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The senescent microenvironment and aged cells per se contribute to tissue remodeling, chronic inflammation, and age-associated dysfunction. However, the metabolic and epigenomic bases of the senescence-associated secretory phenotype (SASP) remain largely unknown. Here, we show that ATP-citrate lyase (ACLY), a key enzyme in acetyl-coenzyme A (CoA) synthesis, is essential for the pro-inflammatory SASP, independent of persistent growth arrest in senescent cells. Citrate-derived acetyl-CoA facilitates the action of SASP gene enhancers. ACLY-dependent de novo enhancers augment the recruitment of the chromatin reader BRD4, which causes SASP activation. Consistently, specific inhibitions of the ACLY-BRD4 axis suppress the STAT1-mediated interferon response, creating the pro-inflammatory microenvironment in senescent cells and tissues. Our results demonstrate that ACLY-dependent citrate metabolism represents a selective target for controlling SASP designed to promote healthy aging.
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RNA sequencing (RNA-seq) is a powerful technique for understanding cellular state and dynamics. However, comprehensive transcriptomic characterization of multiple RNA-seq datasets is laborious without bioinformatics training and skills. To remove the barriers to sequence data analysis in the research community, we have developed "RNAseqChef" (RNA-seq data controller highlighting expression features), a web-based platform of systematic transcriptome analysis that can automatically detect, integrate, and visualize differentially expressed genes and their biological functions. To validate its versatile performance, we examined the pharmacological action of sulforaphane (SFN), a natural isothiocyanate, on various types of cells and mouse tissues using multiple datasets in vitro and in vivo. Notably, SFN treatment upregulated the ATF6-mediated unfolded protein response in the liver and the NRF2-mediated antioxidant response in the skeletal muscle of diet-induced obese mice. In contrast, the commonly downregulated pathways included collagen synthesis and circadian rhythms in the tissues tested. On the server of RNAseqChef, we simply evaluated and visualized all analyzing data and discovered the NRF2-independent action of SFN. Collectively, RNAseqChef provides an easy-to-use open resource that identifies context-dependent transcriptomic features and standardizes data assessment.
Assuntos
Perfilação da Expressão Gênica , Internet , Isotiocianatos , RNA-Seq , Software , Sulfóxidos , Animais , Camundongos , Perfilação da Expressão Gênica/métodos , Perfilação da Expressão Gênica/normas , Isotiocianatos/farmacologia , Sulfóxidos/farmacologia , RNA-Seq/métodos , RNA-Seq/normas , Especificidade de Órgãos/efeitos dos fármacos , Reprodutibilidade dos Testes , Camundongos Obesos , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Fígado/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Antioxidantes/metabolismo , Visualização de DadosRESUMO
Skeletal muscle exhibits remarkable plasticity in response to environmental cues, with stress-dependent effects on the fast-twitch and slow-twitch fibers. Although stress-induced gene expression underlies environmental adaptation, it is unclear how transcriptional and epigenetic factors regulate fiber type-specific responses in the muscle. Here, we show that flavin-dependent lysine-specific demethylase-1 (LSD1) differentially controls responses to glucocorticoid and exercise in postnatal skeletal muscle. Using skeletal muscle-specific LSD1-knockout mice and in vitro approaches, we found that LSD1 loss exacerbated glucocorticoid-induced atrophy in the fast fiber-dominant muscles, with reduced nuclear retention of Foxk1, an anti-autophagic transcription factor. Furthermore, LSD1 depletion enhanced endurance exercise-induced hypertrophy in the slow fiber-dominant muscles, by induced expression of ERRγ, a transcription factor that promotes oxidative metabolism genes. Thus, LSD1 serves as an 'epigenetic barrier' that optimizes fiber type-specific responses and muscle mass under the stress conditions. Our results uncover that LSD1 modulators provide emerging therapeutic and preventive strategies against stress-induced myopathies such as sarcopenia, cachexia, and disuse atrophy.
Assuntos
Glucocorticoides , Doenças Musculares , Camundongos , Animais , Glucocorticoides/metabolismo , Músculo Esquelético/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Fatores de Transcrição/metabolismo , Histona Desmetilases/genética , Histona Desmetilases/metabolismoRESUMO
Nucleus-mitochondria crosstalk is essential for cellular and organismal homeostasis. Although anterograde (nucleus-to-mitochondria) pathways have been well characterized, retrograde (mitochondria-to-nucleus) pathways remain to be clarified. Here, we found that mitochondrial dysfunction triggered a retrograde signaling via unique transcriptional and chromatin factors in hepatic cells. Our transcriptomic analysis revealed that the loss of mitochondrial transcription factor A led to mitochondrial dysfunction and dramatically induced expression of amphiregulin (AREG) and other secretory protein genes. AREG expression was also induced by various mitochondria stressors and was upregulated in murine liver injury models, suggesting that AREG expression is a hallmark of mitochondrial damage. Using epigenomic and informatic approaches, we identified that mitochondrial dysfunction-responsive enhancers of AREG gene were activated by c-JUN/YAP1/TEAD axis and were repressed by chromatin remodeler BRG1. Furthermore, while mitochondrial dysfunction-activated enhancers were enriched with JUN and TEAD binding motifs, the repressed enhancers possessed the binding motifs for hepatocyte nuclear factor 4α, suggesting that both stress responsible and cell type-specific enhancers were reprogrammed. Our study revealed that c-JUN and YAP1-mediated enhancer activation shapes the mitochondrial stress-responsive phenotype, which may shift from metabolism to stress adaptation including protein secretion under such stressed conditions.
Assuntos
Epigenômica , Mitocôndrias , Anfirregulina/metabolismo , Animais , Cromatina/genética , Cromatina/metabolismo , Fatores Nucleares de Hepatócito/metabolismo , Camundongos , Mitocôndrias/genética , Mitocôndrias/metabolismoRESUMO
Cellular senescence is accompanied by metabolic and epigenomic remodeling, but the transcriptional mechanism of this process is unclear. Our previous RNA interference-based screen of chromatin factors found that lysine methyltransferases including SETD8 and NSD2 inhibited the senescence program in cultured fibroblasts. Here, we report that loss of the zinc finger and homeobox protein 3 (ZHX3), a ubiquitously expressed transcription repressor, induced senescence-associated gene expression and mitochondrial-nucleolar activation. Chromatin immunoprecipitation-sequencing analyses of growing cells revealed that ZHX3 was enriched at the transcription start sites of senescence-associated genes such as the cyclin-dependent kinase inhibitor (ARF-p16INK4a) gene and ribosomal RNA (rRNA) coding genes. ZHX3 expression was consistently downregulated in cells with replicative or oncogene-induced senescence. Mass spectrometry-based proteomics identified 28 proteins that interacted with ZHX3, including ATP citrate lyase and RNA metabolism proteins. Loss of ZHX3 or ZHX3-interaction partners by knockdown similarly induced the expression of p16INK4a and rRNA genes. Zhx3-knockout mice showed upregulation of p16INK4a in the testes, thymus and skeletal muscle tissues, together with relatively short survival periods in males. These data suggested that ZHX3 plays an essential role in transcriptional control to prevent cellular senescence.
Assuntos
Nucléolo Celular/genética , Senescência Celular/genética , Regulação da Expressão Gênica/genética , Expressão Gênica/genética , Proteínas de Homeodomínio/genética , Mitocôndrias/genética , Proteínas Repressoras/genética , Animais , Proliferação de Células/genética , Cromatina/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Replicação do DNA/genética , Regulação para Baixo/genética , Epigenômica/métodos , Feminino , Fibroblastos/fisiologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Ribossômico/genética , Sítio de Iniciação de Transcrição/fisiologia , Regulação para Cima/genéticaRESUMO
Senescent cells may possess the intrinsic programs of metabolic and epigenomic remodeling, but the molecular mechanism remains to be clarified. Using an RNAi-based screen of chromatin regulators, we found that knockdown of the NSD2/WHSC1/MMSET methyltransferase induced cellular senescence that augmented mitochondrial mass and oxidative phosphorylation in primary human fibroblasts. Transcriptome analysis showed that loss of NSD2 downregulated the expression of cell cycle-related genes in a retinoblastoma protein (RB)-mediated manner. Chromatin immunoprecipitation analyses further revealed that NSD2 was enriched at the gene bodies of actively transcribed genes, including cell cycle-related genes, and that loss of NSD2 decreased the levels of histone H3 lysine 36 trimethylation (H3K36me3) at these gene loci. Consistent with these findings, oncogene-induced or replicative senescent cells showed reduced NSD2 expression together with lower H3K36me3 levels at NSD2-enriched genes. In addition, we found that NSD2 gene was upregulated by serum stimulation and required for the induction of cell cycle-related genes. Indeed, in both mouse and human tissues and human cancer cell lines, the expression levels of NSD2 were positively correlated with those of cell cycle-related genes. These data reveal that NSD2 plays a pivotal role in epigenomic maintenance and cell cycle control to prevent cellular senescence.
Assuntos
Senescência Celular/fisiologia , Epigenômica/métodos , Histona-Lisina N-Metiltransferase/metabolismo , Metiltransferases/metabolismo , Proteínas Repressoras/metabolismo , Animais , Humanos , Masculino , CamundongosRESUMO
Recycling endosomes are stations that sort endocytic cargoes to their appropriate destinations. Tubular endosomes have been characterized as a recycling endosomal compartment for clathrin-independent cargoes. However, the molecular mechanism by which tubular endosome formation is regulated is poorly understood. In this study, we identified Rab10 as a novel protein localized at tubular endosomes by using a comprehensive localization screen of EGFP-tagged Rab small GTPases. Knockout of Rab10 completely abolished tubular endosomal structures in HeLaM cells. We also identified kinesin motors KIF13A and KIF13B as novel Rab10-interacting proteins by means of in silico screening. The results of this study demonstrated that both the Rab10-binding homology domain and the motor domain of KIF13A are required for Rab10-positive tubular endosome formation. Our findings provide insight into the mechanism by which the Rab10-KIF13A (or KIF13B) complex regulates tubular endosome formation. This article has an associated First Person interview with the first author of the paper.
Assuntos
Endossomos/metabolismo , Cinesinas/metabolismo , Biogênese de Organelas , Proteínas rab de Ligação ao GTP/metabolismo , Endocitose , Endossomos/ultraestrutura , Técnicas de Inativação de Genes , Células HeLa , Humanos , Ligação Proteica , Proteínas rab de Ligação ao GTP/genéticaRESUMO
The Rab family small GTPases are key players in the membrane traffic that underlies various cellular phenomena. Neurite outgrowth, which is a prerequisite for neuronal network formation, also requires membrane traffic from the cell body to the tips of neurites. Although several Rabs have been shown to promote neurite outgrowth, very little is known about Rab involvement in the negative regulation of neurite outgrowth. Here we used nerve growth factor-stimulated PC12 cells to perform siRNA-based comprehensive knockdown screenings for Rabs that negatively regulate neurite outgrowth and succeeded in identifying Rab20 as a novel negative regulator of neurite outgrowth. Our findings showed that knockdown of endogenous Rab20 in PC12 cells promoted neurite outgrowth, whereas overexpression of active Rab20 inhibited it. We also found that the presence of Gly-64 and Cys-70, both of which are conserved only in the switch II region, a putative effector binding domain, of Rab20 is required for the inhibitory effect of Rab20 on neurite outgrowth. These findings indicated that active Rab20 suppresses neurite outgrowth of PC12 cells, possibly through interaction with an unidentified effector molecule that specifically recognizes certain amino acids in the switch II region of Rab20.
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Neuritos/metabolismo , Crescimento Neuronal/fisiologia , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Células PC12 , Transporte Proteico/fisiologia , RatosRESUMO
Small GTPase Rab35 functions as a molecular switch for membrane trafficking, specifically for endocytic recycling, by cycling between a GTP-bound active form and a GDP-bound inactive form. Although Rab35 has been shown to regulate various cellular processes, including cytokinesis, cell migration, and neurite outgrowth, its precise roles in these processes are not fully understood. Since a molecular tool that could be used to measure Rab35 activity would be useful for identifying the mechanisms by which Rab35 mediates membrane trafficking, we recently used a RUN domain-containing region of RUSC2 to develop an active Rab35 trapper, and we named it RBD35 (Rab-binding domain specific for Rab35). Because RBD35 specifically interacts with the GTP-bound active form of Rab35 and does not interact with any of the other 59 Rab proteins identified in humans and mice, RBD35 is a useful tool for measuring the level of active Rab35 by pull-down assays and for inhibiting the function of Rab35 by overexpression. In this chapter, we describe the assay procedures for analyzing Rab35 with RBD35.
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Bioensaio/métodos , Guanosina Trifosfato/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Células PC12 , Ligação Proteica , Estrutura Terciária de Proteína , Ratos , Especificidade por Substrato , Proteínas rab de Ligação ao GTP/antagonistas & inibidores , Proteínas rab de Ligação ao GTP/químicaRESUMO
The small GTPase Rab35 is a molecular switch for membrane trafficking that regulates a variety of cellular events. We previously showed that Rab35 promotes neurite outgrowth of nerve growth factor-stimulated PC12 cells through interaction with centaurin-ß2 (also called ACAP2). Centaurin-ß2 is the only Rab35-binding protein reported thus far that exclusively recognizes Rab35 and does not recognize any of the other 59 Rabs identified in mammals, but the molecular basis for the exclusive specificity of centaurin-ß2 for Rab35 has remained completely unknown. In this study, we performed deletion and mutation analyses and succeeded in identifying the residues of Rab35 and centaurin-ß2 that are crucial for formation of a Rab35·centaurin-ß2 complex. We found that two threonine residues (Thr-76 and Thr-81) in the switch II region of Rab35 are responsible for binding centaurin-ß2 and that the same residues are dispensable for Rab35 recognition by other Rab35-binding proteins. We also determined the minimal Rab35-binding site of centaurin-ß2 and identified two asparagine residues (Asn-610 and Asn-691) in the Rab35-binding site as key residues for its specific Rab35 recognition. We further showed by knockdown-rescue approaches that neither a centaurin-ß2 binding-deficient Rab35(T76S/T81A) mutant nor a Rab35 binding-deficient centaurin-ß2(N610A/N691A) mutant supported neurite outgrowth of PC12 cells, thereby demonstrating the functional significance of the Rab35/centaurin-ß2 interaction during neurite outgrowth of PC12 cells.
Assuntos
Proteínas Ativadoras de GTPase/fisiologia , Neuritos , Proteínas rab de Ligação ao GTP/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Primers do DNA , Proteínas Ativadoras de GTPase/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Células PC12 , Reação em Cadeia da Polimerase , Ratos , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , Proteínas rab de Ligação ao GTP/química , Proteínas rab de Ligação ao GTP/genéticaRESUMO
Small GTPase Rab35 is an important molecular switch for endocytic recycling that regulates various cellular processes, including cytokinesis, cell migration, and neurite outgrowth. We previously showed that active Rab35 promotes nerve growth factor (NGF)-induced neurite outgrowth of PC12 cells by recruiting MICAL-L1, a multiple Rab-binding protein, to Arf6-positive recycling endosomes. However, the physiological significance of the multiple Rab-binding ability of MICAL-L1 during neurite outgrowth remained completely unknown. Here we report that Rab35 and MICAL-L1 promote the recruitment of Rab8, Rab13, and Rab36 to Arf6-positive recycling endosomes during neurite outgrowth. We found that Rab35 functions as a master Rab that determines the intracellular localization of MICAL-L1, which in turn functions as a scaffold for Rab8, Rab13, and Rab36. We further showed by functional ablation experiments that each of these downstream Rabs regulates neurite outgrowth in a non-redundant manner downstream of Rab35 and MICAL-L1, e.g. by showing that knockdown of Rab36 inhibited recruitment of Rab36-specific effector JIP4 to Arf6-positive recycling endosomes, and caused inhibition of neurite outgrowth without affecting accumulation of Rab8 and Rab13 in the same Arf6-positive area. Our findings suggest the existence of a novel mechanism that recruits multiple Rab proteins at the Arf6-positive compartment by MICAL-L1.
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In lymphocytes, the kinase Mst1 is required for the proper organization of integrins in the plasma membrane at the leading edge of migrating cells, which is critical for lymphocyte trafficking. We found a functional link between the small G protein Rab13 and Mst1 in lymphocyte adhesion and migration. In response to stimulation of T lymphocytes with chemokine, Mst1 promoted phosphorylation of the guanine nucleotide exchange factor DENND1C (differentially expressed in normal and neoplastic cells domain 1C), which activated Rab13. Active Rab13 associated with Mst1 to facilitate the delivery of the integrin LFA-1 (lymphocyte function-associated antigen 1) to the leading edge of lymphocytes. Delivery of LFA-1 involved the recruitment of myosin Va along actin filaments, which extended as a result of the localization of the actin regulatory protein VASP to the cell periphery through phosphorylation of VASP at Ser(157) by Mst1. Inhibition of Rab13 function reduced the adhesion and migration of lymphocytes on ICAM-1 (intercellular adhesion molecule-1), the ligand for LFA-1, and inhibited the formation of a ring-like arrangement of LFA-1 at the contact sites between T cells and antigen-presenting cells. The lymphoid tissues of Rab13-deficient mice had reduced numbers of lymphocytes because of the defective trafficking capability of these cells. These results suggest that Rab13 acts with Mst1 to regulate the spatial distribution of LFA-1 and the motility and trafficking of lymphocytes.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fator de Crescimento de Hepatócito/metabolismo , Antígeno-1 Associado à Função Linfocitária/metabolismo , Linfócitos/citologia , Proteínas Proto-Oncogênicas/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Adesão Celular , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Movimento Celular/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Transporte Proteico/imunologia , Linfócitos T/imunologiaRESUMO
Small GTPase Rab35 is a key regulator of neurite outgrowth, and its activation dramatically enhances nerve growth factor (NGF)-induced neurite outgrowth. We recently reported finding that Rab35 and its effector molecules recruit EHD1, a facilitator of vesicle formation, to Arf6-positive perinuclear recycling endosomes (hereafter simply referred to as recycling endosomes) in response to NGF stimulation. Although Rab35 is likely to promote the formation of transport vesicles from recycling endosomes that contributes to neurite outgrowth, the destination of the vesicles during neurite outgrowth remains unknown. Here we report finding that Rab35 is translocated from recycling endosomes to neurite tips in a late phase of NGF stimulation. We found that Rab35 immunofluorescence signals accumulated at recycling endosomes during the first 6 h, i.e., the early phase of NGF stimulation and then translocated to neurite tips during the late phase of NGF stimulation (i.e., >6 h to <36 h after NGF stimulation). These findings suggest that Rab35 regulates membrane trafficking from recycling endosomes to neurite tips during neurite outgrowth.