RESUMEN
Diets rich in saturated fats may contribute to the loss of pancreatic ß-cells in type 2 diabetes. JunB, a member of the activating protein 1 (AP-1) transcription factor family, promotes ß-cell survival and mediates part of the beneficial effects of GLP-1 agonists. In this study we interrogated the molecular mechanisms involved in JunB-mediated ß-cell protection from lipotoxicity. The saturated fatty acid palmitate decreased JunB expression, and this loss may contribute to ß-cell apoptosis, as overexpression of JunB protected cells from lipotoxicity. Array analysis of JunB-deficient ß-cells identified a gene expression signature of a downregulated endoplasmic reticulum (ER) stress response and inhibited AKT signaling. JunB stimulates XBP1 expression via the transcription factor c/EBPδ during ER stress, and forced expression of XBP1s rescued the viability of JunB-deficient cells, constituting an important antiapoptotic mechanism. JunB silencing inhibited AKT activation and activated the proapoptotic Bcl-2 protein BAD via its dephosphorylation. BAD knockdown reversed lipotoxic ß-cell death potentiated by JunB siRNA. Interestingly, XBP1s links JunB and AKT signaling as XBP1 knockdown also reduced AKT phosphorylation. GLP-1 agonists induced cAMP-dependent AKT phosphorylation leading to ß-cell protection against palmitate-induced apoptosis. JunB and XBP1 knockdown or IRE1 inhibition decreased AKT activation by cAMP, leading to ß-cell apoptosis. In conclusion, JunB modulates the ß-cell ER stress response and AKT signaling via the induction of XBP1s. The activation of the JunB gene network and the crosstalk between the ER stress and AKT pathway constitute a crucial defense mechanism by which GLP-1 agonists protect against lipotoxic ß-cell death. These findings elucidate novel ß-cell-protective signal transduction in type 2 diabetes.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Células Secretoras de Insulina/metabolismo , Factores de Transcripción/metabolismo , Animales , Proteínas de Unión al ADN/genética , Diabetes Mellitus Tipo 2/enzimología , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Femenino , Humanos , Células Secretoras de Insulina/enzimología , Masculino , Persona de Mediana Edad , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Factores de Transcripción del Factor Regulador X , Transducción de Señal , Factores de Transcripción/genética , Proteína 1 de Unión a la X-BoxRESUMEN
Apoptosis of pancreatic beta cells is a feature of type 2 diabetes and its prevention may have therapeutic benefit. High glucose concentrations induce apoptosis of islet cells, and this requires the proapoptotic Bcl-2 homology domain 3 (BH3)-only proteins Bim and Puma. We studied the stress pathways induced by glucotoxicity in beta cells that result in apoptosis. High concentrations of glucose or ribose increased expression of the transcription factor CHOP (C/EBP homologous protein) but not endoplasmic reticulum (ER) chaperones, indicating activation of proapoptotic ER stress signaling. Inhibition of ER stress prevented ribose-induced upregulation of Chop and Puma mRNA, and partially protected islets from glucotoxicity. Loss of Bim or Puma partially protected islets from the canonical ER stressor thapsigargin. The antioxidant N-acetyl-cysteine also partially protected islets from glucotoxicity. Islets deficient in both Bim and Puma, but not Bim or Puma alone, were significantly protected from killing induced by the mitochondrial reactive oxygen species donor rotenone. Our data demonstrate that high concentrations of glucose induce ER and oxidative stress, which causes cell death mediated by Bim and Puma. We observed significantly higher Bim and Puma mRNA in islets of human donors with type 2 diabetes. This indicates that inhibition of Bim and Puma, or their inducers, may prevent beta-cell destruction in type 2 diabetes.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis , Estrés del Retículo Endoplásmico , Retículo Endoplásmico/metabolismo , Glucosa/metabolismo , Islotes Pancreáticos/metabolismo , Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Estrés Oxidativo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/deficiencia , Proteínas Reguladoras de la Apoptosis/genética , Proteína 11 Similar a Bcl2 , Línea Celular , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/patología , Estrés del Retículo Endoplásmico/efectos de los fármacos , Humanos , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patología , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/patología , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/efectos de los fármacos , Mitocondrias/patología , Oxidantes/farmacología , Estrés Oxidativo/efectos de los fármacos , Proteínas Proto-Oncogénicas/deficiencia , Proteínas Proto-Oncogénicas/genética , ARN Mensajero/metabolismo , Ribosa/metabolismo , Técnicas de Cultivo de Tejidos , Factor de Transcripción CHOP/deficiencia , Factor de Transcripción CHOP/genética , Proteínas Supresoras de Tumor/deficiencia , Proteínas Supresoras de Tumor/genéticaRESUMEN
The cytokines interleukin (IL)-1ß and tumor necrosis factor (TNF)-α induce ß-cell death in type 1 diabetes via NF-κB activation. IL-1ß induces a more marked NF-κB activation than TNF-α, with higher expression of genes involved in ß-cell dysfunction and death. We show here a differential usage of the IKK complex by IL-1ß and TNF-α in ß-cells. While TNF-α uses IKK complexes containing both IKKα and IKKß, IL-1ß induces complexes with IKKα only; this effect is achieved by induction of IKKß degradation via the proteasome. Both IKKγ and activation of the TRAF6-TAK1-JNK pathway are involved in IL-1ß-induced IKKß degradation.
Asunto(s)
Quinasa I-kappa B/metabolismo , Células Secretoras de Insulina/metabolismo , Interleucina-1beta/metabolismo , FN-kappa B/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Transducción de Señal , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Línea Celular , Células Cultivadas , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Silenciador del Gen , Humanos , Quinasa I-kappa B/antagonistas & inhibidores , Quinasa I-kappa B/genética , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/efectos de los fármacos , Ratones , Terapia Molecular Dirigida , Inhibidores de Proteasas/farmacología , Inhibidores de Proteasoma , Inhibidores de Proteínas Quinasas/farmacología , Subunidades de Proteína/antagonistas & inhibidores , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteolisis/efectos de los fármacos , Ratas , Ratas Wistar , Proteínas Recombinantes/metabolismo , Transducción de Señal/efectos de los fármacos , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Destruction of insulin-producing pancreatic ß-cells by local autoimmune inflammation is a hallmark of type 1 diabetes. Histochemical analysis of pancreases from non-obese diabetic mice indicated activation of the transcription factor JunB/AP-1 (activator protein-1) after autoimmune infiltration of the islets. In vitro studies demonstrated that the cytokines tumor necrosis factor (TNF)-α and interferon (IFN)-γ induce JunB expression as a protective mechanism against apoptosis in both human and rodent ß-cells. The gene network affected was studied by microarray analysis showing that JunB regulates nearly 20% of the cytokine-modified ß-cell genes, including the transcription factor ATF3. Direct transcriptional induction of ATF3 by JunB is a key event for ß-cell survival after TNF-α+IFN-γ treatment. Moreover, pharmacological upregulation of JunB/ATF3 via increased cAMP protected rodent primary ß-cells and human islet cells against pro-inflammatory mediators. These results were confirmed in genetically modified islets derived from Ubi-JunB transgenic mice. Our findings identify ATF3 as a novel downstream target of JunB in the survival mechanism of ß-cells under inflammatory stress.
Asunto(s)
Factor de Transcripción Activador 3/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Inflamación/metabolismo , Células Secretoras de Insulina/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Animales , Técnicas de Silenciamiento del Gen , Humanos , Ratones , Ratones Endogámicos NOD , Ratones Transgénicos , Proteínas Proto-Oncogénicas c-jun/genética , Transducción de Señal , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Chronic inflammation and pro-inflammatory cytokines are important mediators of pancreatic beta-cell destruction in type 1 diabetes (T1D). We presently show that the cytokines IL-1beta+IFN-gamma and different ER stressors activate the Bcl-2 homology 3 (BH3)-only member death protein 5 (DP5)/harakiri (Hrk) resulting in beta-cell apoptosis. Chemical ER stress-induced DP5 upregulation is JNK/c-Jun-dependent. DP5 activation by cytokines also involves JNK/c-Jun phosphorylation and is antagonized by JunB. Interestingly, cytokine-inducted DP5 expression precedes ER stress: mitochondrial release of cytochrome c and ER stress are actually a consequence of enhanced DP5 activation by cytokine-mediated nitric oxide formation. Our findings show that DP5 is central for beta-cell apoptosis after different stimuli, and that it can act up- and downstream of ER stress. These observations contribute to solve two important questions, namely the mechanism by which IL-1beta+IFN-gamma induce beta-cell death and the nature of the downstream signals by which ER stress 'convinces' beta-cells to trigger apoptosis.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis , Retículo Endoplásmico/metabolismo , Células Secretoras de Insulina/citología , Interferón gamma/farmacología , Interleucina-1beta/farmacología , Neuropéptidos/metabolismo , Animales , Citocromos c/metabolismo , Células Secretoras de Insulina/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , ARN Interferente Pequeño/metabolismo , Ratas , Ratas Wistar , Transducción de Señal , Regulación hacia ArribaRESUMEN
The activating protein-1 transcription factor, in particular the Jun proteins play critical roles in the regulation of cell proliferation and tumor progression. To study the potential clinical relevance of interfering with JunB expression, we generated retroviruses expressing short hairpin RNA. Reduction of JunB levels causes increased proliferation and tumorigenicity in wild-type murine fibroblasts, whereas in c-Jun knockout cells p53-independent cell cycle arrest and apoptosis are induced. Using melanoma-derived B16-F10 cancer cells the combination of JunB knockdown and c-Jun/JNK inactivation leads to cell cycle arrest and apoptosis-inducing factor-dependent apoptosis. Furthermore, the combined treatment extends survival of mice inoculated with the tumor cells. These results indicate that in the absence of c-Jun, JunB can act as a tumor promoter and inactivation of both, c-Jun and JunB, could provide a valuable strategy for antitumor intervention.
Asunto(s)
Proliferación Celular , Proteínas Proto-Oncogénicas c-jun/metabolismo , Interferencia de ARN , Animales , Apoptosis , Fibroblastos , Humanos , Melanoma/patología , Ratones , Neoplasias/terapia , Retroviridae , Neoplasias Cutáneas/patología , Células Tumorales CultivadasRESUMEN
Hec1 (highly expressed in cancer) plays an important role in chromosome segregation by interacting with a subset of checkpoint proteins that survey proper chromosome alignment and bipolar spindle attachment. In order to disrupt mitotic progression of tumor cell lines, we have used retrovirus and adenovirus vectors that inhibit Hec1 synthesis. Vector-expressed short hairpin RNAs (shRNAs) caused very efficient depletion of the target protein, cellular arrest and considerable mitotic catastrophe induction 96 h post infection in human cervix-adenocarcinoma (HeLa) and glioblastoma (U-373-MG) cell lines. Furthermore, adenocarcinomas induced in the flanks of nude mice show significant reduction in size compared with control when treated with either Hec1-shRNA retroviruses or adenoviruses. These results indicate that depletion of Hec1 could be used as a new strategy to block the dividing cell, and therefore against cancer.