). Chemicals were of the highest purity grade and, if not otherwise stated, purchased from Sigma or Merck (Darmstadt, Germany). Radiochemicals were from Amersham. Mouse Models and Genotype Determinations. Male BALB/c mice were used for in vivo 32[P]- and 3[H]DMC-LR labeling experiments and for studies on MC-LR-induced effects on keratin solubility. Wildtype (wt), mK82 heterozygous (mK82/1), and mK82 homozygous

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(mK82/2) mice were expanded in the FVB/N mouse strain as reported previously.14 Transgenic TG2 (expressing 17 copies of the wt hK18 gene)41 and F22 (expressing 10 copies of the mutated (arg89=cys) hK18 gene)15 were derived from a FVB/N mouse background. The genotype of each wt, mK82/1, and mK82/2 mouse was determined by Southern blot23 and/or polymerase chain reaction analysis of mouse-tail DNA.14 The absence of mK8 in mK82/2 liver samples was further confirmed by Western blotting of whole tissue extracts (see below) using the K8 antibody. The genotyping of TG2 and F22 mice was performed using dot-blot analysis from tail DNA as described previously.15 Animals were, to the extent practically possible, age- and sex-matched for the various treatments (Table 1). Animals were bred and housed with strict infection controls, and received humane care in compliance with the guidelines of the respective involved animal facilities. Toxin Administration and Sampling. The calculated half-maximal lethal dose (LD50 value) for MC-LR in wt mice is approximately 50 µg/kg.32 The same doses for all mouse strains were used, because LD50 tests did not reveal major differences between the mouse strains (not shown). Various doses reflecting lethal or sublethal doses of MC-LR were administered to mice by intraperitoneal injections. MC-LR was diluted in 0.9% NaCl (from a 1-mg/mL stock in 10% ethanol) freshly each day. Control mice received 0.9% NaCl. Volumes injected varied between 300 to 620 µL depending on mouse weight and dose (the amount of ethanol injected never exceeded 0.5 µL ethanol/30 g mouse). Animals were killed by CO2 inhalation at the indicated times. The abdomen was opened and the gross appearance of the liver was observed in situ; then, a blood sample was drawn (see below). The liver was removed, weighed, and then cut into pieces with a sharp scalpel (the right anterior lobe was used for most sampling). Samples were mainly taken from only one part of the liver, because mouse liver lobules are uniformly affected by microcystins.42 Samples were fixed in 4% formaldehyde in phosphate-buffered saline (PBS) (pH 7.4) (24 hours, 14°C) for histology and 3% glutaraldehyde in 0.15 mol/L sodium cacodylate buffer (pH 7.4) (24 hours, 14°C) for transmission electron microscopy (TEM). Samples for biochemistry were snap-frozen in liquid N2 and stored in N2 or at 270°C. For immunocytochemistry, samples were mounted in O.C.T. compound (4583, Miles Inc., Elkhart, IN) on dry ice and stored at 270°C. Spleens and kidneys were excised and weighed. Blood Plasma Testing. Blood was drawn by cardiac puncture using a 22-gauge needle, collected in an Eppendorf tube containing heparin (2 µL of 25,000 IU/mL), placed on ice, and centrifuged (10,000g, 2 minutes). Plasma was collected and stored at 220°C until analysis. Aspartate transaminase (AST) and alanine transaminase (ALT) levels in plasma were measured according to standard procedures (Standards for Enzyme determination, ECCLS document 3-4:1988, ISSN 1011-6265), using a Hitachi 717 Automatic analyzer. Histology and Electron Microscopy. Hematoxylin-eosin staining was performed according to standard histological procedures on 4-µm sections of formalin-fixed and paraffin-embedded tissue. Lipids were stained with Oil Red O according to standard procedures on 15-µm cryosections of O.C.T. compound-embedded frozen liver

TABLE 1. The Total Number of Animals Used and the Number of Each Sex (female 1 male) for Each Treatment MC-LR Treatment Mouse Strain

Control 60 min

wt mK82/1 mK82/2 TG2 F22

4 (3 1 1) 4 (4 1 0) 4 (3 1 1) 4 (2 1 2) 4 (2 1 2)

Lethal Dose Lethal Dose Sublethal Dose Sublethal Dose 25 min 60 min 3h 3d

3 (2 1 1) 2 (1 1 1) 3 (3 1 0) 3 (2 1 1) 3 (2 1 1)

3 (2 1 1) 3 (2 1 1) 3 (2 1 1) 3 (0 1 3) 3 (0 1 3)

2 (2 1 0) 3 (2 1 1) 2 (1 1 1) 3 (1 1 2) 3 (2 1 1)

NOTE. Lethal dose 5 175 µg/kg; sublethal dose 5 30 µg/kg.

4 (2 1 2) 4 (3 1 1) 2 (1 1 1) 3 (0 1 3) 3 (2 1 1)

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samples fixed in 3% formaldehyde. One-micrometer sections of epon-embedded material (see below) were stained for approximately 10 seconds with 0.1% Toluidine blue in sodium carbonate (2.5 g/100 mL). Samples were analyzed with a Leica microscope. For TEM, glutaraldehyde-fixed samples were postfixed for 120 minutes at 4°C with OsO4 in 0.15 mol/L Na-cacodylate buffer (pH 7.4) and dehydrated in a graded series of acetone before embedding in Epon 812 resin. The ultrathin sections mounted on noncoated grids were stained with uranyl acetate and lead citrate and viewed in a Jeol JSM-35 transmission electron microscope. Immunofluorescence. O.C.T. compound-embedded frozen liver samples were cut on a cryostate (6- to 12-µm thickness), and sections were collected on gelatin-coated slides. Sections were fixed in 220°C water-free methanol for 10 minutes (anti-desmoplakin and anti-K8/K18 localization) or 1% formaldehyde (F-actin and anti-K8/K18 localization) for 20 minutes (22°C). Sections were washed in PBS, blocked with 1% bovine serum albumin, and incubated with the primary antibody in 1% bovine serum albumin (120 minutes, 22°C). Sections were further washed with PBS, incubated with the secondary antibody for 60 minutes in 1% bovine serum albumin, and finally washed before mounting in Mowiol 40-88 (32, 459-0, Aldrich-Chemie, Steinheim, Germany) supplemented with 100 mg/mL 1,4,diazabicyclo[2.2.2]-octane (D 2,780-2, Aldrich-Chemie). Sections fixed in formaldehyde were, before blocking, permeabilized with 0.2% Nonident P-40. Samples were analyzed in a Leitz Aristoplan fluorescence microscope and a Leica TCS40 confocal laser scanning microscope using the program SCANware 4.2a. Metabolic In Vivo 32[P]-Labeling. For metabolic in vivo 32[P]labeling, mice (25 g) were injected in the tail vein with 1 mCi 32[P]-orthophosphate. After 1 hour, the mice received an intraperitoneal dose of MC-LR and were killed after indicated times. Livers were homogenized and treated for separation of detergent-soluble fractions as described below. Tissue Homogenization, Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis, Immunoblotting, and Immunoprecipitation. To separate

Triton X-100 soluble and insoluble fractions, a piece of snap-frozen or fresh liver was homogenized in 3 mL/g tissue TX-100 buffer (20 mmol/L HEPES [pH 7.6], 100 mmol/L NaCl, 5 mmol/l MgCl2, 5 mmol/L EGTA, 1% TX-100, 1 mmol/L phenylmethylsulfonyl fluoride, 10 µg/mL leupeptin, 10 µg/mL antipain). Samples were centrifuged for 15 minutes (15,000g, 4°C) to obtain the TX-100– soluble (supernatant) and –insoluble (pellet) fractions. Both fractions were dissolved in 33 Laemmli sample buffer.43 For whole tissue extracts, a piece of liver was homogenized (20 strokes) in 6 mL/g sodium dodecyl sulfate (SDS) buffer (3% SDS, 0.187 mol/L Tris-HCl [pH 6.8], 3% b-mercaptoethanol, and 5 mmol/L EGTA). Protein samples were further diluted in Laemmli sample buffer, boiled, and separated on SDS polyacrylamide gels.43 Gels were stained with Coomassie Brilliant blue to control for equal loading of proteins, dried, and exposed at 270°C using Kodak XR or Kodak X-Omat AR films. For immunoblotting, proteins were electrotransferred from gels to nitrocellulose membranes. Binding of primary antibody to the proteins was detected using horseradish peroxidase– labeled secondary antibodies and the enhanced chemiluminescence Western blotting detection system (Amersham). K8/K18 were coimmunoprecipitated from TX-100 supernatants as described previously,8 except that NP-40 was omitted from the immunoprecipitation buffer. Specific 32P-labeling of proteins was quantified using a phosphorimager analyzer (Bio-Rad GS-250 Molecular Imager). The enhanced chemiluminescence or 32P signal on films was measured using computerized image analysis (Microcomputer Imaging Device M4, Imaging Research Inc., St. Catherine, Ontario, Canada). Statistical Analyses. Statistical results were obtained by the Student’s t test or one-way ANOVA and the Newman-Keuls post test using the program GraphPad Prism 2.0 (GraphPad Software Inc., San Diego, CA).

HEPATOLOGY July 1998

RESULTS MC-LR Induces Hyperphosphorylation as Well as Disassembly of K8 and K18 In Vivo. To study whether the MC-LR–induced

hyperphosphorylation of K8 and K18 observed in primary rat hepatocyte cultures8 also occurs in vivo in the intact liver, we studied the phosphorylation and disassembly of the liver keratins from MC-LR-treated normal nontransgenic mice. The disassembly was studied by separating liver homogenates in TX-100–soluble and –insoluble fractions. MC-LR induced a time-dependent increase in K8 and K18 phosphorylation in the TX-100–soluble fraction (Fig. 1A and 1D). At a toxin dose corresponding to 2 3 LD50 (100 µg/kg), MC-LR induced a hyperphosphorylation of K18, which was noticeable with increasing treatment times (15, 30, 90 minutes) (Fig. 1A and 1D), and prominent after 65 minutes of a dose corresponding to 6 3 LD50 (300 µg/kg). A less-prominent hyperphosphorylation of K8 was noted after 30 minutes (Fig. 1A), but clearly observed only after 65 minutes following injection of 300 µg/kg MC-LR (Fig. 1A). The identity of these two dominant MC-LR–induced phosphoproteins as K8 and K18 was confirmed by immunoprecipitation (Fig. 1B). Immunoblotting of TX-100 supernatants and pellets with K8/K18 antibodies showed that a time-dependent increase of K18 in the TX1002soluble fraction was already obvious after 15 minutes of MC-LR treatment (Fig. 1C and 1D), corresponding to the increased phosphorylation of the soluble K18 (Fig. 1A). In accordance with the observed hyperphosphorylation (Fig. 1A), K8 was not solubilized until 30 to 60 minutes after MC-LR treatment (Fig. 1C). Plotting the soluble K18 phosphorylation versus its solubilization yields a close correlation. A decrease of K18 could be observed in the pellet fractions, although it did not become apparent until the 90-minute time point (Fig. 1D) because of the large amount of insoluble versus soluble keratins. The treatment of 100 µg/kg MC-LR for 90 minutes left approximately 27% of K18 in the pellet (as measured by densitometric analysis of Coomassie Brilliant blue–stained pelletable protein bands). At this time point, there was a sixfold increase in phosphorylation of soluble K18 (quantified using computerized image analysis). The phosphorylation of K8/K18 in pellet fractions was not markedly altered (results not shown), indicating that the phosphorylated IF subunits are recruited to the disassembled protein pool. Keratin Assembly–Incompetent Mice Show Increased Susceptibility to MC-LR–Induced Liver Lesions. Because K8/K18 are the

major target phosphoproteins affected by MC-LR–induced protein phosphatase inhibition in the intact liver (Fig. 1), we assessed the roles of K8/K18 in the lesion by comparing MC-LR susceptibility in mice with normal keratin expression (wt, mK82/1, and TG2) versus mice lacking the major target proteins altogether (mK82/2) or mice with disrupted filament formation (F22). The different mouse strains were treated with a lethal dose of MC-LR (175 µg/kg) and a sublethal dose (30 µg/kg) to determine possible differences in the induced liver lesions. Mouse behavior to any of the given MC-LR doses did not vary between the mouse strains. Approximately 30 minutes after administration of a lethal dose, mice showed listlessness, a fluffy appearance, and finally pallor of ears, tail, and legs. No symptoms were apparent at sublethal doses, and no obvious gross effects of the toxin were observed on any organ other than the liver. The MC-LR–induced liver gross appearance


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FIG. 1. Identification of K8 and K18 as the main hyperphosphorylated liver phosphoproteins from normal nontransgenic mice treated with MC-LR (intraperitoneally). (A) Autoradiograph shows SDS–polyacrylamide gel electrophoresis–separated 32P-labeled proteins from TX-100–soluble fractions (15,000g, 15 minutes) of liver homogenates obtained from control mice (Con.) and from mice administered with 100 µg/kg MC-LR, 15, 30, and 90 minutes following injection, or with 300 µg/kg 65 minutes following injection. (B) Autoradiograph shows SDS–polyacrylamide gel electrophoresis–separated K8 and K18 immunoprecipitated from 32P-labeled TX-100–soluble fractions of a control mouse (Con.) and a mouse treated with 300 µg/kg MC-LR for 65 minutes. Immunoblots (C) show TX-100–soluble fractions of liver homogenates from a control mouse (Con.) and mice treated with 100 µg/kg MC-LR for 15, 30, 60, and 90 minutes. Blots were probed with anti-K8 and -K18 antibodies. Molecular masses (kDa) are indicated on the left, and migration of K8 and K18 is indicated on the right. Equal loading of proteins was controlled with Coomassie Brilliant blue staining of parallel samples on a different gel (not shown). (D) The keratin IF disassembly was quantified as integrated optical density (IOD) by image analysis–based densitometry of the MC-LR–induced (100 µg/kg) increases in soluble K18 on Western blots (h). These values correlated well with the elevated phosphorylation of soluble K18 (j) and the decreases of K18 in TX-100–resistant pellets (s).

was a dark mosaic pattern caused by hemorrhage (see below) appearing in most mice 25 minutes after treatments with a lethal dose. After 60 minutes, the livers were swollen and the mottling pattern was severe, showing a markedly dark, red color. Mice receiving the sublethal dose and killed 3 hours or 3 days later showed no significant characteristic mottling pattern of the livers. Relative differences in liver, spleen, and kidney weights (calculated as percentage of total body weights) in the different mouse strains were compared at the various MC-LR treatments. MC-LR is known to induce increased liver weights because of a severe hemorrhage of the liver.30 At the lethal MC-LR dose, liver weights increased in all mouse strains in a time-dependent way (Fig. 2). After 25 minutes following MC-LR injection, a minor increase was seen in all mouse strains, whereas in mK82/2 mice, the liver weights already increased prominently at this time point (P , .01) (Fig. 2). After 60 minutes, the liver weights were markedly elevated in all mouse strains (Fig. 2). No major differences in kidney or spleen weights could be detected between controls and MC-LR treatments (not shown). The sublethal dose of MC-LR did not induce any changes in liver weights, except for mK82/2 mice, in which an increase was noticed after 3 days (data not shown). We measured the plasma levels of ALT and AST after

FIG. 2. The effect of MC-LR on the liver weights in mouse strains with variable keratin-specific genotypes. Mouse liver weights, expressed as % of total body weight, are shown for the different mouse strains. Mice were treated with intraperitoneal injections of 0.9% NaCl (controls) or 175 µg/kg MC-LR for 25 or 60 minutes as indicated in the figure (6SEM). The values within each mouse strain were compared with their respective controls using the t test. ***P , .0001; **P , .01; *P , .05. (g), control; (i), 25 minutes; (j), 60 minutes.

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MC-LR treatments as a biochemical reflection of liver injury. ALT was a good marker for the MC-LR–induced damage (Fig. 3). AST showed the same trends as ALT, although the differences between treatments were of a lower magnitude and intergroup variations were higher (data not shown). Basal ALT levels of mK82/2 and F22 mice were slightly higher than in wt mice (Fig. 3A), as shown previously.14,15 MC-LR induced increases in both ALT (Fig. 3A) and AST (not shown) in all mouse strains after 60 minutes of treatment with the lethal dose of MC-LR. In plasma from mK82/2, however, both ALT and AST levels already increased significantly (ALT: P , .05; AST: P , .01) after 25 minutes of treatment (Fig. 3A), when no marked increase was observed in wt, mK82/1, and F22 mice. An unexpected increase of ALT activity in TG2 plasma was observed, which did not correlate with any of the other parameters used as indicators of tissue damage. The high standard deviations in each group at 60 minutes of MC-LR treatment are most likely


caused by the difficulty to draw blood from the heart, because most of the blood accumulated to the liver. Mice were injected with a sublethal dose and killed after 3 hours or 3 days to observe any differences in their ability to recover after mild MC-LR–induced damage. MC-LR induced in all five mouse strains a small damaged area on the lower side of the liver, close to the entrance of the liver (not shown). No obvious damage could be observed in other parts of the liver. At 3 days, all mouse strains showed active lymphocyte infiltration in the areas where hemorrhage was observed after 3 hours. In particular, ALT was a sensitive indicator of sublethal MC-LR treatments in all five mouse strains, because the blood plasma levels of this enzyme increased after 3 hours (Fig. 3B). The enzyme levels returned to control values in all mice after 3 days (Fig. 3), indicating the presence of a sufficient recovery mechanism independent of the various differences in hepatocyte keratins. The same trend was seen in AST levels (data not shown). MC-LR Induced Severe Vacuolization in Keratin Assembly– Incompetent Mice. To further look for functions of keratin IF

FIG. 3. The effects of MC-LR on blood plasma values of liver-specific enzyme activities. Mice were treated with intraperitoneal injections of 0.9% NaCl (controls) or 175 µg/kg MC-LR for 25 or 60 minutes (A) or 30 µg/kg for 3 hours or 3 days (B) as indicated in the figure. Blood samples were drawn by cardiac puncture and plasma separated by centrifugation as described in Materials and Methods. Enzyme values (ALT) are given as U/L 6 SEM. (A) The values within each mouse strain were compared with their respective controls using the t test. (g), control; (i), 25 minutes; (j), 60 minutes. (B) The values at 3 hours for each mouse strain were compared with their respective control values, and the values at 3 days were compared with the respective values at 3 hours using one-way ANOVA and the Newman-Keuls post test. ***P , .0001; **P , .01; *P , .05. (g), control; (i), 3 hours; (j), 3 days.

proteins, the histopathology of MC-LR–induced liver damage was compared between the different transgenic mouse strains. Control mice showed normal liver architectures in wt and TG2 mice (Fig. 4A and 4J), and the livers of mK82/2 and F22 mice showed changes described earlier.14,15 In addition to the necrotic and inflammatory changes, the keratin assembly– incompetent mice also showed in all the examined livers some abnormalitites of liver organization (not shown; Toivola et al., Unpublished data, August 1997). Because most parts of the mK82/2, mK82/1, and F22 livers showed relatively normal liver lobular architecture (Fig. 4D, 4G, 4M), the effects of MC-LR presented here are focused on these apparently normal areas. All the mouse strains showed after 25 minutes of treatment of a lethal dose of MC-LR mild centrilobular hemorrhage (Fig. 4, Table 2). In mK82/2 and F22 mice, however, this effect was more prominent (Fig. 4H and 4N) than in wt, mK82/1, or TG2 mice (Fig. 4B, 4E, 4K, Table 2). All mice studied were severely affected after 60 minutes (Fig. 4C, 4F, 4I, 4L, and 4O), when many of the mice were close to death. In the hemorrhage areas, hepatocytes were dissociated in lacunae of blood, with some of the hepatocytes showing signs of necrosis. The hemorrhage did not extend to the portal areas, which appeared intact in most control wt and TG2 mice. The most striking difference between wt, TG2, and mK82/1, as compared with mK82/2 and F22 mice, was that MC-LR induced severe portal vacuolization at 60 minutes of treatment with a lethal dose in the keratin assembly– incompetent mice (Fig. 4C, 4F, 4I, 4L, and 4O). This effect was clearly more severe in K8-deficient mice than in F22 mice. The round-to-oval-shaped vacuoles were slightly smaller in the hepatocytes in the intimate vicinity to the portal vein, and larger in the outer part of the portal area (Figs. 4I, 4C, 5). The vacuoles seemed empty and did not contain lipids as assessed by Oil Red O stainings (results not shown). Wt and TG2 mice also occasionally showed some portal vacuolization induced by MC-LR, but these vacuoles were generally much smaller and/or less abundant than those in mK82/2 and F22 mice (Figs. 4C, 4L, 5). One third of mK82/1 mice showed vacuolization as extensive as that in mK82/2 mice (shown in Fig. 4F). The vacuolization, which is likely related to the lack of normal IF-protein networks, also occurred in


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FIG. 4. The effects of MC-LR on liver histology. Hematoxylin-eosin staining of paraffin sections of livers from the different mouse strains ([A-C], wt; [D-F], mK82/1; [G-I], mK82/2; [J-L], TG2, and [M-O], F22) is shown. Mice were treated with intraperitoneal injections of 0.9% NaCl (controls) or 175 µg/kg MC-LR for 25 or 60 minutes as indicated in the figure. c, central vein; p, portal vein; h, hemorrhage; v, vacuoles. Bar 5 100 µm.

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TABLE 2. Occurrence of Portal Tract Vacuoles and the Degree of Central Vein Hemorrhage as Judged From Hematoxylin-Eosin–Stained Paraffin Sections of Liver Control Mouse Model

wt mK82/1 mK82/2 TG2 F22

25 min MC-LR

60 min MC-LR

Vacuoles Hemorrhage Vacuoles Hemorrhage Vacuoles Hemorrhage

2 2 2 2 2

2 2 2 2 2

2 2 2 2 2

1 1 11 1 11

2 1 111 2 11

111 111 111 111 111

NOTE. Data are averages of three to four mice per treatment. 2, no vacuoles or blood hemorrhage were observed; 1, mild; 11, moderate; 111, severe vacuolization/hemorrhage.

every young mK82/2 mouse (6-8 weeks of age) treated with doses of 100 µg/kg or higher (data not shown). Detailed Studies of the Portal Vacuolization in Relation to Other Ultrastructural Observations. Further studies were performed

to assess the MC-LR–induced vacuolization in mK82/2 and F22 mice. The vacuoles were membrane-bound and, for the most part, empty, except for scarce membrane-like residuals (Fig. 5B, 5C, 5G, and 5I). It is also clear from the TEM studies that the vacuoles did not contain, for example, glycogen granules (Fig. 5G). TEM studies indicated that these vacuoles represented extensive invaginations of the hepatocyte plasma membrane, both of the lateral domain of the hepatocyte and of the space of Disse (Fig. 5G and 5I). The intracellular organization of hepatocytes in untreated mK82/2 mice and F22 mice livers in ‘‘normal’’ areas appeared different from wt mice, because most cells contained distinct patches of largely unstained (non–electron-dense) areas, devoid of most organelles except glycogen granules (Fig. 5D, 5F, 5H). Consequently, rough endoplasmic reticulum and mitochondria were stacked to ‘‘compartments’’ of the cells (Fig. 5F and 5H). This observation was more prominent in mK82/2 than in F22 mice. In MC-LR–treated mK82/2 or F22 mice, however, this intracellular organization was no longer observed (Fig. 5G and 5I). Common for all mouse strains was a MC-LR–induced dilation and vacuolization of the endoplasmic reticulum, except for a single layer of rough endoplasmic reticulum intimately surrounding each mitochondria (Fig. 5E, 5G, 5I). A widening of the space of Disse was observed in portal areas in all mouse strains, although this effect was more prominent in mK82/2 and F22 livers (Fig. 5G and 5I). After 60 minutes, some of the hepatocytes were observed to undergo degenerative processes such as necrosis (Fig. 5A-C). MC-LR induced in all mouse strains effects on nuclei that turned irregular in shape and experienced swelling (Figs. 4 and 5). In addition, MC-LR induced a widening of the bile canaliculi and a disappearance of microvillae in bile canaliculi and the space of Disse. Most desmosomes appeared intact in portal zones. Further, MC-LR induced in all mice (60 minutes) thick bundles of filaments floating among blood cells in severe hemorrhage areas (not shown). The identity of these filaments was not determined, but they are likely to represent disrupted extracellular matrix proteins such as fibronectin. To that end, plasma levels of fibronectin, which is normally localized only in the pericentral area, becomes elevated in microcystin-treated mice.44

MC-LR–Induced Effects on MFs and IFs. We further examined the MC-LR-induced effects by studying the morphology and distribution of hepatic MFs and IFs. In untreated mice, the distribution of F-actin, which was located mainly at the cell borders and around bile canaliculi, was not remarkably different between the different mouse strains in areas showing normal organization (Fig. 6A, 6I, 6K). Regardless of genotype, MC-LR induced in all mouse strains a similar F-actin reorganization in the hepatocytes surrounding the central veins (Fig. 6B, 6C, 6D, 6J, 6L). Already after 25 minutes of treatment (175 µg/kg), F-actin aggregated to bundles in these cells (Fig. 6B) and finally to intensely stained dots (Fig. 6C). At 60 minutes, the F-actin effects were observed in a lobular gradient with terminal effects seen as small dots in central vein areas (Fig. 6B, 6C, 6D, 6J, 6L), intermediate effects observed as bundles distally from the central veins, and no apparent effect in the hepatocytes surrounding the portal tract (Fig. 6D). The characteristic MC-LR–induced F-actin aggregation was also observed using TEM in hepatocytes in centrilobular regions of all mouse strains (Fig. 7). In the mice expressing normal K8/K18, MC-LR induced a reorganization of the keratin filament networks in centrilobular hepatocytes (Fig. 6E-H). K8/K18 formed short bundled filament segments, before most of the visible filaments accumulated to a cloudy structure in the vicinity of the F-actin accumulation (Fig. 6), similar to that seen earlier in rat primary hepatocyte cultures.8 Simultaneous localization of F-actin and K8/K18 showed that the MC-LR–induced reorganizations of both filament groups occurred approximately simultaneously in the same cells (Fig. 6D and 6H). Obviously, hepatocytes of mK82/2 mice showed no keratin staining, and, in F22 mice, the keratin immunoreactivity appeared as described earlier (Fig. 6M and 6O).23 The Effects on Desmosomal Junctions. We studied the impact of MC-LR on desmosomal integrity in the presence and absence of keratin filaments, because keratins are known to attach to desmosomes, and MC-LR has been shown to induce hyperphosphorylation and dissociation of desmoplakins when hepatocytes detach from each other.8 Apparently normal desmoplakin immunoreactivity was present at cell contacts between hepatocytes in all mouse strains (Fig. 8A-C). Desmoplakin immunoreactivity appeared essentially normal in mK82/2 and F22 mice in areas with normal liver structure (Fig. 8B and 8C). MC-LR induced similar desmoplakin effects in all mouse strains. After 25 minutes of treatment with the lethal dose, desmosomes disintegrated in perivenous hepatocytes in all mouse strains (Fig. 8D-F). In mice treated with MC-LR for 60 minutes, centrilobular areas were devoid of desmoplakin staining, and in the midzonal areas, the immunoreactivity staining was dotty (Fig. 8G). In portal areas, normal-appearing desmoplakin patterns were observed (Fig. 8H). DISCUSSION Keratin 8/18 Filament Assembly Is Regulated by Serine/Threonine Phosphorylation in the Intact Liver. In accordance with previous

studies on primary cultured liver cells,8,45 we show here that inhibition of protein phosphatase types 1 and 2A also leads to hyperphosphorylation of K8 and K18 in the intact liver, indicating that there is a dynamic constitutive regulation by

FIG. 5. MC-LR–induced vacuolization and ultrastructural effects in wt, mK82/2, and F22 mice. Toluidine blue–stained ultrathin epon sections (A-C) and regular electron-microscopic sections of mouse livers (D-I) were prepared as described in Materials and Methods. Wt mice (A, D, E), mK82/2 (B, F, G), and F22 mice (C, H, I) were injected intraperitoneally with 175 µg/kg MC-LR (A, B, C, E, G, I) or with 0.9% NaCl (Controls; D, F, H) and killed 60 minutes later. bc, bile canaliculi; sd, space of Disse; n, nucleus; v, vacuole; g, glycogen-rich areas. Arrow shows invagination of the space of Disse. Bar in (A) (for A-C) 5 50 µm; bars in (D-I) 5 5 µm.

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FIG. 6. MC-LR–induced reorganization of MTs and IFs. Wt (A-H), F22 (I, J, M, N), and mK82/2 (K, L, O) mouse livers were analyzed for F-actin (A-D, I-L) and K8/K18 (E-H, M-O) localization using immunofluorescence techniques. Mice were treated with 0.9% NaCl (controls; A, E, I, K, M, O) or 175 µg/kg MC-LR for 25 minutes (B, F) or 60 minutes (C, D, G, H, J, L, N). (D and H) show a liver area co-stained for both F-actin and K8/K18. Images are maximal projections of eight confocal laser scanning images. All images show central vein areas, except (D) and (H), in which both central vein and portal vein areas are seen. Arrowheads show MF aggregation; arrows show IF aggregations. The positive immunoreactivity in O (which stems from cross-reactivity of the antibodies with keratin 19) is in cells of a bile duct. Bar in (A) (for A-C, E-G, I-O) 5 30 µm; bar in (D) (for D and H) 5 50 µm.

phosphorylation of these proteins in situ. Furthermore, this keratin hyperphosphorylation is accompanied by a disassembly of the keratin IFs as shown in cultured hepatocytes.8 Although the constitutive phosphorylation of K8 in cultured hepatocytes appears to be slightly higher than that of K18,8,16,46 our studies using ser/thr inhibition both in cell culture and in vivo in mice clearly show that the ser/thrspecific phosphate turnover of K18 is more active than that on K8. This is in agreement with a previous study.22 The

indicated higher turnover on K18 may be expected, because we have shown that the phosphorylation of K18 involves more phosphopeptides than K8 phosphorylation.8 Our results demonstrate a clear positive correlation between increased soluble keratin phosphorylation and keratin disassembly (or solubilization). These data correspond well to in vitro studies in primary hepatocyte cultures (see Toivola8) treated with 4 µmol/L MC-LR for 30 minutes, showing a marked decrease in pelletable K18 (13% left in pellet; as measured by


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Coomassie Brilliant blue staining) accompanied with a 31fold increase in phosphorylation of the corresponding soluble K18 (Toivola DM, Eriksson JE, Unpublished observations, September 1997). As noted in primary cultures,8 the MC-LR–induced hyperphosphorylation of K8/K18 also leads to keratin reorganization in intact liver. K8/K18 reorganized in centrilobular hepatocytes in close proximity to the aggregated microfilaments, in a similar way as shown in primary cultures.8 Although a number of studies addressing the regulatory roles of IF phosphorylation have been performed in cell cultures, very little information is available concerning IF phosphorylation in intact animals. Organizational changes of IFs in griseofulvin-fed mice in association with hyperphosphorylation have been described.24 Our results demonstrate that K8 and K18 assembly in intact mouse liver is regulated by dynamic ser/thr phosphorylation. Are Keratin 8/18 Assembly–Incompetent Mice More Susceptible to MC-LR–Induced Toxicity? Because K8 and K18 are major tar-

FIG. 7. The induction of the characteristic microcystin-induced cell blebbing and reorganization of MF. The effects of MC-LR on liver ultrastructure are shown in TEM micrographs from wt (A), mK82/2 (B), and F22 (C) mice, after 175-µg/kg treatment of MC-LR for 60 minutes (A, C) and 25 minutes (B). Arrows show MF aggregations. Bar 5 1 µm.

gets of MC-LR–induced hyperphosphorylation, we used the above-mentioned keratin assembly–incompetent mouse strains as models for studying the lesions induced during specific inhibition of ser/thr protein phosphatases. By subjecting all included mouse strains to a lethal dose of MC-LR, we could assess the possible roles of K8/K18 filaments in the observed liver lesions. The K8-deficient mice responded faster than the other mouse strains to the MC-LR–induced effects, because these mice displayed a marked disruption of the liver structure, an increase of liver weight, as well as elevated plasma levels of liver-specific enzymes 25 minutes following injection. Although the F22 mice also showed signs of increased sensitivity toward the effects of MC-LR (see below), they responded more slowly than the K8-deficient mice. An interpretation of the different behavior of these keratin assembly–incompetent strains could be that the mouse-endogenous wt keratins in F22 mice were sufficient to keep the liver membrane fairly undisturbed, while in mK82/2, the total lack of keratin filaments rendered them more prone to leakage of enzymes at damage. Hence, the mK82/2 mice were at a single dose exceeding the LD50 value clearly more sensitive to MC-LR than wt mice. This increased sensitivity to MC-LR could theoretically be caused by an increased uptake of MC-LR as a result of an altered bile acid transport system in keratin assembly–incompetent mice. However, this assumption is not supported by a study showing that the K8 knockout has no effect on serum bilirubin levels,14 which is indicative of functional retention of this organic anion. Furthermore, a recent study showed a small reduction (20%) in the bile flow of K8-deficient mice,47 which is likely to involve impaired, rather than increased, bile acid transport. Thus, if there in fact is an effect on the bile transporters in the K8 knockout mice, these effects are likely to involve a reduced uptake, rather than an increased uptake. If this is true, these mice would in fact be even more sensitive to microcystin than with equal uptake in mK8 knockout and wt mice, because the inferred reduced uptake still yielded a stronger response in the keratin-deficient mice. Taken together, it is therefore unlikely that the increased sensitivity would be associated with increased uptake of the toxin, but is more likely to be a consequence of some structural defect caused by keratin deficiency. To address the role of keratins in tissue recovery after liver injury, we subjected mice of the various genotypes to suble-

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FIG. 8. The MC-LR–induced dissociation of desmosomal structures in liver. The confocal micrographs show immunolocalization of desmoplakin in livers of wt (A, D, G, H), mK82/2 (B, E), and F22 (C, F) mice. Animals were injected with 0.9% NaCl (control, A-C) or 175 µg/kg MC-LR (1MC) for 25 minutes (D-F) and 60 minutes (G, H). Centrilobular areas (A-G) and portal tract areas (H) are shown. Images are maximal projections of eight confocal laser scanning images. cv, central vein; pv, portal vein. Bar 5 50 µm.

thal doses of MC-LR. All mouse strains seem to have the same ability to recover from the injury, as judged from the return of AST/ALT plasma levels to normal after the sublethal MC-LR treatments. This is in accordance with a study showing normal wound healing in mK82/2 embryos.48 These results also indicate that factors other than IF stability might play a role in membrane integrity of hepatocytes, because mice lacking proper IFs could recover a mild membrane damage. Keratin Assembly–Incompetent Mice Develop Severe Portal Vacuolization on MC-LR Treatments: Evidence for a Function of IFs in Intracellular Organization. The histopathological and ultrastruc-

tural effects induced by MC-LR in wt mice were consistent with those observed in several earlier studies,25,49 consisting, for example, of central vein hemorrhage, enlargement of the space of Disse, and swollen rough endoplasmic reticulum. Although these effects could also be seen in the keratindeficient mice, evidence for IF-dependent structural functions was given by the extensive MC-LR–induced vacuolization of periportal hepatocytes in mK82/2 and F22 mice. Because these large vacuoles appeared empty, it is unlikely that they stemmed from swollen organelles such as mitochondria or endoplasmic reticulum. This electron-microscopic profile suggests that they are hepatocyte plasma membrane invaginations extending from lateral sides of hepatocytes or the space of Disse. TEM studies showed that although most cellular junctions appeared to stay intact in the periportal

zone, at some points, the intercellular space between cells was widened. It is tempting to speculate that the abnormal intracellular organization of organelles in keratin assembly– incompetent mice together with the increased sinusoidal pressure generated by massive blood accumulation would allow for this type of vacuolization to occur. The keratin IFs in hepatocytes thus appear to play a functional role in cellular rigidity. Together, these observations also indicate that in keratin-deficient mice, the liver organization is fragile and more sensitive than in normal mice to a tissue collapse such as could be induced by an increased sinusoidal pressure. This theory is supported by the epidermolysis bullosa simplex skin disease, which harbors a K14 mutation inducing impairment of keratin filament formation, with consequent signs of cytolysis and/or degeneration in the basal layers of the skin. These degenerated ‘‘vacuole’’-like structures4 resemble the vacuoles we observed in mK82/2 and F22 MC-LR–treated mice. In transgenic mice that express K14 in the liver with consequent hepatocyte IF disruption, inflammatory infiltration, ballooning degeneration, increased fat-containing vacuoles, and glycogen accumulation were also observed.50 Liver fragility in mice lacking functional liver IFs has also been observed in a few other studies.23,47 These results together with our present data would strongly imply a role of keratin IFs in intracellular organization.


The less-prominent portal vacuolization in wt mice has been observed earlier in hepatocytes from mice treated with MC-LR, when it was related to membrane invaginations.49,51 Unlike the severe vacuoles in mK82/2, the vacuoles in wt mice contained granular material, which suggests a degenerative process rather than a prominent collapse. Our hypothesis is that the wt vacuoles may be a milder form of those observed in keratin assembly–incompetent mice. Because the MC-LR–induced effects specific for the mK82/2 and F22 mice were more prominent in the K8 knockout, it adds further support for an important organizational and structural role of keratin filaments. MC-LR–Induced Disruption of Cell Junctions and MFs. Previous studies have established that microcystins induce in both cultured hepatocytes and in the intact rat liver a characteristic reorganization of MFs to a condensed pericellular structure.8,3537,52,53 Correspondingly, we show that MFs in mouse liver after MC-LR treatment reorganize to one or several spots in hepatocytes in the centrilobular area, but remain largely intact in the portal tract area. The primary biochemical reason for the F-actin reorganization by MC-LR may be caused by impaired dephosphorylation of some actin-binding proteins. The initiating roles of a-actinin and talin have, however, been ruled out.37 Other possible candidates for this type of effect could be IF-associated proteins such as plectin, a phosphoprotein that links IFs and MTs with each other.9 Some studies have indicated phosphorylation of actin itself, but it is unclear whether this phosphorylation has any consequences in terms of MF organization.54 Based on our previous study in primary rat hepatocyte cultures showing a distinguished and unified rearrangement of IFs and MFs,8 we anticipated a different F-actin effect in mice lacking proper keratin filaments. However, MC-LR induce rearrangements of F-actin in keratin-deficient mouse hepatocytes similar to those in wt. This suggests that observed reorganization of MFs is largely independent of existing keratin networks. We have previously shown a MC-LR–induced hyperphosphorylation of the desmoplakin-proteins and a consequent dissociation of desmosomes in rat primary cultured hepatocytes.8 Also in the intact mouse liver, desmoplakin immunoreactivity is abolished in the centrilobular hepatocytes, showing reorganization of actin and keratins. This effect was similar in the keratin-defective mice, and is therefore not dependent on the presence of keratin IFs. Miura et al.55 observed the absence of desmosomes in MC-LR–treated rat livers in the areas of bile canaliculi, and Theiss et al.56 showed that cyanobacterial toxins alter intercellular attachments. In the intact liver, the cytoskeletal or desmosomal alterations induced by MC-LR were observed only in hepatocytes in the centrilobular zone, while no such effects were observed in the portal zone. There is a functional heterogeneity in periportal and perivenous hepatocytes,57 and also some of the known bile acid carriers are differentially expressed within the lobular structure of the liver.58 Thus, it is plausible that the limited sensitivity of periportal hepatocytes could be caused by a reduced efficiency of MC-LR–specific transport. Taken together, the results in the present study further corroborate the importance of protein phosphatases in maintaining a normal cytoskeletal structure. This view is especially accentuated in regard to regulation of IF proteins. Our data also support the previous concept regarding the impor-

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tance of IF proteins in maintaining the structure of cells and tissues. Acknowledgment: The authors are grateful to David Garrod (School of Biological Sciences, University of Manchester, England) for generously providing us with desmoplakin antibodies. Gunilla Henriksson and Esa Nummelin (Department of Biology, Åbo Akademi University) are acknowledged for skillful technical assistance with sample preparation for electron microscopy and for aid with photograph preparations, respectively. Henna-Riitta Lehti (Department of Biology, Åbo Akademi University) and Markku Kallajoki (Department of Pathology, University of Turku, Finland) are acknowledged for help with preparation of histological samples, and He Tao for assistance with the mouse experiments (Åbo Akademi University, Department of Biochemistry and Pharmacy). The authors further thank Sakari Toikkanen (Department of Pathology, University of Turku) for valuable discussions on liver pathology. REFERENCES 1. van Eyken P, Desmet VJ. Cytokeratins and the liver. Liver 1993;13:113122. 2. Chou Y-H, Skalli O, Goldman RD. Intermediate filaments and cytoplasmic networking: new connections and more functions. Curr Opin Cell Biol 1997;9:49-53. 3. Fuchs E, Clevland DW. A structural scaffolding of intermediate filaments in health and disease. Science 1998;279:14-51. 4. Fuchs E. Of mice and men: genetic disorders of the cytoskeleton [Keith R. Porter Lecture 1996]. Mol Biol Cell 1997;8:189-203. 5. McLean WHI, Lane EB. Intermediate filaments in disease. Curr Opin Cell Biol 1995;7:118-125. 6. Irvin DA, Corden LD, Swensson O, Swensson B, Moore JE, Frazer DG, Smith FJD, et al. Mutations in cornea-specific keratin K3 or K12 genes cause Meesmann’s corneal dystrophy. Nat Genet 1997;16:184-187. 7. Yang Y, Dowling J, Yu Q-C, Kouklis P, Cleveland DW, Fuchs E. An essential cytoskeletal linker protein connecting actin microfilaments to intermediate filaments. Cell 1996;86:655-665. 8. Toivola DM, Goldman RD, Garrod DR, Eriksson JE. Protein phosphatases maintain the organization and structural interactions of hepatic keratin intermediate filaments. J Cell Sci 1997;110:23-33. 9. Svitkina TM, Verkhovsky AB, Borisy GG. Plectin sidearms mediate interaction of intermediate filaments with microtubules and other components of the cytoskeleton. J Cell Biol 1997;135:991-1007. 10. Lazarides E. Intermediate filaments as mechanical integrators of cellular space. Nature 1980;283:249-256. 11. Marceau N, Loranger A. Cytokeratin expression, fibrillar organization, and subtle function in liver cells. Biochem Cell Biol 1995;73:619-625. 12. Goldman RD, Khuon S, Chou YH, Opal P, Steinert PM. The function of intermediate filaments in cell shape and cytoskeletal integrity. J Cell Biol 1996;134:971-983. 13. Omary MB, Ku N-O. Intermediate filament proteins of the liver: emerging disease association and functions. HEPATOLOGY 1997;25:10431048. 14. Baribault H, Penner J, Iozzo RV, Wilson-Heiner M. Colorectal hyperplasia and inflammation in keratin 8-deficient FVB/N mice. Genes Dev 1994;8:2964-2973. 15. Ku N-O, Michie S, Oshima RG, Omary MB. Chronic hepatitis, hepatocyte fragility, and increased soluble phosphoglycokeratins in transgenic mice expressing a keratin 18 conserved arginine mutant. J Cell Biol 1995;131:1303-1314. 16. Kawahara H, Cadrin M, Perry G, Autilio-Gambetti L, Swierenga SHH, Metuzals J, Marceau N, et al. Role of cytokeratin intermediate filaments in transhepatic transport and canalicular secretion. HEPATOLOGY 1990;11: 435-448. 17. Eriksson JE, Opal P, Goldman RD. Intermediate filament dynamics. Curr Opin Cell Biol 1992a;4:99-104. 18. Ku N-O, Liao J, Chou C-F, Omary MB. Implications of intermediate filament protein phosphorylation. Cancer Metastasis Rev 1996a;15:429444. 19. Chou Y-H, Ngai KL, Goldman R. The regulation of intermediate filament reorganization in mitosis. J Biol Chem 1991;266:7325-7328.

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