RESUMEN
The insulin-like growth factor-1 receptor (IGF-1R) signaling pathway is critical for both normal mammary gland development and malignant transformation. It has been reported that the IGF-1 stimulates breast cancer cell proliferation and is upregulated in tumors with BRCA1/2 mutations. We report here that IGF-1 is negatively regulated by BRCA1 at the transcriptional level in human breast cancer cells. BRCA1 knockdown (BRCA1-KD) induces the expression of IGF-1 mRNA in MCF7 cells in an estrogen receptor α (ERα)-dependent manner. We found that both BRCA1 and ERα bind to the endogenous IGF-1 promoter region containing an estrogen-responsive element-like (EREL) site. BRCA1-KD does not significantly affect ERα binding on the IGF-1 promoter. Reporter analysis demonstrates that BRCA1 could regulate IGF-1 transcripts via this EREL site. In addition, enzyme-linked immunosorbent assay revealed that de-repression of IGF-1 transcription by BRCA1-KD increases the level of extracellular IGF-1 protein, and secreted IGF-1 seems to increase the phospho-IGF-1Rß and activate its downstream signaling pathway. Blocking the IGF-1/IGF-1R/phosphoinositide 3-kinase (PI3K)/AKT pathway either by a neutralizing antibody or by small-molecule inhibitors preferentially reduces the proliferation of BRCA1-KD cells. Furthermore, the IGF-1-EREL-Luc reporter assay demonstrates that various inhibitors, which can inhibit the IGF-1R pathway, can suppress this reporter activity. These findings suggest that BRCA1 defectiveness keeps turning on IGF-1/PI3K/AKT signaling, which significantly contributes to increase cell survival and proliferation.
Asunto(s)
Proteína BRCA1/genética , Neoplasias de la Mama/metabolismo , Estrógenos/metabolismo , Factor I del Crecimiento Similar a la Insulina/genética , Proteína BRCA1/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proliferación Celular , Femenino , Humanos , Factor I del Crecimiento Similar a la Insulina/metabolismo , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Elementos de Respuesta , Transducción de SeñalRESUMEN
Besides roles in nucleus mediating the condensation of DNA into chromatin, the involvement of histones in autoimmune diseases, hormone regulation, and killing leukemia cells has been reported. In order to investigate the functions of histones on an autoimmune disease, histone H1 was injected into collagen-induced arthritis (CIA) mice. A dramatic suppression of CIA by histone H1 was observed at a dose of 1 mg/kg bodyweight of mouse. In addition, the increased level of anti-inflammatory cytokine IL-10 was detected in cultured splenocytes from the mouse treated with histone H1. These findings suggest that histone H1 suppresses the collagen-induced arthritis, possibly by increasing the level of IL-10 production.
Asunto(s)
Artritis/prevención & control , Histonas/uso terapéutico , Linfocitos/inmunología , Animales , Anticuerpos/farmacología , Artritis/inducido químicamente , Artritis/patología , Colágeno/inmunología , Colágeno/toxicidad , Dexametasona/uso terapéutico , Interleucina-10/biosíntesis , Articulaciones/efectos de los fármacos , Articulaciones/patología , Linfocitos/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos DBA , Bazo/inmunología , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
The Gadd45gamma (growth arrest and DNA damage-inducible) gene is activated transcriptionally by at least two kinds of agents: DNA damaging agent such as methyl methanesulfonate (MMS) and UV radiation, or cytokines such as interleukin (IL)-6, IL-2 and granulocyte colony-stimulating factor (G-CSF). To investigate the sequences and transcription factors involved in induction of Gadd45gamma after treatment with IL-6, the human gene was cloned and sequenced. We found C/EBP (CCAAT/enhancer-binding protein) family proteins, major transcription factors in the IL-6 signal transduction pathway, could regulate the transcriptional activity of the Gadd45gamma promoter. In addition, a noncanonical C/EBP-binding site within the Gadd45gamma promoter where C/EBPbeta and C/EBPdelta could bind, was identified by electrophoretic mobility shift assay (EMSA) and reporter gene analysis. Furthermore, we found a coordinated expression profile between Gadd45gamma mRNA and C/EBPs (beta and delta) protein during the differentiation of M1 cells: the amount of Gadd45gamma transcripts became maximal when both C/EBPbeta and C/EBPdelta levels were high, on day 1 of differentiation of M1 cells after treatment with IL-6. These findings suggest that mitotic growth arrest coupled to M1 cell differentiation is mediated by C/EBPs stimulation of growth arrest-associated genes such as Gadd45gamma.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Regulación de la Expresión Génica , Regiones Promotoras Genéticas , Proteínas/genética , Células 3T3 , Animales , Secuencia de Bases , Sitios de Unión , Clonación Molecular , Daño del ADN , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Interleucina-6/farmacología , Péptidos y Proteínas de Señalización Intracelular , Ratones , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Isoformas de Proteínas/metabolismo , ARN Mensajero/genética , Proteínas Recombinantes/biosíntesis , Factores de Transcripción/metabolismo , Transcripción Genética , Transfección , Proteinas GADD45RESUMEN
Gadd45 family genes encode nuclear acidic proteins composed of Gadd45, MyD118, and CR6. Sequence analysis showed that Gadd45 family proteins (Gadd45, MyD118, and CR6) contain LXXLL signature motifs considered necessary and sufficient for the binding of several coactivators to nuclear receptors. Interaction between Gadd45 or CR6 and RXR alpha was confirmed by a two-hybrid test in yeast. Results from a series of GST pulldown assays showed that these Gadd45 family proteins interact with several nuclear hormone receptors including RXR alpha, RAR alpha, ER alpha, PPAR alpha, PPAR beta, and PPAR gamma2 in vitro. Interaction between Gadd45 family proteins and nuclear hormone receptors resulted in modest activation of transactivating function of nuclear hormone receptors in reporter systems. When fused to DNA binding domain of GAL4, Gadd45 and CR6 activated the UAS-mediated transcription in mammalian cells. These results suggest that Gadd45 family proteins bind to nuclear hormone receptors and act as nuclear coactivators.
Asunto(s)
Proteínas/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae , Células 3T3 , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Antígenos de Diferenciación/genética , Antígenos de Diferenciación/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de Unión al ADN , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Humanos , Técnicas In Vitro , Péptidos y Proteínas de Señalización Intracelular , Ratones , Datos de Secuencia Molecular , Unión Proteica , Proteínas/genética , Receptores Citoplasmáticos y Nucleares/genética , Receptores de Ácido Retinoico/genética , Receptores de Ácido Retinoico/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Receptores X Retinoide , Homología de Secuencia de Aminoácido , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Activación Transcripcional , Técnicas del Sistema de Dos Híbridos , Proteinas GADD45RESUMEN
The hepatitis B virus (HBV) core/pregenomic promoter is regulated by enhancer I (ENI) and enhancer II (ENII) which are located upstream of the initiation sites of core/pregenomic transcripts. In this study, we identified a negative regulatory element (NRE) (nt 1576 to 1639) upstream of ENII by serial deletion analysis; a 33 kDa cellular protein in HepG2 cells binds to this element. The NRE has a significant activity if it is located upstream of ENII in HepG2 cells. Mutational analysis showed that the sequence (5'-CCAC-3') from nt 1612 to 1615 is responsible for the repression activity of NRE. Southwestern blotting and UV-crosslinking assays with HepG2 nuclear extracts also demonstrated that the 33 kDa protein in HepG2 cells binds to the sequence. It, thus, appears that the 33 kDa protein is responsible for the repression activity of NRE.