RESUMEN
One of the regulatory mechanisms of epigenetic gene expression is the post-translational methylation of arginine residues, which is catalyzed by protein arginine methyltransferases (PRMTs). Abnormal expression of PRMT4/CARM1, one of the PRMTs, is associated with various diseases, including cancers. Here, we designed and synthesized a Förster resonance energy transfer (FRET)-based probe, FRC, which contains coumarin and fluorescein fluorophores at the N-terminus and C-terminus of a peptide containing an arginine residue within an appropriate amino acid sequence to serve as a substrate of CARM1; the two fluorophores act as a FRET donor and a FRET acceptor, respectively. Since trypsin specifically hydrolyzes the arginine residue, but not a monomethylarginine or dimethylarginine residue, CARM1 activity can be evaluated from the change of the coumarin/fluorescein fluorescence ratio of FRC in the presence of trypsin.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia , Colorantes Fluorescentes/química , Proteína-Arginina N-Metiltransferasas/metabolismo , Arginina/genética , Arginina/metabolismo , Cumarinas/química , Fluoresceína/química , Humanos , Estructura Molecular , Procesamiento Proteico-PostraduccionalRESUMEN
Epithelial-to-mesenchymal transition (EMT) is a biological process in which epithelial cells translate into a mesenchymal phenotype with invasive capacities, contributing to tumour progression, metastasis, and the acquisition of chemotherapy resistance. To identify new therapeutic targets for cancers, it is important to clarify the molecular mechanism of induction of EMT. We have previously reported that fad104, a positive regulator of adipocyte differentiation, suppressed the invasion and metastasis of melanoma and breast cancer cells. In this study, we showed that FAD104 functions as a novel suppressor of transforming growth factor-ß (TGF-ß)-mediated EMT in cervical cancer cells. Expression of FAD104 is upregulated during TGF-ß-mediated EMT in human cervical cancer HeLa cells. Reduction of fad104 expression enhanced TGF-ß-mediated EMT and migration in HeLa cells. Conversely, overexpression of FAD104 suppressed TGF-ß-induced EMT. In addition, we showed that FAD104 negatively regulated phosphorylation of Smad2 and Smad3 but positively regulated phosphorylation of Smad1/5/8 via treatment with TGF-ß. These findings demonstrate that FAD104 is a novel suppressor of TGF-ß signalling and represses TGF-ß-mediated EMT in cervical cancer cells.
Asunto(s)
Adipogénesis , Transición Epitelial-Mesenquimal , Fibronectinas/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Neoplasias del Cuello Uterino/metabolismo , Adipogénesis/genética , Biomarcadores , Línea Celular Tumoral , Transición Epitelial-Mesenquimal/genética , Femenino , Fibronectinas/genética , Expresión Génica , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Transducción de Señal , Proteínas Smad/metabolismo , Neoplasias del Cuello Uterino/genéticaRESUMEN
KCNMA1 is a pore-forming α-subunit of the large conductance Ca2+ - and voltage-activated K+ channels, referred to as BK channels, which play key roles in various physiological functions. However, the role of KCNMA1 in mature adipocytes remains unclear. In this study, we reveal that kcnma1 expression is downregulated in white adipose tissue of mice fed a high-fat diet and in hypertrophied adipocytes. Furthermore, inhibition of kcnma1 expression or treatment with a BK channel blocker attenuated insulin-induced Akt phosphorylation in mature adipocytes. These results strongly indicate that KCNMA1 contributes to the regulation of insulin signalling in mature adipocytes.
Asunto(s)
Adipocitos/citología , Insulina/metabolismo , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/metabolismo , Transducción de Señal , Células 3T3 , Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Adipocitos/patología , Tejido Adiposo Blanco/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Hipertrofia/metabolismo , Indoles/farmacología , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/antagonistas & inhibidores , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/deficiencia , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/genética , Ratones , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Anchorage-independent growth is one of the defining characteristics of cancer cells. Many oncogenes and tumor suppressor genes are involved in regulating this type of growth. Factor for adipocyte differentiation 104 gene (fad104) is a regulator of adipogenesis and osteogenesis. Previously, we reported that fad104 suppressed metastasis as well as invasion of melanoma cells. However, it is unclear whether fad104 is involved in malignant transformation, which is associated with metastasis. In this study, we revealed that fad104 negatively regulated the colony forming activity of melanoma cells. The presence of the N-terminal region of FAD104 was required for the regulation of malignant transformation of melanoma cells. In addition, the deletion mutant of FAD104 that contained the N-terminal region and transmembrane domain interacted with signal transducer and activator of transcription 3 (STAT3) and suppressed STAT3 activity. However, the deletion mutant of FAD104 lacking the N-terminal region did not influence the interaction with STAT3 or suppress the STAT3 activity. Moreover, FAD104 interacted with the C-terminal region of STAT3. In summary, we demonstrated that fad104 suppressed anchorage-independent growth of melanoma cells, and that the N-terminal region of FAD104 is essential for inhibiting malignant transformation and STAT3 activity.
Asunto(s)
Transformación Celular Neoplásica/metabolismo , Fibronectinas/metabolismo , Melanoma/metabolismo , Factor de Transcripción STAT3/metabolismo , Adipogénesis , Animales , Línea Celular Tumoral , Fibronectinas/genética , Humanos , Ratones , OsteogénesisRESUMEN
Factor for adipocyte differentiation 24 (fad24) is a positive regulator of adipogenesis. We previously found that human fad24 is abundantly expressed in skeletal muscle. However, the function of fad24 in skeletal muscle remains largely unknown. Because skeletal muscle is a highly regenerative tissue, we focused on the function of fad24 in skeletal muscle regeneration. In this paper, we investigated the role of fad24 in the cell cycle re-entry of quiescent C2C12 myoblasts-mimicked satellite cells. The expression levels of fad24 and histone acetyltransferase binding to ORC1 (hbo1), a FAD24-interacting factor, were elevated at the early phase of the regeneration process in response to cardiotoxin-induced muscle injury. The knockdown of fad24 inhibited the proliferation of quiescent myoblasts, whereas fad24 knockdown did not affect differentiation. S phase entry following serum activation is abrogated by fad24 knockdown in quiescent cells. Furthermore, fad24 knockdown cells show a marked accumulation of p27(Kip1) protein. These results suggest that fad24 may have an important role in the S phase re-entry of quiescent C2C12 cells through the regulation of p27(Kip1) at the protein level.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Mioblastos/metabolismo , Proteínas Nucleares/genética , Adipogénesis/fisiología , Animales , Proteínas de Ciclo Celular , Diferenciación Celular/fisiología , Línea Celular , Ratones Endogámicos C57BL , Músculo Esquelético/metabolismo , Mioblastos/fisiología , Complejo de Reconocimiento del Origen/genética , Fase de Descanso del Ciclo Celular , Fase SRESUMEN
Metastasis is the main cause of death in patients with cancer, and understanding the mechanisms of metastatic processes is essential for the development of cancer therapy. Although the role of several cell adhesion, migration or proliferation molecules in metastasis is established, a novel target for cancer therapy remains to be discovered. Previously, we reported that fad104 (factor for adipocyte differentiation 104), a regulatory factor of adipogenesis, regulates cell adhesion and migration. In this report, we clarify the role of fad104 in the invasion and metastasis of cancer cells. The expression level of fad104 in highly metastatic melanoma A375SM cells was lower than that in poorly metastatic melanoma A375C6 cells. Reduction of fad104 expression enhanced the migration and invasion of melanoma cells, while over-expression of FAD104 inhibited migration and invasion. In addition, melanoma cells stably expressing FAD104 showed a reduction in formation of lung colonization compared with control cells. FAD104 interacted with STAT3 and down-regulated the phosphorylation level of STAT3 in melanoma cells. These findings together demonstrate that fad104 suppressed the invasion and metastasis of melanoma cells by inhibiting activation of the STAT3 signaling pathway. These findings will aid a comprehensive description of the mechanism that controls the invasion and metastasis of cancer cells.
Asunto(s)
Adipocitos/patología , Diferenciación Celular , Fibronectinas/metabolismo , Melanoma/patología , Factor de Transcripción STAT3/antagonistas & inhibidores , Línea Celular Tumoral , Movimiento Celular , Fibronectinas/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Invasividad Neoplásica , Metástasis de la Neoplasia , Fosforilación , Factor de Transcripción STAT3/metabolismo , Transducción de SeñalRESUMEN
KCNK10, a member of tandem pore domain potassium channel family, gives rise to leak K+ currents. It plays important roles in stabilizing the negative resting membrane potential and in counterbalancing depolarization. We previously demonstrated that kcnk10 expression is quickly elevated during the early stage of adipogenesis of 3T3-L1 cells and that reduction of kcnk10 expression inhibits adipocyte differentiation. However, the molecular mechanism of KCNK10 in adipocyte differentiation remains unclear. Here we revealed that kcnk10 is induced by 3-isobutyl-1-methylxanthine, a cyclic nucleotide phosphodiesterase inhibitor and a potent inducer of adipogenesis, during the early stage of adipocyte differentiation. We also demonstrated that KCNK10 functions as a positive regulator of mitotic clonal expansion (MCE), a necessary process for terminal differentiation. The reduction of kcnk10 expression repressed the expression levels of CCAAT/enhancer-binding protein ß (C/EBPß) and C/EBPδ as well as the phosphorylation level of Akt during the early phase of adipogenesis. In addition, knockdown of kcnk10 expression suppressed insulin-induced Akt phosphorylation. These results indicate that KCNK10 contributes to the regulation of MCE through the control of C/EBPß and C/EBPδ expression and insulin signaling.
Asunto(s)
Adipocitos/citología , Adipocitos/metabolismo , Adipogénesis/genética , Diferenciación Celular/genética , Evolución Clonal/genética , Mitosis/genética , Canales de Potasio de Dominio Poro en Tándem/genética , Células 3T3-L1 , Animales , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Proteína delta de Unión al Potenciador CCAAT/metabolismo , Expresión Génica , Técnicas de Silenciamiento del Gen , Insulina/metabolismo , Ratones , Fosforilación , Transducción de SeñalRESUMEN
Osteogenesis is a complex process that is orchestrated by several growth factors, extracellular cues, signaling molecules, and transcriptional factors. Understanding the mechanisms of bone formation is pivotal for clarifying the pathogenesis of bone diseases. Previously, we reported that fad104 (factor for adipocyte differentiation 104), a novel positive regulator of adipocyte differentiation, negatively regulated the differentiation of mouse embryonic fibroblasts into osteocytes. However, the physiological role of fad104 in bone formation has not been elucidated. Here, we clarified the role of fad104 in bone formation in vivo and in vitro. fad104 disruption caused craniosynostosis-like premature ossification of the calvarial bone. Furthermore, analyses using primary calvarial cells revealed that fad104 negatively regulated differentiation and BMP/Smad signaling pathway. FAD104 interacted with Smad1/5/8. The N-terminal region of FAD104, which contains a proline-rich motif, was capable of binding to Smad1/5/8. We demonstrated that down-regulation of Smad1/5/8 phosphorylation by FAD104 is dependent on the N-terminal region of FAD104 and that fad104 functions as a novel negative regulator of BMP/Smad signaling and is required for proper development for calvarial bone. These findings will aid a comprehensive description of the mechanism that controls normal and premature calvarial ossification.
Asunto(s)
Diferenciación Celular/fisiología , Fibronectinas/biosíntesis , Regulación del Desarrollo de la Expresión Génica/fisiología , Osteogénesis/fisiología , Transducción de Señal/fisiología , Cráneo/embriología , Adipogénesis/fisiología , Animales , Células Cultivadas , Craneosinostosis/embriología , Craneosinostosis/genética , Craneosinostosis/patología , Regulación hacia Abajo/fisiología , Fibronectinas/genética , Metaloproteinasas de la Matriz Secretadas/genética , Metaloproteinasas de la Matriz Secretadas/metabolismo , Ratones , Ratones Noqueados , Fosforilación/fisiología , Estructura Terciaria de Proteína , Proteínas Smad/genética , Proteínas Smad/metabolismoRESUMEN
Aberrant expression of regulators for epigenetics is involved in tumorigenesis. There is an urgent need to identify and characterize regulators concerned with epigenetics in the early stages of hepatocarcinogenesis. In the present study, we found that the expression of coactivator-associated arginine methyltransferase 1 (CARM1), a histone methyltransferase that functions as a cofactor for nuclear hormone receptors and several transcription factors, was elevated in adenomas and aberrant in carcinomas during hepatocellular carcinogenesis. In addition to RNA expression, immunohistochemical staining of liver sections revealed that CARM1 was highly expressed in the nucleus of tumor marker glutathione S-transferase placental form (GST-P)-positive foci. Neoplastic transformation of GST-P-positive foci guides the formation of hepatocellular carcinomas. CARM1 expression was not elevated in GST-P-negative regions. Furthermore, a luciferase reporter analysis revealed that CARM1 activated the Gst-p promoter in H4IIE, a hepatocellular carcinoma cell line. This activation was mediated by the enhancer element responsible for the carcinogenic-specific expression of Gst-p and nuclear factor E2-related factor 2. Knockdown of Carm1 by shRNA in H4IIE cells inhibited cell proliferation. These findings suggest that aberrantly expressed CARM1 in tumor marker-positive cells promotes tumorigenesis in the early stages of hepatocarcinogenesis.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Transformación Celular Neoplásica/metabolismo , Neoplasias Hepáticas Experimentales/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Proteína-Arginina N-Metiltransferasas/metabolismo , Animales , Apoptosis/genética , Línea Celular Tumoral , Proliferación Celular , Glutatión Transferasa/biosíntesis , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Masculino , Regiones Promotoras Genéticas , Proteína-Arginina N-Metiltransferasas/biosíntesis , Proteína-Arginina N-Metiltransferasas/genética , Interferencia de ARN , ARN Mensajero/biosíntesis , ARN Interferente Pequeño , Ratas , Ratas Endogámicas F344RESUMEN
In previous studies, we identified a novel gene, factor for adipocyte differentiation 24 (fad24), which plays an important role during the early stages of adipogenesis in mouse 3T3-L1 cells. Moreover, overexpression of fad24 increased the number of smaller adipocytes in white adipose tissue and improved glucose metabolic activity in mice, thus indicating that fad24 functions as a regulator of adipogenesis in vivo. However, the physiological roles of fad24 in vivo are largely unknown. In this study, we attempted to generate fad24-deficient mice by gene targeting. No fad24-null mutants were recovered after embryonic day 9.5 (E9.5). Although fad24-null embryos were detected in an expected Mendelian ratio of genotypes at E3.5, none of the homozygous mutants developed into blastocysts. In vitro culture experiments revealed that fad24-null embryos develop normally to the morula stage but acquire growth defects during subsequent stages. The number of nuclei decreased in fad24-deficient morulae compared with that in wild-type ones. These results strongly suggested that fad24 is essential for pre-implantation in embryonic development, particularly for the progression to the blastocyst stage.
Asunto(s)
Adipogénesis , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/fisiología , Blastocisto/citología , Regulación del Desarrollo de la Expresión Génica , Proteínas Nucleares/genética , Proteínas Nucleares/fisiología , Células 3T3-L1 , Animales , Proteínas de Ciclo Celular , Cruzamientos Genéticos , Células Madre Embrionarias/citología , Femenino , Genotipo , Glucosa/metabolismo , Heterocigoto , Homocigoto , Masculino , Ratones , Ratones Endogámicos C57BL , Mórula/metabolismo , Mutación , Factores de TiempoRESUMEN
Human mannose-binding lectin (MBL) is encoded by the MBL2 gene and is a key player in innate immunity. However, the mechanism of the transcriptional regulation of MBL2 is largely unknown. The peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that play an important role in a number of biological responses, including lipid homeostasis, immune function and adipogenesis. In this study, we showed that PPARα and PPARγ up-regulate the expression of human MBL2. Using a luciferase assay, electrophoretic mobility-shift assay and chromatin immunoprecipitation assay, we demonstrated that PPARs regulate the expression of human MBL2 via the peroxisome proliferator responsive element (PPRE). On the other hand, MBL2 mRNA expression was not affected by the PPARα ligand both in vivo in rat liver and in vitro in rat H4IIE hepatoma cells. Thus, there is a species difference in regulation of MBL2 gene expression by PPARs between humans and rodents. We also show that the species differences in response to PPAR could be due in part to sequence-specific differences in the PPRE in the promoter region of MBL2. These results indicate that human, but not rat, MBL2 expression is regulated by PPARs via a PPRE.
Asunto(s)
Regulación de la Expresión Génica , Hepatocitos/metabolismo , Lectina de Unión a Manosa/genética , PPAR alfa/genética , PPAR gamma/genética , Elementos de Respuesta , Animales , Secuencia de Bases , Línea Celular Tumoral , Genes Reporteros , Hepatocitos/patología , Humanos , Hígado/citología , Hígado/metabolismo , Luciferasas/genética , Luciferasas/metabolismo , Masculino , Lectina de Unión a Manosa/metabolismo , Datos de Secuencia Molecular , PPAR alfa/metabolismo , PPAR gamma/metabolismo , Ratas , Ratas Endogámicas F344 , Transducción de Señal , Especificidad de la EspecieRESUMEN
We previously identified Ku proteins and interleukin enhancer binding factor 3 (ILF3) as cofactors for the nuclear receptor farnesoid X receptor and liver receptor homolog-1, respectively. Here we provide further evidence that these cofactors modulate the promoter activity of the nuclear receptor thyroid hormone receptor (TR) target gene, thyroid-stimulating hormone alpha (TSHα), which is negatively regulated by the TR ligand triiodothyronine (T(3)). Ku proteins suppressed TSHα promoter activity independent of T(3), whereas ILF3 enhanced TSHα activity, especially in the presence of T(3). Taken together, our results suggest that Ku proteins and ILF3 function as co-regulators for TR-mediated TSHα expression.
Asunto(s)
ADN Helicasas/metabolismo , Hormonas Glicoproteicas de Subunidad alfa/metabolismo , Proteínas del Factor Nuclear 90/metabolismo , Receptores de Hormona Tiroidea/metabolismo , Proteína Quinasa Activada por ADN/metabolismo , Glutatión Transferasa/metabolismo , Hormonas Glicoproteicas de Subunidad alfa/genética , Células HEK293 , Células HeLa , Humanos , Autoantígeno Ku , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas , Triyodotironina/metabolismoRESUMEN
Histone eviction and deposition are critical steps in many nuclear processes. The histone H3/H4 chaperone Asf1p is highly conserved and is involved in DNA replication, DNA repair, and transcription. To identify the factors concerned with anti-silencing function 1 (ASF1), we purified Asf1p-associated factors from the yeast Saccharomyces cerevisiae by a GST pull-down experiment, and mass spectrometry analysis was performed. Several factors are specifically associated with Asf1p, including Vip1p. VIP1 is conserved from yeast to humans and encodes inositol hexakisphoshate and inositol heptakisphosphate kinase. Vip1p interacted with Asf1p as a dimer or in a complex with another protein(s). Deletion of VIP1 did not affect the interaction between Asf1p and other Asf1p-associated factors. An in vitro GST pull-down assay indicated a direct interaction between Asf1p and Vip1p, and the interaction between the two factors in vivo was detected by an immunoprecipitation experiment. Furthermore, genetic experiments revealed that VIP1 disruption increased sensitivity to 6-azauracil (6-AU), but not to DNA-damaging reagents in wild-type and ASF1-deleted strains. It is thought that 6-AU decreases nucleotide levels and reduces transcription elongation. These observations suggest that the association of Asf1p and Vip1p may be implicated in transcription elongation.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Chaperonas de Histonas/metabolismo , Chaperonas Moleculares/metabolismo , Fosfotransferasas (Aceptor del Grupo Fosfato)/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Daño del ADN , Replicación del ADN , Eliminación de Gen , Unión Proteica , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Transcripción GenéticaRESUMEN
To clarify the molecular mechanism of adipocyte differentiation, we previously isolated a novel gene, factor for adipocyte differentiation (fad) 158, whose expression was induced during the earliest stages of adipogenesis, and its product was localized to the endoplasmic reticulum. We found that the knockdown of fad158 expression prevented the differentiation of 3T3-L1 cells into adipocytes. In addition, over-expression of fad158 promoted the differentiation of NIH-3T3 cells, which do not usually differentiate into adipocytes. Although these findings strongly suggest that fad158 has a crucial role in regulating adipocyte differentiation, the physiological role of the gene is still unclear. In this study, we generated mice in which fad158 expression was deleted. The fad158-deficient mice did not show remarkable changes in body weight or the weight of white adipose tissue on a chow diet, but had significantly lower body weights and fat mass than wild-type mice when fed a high-fat diet. Furthermore, although the disruption of fad158 did not influence insulin sensitivity on the chow diet, it improved insulin resistance induced by the high-fat diet. These results indicate that fad158 is a key factor in the development of obesity and insulin resistance caused by a high-fat diet.
Asunto(s)
Adipocitos/citología , Adipogénesis/genética , Tejido Adiposo Blanco/metabolismo , Grasas de la Dieta/efectos adversos , Resistencia a la Insulina/genética , Proteínas de la Membrana/genética , Obesidad/genética , Aumento de Peso/genética , Células 3T3-L1 , Animales , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Obesidad/metabolismoRESUMEN
Factor for adipocyte differentiation 104 (fad104) is a regulator of adipogenesis and osteogenesis. Our previous study showed that fad104-deficient mice died immediately after birth, suggesting fad104 to be essential for neonatal survival. However, the cause of this rapid death is unclear. Here, we demonstrate the role of fad104 in neonatal survival. Phenotypic and morphological analyses showed that fad104-deficient mice died due to cyanosis-associated lung dysplasia including atelectasis. Furthermore, immunohistochemistry revealed that FAD104 was strongly expressed in ATII cells in the developing lung. Most importantly, the ATII cells in lungs were immature, and impaired the expression of surfactant-associated proteins. Collectively, these results indicate that fad104 has an indispensable role in lung maturation, especially the maturation and differentiation of ATII cells.
Asunto(s)
Fibronectinas/fisiología , Pulmón/embriología , Adipogénesis , Animales , Diferenciación Celular , Embrión de Mamíferos/metabolismo , Fibronectinas/metabolismo , Inmunohistoquímica , Pulmón/citología , Pulmón/metabolismo , Ratones , Ratones NoqueadosRESUMEN
LRH-1 (liver receptor homologue-1), a transcription factor and member of the nuclear receptor superfamily, regulates the expression of its target genes, which are involved in bile acid and cholesterol homoeostasis. However, the molecular mechanisms of transcriptional control by LRH-1 are not completely understood. Previously, we identified Ku80 and Ku70 as LRH-1-binding proteins and reported that they function as co-repressors. In the present study, we identified an additional LRH-1-binding protein, ILF3 (interleukin enhancer-binding factor 3). ILF3 formed a complex with LRH-1 and the other two nuclear receptor co-activators PRMT1 (protein arginine methyltransferase 1) and PGC-1α (peroxisome proliferator-activated receptor γ co-activator-1α). We demonstrated that ILF3, PRMT1 and PGC-1α were recruited to the promoter region of the LRH-1-regulated SHP (small heterodimer partner) gene, encoding one of the nuclear receptors. ILF3 enhanced SHP gene expression in co-operation with PRMT1 and PGC-1α through the C-terminal region of ILF3. In addition, we found that the small interfering RNA-mediated down-regulation of ILF3 expression led to a reduction in the occupancy of PGC-1α at the SHP promoter and SHP expression. Taken together, our results suggest that ILF3 functions as a novel LRH-1 co-activator by acting synergistically with PRMT1 and PGC-1α, thereby promoting LRH-1-dependent gene expression.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Proteínas de Choque Térmico/metabolismo , Proteínas del Factor Nuclear 90/metabolismo , Proteína-Arginina N-Metiltransferasas/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Inmunoprecipitación de Cromatina , Células HEK293 , Células HeLa , Proteínas de Choque Térmico/genética , Células Hep G2 , Humanos , Proteínas del Factor Nuclear 90/genética , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Regiones Promotoras Genéticas , Unión Proteica , Proteína-Arginina N-Metiltransferasas/genética , Receptores Citoplasmáticos y Nucleares/genética , Proteínas Represoras/genética , Factores de Transcripción/genéticaRESUMEN
To elucidate molecular mechanisms of adipocyte differentiation, we previously isolated TC10-like/ TC10betaLong (TCL/TC10betaL), which was transiently expressed in the early phase of adipogenesis of 3T3-L1 cells and seemed to be a positive regulator of adipogenesis. By using TCL/TC10betaL-overexpressing NIH-3T3 cells, we also isolated gelsolin as a gene whose expression was up-regulated by TCL/TC10betaL. However, the roles of gelsolin in adipocyte differentiation are unclear. In this paper we characterized the function of gelsolin in adipogenesis in 3T3-L1 cells. The level of gelsolin changed during adipocyte differentiation. Knockdown of the expression of gelsolin using RNAi inhibited adipocyte differentiation, and impaired the expression of peroxisome proliferator-activated receptor gamma (PPARgamma) and CCAAT/enhancer-binding protein (C/EBP) alpha. Interestingly, the knockdown also impaired mitotic clonal expansion (MCE), and increased cell size, though it reduced levels of C/EBPbeta and C/EBPdelta, markers for the early stage of adipogenesis, only slightly. Gelsolin plays a crucial role in the differentiation of 3T3-L1 cells into adipocytes.
Asunto(s)
Adipocitos/metabolismo , Adipogénesis/fisiología , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Diferenciación Celular/fisiología , Gelsolina/metabolismo , Regulación de la Expresión Génica , PPAR gamma/metabolismo , Células 3T3-L1 , Actinas/metabolismo , Adipogénesis/genética , Animales , Proteínas Potenciadoras de Unión a CCAAT/genética , Diferenciación Celular/genética , Gelsolina/genética , Expresión Génica , Ratones , Mitosis , PPAR gamma/genética , Interferencia de ARNRESUMEN
Nuclear receptor liver receptor homolog-1 (LRH-1; NR5A2) plays a crucial role in the homeostasis of bile acids and cholesterol by controlling the expression of genes central to bile acid synthesis and efflux, reverse cholesterol transport, and high density lipoprotein-remodeling. However, the molecular mechanisms that modulate the transactivation activity of LRH-1 remain unclear. It is proposed that LRH-1's activity is regulated by post-modifications, the binding of small heterodimer partner (SHP), or the binding of coregulators. To search for cofactors that regulate the transactivation activity of LRH-1, we performed a pull-down assay using glutathione S-transferase (GST) fused to the N-terminal portion of LRH-1 and nuclear extracts from HeLa cells, and identified Ku proteins as interacting proteins with LRH-1. We also found that Ku proteins associate with LRH-1 through its DNA-binding domain and hinge region. Luciferase reporter assays revealed that Ku proteins repressed the SHP promoter activity mediated by LRH-1. Furthermore, Ku proteins suppressed the coactivating effect of peroxisome proliferator-activated receptor (PPAR) gamma coactivator-1alpha (PGC-1alpha), an LRH-1 coactivator, on the LRH-1-mediated SHP promoter activity. Previously, we showed that Ku proteins interacted with nuclear receptor farnesoid X receptor (FXR; NR1H4) and decreased the expression of its target gene. In this study, we demonstrated that Ku proteins also interacted with not only LRH-1 but various nuclear receptors, such as the estrogen receptor, PPAR, and Rev-erb. Ku proteins may function as corepressors for various nuclear receptors including LRH-1.
Asunto(s)
Proteínas Co-Represoras/metabolismo , ADN Helicasas/metabolismo , Regulación de la Expresión Génica , Expresión Génica , Regiones Promotoras Genéticas , Receptores Citoplasmáticos y Nucleares/metabolismo , Línea Celular , ADN , ADN Helicasas/genética , Dimerización , Glutatión Transferasa , Células HeLa , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Autoantígeno Ku , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Activación TranscripcionalRESUMEN
Fad104 (factor for adipocyte differentiation 104) is a novel gene expressed temporarily in the early stages of adipocyte differentiation. Previously, we showed that fad104 promotes adipocyte differentiation in mouse 3T3-L1 cells and mouse embryonic fibroblasts (MEFs). Furthermore, we reported that implanted wild-type MEFs could develop into adipocytes, whereas fad104-deficient MEFs could not. Interestingly, bone-like tissues were only observed in the implants derived from fad104-deficient MEFs. This result implies that fad104 is involved in osteoblast differentiation. However, the functions of fad104 during osteogenesis are unknown. In this paper, we show that fad104 negatively regulates osteoblast differentiation. During the differentiation process, the level of fad104 expression decreased. Deletion of fad104 facilitated osteoblast differentiation in MEFs, and elevated the level of runx2, a master regulator of osteoblast differentiation. Disruption of fad104 suppressed BMP-2-mediated adipocyte differentiation in MEFs. In conclusion, we demonstrate that fad104 reciprocally regulates differentiation of adipocytes and osteoblast; functions as a positive regulator in adipocyte differentiation and as a negative regulator in osteoblast differentiation.
Asunto(s)
Adipogénesis/genética , Diferenciación Celular/genética , Fibronectinas/fisiología , Osteoblastos/citología , Animales , Células Cultivadas , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Fibronectinas/genética , Eliminación de Gen , Ratones , Ratones MutantesRESUMEN
To elucidate molecular mechanisms of adipocyte differentiation, we previously isolated TC10-like/TC10betaLong (TCL/TC10betaL), regulators of G protein signaling 2 (RGS2), factor for adipocyte differentiation (fad) 104 and fad158, which were transiently expressed in the early phase of adipogenesis. These four genes seem to be positive regulators of adipogenesis, since their knockdown resulted in the inhibition of adipocyte differentiation. When growth-arrested 3T3-L1 cells were induced to differentiate, they first reentered the cell cycle and underwent several rounds of cell division, a process known as mitotic clonal expansion (MCE). Although MCE is required for completion of the differentiation program, its molecular mechanisms are not fully understood. We examined the roles of these four genes during MCE. Knockdown of the expression of TCL/TC10betaL impaired MCE, while that of RGS2 or fad104 had a rather weak effect and that of fad158 had no effect. The suppression of TCL/TC10betaL inhibited the incorporation of bromodeoxyuridine (BrdU), indicating that DNA synthesis was prevented by the knockdown. Interestingly, the knockdown of TCL/TC10betaL inhibited the expression of the CCAAT/enhancer-binding protein (C/EBP) family, C/EBPbeta and C/EBPdelta, during MCE. The results strongly suggest that TCL/TC10betaL regulates adipocyte differentiation by controlling MCE and this regulatory effect is closely linked to C/EBPbeta and C/EBPdelta expression.