RESUMEN
BACKGROUND: Ischemic stroke remains the predominant contributor to mortality and disability globally. Microglia undergo rapid activation and initiate inflammatory cascade reactions by phenotypic polarization, participating in the regulation of inflammatory injury and tissue repair post-ischemic stroke. Regulating microglia-mediated neuroinflammation is a promising therapeutic strategy for ischemic stroke. Previously, we designed and synthesized a novel p55PIK inhibitor, TAT-N15 polypeptide, which presents inhibitive activity on NF-κB signaling-mediated inflammation in acute conjunctivitis and allergic rhinitis. The present study aimed to explore the therapeutic effect and mechanism of TAT-N15 on ischemia stroke. METHODS: The mouse model of transient cerebral ischemia was made using the intraluminal filament method. After being treated with daily intraperitoneal injections of TAT-N15 (10 mg/kg) for 7 d, the neurological outcomes and the cerebral infarction volume were evaluated. Histopathology of the ischemia cerebral hemisphere was observed by H&E and Nissl staining. Neuronal survival, astrogliosis, and co-labeling of CD86/Iba1 and CD206/Iba1 were detected by immunofluorescence. The cell apoptosis was estimated by TUNEL staining. The expression levels of apoptosis-associated proteins, proinflammatory cytokines, protein markers of M1 and M2 microglia, and the phosphorylation of NF-κB and STAT3 proteins in the ischemic penumbra were detected by Western blot. RESULTS: TAT-N15 treatment significantly decreased the infarct volume and alleviated neurological functional impairment, neuronal injury, and neuron apoptosis. Meanwhile, TAT-N15 treatment restrained the activation of microglia and astrocytes as well as the protein expression of proinflammatory cytokine in ischemic penumbra. Additionally, the administration of TAT-N15 treatment resulted in a significant reduction in the density of M1 phenotype microglia while concurrently increasing the density of M2 phenotype microglia within the ischemic penumbra. Finally, mechanical analysis unveiled that TAT-N15 exerted a substantial inhibitory effect on the protein expression of phosphorylated STAT3 and NF-κB. CONCLUSION: TAT-N15 may inhibit neuroinflammation via regulating microglia activation and polarization through the STAT3/NF-κB pathway, which exhibits the neuroprotection effect in ischemic stroke.
Asunto(s)
Antiinflamatorios , Apoptosis , Modelos Animales de Enfermedad , Mediadores de Inflamación , Ratones Endogámicos C57BL , Microglía , FN-kappa B , Enfermedades Neuroinflamatorias , Fármacos Neuroprotectores , Factor de Transcripción STAT3 , Transducción de Señal , Animales , Factor de Transcripción STAT3/metabolismo , Factor de Transcripción STAT3/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Microglía/efectos de los fármacos , Microglía/metabolismo , Microglía/patología , FN-kappa B/metabolismo , FN-kappa B/antagonistas & inhibidores , Enfermedades Neuroinflamatorias/tratamiento farmacológico , Enfermedades Neuroinflamatorias/metabolismo , Fármacos Neuroprotectores/farmacología , Masculino , Antiinflamatorios/farmacología , Apoptosis/efectos de los fármacos , Mediadores de Inflamación/metabolismo , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Ataque Isquémico Transitorio/tratamiento farmacológico , Ataque Isquémico Transitorio/metabolismo , Ataque Isquémico Transitorio/patología , Accidente Cerebrovascular Isquémico/tratamiento farmacológico , Accidente Cerebrovascular Isquémico/metabolismo , Accidente Cerebrovascular Isquémico/patología , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Infarto de la Arteria Cerebral Media/metabolismo , Infarto de la Arteria Cerebral Media/patologíaRESUMEN
PURPOSE: Previously we developed TAT-N24 as a synthetic cell-permeable peptide inhibitor of p55PIK signaling and demonstrated its anti-inflammatory effects. This study aimed to evaluate the potential of TAT-N24 as a new agent for the treatment of ocular inflammatory diseases. METHODS: The endotoxin-induced uveitis (EIU) model was established by intravitreal injection of lipopolysaccharide (LPS) in BALB/c mice and experimental autoimmune uveitis (EAU) model was established by subcutaneous injection of a peptide spanning amino acid residues 161-180 of interphotoreceptor retinoid binding protein (IRBP161-180) with complete Freund's adjuvant (CFA) in B10.RIII mice. TAT-N24 was topically administered in EIU model and intraperitoneally administered in EAU model. The severity levels of uveitis were assessed by clinical and histopathological scores. The mRNA levels of inflammatory cytokines in iris-ciliary body (ICB) and retina were analyzed by reverse transcription quantitative polymerase chain reaction (RT-qPCR). The protein levels of inflammatory factors were determined by ELISA or Western blotting. RESULTS: The results showed that TAT-N24 alleviated clinical signs, decreased inflammatory cell infiltration and the expression of inflammatory cytokines in both EIU and EAU models. Furthermore, protein levels of tumor necrosis factor-alpha (TNF-α), interleukin-1ß (IL-1ß) and interleukin-6 (IL-6) in aqueous humor and mRNA and protein levels of NF-κB p65 in the ICB significantly decreased in EIU model. In EAU model, TAT-N24 application induced a significant decrease of IFN-gamma (IFN-γ) and interleukin-17 (IL-17) in the retina, which were secreted by Th1 and Th17 cells, respectively. CONCLUSION: In conclusion, TAT-N24 suppressed intraocular inflammation in both EIU and EAU models, and the anti-inflammatory effects were mediated by suppressing the expression of inflammatory cytokines by PI3K/NF-κB signaling pathway. TAT-N24 could be potential candidate for the treatment of ocular inflammatory diseases.
Asunto(s)
Citocinas/metabolismo , Productos del Gen tat/farmacología , Fosfatidilinositol 3-Quinasas/efectos de los fármacos , Uveítis/metabolismo , Animales , Western Blotting , Modelos Animales de Enfermedad , Femenino , Ratones , Ratones Endogámicos BALB C , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de Señal , Uveítis/tratamiento farmacológico , Uveítis/patologíaRESUMEN
PURPOSE: Phosphatidylinositol 3-kinase (PI3K) plays an important role in tumorigenesis by cross-talking with several signaling pathways. p55PIK is a unique regulatory subunit of PI3K and contains an extra 24-residue N-terminal domain (N24). This study aimed to explore the interaction of p55PIK with p53 and the role of p55PIK in regulating p53-dependent apoptosis in cancer cells. MATERIALS AND METHODS: The expression of p55PIK was detected in cancer cells, and the interaction of p55PIK with p53 was examined by immunoprecipitation and pull-down assay. The expression of p53-dependent apoptosis-related genes was detected by PCR. RESULTS: N24 domain of p55PIK interacted with DNA-specific binding domain (DBD) of p53. The increase or decrease of p55PIK expression led to the change of the expression of p53 and p53-regulated genes in cancer cells. Moreover, N24 peptide led to the change of the expression of p53-regulated genes. Moreover, a membrane-permeable N24 peptide enhanced p53-dependent apoptosis induced by methyl methanesulfonate. CONCLUSION: Our results reveal a novel mechanism that regulates p53-dependent apoptosis in cancer cells via p55PIK-p53 interaction.
RESUMEN
A pot experiment was conducted to study the selenium (Se) accumulation characteristics and the tolerance of Cyphomandra betacea (Solanum betaceum) seedlings under different soil Se concentrations. The 5 mg/kg soil Se concentration increased the C. betacea seedling biomass and photosynthetic pigment contents (chlorophyll a, chlorophyll b, total chlorophyll, and carotenoid), whereas the other soil Se concentrations (10, 25, and 50 mg/kg) inhibited seedling growth. Increases in the soil Se concentrations tended to decrease the superoxide dismutase activity and soluble protein content, but had the opposite effect on the peroxidase and catalase activities. The 5, 10, and 25 mg/kg soil Se concentrations decreased the DNA methylation levels of C. betacea seedlings because of an increase in demethylation patterns (versus 0 mg/kg), whereas the 50 mg/kg soil Se concentration increased the DNA methylation levels because of an increase in hypermethylation patterns (versus 0 mg/kg). Increases in the soil Se concentrations were accompanied by an increasing trend in the Se content of C. betacea seedlings. Moreover, the amount of Se extracted by the shoots was highest for the 25 mg/kg soil Se concentration. Therefore, C. betacea may be able to accumulate relatively large amounts of Se and its growth may be promoted in 5 mg/kg soil Se.
RESUMEN
AIMS: Alpha-fetoprotein (AFP) is regarded as a diagnostic and prognostic biomarker and a potential therapeutic target for hepatocellular carcinoma (HCC). However, the regulation of AFP expression in HCC remains poorly understood. This study aimed to investigate the mechanism by which AFP expression is regulated by p55PIK, an isoform of PI3K. MAIN METHODS: Human HCC cell lines (HepG2 and Huh-7) were treated with p55PIK specific competitive inhibitor or shRNA, or p55PIK overexpression vector, in the absence or presence of NF-κB inhibitor PDTC. AFP expression was detected by quantitative real-time PCR and Western blotting. NF-κB responsive elements in AFP enhancer region were characterized by luciferase reporter assay. KEY FINDINGS: p55PIK significantly stimulated the expression of AFP by activating NF-κB signaling pathway in HCC cells. Furthermore, two NF-κB binding sites in AFP enhancer region were identified to be primarily responsible for p55PIK mediated upregulation of AFP expression. SIGNIFICANCE: p55PIK/NF-κB signaling plays an important role in the upregulation of AFP expression in HCC.
Asunto(s)
Carcinoma Hepatocelular/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Hepáticas/genética , FN-kappa B/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de Señal , alfa-Fetoproteínas/genética , Carcinoma Hepatocelular/metabolismo , Línea Celular Tumoral , Células Hep G2 , Humanos , Neoplasias Hepáticas/metabolismo , Regulación hacia ArribaRESUMEN
Manganese (Mn) is required in various human physiological processes. Excessive Mn exposure causes manganism, a progressive neurodegenerative disorder similar to idiopathic Parkinson's disease (IPD). However, the detailed mechanism of Mn-induced neurotoxicity is not yet fully understood. MicroRNAs (miRNAs) play important roles in gene expression regulation, and miRNA expression profile provides additional biological and prognostic information of diseases. In our study, RNA sequencing was performed to profile miRNAs in the SH-SY5Y cells following MnCl2 treatment. Expressions of 73 miRNAs were altered following excessive Mn treatment. Furthermore, has-miR-4306 was identified to target 3'UTR of ATP13A2 (PARK9) directly. Inhibition of has-miR-4306 efficiently restored Mn-induced cytotoxicity. Thus, for the first time, we revealed the miRNA effects of Mn ions to neuron cells, highlighted the involvement of miRNA regulation in neurodegeneration caused by Mn exposure, and provided a potential application of miRNAs in future therapeutic intervention.
Asunto(s)
Cloruros/toxicidad , Regulación de la Expresión Génica , MicroARNs/genética , Neuronas/efectos de los fármacos , ATPasas de Translocación de Protón/genética , Secuencia de Bases , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Perfilación de la Expresión Génica , Humanos , Compuestos de Manganeso , MicroARNs/metabolismo , Modelos Biológicos , Neuronas/metabolismo , Neuronas/patología , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , ATPasas de Translocación de Protón/antagonistas & inhibidores , ATPasas de Translocación de Protón/metabolismo , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , ARN Nucleolar Pequeño/genética , ARN Nucleolar Pequeño/metabolismo , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Transducción de SeñalRESUMEN
Imatinib is the first-line drug for gastrointestinal stromal tumors (GISTs), as mutated KIT is closely associated with the occurrence of GIST. However, Imatinib resistance (IMA-resistance) occurs inevitably in most GIST patients. Although the over-expression of KIT in GIST is one of the major factors contributing to IMA-resistance, the underlying mechanism is still unclear. In this study, we demonstrate that p55PIK, an isoform of phosphoinositide 3-kinase (PI3K), increases KIT expression, leading to IMA-resistance in GISTs by activating NF-κB signaling pathway. Furthermore, down-regulation of p55PIK significantly decreases KIT expression and re-sensitizes IMA-resistance-GIST cells to Imatinib in vitro and in vivo. Interestingly, the expression of both p55PIK and KIT proteins is significantly increased in tumor samples from IMA-resistance-GIST patients, suggesting that p55PIK up-regulation may be important for IMA-resistance in the clinical setting. Altogether, our data provide evidence that p55PIK-PI3K signaling can contribute to IMA-resistance in GIST by increasing KIT expression. Moreover, p55PIK may be a novel potential drug target for treating tumors that develop IMA-resistance.
Asunto(s)
Antineoplásicos/farmacología , Resistencia a Antineoplásicos , Neoplasias Gastrointestinales/tratamiento farmacológico , Tumores del Estroma Gastrointestinal/tratamiento farmacológico , Mesilato de Imatinib/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-kit/metabolismo , Animales , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Resistencia a Antineoplásicos/genética , Neoplasias Gastrointestinales/enzimología , Neoplasias Gastrointestinales/genética , Neoplasias Gastrointestinales/patología , Tumores del Estroma Gastrointestinal/enzimología , Tumores del Estroma Gastrointestinal/genética , Tumores del Estroma Gastrointestinal/patología , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones Desnudos , Mutación , FN-kappa B/metabolismo , Proteínas Proto-Oncogénicas c-kit/genética , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Transfección , Regulación hacia Arriba , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Previously, we have shown that p55PIK, an isoform of class I(A) phosphoinositide 3-kinase (PI3K), specifically interacts with important cell-cycle regulators, such as retinoblastoma (Rb), to promote cell-cycle progression. Here, we used the glutathione S-transferase pull-down assay to identify other p55PIK-interacting proteins besides Rb in a Rb-deficient cell line and found that p55PIK interacted with proliferation cell nuclear antigen (PCNA), which plays a key role in coordinating both initiation of the leading strand DNA replication and discontinuous lagging strand synthesis. Overexpression of p55PIK increased, and knockdown decreased, DNA synthesis and DNA replication by modulating the binding of DNA polymerase δ (Polδ) to PCNA. Moreover, a cell-permeable peptide containing the N-terminal-binding domain of p55PIK (TAT-N24) disrupted the p55PIK-PCNA interaction in cancer cells, and also inhibited the DNA synthesis and tumor growth in cell culture and in vivo. Altogether, our results show that the p55PIK-PCNA interaction is important in regulating DNA synthesis and contributes to tumorigenesis. Furthermore, the p55PIK-PCNA interaction provides a potential new target for anticancer drug development.
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ADN/biosíntesis , Neoplasias/genética , Fosfatidilinositol 3-Quinasas/genética , Antígeno Nuclear de Célula en Proliferación/genética , Carcinogénesis/genética , Puntos de Control del Ciclo Celular/genética , Línea Celular Tumoral , Proliferación Celular , Replicación del ADN/genética , Elafina/genética , Elafina/metabolismo , Glutatión Transferasa/química , Glutatión Transferasa/genética , Humanos , Neoplasias/patología , Fosfatidilinositol 3-Quinasas/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Unión Proteica , Mapas de Interacción de Proteínas/genéticaRESUMEN
p55PIK, regulatory subunit of class IA phosphatidylinositol 3-kinase (PI3K), plays a crucial role in cell cycle progression by interaction with tumor repressor retinoblastoma (Rb) protein. A recent study showed that Rb protein can localize to the mitochondria in proliferative cells. Aberrant p55PIK expression may contribute to mitochondrial dysfunction in cancer progression. To reveal the mechanisms of p55PIK transcriptional regulation, the p55PIK promoter characteristics were analyzed. The data show that myeloid zinc finger 1, MZF1, is necessary for p55PIK gene transcription activation. ChIP (Chromatin immuno-precipitation) assay shows that MZF1 binds to the cis-element "TGGGGA" in p55PIK promoter. In MZF1 overexpressed cells, the promoter activity, expression of p55PIK, and cell proliferation rate were observed to be significantly enhanced. Whereas in MZF1-silenced cells, the promoter activity and expression of p55PIK and cell proliferation level was statistically decreased. In CRC tissues, MZF1 and p55PIK mRNA expression were increased (P = 0.046, P = 0.047, resp.). A strong positive correlation (Rs = 0.94) between MZF1 and p55PIK mRNA expression was observed. Taken together, we concluded that p55PIK is transcriptionally activated by MZF1, resulting in increased proliferation of colorectal cancer cells.
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Neoplasias Colorrectales/metabolismo , Regulación Neoplásica de la Expresión Génica , Factores de Transcripción de Tipo Kruppel/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Sitios de Unión , Línea Celular Tumoral , Proliferación Celular , Neoplasias Colorrectales/genética , Genes Reporteros , Humanos , Mutagénesis Sitio-Dirigida , Fosfatidilinositol 3-Quinasas/genética , Regiones Promotoras Genéticas , ARN Interferente Pequeño/metabolismo , Proteína de Retinoblastoma/metabolismoRESUMEN
Vascular growth factor (VEGF) is an important mediator of angiogenesis. PI3K plays essential roles in angiogenesis; however, the mechanisms and specific functions of individual isoforms of PI3K members in tumor angiogenesis regulation are still not fully understood. In this study, we evaluate the role of p55PIK, a PI3K regulatory subunit encoded by PIK3R3 gene, in tumor angiogenesis. We reported that overexpression of p55PIK in cancer cells up-regulated HIF-1α expression and increased VEGF expression. Furthermore, overexpression of p55PIK increased tumor angiogenesis in vivo and in vitro. Moreover, data indicated enhanced HIF-1α expression by p55PIK-PI3K depended on its ability to activate NF-кB signaling pathways, especially to increase the phosphorylation of p65 subunits of NF-κB. Our study suggested that p55PIK-PI3K was essential in regulating cancer cell-mediated angiogenesis and contributed to tumor growth and that the p55PIK provides a potential and specific target for new anti-angiogenesis drug development.
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Neoplasias Colorrectales/fisiopatología , Regulación Neoplásica de la Expresión Génica/fisiología , FN-kappa B/metabolismo , Neovascularización Patológica/fisiopatología , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de Señal/fisiología , Western Blotting , Línea Celular Tumoral , Cartilla de ADN/genética , Células Endoteliales de la Vena Umbilical Humana , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Análisis por Micromatrices , Reacción en Cadena en Tiempo Real de la Polimerasa , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
BACKGROUND: While there is strong evidence for phosphatidylinositol 3-kinase (PI3K) involvement in cancer development, there is limited information about the role of PI3K regulatory subunits. PIK3R3, the gene that encodes the PI3K regulatory subunit p55γ, is over-expressed in glioblastoma and ovarian cancers, but its expression in gastric cancer (GC) is not known. We thus used genetic and bioinformatic approaches to examine PIK3R3 expression and function in GC, the second leading cause of cancer mortality world-wide and highly prevalent among Asians. METHODS: Primary GC and matched non-neoplastic mucosa tissue specimens from a unique Asian patient gastric cancer library were comprehensively profiled with platforms that measured genome-wide mRNA expression, DNA copy number variation, and DNA methylation status. Function of PIK3R3 was predicted by IPA pathway analysis of co-regulated genes with PIK3R3, and further investigated by siRNA knockdown studies. Cell proliferation was estimated by crystal violet dye elution and BrdU incorporation assay. Cell cycle distribution was analysed by FACS. RESULTS: PIK3R3 was significantly up-regulated in GC specimens (n = 126, p < 0.05), and 9.5 to 15% tumors showed more than 2 fold increase compare to the paired mucosa tissues. IPA pathway analysis showed that PIK3R3 promoted cellular growth and proliferation. Knockdown of PIK3R3 decreased the growth of GC cells, induced G0/G1 cell cycle arrest, decreased retinoblastoma protein (Rb) phosphorylation, cyclin D1, and PCNA expression. CONCLUSION: Using a combination of genetic, bioinformatic, and molecular biological approaches, we showed that PIK3R3 was up-regulated in GC and promoted cell cycle progression and proliferation; and thus may be a potential new therapeutic target for GC.
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Pueblo Asiatico/genética , Biología Computacional/métodos , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Fosfatidilinositol 3-Quinasas/genética , Neoplasias Gástricas/enzimología , Neoplasias Gástricas/genética , Proliferación Celular , Ciclina D1/genética , Ciclina D1/metabolismo , Puntos de Control de la Fase G1 del Ciclo Celular/genética , Mucosa Gástrica/metabolismo , Mucosa Gástrica/patología , Técnicas de Silenciamiento del Gen , Humanos , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación , Subunidades de Proteína/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Fase de Descanso del Ciclo Celular/genética , Proteína de Retinoblastoma/metabolismo , Transducción de Señal/genética , Neoplasias Gástricas/patologíaRESUMEN
BACKGROUND: Hormonally-regulated histone modifications that govern positive versus negative transcription of target genes are poorly characterized despite their importance for normal and pathological endocrine function. There have been only a few studies examining chromatin modifications on target gene promoters by nuclear hormone receptors. Moreover, these studies have focused on positively-regulated target genes. TSHalpha, a heterodimer partner for thyrotropin (TSH), is secreted by the pituitary gland. T(3) negatively regulates TSHalpha gene expression via thyroid hormone receptors (TRs) which belong to the nuclear hormone receptor superfamily, whereas thyrotropin releasing hormone (TRH) positively regulates via the TRH receptor, a G protein-coupled receptor. METHODOLOGY/PRINCIPAL FINDINGS: We studied regulation of the TSHalpha gene by cAMP and T(3) using chromatin immunoprecipitation (ChIP) assays in stably-transfected rat pituitary cells containing the human TSHalpha promoter. Interestingly, cAMP selectively increased histone H4 acetylation whereas, as previously reported, T(3) induced histone H3 acetylation. In particular, cAMP increased H4K5 and H4K8 acetylation and decreased H4K20 trimethylation, modifications associated with transcriptional activation. T(3) increased H3K9 and H3K18 acetylation and H3K4 trimethylation; however, it also decreased H3K27 acetylation and increased H3K27 trimethylation which are associated with transcriptional repression. Of note, cAMP recruited pCREB, CBP/p300, and PCAF to the promoter whereas T(3) caused dissociation of NCoR/SMRT and HDAC3. Overexpression of a dominant negative mutant thyroid hormone receptor (TR) from a patient with resistance to thyroid hormone (RTH) led to less T(3)-dependent negative regulation and partially blocked histone H3 modifications of the TSHalpha promoter. CONCLUSIONS/SIGNIFICANCE: Our findings show that non-overlapping and specific histone modifications determine positive versus negative transcriptional regulation, and integrate opposing hormonal and intracellular signals at the TSHalpha promoter. A mutant TR from a patient with RTH exerted dominant negative activity by blocking the histone modifications induced by T(3) on the TSHalpha promoter and likely contributes to the inappropriate TSH production observed in RTH.
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Regulación de la Expresión Génica , Hormonas Glicoproteicas de Subunidad alfa/metabolismo , Regiones Promotoras Genéticas , Transcripción Genética , Acetilación , Animales , Núcleo Celular/metabolismo , Cromatina/química , Inmunoprecipitación de Cromatina , AMP Cíclico/metabolismo , Histonas/metabolismo , Humanos , Metilación , Hipófisis/citología , RatasRESUMEN
Currently, little is known about histone modifications and molecular mechanisms of negatively regulated transcription. In pituitary cells, thyroid hormone (T(3)) decreased transcription, and surprisingly increased histone acetylation, of TSHalpha promoter. This increase was mediated directly by thyroid hormone receptor. Histone acetylation of H3K9 and H3K18 sites, two modifications usually associated with transcriptional activation, occur in negative regulation of TSHalpha promoter. T(3) also caused release of a corepressor complex composed of histone deacetylase 3 (HDAC3), transducin beta-like protein 1, and nuclear receptor coprepressor (NCoR)/ silencing mediator for retinoic and thyroid hormone receptor from TSHalpha promoter in chromatin immunoprecipitation assays. NCoR and HDAC3 overexpression selectively increased ligand-independent basal transcription. Two histone acetyltransferase inhibitors increased overall transcription but did not abrogate negative regulation or NCoR/HDAC3 complex release by T(3). Chromatin immunoprecipitation analyses of an endogenous positively regulated target gene showed increased histone acetylation and corepressor complex release with T(3) treatment. Finally, microarray analyses suggested there is a subset of negatively regulated genes with increased histone acetylation. These findings demonstrate the critical role of NCoR/HDAC3 complex in negative regulation of TSHalpha gene expression and show that similar complexes and overlapping epigenetic modifications can participate in both negative and positive transcriptional regulation.
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Regulación de la Expresión Génica/efectos de los fármacos , Hormonas Glicoproteicas de Subunidad alfa/genética , Histonas/metabolismo , Proteínas Represoras/metabolismo , Triyodotironina/farmacología , Acetilación/efectos de los fármacos , Animales , Línea Celular , Inmunoprecipitación de Cromatina , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas/genética , Unión Proteica/efectos de los fármacos , Ratas , Receptores de Hormona Tiroidea/metabolismo , Transducina/metabolismoRESUMEN
p55PIK, a regulatory subunit of phosphatidylinositol 3-kinase (PI3K), specifically interacts with retinoblastoma protein (Rb) through the unique NH2 terminus of p55PIK, N24. This interaction is critical for cell proliferation and cell cycle progression. To examine p55PIK as a potential target for cancer therapy, we generated an adenovirus expressing N24 (Ad-N24-GFP) and studied its effects on the proliferation of cultured cancer cells, including human colon (HT29) and thyroid (FTC236) cancer cells. Ad-N24-GFP blocked cell proliferation and induced cell cycle arrest in all cancer cell lines tested. N24 induced cell cycle arrest at G0-G1 phase in cell lines that expressed Rb. Interestingly, N24 inhibited cell proliferation by blocking cell cycle transition at both S and G2-M phases in FTC236 cells, which did not express Rb. When Rb was knocked down by short hairpin RNA in HT29 cells, N24 also inhibited cell cycle progression at S and G2-M phases, suggesting that p55PIK regulates cell cycle progression by Rb-dependent and Rb-independent mechanisms. Finally, Ad-N24-GFP markedly decreased the growth of xenograft tumors derived from HT29 and FTC236 cancer cells in athymic nude mice. Our data strongly suggest that N24 peptide is an effective anticancer therapy, which specifically inhibits PI3K signaling pathways mediated by p55PIK. Moreover, they show that the regulatory subunit of an enzyme, in addition to its catalytic subunit, can be an important target for drug development.
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Antineoplásicos/farmacología , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Fragmentos de Péptidos/farmacología , Péptidos/farmacología , Fosfatidilinositol 3-Quinasas/farmacología , Fosfatidilinositol 3-Quinasas/fisiología , Animales , Línea Celular Tumoral , Inhibidores Enzimáticos/farmacología , Humanos , Masculino , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Neoplasias/enzimología , Neoplasias/patología , Fragmentos de Péptidos/química , Fosfatidilinositol 3-Quinasas/química , Fosfatidilinositol 3-Quinasas/metabolismo , Proteína de Retinoblastoma/metabolismoRESUMEN
Thyroid hormone (TH) plays important roles in metabolism, growth and differentiation. Thyroid hormone receptors (TRs) are ligand-regulatable transcription factors that bind both TH and DNA enhancer sequences in the promoter region of target genes where they can interact with co-repressor and co-activator complexes. These interactons, in turn, have consequent effects on transcription. This review describes studies on TH action from our laboratory examining the cellular localization and motility of TRs using green fluorescent fusion proteins, gene expression profiles of TH in WT and TRalpha and TRbeta KO mice, as well as general transcription factor and co-activator recruitment on the promoters of target genes by TH in chromatin immunoprecipitation assays.
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Regiones Promotoras Genéticas/efectos de los fármacos , Receptores de Hormona Tiroidea/metabolismo , Hormonas Tiroideas/metabolismo , Animales , Células HeLa , Humanos , Ratones , Ratones Noqueados , Modelos Moleculares , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiología , Receptores de Hormona Tiroidea/genética , Receptores de Hormona Tiroidea/fisiología , Hormonas Tiroideas/farmacología , Transcripción GenéticaRESUMEN
Thyroid hormone receptors (TRs) are ligand-regulated transcription factors that bind to thyroid hormone response elements of target genes. Upon ligand binding, they recruit coactivator complexes that increase histone acetylation and recruit RNA polymerase II (Pol II) to activate transcription. Recent studies suggest that nuclear receptors and coactivators may have temporal recruitment patterns on hormone response elements, yet little is known about the nature of the patterns at multiple endogenous target genes. We thus performed chromatin immunoprecipitation assays to investigate coactivator recruitment and histone acetylation patterns on the thyroid hormone response elements of four endogenous target genes (GH, sarcoplasmic endoplasmic reticulum calcium-adenosine triphosphatase, phosphoenolpyruvate carboxykinase, and cholesterol 7alpha-hydroxylase) in a rat pituitary cell line that expresses TRs. We found that TRbeta, several associated coactivators (steroid receptor coactivator-1, glucocorticoid receptor interacting protein-1, and TR-associated protein 220), and RNA Pol II were rapidly recruited to thyroid hormone response elements as early as 15 min after T3 addition. When the four target genes were compared, we observed differences in the types and temporal patterns of recruited coactivators and histone acetylation. Interestingly, the temporal pattern of RNA Pol II was similar for three genes studied. Our findings suggest that thyroid hormone-regulated target genes may have distinct patterns of coactivator recruitment and histone acetylation that may enable highly specific regulation.
Asunto(s)
Regulación de la Expresión Génica , Histonas/metabolismo , Transactivadores/metabolismo , Triyodotironina/metabolismo , Acetilación , Animales , ATPasas Transportadoras de Calcio/genética , ATPasas Transportadoras de Calcio/metabolismo , Células Cultivadas , Colesterol 7-alfa-Hidroxilasa/genética , Colesterol 7-alfa-Hidroxilasa/metabolismo , Hormona del Crecimiento/genética , Hormona del Crecimiento/metabolismo , Histona Acetiltransferasas , Subunidad 1 del Complejo Mediador , Coactivador 1 de Receptor Nuclear , Coactivador 2 del Receptor Nuclear/genética , Coactivador 2 del Receptor Nuclear/metabolismo , Fosfoenolpiruvato Carboxiquinasa (GTP)/genética , Fosfoenolpiruvato Carboxiquinasa (GTP)/metabolismo , Hipófisis/citología , Hipófisis/efectos de los fármacos , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Ratas , Elementos de Respuesta , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico , Receptores beta de Hormona Tiroidea/efectos de los fármacos , Receptores beta de Hormona Tiroidea/genética , Receptores beta de Hormona Tiroidea/metabolismo , Transactivadores/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Triyodotironina/farmacologíaRESUMEN
pRb and its family members p130 and p107 regulate cell cycle progression and direct G1/S transition in mammalian cells through interaction with the transcription factor E2F. Phosphatidylinositol 3-kinase (PI3K) is an essential component of growth factor-regulated pathways and plays a crucial role in the regulation of cellular proliferation and differentiation. It has been demonstrated that PI3K can regulate cell cycle progression via Akt-mediated pathway. However, the possible interactions between PI3K and Rb pathways remain to be defined. It was reported that the unique 24-amino-acid N-terminal end of the p55 regulatory subunits of PI3K is an Rb-binding domain and affects Rb action or Rb-E2F interaction. The 24 N-terminal amino acids of p55gamma encoded by a cDNA construct could compete with the endogenous p55gamma for binding to Rb, which influences Rb-mediated signaling and blocks cell cycle progression. In the current study, we investigated the effects of this 24-peptide on cell proliferation in human gastric carcinoma MKN-28 cells by means of cell cycle analysis, BrdU incorporation, and determining the levels of cell cycle regulatory molecule expression. Our results showed that p55gammaPI3K and the Rb family members p130 and p107 exist in MKN-28 cells, while the p110PI3K was not detected. Moreover, p55gammaPI3K was found binding to p130/p107 in these cells. We demonstrated that the introduction of the plasmid N24p55-GFP (harboring the cDNA for the 24 N-terminal amino acids of p55gamma) into MKN-28 cells caused cell cycle arrest at G1. Furthermore, we showed that the over-expression of the 24-peptide in MKN-28 cells decreased the population of cells incorporating BrdU and reduced the levels of cyclin D1 and cyclin A. These observations suggest that PI3K can regulate cell cycle progression and cell proliferation in human gastric tumor cells via Rb-mediated pathway, and that this effect of PI3K is mediated through a direct association with Rb via the N-terminal end of its p55 kDa regulatory subunits and modulating Rb-E2F interactions. Taken together with previous studies, our data provide a new therapeutic target in human stomach cancer. Strategies targeting PI3K signal transduction or the association of PI3K with Rb, or regulating PI3K-Rb interactions could be employed for gene therapy or chemotherapy of gastric cancer and other tumors.
Asunto(s)
Carcinoma/genética , Carcinoma/patología , Ciclo Celular/genética , Ciclo Celular/fisiología , Fosfatidilinositol 3-Quinasas/biosíntesis , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Proliferación Celular , ADN Complementario/genética , Humanos , Fragmentos de Péptidos , Proteína de Retinoblastoma/metabolismo , Transducción de Señal , Células Tumorales Cultivadas , Regulación hacia ArribaRESUMEN
Currently, little is known about the direct interactions of general transcription factors and nuclear hormone receptors. To investigate the potential role of the general transcription factor, TFIIH, in T3-mediated transcriptional activation, we examined thyroid hormone receptor (TR) interaction with individual TFIIH subunits in a yeast-two hybrid system. Among the nine subunits of TFIIH studied, only p62 subunit interacted with TRbeta in a ligand-dependent manner. Glutathione-S-transferase pull-down and in vivo coimmunoprecipitation studies also demonstrated direct TR/p62 interaction. Using chromatin immunoprecipitation assays, we showed that TFIIH subunits were corecruited on or near an endogenous thyroid hormone response element upon T3 addition. Cotransfection studies with TSA201 cells showed that p62 increased T3-mediated transcription, which could be further enhanced when p62 and another TFIIH subunit, p44, were cotransfected simultaneously. Taken together, these data suggest that TRs can interact directly with a subunit of TFIIH and may provide an alternative pathway for nuclear receptor communication with the general transcription machinery that circumvents coactivators.
Asunto(s)
Fosfoproteínas/metabolismo , Receptores beta de Hormona Tiroidea/metabolismo , Factores de Transcripción TFII/metabolismo , Activación Transcripcional , Triyodotironina/farmacología , Células Cultivadas , Inmunoprecipitación de Cromatina , Humanos , Ligandos , Fosfoproteínas/genética , Subunidades de Proteína/metabolismo , Elementos de Respuesta , Factor de Transcripción TFIIH , Factores de Transcripción TFII/genética , Transcripción Genética , Técnicas del Sistema de Dos HíbridosRESUMEN
Ebp1, an ErbB3 binding protein that is a member of the proliferation-associated PA2G4 family, inhibits the proliferation and induces the differentiation of human ErbB positive breast and prostate cancer cell lines. Ebp1 binds the tumor suppressor retinoblastoma protein (Rb) both in vivo and in vitro, and Rb and Ebp1 cooperate to inhibit the transcription of the E2F1-regulated cyclin E promoter. We show here that Ebp1 can inhibit the transcription of other E2F-regulated reporter genes and of several endogenous E2F-regulated genes important in cell cycle progression in both Rb positive and Rb null cells. The Ebp1-mediated transcriptional repression depended on the presence of an E2F1 consensus element in the promoters. A fusion of Ebp1 with the GAL4 DNA binding domain protein had independent transcriptional repression activity that mapped to the C-terminal region of Ebp1. This C-terminal region of Ebp1 bound functional histone deacetylase (HDAC) activity and inhibitors of HDAC significantly reduced Ebp1-mediated repression. Ebp1 bound HDAC2, but not HDAC1, in vitro. An Ebp1 mutant lacking the HDAC binding domain failed to inhibit transcription. Our results suggest that Ebp1 can repress transcription of some E2F-regulated promoters and that one mechanism of Ebp1- mediated transcriptional repression is via its ability to recruit HDAC activity.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular , Proteínas de Unión al ADN , Histona Desacetilasas/metabolismo , Factores de Transcripción/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Sitios de Unión/genética , Butiratos/farmacología , Células COS , Proteínas Portadoras/genética , Factores de Transcripción E2F , Factor de Transcripción E2F1 , Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Células HeLa , Inhibidores de Histona Desacetilasas , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Masculino , Mutación , Plásmidos/genética , Unión Proteica , Proteínas de Unión al ARN , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factores de Transcripción/genética , Transcripción Genética/genética , Transfección , Células Tumorales CultivadasRESUMEN
Although phosphoinositide 3-kinase (PI 3-kinase) is essential for cell cycle progression, the molecular mechanisms that regulate its diverse biological effects are poorly understood. We demonstrate here that Rb, a key regulator of cell cycle progression, associates with p55 kDa (p55alpha and p55gamma) regulatory subunits of PI 3-kinase in vivo and in vitro. Both confocal microscopy and biochemical analysis demonstrated the presence of p55gamma in the nucleus. The 24-amino-acid N-terminal end of p55gamma, which is unique among PI 3-kinase regulatory subunits, was sufficient to bind Rb. Addition of serum or growth factors to quiescent cells triggered the dissociation of Rb from p55. Ectopic expression of the 24-amino-acid N-terminal end of p55gamma inhibited cell cycle progression, as evidenced by induction of cell growth arrest at the G0/G1 phase, inhibition of DNA synthesis, inhibition of cyclin D and cyclin E promoter activity, and changes in the expression of cell cycle-related proteins. The inhibitory effects of the N-terminal end of p55gamma on cell cycle progression depended on the presence of functional Rb. These data demonstrate for the first time an association of p55gamma with Rb and show that modification of this association can lead to cell cycle arrest.