Biomedical subjects

M B Omary

Publications and source records attributed to M B Omary.

At least 19 recordsLinked to original sources

PubMed2003

An in vitro model of epithelial cell growth stimulation in the rodent mammary gland.

Mouse mammary epithelial cell cultures previously described bring about extensive proliferation and a cell population with the appropriate markers for luminal ductal epithelial cells, and also the ability to form normal tissue after implantation into mice. This success may result from a culture environment that resembles certain aspects of the environment in the mammary gland. Mouse mammary epithelial cells, whose proliferation is limited when plated alone, can be stimulated to multiply by contact with lethally irradiated cells of the LA7 rat mammary tumour line. Most of the proliferative stimulus is imparted by direct cell contact between LA7 and mouse mammary cells. Junctions, including adherens junctions, form among all cells in the culture, much as junctions form in the mammary gland. LA7 cells secrete TGFalpha and bFGF, factors found in the mammary gland, and factors to which mouse mammary cells respond in culture. Mouse mammary cells express keratins 8 and 18, markers for luminal cells of the mammary duct. LA7 cells express keratin 14 and vimentin, markers for myoepithelial cells. These facts, taken together, fit a model of cell replacement in an epithelial tissue and also imitate the relationship between luminal ductal cells and myoepithelial cells in the mammary gland. This method of culturing cells is useful, not only for in vitro-in vivo carcinogenesis studies, but also for the study of mechanisms by which growth signals are imparted from one cell to another.

Animals↗

PubMed2001

Keratin 8 mutations in patients with cryptogenic liver disease.

BACKGROUND: About 10 percent of patients who undergo liver transplantation have cryptogenic liver disease. In animal models, the absence of heteropolymeric keratins 8 and 18 or the presence of mutant keratins in hepatocytes causes or promotes liver disease. We have previously described a mutation in the keratin 18 gene in a patient with cryptogenic cirrhosis, but the importance of mutations in the keratin 8 and keratin 18 genes in such patients is unclear. METHODS: We tested for mutations in the keratin 8 and keratin 18 genes in purified genomic DNA isolated from 150 explanted livers and 89 peripheral-blood specimens from three groups of patients: 55 patients with cryptogenic liver disease; 98 patients with noncryptogenic liver disease, with causes that included alcohol use, autoimmunity, drug use, and viral infections; and 86 randomly selected inpatients and outpatients who provided blood to the hematology laboratory. RESULTS: Of the 55 patients with cryptogenic liver disease, 3 had glycine-to-cysteine mutations at position 61 (a highly conserved glycine) of keratin 8, and 2 had tyrosine-to-histidine mutations at position 53 of keratin 8. These mutations were not detected in the patients with other liver diseases or in the randomly selected patients. We verified the presence of the mutations in specimens of explanted livers by protein analysis and by the detection of unique restriction-enzyme cleavage sites. In transfected cells, the glycine-to-cysteine mutation limited keratin-filament reorganization when the cells were exposed to oxidative stress. In contrast, the tyrosine-to-histidine mutation destabilized keratin filaments when transfected cells were exposed to heat or okadaic acid stress. CONCLUSIONS: Mutations in the keratin 8 gene may predispose people to liver disease and may account for cryptogenic liver disease in some patients.

Amino Acid Sequence↗

PubMed2001

Effect of mutation and phosphorylation of type I keratins on their caspase-mediated degradation.

Type I keratins K18 and K19 undergo caspase-mediated degradation during apoptosis. Two known K18 caspase cleavage sites are aspartates in the consensus sequences VEVDA and DALDS, located within the rod domain and tail domain, respectively. Several K14 (another type I keratin) mutations within the caspase cleavage motif have been described in patients with epidermolysis bullosa simplex. Here we use extensive mutational analysis to show that K19 and K14 are caspase substrates and that the ability to undergo caspase-mediated digestion of K18, K19, or K14 is highly dependent on the location and nature of the mutation within the caspase cleavage motif. Caspase cleavage of K14 occurs at the aspartate of VEMDA, a consensus sequence found in type I keratins K12-17 with similar but not identical sequences in K18 and K19. For K18, apoptosis-induced cleavage occurs sequentially, first at (393)DALD and then at (234)VEVD. Hyperphosphorylation of K18 protects from caspase-3 in vitro digestion at (234)VEVD but not at (393)DALD. Hence, keratins K12-17 are likely caspase substrates during apoptosis. Keratin hyperphosphorylation, which occurs early in apoptosis, protects from caspase-mediated K18 digestion in a cleavage site-specific manner. Mutations in epidermolysis bullosa simplex patients could interfere with K14 degradation during apoptosis, depending on their location.

Amino Acid Sequence↗

PubMed2001

Disturbances in hepatic cell-cycle regulation in mice with assembly-deficient keratins 8/18.

Simple epithelial tissues such as liver and pancreas express keratins 8 (K8) and 18 (K18) as their major intermediate filament proteins. K8 and K18 null mice and transgenic mice that express mutant K18 (K18C) manifest several hepatocyte abnormalities and demonstrate that K8/18 are important in maintaining liver tissue and cell integrity, although other potential functions remain uncharacterized. Here, we report an additional abnormal liver phenotype, which is similar in K8 null, K18 null, and K18C mouse models. Liver histologic examination showed large polynuclear areas that lacked cell membranes, desmosomal structures, and filamentous actin. Similar, but less prominent, areas were observed in the pancreas. The parenchyma outside the polynuclear areas displayed irregular sinusoidal structures and markedly enlarged nuclei. Most K8 null hepatocytes were positive for the proliferating cell nuclear antigen (PCNA) with a doubled DNA content in comparison with the predominantly PCNA-negative wild-type hepatocytes. The distribution of the 14-3-3zeta protein was also altered in K8 null mice. Taken together, our results indicate that absence of keratin filaments causes disturbances in cell-cycle regulation, driving cells into the S-G2 phase and causing aberrant cytokinesis. These effects could stem from disturbed functions of K8/18-dependent cell-cycle regulators, such as the signaling integrator, 14-3-3.

Actins↗

PubMed2000

Interferon-alpha induces nmi-IFP35 heterodimeric complex formation that is affected by the phosphorylation of IFP35.

Nmi and IFP35 are interferon (IFN)-induced proteins. In cells treated with IFN-gamma, Nmi enhances the association of transcription co-activator CBP/p300 with signal transducer and activator of transcription proteins, and IFP35 forms a high molecular weight cytosolic complex of unknown constituents. Here we show that Nmi and IFP35 co-immunoprecipitate with an anti-keratin 19 antibody, which is due to cross-reaction of the antibody with Nmi, and suggests an Nmi-IFP35 physical association. In support of this, Nmi and IFP35 co-immunoprecipitate using anti-Nmi and anti-IFP35 antibodies, manifest enhanced colocalization as determined by immunofluorescence staining of IFN-treated cells, and form heterodimers as determined by chemical cross-linking. Nmi and IFP35 are primarily cytosolic proteins, and their interaction is increased after IFN-alpha treatment of cells as early as 1 h after exposure. Sucrose gradient sedimentation and size fractionation showed a shift of Nmi-IFP35 heterodimers toward a heavier fraction (100-200 kDa) in IFN-alpha-treated cells. This dynamic complex formation is reversed by pretreatment with okadaic acid. Two-dimensional gel analysis indicates that the IFN-induced complex formation correlates with IFP35 dephosphorylation. Our data demonstrate Nmi-IFP35 cytosolic localization and heterodimerization, and an IFN-alpha-regulated molecular event in which Nmi and IFP35 participate, reversibly and by a dephosphorylation dependent fashion, in a 100-200-kDa molecular complex formation.

Antibodies↗

PubMed2000

Keratins turn over by ubiquitination in a phosphorylation-modulated fashion.

Keratin polypeptides 8 and 18 (K8/18) are intermediate filament (IF) proteins that are expressed in glandular epithelia. Although the mechanism of keratin turnover is poorly understood, caspase-mediated degradation of type I keratins occurs during apoptosis and the proteasome pathway has been indirectly implicated in keratin turnover based on colocalization of keratin-ubiquitin antibody staining. Here we show that K8 and K18 are ubiquitinated based on cotransfection of His-tagged ubiquitin and human K8 and/or K18 cDNAs, followed by purification of ubiquitinated proteins and immunoblotting with keratin antibodies. Transfection of K8 or K18 alone yields higher levels of keratin ubiquitination as compared with cotransfection of K8/18, likely due to stabilization of the keratin heteropolymer. Most of the ubiquitinated species partition with the noncytosolic keratin fraction. Proteasome inhibition stabilizes K8 and K18 turnover, and is associated with accumulation of phosphorylated keratins, which indicates that although keratins are stable they still turnover. Analysis of K8 and K18 ubiquitination and degradation showed that K8 phosphorylation contributes to its stabilization. Our results provide direct evidence for K8 and K18 ubiquitination, in a phosphorylation modulated fashion, as a mechanism for regulating their turnover and suggest that other IF proteins could undergo similar regulation. These and other data offer a model that links keratin ubiquitination and hyperphosphorylation that, in turn, are associated with Mallory body deposits in a variety of liver diseases.

Cell Line↗

PubMed2000

Effects of keratin filament disruption on exocrine pancreas-stimulated secretion and susceptibility to injury.

Disruption or absence of hepatocyte keratins 8 and 18 is associated with chronic hepatitis, marked hepatocyte fragility, and a significant predisposition to stress-induced liver injury. In contrast, pancreatic keratin disruption in transgenic mice that express keratin 18 Arg89 --> Cys (K18C) is not associated with an obvious pancreatic pathology. We compared the effects of keratin filament disruption on pancreatic acini or acinar cell viability, and on cholecystokinin (CCK)-stimulated secretion, in transgenic mice that overexpress wild-type keratin 18 and harbor normal extended keratin filaments (TG2) and K18C mice. We also compared the response of these mice to pancreatitis induced by a choline-deficient ethionine-supplemented diet or by caerulein. Despite extensive cytoplasmic keratin filament disruption, the apicolateral keratin filament bundles appear intact in the acinar pancreas of K18C mice, as determined ultrastructurally and by light microscopy. No significant pancreatitis-associated histologic, serologic, or F-actin/keratin apicolateral redistribution differences were noted between TG2 and K18C mice. Acinar cell viability and yield after collagenase digestion were lower in K18C than in TG2 mice, but the yields of intact acini and their (125)I-CCK uptake and responses to CCK-stimulated secretion were similar. Our results indicate that keratin filament reorganization is a normal physiologic response to pancreatic cell injury, but an intact keratin cytoplasmic filament network is not as essential in protection from cell injury as in the liver. These findings raise the possibility that the abundant apicolateral acinar keratin filaments, which are not as evident in hepatocytes, may play the cytoprotective role that is seen in liver and other tissues. Alternatively, identical keratins may function differently in different tissues.

Actin Cytoskeleton↗

PubMed2000

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.

Animals↗

PubMed2000

Cyclooxygenase 2 expression in Barrett's esophagus and adenocarcinoma: Ex vivo induction by bile salts and acid exposure.

BACKGROUND & AIMS: Barrett's esophagus (BE) results from chronic, severe gastroesophageal reflux and predisposes to esophageal adenocarcinoma. Cyclooxygenase (COX)-2 is involved in chronic inflammation and epithelial cell growth. We investigated COX-2 expression in BE and esophageal adenocarcinoma to explore a potential relation between COX-2 expression and metaplasia or carcinogenesis. METHODS: Endoscopic mucosal biopsy specimens of Barrett's intestinal metaplasia (n = 30), Barrett's dysplasia (n = 11), and esophageal adenocarcinoma (n = 5) were compared with normal esophagus (n = 46) and duodenum (n = 46) and analyzed by Western blotting and immunohistochemistry. RESULTS: Immunoblots revealed constitutive expression of COX-2 in normal esophagus and duodenum. COX-2 protein expression was significantly higher in patients with Barrett's metaplasia, dysplasia, and adenocarcinoma compared with normal squamous esophageal or columnar duodenal epithelia and was heterogenous in different regions of the BE surface. Immunohistochemistry revealed prominent staining in the glands of BE, dysplasia, and adenocarcinoma and faint staining in the basal layers of squamous esophagus and the surface of the duodenum. In response to pulses of acid or bile salts in an ex vivo organ culture system, COX-2 expression increased significantly in BE tissues, and this effect was attenuated by the selective COX-2 inhibitor NS-398. CONCLUSIONS: The results show COX-2 expression in normal esophagus, which increases significantly in BE and esophageal adenocarcinoma. COX-2 is regulated ex vivo by exposure to acid or bile salts.

Acids↗

PubMed2000

Bile salts induce or blunt cell proliferation in Barrett's esophagus in an acid-dependent fashion.

Barrett's esophagus (BE) results from acid and bile reflux and predisposes to cancer. We investigated the effect of bile salts, with or without acid, on cell proliferation in BE and assessed mechanism(s) involved. To mimic physiological conditions, biopsies of esophagus, BE, and duodenum were exposed to a bile salt mixture, either continuously or as a 1-h pulse, and were compared with control media without bile salts (pH 7.4) for < or =24 h. Similar experiments were also performed with acidified media (pH 3.5) combined with the bile salt mixture as a 1-h pulse. Cell proliferation was assessed by a [(3)H]thymidine incorporation assay with or without bisindolylmaleimide (BIM), a selective protein kinase C inhibitor. Bile salt pulses enhanced cell proliferation in BE without affecting cell proliferation in esophageal or duodenal epithelia. In the presence of BIM, there was complete obliteration of the bile salt-induced BE hyperproliferation. In contrast, 1-h pulses of bile salts in combination with acid significantly inhibited proliferation in BE but had no effect on esophagus or duodenum. We conclude that in BE explants, brief exposure to bile salts, in the absence of acid, increases proliferation, whereas exposure to a combination of bile salts and acid together inhibits proliferation.

Acids↗

PubMed2000

Simple epithelial keratins are dispensable for cytoprotection in two pancreatitis models.

Pancreatic acinar cells express keratins 8 and 18 (K8/18), which form cytoplasmic filament (CF) and apicolateral filament (ALF) pools. Hepatocyte K8/18 CF provide important protection from environmental stresses, but disruption of acinar cell CF has no significant impact. We asked whether acinar cell ALF are important in providing cytoprotective roles by studying keratin filaments in pancreata of K8- and K18-null mice. K8-null pancreas lacks both keratin pools, but K18-null pancreas lacks only CF. Mouse but not human acinar cells also express apicolateral keratin 19 (K19), which explains the presence of apicolateral keratins in K18-null pancreas. K8- and K18-null pancreata are histologically normal, and their acini respond similarly to stimulated secretion, although K8-null acini viability is reduced. Absence of total filaments (K8-null) or CF (K18-null) does not increase susceptibility to pancreatitis induced by caerulein or a choline-deficient diet. In normal and K18-null acini, K19 is upregulated after caerulein injury and, unexpectedly, forms CF. As in hepatocytes, acinar injury is also associated with keratin hyperphosphorylation. Hence, K19 forms ALF in mouse acinar cells and helps define two distinct ALF and CF pools. On injury, K19 forms CF that revert to ALF after healing. Acinar keratins appear to be dispensable for cytoprotection, in contrast to hepatocyte keratins, despite similar hyperphosphorylation patterns after injury.

Actins↗

PubMed1999

Non-invasive transgenic mouse genotyping using stool analysis.

Commonly applied genotyping of transgenic mice involves using tail or ear biopsies which may cause discomfort to the animal. We tested the possibility of polymerase chain reaction (PCR)-based mouse genotyping using stool specimens from three transgenic mouse lines that overexpress 10-18 transgene copies of human keratin polypeptide 18, as compared to genotyping using tail biopsies. Stool specimens were obtained with ease and provided easy detection of the human transgene product. The method was also able to detect endogenous mouse actin and keratin genes which presumably are present at two copies each. Nested PCR was not necessary for genotyping using stool-derived genomic material but did increase the relative magnitude of the signal obtained. The non-invasive genotyping method described herein offers a reproducible, sensitive and effective modality that could replace invasive tissue sampling procedures currently used to test thousands of genetically altered mice.

Animals↗

PubMed1999

Characterization of the major physiologic phosphorylation site of human keratin 19 and its role in filament organization.

Keratin polypeptide 19 (K19) is a type I intermediate filament protein that is expressed in stratified and simple-type epithelia. Little is known regarding K19 regulation or function, and the only other type I keratin that has been studied in terms of regulation is keratin 18 (K18). We characterized K19 phosphorylation as a handle to study its function. In vivo, serine is the major phosphorylated residue, and phosphopeptide mapping of 32PO4-labeled K19 generates one major phosphopeptide. Edman degradation suggested that the radiolabeled phosphopeptide represents K19 Ser-10 and/or Ser-35 phosphorylation. Mutation of Ser-10 or Ser-35 followed by transfection confirmed that Ser-35 is the major K19 phosphorylation site. Transfection of Ser-35 --> Ala K19 showed a filament assembly defect as compared with normal or with Ser-10 --> Ala K19. Comparison of K18 and K19 phosphorylation features in interphase cells showed that both are phosphorylated primarily at a single site, preferentially in the soluble versus the insoluble keratin fractions. K19 has higher basal phosphorylation, whereas K18 phosphorylation is far more sensitive to phosphatase type I and IIA inhibition. Our results demonstrate that Ser-35 is the major K19 interphase phosphorylation site and that it plays a role in keratin filament assembly. K19 and K18 phosphorylations share some features but also have distinct properties that suggest different regulation of type I keratins within the same cells.

Animals↗

PubMed1999

The cytoskeleton of digestive epithelia in health and disease.

The mammalian cell cytoskeleton consists of a diverse group of fibrillar elements that play a pivotal role in mediating a number of digestive and nondigestive cell functions, including secretion, absorption, motility, mechanical integrity, and mitosis. The cytoskeleton of higher-eukaryotic cells consists of three highly abundant major protein families: microfilaments (MF), microtubules (MT), and intermediate filaments (IF), as well as a growing number of associated proteins. Within digestive epithelia, the prototype members of these three protein families are actins, tubulins, and keratins, respectively. This review highlights the important structural, regulatory, functional, and unique features of the three major cytoskeletal protein groups in digestive epithelia. The emerging exciting biological aspects of these protein groups are their involvement in cell signaling via direct or indirect interaction with a growing list of associated proteins (MF, MT, IF), the identification of several disease-causing mutations (IF, MF), the functional role that they play in protection from environmental stresses (IF), and their functional integration via several linker proteins that bridge two or potentially all three of these groups together. The use of agents that target specific cytoskeletal elements as therapeutic modalities for digestive diseases offers potential unique areas of intervention that remain to be fully explored.

Animals↗

PubMed1999

Pervanadate-mediated tyrosine phosphorylation of keratins 8 and 19 via a p38 mitogen-activated protein kinase-dependent pathway.

Glandular epithelia express the keratin intermediate filament (IF) polypeptides 8, 18 and 19 (K8/18/19). These proteins undergo significant serine phosphorylation upon stimulation with growth factors and during mitosis, with subsequent modulation of their organization and interaction with associated proteins. Here we demonstrate reversible and dynamic tyrosine phosphorylation of K8 and K19, but not K18, upon exposure of intact mouse colon or cultured human cells to pervanadate. K8/19 tyrosine phosphorylation was confirmed by metabolic 32PO4-labeling followed by phosphoamino acid analysis, and by immunoblotting with anti-phosphotyrosine antibodies. Pervanadate treatment increases keratin solubility and also indirectly increases K8/18 serine phosphorylation at several known sites, some of which were previously shown to be associated with EGF stimulation, extracellular signal-regulated kinase (ERK), or p38 kinase activation. However, K8/19 tyrosine phosphorylation is independent of EGF signaling or ERK activation while inhibition of p38 kinase activity blocks pervanadate-induced K8/19 tyrosine phosphorylation. Our results demonstrate tyrosine phosphatase inhibitor-mediated in vivo tyrosine phosphorylation of K8/19, but not K18, and suggest that tyrosine phosphorylation may be a general modification of other IF proteins. K8/19 tyrosine phosphorylation involves a pathway that utilizes the p38 mitogen-activated protein kinase, but appears independent of EGF signaling or ERK kinase activation.

Animals↗

PubMed1998

Mutation of a major keratin phosphorylation site predisposes to hepatotoxic injury in transgenic mice.

Simple epithelia express keratins 8 (K8) and 18 (K18) as their major intermediate filament (IF) proteins. One important physiologic function of K8/18 is to protect hepatocytes from drug-induced liver injury. Although the mechanism of this protection is unknown, marked K8/18 hyperphosphorylation occurs in association with a variety of cell stresses and during mitosis. This increase in keratin phosphorylation involves multiple sites including human K18 serine-(ser)52, which is a major K18 phosphorylation site. We studied the significance of keratin hyperphosphorylation and focused on K18 ser52 by generating transgenic mice that overexpress a human genomic K18 ser52--> ala mutant (S52A) and compared them with mice that overexpress, at similar levels, wild-type (WT) human K18. Abrogation of K18 ser52 phosphorylation did not affect filament organization after partial hepatectomy nor the ability of mouse livers to regenerate. However, exposure of S52A-expressing mice to the hepatotoxins, griseofulvin or microcystin, which are associated with K18 ser52 and other keratin phosphorylation changes, resulted in more dramatic hepatotoxicity as compared with WT K18-expressing mice. Our results demonstrate that K18 ser52 phosphorylation plays a physiologic role in protecting hepatocytes from stress-induced liver injury. Since hepatotoxins are associated with increased keratin phosphorylation at multiple sites, it is likely that unique sites aside from K18 ser52, and phosphorylation sites on other IF proteins, also participate in protection from cell stress.

3T3 Cells↗

PubMed1998

Phosphorylation of human keratin 18 serine 33 regulates binding to 14-3-3 proteins.

Members of the 14-3-3 protein family bind the human intermediate filament protein keratin 18 (K18) in vivo, in a cell-cycle- and phosphorylation-dependent manner. We identified K18 Ser33 as an interphase phosphorylation site, which increases its phosphorylation during mitosis in cultured cells and regenerating liver, and as an in vitro cdc2 kinase phosphorylation site. Comparison of wild-type versus K18 Ser33-->Ala/Asp transfected cells showed that K18 Ser33 phosphorylation is essential for the association of K18 with 14-3-3 proteins, and plays a role in keratin organization and distribution. Mutation of another K18 major phosphorylation site (Ser52) or K18 glycosylation sites had no effect on the binding of K18 to 14-3-3 proteins. The K18 phospho-Ser33 motif is different from several 14-3-3-binding phosphomotifs already described. Antibodies that are specific to K18 phospho-Ser33 or phospho-Ser52 show that although Ser52 and Ser33 phosphorylated K18 molecules manifest partial colocalization, these phosphorylation events reside predominantly on distinct K18 molecules. Our results demonstrate a unique K18 phosphorylation site that is necessary but not sufficient for K18 binding to 14-3-3 proteins. This binding is likely to involve one or more mitotic events coupled to K18 Ser33 phosphorylation, and plays a role in keratin subcellular distribution. Physiological Ser52 or Ser33 phosphorylation on distinct K18 molecules suggests functional compartmentalization of these modifications.

14-3-3 Proteins↗

PubMed1998

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.

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 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.

Animals↗