RESUMO
Hematopoietic stem cell (HSC) divisional fate and function are determined by cellular metabolism, yet the contribution of specific cellular organelles and metabolic pathways to blood maintenance and stress-induced responses in the bone marrow remains poorly understood. The outer mitochondrial membrane-localized E3 ubiquitin ligase MITOL/MARCHF5 (encoded by the Mitol gene) is known to regulate mitochondrial and endoplasmic reticulum (ER) interaction and to promote cell survival. Here, we investigated the functional involvement of MITOL in HSC maintenance by generating MX1-cre inducible Mitol knockout mice. MITOL deletion in the bone marrow resulted in HSC exhaustion and impairment of bone marrow reconstitution capability in vivo. Interestingly, MITOL loss did not induce major mitochondrial dysfunction in hematopoietic stem and progenitor cells. In contrast, MITOL deletion induced prolonged ER stress in HSCs, which triggered cellular apoptosis regulated by IRE1α. In line, dampening of ER stress signaling by IRE1α inihibitor KIRA6 partially rescued apoptosis of long-term-reconstituting HSC. In summary, our observations indicate that MITOL is a principal regulator of hematopoietic homeostasis and protects blood stem cells from cell death through its function in ER stress signaling.
Assuntos
Endorribonucleases , Proteínas Serina-Treonina Quinases , Animais , Camundongos , Apoptose , Células-Tronco Hematopoéticas/metabolismo , Proteínas Serina-Treonina Quinases/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Hematopoietic stem cells (HSCs) are produced from the blood vessel walls and circulate in the blood during the perinatal period. However, the migration dynamics of how HSCs enter the bone marrow remain elusive. To observe the dynamics of HSCs over time, the present study develops an intravital imaging method to visualize bone marrow in neonatal long bones formed by endochondral ossification which is essential for HSC niche formation. Endogenous HSCs are labeled with tdTomato under the control of an HSC marker gene Hlf, and a customized imaging system with a bone penetrating laser is developed for intravital imaging of tdTomato-labeled neonatal HSCs in undrilled tibia, which is essential to avoid bleeding from fragile neonatal tibia by bone drilling. The migration speed of neonatal HSCs is higher than that of adult HSCs. Neonatal HSCs migrate from outside to inside the tibia via the blood vessels that penetrate the bone, which is a transient structure during the neonatal period, and settle on the blood vessel wall in the bone marrow. The results obtained from direct observations in vivo reveal the motile dynamics and colonization process of neonatal HSCs during bone marrow formation.
Assuntos
Medula Óssea , Nicho de Células-Tronco , Osso e Ossos , Diagnóstico por Imagem , Células-Tronco Hematopoéticas , Humanos , Recém-NascidoRESUMO
In order to support bone marrow regeneration after myeloablation, hematopoietic stem cells (HSCs) actively divide to provide both stem and progenitor cells. However, the mechanisms regulating HSC function and cell fate choice during hematopoietic recovery remain unclear. We herein provide novel insights into HSC regulation during regeneration by focusing on mitochondrial metabolism and ATP citrate lyase (ACLY). After 5-fluorouracil-induced myeloablation, HSCs highly expressing endothelial protein C receptor (EPCRhigh ) were enriched within the stem cell fraction at the expense of more proliferative EPCRLow HSCs. These EPCRHigh HSCs were initially more primitive than EPCRLow HSCs and enabled stem cell expansion by enhancing histone acetylation, due to increased activity of ACLY in the early phase of hematopoietic regeneration. In the late phase of recovery, HSCs enhanced differentiation potential by increasing the accessibility of cis-regulatory elements in progenitor cell-related genes, such as CD48. In conditions of reduced mitochondrial metabolism and ACLY activity, these HSCs maintained stem cell phenotypes, while ACLY-dependent histone acetylation promoted differentiation into CD48+ progenitor cells. Collectively, these results indicate that the dynamic control of ACLY-dependent metabolism and epigenetic alterations is essential for HSC regulation during hematopoietic regeneration.
Assuntos
ATP Citrato (pro-S)-Liase , Medula Óssea , ATP Citrato (pro-S)-Liase/genética , ATP Citrato (pro-S)-Liase/metabolismo , Receptor de Proteína C Endotelial/metabolismo , Células-Tronco Hematopoéticas/fisiologia , Histonas/metabolismoRESUMO
Brown and beige adipocytes, which express thermogenic uncoupling protein-1 (UCP1), stimulate glucose and lipid metabolism, improving obesity and metabolic diseases such as type 2 diabetes and hyperlipidemia. Overexpression of cellular repressor of E1A-stimulated genes 1 (CREG1) promotes adipose tissue browning and inhibits diet-induced obesity (DIO) in mice. In this study, we investigated the effects of CREG1 administration on DIO inhibition and adipose browning. Subcutaneous administration of recombinant CREG1 protein to C57BL/6 mice stimulated UCP1 expression in interscapular brown adipose tissue (IBAT) and improved DIO, glucose tolerance and fatty liver compared with those in phosphate-buffered saline-treated mice. Injection of Creg1-expressing adenovirus into inguinal white adipose tissue (IWAT) significantly increased browning and mRNA expression of beige adipocyte marker genes compared with that in mice injected with control virus. The effect of Creg1 induction on beige adipocyte differentiation was supported in primary culture using preadipocytes isolated from IWAT of Creg1-transgenic mice compared with that of wild-type mice. Our results indicate a therapeutic effect of CREG1 on obesity and its associated pathology and a potential of CREG1 to stimulate brown/beige adipocyte formation.
Assuntos
Diabetes Mellitus Tipo 2 , Animais , Dieta , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/tratamento farmacológico , Obesidade/etiologia , TermogêneseRESUMO
Hematopoietic stem cells (HSCs) undergo self-renewal or differentiation to sustain lifelong hematopoiesis. HSCs are preserved in quiescence with low mitochondrial activity. Recent studies indicate that autophagy contributes to HSC quiescence through suppressing mitochondrial metabolism. However, it remains unclear whether autophagy is involved in the regulation of neonatal HSCs, which proliferate actively. In this study, we clarified the role of autophagy in neonatal HSCs using 2 types of autophagy-related gene 7 (Atg7)-conditional knockout mice: Mx1-Cre inducible system and Vav-Cre system. Atg7-deficient HSCs exhibited excess cell divisions with enhanced mitochondrial metabolism, leading to bone marrow failure at adult stage. However, Atg7 deficiency minimally affected hematopoiesis and metabolic state in HSCs at neonatal stage. In addition, Atg7-deficient neonatal HSCs exhibited long-term reconstructing activity, equivalent to wild-type neonatal HSCs. Taken together, autophagy is dispensable for stem cell function and hematopoietic homeostasis in neonates and provide a novel aspect into the role of autophagy in the HSC regulation.
Assuntos
Hematopoese , Células-Tronco Hematopoéticas , Animais , Autofagia , Transtornos da Insuficiência da Medula Óssea , Diferenciação Celular , CamundongosRESUMO
Cellular repressor of E1A-stimulated genes 1 (CREG1) is a secreted glycoprotein that accelerates p16-dependent cellular senescence in vitro. We recently reported the ability of CREG1 to stimulate brown adipogenesis using adipocyte P2-CREG1-transgenic (Tg) mice; however, little is known about the effect of CREG1 on aging-associated phenotypes. In this study, we investigated the effects of CREG1 on age-related obesity and renal dysfunction in Tg mice. Increased brown fat formation was detected in aged Tg mice, in which age-associated metabolic phenotypes such as body weight gain and increases in blood glucose were improved compared with those in wild-type (WT) mice. Blood CREG1 levels increased significantly in WT mice with age, whereas the age-related increase was suppressed, and its levels were reduced, in the livers and kidneys of Tg mice relative to those in WT mice at 25 months. Intriguingly, the mRNA levels of Ink4a, Arf, and senescence-associated secretory phenotype (SASP)-related genes and p38MAPK activity were significantly lowered in the aged kidneys of Tg mice, in which the morphological abnormalities of glomeruli as well as filtering function seen in WT kidneys were alleviated. These results suggest the involvement of CREG1 in kidney aging and its potential as a target for improving age-related renal dysfunction.
Assuntos
Tecido Adiposo Marrom/metabolismo , Envelhecimento/genética , Rim/metabolismo , Obesidade/genética , Proteínas Repressoras/genética , Adipócitos Marrons/metabolismo , Adipócitos Marrons/patologia , Adipogenia/genética , Tecido Adiposo Marrom/patologia , Envelhecimento/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica , Rim/patologia , Testes de Função Renal , Masculino , Camundongos , Camundongos Transgênicos , Obesidade/metabolismo , Obesidade/patologia , Fenótipo , Proteínas Repressoras/metabolismo , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína Desacopladora 1/genética , Proteína Desacopladora 1/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Oxidative stress is one of the major causes of cellular senescence in mammalian cells. The excess amount of reactive oxygen species generated by oxygen metabolism is pathogenic and facilitates tissue aging. Lung tissue is more susceptible to oxidative stress than other organs because it is directly exposed to environmental stresses. The aging of lung tissues increases the risk of chronic diseases. Senescent cells accumulate in tissues during aging and contribute to aging-associated morbidity; however, the roles of cellular senescence in lung aging and diseases have not yet been elucidated in detail. To clarify the physiological role of oxidative stress-induced cellular senescence in aging-associated declines in pulmonary function, we herein investigated the effects of the antioxidant N-acetyl-L-cysteine (NAC) on lung cellular senescence and aging in mice. The administration of NAC to 1-year-old mice reduced the expression of senescence-associated genes in lung tissue. Pulmonary function and lung morphology were partly restored in mice administered NAC. Collectively, these results suggest that oxidative stress is a major inducer of cellular senescence in vivo and that the control of oxidative stress may prevent lung aging and diseases.
Assuntos
Acetilcisteína/farmacologia , Envelhecimento , Antioxidantes/farmacologia , Senescência Celular/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Pulmão/fisiologia , Animais , Células Cultivadas , Senescência Celular/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Feminino , Perfilação da Expressão Gênica , Pulmão/anatomia & histologia , Pulmão/citologia , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo/efeitos dos fármacosRESUMO
The tumor suppressor folliculin (FLCN) suppresses nuclear translocation of TFE3, a master transcription factor for lysosomal biogenesis, via regulation of amino-acid-sensing Rag GTPases. However, the importance of this lysosomal regulation in mammalian physiology remains unclear. Following hematopoietic-lineage-specific Flcn deletion in mice, we found expansion of vacuolated phagocytes that accumulate glycogen in their cytoplasm, phenotypes reminiscent of lysosomal storage disorder (LSD). We report that TFE3 acts in a feedback loop to transcriptionally activate FLCN expression, and FLCN loss disrupts this loop, augmenting TFE3 activity. Tfe3 deletion in Flcn knockout mice reduces the number of phagocytes and ameliorates LSD-like phenotypes. We further reveal that TFE3 stimulates glycogenesis by promoting the expression of glycogenesis genes, including Gys1 and Gyg, upon loss of Flcn. Taken together, we propose that the FLCN-TFE3 feedback loop acts as a rheostat to control lysosome activity and prevents excessive glycogenesis and LSD-like phagocyte activation.
Assuntos
Lisossomos/metabolismo , Fagócitos/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Humanos , Camundongos , Camundongos KnockoutRESUMO
Monocyte-derived fibrocytes recently garnered attention because the novel pathogenesis of myelofibrosis (MF), and suppression of fibrocyte differentiation by serum amyloid P remarkably improved MF. We previously revealed that human fibrocytes highly expressed signaling lymphocytic activation molecule F7 (SLAMF7) compared with macrophages and that SLAMF7high monocytes in the peripheral blood (PB) of MF patients were significantly elevated relative to those in healthy controls (HCs). In this study, we evaluated SLAMF7high monocyte percentage in the PB of HCs, myeloproliferative neoplasm (MPN) patients with MF, and MPN patients without MF by using a cross-sectional approach. We found that MPN patients with MF who harbored JAK2V617F had a significantly elevated SLAMF7high monocyte percentage, which correlated positively with the JAK2V617F allele burden. In addition, the serum concentration of interleukin-1ra (IL-1ra) was significantly correlated with the SLAMF7high monocyte percentage and JAK2V617F allele burden. These findings suggest that both SLAMF7high monocytes and IL-1ra could be useful noninvasive markers of MF onset. Furthermore, the JAK2V617F allele burden of SLAMF7high monocytes was significantly higher than that of SLAMF7low monocytes and could be a potential target of elotuzumab (Elo), an anti-SLAMF7 antibody used for treating multiple myeloma. Elo independently inhibited differentiation of fibrocytes derived not only from HCs but also from MF patients in vitro. Elo also ameliorated MF and splenomegaly induced by romiplostim administration in humanized NOG mice. In conclusion, an increase of SLAMF7high monocytes with higher JAK2V617F allele burden was associated with the onset of MF in MPN patients harboring JAK2V617F, and Elo could be a therapeutic agent for MPN patients with MF who harbor JAK2V617F.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Janus Quinase 2/genética , Monócitos/patologia , Mielofibrose Primária/tratamento farmacológico , Mielofibrose Primária/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Substituição de Aminoácidos , Contagem de Células Sanguíneas , Proliferação de Células , Estudos Transversais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Monócitos/metabolismo , Mutação de Sentido Incorreto , Fenilalanina/genética , Mielofibrose Primária/sangue , Mielofibrose Primária/patologia , Família de Moléculas de Sinalização da Ativação Linfocitária/metabolismo , Valina/genéticaRESUMO
Increased formation of brown and beige adipocytes is critical for adaptive thermogenesis to maintain homeothermy in cold or to circumvent diet-induced obesity (DIO). Cellular repressor of adenovirus early region 1A-stimulated genes 1 (CREG1) exhibits the ability to stimulate brown adipogenesis, including the induction of uncoupling protein 1 (UCP1), in vitro. Thus, we aimed to clarify whether CREG1 promotes brown adipocyte formation and inhibits DIO at the whole-animal level. In mouse brown adipose tissue (BAT), CREG1 expression was markedly increased in cold but was decreased under thermoneutrality, suggesting CREG1 involvement in BAT thermogenesis. Moreover, in BAT and white adipose tissue, expression of UCP1 and fibroblast growth factor-21 and browning were both significantly higher in adipocyte P2-Creg1-transgenic (Tg) mice than in wild-type (WT) littermates. Following stimulation with a ß3-adrenergic agonist, energy consumption was elevated in the Tg mice, which showed increased resistance to DIO and improvement of obesity-associated complications including fatty liver relative to WT mice. The CREG1 stimulatory effect on brown adipogenesis was confirmed in Tg-BAT primary cultures. It was also found that CREG1 binds to retinoid X receptor α, which interacts with thyroid hormone receptor for brown adipogenesis. Our findings demonstrate that CREG1 stimulates brown adipocyte formation and browning, ameliorating obesity and its related pathology in vivo.-Hashimoto, M., Kusudo, T., Takeuchi, T., Kataoka, N., Mukai, T., Yamashita, H. CREG1 stimulates brown adipocyte formation and ameliorates diet-induced obesity in mice.
Assuntos
Adipócitos Marrons/metabolismo , Tecido Adiposo Marrom/metabolismo , Dieta Hiperlipídica/efeitos adversos , Obesidade/metabolismo , Proteínas Repressoras/metabolismo , Adipócitos Marrons/patologia , Tecido Adiposo Marrom/patologia , Animais , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Obesidade/induzido quimicamente , Obesidade/genética , Obesidade/patologia , Proteínas Repressoras/genética , Termogênese , Proteína Desacopladora 1/biossínteseRESUMO
Erythropoiesis is a highly coordinated stepwise process involving the progressive clearance of mitochondria via mitophagy. Based on the expression of several macroautophagy and mitophagy specific genes, we identified a sequential change in the transcriptional pattern during terminal erythroid differentiation. Because erythroid cells are a major source of serum sphingosine-1-phosphate, we analyzed the role of sphingolipid signaling in erythropoiesis and demonstrate that sphingosine kinase activity promotes terminal erythroid differentiation by regulating the expression of key mitophagy genes Pink1 and Bnip3l/Nix. Sphingosine kinase 1 (Sphk1) inhibition also disrupted Pink1-p62 mediated mitochondria clearance in late erythroblasts. Notably, we show that supplementing sphingosine-1-phosphate in vitro can promote erythroid differentiation. Our study clarifies the role of sphingolipid signaling in regulating mitophagy during terminal erythroid differentiation and highlights the potential utility of modulating sphingolipid signaling to facilitate the large-scale production of transfusable red blood cells.
Assuntos
Diferenciação Celular/fisiologia , Eritropoese/fisiologia , Lisofosfolipídeos/metabolismo , Mitofagia/fisiologia , Transdução de Sinais/fisiologia , Esfingosina/análogos & derivados , Animais , Lisofosfolipídeos/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Esfingosina/genética , Esfingosina/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
Most of the hematopoietic stem cells (HSCs) within the bone marrow (BM) show quiescent state with a low mitochondrial membrane potential (ΔΨm). In contrast, upon stress hematopoiesis, HSCs actively start to divide. However, the underlying mechanism for the initiation of HSC division still remains unclear. To elucidate the mechanism underlying the transition of cell cycle state in HSCs, we analyzed the change of mitochondria in HSCs after BM suppression induced by 5-fluoruracil (5-FU). We found that HSCs initiate cell division after exhibiting enhanced ΔΨm as a result of increased intracellular Ca2+ level. Although further activation of Ca2+-mitochondria pathway led to loss of HSCs after cell division, the appropriate suppression of intracellular Ca2+ level by exogenous adenosine or Nifedipine, a Ca2+ channel blocker, prolonged cell division interval in HSCs, and simultaneously achieved both cell division and HSC maintenance. Collectively, our results indicate that the Ca2+-mitochondria pathway induces HSC division critically to determine HSC cell fate.
Assuntos
Cálcio/metabolismo , Divisão Celular , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Mitocôndrias/metabolismo , Adenosina/farmacologia , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Fluoruracila/administração & dosagem , Fluoruracila/farmacologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Nifedipino/farmacologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
Osteoclast differentiation is a dynamic differentiation process, which is accompanied by dramatic changes in metabolic status as well as in gene expression. Recent findings have revealed an essential connection between metabolic reprogramming and dynamic gene expression changes during osteoclast differentiation. However, the upstream regulatory mechanisms that drive these metabolic changes in osteoclastogenesis remain to be elucidated. Here, we demonstrate that induced deletion of a tumor suppressor gene, Folliculin (Flcn), in mouse osteoclast precursors causes severe osteoporosis in 3 weeks through excess osteoclastogenesis. Flcn-deficient osteoclast precursors reveal cell autonomous accelerated osteoclastogenesis with increased sensitivity to receptor activator of NF-κB ligand (RANKL). We demonstrate that Flcn regulates oxidative phosphorylation and purine metabolism through suppression of nuclear localization of the transcription factor Tfe3, thereby inhibiting expression of its target gene Pgc1. Metabolome studies revealed that Flcn-deficient osteoclast precursors exhibit significant augmentation of oxidative phosphorylation and nucleotide production, resulting in an enhanced purinergic signaling loop that is composed of controlled ATP release and autocrine/paracrine purinergic receptor stimulation. Inhibition of this purinergic signaling loop efficiently blocks accelerated osteoclastogenesis in Flcn-deficient osteoclast precursors. Here, we demonstrate an essential and novel role of the Flcn-Tfe3-Pgc1 axis in osteoclastogenesis through the metabolic reprogramming of oxidative phosphorylation and purine metabolism. © 2018 The Authors Journal of Bone and Mineral Research published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research (ASBMR).
Assuntos
Osteoclastos/metabolismo , Osteogênese , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Medula Óssea/patologia , Camundongos , Camundongos Knockout , Biogênese de Organelas , Osteoclastos/patologia , Osteoporose/metabolismo , Osteoporose/patologia , Fosforilação Oxidativa , Purinas/metabolismo , Células RAW 264.7 , Transdução de Sinais , Fatores de Transcrição/metabolismo , Regulação para CimaRESUMO
Hematopoietic homeostasis depends on the maintenance of hematopoietic stem cells (HSCs), which are regulated within a specialized bone marrow (BM) niche. When HSC sense external stimuli, their adhesion status may be critical for determining HSC cell fate. The cell surface molecule, integrin αvß3, is activated through HSC adhesion to extracellular matrix and niche cells. Integrin ß3 signaling maintains HSCs within the niche. Here, we showed the synergistic negative regulation of the pro-inflammatory cytokine interferon-γ (IFNγ) and ß3 integrin signaling in murine HSC function by a novel definitive phenotyping of HSCs. Integrin αvß3 suppressed HSC function in the presence of IFNγ and impaired integrin ß3 signaling mitigated IFNγ-dependent negative action on HSCs. During IFNγ stimulation, integrin ß3 signaling enhanced STAT1-mediated gene expression via serine phosphorylation. These findings show that integrin ß3 signaling intensifies the suppressive effect of IFNγ on HSCs, which indicates that cell adhesion via integrin αvß3 within the BM niche acts as a context-dependent signal modulator to regulate the HSC function under both steady-state and inflammatory conditions.
Assuntos
Proliferação de Células , Células-Tronco Hematopoéticas/fisiologia , Integrina alfaVbeta3/metabolismo , Interferon gama/metabolismo , Animais , Regulação da Expressão Gênica , Camundongos , Fosforilação , Processamento de Proteína Pós-Traducional , Fator de Transcrição STAT1/metabolismo , Transdução de SinaisRESUMO
Senescent cells accumulate in many tissues as animals age and are considered to underlie several aging-associated pathologies. The tumor suppressors p19ARF and p16INK4a, both of which are encoded in the CDKN2A locus, play critical roles in inducing and maintaining permanent cell cycle arrest during cellular senescence. Although the elimination of p16INK4a-expressing cells extends the life span of the mouse, it is unclear whether tissue function is restored by the elimination of senescent cells in aged animals and whether and how p19ARF contributes to tissue aging. The aging-associated decline in lung function is characterized by an increase in compliance as well as pathogenic susceptibility to pulmonary diseases. We herein demonstrated that pulmonary function in 12-month-old mice was reversibly restored by the elimination of p19ARF-expressing cells. The ablation of p19ARF-expressing cells using a toxin receptor-mediated cell knockout system ameliorated aging-associated lung hypofunction. Furthermore, the aging-associated gene expression profile was reversed after the elimination of p19ARF. Our results indicate that the aging-associated decline in lung function was, at least partly, attributed to p19ARF and was recovered by eliminating p19ARF-expressing cells.
Assuntos
Envelhecimento , Senescência Celular/genética , Pulmão/fisiologia , Proteína Supressora de Tumor p14ARF/genética , Animais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Testes de Função RespiratóriaRESUMO
Tunneling nanotubes (TNTs), the long membrane extensions connecting distant cells, have emerged as a novel form of cell-to-cell communication. However, it is not fully understood how and to what extent TNTs contribute to intercellular spread of pathogens including HIV-1. In this study, we show that HIV-1 promotes TNT formation per se via its protein Nef and a cellular protein M-Sec, which appears to mediate approximately half of viral spread among monocyte-derived macrophages (MDMs). A small compound that inhibits M-Sec-induced TNT formation reduced HIV-1 production by almost half in MDMs. Such inhibition was not observed with Nef-deficient mutant HIV-1 that fails to promote TNT formation and replicates less efficiently than the wild-type HIV-1 in MDMs. The TNT inhibitor-sensitive/Nef-promoting viral production was also observed in a T cell line ectopically expressing M-Sec, but not in another M-Sec(-) T cell line. Our results suggest the importance of TNTs in HIV-1 spread among MDMs and might answer the long-standing question how Nef promotes HIV-1 production in a cell type-specific manner.
Assuntos
Comunicação Celular/fisiologia , HIV-1/metabolismo , HIV-1/patogenicidade , Macrófagos/virologia , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo , Western Blotting , Linhagem Celular , Citocinas/metabolismo , Citometria de Fluxo , Imunofluorescência , Humanos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
Fibrocytes (fibroblastic leukocytes) are recently identified as unique hematopoietic cells with features of both macrophages and fibroblasts. Fibrocytes are known to contribute to the remodeling or fibrosis of various injured tissues. However, their role in viral infection is not fully understood. In this study, we show that differentiated fibrocytes are phenotypically distinguishable from macrophages but can be infected with HIV-1. Importantly, fibrocytes exhibited persistently infected cell-like phenotypes, the degree of which was more apparent than macrophages. The infected fibrocytes produced replication-competent HIV-1, but expressed HIV-1 mRNA at low levels and strongly resisted HIV-1-induced cell death, which enabled them to support an extremely long-term HIV-1 production at low but steady levels. More importantly, our results suggested that fibrocytes were susceptible to HIV-1 regardless of their differentiation state, in contrast to the fact that monocytes become susceptible to HIV-1 after the differentiation into macrophages. Our findings indicate that fibrocytes are the previously unreported HIV-1 host cells, and they suggest the importance of considering fibrocytes as one of the long-lived persistently infected cells for curing HIV-1.
Assuntos
Fibroblastos/virologia , HIV-1/fisiologia , Leucócitos/virologia , Macrófagos/virologia , Forma Celular/genética , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação Viral da Expressão Gênica , Infecções por HIV/sangue , HIV-1/genética , Interações Hospedeiro-Patógeno/genética , Humanos , Leucócitos/citologia , Leucócitos/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Microscopia Confocal , Monócitos/citologia , Monócitos/metabolismo , Monócitos/virologia , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Transcriptoma , Replicação Viral/genéticaRESUMO
BACKGROUND: HuR (human antigen R) is a ubiquitously expressed member of the Hu/ELAV family of proteins that is involved in diverse biological processes. HuR has also been shown to play an important role in cell cycle arrest during replicative senescence in both human and mouse cells. Senescent cells not only halt their proliferation, but also activate the secretion of proinflammatory cytokines. A persistent DNA damage response is essential for the senescence-associated secretory phenotype (SASP), and increasing evidence has suggested that the SASP is associated with malignancy. METHODS: Senescence-associated phenotypes were analyzed in MEFs and other cell line in which HuR expression is inhibited by sh-RNA-mediated knockdown. RESULTS: RNAi-mediated HuR inhibition resulted in an increase in SASP-related cytokines. The induction of SASP factors did not depend on ARF-p53 pathway-mediated cell cycle arrest, but required NF-κB activity. In the absence of HuR, cells were defective in the DNA-damage response, and single strand DNA breaks accumulated, which may have caused the activation of NF-κB and subsequent cytokine induction. CONCLUSIONS: In the absence of HuR, cells exhibit multiple senescence-associated phenotypes. Our findings suggest that HuR regulates not only the replicative lifespan, but also the expression of SASP-related cytokines in mouse fibroblasts. GENERAL SIGNIFICANCE: RNA-binding protein HuR protects cells from undergoing senescence. Senescence-associated phenotypes are accelerated in HuR-deficient cells.
Assuntos
Senescência Celular/fisiologia , Citocinas/metabolismo , Proteínas ELAV/metabolismo , Fibroblastos/metabolismo , NF-kappa B/metabolismo , Fatores de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/metabolismo , Animais , Citocinas/genética , Proteínas ELAV/genética , Fibroblastos/citologia , Células HEK293 , Humanos , Camundongos , NF-kappa B/genética , Células NIH 3T3 , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
M-CSF promotes the differentiation and survival of macrophages, and preferentially induces anti-inflammatory M2, rather than proinflammatory M1 macrophages. Recently, another cytokine, IL-32, was also shown to promote macrophage differentiation. In this article, we provide the first evidence, to our knowledge, that M-CSF has both additive and inhibitory effects on the macrophage-related activities of IL-32. When added to M-CSF-derived macrophages, M-CSF and IL-32 promoted macrophage survival, which was further enhanced by their combination. However, they had different effects on HIV-1 replication; that is, it was stimulated by M-CSF and inhibited by IL-32. Interestingly, the anti-HIV-1 activity of IL-32 was counteracted by M-CSF. Such inhibitory effect of M-CSF was not observed with IL-32-induced M1-like features including high cytokine/chemokine production and strong expression of the costimulatory molecule CD80. However, IL-32-treated macrophages unexpectedly showed also M2-like features including increased phagocytic activity, and high expression of CD14 and the scavenger receptor CD163, and the expression of CD14 and CD163 was further upregulated by cotreatment with M-CSF. The findings of this study regarding the unique functional interplay between M-CSF and IL-32 increase our understanding of the mechanisms that regulate the survival and M1/M2 ratio of macrophages, as well as HIV-1 replication in macrophages.