RESUMEN
Multiple embryonic precursors give rise to leukocytes in adults while the lineage-based functional impacts are underappreciated. Mesodermal precursors expressing PDGFRα appear transiently during E7.5-8.5 descend to a subset of Lin- Sca1+ Kit+ hematopoietic progenitors found in adult BM. By analyzing a PDGFRα-lineage tracing mouse line, we here report that PDGFRα-lineage BM F4/80+ SSClo monocytes/macrophages are solely Ly6C+ LFA-1hi Mac-1hi monocytes enriched on the abluminal sinusoidal endothelium while Ly6C- LFA-1lo Mac-1lo macrophages are mostly from non-PDGFRα-lineage in vivo. Monocytes with stronger integrin profiles outcompete macrophages for adhesion on an endothelial monolayer or surfaces coated with ICAM-1-Fc or VCAM-1-Fc. Egress of PDGFRα-lineage-rich monocytes and subsequent differentiation to peripheral macrophages spatially segregates them from non-PDGFRα-lineage BM-resident macrophages and allows functional specialization since macrophages derived from these egressing monocytes differ in morphology, phenotype, and functionality from BM-resident macrophages in culture. Extravasation preference for blood PDGFRα-lineage monocytes varies by tissues and governs the local lineage composition of macrophages. More PDGFRα-lineage classical monocytes infiltrated into skin and colon but not into peritoneum. Accordingly, transcriptomic analytics indicated augmented inflammatory cascades in dermatitis skin of BM-chimeric mice harbouring only PDGFRα-lineage leukocytes. Thus, the PDGFRα-lineage origin biasedly generates monocytes predestined for BM exit to support peripheral immunity following extravasation and macrophage differentiation.
Asunto(s)
Linaje de la Célula/inmunología , Movimiento Celular/inmunología , Endotelio Vascular/inmunología , Macrófagos/inmunología , Monocitos/inmunología , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/inmunología , Animales , Linaje de la Célula/genética , Movimiento Celular/genética , Ratones , Ratones Transgénicos , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/genéticaRESUMEN
Platelet-derived growth factor receptor alpha (PDGFRα) is a dominant marker of mesodermal mesenchymal cells in mice. Previous studies demonstrated that PDGFRα-positive (PDGFRα+) mesodermal cells develop not only into mesenchymal cells but also into a subset of total hematopoietic cells (HCs) in the limited period during mouse embryogenesis. However, the precise characteristics of the PDGFRα lineage positive (PDGFRα Lin+) HCs in adult mouse hematopoiesis are largely unknown. In this study, we systematically evaluated the characteristics of PDGFRα Lin+ HCs in the bone marrow and peripheral blood using PDGFRα-CRE; ROSAtdTomato mice. Flow cytometry analysis revealed that PDGFRα Lin+ HCs accounted for approximately 20% of total HCs in both the bone marrow and peripheral blood in adult mice. Compositions of myeloid and lymphoid subpopulations among CD45+ mononuclear cells were almost identical in both PDGFRα Lin+ and PDGFRα Lin- cells. Single-cell RNA-sequencing analysis also demonstrated that the transcriptomic signatures of the PDGFRα Lin+ HCs in the peripheral blood largely overlapped with those of the PDGFRα Lin- HCs, suggesting equivalent functions of the PDGFRα Lin+ and PDGFRα Lin- HCs. Although pathophysiological activities of the PDGFRα Lin + HCs were not evaluated, our data clearly demonstrate a significant role of the PDGFRα Lin + HCs in physiological hematopoiesis in adult mice.
Asunto(s)
Hematopoyesis , Células Madre Hematopoyéticas/fisiología , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Animales , Linaje de la Célula , Femenino , Hematopoyesis/genética , Células Madre Hematopoyéticas/metabolismo , Masculino , Mesodermo/citología , Ratones , RNA-Seq , Análisis de la Célula IndividualRESUMEN
Transcription factor access to regulatory elements is prevented by the nucleosome. Heat shock factor 1 (HSF1) is a winged helix transcription factor that plays roles in control and stressed conditions by gaining access to target elements, but mechanisms of HSF1 access are not well known in mammalian cells. Here, we show the physical interaction between the wing motif of human HSF1 and replication protein A (RPA), which is involved in DNA metabolism. Depletion of RPA1 abolishes HSF1 access to the promoter of HSP70 in unstressed condition and delays its rapid activation in response to heat shock. The HSF1-RPA complex leads to preloading of RNA polymerase II and opens the chromatin structure by recruiting a histone chaperone, FACT. Furthermore, this interaction is required for melanoma cell proliferation. These results provide a mechanism of constitutive HSF1 access to nucleosomal DNA, which is important for both basal and inducible gene expression.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Proteínas del Grupo de Alta Movilidad , Elementos Reguladores de la Transcripción , Proteína de Replicación A/metabolismo , Factores de Transcripción/metabolismo , Factores de Elongación Transcripcional , Secuencia de Aminoácidos , Secuencia de Bases , Cromatina/genética , ADN/genética , ADN/metabolismo , Proteínas de Unión al ADN/genética , Células HEK293 , Factores de Transcripción del Choque Térmico , Proteínas del Grupo de Alta Movilidad/genética , Proteínas del Grupo de Alta Movilidad/metabolismo , Humanos , Datos de Secuencia Molecular , Nucleosomas/genética , Regiones Promotoras Genéticas , Unión Proteica , Dominios y Motivos de Interacción de Proteínas/genética , ARN Polimerasa II/metabolismo , Factores de Transcripción/genética , Factores de Elongación Transcripcional/genética , Factores de Elongación Transcripcional/metabolismoRESUMEN
BACKGROUND: Deciphering the 3D structure of the genome is essential for elucidating the regulatory mechanisms of gene expression in detail. Existing methods, such as chromosome conformation capture (3C) and Hi-C have enabled the identification of novel aspects of chromatin structure. Further identification of protein-centric chromatin conformation is enabled by coupling the Hi-C procedure with a conventional chromatin immunoprecipitation assay. However, these methods are time-consuming and require independent methods for validation. RESULTS: To simultaneously identify protein-centric chromatin conformation and target protein localization, we have developed Cut-C, a method that combines antibody-mediated cleavage by tethered nuclease with chromosome conformation capture to identify chromatin interactions mediated by a protein of interest. Applying Cut-C to H3K4me3, a histone modification enriched at active gene promoters, we have successfully identified chromatin loops mediated by H3K4me3 along with the genome-wide distribution of H3K4me3. Cut-C also identified chromatin loops mediated by CTCF, validating the general applicability of the method. CONCLUSIONS: Cut-C identifies protein-centric chromatin conformations along with the genome-wide distribution of target proteins using simple procedures. The simplified protocol will improve the efficiency of analysing chromatin conformation using precious materials, such as clinical samples.
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Cromatina/química , Cromatina/metabolismo , Desoxirribonucleasas/metabolismo , Genómica , Células HEK293 , Histonas/química , Histonas/metabolismo , Humanos , Lisina/metabolismo , Conformación ProteicaRESUMEN
Umbilical cord blood contains mesenchymal stem/stromal cells (MSCs) in addition to hematopoietic stem cells, serving as an attractive tool for regenerative medicine. As umbilical cord blood originates from fetus, abundant MSCs are expected to circulate in fetus. However, the properties of circulating MSCs in fetus have not been fully examined. In the present study, we aimed to analyze circulating MSCs, marked by the expression of platelet-derived growth factor receptor α (PDGFRα), during fetal development. Using PDGFRα GFP knock-in mice, we quantified the number of circulating PDGFRα positive MSCs during development. We further performed whole transcriptome analysis of circulating MSCs at single cell levels. We found that abundant PDGFRα positive cells circulate in embryo and diminish immediately after birth. In addition, single cell RNA-sequencing revealed transcriptional heterogeneity of MSCs in fetal circulation. These data lay a foundation to analyze the function of circulating MSCs during development.
Asunto(s)
Sangre Fetal/citología , Sangre Fetal/metabolismo , Feto/citología , Feto/metabolismo , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Animales , Recuento de Células , Femenino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Embarazo , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/genética , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Medicina Regenerativa , Análisis de la Célula Individual , Transcripción GenéticaRESUMEN
Ovarian aging is characterized by gradual declines in oocyte quantity and quality. Melatonin is considered an anti-aging agent due to its cytoprotective actions as an antioxidant. This study examined whether long-term melatonin treatment would delay ovarian aging in mice. Female ICR mice (10 weeks old) were given melatonin-containing water (100 µg/mL; melatonin) or water only until 43 weeks of age. Their oocytes were recovered from the oviduct, and in vitro fertilization was performed. The ovaries were used for a histological analysis of the number of follicles. The mRNA expression of the aging-related sirtuin genes (SIRT1, SIRT3) and the autophagy-related gene (LC3) and the telomere length of the ovarian chromosomes were analyzed. Transcriptome changes in the ovaries were also characterized using microarray. The number of ovulated oocytes decreased with age; however, it was greater in melatonin-treated mice than that from control animals. The decreased fertilization rate and blastocyst rate during aging also were higher in the melatonin-treated mice than in the controls, as were the numbers of primordial, primary, and antral follicles. The mRNA expression of SIRT1 and LC3 and telomere length were enhanced due to melatonin treatment. Seventy-eight genes that were downregulated during aging and upregulated by melatonin were identified by a microarray analysis. Forty of these 78 genes were ribosome-related genes, and a free radical scavenging network was identified. The present results indicate that melatonin delays ovarian aging by multiple mechanisms including antioxidant action, maintaining telomeres, stimulating SIRT expression and ribosome function, and by reducing autophagy.
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Envejecimiento/efectos de los fármacos , Antioxidantes/farmacología , Fertilidad/efectos de los fármacos , Melatonina/farmacología , Ovario/efectos de los fármacos , Animales , Femenino , Ratones , Ratones Endogámicos ICR , Modelos Animales , Análisis de Secuencia por Matrices de Oligonucleótidos , Oocitos/efectos de los fármacos , Distribución Aleatoria , Reacción en Cadena en Tiempo Real de la Polimerasa , Transcriptoma/efectos de los fármacosRESUMEN
Fatty acid-binding proteins (FABPs) bind and solubilize long-chain fatty acids, controlling intracellular lipid dynamics. FABP7 is expressed by astrocytes in the developing brain, and suggested to be involved in the control of astrocyte lipid homeostasis. In this study, we sought to examine the role of FABP7 in astrocytes, focusing on plasma membrane lipid raft function, which is important for receptor-mediated signal transduction in response to extracellular stimuli. In FABP7-knockout (KO) astrocytes, the ligand-dependent accumulation of Toll-like receptor 4 (TLR4) and glial cell-line-derived neurotrophic factor receptor alpha 1 into lipid raft was decreased, and the activation of mitogen-activated protein kinases and nuclear factor-κB was impaired after lipopolysaccharide (LPS) stimulation when compared with wild-type astrocytes. In addition, the expression of caveolin-1, not cavin-1, 2, 3, caveolin-2, and flotillin-1, was found to be decreased at the protein and transcriptional levels. FABP7 re-expression in FABP7-KO astrocytes rescued the decreased level of caveolin-1. Furthermore, caveolin-1-transfection into FABP7-KO astrocytes significantly increased TLR4 recruitment into lipid raft and tumor necrosis factor-α production after LPS stimulation. Taken together, these data suggest that FABP7 controls lipid raft function through the regulation of caveolin-1 expression and is involved in the response of astrocytes to the external stimuli. GLIA 2015;63:780-794.
Asunto(s)
Astrocitos/citología , Caveolas/metabolismo , Caveolina 1/metabolismo , Proteínas de Unión a Ácidos Grasos/metabolismo , Regulación de la Expresión Génica/genética , Proteínas del Tejido Nervioso/metabolismo , Animales , Animales Recién Nacidos , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Caveolas/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Corteza Cerebral/citología , Colesterol/metabolismo , Citocinas/metabolismo , Proteína de Unión a los Ácidos Grasos 7 , Proteínas de Unión a Ácidos Grasos/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Factor Neurotrófico Derivado de la Línea Celular Glial/farmacología , Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Transducción GenéticaRESUMEN
Down syndrome (DS) is the most common cause of mental retardation. Although structural and neurogenic abnormalities have been shown in the brains of DS patients, the molecular etiology is still unknown. To define it, we have performed structural and histological examinations of the brains of Ts1Cje and Ts2Cje, 2 mouse models for DS. These mice carry different length of trisomic segments of mouse chromosome 16 that are orthologous to human chromosome 21. At 3 months of age, ventricular enlargements were observed in both Ts1Cje and Ts2Cje brains at a similar degree. Both mice also showed decreases of the number of doublecortin-positive neuroblasts and thymidine-analog BrdU-labeled proliferating cells in the subventricular zone of the lateral ventricles (LVs) and in the hippocampal dentate gyrus at a similar degree, suggesting impaired adult neurogenesis. Additionally, at embryonic day 14.5, both strains of mice, when compared with diploid littermates, had smaller brains and decreased cortical neurogenesis that could possibly contribute to the ventricular enlargements observed in adulthood. Our findings suggest that the trisomic segment of the Ts1Cje mouse, which is shared with Ts2Cje, contains the genes that are responsible for these abnormal phenotypes and could be relevant to the mental retardation associated with DS.
Asunto(s)
Ventrículos Cerebrales/patología , Cromosomas de los Mamíferos/genética , Síndrome de Down/genética , Neurogénesis , Trisomía/genética , Trisomía/fisiopatología , Animales , Proliferación Celular , Ventrículos Cerebrales/embriología , Ventrículos Cerebrales/crecimiento & desarrollo , Ventrículos Cerebrales/metabolismo , Cromosomas Humanos Par 21 , Modelos Animales de Enfermedad , Proteínas de Dominio Doblecortina , Síndrome de Down/patología , Síndrome de Down/fisiopatología , Femenino , Humanos , Imagen por Resonancia Magnética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Asociadas a Microtúbulos/metabolismo , Neuropéptidos/metabolismo , EmbarazoRESUMEN
BACKGROUND & AIMS: Proper resolution of inflammation is essential to maintaining homeostasis, which is important as a dysregulated inflammatory response has adverse consequences, even being regarded as a hallmark of cancer. However, our picture of dynamic changes during inflammation remains far from comprehensive. METHODS: Here we used single-cell transcriptomics to elucidate changes in distinct cell types and their interactions in a mouse model of chemically induced colitis. RESULTS: Our analysis highlights the stromal cell population of the colon functions as a hub with dynamically changing roles over time. Importantly, we found that Serpina3n, a serine protease inhibitor, is specifically expressed in stromal cell clusters as inflammation resolves, interacting with a potential target, elastase. Indeed, genetic ablation of the Serpina3n gene delays resolution of induced inflammation. Furthermore, systemic Serpina3n administration promoted the resolution of inflammation, ameliorating colitis symptoms. CONCLUSIONS: This study provides a comprehensive, single-cell understanding of cell-cell interactions during colorectal inflammation and reveals a potential therapeutic target that leverages inflammation resolution.
Asunto(s)
Proteínas de Fase Aguda/metabolismo , Colitis/genética , Colitis/patología , Inflamación/genética , Inflamación/patología , Serpinas/metabolismo , Análisis de la Célula Individual , Transcriptoma/genética , Animales , Comunicación Celular , Colon/patología , Sulfato de Dextran , Modelos Animales de Enfermedad , Predisposición Genética a la Enfermedad , Enfermedades Inflamatorias del Intestino/genética , Enfermedades Inflamatorias del Intestino/patología , Ratones Endogámicos C57BL , Fenotipo , RNA-Seq , Factores de Riesgo , Células del Estroma/metabolismoRESUMEN
Elevated oxidative stress has been suggested to be associated with the features of Down's syndrome (DS). We previously reported increased oxidative stress in cultured cells from the embryonic brain of Ts1Cje, a mouse genetic DS model. However, since in vivo evidence for increased oxidative stress is lacking, we here examined lipid peroxidation, a typical marker of oxidative stress, in the brains of Ts1Cje and another DS mouse model Ts2Cje with an overlapping but larger trisomic segment. Accumulations of proteins modified with the lipid peroxidation-derived products, 13-hydroperoxy-9Z,11E-octadecadienoic acid and 4-hydroxy-2-nonenal were markedly increased in Ts1Cje and Ts2Cje brains. Analysis with oxidation-sensitive fluorescent probe also showed that reactive oxygen species themselves were increased in Ts1Cje brain. However, electron spin resonance analysis of microdialysate from the hippocampus of Ts1Cje showed that antioxidant activity remained unaffected, suggesting that the reactive oxygen species production was accelerated in Ts1Cje. Proteomics approaches with mass spectrometry identified the proteins modified with 13-hydroperoxy-9Z,11E-octadecadienoic acid and/or 4-hydroxy-2-nonenal to be involved in either ATP generation, the neuronal cytoskeleton or antioxidant activity. Structural or functional impairments of these proteins by such modifications may contribute to the DS features such as cognitive impairment that are present in the Ts1Cje mouse.
Asunto(s)
Encéfalo/metabolismo , Síndrome de Down/metabolismo , Síndrome de Down/fisiopatología , Peroxidación de Lípido/fisiología , Factores de Edad , Aldehídos/metabolismo , Animales , Encéfalo/patología , Modelos Animales de Enfermedad , Síndrome de Down/genética , Síndrome de Down/patología , Electroforesis en Gel Bidimensional/métodos , Femenino , Regulación de la Expresión Génica/genética , Humanos , Ácidos Linoleicos/metabolismo , Peróxidos Lipídicos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microdiálisis , Especies Reactivas de Oxígeno/metabolismo , Trisomía/genéticaRESUMEN
Exercise-induced cardiac hypertrophy has been reported to have better prognosis than pressure overload-induced cardiac hypertrophy. Cardiac hypertrophy induced by exercise was associated with less cardiac fibrosis and better systolic function, suggesting that the adaptive mechanisms may exist in exercise-induced hypertrophy. Here, we showed a critical role of heat shock transcription factor 1 (HSF1), an important transcription factor for heat shock proteins, in the adaptive mechanism of cardiac hypertrophy. We examined expression of 8800 genes in the heart of exercise-induced hypertrophy model using DNA chip technique and compared with pressure overload-induced hypertrophy. Expression of HSF1 and its target molecule heat shock proteins was significantly upregulated in the heart by exercise but not by chronic pressure overload. Constitutive activation of HSF1 in the heart significantly ameliorated death of cardiomyocytes and cardiac fibrosis and thereby prevented cardiac dysfunction as well as hypertrophy induced by chronic pressure overload. Conversely, decreased activity of HSF1 in the heart promoted cardiac dysfunction in response to exercise, a load that normally leads to adaptive hypertrophy with preserved systolic function. Likewise, cardiac function was significantly impaired from the early phase of pressure overload, when HSF1 activation was inhibited. These results suggest that HSF1 plays a critical role in the transition between adaptive and maladaptive hypertrophy.
Asunto(s)
Adaptación Fisiológica/fisiología , Cardiomegalia/fisiopatología , Proteínas de Unión al ADN/genética , Insuficiencia Cardíaca/fisiopatología , Factores de Transcripción/genética , Animales , Aorta Abdominal , Presión Sanguínea , Cardiomegalia/patología , Proteínas de Unión al ADN/metabolismo , Modelos Animales de Enfermedad , Fibrosis , Expresión Génica , Proteínas del Choque Térmico HSP72/genética , Proteínas del Choque Térmico HSP72/metabolismo , Insuficiencia Cardíaca/patología , Factores de Transcripción del Choque Térmico , Ligadura , Masculino , Miocardio/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Tamaño de los Órganos , Esfuerzo Físico , Ratas , Ratas Wistar , Factores de Transcripción/metabolismo , Regulación hacia ArribaRESUMEN
Mesenchymal stem cells (MSCs), which can differentiate into tri-lineage (osteoblast, adipocyte, and chondrocyte) and suppress inflammation, are promising tools for regenerative medicine. MSCs are phenotypically diverse based on their tissue origins. However, the mechanisms underlying cell-type-specific gene expression patterns are not fully understood due to the lack of suitable strategy to identify the diversity. In this study, we investigated gene expression programs and chromatin accessibilities of MSCs by whole-transcriptome RNA-seq analysis and an assay for transposase-accessible chromatin using sequencing (ATAC-seq). We isolated MSCs from four tissues (femoral and vertebral bone marrow, adipose tissue, and lung) and analysed their molecular signatures. RNA-seq identified the expression of MSC markers and both RNA-seq and ATAC-seq successfully clustered the MSCs based on their tissue origins. Interestingly, clustering based on tissue origin was more accurate with chromatin accessibility signatures than with transcriptome profiles. Furthermore, we identified transcription factors potentially involved in establishing cell-type specific chromatin structures. Thus, epigenome analysis is useful to analyse MSC identity and can be utilized to characterize these cells for clinical use.
Asunto(s)
Cromatina/genética , Cromatina/metabolismo , Células Madre Mesenquimatosas/metabolismo , Adipocitos/metabolismo , Adipocitos/fisiología , Tejido Adiposo/metabolismo , Tejido Adiposo/fisiología , Animales , Biomarcadores/metabolismo , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/fisiología , Análisis por Conglomerados , Fémur/metabolismo , Fémur/fisiología , Expresión Génica/genética , Expresión Génica/fisiología , Perfilación de la Expresión Génica/métodos , Pulmón/metabolismo , Pulmón/fisiología , Ratones , Ratones Endogámicos C57BL , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcriptoma/genética , Transcriptoma/fisiologíaRESUMEN
Poly(ADP-ribose) polymerase 1 (PARP1) is involved in DNA repair, chromatin structure, and transcription. However, the mechanisms that regulate PARP1 distribution on DNA are poorly understood. Here, we show that heat shock transcription factor 1 (HSF1) recruits PARP1 through the scaffold protein PARP13. In response to DNA damage, activated and auto-poly-ADP-ribosylated PARP1 dissociates from HSF1-PARP13, and redistributes to DNA lesions and DNA damage-inducible gene loci. Histone deacetylase 1 maintains PARP1 in the ternary complex by inactivating PARP1 through deacetylation. Blocking ternary complex formation impairs redistribution of PARP1 during DNA damage, which reduces gene expression and DNA repair. Furthermore, ternary complex formation and PARP1 redistribution protect cells from DNA damage by promoting DNA repair, and support growth of BRCA1-null mammary tumors, which are sensitive to PARP inhibitors. Our findings identify HSF1 as a regulator of genome integrity and define this function as a guarding mechanism for a specific type of mammary tumorigenesis.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Carcinogénesis/metabolismo , Reparación del ADN , Factores de Transcripción del Choque Térmico/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Neoplasias de la Mama/patología , Carcinogénesis/genética , Daño del ADN , Femenino , Inestabilidad Genómica , Factores de Transcripción del Choque Térmico/genética , Humanos , Ratones , Poli(ADP-Ribosa) Polimerasa-1/genética , Unión Proteica , Proteínas de Unión al ARN/genéticaRESUMEN
Cells cope with temperature elevations, which cause protein misfolding, by expressing heat shock proteins (HSPs). This adaptive response is called the heat shock response (HSR), and it is regulated mainly by heat shock transcription factor (HSF). Among the four HSF family members in vertebrates, HSF1 is a master regulator of HSP expression during proteotoxic stress including heat shock in mammals, whereas HSF3 is required for the HSR in birds. To examine whether only one of the HSF family members possesses the potential to induce the HSR in vertebrate animals, we isolated cDNA clones encoding lizard and frog HSF genes. The reconstructed phylogenetic tree of vertebrate HSFs demonstrated that HSF3 in one species is unrelated with that in other species. We found that the DNA-binding activity of both HSF1 and HSF3 in lizard and frog cells was induced in response to heat shock. Unexpectedly, overexpression of lizard and frog HSF3 as well as HSF1 induced HSP70 expression in mouse cells during heat shock, indicating that the two factors have the potential to induce the HSR. Furthermore, knockdown of either HSF3 or HSF1 markedly reduced HSP70 induction in lizard cells and resistance to heat shock. These results demonstrated that HSF1 and HSF3 cooperatively regulate the HSR at least in lizards, and suggest complex mechanisms of the HSR in lizards as well as frogs.
Asunto(s)
Evolución Molecular , Proteínas de Choque Térmico/genética , Respuesta al Choque Térmico/genética , Lagartos/genética , Animales , Anuros/genética , Anuros/fisiología , Proteínas Aviares/genética , ADN Complementario/genética , Proteínas de Unión al ADN/genética , Factores de Transcripción del Choque Térmico , Calor , Lagartos/fisiología , Filogenia , Transactivadores/genética , Factores de Transcripción/genéticaRESUMEN
OBJECTIVES: This study used Tsumura Suzuki Obese Diabetes (TSOD) mice as a spontaneous type 2 diabetes model and Tsumura Suzuki Non-obesity (TSNO) mice as controls to investigate factors involved in the onset of hearing impairment. METHOD: Body weight, blood glucose levels, and auditory brainstem responses (ABRs) were measured. The cochleae were excised and evaluated histopathologically. RESULTS: The TSOD mice showed significant hyperglycemia at 2-7 months and severe obesity at 5-10 months; significantly elevated ABR thresholds at 8-10 months; and the capillary lumens in the cochlea stria vascularis were narrower in the TSOD mice than in the TSNO mice. At 17 months, India ink vascular staining of the TSOD mice's cochleae revealed decreased capillary density in the stria vascularis. The vascular area of capillaries in the stria vascularis and the vascular area were significantly smaller in TSOD mice. Histopathological analysis showed vessel wall thickening in the modiolus and narrowed capillaries in the stria vascularis, suggesting reduced blood flow to the inner ear. CONCLUSION: The diabetes mice model used in our study showed early age-associated hearing loss, and histopathology showed findings of vessel wall thickening in the modiolus, narrowing of capillaries in the stria vascularis, and chronically reduced blood flow in the cochlea.
Asunto(s)
Cóclea/patología , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Tipo 2/complicaciones , Pérdida Auditiva/etiología , Síndrome Metabólico/complicaciones , Animales , Glucemia , Peso Corporal , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Potenciales Evocados Auditivos del Tronco Encefálico , Expresión Génica , Audición , Síndrome Metabólico/metabolismo , Síndrome Metabólico/patología , Ratones , Obesidad/complicacionesRESUMEN
BACKGROUND: Renal ischemia-reperfusion (I/R) injury is associated with delayed graft function and results in poor long-term graft survival. We previously showed that splenectomy (SPLN) protects the kidney from I/R injury and reduces serum TNF-α levels. Herein, we further investigated the effects of SPLN on inflammatory responses and tissue injury in renal I/R by examining the expression of major inflammatory cytokines and heat shock protein 70 (HSP70). Because it was shown previously that the anti-TNF-α agent infliximab (IFX) attenuated renal I/R injury, we also investigated whether IFX administration mimics the effects of SPLN. METHODS: The left renal pedicles of adult male Wistar rats were clamped for 45 minutes and then reperfused for 24 hours; right nephrectomy and SPLN were performed immediately. A separate cohort was administered IFX 1 hour before surgery in lieu of SPLN. RESULTS: Serum creatinine and blood urea nitrogen levels were markedly elevated by I/R injury; these increases were significantly reversed by IFX. Furthermore, IFX inhibited the induction of inflammatory cytokines and HSP70 during renal I/R injury. Time-dependent profiles revealed that the expression of inflammatory cytokines was elevated immediately after I/R, whereas levels of HSP70, serum creatinine, and blood urea nitrogen began to rise 3 hours postreperfusion. Macrophages/monocytes were significantly increased in I/R-injured kidneys, but not in those administered IFX. The outcomes of SPLN mirrored those of IFX administration. CONCLUSIONS: Splenectomy and TNF-α inhibition both protect the kidney from I/R injury by reducing the accumulation of renal macrophages/monocytes and induction of major inflammatory cytokines.
Asunto(s)
Antiinflamatorios/farmacología , Funcionamiento Retardado del Injerto/prevención & control , Infliximab/farmacología , Trasplante de Riñón/efectos adversos , Riñón/efectos de los fármacos , Daño por Reperfusión/prevención & control , Esplenectomía , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Animales , Biomarcadores/sangre , Nitrógeno de la Urea Sanguínea , Creatinina/sangre , Funcionamiento Retardado del Injerto/sangre , Funcionamiento Retardado del Injerto/inmunología , Funcionamiento Retardado del Injerto/patología , Modelos Animales de Enfermedad , Proteínas HSP70 de Choque Térmico/sangre , Riñón/inmunología , Riñón/metabolismo , Riñón/patología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Monocitos/efectos de los fármacos , Monocitos/inmunología , Monocitos/metabolismo , Ratas Wistar , Daño por Reperfusión/sangre , Daño por Reperfusión/inmunología , Daño por Reperfusión/patología , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Factor de Necrosis Tumoral alfa/sangre , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Heat-shock response is an adaptive response to proteotoxic stresses including heat shock, and is regulated by heat-shock factor 1 (HSF1) in mammals. Proteotoxic stresses challenge all subcellular compartments including the mitochondria. Therefore, there must be close connections between mitochondrial signals and the activity of HSF1. Here, we show that heat shock triggers nuclear translocation of mitochondrial SSBP1, which is involved in replication of mitochondrial DNA, in a manner dependent on the mitochondrial permeability transition pore ANT-VDAC1 complex and direct interaction with HSF1. HSF1 recruits SSBP1 to the promoters of genes encoding cytoplasmic/nuclear and mitochondrial chaperones. HSF1-SSBP1 complex then enhances their induction by facilitating the recruitment of a chromatin-remodelling factor BRG1, and supports cell survival and the maintenance of mitochondrial membrane potential against proteotoxic stresses. These results suggest that the nuclear translocation of mitochondrial SSBP1 is required for the regulation of cytoplasmic/nuclear and mitochondrial proteostasis against proteotoxic stresses.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas Mitocondriales/metabolismo , Factores de Transcripción/metabolismo , Transporte Activo de Núcleo Celular , Secuencia de Aminoácidos , Animales , Núcleo Celular/metabolismo , Supervivencia Celular , Cromatina/química , Cromatina/metabolismo , Citoplasma/metabolismo , ADN Helicasas/metabolismo , ADN Mitocondrial/metabolismo , Células HEK293 , Proteínas HSP70 de Choque Térmico/metabolismo , Células HeLa , Factores de Transcripción del Choque Térmico , Humanos , Potencial de la Membrana Mitocondrial , Ratones , Mitocondrias/metabolismo , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Transporte de Proteínas , Homología de Secuencia de Aminoácido , Temperatura , Transcripción GenéticaRESUMEN
The heat shock response is an evolutionally conserved adaptive response to high temperatures that controls proteostasis capacity and is regulated mainly by an ancient heat shock factor (HSF). However, the regulation of target genes by the stress-inducible HSF1 transcription complex has not yet been examined in detail in mammalian cells. In the present study, we demonstrated that HSF1 interacted with members of the ATF1/CREB family involved in metabolic homeostasis and recruited them on the HSP70 promoter in response to heat shock. The HSF1 transcription complex, including the chromatin-remodeling factor BRG1 and lysine acetyltransferases p300 and CREB-binding protein (CBP), was formed in a manner that was dependent on the phosphorylation of ATF1. ATF1-BRG1 promoted the establishment of an active chromatin state and HSP70 expression during heat shock, whereas ATF1-p300/CBP accelerated the shutdown of HSF1 DNA-binding activity during recovery from acute stress, possibly through the acetylation of HSF1. Furthermore, ATF1 markedly affected the resistance to heat shock. These results revealed the unanticipated complexity of the primitive heat shock response mechanism, which is connected to metabolic adaptation.
Asunto(s)
Factor de Transcripción Activador 1/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Respuesta al Choque Térmico , Factores de Transcripción/metabolismo , Animales , Proteína de Unión a CREB/metabolismo , Células Cultivadas , Cromatina/metabolismo , ADN Helicasas/metabolismo , Fibroblastos/metabolismo , Factores de Transcripción del Choque Térmico , Proteínas de Choque Térmico/metabolismo , Calor , Ratones , Proteínas Nucleares/metabolismo , Fosforilación , Regiones Promotoras Genéticas , Unión Proteica , Interferencia de ARNRESUMEN
BACKGROUND: Despite endometriosis is common estrogen dependent disease afflicting women in reproductive age, the pathogenesis has not been fully elucidated. Retinoic acid has various functions in cells as biologic modulator, and aberrant retinoid metabolism seems to be involved in the lesions of endometriosis. In order to evaluate the potential of all-trans retinoic acid (ATRA) for therapeutic treatment, a transcriptome analysis and estradiol measurements in cultured endometriotic cells and tissues were conducted. METHODS: The mRNA expression levels in ATRA-treated endometriotic stromal cells (ESC) isolated from ovarian endometrial cysts (OEC) were investigated. Estradiol production in OEC tissues was also investigated. RESULTS: In the isolated ESC culture supplemented with ATRA for four days, total RNA was extracted followed by a transcriptome analysis using GeneChip. Forty-nine genes were upregulated and four genes were down-regulated by the ATRA treatment. Many upregulated genes were associated with the negative regulation of cellular proliferation. In addition, ATRA treatment decreased the mRNA expression of 17-beta-dehydrogenase 2 (HSD17B2) which converts estradiol into estrone in a dose-dependent manner, and the ELISA measurements indicated that estradiol production in the OEC tissue was inhibited by ATRA treatment. CONCLUSIONS: Retinoic acid has the potential to suppress endometriosis development.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Endometriosis/tratamiento farmacológico , Estradiol Deshidrogenasas/biosíntesis , Tretinoina/administración & dosificación , Adulto , Endometriosis/genética , Endometriosis/patología , Estradiol/genética , Estradiol Deshidrogenasas/genética , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , ARN Mensajero/biosíntesis , Células del Estroma/efectos de los fármacos , Células del Estroma/patología , Transcriptoma/genéticaRESUMEN
Heat shock proteins (Hsps) are induced in response to various kinds of environmental and physiological stresses. However, it is unclear whether Hsps play roles in protecting cells in the digestive organs against xenobiotic chemicals. Here, we found that feeding induces expression of a set of Hsps specifically in the mouse liver and intestine by activating heat shock transcription factor 1 (HSF1). In the liver, HSF1 is required to suppress toxic effects of electrophiles, which are xenobiotic chemicals causing oxidative stress. We found that overexpression of Hsp27, which elevates cellular glutathione level, promotes survival of culture cells exposed to electrophiles. These results suggest a novel mechanism of cell protection against xenobiotic chemicals in the food.