RESUMEN
Wilson disease is a genetic disorder characterized by the accumulation of copper in the body by defective biliary copper excretion. Wilson disease gene product (ATP7B) functions in copper incorporation to ceruloplasmin (Cp) and biliary copper excretion. However, copper metabolism in hepatocytes has been still unclear. Niemann-Pick disease type C (NPC) is a lipid storage disorder and the most commonly mutated gene is NPC1 and its gene product NPC1 is a late endosome protein and regulates intracellular vesicle traffic. In the present study, we induced NPC phenotype and examined the localization of ATP7B and secretion of holo-Cp, a copper-binding mature form of Cp. The vesicle traffic was modulated using U18666A, which induces NPC phenotype, and knock down of NPC1 by RNA interference. ATP7B colocalized with the late endosome markers, but not with the trans-Golgi network markers. U18666A and NPC1 knock down decreased holo-Cp secretion to culture medium, but did not affect the secretion of other secretory proteins. Copper accumulated in the cells after the treatment with U18666A. These findings suggest that ATP7B localizes in the late endosomes and that copper in the late endosomes is transported to the secretory compartment via NPC1-dependent pathway and incorporated into apo-Cp to form holo-Cp.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas Portadoras/fisiología , Proteínas de Transporte de Catión/metabolismo , Cobre/metabolismo , Endosomas/metabolismo , Glicoproteínas de Membrana/fisiología , Subunidades gamma de Complejo de Proteína Adaptadora/metabolismo , Adenosina Trifosfatasas/genética , Androstenos/farmacología , Anticolesterolemiantes/farmacología , Transporte Biológico/efectos de los fármacos , Transporte Biológico/fisiología , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Proteínas Portadoras/genética , Proteínas de Transporte de Catión/genética , Línea Celular Tumoral , Ceruloplasmina/metabolismo , ATPasas Transportadoras de Cobre , Humanos , Péptidos y Proteínas de Señalización Intracelular , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Proteína 2 de la Membrana Asociada a los Lisosomas , Proteínas de Membrana de los Lisosomas/metabolismo , Glicoproteínas de Membrana/genética , Mutación , Proteína Niemann-Pick C1 , Interferencia de ARN , ARN Interferente Pequeño/genética , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión a GTP rab7RESUMEN
Microtubules (MTs) and microfilaments (MFs) are known to modulate mitochondrial morphology, distribution and function. However, little is known evidence about the role of intermediate filaments (IFs) in modulating mitochondria except desmin. To investigate whether or not the IFs regulate mitochondrial morphology, distribution, and function, we manipulated the IFs of cultured epithelial cells to express a mutant keratin 18 (K18). In contrast to the filamentous expression of wild K18, mutant K18 induced aggregation of K8/18, showing no fine IF network in the cells. In mutant K18-transfected cells, the mitochondria were fragmented into small spheroids, although they were observed as mitochondrial fibers in un-transfected or wild K18-transfected cells. Fluorescence recovery after photobleaching of fluorescence-labeled mitochondria was markedly less in the mutant K18-transfected cells, although a significant recovery was confirmed in wild K18-transfected cells. These findings suggest that the IFs are important for the maintenance of normal mitochondrial structures.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Células Epiteliales/patología , Queratina-18/genética , Hepatopatías/patología , Mitocondrias Hepáticas/metabolismo , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/patología , Animales , Células Cultivadas , Células Epiteliales/metabolismo , Colorantes Fluorescentes/química , Humanos , Hepatopatías/genética , Hepatopatías/metabolismo , Microscopía Confocal , Microscopía Fluorescente , Mitocondrias Hepáticas/genética , Mitocondrias Hepáticas/patología , Mutación , TransfecciónRESUMEN
AIM: Mallory bodies have been observed in various liver diseases, however, the precise mechanism and significance of these structures have yet to be determined. METHODS: Previously we reported on the redistribution of cytosolic proteins to keratin inclusions in mutant keratin 18-transfected cells. In this study, we treated green fluorescent protein-tagged wild-type keratin 18-transfected cells with several proteasome inhibitors and performed immunofluorescent analyses. RESULTS: Proteasome inhibitors induced intracellular keratin inclusions, and desmoplakin, zonula occludens-1 and beta-catenin were relocated to keratin inclusions, while theintegral membrane proteins were intact. The cytosolic proteins, 14-3-3 zeta protein and glucose-6-phosphate dehydrogenase were also relocated to inclusions. Moreover, E-cadherin, a basolateral membrane protein, was present on both the apical and basolateral domains in inclusion-containing cells. CONCLUSION: These data are identical to those in the mutant keratin 18 transfection study and suggest that keratin inclusions induced by different treatments affect localization of various cytosolic components, which may influence cellular functions performed by these proteins.
RESUMEN
BACKGROUND: The aim of this study was to evaluate the efficacy of daclatasvir plus asunaprevir therapy in patients infected with hepatitis C virus and determine its relevance to resistant variants. METHODS: A total of 629 consecutive patients infected with hepatitis C virus genotype 1 were assessed. Daclatasvir (60 mg/day) plus asunaprevir (200 mg/day) was given for 24 weeks. The virological responses and resistance-associated substitutions of hepatitis C virus mutants were examined by the direct sequence and cycleave methods were evaluated. RESULTS: Overall, 89.4% (555/621) of patients exhibited a sustained virological response (SVR). The SVR rates in the patients with wild type, mixed, and mutant type Y93 by direct sequencing were 92.5% (520/562), 70.3% (26/37), and 42.9% (9/21), respectively. The SVR rates in the patients with 100%, 90%, 80%-30%, and 20%-0% Y93 wild by the cycleave method were 93.4% (456/488), 88.2%(30/34), 56.0%(14/25), and 36.8%(7/19), respectively. In contrast, the SVR rates for the wild type and mixed/mutant type L31 by direct sequencing were 90.2% (534/592) and 72.4% (21/29), respectively. In the multivariate analyses, the wild type Y93, no history of simeprevir therapy, the wild type L31, and low HCV RNA level were independent factors of SVR. CONCLUSION: NS5A resistance-associated substitutions, especially Y93H, were major factors predicting the SVR. Although direct sequencing can predict the SVR rate, the cycleave method is considered to be more useful for predicting the SVR when used in combination.
RESUMEN
A 47-year-old woman with primary biliary cirrhosis and scleroderma was examined at our hospital for a 1-week history of non-resolving fever, arthralgia, myalgia, muscle weakness and fatigue. A diagnosis of systemic lupus erythematosus was made based on arthralgia, low leukocyte count, low lymphocyte count, low serum concentration of complements, positive anti-nuclear antibody and positive anti-double-strand-DNA antibody. She was negative for anti-U1RNP antibody, but positive for anti-Jo1 antibody, and her initial serum concentration of creatine phosphokinase was elevated. We diagnosed her as having overlap syndrome with scleroderma, systemic lupus erythematosus and possible polymyositis associated with primary biliary cirrhosis. Prednisolone rapidly improved her symptoms. Lobulated leukocytes were observed in her peripheral blood specimen. She was positive for anti-HTLV-1 antibody, but Southern blot hybridization did not confirm monoclonal integration of HTLV-I proviral DNA in her peripheral blood. This suggests the possibility of a relationship between HTLV-1 infection and various autoimmune disorders including primary biliary cirrhosis.
RESUMEN
Angioedema associated with eosinophilia is a disorder characterized by angioedema and eosinophilia. However, the pathogenesis of this disorder has not been fully understood. We experienced 4 patients with angioedema associated with eosinophilia. All patients were young, 3 were female and 1 was male. All patients revealed edema of the limbs and eosinophilia. The symptom was rapidly improved after the initiation of low or medium dose of prednisolone. We evaluated the serum concentration of interleukin 5 (IL-5) and the plasma concentration of vascular endothelial growth factor (VEGF) in 1 patient. Both cytokines were markedly elevated before the treatment and decreased after the treatment. Angioedema associated with eosinophilia is not so rare, and IL-5 and VEGF are involved in the pathogenesis of this disease.
Asunto(s)
Angioedema/etiología , Eosinofilia/etiología , Factor A de Crecimiento Endotelial Vascular/fisiología , Adulto , Femenino , Humanos , Interleucina-5/sangre , Interleucina-5/fisiología , Masculino , Factor A de Crecimiento Endotelial Vascular/sangreRESUMEN
A 33-year-old woman was referred to our hospital due to repeated spontaneous abortions and positive autoantibodies. She had noticed Raynaud's phenomenon 13 years earlier. We diagnosed scleroderma based on the presence of Raynaud's phenomenon, proximal scleroderma, presence of anti-centromere antibodies, and histological findings on skin biopsy. Neither lupus anticoagulant nor anti-cardiolipin-beta2-glycoprotein 1 antibody was detected. We administered tocopherol nicotinate. Five months after the initiation of the treatment, she became pregnant and later delivered a healthy baby.
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Aborto Habitual/etiología , Complicaciones del Embarazo/tratamiento farmacológico , Esclerodermia Sistémica/tratamiento farmacológico , Vitamina E/uso terapéutico , Adulto , Autoantígenos/sangre , Femenino , Humanos , Embarazo , ARN Citoplasmático Pequeño/sangre , Ribonucleoproteínas/sangre , Esclerodermia Sistémica/complicacionesRESUMEN
Primary sclerosing cholangitis (PSC) is known to be frequently associated with inflammatory bowel diseases. In a rat with self-filling blind loop (SFBL), a proposed animal model for PSC, hepatobiliary inflammation has previously been demonstrated. In this study, we assessed the involvement of lipopolysaccharide (LPS), a bacterial endotoxin, in the pathogenesis of hepatobiliary inflammation of the SFBL model. The hepatic localization of LPS was examined by immunohistochemistry using an anti-lipid A antibody. The portal blood concentration of LPS was measured by an endotoxin-specific chromogenic Limulus test (Endospecy test). LPS was localized in the biliary epithelial cells (BECs) of rats with SFBL, and the portal blood concentration of LPS was significantly higher than that of sham-operated rats. Development of hepatobiliary inflammation, peribiliary fibrosis, and injury to the intestinal mucosa were histologically confirmed. Constriction in the biliary trees was radiologically demonstrated. These findings suggested that abnormal accumulation of LPS, which may be derived from portal blood, in BECs was involved in the pathogenesis of hepatobiliary inflammation with intestinal injury.
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Conductos Biliares/anatomía & histología , Conductos Biliares/patología , Células Epiteliales/metabolismo , Lipopolisacáridos/metabolismo , Animales , Colangiografía , Inmunohistoquímica , Inflamación , Lípido A/inmunología , Lipopolisacáridos/sangre , Masculino , Microscopía Electrónica , Unión Proteica , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
Wilson disease is a genetic disorder characterized by the accumulation of copper in the body due to a defect of biliary copper excretion. The gene responsible for Wilson disease has been cloned, however, the precise localization of this gene product ATP7B, a copper-transporting ATPase, is still controversial. We examined the localization of ATP7B by expressing a chimeric protein, ATP7B-tagged with green fluorescent protein (GFP) (GFP-ATP7B), in HEK293, Hep3B and a highly polarized human hepatocyte line (OUMS29). Intracellular organelles were visualized by immunofluorescence microscopy. The effects of bathocuproine disulfonate, a copper chelator, and copper sulfate were examined. GFP-ATP7B colocalized with a late endosome marker, but not with endoplasmic reticulum, Golgi, or lysosome markers in a copper-depleting condition. Treatment with copper sulfate did not affect the localization of ATP7B. ATP7B is localized in the late endosomes in both copper-depleting and copper-loaded conditions. ATP7B seems to translocate copper from the cytosol into the late endosomes, and copper may be excreted to bile via lysosomes. We believe that the disturbed incorporation of copper into the late endosomes caused by mutated ATP7B is the main defect in Wilson disease.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas de Transporte de Catión/metabolismo , Endosomas/metabolismo , Hepatocitos/metabolismo , Adenosina Trifosfatasas/efectos de los fármacos , Adenosina Trifosfatasas/genética , Proteínas de Transporte de Catión/efectos de los fármacos , Proteínas de Transporte de Catión/genética , Línea Celular , Polaridad Celular , Quelantes/metabolismo , Quelantes/farmacología , Cobre/metabolismo , Sulfato de Cobre/farmacología , ATPasas Transportadoras de Cobre , Endosomas/efectos de los fármacos , Proteínas Fluorescentes Verdes , Humanos , Proteínas Luminiscentes/metabolismo , Mutación , Fenantrolinas/farmacología , Proteínas Recombinantes/metabolismo , TransfecciónRESUMEN
A 62-year-old woman with primary biliary cirrhosis (PBC) and rheumatoid arthritis (RA) was examined at our hospital for a 2-week history of non-resolving fever, cough and malaise. Her chest radiograph revealed left lower lobe opacity. Various kinds of antibiotics were not effective and transbronchial biopsy revealed non-specific inflammatory alveolar lesions. Chest radiograph at 14 days after admission revealed migration of the pulmonary opacity, suggesting bronchiolitis obliterans organizing pneumonia (BOOP), which responded well clinically and radiologically to oral corticosteroid therapy. BOOP may be one of the possible non-hepatic complications of PBC especially in patients associated with other connective tissue diseases.
RESUMEN
A 77-year-old man with pneumonia associated with acute myeloid leukemia was introduced to the hepatology unit at our hospital for hyperbilirubinemia. He had been suffering from a high fever because of pneumonia. He was icteric and his serum concentrations of total and direct bilirubin were 13.1 and 7.9 mg/dl, respectively. However, the other standard biochemical examinations for hepatic function, such as serum concentrations of aspartate aminotransferase, alanine aminotransferase, gamma-glutamyl transpeptidase and alkaline phosphatase were normal except for lactate dehydrogenase. Lactate dehydrogenase isoenzyme analysis revealed that the high concentration was derived from leukemia cells. Ultrasonography of the abdomen revealed no abnormality in the liver or biliary tract. Administration of antibiotics for pneumonia decreased the serum bilirubin concentration, however, he died because of respiratory failure caused by the progression of pneumonia at 33 days after the admission. It was suggested that a disturbance in the bilirubin metabolism without hepatocyte necrosis or mechanical cholestasis might be involved in the pathogenesis of hyperbilirubinemia in patients with infectious diseases.
RESUMEN
AIM: Wilson disease is a genetic disorder of copper metabolism characterized by impaired biliary copper excretion. Wilson disease gene product (ATP7B) functions in copper incorporation to ceruloplasmin (Cp) and biliary copper excretion. Our previous study showed the late endosome localization of ATP7B and described the copper transport pathway from the late endosome to trans-Golgi network (TGN). However, the cellular localization of ATP7B and copper metabolism in hepatocytes remains controversial. The present study was performed to evaluate the role of Niemann-Pick type C (NPC) gene product NPC1 on intracellular copper transport in hepatocytes. METHODS: We induced the NPC phenotype using U18666A to modulate the vesicle traffic from the late endosome to TGN. Then, we examined the effect of NPC1 overexpression on the localization of ATP7B and secretion of holo-Cp, a copper-binding mature form of Cp. RESULTS: Overexpression of NPC1 increased holo-Cp secretion to culture medium of U18666A-treated cells, but did not affect the secretion of albumin. Manipulation of NPC1 function affected the localization of ATP7B and late endosome markers, but did not change the localization of a TGN marker. ATP7B co-localized with the late endosome markers, but not with the TGN marker. CONCLUSION: These findings suggest that ATP7B localizes in the late endosomes and that copper in the late endosomes is transported to the secretory compartment via an NPC1-dependent pathway and incorporated into Cp.
RESUMEN
BACKGROUND/AIMS: The precise mechanism of formation and significance of Mallory bodies (MBs) are poorly understood. The endoplasmic reticulum (ER) is the organelle responsible for proper folding and elimination of unfolded proteins. Therefore, failure of this function increases defective proteins in the cell. METHODS: We examined the effects of oxidative stress on induction of ER stress and keratin 8 and 18 (K8/18)-containing inclusion formation in cultured human hepatoma cells and hepatocytes by immunofluorescence and immunoblot analyses. RESULTS: Generation of H(2)O(2) was detected in glucose oxidase (GO)-treated cells by 2',7'-dichlorodihydrofluorescein diacetate and co-treatment with GO and acetyl-leucyl-leucyl-norleucinal (ALLN), a proteasome inhibitor, induced formation of extensive keratin inclusions that were inhibited by pre-treatment with N-acetyl-cysteine. These inclusions shared similar features with MBs by immunofluorescence analysis. Electron microscopy showed that these structures appeared near the nuclei, surrounded by filamentous structures. GO and ALLN upregulated the expression of ER stress markers, however, 4-phenylbutyrate, a chemical chaperone, reduced formation of inclusions and expression of the ER stress markers. CONCLUSIONS: The oxidative stress coupled with limited inhibition of the proteasome induces dysfunction of the ER and results in inclusion formation in cultured cells. This suggests that ER stress plays a role in MB formation in liver disease.
Asunto(s)
Retículo Endoplásmico/metabolismo , Cuerpos de Inclusión/metabolismo , Hepatopatías/metabolismo , Estrés Oxidativo , Inhibidores de Cisteína Proteinasa/farmacología , Retículo Endoplásmico/ultraestructura , Fluoresceínas/farmacología , Glucosa Oxidasa/farmacología , Humanos , Peróxido de Hidrógeno/metabolismo , Cuerpos de Inclusión/química , Cuerpos de Inclusión/ultraestructura , Queratina-18/análisis , Queratina-8/análisis , Leupeptinas/farmacología , Hepatopatías/patología , Fenilbutiratos/metabolismo , Inhibidores de Proteasoma , Células Tumorales CultivadasRESUMEN
BACKGROUND & AIMS: Neovascularization, which is vital to the healing of injured tissues, recently has been found to include both angiogenesis, which involves in mature endothelial cells, and vasculogenesis, involving endothelial progenitor cells. The aim of this study was to clarify the possible roles of endothelial progenitor cells during postnatal liver regeneration. METHODS: To determine how endothelial progenitor cells participate in liver regeneration, human or mouse endothelial progenitor cells were transplanted into the mice with carbon tetrachloride-induced acute liver injury. Survival rate of the mice in endothelial progenitor cell-transplanted and control groups was calculated. Separately, livers removed temporally from both groups were examined. RESULTS: At an early stage, transplanted human endothelial progenitor cells were seen mainly surrounding hepatic central veins where hepatocytes showed extensive necrosis; later, the transplanted cells formed tubular structures. More of these cells were observed along hepatic sinusoids. Transplantation of human or mouse endothelial progenitor cells improved survival of the mice following liver injury (from 28.6% to 85.7%, P < .0005 and from 33.3% to 80.0%, P < .001, respectively), accompanied by greater proliferation of hepatocytes. Human endothelial progenitor cells produced several growth factors, such as hepatocyte growth factor, transforming growth factor-alpha, heparin-binding epidermal growth factor-like growth factor, and vascular endothelial growth factor, and also elicited endogenous growth factors. CONCLUSIONS: Endogenous and exogenous growth factors and direct neovascularization after endothelial progenitor cell transplantation promoted liver regeneration, thus improving survival after liver injury. Transplantation of endothelial progenitor cells could represent a new therapeutic strategy for promoting liver regeneration.
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Endotelio Vascular/trasplante , Regeneración Hepática/fisiología , Hígado/lesiones , Trasplante de Células Madre , Animales , Modelos Animales de Enfermedad , Endotelio Vascular/embriología , Humanos , Hígado/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Modelos Animales , Neovascularización Fisiológica , Análisis de SupervivenciaRESUMEN
Many neurodegenerative diseases are characterized by the presence of protein aggregates bundled with intermediate filaments (IFs) and similar structures, known as Mallory bodies (MBs), are observed in various liver diseases. IFs are anchored at desmosomes and hemidesmosomes, however, interactions with other intercellular junctions have not been determined. We investigated the effect of IF inclusions on junction-associated and cytosolic proteins in various cultured cells. We performed gene transfection of the green fluorescent protein (GFP)-tagged cytokeratin (CK) 18 mutant arg89cys (GFP-CK18 R89C) in cultured cells and observed CK aggregations as well as loss of IF networks. Among various junction-associated proteins, zonula occludens-1 and beta-catenin were colocalized with CK aggregates on immunofluorescent analyses. Similar results were obtained on immunostaining for cytosolic proteins, 14-3-3 zeta protein, glucose-6-phosphate dehydrogenase and DsRed. E-cadherin, a basolateral membrane protein in polarized epithelia, was present on both the apical and basolateral domains in GFP-CK18 R89C-transfected cells. Furthermore, cells containing CK aggregates were significantly larger than GFP-tagged wild type CK18 (GFP-WT CK18)-transfected or non-transfected cells (P < 0.01) and sometimes their morphology was significantly altered. Our data indicate that CK aggregates affect not only cell morphology but also the localization of various cytosolic components, which may affect the cellular function.
Asunto(s)
Citosol/metabolismo , Células Epiteliales/metabolismo , Cuerpos de Inclusión/metabolismo , Uniones Intercelulares/metabolismo , Filamentos Intermedios/metabolismo , Queratinas/metabolismo , Proteínas 14-3-3/metabolismo , Animales , Línea Celular , Línea Celular Tumoral , Forma de la Célula/fisiología , Tamaño de la Célula , Citosol/patología , Citosol/ultraestructura , Perros , Células Epiteliales/patología , Células Epiteliales/ultraestructura , Técnica del Anticuerpo Fluorescente , Glucosafosfato Deshidrogenasa/metabolismo , Proteínas Fluorescentes Verdes , Humanos , Cuerpos de Inclusión/patología , Cuerpos de Inclusión/ultraestructura , Uniones Intercelulares/patología , Uniones Intercelulares/ultraestructura , Filamentos Intermedios/patología , Filamentos Intermedios/ultraestructura , Queratinas/genética , Proteínas de la Membrana/metabolismo , Microscopía Electrónica de Transmisión , Mutación/genética , TransfecciónRESUMEN
Wilson disease is a genetic disorder characterized by the accumulation of copper in the body due to a defect of biliary copper excretion. Although the Wilson disease gene has been cloned, the cellular localization of the gene product (ATP7B) has not been fully clarified. Therefore, the precise physiological action of ATP7B is still unknown. We examined the distribution of ATP7B using an anti-ATP7B antibody, green fluorescent protein (GFP)-ATP7B (GFP-ATP7B) and ATP7B-DsRed in various cultured cells. Intracellular organelles were visualized by fluorescence microscopy. The distribution of ATP7B was compared with that of Rab7 and Niemann-Pick C1 (NPC1), proteins that localize in the late endosomes. U18666A, which induces the NPC phenotype, was used to modulate the intracellular vesicle traffic. GFP-ATP7B colocalized with various late endosome markers including Rab7 and NPC1 but not with Golgi or lysosome markers. U18666A induced the formation of late endosome-lysosome hybrid organelles, with GFP-ATP7B localized with NPC1 in these structures. We have confirmed that ATP7B is a late endosome-associated membrane protein. ATP7B appears to translocate copper from the cytosol to the late endosomal lumen, thus participating in biliary copper excretion via lysosomes. Thus, defective copper ATPase activity of ATP7B in the late endosomes appears to be the main defect of Wilson disease.
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Adenosina Trifosfatasas/biosíntesis , Proteínas Portadoras/biosíntesis , Proteínas de Transporte de Catión/biosíntesis , Endosomas/metabolismo , Glicoproteínas de Membrana/biosíntesis , Proteínas de Unión al GTP rab/biosíntesis , Conductos Biliares/metabolismo , Línea Celular Tumoral , Quelantes/farmacología , Cobre/metabolismo , Sulfato de Cobre/farmacología , ATPasas Transportadoras de Cobre , Citosol/metabolismo , ADN Complementario/metabolismo , Aparato de Golgi/metabolismo , Hepatocitos/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Lisosomas/metabolismo , Microscopía Confocal , Microscopía Electrónica , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Mutación , Proteína Niemann-Pick C1 , Fenotipo , Transporte de Proteínas , Proteínas de Unión a GTP rab7RESUMEN
Intermediate filaments are one of the three major cytoskeletons. Some roles of intermediate filaments in cellular functions have emerged based on various diseases associated with mutations of cytokeratins. However, the precise functions of intermediate filament are still unclear. To resolve this, we manipulated intermediate filaments of cultured cells by expressing a mutant cytokeratin. Arginine 89 of cytokeratin18 plays an important role in intermediate filament assembly. The expression of green fluorescent protein-tagged cytokeratin18 arg89cys induced aggregations and loss of the intermediate filament network composed of cytokeratins in liver-derived epithelial cells, Huh7 and OUMS29, but only induced the formation of cytokeratin aggregates and did not affect the intermediate filament network of endogenous vimentin in HEK293. The expression of this mutant affected the distribution of Golgi apparatus and the reassembly of Golgi apparatus after perturbations by nocodazole or brefeldin A in both Huh7 and OUMS29, but not in HEK293. Our data show that loss of the original intermediate filament network, but not the existence of cytokeratin aggregates, induces redistribution of the Golgi apparatus. The original intact intermediate filament network is necessary for the organization of Golgi apparatus.
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Células Epiteliales/metabolismo , Aparato de Golgi/metabolismo , Hepatocitos/metabolismo , Filamentos Intermedios/patología , Queratinas/genética , Hígado/metabolismo , Arginina/metabolismo , Brefeldino A/farmacología , Línea Celular , Células Epiteliales/ultraestructura , Aparato de Golgi/patología , Aparato de Golgi/ultraestructura , Proteínas Fluorescentes Verdes , Hepatocitos/ultraestructura , Humanos , Filamentos Intermedios/genética , Hígado/fisiopatología , Hígado/ultraestructura , Proteínas Luminiscentes , Microscopía Confocal , Microscopía Electrónica , Mutación/genética , Nocodazol/farmacología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transfección , Vimentina/metabolismoRESUMEN
The ubiquitin-proteasome system is involved in a variety of biological processes. Inclusion bodies associated with intermediate filaments (IFs) and ubiquitin are observed in various diseases; however, the precise mechanisms of formation and the pathological significance of inclusion bodies have not been fully understood. We examined the effect of proteasome inhibitors on the structure of IF using anti-cytokeratin antibodies or transfection of green fluorescent protein-fused cytokeratin 18 in a hepatoma cell line, Huh7. Intracellular organelles were visualized by immunofluorescent and electron microscopies. Proteasome inhibitors induced IF inclusions associated with ubiquitin. Electron microscopic examination revealed inclusion bodies surrounded by filamentous structures. Autophagic vacuoles and lysosomes were frequently observed, and the organization of the Golgi apparatus was disrupted in these cells. After the removal of the proteasome inhibitors, the IF network and organization of the Golgi apparatus were restored. The IF inclusions could be induced by inhibition of the proteasome function. IF inclusions induced fragmentation of the Golgi apparatus and might inhibit the function of this important station of membrane traffic. The IF inclusions disappeared by restoring proteasome function, and autophagy and lysosomal degradation might be, at least in part, associated with the elimination of inclusion bodies.
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Aparato de Golgi/efectos de los fármacos , Cuerpos de Inclusión/efectos de los fármacos , Filamentos Intermedios/efectos de los fármacos , Complejos Multienzimáticos/antagonistas & inhibidores , Inhibidores de Proteasas/farmacología , Autofagia , Cisteína Endopeptidasas , Aparato de Golgi/ultraestructura , Humanos , Filamentos Intermedios/ultraestructura , Lisosomas/metabolismo , Microscopía Electrónica , Microscopía Fluorescente , Complejo de la Endopetidasa Proteasomal , Células Tumorales CultivadasRESUMEN
BACKGROUND/AIMS: Uncoupling proteins are thought to protect cells from oxidative stresses. Because uncoupling protein-2 is expressed in liver and reactive oxygen species are involved in pathogenesis of various liver diseases, this protein may protect liver cells from disease-associated oxidative stress. However, uncoupling protein-2 expression in human liver has not been examined. METHODS: We investigated hepatic uncoupling protein-2 distribution in various liver diseases including primary biliary cirrhosis, autoimmune hepatitis, chronic viral hepatitis, and histologically normal liver by immunohistochemistry. RESULTS: Uncoupling protein-2 was expressed in some hepatocytes, however, the degree of hepatocytic uncoupling protein-2 expression did not differ significantly among liver diseases and normal liver. Uncoupling protein-2 was abundant in biliary epithelial cells in primary biliary cirrhosis but not in other liver specimens. Enhanced uncoupling protein-2 expression in biliary epithelial cells was specific for primary biliary cirrhosis and did not result simply from cholestasis. The percentage of uncoupling protein-2 positive bile ducts in primary biliary cirrhosis patients treated with ursodeoxycholic acid was significantly lower than in untreated patients. CONCLUSIONS: These results suggest that uncoupling protein-2 is involved in the pathogenesis of primary biliary cirrhosis.
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Cirrosis Hepática Biliar/metabolismo , Proteínas de Transporte de Membrana , Proteínas Mitocondriales , Biosíntesis de Proteínas , Desacopladores , Conductos Biliares/metabolismo , Conductos Biliares/patología , Epitelio/metabolismo , Epitelio/patología , Hepatitis Autoinmune/metabolismo , Hepatitis Autoinmune/patología , Hepatitis Viral Humana/metabolismo , Hepatitis Viral Humana/patología , Hepatocitos/metabolismo , Hepatocitos/patología , Humanos , Técnicas para Inmunoenzimas , Canales Iónicos , Hígado/metabolismo , Hígado/patología , Cirrosis Hepática Biliar/patología , Proteína Desacopladora 2RESUMEN
Troglitazone has been withdrawn from therapeutic options for diabetes mellitus because of its severe hepatocyte toxicity of unknown pathogenesis. The aim of the present study was to assess both morphologic and functional alterations in the mitochondria of troglitazone-treated hepatocytes. A polarized human hepatocyte cell line, OUMS-29, was used in this study. The mitochondrial volume and the mitochondrial transmembrane potential (DeltaPsi(m)) were examined using flow cytometry with nonylacridine orange (NAO) and rhodamine-123, respectively. An ultrastructural examination of the troglitazone-treated OUMS-29 cells was performed using transmission electron microscopy (TEM). Reactive oxygen species (ROS) production was assessed using flow cytometry with dihydroethidium and 2',7'-dichlorodihydrofluorescein diacetate. A significant increase in the mitochondrial volume of the troglitazone-treated cells was found by the NAO analysis, in comparison with pioglitazone-treated and ciglitazone-treated cells. The increase in volume was due to a marked enlargement in the mitochondria. The markedly enlarged mitochondria with intramitochondrial electron-dense deposits were confirmed on TEM, which showed myelin-like structures, indicating degraded membrane constituents. The troglitazone-treated cells showed a significant decline in the DeltaPsi(m) per unit mitochondrial volume but resulted in no clear cell death. ROS analysis revealed a significant production of hydrogen peroxide in the troglitazone-treated hepatocytes. This production was attenuated using an antioxidant, N-acetyl-L-cysteine. In conclusion, troglitazone caused overproduction of hydrogen peroxide, which deteriorated both mitochondrial membrane structures and mitochondrial function, leading to a possible priming for the severe hepatocyte toxicity.