RESUMEN
Aberrant regulation of the Wnt/ß-catenin signaling pathway is one of the major causes of colorectal cancer (CRC). In this study, we examined the effect of polymethoxyflavones present in citrus peels on Wnt/ß-catenin signaling in the HCT116 CRC cell line. We found that 5,7,3',4'-tetra-methoxyflavone (TMF) and 7,8,3',4'-TMF inhibited the expression of target genes of Wnt/ß-catenin signaling and the transcriptional activities of ß-catenin/Tcf and suppressed the motility of HCT116 cells. Because the binding of ß-catenin to Tcf-4 was disrupted by 5,7,3',4'-TMF and 7,8,3',4'- TMF, we suggest that they are inhibitors of the Wnt/ß-catenin signaling and may have potential applications in CRC prevention.
Asunto(s)
Neoplasias del Colon , Neoplasias Colorrectales , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Neoplasias del Colon/metabolismo , Neoplasias Colorrectales/metabolismo , Regulación Neoplásica de la Expresión Génica , Células HCT116 , Humanos , Vía de Señalización Wnt , beta Catenina/metabolismoRESUMEN
Estrogen affects mitochondrial function in various tissues, but the precise mechanism remains unclear. We, therefore investigated the effect on estrogen-regulated mitochondrial morphology by dynamin-related protein 1 (Drp1) and its Ser616-phosphorylated derivative (pDrp1Ser616) are involved in mitochondrial fission. MCF7 human breast cancer cells were treated with 17ß-estradiol (E2), an estrogen receptor (ER) α and ß antagonist (ICI 182, 780), an ERα antagonist (MPP), and an ERß antagonist (PHTPP) for 24 hr. The expression of Drp1 and pDrp1Ser616 was analyzed by western blotting and immunohistochemistry. Mitochondrial morphology was analyzed by transmission electron microscopy (TEM). In control cells, Drp1 was detected in the cytoplasm of all cells while pDrp1 was observed in the cytoplasm of 3.4 ± 1.0% of the total population. After E2 treatment, pDrp1Ser616-positive cells comprised 30.6 ± 5.6% of the total population, 10.5 ± 1.7% after E2 + ICI treatment, 12.4 ± 4.2% after E2 + MPP treatment, and 24.0 ± 2.2% after E2 + PHTPP treatment. In ERα knockdown MCF7 cells, pDrp1 expression was decreased after E2 treatment compared to E2-treated wild type cells. Tubular pattern mitochondria were found in the control cells but the number of short and small pattern mitochondria (< 0.5 µm2) was significantly increased after E2 treatment (as observed by TEM). We, therefore concluded that the phosphorylation of Drp1 is important for E2-dependent mitochondrial morphological changes through ERα.
RESUMEN
Estrogen is considered to be involved in duodenal function; however, the details of its receptor expression are largely unknown. The purpose of this study was to determine the expression and localization of estrogen receptors (ERs) in mouse duodenum. Male and female C57BL/6J mouse intestinal tissues were used to investigate the expression of ERα and ERß by RT-PCR, western blotting, immunohistochemistry, and Southwestern histochemistry. ERß, but not ERα, was expressed in proximal duodenal epithelium, but not in jejunum and ileum. The expression of ERß mRNA and protein were confirmed by RT-PCR and western blotting, respectively. At postnatal day 20, the transit period of suckling to weaning, the distribution of ERß-positive cells was changed in the crypt-villus axis, and cytoplasm/nuclear staining changed to only nuclear staining. Moreover, Southwestern histochemistry was used to detect estrogen response element (ERE)-binding proteins, and their expression pattern was highly similar to that of ERß. These results suggested that ERß is the predominant ER type in mouse small intestine, and the highly similar co-localization of ERE-binding proteins reveals that ERß is functionally active in mouse duodenum. The ERß expression changes during postnatal development indicate that ERß may be involved in the differentiation of duodenal epithelium.
Asunto(s)
Duodeno/metabolismo , Receptor beta de Estrógeno/metabolismo , Mucosa Intestinal/metabolismo , Animales , Animales Recién Nacidos , Western Blotting , Receptor beta de Estrógeno/genética , Femenino , Inmunohistoquímica , Masculino , Ratones Endogámicos C57BL , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Old astrocyte specifically induced substance (OASIS), which is a new type of endoplasmic reticulum (ER) stress transducer, is a basic leucine zipper transcription factor of the CREB/ATF family that contains a transmembrane domain and is processed by regulated intramembrane proteolysis in response to ER stress. OASIS is selectively expressed in certain types of cells such as astrocytes and osteoblasts. We have previously demonstrated that OASIS activates transcription of the type I collagen gene Col1a1 and contributes to the secretion of bone matrix proteins in osteoblasts, and that OASIS-/- mice exhibit osteopenia and growth retardation. In the present study, we examined whether osteopenia in OASIS-/- mice is rescued by OASIS introduction into osteoblasts. We generated OASIS-/- mice that specifically expressed OASIS in osteoblasts using a 2.3-kb osteoblast-specific type I collagen promoter (OASIS-/-;Tg mice). Histological analysis of OASIS-/-;Tg mice revealed that osteopenia in OASIS-/- mice was rescued by osteoblast-specific expression of the OASIS transgene. The decreased expression levels of type I collagen mRNAs in the bone tissues of OASIS-/- mice were recovered by the OASIS transgene accompanied by the rescue of an abnormal expansion of the rough ER in OASIS-/- osteoblasts. In contrast, growth retardation in OASIS-/- mice did not improve in OASIS-/-;Tg mice. Interestingly, the serum levels of growth hormone (GH) and insulin-like growth factor (IGF)-1 were downregulated in OASIS-/- mice compared with those in wild-type mice. These decreased GH and IGF-1 levels in OASIS-/- mice did not change when OASIS was introduced into osteoblasts. Taken together, these results indicate that OASIS regulates skeletal development by osteoblast-dependent and -independent mechanisms.
Asunto(s)
Enfermedades Óseas Metabólicas/patología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/deficiencia , Crecimiento y Desarrollo , Proteínas del Tejido Nervioso/deficiencia , Animales , Enfermedades Óseas Metabólicas/metabolismo , Huesos/efectos de los fármacos , Huesos/metabolismo , Huesos/patología , Huesos/ultraestructura , Agregación Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Condrocitos/efectos de los fármacos , Condrocitos/patología , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Hormona del Crecimiento/farmacología , Crecimiento y Desarrollo/efectos de los fármacos , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/metabolismo , Especificidad de Órganos/efectos de los fármacos , Osteoblastos/metabolismo , Osteoblastos/patología , Osteoblastos/ultraestructura , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transgenes/genéticaRESUMEN
Endoplasmic reticulum (ER) stress has been reported to be linked to various diseases such as diabetes, neurodegenerative diseases, and osteogenesis imperfecta (OI). Old astrocyte specifically induced substance (OASIS), a novel type of ER stress transducer, is a basic leucine zipper transcription factor belonging to the CREB/ATF family and is markedly expressed in osteoblasts. Recently, we demonstrated that OASIS activates the transcription of the gene for type I collagen, Col1a1, and contributes to the secretion of bone matrix proteins in osteoblasts. OASIS-/- mice exhibit severe osteopenia involving a decrease in type I collagen in the bone matrix and a dysfunction of osteoblasts, which show abnormal expansion of the rough ER. These phenotypic features of osteopenia are similar to those observed in OI type I. In this study, we investigated whether administration of the third-generation bisphosphonate risedronate (RIS) is effective for treating osteopenia in OASIS-/- mice. Histological and histomorphometric analyses revealed that the trabecular bones increased dramatically in OASIS-/- mice treated with RIS, owing to the inhibition of bone resorption. Intriguingly, the abnormal expansion of the rough ER in OASIS-/- osteoblasts was improved by the treatment with RIS. Taken together, we conclude that OASIS-/- mice will be useful as new model mice for evaluating the medicinal effects of osteopenia treatments and developing new drugs for the osteopenia associated with diseases such as OI and osteoporosis.
Asunto(s)
Conservadores de la Densidad Ósea/uso terapéutico , Enfermedades Óseas Metabólicas/tratamiento farmacológico , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/deficiencia , Ácido Etidrónico/análogos & derivados , Proteínas del Tejido Nervioso/deficiencia , Animales , Enfermedades Óseas Metabólicas/genética , Huesos/efectos de los fármacos , Huesos/patología , Huesos/ultraestructura , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Difosfonatos/farmacología , Difosfonatos/uso terapéutico , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/patología , Retículo Endoplásmico/ultraestructura , Ácido Etidrónico/farmacología , Ácido Etidrónico/uso terapéutico , Immunoblotting , Ratones , Ratones Noqueados , Microscopía Electrónica , Proteínas del Tejido Nervioso/genética , Ácido RisedrónicoRESUMEN
Endoplasmic reticulum (ER) stress response is important for protein maturation in the ER. Some murine models for bone diseases have provided significant insight into the possibility that pathogenesis of osteoporosis is related to ER stress response of osteoblasts. We examined a possible correlation between osteoporosis and ER stress response. Bone specimens from 8 osteoporosis patients and 8 disease-controls were used for immunohistochemical analysis. We found that ER molecular chaperones, such as BiP (immunoglobulin heavy-chain binding protein) and PDI (protein-disulfide isomerase) are down-regulated in osteoblasts from osteoporosis patients. Based on this result, we hypothesized that up-regulation of ER molecular chaperones in osteoblasts could restore decreased bone formation in osteoporosis. Therefore, we investigated whether treatment of murine model for osteoporosis with BIX (BiP inducer X), selective inducer BiP, could prevent bone loss. We found that oral administration of BIX effectively improves decline in bone formation through the activation of folding and secretion of bone matrix proteins. Considering these results together, BIX may be a potential therapeutic agent for the prevention of bone loss in osteoporosis patients.
Asunto(s)
Conservadores de la Densidad Ósea/uso terapéutico , Regulación de la Expresión Génica/efectos de los fármacos , Chaperonas Moleculares/metabolismo , Osteoporosis/prevención & control , Tiocianatos/uso terapéutico , Anciano , Anciano de 80 o más Años , Animales , Animales Recién Nacidos , Densidad Ósea/efectos de los fármacos , Conservadores de la Densidad Ósea/administración & dosificación , Huesos/efectos de los fármacos , Huesos/patología , Células Cultivadas , Retículo Endoplásmico/metabolismo , Chaperón BiP del Retículo Endoplásmico , Femenino , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Persona de Mediana Edad , Chaperonas Moleculares/genética , Osteoblastos/efectos de los fármacos , Osteoblastos/patología , Osteopontina/metabolismo , Osteoporosis/metabolismo , Osteoporosis/patología , Proteína Disulfuro Isomerasas/metabolismo , Tiocianatos/administración & dosificación , Factores de TiempoRESUMEN
Eukaryotic cells have signalling pathways from the endoplasmic reticulum (ER) to cytosol and nuclei, to avoid excess accumulation of unfolded proteins in the ER. We previously identified a new type of ER stress transducer, OASIS, a bZIP (basic leucine zipper) transcription factor, which is a member of the CREB/ATF family and has a transmembrane domain. OASIS is processed by regulated intramembrane proteolysis (RIP) in response to ER stress, and is highly expressed in osteoblasts. OASIS(-/-) mice exhibited severe osteopenia, involving a decrease in type I collagen in the bone matrix and a decline in the activity of osteoblasts, which showed abnormally expanded rough ER, containing of a large amount of bone matrix proteins. Here we identify the gene for type 1 collagen, Col1a1, as a target of OASIS, and demonstrate that OASIS activates the transcription of Col1a1 through an unfolded protein response element (UPRE)-like sequence in the osteoblast-specific Col1a1 promoter region. Moreover, expression of OASIS in osteoblasts is induced by BMP2 (bone morphogenetic protein 2), the signalling of which is required for bone formation. Additionally, RIP of OASIS is accelerated by BMP2 signalling, which causes mild ER stress. Our studies show that OASIS is critical for bone formation through the transcription of Col1a1 and the secretion of bone matrix proteins, and they reveal a new mechanism by which ER stress-induced signalling mediates bone formation.
Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico/fisiología , Retículo Endoplásmico/fisiología , Proteínas del Tejido Nervioso/fisiología , Osteogénesis/genética , Animales , Proteína Morfogenética Ósea 2/genética , Proteína Morfogenética Ósea 2/metabolismo , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Cadena alfa 1 del Colágeno Tipo I , Retículo Endoplásmico/ultraestructura , Femenino , Inmunohistoquímica , Hibridación in Situ , Masculino , Ratones , Ratones Noqueados , Regiones Promotoras Genéticas , Pliegue de Proteína , ARN Mensajero/metabolismo , Transducción de SeñalRESUMEN
Many tissues have a specific signal transduction system for endoplasmic reticulum (ER) dysfunction; however, the mechanisms underlying the ER stress response in cartilage remain unclear. BBF2H7 (BBF2 human homologue on chromosome 7), an ER-resident basic leucine zipper transcription factor, is activated in response to ER stress and is highly expressed in chondrocytes. In this study, we generated Bbf2h7(-/-) mice to assess the in vivo function of BBF2H7. The mice showed severe chondrodysplasia and died by suffocation shortly after birth because of an immature chest cavity. The cartilage showed a lack of typical columnar structure in the proliferating zone and a decrease in the size of the hypertrophic zone, resulting in a significant reduction of extracellular matrix proteins. Interestingly, proliferating chondrocytes showed abnormally expanded ER, containing aggregated type II collagen (Col2) and cartilage oligomeric matrix protein (COMP). We identified Sec23a, which encodes a coat protein complex II component responsible for protein transport from the ER to the Golgi, as a target of BBF2H7, which directly bound to a CRE-like sequence in the promoter region of Sec23a to activate its transcription. When Sec23a was introduced to Bbf2h7(-/-) chondrocytes, the impaired transport and secretion of cartilage matrix proteins was totally restored, indicating that by activating protein secretion the BBF2H7-Sec23a pathway has a crucial role in chondrogenesis. Our findings provide a new link by which ER stress is converted to signalling for the activation of ER-to-Golgi trafficking.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Condrogénesis , Retículo Endoplásmico/fisiología , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Animales , Proteína de la Matriz Oligomérica del Cartílago , Cartílago Articular/citología , Células Cultivadas , Condrocitos/fisiología , Condrocitos/ultraestructura , Colágeno Tipo II/metabolismo , Embrión de Mamíferos , Retículo Endoplásmico/ultraestructura , Proteínas de la Matriz Extracelular/metabolismo , Regulación del Desarrollo de la Expresión Génica , Glicoproteínas/metabolismo , Aparato de Golgi/metabolismo , Inmunohistoquímica , Hibridación in Situ , Proteínas Matrilinas , Ratones , Ratones Noqueados , Transporte de Proteínas , Costillas/citología , Proteínas de Transporte Vesicular/biosíntesisRESUMEN
The endoplasmic reticulum (ER) stress response is a defense system for dealing with the accumulation of unfolded proteins in the ER lumen. Old astrocyte specifically induced substance (OASIS) is known to be expressed in astrocytes and involved in the ER stress response; however the function of OASIS in the injured brain has remained unclear. In this study, we examined the roles of OASIS in neuronal degeneration in the hippocampi of mice intraperitoneally injected with kainic acid (KA). OASIS mRNA was strongly induced in response to KA injection, with a similar time course to the induction of ER molecular chaperone immunoglobulin heavy chain binding protein mRNA. In situ hybridization showed that KA injection causes induction of immunoglobulin heavy chain binding protein mRNA in glial fibrillary acidic protein-positive astrocytes as well as in pyramidal neurons, although up-regulation of OASIS mRNA was only detected in glial fibrillary acidic protein-positive astrocytes. Primary cultured astrocytes, but not the neurons of OASIS-/- mice, revealed reduced vulnerability to ER stress. Furthermore, pyramidal neurons in the hippocampi of OASIS-/- mice were more susceptible to the toxicity induced by KA than those of wild-type mice. Taken together, these data suggest that OASIS expressed in astrocytes plays important roles in protection against the neuronal damage induced by KA.
Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico/deficiencia , Hipocampo/patología , Ácido Kaínico/toxicidad , Proteínas del Tejido Nervioso/deficiencia , Neuronas/patología , Células Piramidales/metabolismo , Células Piramidales/patología , Animales , Células Cultivadas , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/fisiología , Femenino , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/fisiología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Células Piramidales/efectos de los fármacosRESUMEN
Myotonic dystrophy type 1 (DM1) is an autosomal dominant neuromuscular disorder associated with an expansion of CTG trinucleotide repeats in the 3'-untranslated region of the myotonic dystrophy protein kinase (DMPK) gene. The RNA gain-of-function hypothesis proposes that mutant DMPK mRNA alters the function and localization of alternative splicing regulators, which are critical for normal RNA processing. Previously, we found alternative splicing variants of sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase 1 (SERCA1), which excluded exon 22, in skeletal muscle of DM1 patients. In the present study, we analyzed the molecular mechanisms responsible for the splicing dysregulation of SERCA1. Five 'YGCU(U/G)Y' motifs that could potentially serve as Muscleblind-like 1, (MBNL1)-binding motifs, are included downstream from the SERCA1 exon 22. Exon trapping experiments showed that MBNL1 acts on the 'YGCU(U/G)Y' motif, and positively regulates exon 22 splicing. Of the five MBNL1 motifs in intron 22, the second and third sites were important for regulation of exon 22 splicing, but the other three binding sites were not required. Overexpression of the CUG repeat expansion of DMPK mRNA resulted in exclusion of exon 22 of SERCA1. These results suggest that sequestration of MBNL1 into the CUG repeat expansion of DMPK mRNA could cause the exclusion of SERCA1 exon 22, and the expression of this aberrant splicing form of SERCA1 could affect the regulation of Ca(2+) concentration of sarcoplasmic reticulum in DM patients.
Asunto(s)
Empalme Alternativo , Distrofia Miotónica/enzimología , Distrofia Miotónica/genética , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , Regiones no Traducidas 3' , Animales , Secuencia de Bases , Sitios de Unión/genética , Línea Celular , ADN/genética , Exones , Humanos , Intrones , Ratones , Datos de Secuencia Molecular , Distrofia Miotónica/clasificación , Proteína Quinasa de Distrofia Miotónica , Proteínas Serina-Treonina Quinasas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Transfección , Expansión de Repetición de TrinucleótidoRESUMEN
Secretory and transmembrane proteins are correctly folded or processed in the endoplasmic reticulum (ER). Various stresses disturb ER function and provoke the accumulation of unfolded proteins in the ER lumen. This condition is termed ER stress. Recently, ER stress has been linked to neuronal death in various neurodegenerative diseases. Among the cell populations in the nervous system, which comprises heterogeneous cell types including neuronal and glial cells, astrocytes have the unique ability of being able to tolerate and even proliferate under ischemic and hypoxic conditions that lead to ER stress. This review introduces a novel ER stress transducer, old astrocyte specifically induced substance (OASIS), that regulates the signaling of the unfolded protein response specifically in astrocytes and contributes to resistance to ER stress. In addition, current information is summarized regarding new types of ER stress transducers homologous to OASIS that are involved in cell type-specific ER stress responses.
Asunto(s)
Astrocitos/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Activación Transcripcional , Factor de Transcripción Activador 6/química , Factor de Transcripción Activador 6/metabolismo , Animales , Membrana Celular/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/química , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/fisiología , Retículo Endoplásmico/fisiología , Regulación de la Expresión Génica , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/fisiología , Pliegue de Proteína , Transducción de SeñalRESUMEN
Endoplasmic reticulum (ER) stress transducers IRE1 (inositol requiring 1), PERK (PKR-like endoplasmic reticulum kinase), and ATF6 (activating transcription factor 6) are well known to transduce signals from the ER to the cytoplasm and nucleus when unfolded proteins accumulate in the ER. Recently, we identified OASIS (old astrocyte specifically induced substance) as a novel ER stress transducer expressed in astrocytes. We report here that BBF2H7 (BBF2 human homolog on chromosome 7), an ER-resident transmembrane protein with the bZIP domain in the cytoplasmic portion and structurally homologous to OASIS, is cleaved at the membrane in response to ER stress. The cleaved fragments of BBF2H7 translocate into the nucleus and can bind directly to cyclic AMP-responsive element sites to activate transcription of target genes. Interestingly, although BBF2H7 protein is not expressed under normal conditions, it is markedly induced at the translational level during ER stress, suggesting that BBF2H7 might contribute to only the late phase of unfolded protein response signaling. In a mouse model of focal brain ischemia, BBF2H7 protein is prominently induced in neurons in the peri-infarction region. Furthermore, in a neuroblastoma cell line, BBF2H7 overexpression suppresses ER stress-induced cell death, while small interfering RNA knockdown of BBF2H7 promotes ER stress-induced cell death. Taken together, our results suggest that BBF2H7 is a novel ER stress transducer and could play important roles in preventing accumulation of unfolded proteins in damaged neurons.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/fisiología , Retículo Endoplásmico/fisiología , Animales , Isquemia Encefálica/etiología , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patología , Línea Celular , Línea Celular Tumoral , Ensayo de Cambio de Movilidad Electroforética , Fibroblastos/citología , Técnica del Anticuerpo Fluorescente Indirecta , Genes Reporteros , Glioma/patología , Células HeLa , Humanos , Inmunohistoquímica , Luciferasas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Ratas , Estrés MecánicoRESUMEN
Eukaryotic cells deal with accumulation of unfolded proteins in the endoplasmic reticulum (ER) by the unfolded protein response, involving the induction of molecular chaperones, translational attenuation, and ER-associated degradation, to prevent cell death. Here, we found that the autophagy system is activated as a novel signaling pathway in response to ER stress. Treatment of SK-N-SH neuroblastoma cells with ER stressors markedly induced the formation of autophagosomes, which were recognized at the ultrastructural level. The formation of green fluorescent protein (GFP)-LC3-labeled structures (GFP-LC3 "dots"), representing autophagosomes, was extensively induced in cells exposed to ER stress with conversion from LC3-I to LC3-II. In IRE1-deficient cells or cells treated with c-Jun N-terminal kinase (JNK) inhibitor, the autophagy induced by ER stress was inhibited, indicating that the IRE1-JNK pathway is required for autophagy activation after ER stress. In contrast, PERK-deficient cells and ATF6 knockdown cells showed that autophagy was induced after ER stress in a manner similar to the wild-type cells. Disturbance of autophagy rendered cells vulnerable to ER stress, suggesting that autophagy plays important roles in cell survival after ER stress.
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Autofagia/fisiología , Retículo Endoplásmico/patología , Estrés Fisiológico/patología , Línea Celular Tumoral , Supervivencia Celular/fisiología , Retículo Endoplásmico/enzimología , Retículo Endoplásmico/ultraestructura , Activación Enzimática/fisiología , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/fisiología , Pliegue de Proteína , Transducción de Señal , Estrés Fisiológico/enzimología , Estrés Fisiológico/metabolismo , Factores de TiempoRESUMEN
h-prune, which has been suggested to be involved in cell migration, was identified as a glycogen synthase kinase 3 (GSK-3)-binding protein. Treatment of cultured cells with GSK-3 inhibitors or small interfering RNA (siRNA) for GSK-3 and h-prune inhibited their motility. The kinase activity of GSK-3 was required for the interaction of GSK-3 with h-prune. h-prune was localized to focal adhesions, and the siRNA for GSK-3 or h-prune delayed the disassembly of paxillin. The tyrosine phosphorylation of focal adhesion kinase (FAK) and the activation of Rac were suppressed in GSK-3 or h-prune knocked-down cells. GSK-3 inhibitors suppressed the disassembly of paxillin and the activation of FAK and Rac. Furthermore, h-prune was highly expressed in colorectal and pancreatic cancers, and the positivity of the h-prune expression was correlated with tumor invasion. These results suggest that GSK-3 and h-prune cooperatively regulate the disassembly of focal adhesions to promote cell migration and that h-prune is useful as a marker for tumor aggressiveness.
Asunto(s)
Proteínas Portadoras/metabolismo , Movimiento Celular , Adhesiones Focales/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Neoplasias/patología , Animales , Proteínas Portadoras/análisis , Proteínas Portadoras/genética , Células Cultivadas , Activación Enzimática , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Adhesiones Focales/química , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/genética , Humanos , Invasividad Neoplásica , Neoplasias/metabolismo , Paxillin/metabolismo , Monoéster Fosfórico Hidrolasas , Mapeo de Interacción de Proteínas , Proteínas Proto-Oncogénicas c-akt/metabolismo , Interferencia de ARNRESUMEN
The mechanism of cross talk between the Wnt signaling and cyclic AMP (cAMP)-dependent protein kinase (protein kinase A [PKA]) pathways was studied. Prostaglandin E(1) (PGE(1)), isoproterenol, and dibutyryl cAMP (Bt(2)cAMP), all of which activate PKA, increased the cytoplasmic and nuclear beta-catenin protein level, and these actions were suppressed by a PKA inhibitor and RNA interference for PKA. PGE(1) and Bt(2)cAMP also increased T-cell factor (Tcf)-dependent transcription through beta-catenin. Bt(2)cAMP suppressed degradation of beta-catenin at the protein level. Although PKA did not affect the formation of a complex between glycogen synthase kinase 3beta (GSK-3beta), beta-catenin, and Axin, phosphorylation of beta-catenin by PKA inhibited ubiquitination of beta-catenin in intact cells and in vitro. Ser675 was found to be a site for phosphorylation by PKA, and substitution of this serine residue with alanine in beta-catenin attenuated inhibition of the ubiquitination of beta-catenin by PKA, PKA-induced stabilization of beta-catenin, and PKA-dependent activation of Tcf. These results indicate that PKA inhibits the ubiquitination of beta-catenin by phosphorylating beta-catenin, thereby causing beta-catenin to accumulate and the Wnt signaling pathway to be activated.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Alprostadil/farmacología , Animales , Proteína Axina , Secuencia de Bases , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Sitios de Unión , Bucladesina/farmacología , Células COS , Línea Celular , Núcleo Celular/metabolismo , Chlorocebus aethiops , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Citoplasma/metabolismo , Estabilidad de Medicamentos , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Humanos , Técnicas In Vitro , Isoproterenol/farmacología , Células L , Ratones , Mutagénesis Sitio-Dirigida , Proteínas del Tejido Nervioso/metabolismo , Fosforilación , Interferencia de ARN , Proteínas Represoras/metabolismo , Serina/química , Transducción de Señal , Factores de Transcripción TCF/metabolismo , Factor de Transcripción 4 , Ubiquitina/metabolismoRESUMEN
We demonstrate that Dvl-1, casein kinase I epsilon (CKI epsilon), and Frat-1 activate the Wnt signaling pathway cooperatively. The amino acid region 228-250 of Dvl-1 was necessary for its binding to Frat-1, and the interaction of Dvl-1 with Frat-1 was enhanced by CKI epsilon. Coexpression of Dvl-1 and Frat-1 caused accumulation of beta-catenin synergistically in L cells. Both proteins also activated the transcriptional activity of T-cell factor-4 (Tcf-4) synergistically in human embryonic kidney 293 cells, but coexpression of Dvl-1-(Delta 228-250), which lacks the amino acid region 228-250 from Dvl-1, and Frat-1 did not. Dvl-1, but not Dvl-1-(Delta 228-250), acted synergistically with CKI epsilon to activate Tcf-4. Depletion of CKI epsilon by double-stranded RNA interference in HeLa S3 cells led to the inhibition of Wnt-3a-induced phosphorylation of Dvl and the binding of Dvl-1 to Frat-1. Furthermore, depletion of CKI epsilon reduced the Wnt-3a-induced accumulation of beta-catenin, although it did not affect the basal level of beta-catenin. These results indicate that CKI epsilon-dependent phosphorylation of Dvl enhances the formation of a complex of Dvl-1 with Frat-1 and that this complex leads to the activation of the Wnt signaling pathway.