RESUMEN
Akt, a downstream effector kinase of insulin receptor and insulin-like growth factor-I receptor (IGF-IR), is critically involved in epithelial-mesenchymal transition (EMT). The aim of this study was to assess the impact of SLUG in the IGF/IGF-IR/Akt axis. The SLUG-overexpressing MDCK (SLUG-MDCK) cell clones were used as the EMT model. In contrast to the parental cells and mock-transfected MDCK cells, SLUG-MDCK cells were markedly sensitive to IGFs, showing a clear tyrosine-phosphorylation in IGF-IR under serum-starved conditions. The IGF-IR of hepatocytes was highly activated by culture supernatants from SLUG-MDCK cells. This activation was inhibited by IGF-binding protein (IGFBP)-3, and by the IGF-IR inhibitor PQ401, leading to inactivation of Akt. This finding suggested establishment of autocrine IGF-IR signaling in the SLUG-MDCK cells. It is known that cells overexpressing receptor tyrosine kinases have an increased activity in Src kinase, which constitutively phosphorylates IGF-IR. In the present study, we found an increased phosphorylation of Src in SLUG-MDCK cells, and not in mock-MDCK cells; however, this Src activation was not always coupled with the constitutive activation of IGF-IR, since the specific Src inhibitor PP2 failed to decrease the IGF-IR phosphorylation. PP2 just attenuated the phosphorylation in Akt, not through IGF-IR inactivation, leading to apoptosis in SLUG-MDCK cells. Of interest, the inactivation of Akt by IGFBP-3 was dramatically enhanced in combination with the use of PP2, resulting in a significant apoptosis in SLUG-MDCK cells. These findings suggested that dual targeting for IGF-IR and Src might be a potential therapeutic strategy in EMT-driven aggressive cancers.
Asunto(s)
Células Epiteliales/citología , Mesodermo/citología , Osteoblastos/fisiología , Receptor IGF Tipo 1/fisiología , Factores de Transcripción/genética , Factores de Transcripción/fisiología , Animales , Línea Celular , Perros , Hepatocitos/citología , Hepatocitos/fisiología , Humanos , Riñón/fisiología , Fosforilación , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Factores de Transcripción de la Familia SnailRESUMEN
BACKGROUND: Acquired features of cells under epithelial-mesenchymal transition (EMT) have not yet been fully identified. The current study was conducted to assess alterations in both the proliferative potential and the responsiveness to extracellular matrices (ECMs) in EMT. METHODS: MDCK cells and SLUG-transfected MDCK clones (SLUG-MDCK) were used in this study. The cell cycle was analyzed by using flow cytometry and Western blotting. ECM-stimulated cell proliferation was examined by using the following ECMs, type I collagen, type IV collagen, fibronectin, and laminin. Protein phosphorylation was detected by immunoprecipitation-Western by using the 4G10 antibody. RESULTS: Both G1 and G2/M arrest were found in the SLUG-MDCK cells, and the responsible molecules for the cell-cycle arrests were, at least in part, p21WAF1/Cip1 and Wee1. Once in contact with type I collagen, the SLUG-MDCK cells, showing the Wee1 degradation, dramatically started to proliferate up to 6-fold in cell number at Day 5, in contrast to only a 2-fold increase in the control. The analysis of the collagen receptors in the SLUG-MDCK cells disclosed a striking increase in the discoid domain receptor (DDR) 2 expression and a clear decrease in the DDR1 expression. The immunoprecipitated DDR2 protein extracted from SLUG-MDCK cells, which were cultured on collagen for 30 minutes, was tyrosine-phosphorylated, indicating valid functionality of the up-regulated receptor. The altered expression from DDR1 to DDR2 was also found in the naturally dedifferentiated sister cell lines of human liver cancer. CONCLUSIONS: Collectively, SLUG-induced EMT may alter the expression profile of receptor tyrosine kinases, including DDRs.
Asunto(s)
Diferenciación Celular/fisiología , Transformación Celular Neoplásica/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Regulación de la Expresión Génica/fisiología , Proteínas Tirosina Quinasas Receptoras/biosíntesis , Receptores Mitogénicos/biosíntesis , Factores de Transcripción/metabolismo , Animales , Western Blotting , Ciclo Celular/fisiología , Línea Celular , Proliferación Celular , Receptores con Dominio Discoidina , Perros , Epitelio/metabolismo , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Expresión Génica , Humanos , Inmunoprecipitación , Mesodermo/metabolismo , Microscopía Confocal , Factores de Transcripción de la Familia Snail , 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/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
In this study, we have investigated the underlying molecular mechanism for the potent proapoptotic effect of luteolin on human hepatoma cells both in vitro and in vivo, focusing on the signal transducer and activator of transcription 3 (STAT3)/Fas signaling. A clear apoptosis was found in the luteolin-treated HLF hepatoma cells in a time- and dosage-dependent manner. In concert with the caspase-8 activation by luteolin, an enhanced expression in functional Fas/CD95 was identified. Consistent with the increased Fas/CD95 expression, a drastic decrease in the Tyr(705) phosphorylation of STAT3, a known negative regulator of Fas/CD95 transcription, was found within 20 minutes in the luteolin-treated cells, leading to down-regulation in the target gene products of STAT3, such as cyclin D1, survivin, Bcl-xL, and vascular endothelial growth factor. Of interest, the rapid down-regulation in STAT3 was consistent with an accelerated ubiquitin-dependent degradation in the Tyr(705)-phosphorylated STAT3, but not the Ser(727)-phosphorylated one, another regulator of STAT3 activity. The expression level of Ser(727)-phosphorylated STAT3 was gradually decreased by the luteolin treatment, followed by a fast and clear down-regulation in the active forms of CDK5, which can phosphorylate STAT3 at Ser(727). An overexpression in STAT3 led to resistance to luteolin, suggesting that STAT3 was a critical target of luteolin. In nude mice with xenografted tumors using HAK-1B hepatoma cells, luteolin significantly inhibited the growth of the tumors in a dosage-dependent manner. These data suggested that luteolin targeted STAT3 through dual pathways-the ubiquitin-dependent degradation in Tyr(705)-phosphorylated STAT3 and the gradual down-regulation in Ser(727)-phosphorylated STAT3 through inactivation of CDK5, thereby triggering apoptosis via up-regulation in Fas/CD95.
Asunto(s)
Carcinoma Hepatocelular/tratamiento farmacológico , Neoplasias Hepáticas/tratamiento farmacológico , Luteolina/farmacología , Factor de Transcripción STAT3/metabolismo , Animales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Procesos de Crecimiento Celular/efectos de los fármacos , Línea Celular Tumoral , Regulación hacia Abajo , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Fosforilación/efectos de los fármacos , Receptores Tipo I de Factores de Necrosis Tumoral/biosíntesis , Factor de Transcripción STAT3/biosíntesis , Factor de Transcripción STAT3/genética , Ubiquitina/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Receptor fas/biosíntesisRESUMEN
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.
Asunto(s)
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.
Asunto(s)
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
The pathogenesis of hepatitis C virus (HCV)-associated insulin resistance remains unclear. Therefore, we investigated mechanisms for HCV-associated insulin resistance. Homeostasis model assessment for insulin resistance was increased in patients with HCV infection. An increase in fasting insulin levels was associated with the presence of serum HCV core, the severity of hepatic fibrosis and a decrease in expression of insulin receptor substrate (IRS) 1 and IRS2, central molecules of the insulin-signaling cascade, in patients with HCV infection. Down-regulation of IRS1 and IRS2 was also seen in HCV core-transgenic mice livers and HCV core-transfected human hepatoma cells. Carbobenzoxy-l-leucyl-l-leucyl-l-leucinal, a potent proteosomal proteolysis inhibitor, blocked down-regulation of IRS1 and IRS2 in HCV core-transfected hepatoma cells. In human hepatoma cells, HCV core up-regulated suppressor of cytokine signaling (SOCS) 3 and caused ubiquitination of IRS1 and IRS2. HCV core-induced down-regulation of IRS1 and IRS2 was not seen in SOCS3(-/-) mouse embryonic fibroblast cells. Furthermore, HCV core suppressed insulin-induced phosphorylation of p85 subunit of phosphatidylinositol 3-kinase and Akt, activation of 6-phosphofructo-2-kinase, and glucose uptake. In conclusion, HCV infection changes a subset of hepatic molecules regulating glucose metabolism. A possible mechanism is that HCV core-induced SOCS3 promotes proteosomal degradation of IRS1 and IRS2 through ubiquitination.