RESUMEN
AIMS/HYPOTHESIS: Pro-inflammatory cytokines involved in the pathogenesis of type 1 diabetes deplete endoplasmic reticulum (ER) Ca2+ stores, leading to ER-stress and beta cell apoptosis. However, the cytokine-induced ER-stress response in beta cells is atypical and characterised by induction of the pro-apoptotic PKR-like ER kinase (PERK)-C/EBP homologous protein (CHOP) branch of the unfolded protein response, but defective X-box binding protein 1 (XBP1) splicing and activating transcription factor 6 activation. The purpose of this study was to overexpress spliced/active Xbp1 (XBP1s) to increase beta cell resistance to cytokine-induced ER-stress and apoptosis. METHODS: Xbp1s was overexpressed using adenoviruses and knocked down using small interference RNA in rat islet cells. In selected experiments, Xbp1 was also knocked down in FACS-purified rat beta cells and rat fibroblasts. Expression and production of XBP1s and key downstream genes and proteins was measured and beta cell function and viability were evaluated. RESULTS: Adenoviral-mediated overproduction of Xbp1s resulted in increased XBP1 activity and induction of several XBP1s target genes. Surprisingly, XBP1s overexpression impaired glucose-stimulated insulin secretion and increased beta cell apoptosis, whereas it protected fibroblasts against cell death induced by ER-stress. mRNA expression of Pdx1 and Mafa was inhibited in cells overproducing XBP1s, leading to decreased insulin expression. XBP1s knockdown partially restored cytokine/ER-stress-driven insulin and Pdx1 inhibition but had no effect on cytokine-induced ER-stress and apoptosis. CONCLUSIONS/INTERPRETATION: XBP1 has a distinct inhibitory role in beta cell as compared with other cell types. Prolonged XBP1s production hampers beta cell function via inhibition of insulin, Pdx1 and Mafa expression, eventually leading to beta cell apoptosis.
Asunto(s)
Apoptosis/fisiología , Proteínas de Unión al ADN/metabolismo , Retículo Endoplásmico/metabolismo , Células Secretoras de Insulina/metabolismo , Factores de Transcripción/metabolismo , Análisis de Varianza , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Recuento de Células , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Proteínas de Unión al ADN/genética , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/genética , Técnica del Anticuerpo Fluorescente , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Indoles/farmacología , Insulina/genética , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/efectos de los fármacos , Interferón gamma/farmacología , Interleucina-8/farmacología , Factores de Transcripción Maf/genética , Factores de Transcripción Maf/metabolismo , Masculino , Interferencia de ARN , ARN Interferente Pequeño , Ratas , Ratas Wistar , Factores de Transcripción del Factor Regulador X , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/genética , Transfección , Proteína 1 de Unión a la X-BoxRESUMEN
AIMS/HYPOTHESIS: IL-1beta and TNF-alpha contribute to pancreatic beta cell death in type 1 diabetes. Both cytokines activate the transcription factor nuclear factor-kappaB (NF-kappaB), but recent observations suggest that NF-kappaB blockade prevents IL-1beta + IFN-gamma- but not TNF-alpha + IFN-gamma-induced beta cell apoptosis. The aim of the present study was to compare the effects of IL-1beta and TNF-alpha on cell death and the pattern of NF-kappaB activation and global gene expression in beta cells. METHODS: Cell viability was measured after exposure to IL-1beta or to TNF-alpha alone or in combination with IFN-gamma, and blockade of NF-kappaB activation or protein synthesis. INS-1E cells exposed to IL-1beta or TNF-alpha in time course experiments were used for IkappaB kinase (IKK) activation assay, detection of p65 NF-kappaB by immunocytochemistry, real-time RT-PCR and microarray analysis. RESULTS: Blocking NF-kappaB activation protected beta cells against IL-1beta + IFNgamma- or TNFalpha + IFNgamma-induced apoptosis. Blocking de novo protein synthesis did not increase TNF-alpha- or IL-1beta-induced beta cell death, in line with the observations that cytokines induced the expression of the anti-apoptotic genes A20, Iap-2 and Xiap to a similar extent. Microarray analysis of INS-1E cells treated with IL-1beta or TNF-alpha showed similar patterns of gene expression. IL-1beta, however, induced a higher rate of expression of NF-kappaB target genes putatively involved in beta cell dysfunction and death and a stronger activation of the IKK complex, leading to an earlier translocation of NF-kappaB to the nucleus. CONCLUSIONS/INTERPRETATION: NF-kappaB activation in beta cells has a pro-apoptotic role following exposure not only to IL-1beta but also to TNF-alpha. The more marked beta cell death induced by IL-1beta is explained at least in part by higher intensity NF-kappaB activation, leading to increased transcription of key target genes.
Asunto(s)
Apoptosis/fisiología , Diabetes Mellitus Tipo 1/fisiopatología , Células Secretoras de Insulina/fisiología , Interleucina-1beta/metabolismo , FN-kappa B/genética , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Apoptosis/efectos de los fármacos , Núcleo Celular/metabolismo , Células Cultivadas , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/patología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/inmunología , Quinasa I-kappa B/metabolismo , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/efectos de los fármacos , Interleucina-1beta/farmacología , FN-kappa B/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Ratas , Ratas Wistar , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
AIMS/HYPOTHESIS: Pancreatic beta cells respond to endoplasmic reticulum (ER) stress by activating the unfolded protein response. If the stress is prolonged, or the adaptive response fails, apoptosis is triggered. We used a 'homemade' microarray specifically designed for the study of beta cell apoptosis (the APOCHIP) to uncover mechanisms regulating beta cell responses to ER stress. MATERIALS AND METHODS: A time course viability and microarray analysis was performed in insulin-producing INS-1E cells exposed to the reversible ER stress inducer cyclopiazonic acid (CPA). Modification of selected genes was confirmed by real-time RT-PCR, and the observed inhibition of expression of the insulin-1 (Ins1) and insulin-2 (Ins2) genes was further characterised in primary beta cells exposed to a diverse range of agents that induce ER stress. RESULTS: CPA-induced ER stress modified the expression of 183 genes at one or more of the time points studied. The expression of most of these genes returned to control levels after a 3 h recovery period following CPA removal, with all cells surviving. Two groups of genes were particularly affected by CPA, namely, those related to cellular responses to ER stress, which were mostly upregulated, and those related to differentiated beta cell functions, which were downregulated. Levels of Ins1 and Ins2 mRNAs were severely decreased in response to CPA treatment as a result of degradation, and there was a concomitant increase in the level of IRE1 activation. CONCLUSIONS/INTERPRETATION: In this study we provide the first global analysis of beta cell molecular responses to a severe ER stress, and identify the early degradation of mRNA transcripts of the insulin genes as an important component of this response.
Asunto(s)
Retículo Endoplásmico/fisiología , Perfilación de la Expresión Génica , Células Secretoras de Insulina/fisiología , Insulina/genética , ARN Mensajero/metabolismo , Línea Celular , Supervivencia Celular , Células Cultivadas , Regulación de la Expresión Génica , Humanos , Indoles/farmacología , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/efectos de los fármacos , Cinética , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Estrés MecánicoRESUMEN
AIMS/HYPOTHESIS: A tight control of endoplasmic reticulum homeostasis is crucial for beta cell function and survival. We recently described that IL-1beta plus IFN-gamma deplete endoplasmic reticulum Ca2+ stores in beta cells, leading to endoplasmic reticulum stress and apoptosis. IL-1beta alone induced endoplasmic reticulum stress but failed to induce beta cell death, while IFN-gamma alone neither caused endoplasmic reticulum stress nor induced beta cell death. This suggests that IFN-gamma aggravates endoplasmic reticulum stress induced by IL-1beta, eventually triggering apoptosis. Here we tested this hypothesis and the mechanisms involved, by investigating the effects of IFN-gamma on endoplasmic reticulum-stress-induced beta cell apoptosis caused by a specific blocker of the sarcoendoplasmic-reticulum pump Ca2+-ATPase (SERCA). MATERIALS AND METHODS: INS-1E cells or beta cells were pretreated with IFN-gamma and then exposed to the SERCA blocker cyclopiazonic acid (CPA) for induction of endoplasmic reticulum stress. Cell death was evaluated by Hoechst 342 and propidium iodide staining. Expression of genes related to endoplasmic reticulum stress was determined by real-time RT-PCR, while activation of the endoplasmic reticulum stress response was determined by analysing X-box binding protein-1 (Xbp1) splicing and using a reporter construct containing five copies of the unfolded protein response element (UPRE). RESULTS: CPA induces endoplasmic reticulum stress and apoptosis in insulin-producing cells. Pretreatment with IFN-gamma decreased the basal level of spliced Xbp1 mRNA, the basal and CPA-induced activity of the UPRE reporter, and the mRNA expression of several endoplasmic reticulum chaperones (Bip, Grp94 and Orp 150) and Sec61a. Furthermore, CPA-induced Chop mRNA expression and beta cell apoptosis were potentiated in cells that had been pretreated with IFN-gamma. CONCLUSIONS/INTERPRETATION: CPA-induced endoplasmic reticulum stress and apoptosis is enhanced in IFN-gamma-treated beta cells. These effects are mediated via downregulation of the expression of genes involved in beta cell defence against endoplasmic reticulum stress.