RESUMEN
Telomerase is intimately associated with stem cells and cancer, because it catalytically elongates telomeres-nucleoprotein caps that protect chromosome ends1. Overexpression of telomerase reverse transcriptase (TERT) enhances the proliferation of cells in a telomere-independent manner2-8, but so far, loss-of-function studies have provided no evidence that TERT has a direct role in stem cell function. In many tissues, homeostasis is shaped by stem cell competition, a process in which stem cells compete on the basis of inherent fitness. Here we show that conditional deletion of Tert in the spermatogonial stem cell (SSC)-containing population in mice markedly impairs competitive clone formation. Using lineage tracing from the Tert locus, we find that TERT-expressing SSCs yield long-lived clones, but that clonal inactivation of TERT promotes stem cell differentiation and a genome-wide reduction in open chromatin. This role for TERT in competitive clone formation occurs independently of both its reverse transcriptase activity and the canonical telomerase complex. Inactivation of TERT causes reduced activity of the MYC oncogene, and transgenic expression of MYC in the TERT-deleted pool of SSCs efficiently rescues clone formation. Together, these data reveal a catalytic-activity-independent requirement for TERT in enhancing stem cell competition, uncover a genetic connection between TERT and MYC and suggest that a selective advantage for stem cells with high levels of TERT contributes to telomere elongation in the male germline during homeostasis and ageing.
Asunto(s)
Competencia Celular , Células Clonales , Células Madre , Telomerasa , Animales , Masculino , Ratones , Diferenciación Celular , Linaje de la Célula , Cromatina/metabolismo , Cromatina/genética , Células Clonales/citología , Células Clonales/enzimología , Células Clonales/metabolismo , Eliminación de Gen , Genes myc , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Espermatogonias/citología , Espermatogonias/metabolismo , Células Madre/citología , Células Madre/enzimología , Células Madre/metabolismo , Telomerasa/deficiencia , Telomerasa/genética , Telomerasa/metabolismo , Transcripción Reversa , Biocatálisis , Homeostasis , EnvejecimientoRESUMEN
Telomere length control is critical for cellular lifespan and tumor suppression. Telomerase is transiently activated in the inner cell mass of the developing blastocyst to reset telomere reserves. Its silencing upon differentiation leads to gradual telomere shortening in somatic cells. Here, we report that transcriptional regulation through cis-regulatory elements only partially accounts for telomerase activation in pluripotent cells. Instead, developmental control of telomerase is primarily driven by an alternative splicing event, centered around hTERT exon 2. Skipping of exon 2 triggers hTERT mRNA decay in differentiated cells, and conversely, its retention promotes telomerase accumulation in pluripotent cells. We identify SON as a regulator of exon 2 alternative splicing and report a patient carrying a SON mutation and suffering from insufficient telomerase and short telomeres. In summary, our study highlights a critical role for hTERT alternative splicing in the developmental regulation of telomerase and implicates defective splicing in telomere biology disorders.
Asunto(s)
Empalme Alternativo , Proteínas de Unión al ADN/genética , Elementos de Facilitación Genéticos , Antígenos de Histocompatibilidad Menor/genética , Telomerasa/genética , Homeostasis del Telómero , Telómero/metabolismo , Blastocisto/metabolismo , Blastocisto/patología , Diferenciación Celular , Preescolar , Proteínas de Unión al ADN/deficiencia , Femenino , Células Madre Embrionarias Humanas/metabolismo , Células Madre Embrionarias Humanas/patología , Humanos , Linaje , Células Madre Pluripotentes/metabolismo , Células Madre Pluripotentes/patología , Cultivo Primario de Células , Estabilidad del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Telomerasa/deficiencia , Telómero/patologíaRESUMEN
Alternative lengthening of telomeres (ALT) in human cells is a conserved process that is often activated in telomerase-deficient human cancers. This process exploits components of the recombination machinery to extend telomere ends, thus allowing for increased proliferative potential. Human MUS81 (Mus81 in Saccharomyces cerevisiae) is the catalytic subunit of structure-selective endonucleases involved in recombination and has been implicated in the ALT mechanism. However, it is unclear whether MUS81 activity at the telomere is specific to ALT cells or if it is required for more general aspects of telomere stability. In this study, we use S. cerevisiae to evaluate the contribution of the conserved Mus81-Mms4 endonuclease in telomerase-deficient yeast cells that maintain their telomeres by mechanisms akin to human ALT. Similar to human cells, we find that yeast Mus81 readily localizes to telomeres and its activity is important for viability after initial loss of telomerase. Interestingly, our analysis reveals that yeast Mus81 is not required for the survival of cells undergoing recombination-mediated telomere lengthening, i.e. for ALT itself. Rather we infer from genetic analysis that Mus81-Mms4 facilitates telomere replication during times of telomere instability. Furthermore, combining mus81 mutants with mutants of a yeast telomere replication factor, Rrm3, reveals that the two proteins function in parallel to promote normal growth during times of telomere stress. Combined with previous reports, our data can be interpreted in a consistent model in which both yeast and human MUS81-dependent nucleases participate in the recovery of stalled replication forks within telomeric DNA. Furthermore, this process becomes crucial under conditions of additional replication stress, such as telomere replication in telomerase-deficient cells.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Endonucleasas/metabolismo , Endonucleasas de ADN Solapado/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Telomerasa/deficiencia , Replicación del ADN , Proteínas de Unión al ADN/genética , Endonucleasas/genética , Endonucleasas de ADN Solapado/genética , Viabilidad Microbiana , Recombinación Genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Telómero/metabolismo , Homeostasis del TelómeroRESUMEN
Ventilation with gases containing enhanced fractions of oxygen is the cornerstone of therapy for patients with hypoxia and acute respiratory distress syndrome. Yet, hyperoxia treatment increases free reactive oxygen species (ROS)-induced lung injury, which is reported to disrupt autophagy/mitophagy. Altered extranuclear activity of the catalytic subunit of telomerase, telomerase reverse transcriptase (TERT), plays a protective role in ROS injury and autophagy in the systemic and coronary endothelium. We investigated interactions between autophagy/mitophagy and TERT that contribute to mitochondrial dysfunction and pulmonary injury in cultured rat lung microvascular endothelial cells (RLMVECs) exposed in vitro, and rat lungs exposed in vivo to hyperoxia for 48 h. Hyperoxia-induced mitochondrial damage in rat lungs [TOMM20, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)], which was paralleled by increased markers of inflammation [myeloperoxidase (MPO), IL-1ß, TLR9], impaired autophagy signaling (Beclin-1, LC3B-II/1, and p62), and decreased the expression of TERT. Mitochondrial-specific autophagy (mitophagy) was not altered, as hyperoxia increased expression of Pink1 but not Parkin. Hyperoxia-induced mitochondrial damage (TOMM20) was more pronounced in rats that lack the catalytic subunit of TERT and resulted in a reduction in cellular proliferation rather than cell death in RLMVECs. Activation of TERT or autophagy individually offset mitochondrial damage (MTT). Combined activation/inhibition failed to alleviate hyperoxic-induced mitochondrial damage in vitro, whereas activation of autophagy in vivo decreased mitochondrial damage (MTT) in both wild type (WT) and rats lacking TERT. Functionally, activation of either TERT or autophagy preserved transendothelial membrane resistance. Altogether, these observations show that activation of autophagy/mitophagy and/or TERT mitigate loss of mitochondrial function and barrier integrity in hyperoxia.NEW & NOTEWORTHY In cultured pulmonary artery endothelial cells and in lungs exposed in vivo to hyperoxia, autophagy is activated, but clearance of autophagosomes is impaired in a manner that suggests cross talk between TERT and autophagy. Stimulation of autophagy prevents hyperoxia-induced decreases in mitochondrial metabolism and sustains monolayer resistance. Hyperoxia increases mitochondrial outer membrane (TOMM20) protein, decreases mitochondrial function, and reduces cellular proliferation without increasing cell death.
Asunto(s)
Células Endoteliales/enzimología , Hiperoxia/complicaciones , Lesión Pulmonar/enzimología , Pulmón/irrigación sanguínea , Microvasos/enzimología , Mitocondrias/enzimología , Mitofagia , Telomerasa/metabolismo , Animales , Proteínas Relacionadas con la Autofagia/metabolismo , Permeabilidad Capilar , Células Cultivadas , Modelos Animales de Enfermedad , Células Endoteliales/patología , Femenino , Técnicas de Inactivación de Genes , Mediadores de Inflamación/metabolismo , Lesión Pulmonar/etiología , Lesión Pulmonar/genética , Lesión Pulmonar/patología , Masculino , Proteínas de Transporte de Membrana/metabolismo , Microvasos/patología , Mitocondrias/genética , Mitocondrias/patología , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Ratas Sprague-Dawley , Ratas Transgénicas , Receptores de Superficie Celular/metabolismo , Telomerasa/deficiencia , Telomerasa/genética , Receptor Toll-Like 4/metabolismo , Receptor Toll-Like 9/metabolismoRESUMEN
Mutations in the genes encoding telomerase reverse transcriptase (TERT) and telomerase's RNA components as well as shortened telomeres are risk factors for idiopathic pulmonary fibrosis, where repetitive injury to the alveolar epithelium is considered a key factor in pathogenesis. Given the importance of TERT in stem cells, we hypothesized that TERT plays an important role in epithelial repair and that its deficiency results in exacerbation of fibrosis by impairing this repair/regenerative process. To evaluate the role of TERT in epithelial cells, we generated type II alveolar epithelial cell (AECII)-specific TERT conditional knockout (SPC-Tert cKO) mice by crossing floxed Tert mice with inducible SPC-driven Cre mice. SPC-Tert cKO mice did not develop pulmonary fibrosis spontaneously up to 9 months of TERT deficiency. However, upon bleomycin treatment, they exhibited enhanced lung injury, inflammation, and fibrosis compared with control mice, accompanied by increased pro-fibrogenic cytokine expression but without a significant effect on AECII telomere length. Moreover, selective TERT deficiency in AECII diminished their proliferation and induced cellular senescence. These findings suggest that AECII-specific TERT deficiency enhances pulmonary fibrosis by heightening susceptibility to bleomycin-induced epithelial injury and diminishing epithelial regenerative capacity because of increased cellular senescence. We confirmed evidence for increased AECII senescence in idiopathic pulmonary fibrosis lungs, suggesting potential clinical relevance of the findings from our animal model. Our results suggest that TERT has a protective role in AECII, unlike its pro-fibrotic activity, observed previously in fibroblasts, indicating that TERT's role in pulmonary fibrosis is cell type-specific.
Asunto(s)
Senescencia Celular , Fibrosis Pulmonar/etiología , Telomerasa/genética , Células Epiteliales Alveolares/citología , Células Epiteliales Alveolares/metabolismo , Animales , Bleomicina/farmacología , Proliferación Celular , Células Cultivadas , Senescencia Celular/efectos de los fármacos , Citocinas/metabolismo , Modelos Animales de Enfermedad , Lesión Pulmonar/etiología , Lesión Pulmonar/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fibrosis Pulmonar/metabolismo , Linfocitos T/citología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Telomerasa/deficiencia , Telomerasa/metabolismo , Telómero/metabolismo , Acortamiento del TelómeroRESUMEN
PURPOSE: The acquisition of pathogenic variants in the TERT promoter (TERTp) region is a mechanism of tumorigenesis. In nonmalignant diseases, TERTp variants have been reported only in patients with idiopathic pulmonary fibrosis (IPF) due to germline variants in telomere biology genes. METHODS: We screened patients with a broad spectrum of telomeropathies (n = 136), their relatives (n = 52), and controls (n = 195) for TERTp variants using a customized massively parallel amplicon-based sequencing assay. RESULTS: Pathogenic -124 and -146 TERTp variants were identified in nine (7%) unrelated patients diagnosed with IPF (28%) or moderate aplastic anemia (4.6%); five of them also presented cirrhosis. Five (10%) relatives were also found with these variants, all harboring a pathogenic germline variant in telomere biology genes. TERTp clone selection did not associate with peripheral blood counts, telomere length, and response to danazol treatment. However, it was specific for patients with telomeropathies, more frequently co-occurring with TERT germline variants and associated with aging. CONCLUSION: We extend the spectrum of nonmalignant diseases associated with pathogenic TERTp variants to marrow failure and liver disease due to inherited telomerase deficiency. Specificity of pathogenic TERTp variants for telomerase dysfunction may help to assess the pathogenicity of unclear constitutional variants in the telomere diseases.
Asunto(s)
Regiones Promotoras Genéticas , Telomerasa/genética , Telómero/patología , Adolescente , Adulto , Anciano , Anemia Aplásica/genética , Recuento de Células Sanguíneas , Enfermedades de la Médula Ósea/genética , Niño , Preescolar , Estudios de Cohortes , Femenino , Humanos , Fibrosis Pulmonar Idiopática/genética , Hepatopatías/genética , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Telomerasa/deficiencia , Adulto JovenRESUMEN
OBJECTIVE: Atherosclerosis is an age-related disease characterized by systemic oxidative stress and low-grade inflammation. The role of telomerase and telomere length in atherogenesis remains contentious. Short telomeres of peripheral leukocytes are predictive for coronary artery disease. Conversely, attenuated telomerase has been demonstrated to be protective for atherosclerosis. Hence, a potential causative role of telomerase in atherogenesis is critically debated. APPROACH AND RESULTS: In this study, we used multiple mouse models to investigate the regulation of telomerase under oxidative stress as well as its impact on atherogenesis in vitro and in vivo. Using primary lymphocytes and myeloid cell cultures, we demonstrate that cultivation under hyperoxic conditions induced oxidative stress resulting in chronic activation of CD4+ cells and significantly reduced CD4+ T-cell proliferation. The latter was telomerase dependent because oxidative stress had no effect on the proliferation of primary lymphocytes isolated from telomerase knockout mice. In contrast, myeloid cell proliferation was unaffected by oxidative stress nor reliant on telomerase. Telomerase reverse transcriptase deficiency had no effect on regulatory T-cell (Treg) numbers in vivo or suppressive function ex vivo. Adoptive transfer of telomerase reverse transcriptase-/- Tregs into Rag2-/- ApoE-/- (recombination activating gene 2/apolipoprotein E) double knockout mice demonstrated that telomerase function was not required for the ability of Tregs to protect against atherosclerosis. However, telomere length was critical for Treg function. CONCLUSIONS: Telomerase contributes to lymphocyte proliferation but plays no major role in Treg function, provided that telomere length is not critically short. We suggest that oxidative stress may contribute to atherosclerosis via suppression of telomerase and acceleration of telomere attrition in Tregs.
Asunto(s)
Aterosclerosis/enzimología , Linfocitos T CD4-Positivos/enzimología , Proliferación Celular , Activación de Linfocitos , Linfocitos T Reguladores/enzimología , Telomerasa/metabolismo , Traslado Adoptivo , Animales , Aterosclerosis/genética , Aterosclerosis/inmunología , Aterosclerosis/prevención & control , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/trasplante , Células Cultivadas , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Modelos Animales de Enfermedad , Femenino , Masculino , Ratones Noqueados , Ratones Noqueados para ApoE , Estrés Oxidativo , Transducción de Señal , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/trasplante , Telomerasa/deficiencia , Telomerasa/genética , Homeostasis del TelómeroRESUMEN
A rise in reactive oxygen species (ROS) may contribute to cardiovascular disease by reducing nitric oxide (NO) levels, leading to loss of NO's vasodilator and anti-inflammatory effects. Although primarily studied in larger conduit arteries, excess ROS release and a corresponding loss of NO also occur in smaller resistance arteries of the microcirculation, but the underlying mechanisms and therapeutic targets have not been fully characterized. We examined whether either of the two subunits of telomerase, telomerase reverse transcriptase (TERT) or telomerase RNA component (TERC), affect microvascular ROS production and peak vasodilation at baseline and in response to in vivo administration to angiotensin II (ANG II). We report that genetic loss of TERT [maximal dilation: 52.0 ± 6.1% with vehicle, 60.4 ± 12.9% with Nω-nitro-l-arginine methyl ester (l-NAME), and 32.2 ± 12.2% with polyethylene glycol-catalase (PEG-Cat) ( P < 0.05), means ± SD, n = 9-19] but not TERC [maximal dilation: 79 ± 5% with vehicle, 10.7 ± 9.8% with l-NAME ( P < 0.05), and 86.4 ± 8.4% with PEG-Cat, n = 4-7] promotes flow-induced ROS formation. Moreover, TERT knockout exacerbates the microvascular dysfunction resulting from in vivo ANG II treatment, whereas TERT overexpression is protective [maximal dilation: 88.22 ± 4.6% with vehicle vs. 74.0 ± 7.3% with ANG II (1,000 ng·kg-1·min-1) ( P = not significant), n = 4]. Therefore, loss of TERT but not TERC may be a key contributor to the elevated microvascular ROS levels and reduced peak dilation observed in several cardiovascular disease pathologies. NEW & NOTEWORTHY This study identifies telomerase reverse transcriptase (TERT) but not telomerase RNA component as a key factor regulating endothelium-dependent dilation in the microcirculation. Loss of TERT activity leads to microvascular dysfunction but not conduit vessel dysfunction in first-generation mice. In contrast, TERT is protective in the microcirculation in the presence of prolonged vascular stress. Understanding the mechanism of how TERT protects against vascular stress represents a novel target for the treatment of vascular disorders.
Asunto(s)
Angiotensina II/toxicidad , Vasos Coronarios/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Arterias Mesentéricas/efectos de los fármacos , Microvasos/efectos de los fármacos , Telomerasa/metabolismo , Vasodilatación/efectos de los fármacos , Animales , Vasos Coronarios/enzimología , Vasos Coronarios/fisiopatología , Endotelio Vascular/enzimología , Endotelio Vascular/fisiopatología , Femenino , Peróxido de Hidrógeno/metabolismo , Masculino , Arterias Mesentéricas/enzimología , Arterias Mesentéricas/fisiopatología , Ratones Endogámicos C57BL , Ratones Noqueados , Microvasos/enzimología , Microvasos/fisiopatología , Óxido Nítrico/metabolismo , ARN/genética , ARN/metabolismo , Telomerasa/deficiencia , Telomerasa/genéticaRESUMEN
BACKGROUND & AIMS: Short telomeres and genetic telomerase defects are risk factors for some human liver diseases, ranging from non-alcoholic fatty liver disease and non-alcoholic steatohepatitis to cirrhosis. In murine models, telomere dysfunction has been shown to metabolically compromise hematopoietic cells, liver and heart via the activation of the p53-PGC axis. METHODS: Tert- and Terc-deficient mice were challenged with liquid high-fat diet. Liver metabolic contents were analysed by CE-TOFMS and liver fat content was confirmed by confocal and electronic microscopy. RESULTS: Tert-deficient but not Terc-deficient mice develop hepatocyte injury and frank steatosis when challenged with liquid high-fat diet. Upon high-fat diet, Tert-/- hepatocytes fail to engage the citric acid cycle (TCA), with an imbalance of NADPH/NADP+ and NADH/NAD+ ratios and depletion of intermediates of TCA cycle, such as cis-aconitic acid. Telomerase deficiency caused an intrinsic metabolic defect unresponsive to environmental challenge. Chemical inhibition of telomerase by zidovudine recapitulated the abnormal Tert-/- metabolic phenotype in Terc-/- hepatocytes. CONCLUSIONS: Our findings indicate that in telomeropathies short telomeres are not the only molecular trigger and telomerase enzyme deficiency provokes hepatocyte metabolic dysfunction, abrogates response to environmental challenge, and causes cellular injury and steatosis, providing a mechanism for liver damage in telomere diseases.
Asunto(s)
Dieta Alta en Grasa , Metabolismo Energético , Hígado Graso/enzimología , Hepatocitos/enzimología , Metabolismo de los Lípidos , Hígado/enzimología , Telomerasa/deficiencia , Acortamiento del Telómero , Animales , Glucemia/metabolismo , Modelos Animales de Enfermedad , Metabolismo Energético/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Hígado Graso/sangre , Hígado Graso/genética , Hígado Graso/patología , Hepatocitos/efectos de los fármacos , Hepatocitos/patología , Metabolismo de los Lípidos/efectos de los fármacos , Metabolismo de los Lípidos/genética , Hígado/efectos de los fármacos , Hígado/patología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , ARN/genética , Telomerasa/antagonistas & inhibidores , Telomerasa/genética , Zidovudina/farmacologíaRESUMEN
OBJECTIVE: Aberrant proliferation of smooth muscle cells (SMC) in response to injury induces pathological vascular remodeling during atherosclerosis and neointima formation. Telomerase is rate limiting for tissue renewal and cell replication; however, the physiological role of telomerase in vascular diseases remains to be determined. The goal of the present study was to determine whether telomerase reverse transcriptase (TERT) affects proliferative vascular remodeling and to define the molecular mechanism by which TERT supports SMC proliferation. APPROACH AND RESULTS: We first demonstrate high levels of TERT expression in replicating SMC of atherosclerotic and neointimal lesions. Using a model of guidewire-induced arterial injury, we demonstrate decreased neointima formation in TERT-deficient mice. Studies in SMC isolated from TERT-deficient and TERT overexpressing mice with normal telomere length established that TERT is necessary and sufficient for cell proliferation. TERT deficiency did not induce a senescent phenotype but resulted in G1 arrest albeit hyperphosphorylation of the retinoblastoma protein. This proliferative arrest was associated with stable silencing of the E2F1-dependent S-phase gene expression program and not reversed by ectopic overexpression of E2F1. Finally, chromatin immunoprecipitation and accessibility assays revealed that TERT is recruited to E2F1 target sites and promotes chromatin accessibility for E2F1 by facilitating the acquisition of permissive histone modifications. CONCLUSIONS: These data indicate a previously unrecognized role for TERT in neointima formation through epigenetic regulation of proliferative gene expression in SMC.
Asunto(s)
Aterosclerosis/enzimología , Ensamble y Desensamble de Cromatina , Factor de Transcripción E2F1/metabolismo , Silenciador del Gen , Músculo Liso Vascular/enzimología , Neointima , Telomerasa/deficiencia , Telomerasa/metabolismo , Lesiones del Sistema Vascular/enzimología , Acetilación , Animales , Aterosclerosis/genética , Aterosclerosis/patología , Sitios de Unión , Proliferación Celular , Células Cultivadas , Modelos Animales de Enfermedad , Factor de Transcripción E2F1/genética , Arteria Femoral/enzimología , Arteria Femoral/lesiones , Arteria Femoral/patología , Puntos de Control de la Fase G1 del Ciclo Celular , Predisposición Genética a la Enfermedad , Histonas/metabolismo , Humanos , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Músculo Liso Vascular/lesiones , Músculo Liso Vascular/patología , Fenotipo , Fosforilación , Unión Proteica , Interferencia de ARN , Proteína de Retinoblastoma/metabolismo , Transducción de Señal , Telomerasa/genética , Factores de Tiempo , Transfección , Remodelación Vascular , Lesiones del Sistema Vascular/genética , Lesiones del Sistema Vascular/patologíaRESUMEN
BACKGROUND: In the course of replication of eukaryotic chromosomes, the telomere length is maintained due to activity of telomerase, the ribonucleoprotein reverse transcriptase. Abolishing telomerase function causes progressive shortening of telomeres and, ultimately, cell cycle arrest and replicative senescence. To better understand the cellular response to telomerase deficiency, we performed a transcriptomic study for the thermotolerant methylotrophic yeast Hansenula polymorpha DL-1 lacking telomerase activity. RESULTS: Mutant strain of H. polymorpha carrying a disrupted telomerase RNA gene was produced, grown to senescence and analyzed by RNA-seq along with wild type strain. Telomere shortening induced a transcriptional response involving genes relevant to telomere structure and maintenance, DNA damage response, information processing, and some metabolic pathways. Genes involved in DNA replication and repair, response to environmental stresses and intracellular traffic were up-regulated in senescent H. polymorpha cells, while strong down-regulation was observed for genes involved in transcription and translation, as well as core histones. CONCLUSIONS: Comparison of the telomerase deletion transcription responses by Saccharomyces cerevisiae and H. polymorpha demonstrates that senescence makes different impact on the main metabolic pathways of these yeast species but induces similar changes in processes related to nucleic acids metabolism and protein synthesis. Up-regulation of a subunit of the TORC1 complex is clearly relevant for both types of yeast.
Asunto(s)
Genómica , Pichia/enzimología , Pichia/genética , Telomerasa/deficiencia , Termotolerancia , Transcripción Genética , Autofagia/genética , Metabolismo de los Hidratos de Carbono/genética , Daño del ADN/genética , Metabolismo Energético/genética , Ambiente , Genes Fúngicos/genética , Espacio Intracelular/metabolismo , Pichia/citología , Pichia/fisiología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Estrés Fisiológico/genética , Acortamiento del Telómero/genéticaRESUMEN
Telomere dysfunction activates p53-mediated cellular growth arrest, senescence and apoptosis to drive progressive atrophy and functional decline in high-turnover tissues. The broader adverse impact of telomere dysfunction across many tissues including more quiescent systems prompted transcriptomic network analyses to identify common mechanisms operative in haematopoietic stem cells, heart and liver. These unbiased studies revealed profound repression of peroxisome proliferator-activated receptor gamma, coactivator 1 alpha and beta (PGC-1α and PGC-1ß, also known as Ppargc1a and Ppargc1b, respectively) and the downstream network in mice null for either telomerase reverse transcriptase (Tert) or telomerase RNA component (Terc) genes. Consistent with PGCs as master regulators of mitochondrial physiology and metabolism, telomere dysfunction is associated with impaired mitochondrial biogenesis and function, decreased gluconeogenesis, cardiomyopathy, and increased reactive oxygen species. In the setting of telomere dysfunction, enforced Tert or PGC-1α expression or germline deletion of p53 (also known as Trp53) substantially restores PGC network expression, mitochondrial respiration, cardiac function and gluconeogenesis. We demonstrate that telomere dysfunction activates p53 which in turn binds and represses PGC-1α and PGC-1ß promoters, thereby forging a direct link between telomere and mitochondrial biology. We propose that this telomere-p53-PGC axis contributes to organ and metabolic failure and to diminishing organismal fitness in the setting of telomere dysfunction.
Asunto(s)
Mitocondrias/metabolismo , Mitocondrias/patología , Telómero/metabolismo , Telómero/patología , Adenosina Trifosfato/biosíntesis , Envejecimiento/metabolismo , Envejecimiento/patología , Animales , Cardiomiopatías/inducido químicamente , Cardiomiopatías/metabolismo , Cardiomiopatías/patología , Cardiomiopatías/fisiopatología , Proliferación Celular , ADN Mitocondrial/análisis , Doxorrubicina/toxicidad , Gluconeogénesis , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/patología , Hígado/citología , Hígado/metabolismo , Ratones , Miocardio/citología , Miocardio/metabolismo , ARN/genética , Especies Reactivas de Oxígeno/metabolismo , Telomerasa/deficiencia , Telomerasa/genética , Telómero/enzimología , Telómero/genética , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/metabolismo , Proteína p53 Supresora de Tumor/deficiencia , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
An ageing world population has fuelled interest in regenerative remedies that may stem declining organ function and maintain fitness. Unanswered is whether elimination of intrinsic instigators driving age-associated degeneration can reverse, as opposed to simply arrest, various afflictions of the aged. Such instigators include progressively damaged genomes. Telomerase-deficient mice have served as a model system to study the adverse cellular and organismal consequences of wide-spread endogenous DNA damage signalling activation in vivo. Telomere loss and uncapping provokes progressive tissue atrophy, stem cell depletion, organ system failure and impaired tissue injury responses. Here, we sought to determine whether entrenched multi-system degeneration in adult mice with severe telomere dysfunction can be halted or possibly reversed by reactivation of endogenous telomerase activity. To this end, we engineered a knock-in allele encoding a 4-hydroxytamoxifen (4-OHT)-inducible telomerase reverse transcriptase-oestrogen receptor (TERT-ER) under transcriptional control of the endogenous TERT promoter. Homozygous TERT-ER mice have short dysfunctional telomeres and sustain increased DNA damage signalling and classical degenerative phenotypes upon successive generational matings and advancing age. Telomerase reactivation in such late generation TERT-ER mice extends telomeres, reduces DNA damage signalling and associated cellular checkpoint responses, allows resumption of proliferation in quiescent cultures, and eliminates degenerative phenotypes across multiple organs including testes, spleens and intestines. Notably, somatic telomerase reactivation reversed neurodegeneration with restoration of proliferating Sox2(+) neural progenitors, Dcx(+) newborn neurons, and Olig2(+) oligodendrocyte populations. Consistent with the integral role of subventricular zone neural progenitors in generation and maintenance of olfactory bulb interneurons, this wave of telomerase-dependent neurogenesis resulted in alleviation of hyposmia and recovery of innate olfactory avoidance responses. Accumulating evidence implicating telomere damage as a driver of age-associated organ decline and disease risk and the marked reversal of systemic degenerative phenotypes in adult mice observed here support the development of regenerative strategies designed to restore telomere integrity.
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Envejecimiento/metabolismo , Envejecimiento/patología , Telomerasa/deficiencia , Telomerasa/metabolismo , Envejecimiento/efectos de los fármacos , Animales , Reacción de Prevención/efectos de los fármacos , Encéfalo/anatomía & histología , Encéfalo/citología , Encéfalo/efectos de los fármacos , Encéfalo/patología , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Daño del ADN/efectos de los fármacos , Proteína Doblecortina , Activación Enzimática/efectos de los fármacos , Reactivadores Enzimáticos/farmacología , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Vaina de Mielina/metabolismo , Células-Madre Neurales/citología , Células-Madre Neurales/efectos de los fármacos , Células-Madre Neurales/enzimología , Células-Madre Neurales/patología , Tamaño de los Órganos/efectos de los fármacos , Fenotipo , Receptores de Estrógenos/genética , Receptores de Estrógenos/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Medicina Regenerativa , Olfato/efectos de los fármacos , Olfato/fisiología , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacología , Telomerasa/genética , Telómero/efectos de los fármacos , Telómero/metabolismo , Telómero/patologíaRESUMEN
In human somatic cells or yeast cells lacking telomerase, telomeres are shortened upon each cell division. This gradual shortening of telomeres eventually leads to senescence. However, a small population of telomerase-deficient cells can survive by bypassing senescence through the activation of alternative recombination pathways to maintain their telomeres. Although genes involved in telomere recombination have been identified, mechanisms that trigger telomere recombination are less known. The THO (suppressor of the transcriptional defects of Hpr1 mutants by overexpression) complex is involved in transcription elongation and mRNA export. Here we demonstrate that mutations in THO complex components can stimulate early senescence and type II telomere recombination in cells lacking telomerase. The accumulation of telomere-associated noncoding telomere repeat-containing RNA (TERRA) is required for the observed telomere effects in THO complex mutants; reduced transcriptional efficiency, or overexpression of RNase H or C(1-3)A RNA can severely impair the type II telomere recombination. The results highlight a unique function for telomere-associated TERRA, in the formation of type II survivors. Moreover, because TERRA is a long noncoding RNA, these results reveal a function for long noncoding RNA in regulating recombination.
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Senescencia Celular/fisiología , Complejos Multiproteicos/genética , ARN Largo no Codificante/genética , Recombinación Genética/genética , Telomerasa/deficiencia , Homeostasis del Telómero/genética , Telómero/genética , Senescencia Celular/genética , Inmunoprecipitación de Cromatina , Mutación/genética , ARN Largo no Codificante/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Saccharomyces cerevisiaeRESUMEN
Mutations of human telomerase RNA component (TERC) and telomerase reverse transcriptase (TERT) are associated with a subset of lung aging diseases, but the mechanisms by which TERC and TERT participate in lung diseases remain unclear. In this report, we show that knock-out (KO) of the mouse gene Terc or Tert causes pulmonary alveolar stem cell replicative senescence, epithelial impairment, formation of alveolar sacs, and characteristic inflammatory phenotype. Deficiency in TERC or TERT causes a remarkable elevation in various proinflammatory cytokines, including IL-1, IL-6, CXCL15 (human IL-8 homolog), IL-10, TNF-α, and monocyte chemotactic protein 1 (chemokine ligand 2 (CCL2)); decrease in TGF-ß1 and TGFßRI receptor in the lungs; and spillover of IL-6 and CXCL15 into the bronchoalveolar lavage fluids. In addition to increased gene expressions of α-smooth muscle actin and collagen 1α1, suggesting myofibroblast differentiation, TERC deficiency also leads to marked cellular infiltrations of a mononuclear cell population positive for the leukocyte common antigen CD45, low-affinity Fc receptor CD16/CD32, and pattern recognition receptor CD11b in the lungs. Our data demonstrate for the first time that telomerase deficiency triggers alveolar stem cell replicative senescence-associated low-grade inflammation, thereby driving pulmonary premature aging, alveolar sac formation, and fibrotic lesion.
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Enfermedades Pulmonares/inmunología , Alveolos Pulmonares/enzimología , Células Madre/citología , Telomerasa/deficiencia , Animales , Senescencia Celular , Femenino , Humanos , Interleucina-1/genética , Interleucina-1/inmunología , Interleucina-6/genética , Interleucina-6/inmunología , Pulmón/citología , Pulmón/inmunología , Enfermedades Pulmonares/enzimología , Enfermedades Pulmonares/genética , Enfermedades Pulmonares/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Alveolos Pulmonares/citología , Alveolos Pulmonares/inmunología , ARN/genética , Células Madre/inmunología , Telomerasa/genética , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
BACKGROUND: Cells exhibiting dysregulated growth may express telomerase reverse transcriptase (TERT), the dual function of which consists of maintaining telomere length, in association with the RNA template molecule TERC, and controlling cell growth. Here, we investigated lung TERT in human and experimental pulmonary hypertension (PH) and its role in controlling pulmonary artery smooth muscle cell (PA-SMC) proliferation. METHODS AND RESULTS: Marked TERT expression or activity was found in lungs from patients with idiopathic PH and from mice with PH induced by hypoxia or serotonin-transporter overexpression (SM22-5HTT(+) mice), chiefly within PA-SMCs. In cultured mouse PA-SMCs, TERT was expressed on growth stimulation by serum. The TERT inhibitor imetelstat and the TERT activator TA65 abrogated and stimulated PA-SMC growth, respectively. PA-SMCs from PH mice showed a heightened proliferative phenotype associated with increased TERT expression, which was suppressed by imetelstat treatment. TERC(-/-) mice at generation 2 and TERT(-/-) mice at generations 2, 3, and 4 developed less severe PH than did wild-type mice exposed to chronic hypoxia, with less distal pulmonary artery muscularization and fewer Ki67-stained proliferating PA-SMCs. Telomere length differed between TERC(-/-) and TERT(-/-) mice, whereas PH severity was similar in the 2 strains and across generations. Chronic imetelstat treatment reduced hypoxia-induced PH in wild-type mice or partially reversed established PH in SM22-5HTT(+) mice while simultaneously decreasing TERT expression. Opposite effects occurred in mice treated with TA65. CONCLUSIONS: Telomerase exerts telomere-independent effects on PA-SMC growth in PH and may constitute a treatment target for PH.
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Hipertensión Pulmonar/fisiopatología , Músculo Liso Vascular/fisiopatología , Arteria Pulmonar/fisiopatología , Telomerasa/fisiología , Adulto , Animales , Proliferación Celular/fisiología , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Humanos , Hipertensión Pulmonar/etiología , Hipertensión Pulmonar/patología , Hipoxia/complicaciones , Indoles/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Mutantes , Persona de Mediana Edad , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/patología , Niacinamida/análogos & derivados , Niacinamida/farmacología , Oligonucleótidos , Arteria Pulmonar/efectos de los fármacos , Arteria Pulmonar/patología , Proteínas de Transporte de Serotonina en la Membrana Plasmática/genética , Proteínas de Transporte de Serotonina en la Membrana Plasmática/fisiología , Telomerasa/deficiencia , Telomerasa/genéticaRESUMEN
To counteract replication-dependent telomere shortening most eukaryotic cells rely on the telomerase pathway, which is crucial for the maintenance of proliferative potential of germ and stem cell populations of multicellular organisms. Likewise, cancer cells usually engage the telomerase pathway for telomere maintenance to gain immortality. However, in â¼10% of human cancers telomeres are maintained through telomerase-independent alternative lengthening of telomeres (ALT) pathways. Here, we describe the generation and characterization of C. elegans survivors in a strain lacking the catalytic subunit of telomerase and the nematode telomere-binding protein CeOB2. These clonal strains, some of which have been propagated for >180 generations, represent the first example of a multicellular organism with canonical telomeres that can survive without a functional telomerase pathway. The animals display the heterogeneous telomere length characteristic for ALT cells, contain single-stranded C-circles, a transcription profile pointing towards an adaptation to chronic stress and are therefore a unique and valuable tool to decipher the ALT mechanism.
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Caenorhabditis elegans/enzimología , Caenorhabditis elegans/crecimiento & desarrollo , Telomerasa/deficiencia , Proteínas de Unión a Telómeros/deficiencia , Telómero/metabolismo , Animales , Caenorhabditis elegans/genética , Análisis de SupervivenciaRESUMEN
The aged population suffers increased morbidity and higher mortality in response to episodes of acute kidney injury (AKI). Aging is associated with telomere shortening, and both telomerase reverse transcriptase (TerT) and RNA (TerC) are essential to maintain telomere length. To define a role of telomerase deficiency in susceptibility to AKI, we used ischemia/reperfusion injury in wild-type mice or mice with either TerC or TerT deletion. Injury induced similar renal impairment at day 1 in each genotype, as assessed by azotemia, proteinuria, acute tubular injury score, and apoptotic tubular epithelial cell index. However, either TerC or TerT knockout significantly delayed recovery compared with wild-type mice. Electron microscopy showed increased autophagosome formation in renal tubular epithelial cells in wild-type mice but a significant delay of their development in TerC and TerT knockout mice. There were also impeded increases in the expression of the autophagosome marker LC3 II, prolonged accumulation of the autophagosome protein P62, an increase of the cell cycle regulator p16, and greater activation of the mammalian target of rapamycin (mTOR) pathway. The mTORC1 inhibitor, rapamycin, partially restored the ischemia/reperfusion-induced autophagy response, without a significant effect on either p16 induction or tubule epithelial cell proliferation. Thus, muting the maintenance of normal telomere length in mice impaired recovery from AKI, owing to an increase in tubule cell senescence and impairment of mTOR-mediated autophagy.
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Lesión Renal Aguda/enzimología , Autofagia , Riñón/fisiología , Regeneración/fisiología , Telomerasa/deficiencia , Lesión Renal Aguda/etiología , Lesión Renal Aguda/patología , Envejecimiento/fisiología , Animales , Apoptosis , Nitrógeno de la Urea Sanguínea , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Modelos Animales de Enfermedad , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Túbulos Renales Proximales/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , ARN/genética , Daño por Reperfusión/complicaciones , Transducción de Señal , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/metabolismo , Telomerasa/genética , Acortamiento del TelómeroRESUMEN
The contribution of deficient telomerase activity to age-related decline in osteoblast functions and bone formation is poorly studied. We have previously demonstrated that telomerase over-expression led to enhanced osteoblast differentiation of human bone marrow skeletal (stromal) stem cells (hMSC) in vitro and in vivo. Here, we investigated the signaling pathways underlying the regulatory functions of telomerase in osteoblastic cells. Comparative microarray analysis and Western blot analysis of telomerase-over expressing hMSC (hMSC-TERT) versus primary hMSC revealed significant up-regulation of several components of insulin-like growth factor (IGF) signaling. Specifically, a significant increase in IGF-induced AKT phosphorylation and alkaline phosphatase (ALP) activity were observed in hMSC-TERT. Enhanced ALP activity was reduced in presence of IGF1 receptor inhibitor: picropodophyllin. In addition, telomerase deficiency caused significant reduction in IGF signaling proteins in osteoblastic cells cultured from telomerase deficient mice (Terc(-/-)). The low bone mass exhibited by Terc(-/-) mice was associated with significant reduction in serum levels of IGF1 and IGFBP3 as well as reduced skeletal mRNA expression of Igf1, Igf2, Igf2r, Igfbp5 and Igfbp6. IGF1-induced osteoblast differentiation was also impaired in Terc(-/-) MSC. In conclusion, our data demonstrate that impaired IGF/AKT signaling contributes to the observed decreased bone mass and bone formation exhibited by telomerase deficient osteoblastic cells.
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Osteoblastos/citología , Osteoblastos/metabolismo , ARN/metabolismo , Somatomedinas/metabolismo , Telomerasa/metabolismo , Animales , Diferenciación Celular , Células Cultivadas , Senescencia Celular/fisiología , Activación Enzimática , Humanos , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/sangre , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Noqueados , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN/genética , Transducción de Señal , Telomerasa/deficiencia , Telomerasa/genéticaRESUMEN
Cell-intrinsic checkpoints limit the proliferative capacity of primary cells in response to telomere dysfunction. It is not known, however, whether telomere dysfunction contributes to cell-extrinsic alterations that impair stem cell function and organ homeostasis. Here we show that telomere dysfunction provokes defects of the hematopoietic environment that impair B lymphopoiesis but increase myeloid proliferation in aging telomerase knockout (Terc(-/-)) mice. Moreover, the dysfunctional environment limited the engraftment of transplanted wild-type hematopoietic stem cells (HSCs). Dysfunction of the hematopoietic environment was age dependent and correlated with progressive telomere shortening in bone marrow stromal cells. Telomere dysfunction impaired mesenchymal progenitor cell function, reduced the capacity of bone marrow stromal cells to maintain functional HSCs, and increased the expression of various cytokines, including granulocyte colony-stimulating factor (G-CSF), in the plasma of aging mice. Administration of G-CSF to wild-type mice mimicked some of the defects seen in aging Terc(-/-) mice, including impairment of B lymphopoiesis and HSC engraftment. Conversely, inhibition of G-CSF improved HSC engraftment in aged Terc(-/-) mice. Taken together, these results show that telomere dysfunction induces alterations of the environment that can have implications for organismal aging and cell transplantation therapies.