RESUMO
Receptor-associated coactivator 3 (RAC3) is a nuclear receptor coactivator usually overexpressed in tumors that exerts oncogenic functions in the cytoplasm and the nucleus. Although as part of its oncogenic actions it was previously identified as an inhibitor of apoptosis and autophagy, its expression is required in order to preserve the pluripotency and embryonic stem cell self-renewal. In this work we investigated its role in cellular senescence. We found that RAC3 overexpression in the nontumoral HEK293 cells inhibits the premature senescence induced by hydrogen peroxide or rapamycin. The mechanism involves not only the inhibition of autophagy early induced by these stimuli in the pathway to senescence, but also the increase in levels and nuclear localization of both the cell cycle suppressors p53/p21 and the longevity promoters FOXO1A, FOXO3A and SIRT1. Furthermore, we found that RAC3 overexpression is required in order to maintain the telomerase activity. In tumoral HeLa cells its activity was inhibited by depletion of RAC3 inducing replicative senescence. Moreover, we demonstrated that in vivo, levels of RAC3 are downregulated in the liver from aged as compared with young rats, whereas the levels of p21 are increased, correlating with the expected senescent cell contents in aged tissues. A similar downregulation of RAC3 was observed in the premature and replicative senescence of human fetal WI-38 cells and premature senescence of hepatocyte HepG2 cell line. Taken together, all these results demonstrate that RAC3 is an inhibitor of senescence whose downregulation in aged individuals could be probably a tumor suppressor mechanism, avoiding the clonal expansion of risky old cells having damaged DNA.
Assuntos
Proteínas rac de Ligação ao GTP/fisiologia , Envelhecimento , Animais , Proliferação de Células , Senescência Celular , Regulação para Baixo , Feminino , Expressão Gênica , Técnicas de Silenciamento de Genes , Células HEK293 , Células HeLa , Humanos , Peróxido de Hidrogênio/farmacologia , Ratos Wistar , Sirolimo/farmacologiaRESUMO
The p160 nuclear receptor co-activators represent a family of molecules, which are recruited by steroid nuclear receptors as well as other transcription factors that are overexpressed in several tumors. We investigated the role of one member of this family on the sensitivity of cells to apoptosis. We observed that overexpression of the RAC3 (receptor-associated co-activator-3) p160 co-activator inhibits hydrogen peroxide-induced cell death in human embryonic kidney 293 (HEK293) cells. The mechanism involves the activation of anti-apoptotic pathways mediated through enhanced nuclear factor kappa B (NF-kappaB) activity, inhibition of caspase-9 activation, diminished apoptotic-inducing factor (AIF) nuclear localization and a change in the activation pattern of several kinases, including an increase in both AKT and p38 kinase activities, and inhibition of ERK2. Moreover, RAC3 has been found associated with a protein complex containing AIF, Hsp90 and dynein, suggesting a role for the co-activator in the cytoplasmatic nuclear transport of these proteins associated with cytoskeleton. These results demonstrate that there are several molecular pathways that could be affected by their overexpression, including those not restricted to steroid regulation or the nuclear action of co-activators, which results in diminished sensitivity to apoptosis. Furthermore, this could represent one mechanism by which co-activators contribute to tumor development.
Assuntos
Apoptose , Citosol/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Transporte Ativo do Núcleo Celular , Apoptose/efeitos dos fármacos , Fator de Indução de Apoptose/metabolismo , Caspase 9/metabolismo , Linhagem Celular , Dineínas/metabolismo , Ativação Enzimática , Regulação da Expressão Gênica , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Peróxido de Hidrogênio/farmacologia , NF-kappa B/metabolismo , Ligação Proteica , Proteínas Quinases/metabolismo , Proteínas de Ligação a Tacrolimo/metabolismo , Proteínas rac de Ligação ao GTP/genéticaRESUMO
Breast tumors are usually classified according to their response to estrogens as hormone-dependent or -independent. In this work, we investigated the role of the proinflammatory cytokine TNF-alpha on the estrogen-receptor-positive T47D breast ductal tumor cells. We have found that TNF-alpha exerts a mitogenic effect, inducing cyclin D1 expression and activation of the transcription factor NF-kappaB. Importantly, activation of NF-kappaB was required for estrogen-induced proliferation and cyclin D1 expression. TNF-alpha enhanced the estrogen response by increasing the levels and availability of NF-kappaB. Chromatin immunoprecipitation analysis suggested that the action of estrogens is mediated by a protein complex that contains the activated estrogen receptor, the nuclear receptor coactivator RAC3 and a member of the NF-kappaB family. Finally, our results demonstrate that activation of this transcription factor could be one of the key signals for estrogen-mediated response.
Assuntos
Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/patologia , Proliferação de Células , NF-kappa B/fisiologia , Receptores de Estrogênio/fisiologia , Fator de Necrose Tumoral alfa/fisiologia , Animais , Imunoprecipitação da Cromatina , Ciclina D1/biossíntese , Estrogênios/fisiologia , Feminino , Humanos , Camundongos , Células Tumorais CultivadasRESUMO
We have previously shown that nuclear receptor coactivator overexpression significantly enhanced NF-kappaB activity in a dose response manner. We studied the mechanism by which TIF2 regulates NF-kappaB activity. We determined that: 1) the p38 specific inhibitor reduces 50% NF-kappaB transcriptional activity, even in cells that overexpress distinct TIF2 deletions; 2) there is a physical interaction between TIF2 and p38 and RelA determined through in vitro translated protein binding assays; 3) TIF2 is a p38 substrate; 4) there is a physical interaction between TIF2 and IKK in TNF-alpha 20 ng/ml stimulated or not HEK 293 cell protein extract, and IkappaB only in basal conditions, determined by binding pull down assays. This NF-kappaB complex regulates its activity and targets gene expression in a determined physiologic context depending on the coactivator complex content.
Demonstramos previamente que la sobreeexpresión de coactavadores de receptores nucleares aumenta la actividad NF-kB en forma de dosis depepndiente. Se estudió el mecanismo por el cual el coactavador TIF2 regula la actividad de NF-kB. Determinamos que: 1)el inhibidor específico de p38 disminuye al 50% la actividad transcripcional de NF-kB, aún en células que sobreexpresan distitntas deleciones de TIF2; 2) existe interacción físca directa de TIF2 con p38; y RelA determinada a trav[es de ensayos de unión con proteína traducida in vitro; 3) TIF2 ES SUSTRATO DE P38; 4) mediante ensayos de unión con extractos proteicos de células...
Assuntos
Humanos , Fatores de Transcrição/fisiologia , NF-kappa B/metabolismo , /metabolismo , Citoplasma/metabolismo , Ativação Enzimática , Núcleo Celular/metabolismo , Fosforilação , /antagonistas & inibidores , Ativação TranscricionalRESUMO
We have previously shown that nuclear receptor coactivator overexpression significantly enhanced NF-kappaB activity in a dose response manner. We studied the mechanism by which TIF2 regulates NF-kappaB activity. We determined that: 1) the p38 specific inhibitor reduces 50% NF-kappaB transcriptional activity, even in cells that overexpress distinct TIF2 deletions; 2) there is a physical interaction between TIF2 and p38 and RelA determined through in vitro translated protein binding assays; 3) TIF2 is a p38 substrate; 4) there is a physical interaction between TIF2 and IKK in TNF-alpha 20 ng/ml stimulated or not HEK 293 cell protein extract, and IkappaB only in basal conditions, determined by binding pull down assays. This NF-kappaB complex regulates its activity and targets gene expression in a determined physiologic context depending on the coactivator complex content.(AU)
Demonstramos previamente que la sobreeexpresión de coactavadores de receptores nucleares aumenta la actividad NF-kB en forma de dosis depepndiente. Se estudió el mecanismo por el cual el coactavador TIF2 regula la actividad de NF-kB. Determinamos que: 1)el inhibidor específico de p38 disminuye al 50% la actividad transcripcional de NF-kB, aún en células que sobreexpresan distitntas deleciones de TIF2; 2) existe interacción físca directa de TIF2 con p38; y RelA determinada a trav[es de ensayos de unión con proteína traducida in vitro; 3) TIF2 ES SUSTRATO DE P38; 4) mediante ensayos de unión con extractos proteicos de células...(AU)
Assuntos
Humanos , NF-kappa B/metabolismo , Fatores de Transcrição/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Ativação Enzimática , Coativador 2 de Receptor Nuclear , Fosforilação , Ativação Transcricional , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
Most cells are naturally resistant to TNF-alpha-induced cell death and become sensitized when NF-kappaB transactivation is blocked or in the presence of protein synthesis inhibitors that prevent the expression of anti-apoptotic genes. In this report we analyzed the role of osmotic stress on TNF-alpha-induced cell death. We found that it sensitizes the naturally resistant HeLa cells to TNF-alpha-induced apoptosis, with the involvement of an increase in the activity of several kinases, the inhibition of Bcl-2 expression, and a late increase on NF-kappaB activation. Cell death occurs regardless of the enhanced NF-kappaB activity, whose inhibition produces an increase in apoptosis. The inhibition of p38 kinase, also involved in NF-kappaB activation, significantly increases the effect of osmotic stress on TNF-alpha-induced cell death.
Assuntos
Apoptose/genética , Células Eucarióticas/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , NF-kappa B/metabolismo , Estresse Fisiológico/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Apoptose/efeitos dos fármacos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Células Eucarióticas/efeitos dos fármacos , Células HeLa , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteína Quinase 12 Ativada por Mitógeno , Proteína Quinase 8 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/efeitos dos fármacos , NF-kappa B/genética , Pressão Osmótica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Solução Salina Hipertônica/farmacologia , Estresse Fisiológico/genética , Sulfassalazina/farmacologia , Fator de Transcrição RelA , Fator de Necrose Tumoral alfa/farmacologia , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
It has been shown that the molecular mechanism by which cytokines and glucocorticoids mutually antagonize their functions involves a mutual glucocorticoid receptor (GR)/nuclear factor-kappa B (NF-kappa B) transrepression. Here we report a role for the nuclear receptor coactivator RAC3, in modulating NF-kappa B transactivation. We found that RAC3 functions as a coactivator by binding to the active form of NF-kappa B and that overexpression of RAC3 restores GR-dependent transcription neglecting GR/NF-kappa B transrepression. The competition between GR and NF-kappa B for binding to RAC3 may represent a general mechanism by which both transcription factors mutually antagonize their activity.
Assuntos
NF-kappa B/metabolismo , Receptores de Glucocorticoides/metabolismo , Transativadores/farmacologia , Fatores de Transcrição , Ligação Competitiva , Glucocorticoides/farmacologia , Células HeLa , Humanos , Técnicas de Imunoadsorção , NF-kappa B/análise , Coativador 3 de Receptor Nuclear , Elementos de Resposta , Transativadores/genética , Transativadores/metabolismo , Fator de Transcrição RelA , Transcrição Gênica , Transfecção , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
We have previously shown that TNF-alpha and IL-1 may enhance the glucocorticoid (GC)-induced transcriptional activity of glucocorticoid receptor (GR) in different cell lines transfected with a reporter plasmid carrying GC response elements (GRE). In TNF-alpha and GC target cell lines, it was found that: 1) TNF-alpha enhanced GR number in L-929 cells, and 2) by transfection of these cells with a reporter plasmid carrying the GR promoter, that TNF-alpha-induced increase in GR is at the transcriptional level, 3) by electrophoretic mobility shift assay, using nuclear extracts of TNF-alpha (0.02 ng/ml) or TNF-alpha plus DEX (10 nM) stimulated L-929 cells, that cytokines can increase the binding of GR to GRE (45 min, 1.8 x), while the TNF-alpha-induced NFkB factor expression was not affected by GC. 4) As a biological correlate of this mechanism, priming of L-929 cells with TNF-alpha significantly increased (p < 0.001) the sensitivity to GC inhibition of TNF-alpha-induced apoptosis. The organism protects itself from an immune overreaction, not only via the HPA axis induction and an increase in GC by cytokines, but also enhancing the sensitivity to GC: by an increase in GR number, the binding to GRE and the transcription of GC target genes (e.g. TNF-alpha-induced apoptosis inhibitory genes). These mechanisms contribute to enhance the immunosuppressive and antiinflammatory GC activity, in order to maintain homeostasis.
Assuntos
Citocinas/fisiologia , Glucocorticoides/fisiologia , Receptores de Glucocorticoides/fisiologia , Apoptose , Homeostase , Interleucina-1/fisiologia , Transcrição Gênica , Transfecção , Fator de Necrose Tumoral alfa/fisiologiaRESUMO
We observed that Sendai virus preinduction of peripheral blood mononuclear cells and subsequent mitogenic stimulation resulted in: (i) Superproduction of interferon-gamma, (IFN-gamma) (ii) an increase in interleukin-2 (IL-2) synthesis that correlates with DNA synthesis when stimulated with phytohemagglutinin (PHA) or pokeweed mitogen (PWM) after treatment with the Sendai virus, while stimulation with Protein A from Staphylococcus aureus was not affected, and (iii) enhanced tumor necrosis factor-alpha (TNF-alpha) production in response to bacterial lipopolysaccharide (LPS). Treatment of monocyte cultures with LPS and cycloheximide or actinomycin-D inhibited the superinduction phenomenon. When cycloheximide was added at the viral induction time, the inhibition of TNF-alpha superproduction and DNA synthesis was still observed. These results suggest that Sendai virus lymphocyte superinduction is specific for a particular stimulatory pathway, not dependent on mRNA accumulation, and probably mediated by induction of an activating protein.