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1.
Am J Physiol Regul Integr Comp Physiol ; 321(2): R228-R237, 2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-34189960

RESUMO

The mechanistic target of rapamycin complex 1 (mTORC1) signaling complex is emerging as a critical regulator of cardiovascular function with alterations in this pathway implicated in cardiovascular diseases. In this study, we used animal models and human tissues to examine the role of vascular mTORC1 signaling in the endothelial dysfunction associated with obesity. In mice, obesity induced by high-fat/high-sucrose diet feeding for ∼2 mo resulted in aortic endothelial dysfunction without appreciable changes in vascular mTORC1 signaling. On the other hand, chronic high-fat diet feeding (45% or 60% kcal: ∼9 mo) in mice resulted in endothelial dysfunction associated with elevated vascular mTORC1 signaling. Endothelial cells and visceral adipose vessels isolated from obese humans display a trend toward elevated mTORC1 signaling. Surprisingly, genetic disruption of endothelial mTORC1 signaling through constitutive or tamoxifen inducible deletion of endothelial Raptor (critical subunit of mTORC1) did not prevent or rescue the endothelial dysfunction associated with high-fat diet feeding in mice. Endothelial mTORC1 deficiency also failed to reverse the endothelial dysfunction evoked by a high-fat/high-sucrose diet in mice. Taken together, these data show increased vascular mTORC1 signaling in obesity, but this vascular mTORC1 activation appears not to be required for the development of endothelial impairment in obesity.


Assuntos
Endotélio Vascular/enzimologia , Alvo Mecanístico do Complexo 1 de Rapamicina/deficiência , Obesidade/prevenção & controle , Gordura Subcutânea/irrigação sanguínea , Vasodilatação , Animais , Aorta Torácica/enzimologia , Aorta Torácica/fisiopatologia , Estudos de Casos e Controles , Dieta Hiperlipídica , Sacarose Alimentar , Modelos Animais de Doenças , Endotélio Vascular/fisiopatologia , Humanos , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Artérias Mesentéricas/enzimologia , Artérias Mesentéricas/fisiopatologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/enzimologia , Obesidade/genética , Obesidade/fisiopatologia , Proteína Regulatória Associada a mTOR/deficiência , Proteína Regulatória Associada a mTOR/genética , Transdução de Sinais
2.
FEBS Lett ; 595(3): 360-369, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33247956

RESUMO

The morphological structure and metabolic activity of mitochondria are coordinately regulated by circadian mechanisms. However, the mechanistic interplay between circadian mechanisms and mitochondrial architecture remains poorly understood. Here, we demonstrate circadian rhythmicity of Rheb protein in liver, in line with that of Per2. Using genetic mouse models, we show that Rheb, a small GTPase that binds mTOR, is critical for circadian oscillation of mTORC1 activity in liver. Disruption of Rheb oscillation in hepatocytes by persistent expression of Rheb transgene interrupted mTORC1 oscillation. We further show that Rheb-regulated mTORC1 altered mitochondrial fission factor DRP1 in liver, leading to altered mitochondrial dynamics. Our results suggest that Rheb/mTORC1 regulated DRP1 oscillation involves ubiquitin-mediated proteolysis. This study identifies Rheb as a nodal point that couples circadian clock and mitochondrial architecture for optimal mitochondrial metabolism.


Assuntos
Relógios Circadianos/genética , Dinaminas/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Proteínas Circadianas Period/genética , Proteína Enriquecida em Homólogo de Ras do Encéfalo/genética , Serina-Treonina Quinases TOR/genética , Animais , Dinaminas/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Hepatócitos/metabolismo , Hepatócitos/ultraestrutura , Humanos , Fígado/citologia , Fígado/metabolismo , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Camundongos Transgênicos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Dinâmica Mitocondrial/genética , Proteínas Circadianas Period/metabolismo , Ligação Proteica , Proteína Enriquecida em Homólogo de Ras do Encéfalo/deficiência , Proteína Regulatória Associada a mTOR/deficiência , Proteína Regulatória Associada a mTOR/genética , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo
3.
Elife ; 92020 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-32406817

RESUMO

The transcriptional activation and repression during NK cell ontology are poorly understood. Here, using single-cell RNA-sequencing, we reveal a novel role for T-bet in suppressing the immature gene signature during murine NK cell development. Based on transcriptome, we identified five distinct NK cell clusters and define their relative developmental maturity in the bone marrow. Transcriptome-based machine-learning classifiers revealed that half of the mTORC2-deficient NK cells belongs to the least mature NK cluster. Mechanistically, loss of mTORC2 results in an increased expression of signature genes representing immature NK cells. Since mTORC2 regulates the expression of T-bet through AktS473-FoxO1 axis, we further characterized the T-bet-deficient NK cells and found an augmented immature transcriptomic signature. Moreover, deletion of Foxo1 restores the expression of T-bet and corrects the abnormal expression of immature NK genes. Collectively, our study reveals a novel role for mTORC2-AktS473-FoxO1-T-bet axis in suppressing the transcriptional signature of immature NK cells.


Assuntos
Células da Medula Óssea/metabolismo , Perfilação da Expressão Gênica , Células Matadoras Naturais/metabolismo , Aprendizado de Máquina , RNA-Seq , Análise de Célula Única , Proteínas com Domínio T/genética , Transcriptoma , Animais , Células da Medula Óssea/imunologia , Análise por Conglomerados , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Regulação da Expressão Gênica , Genótipo , Células Matadoras Naturais/imunologia , Alvo Mecanístico do Complexo 2 de Rapamicina/deficiência , Alvo Mecanístico do Complexo 2 de Rapamicina/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína Companheira de mTOR Insensível à Rapamicina/deficiência , Proteína Companheira de mTOR Insensível à Rapamicina/genética , Proteína Regulatória Associada a mTOR/deficiência , Proteína Regulatória Associada a mTOR/genética , Proteínas com Domínio T/metabolismo
4.
Endocrinology ; 161(5)2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32154868

RESUMO

The mechanistic target of the rapamycin (mTOR) pathway plays a role in features common to both excess salt/aldosterone and cardiovascular/renal diseases. Dietary sodium can upregulate mTORC1 signaling in cardiac and renal tissue, and the inhibition of mTOR can prevent aldosterone-associated, salt-induced hypertension. The impact of sex and age on mTOR's role in volume homeostasis and the regulation of aldosterone secretion is largely unknown. We hypothesize that both age and sex modify mTOR's interaction with volume homeostatic mechanisms. The activity of 3 volume homeostatic mechanisms-cardiovascular, renal, and hormonal (aldosterone [sodium retaining] and brain natriuretic peptide [BNP; sodium losing])-were assessed in mTORC1 deficient (Raptor+/-) and wild-type male and female littermates at 2 different ages. The mice were volume stressed by being given a liberal salt (LibS) diet. Raptor+/-mice of both sexes when they aged: (1) reduced their blood pressure, (2) increased left ventricular internal diameter during diastole, (3) decreased renal blood flow, and (4) increased mineralocorticoid receptor expression. Aldosterone levels did not differ by sex in young Raptor+/- mice. However, as they aged, compared to their littermates, aldosterone decreased in males but increased in females. Finally, given the level of Na+ intake, BNP was inappropriately suppressed, but only in Raptor+/- males. These data indicate that Raptor+/- mice, when stressed with a LibS diet, display inappropriate volume homeostatic responses, particularly with aging, and the mechanisms altered, differing by sex.


Assuntos
Homeostase/efeitos dos fármacos , Rim/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/deficiência , Miocárdio/metabolismo , Proteína Regulatória Associada a mTOR/deficiência , Sódio na Dieta/farmacologia , Aldosterona/metabolismo , Animais , Pressão Sanguínea/efeitos dos fármacos , Feminino , Hipertensão/fisiopatologia , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Proteína Regulatória Associada a mTOR/genética , Fatores Sexuais , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Fatores de Tempo
5.
Cell Death Differ ; 27(7): 2248-2262, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32001780

RESUMO

The metabolism-controlled differentiation of αß T cells has been well documented; however, the role of a metabolism program in γδ T cell differentiation and function has not been clarified. Here, using CD2-cre; mTORC1 Raptor-f/f, and mTORC2 Rictor-f/f mice (KO mice), we found that mTORC1, but not mTORC2, was required for the proliferation and survival of peripheral γδ T cells, especially Vγ4 γδ T cells. Moreover, mTORC1 was essential for both γδ T1 and γδ Τ17 differentiation, whereas mTORC2 was required for γδ T17, but not for γδ Τ1, differentiation. We further studied the underlying molecular mechanisms and found that depletion of mTORC1 resulted in the increased expression of SOCS1, which in turn suppressed the key transcription factor Eomes, consequentially reducing IFN-γ production. Whereas the reduced glycolysis resulted in impaired γδ Τ17 differentiation in Raptor KO γδ T cells. In contrast, mTORC2 potentiated γδ Τ17 induction by suppressing mitochondrial ROS (mitoROS) production. Consistent with their cytokine production profiles, the Raptor KO γδ T cells lost their anti-tumor function both in vitro and in vivo, whereas both Raptor and Rictor KO mice were resistant to imiquimod (IMQ)-induced psoriasis-like skin pathogenesis. In summary, we identified previously unknown functions of mTORC1 and mTORC2 in γδ T cell differentiation and clarified their divergent roles in mediating the activity of γδ T cells in tumors and autoimmunity.


Assuntos
Diferenciação Celular , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Células Th1/citologia , Células Th1/imunologia , Células Th17/citologia , Células Th17/imunologia , Animais , Modelos Animais de Doenças , Glicólise , Interferon gama/biossíntese , Contagem de Linfócitos , Camundongos Knockout , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Neoplasias/imunologia , Psoríase/patologia , Proteína Regulatória Associada a mTOR/deficiência , Proteína Regulatória Associada a mTOR/metabolismo , Transdução de Sinais , Proteína 1 Supressora da Sinalização de Citocina/metabolismo , Proteínas com Domínio T/metabolismo , Regulação para Cima
6.
Prostate ; 80(5): 412-423, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31995655

RESUMO

BACKGROUND: Mammalian target of rapamycin (mTOR) is a downstream substrate activated by PI3K/AKT pathway and it is essential for cell migration. It exists as two complexes: mTORC1 and mTORC2. mTORC1 is known to be regulated by active AKT, but the activation of mTORC2 is poorly understood. In this study, we investigated the roles and differential activation of the two mTOR complexes during cell migration in prostate cancer cells. METHODS: We used small interfering RNA to silence the expression of Rac1 and the main components of mTOR complexes (regulatory associated protein of mTOR [RAPTOR] and rapamycin-insensitive companion of mTOR [RICTOR]) in LNCaP, DU145, and PC3 prostate cancer cell lines. We performed transwell migration assay to evaluate the migratory capability of the cells, and Western blot analysis to study the activation levels of mTOR complexes. RESULTS: Specific knockdown of RAPTOR and RICTOR caused a decrease of cell migration, suggesting their essential role in prostate cancer cell movement. Furthermore, epidermal growth factor (EGF) treatments induced the activation of both the mTOR complexes. Lack of Rac1 activity in prostate cancer cells blocked EGF-induced activation of mTORC2, but had no effect on mTORC1 activation. Furthermore, the overexpression of constitutively active Rac1 resulted in significant increase in cell migration and activation of mTORC2 in PC3 cells, but had no effect on mTORC1 activation. Active Rac1 was localized in the plasma membrane and was found to be in a protein complex, with RICTOR, but not RAPTOR. CONCLUSION: We suggest that EGF-induced activation of Rac1 causes the activation of mTORC2 via RICTOR. This mechanism plays a critical role in prostate cancer cell migration.


Assuntos
Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Aminoquinolinas/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Fator de Crescimento Epidérmico/farmacologia , Técnicas de Silenciamento de Genes , Humanos , Masculino , Células PC-3 , Pirimidinas/farmacologia , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Proteína Companheira de mTOR Insensível à Rapamicina/deficiência , Proteína Companheira de mTOR Insensível à Rapamicina/genética , Proteína Companheira de mTOR Insensível à Rapamicina/metabolismo , Proteína Regulatória Associada a mTOR/deficiência , Proteína Regulatória Associada a mTOR/genética , Proteína Regulatória Associada a mTOR/metabolismo , Sirolimo/farmacologia , Proteínas rac1 de Ligação ao GTP/antagonistas & inibidores , Proteínas rac1 de Ligação ao GTP/metabolismo
7.
Autophagy ; 16(9): 1668-1682, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-31840569

RESUMO

Dysregulation of macroautophagy/autophagy is implicated in obesity and insulin resistance. However, it remains poorly defined how autophagy regulates adipocyte development. Using adipose-specific rptor/raptor knockout (KO), atg7 KO and atg7 rptor double-KO mice, we show that inhibiting MTORC1 by RPTOR deficiency led to autophagic sequestration of lipid droplets, formation of LD-containing lysosomes, and elevation of basal and isoproterenol-induced lipolysis in vivo and in primary adipocytes. Despite normal differentiation at an early phase, progressive degradation and shrinkage of cellular LDs and downregulation of adipogenic markers PPARG and PLIN1 occurred in terminal differentiation of rptor KO adipocytes, which was rescued by inhibiting lipolysis or lysosome. In contrast, inactivating autophagy by depletion of ATG7 protected adipocytes against RPTOR deficiency-induced formation of LD-containing lysosomes, LD degradation, and downregulation of adipogenic markers in vitro. Ultimately, atg7 rptor double-KO mice displayed decreased lipolysis, restored adipose tissue development, and upregulated thermogenic gene expression in brown and inguinal adipose tissue compared to RPTOR-deficient mice in vivo. Collectively, our study demonstrates that autophagy plays an important role in regulating adipocyte maturation via a lipophagy and lipolysis-dependent mechanism. ABBREVIATIONS: ATG7: autophagy related 7; BAT: brown adipose tissue; CEBPB/C/EBPß: CCAAT enhancer binding protein beta; DGAT1: diacylglycerol O-acyltransferase 1; eWAT: epididymal white adipose tissue; iWAT: inguinal white adipose tissue; KO: knockout; LD: lipid droplet; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; MTORC1: mechanistic target of rapamycin kinase complex 1; PLIN1: perepilin 1; PNPLA2/ATGL: patatin-like phospholipase domain containing 2; PPARG/PPARγ: peroxisome proliferator activated receptor gamma; RPTOR: regulatory associated protein of MTOR complex1; TG: triglyceride; ULK1: unc-51 like kinase 1; UCP1: uncoupling protein 1; WAT: white adipose tissue.


Assuntos
Adipócitos/citologia , Adipócitos/metabolismo , Autofagia , Lipólise , Adipócitos/efeitos dos fármacos , Adipócitos/ultraestrutura , Tecido Adiposo Marrom/efeitos dos fármacos , Tecido Adiposo Marrom/metabolismo , Animais , Autofagia/efeitos dos fármacos , Proteína 7 Relacionada à Autofagia/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Isoproterenol/farmacologia , Gotículas Lipídicas/metabolismo , Lipólise/efeitos dos fármacos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos Knockout , Proteína Regulatória Associada a mTOR/deficiência , Proteína Regulatória Associada a mTOR/metabolismo , Termogênese/efeitos dos fármacos , Termogênese/genética
8.
Cell Mol Gastroenterol Hepatol ; 7(1): 211-231, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30539788

RESUMO

BACKGROUND & AIMS: Nonalcoholic steatohepatitis (NASH) is an increasingly prevalent nonalcoholic fatty liver disease, characterized by inflammatory cell infiltration and hepatocellular damage. Mammalian target of rapamycin complex 1 (mTORC1) has been investigated extensively in the context of cancer, including hepatocellular carcinoma. However, the role of mTORC1 in NASH remains largely unknown. METHODS: mTORC1 activity in macrophages in human mild and severe NASH liver was compared. Mice with macrophage-specific deletion of the regulatory-associated protein of mTOR (Raptor) subunit and littermate controls were fed a high-fructose, palmitate, and cholesterol diet for 24 weeks or a methionine- and choline-deficient diet for 4 weeks to develop NASH. RESULTS: We report that in human beings bearing NASH, macrophage mTORC1 activity was lower in livers experiencing severe vs mild NASH liver. Moreover, macrophage mTORC1 disruption exacerbated the inflammatory response in 2 diet-induced NASH mouse models. Mechanistically, in response to apoptotic hepatocytes (AHs), macrophage polarization toward a M2 anti-inflammatory phenotype was inhibited in Raptor-deficient macrophages. During the digestion of AHs, macrophage mTORC1 was activated and coupled with dynamin-related protein 1 to facilitate the latter's phosphorylation, leading to mitochondrial fission-mediated calcium release. Ionomycin or A23187, calcium ionophores, prevented Raptor deficiency-mediated failure of lysosome acidification and subsequent lipolysis. Blocking dynamin-related protein 1-dependent mitochondria fission impaired lysosome function, resulting in reduced production of anti-inflammatory factors such as interleukins 10 and 13. CONCLUSIONS: Persistent mTORC1 deficiency in macrophages contributes to the progression of NASH by causing lysosome dysfunction and subsequently attenuating anti-inflammatory M2-like response in macrophages during clearance of AHs.


Assuntos
Progressão da Doença , Lisossomos/patologia , Macrófagos Peritoneais/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Proteína Regulatória Associada a mTOR/deficiência , Animais , Apoptose , Cálcio/metabolismo , Colesterol , Colina , Dieta , Dinaminas/metabolismo , Comportamento Alimentar , Frutose , GTP Fosfo-Hidrolases/metabolismo , Deleção de Genes , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Inflamação/patologia , Lipólise , Fígado/metabolismo , Fígado/patologia , Lisossomos/metabolismo , Macrófagos Peritoneais/patologia , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Metionina/deficiência , Camundongos Endogâmicos C57BL , Camundongos Knockout , Palmitatos , Fagossomos/metabolismo , Fosforilação , Fosfosserina/metabolismo , Proteína Regulatória Associada a mTOR/metabolismo
9.
Nature ; 565(7737): 101-105, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30568299

RESUMO

A defining feature of adaptive immunity is the development of long-lived memory T cells to curtail infection. Recent studies have identified a unique stem-like T-cell subset amongst exhausted CD8-positive T cells in chronic infection1-3, but it remains unclear whether CD4-positive T-cell subsets with similar features exist in chronic inflammatory conditions. Amongst helper T cells, TH17 cells have prominent roles in autoimmunity and tissue inflammation and are characterized by inherent plasticity4-7, although how such plasticity is regulated is poorly understood. Here we demonstrate that TH17 cells in a mouse model of autoimmune disease are functionally and metabolically heterogeneous; they contain a subset with stemness-associated features but lower anabolic metabolism, and a reciprocal subset with higher metabolic activity that supports transdifferentiation into TH1-like cells. These two TH17-cell subsets are defined by selective expression of the transcription factors TCF-1 and T-bet, and by discrete levels of CD27 expression. We also identify signalling via the kinase complex mTORC1 as a central regulator of TH17-cell fate decisions by coordinating metabolic and transcriptional programmes. TH17 cells with disrupted mTORC1 signalling or anabolic metabolism fail to induce autoimmune neuroinflammation or to develop into TH1-like cells, but instead upregulate TCF-1 expression and acquire stemness-associated features. Single-cell RNA sequencing and experimental validation reveal heterogeneity in fate-mapped TH17 cells, and a developmental arrest in the TH1 transdifferentiation trajectory upon loss of mTORC1 activity or metabolic perturbation. Our results establish that the dichotomy of stemness and effector function underlies the heterogeneous TH17 responses and autoimmune pathogenesis, and point to previously unappreciated metabolic control of plasticity in helper T cells.


Assuntos
Transdiferenciação Celular , Células-Tronco/citologia , Células-Tronco/metabolismo , Células Th17/citologia , Células Th17/metabolismo , Animais , Doenças Autoimunes/imunologia , Doenças Autoimunes/metabolismo , Doenças Autoimunes/patologia , Modelos Animais de Doenças , Feminino , Memória Imunológica/imunologia , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Proteína Regulatória Associada a mTOR/deficiência , Proteína Regulatória Associada a mTOR/genética , Análise de Sequência de RNA , Transdução de Sinais , Análise de Célula Única , Células-Tronco/imunologia , Fator 1 de Transcrição de Linfócitos T/biossíntese , Fator 1 de Transcrição de Linfócitos T/metabolismo , Proteínas com Domínio T/biossíntese , Proteínas com Domínio T/metabolismo , Células Th17/imunologia , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo
10.
Nat Commun ; 9(1): 4874, 2018 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-30451838

RESUMO

The metabolic checkpoint kinase mechanistic/mammalian target of rapamycin (mTOR) regulates natural killer (NK) cell development and function, but the exact underlying mechanisms remain unclear. Here, we show, via conditional deletion of Raptor (mTORC1) or Rictor (mTORC2), that mTORC1 and mTORC2 promote NK cell maturation in a cooperative and non-redundant manner, mainly by controlling the expression of Tbx21 and Eomes. Intriguingly, mTORC1 and mTORC2 regulate cytolytic function in an opposing way, exhibiting promoting and inhibitory effects on the anti-tumor ability and metabolism, respectively. mTORC1 sustains mTORC2 activity by maintaining CD122-mediated IL-15 signaling, whereas mTORC2 represses mTORC1-modulated NK cell effector functions by restraining STAT5-mediated SLC7A5 expression. These positive and negative crosstalks between mTORC1 and mTORC2 signaling thus variegate the magnitudes and kinetics of NK cell activation, and help define a paradigm for the modulation of NK maturation and effector functions.


Assuntos
Células Matadoras Naturais/imunologia , Proteína Companheira de mTOR Insensível à Rapamicina/genética , Proteína Regulatória Associada a mTOR/genética , Proteínas com Domínio T/genética , Animais , Diferenciação Celular , Regulação da Expressão Gênica , Humanos , Interleucina-15/genética , Interleucina-15/imunologia , Subunidade beta de Receptor de Interleucina-2/genética , Subunidade beta de Receptor de Interleucina-2/imunologia , Células Matadoras Naturais/citologia , Transportador 1 de Aminoácidos Neutros Grandes/genética , Transportador 1 de Aminoácidos Neutros Grandes/imunologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteína Companheira de mTOR Insensível à Rapamicina/deficiência , Proteína Companheira de mTOR Insensível à Rapamicina/imunologia , Proteína Regulatória Associada a mTOR/deficiência , Proteína Regulatória Associada a mTOR/imunologia , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/imunologia , Transdução de Sinais , Proteínas com Domínio T/imunologia
11.
Sci Rep ; 8(1): 14501, 2018 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-30266921

RESUMO

Skeletal osteoblasts are important regulators of B-lymphopoiesis, serving as a rich source of factors such as CXCL12 and IL-7 which are crucial for B-cell development. Recent studies from our laboratory and others have shown that deletion of Rptor, a unique component of the mTORC1 nutrient-sensing complex, early in the osteoblast lineage development results in defective bone development in mice. In this study, we now demonstrate that mTORC1 signalling in pre-osteoblasts is required for normal B-lymphocyte development in mice. Targeted deletion of Rptor in osterix-expressing pre-osteoblasts (Rptorob-/-) leads to a significant reduction in the number of B-cells in the bone marrow, peripheral blood and spleen at 4 and 12 weeks of age. Rptorob-/- mice also exhibit a significant reduction in pre-B and immature B-cells in the BM, indicative of a block in B-cell development from the pro-B to pre-B cell stage. Circulating levels of IL-7 and CXCL12 are also significantly reduced in Rptorob-/- mice. Importantly, whilst Rptor-deficient osteoblasts are unable to support HSC differentiation to B-cells in co-culture, this can be rescued by the addition of exogenous IL-7 and CXCL12. Collectively, these findings demonstrate that mTORC1 plays an important role in extrinsic osteoblastic regulation of B-cell development.


Assuntos
Linfócitos B/citologia , Linfopoese/fisiologia , Alvo Mecanístico do Complexo 1 de Rapamicina/fisiologia , Osteoblastos/metabolismo , Animais , Linfócitos B/metabolismo , Quimiocina CXCL12/biossíntese , Quimiocina CXCL12/sangue , Quimiocina CXCL12/farmacologia , Técnicas de Cocultura , Regulação para Baixo , Genes Reporter , Interleucina-7/sangue , Interleucina-7/farmacologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , RNA Mensageiro/biossíntese , Proteína Regulatória Associada a mTOR/deficiência , Proteína Regulatória Associada a mTOR/genética , Proteína Regulatória Associada a mTOR/fisiologia , Fator de Transcrição Sp7/metabolismo
12.
Sci Rep ; 7(1): 5580, 2017 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-28717211

RESUMO

Intestinal tuft cells are one of 4 secretory cell linages in the small intestine and the source of IL-25, a critical initiator of the type 2 immune response to parasite infection. When Raptor, a critical scaffold protein for mammalian target of rapamycin complex 1 (mTORC1), was acutely deleted in intestinal epithelium via Tamoxifen injection in Tritrichomonas muris (Tm) infected mice, tuft cells, IL-25 in epithelium and IL-13 in the mesenchyme were significantly reduced, but Tm burden was not affected. When Tm infected mice were treated with rapamycin, DCLK1 and IL-25 expression in enterocytes and IL-13 expression in mesenchyme were diminished. After massive small bowel resection, tuft cells and Tm were diminished due to the diet used postoperatively. The elimination of Tm and subsequent re-infection of mice with Tm led to type 2 immune response only in WT, but Tm colonization in both WT and Raptor deficient mice. When intestinal organoids were stimulated with IL-4, tuft cells and IL-25 were induced in both WT and Raptor deficient organoids. In summary, our study reveals that enterocyte specific Raptor is required for initiating a type 2 immune response which appears to function through the regulation of mTORC1 activity.


Assuntos
Enterócitos/citologia , Intestino Delgado/citologia , Infecções Protozoárias em Animais/imunologia , Proteína Regulatória Associada a mTOR/deficiência , Sirolimo/administração & dosagem , Tritrichomonas/imunologia , Animais , Quinases Semelhantes a Duplacortina , Regulação para Baixo , Enterócitos/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Imunidade nas Mucosas/efeitos dos fármacos , Interleucina-13/genética , Interleucina-13/metabolismo , Interleucinas/genética , Interleucinas/metabolismo , Intestino Delgado/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Infecções Protozoárias em Animais/tratamento farmacológico , Sirolimo/farmacologia , Tamoxifeno/administração & dosagem , Tamoxifeno/farmacologia
13.
Cell Death Differ ; 24(11): 1886-1899, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28686577

RESUMO

The mammalian target of rapamycin (mTOR)/regulatory-associated protein of mTOR (Raptor) pathway transmits and integrates different signals including growth factors, nutrients, and energy metabolism. Nearly all these signals have been found to play roles in skeletal biology. However, the contribution of mTOR/Raptor to osteoblast biology in vivo remains to be elucidated as the conclusions of recent studies are controversial. Here we report that mice with a deficiency of either mTOR or Raptor in preosteoblasts exhibited clavicular hypoplasia and delayed fontanelle fusion, similar to those found in human patients with cleidocranial dysplasia (CCD) haploinsufficient for the transcription factor runt-related transcription factor 2 (Runx2) or those identified in Runx2+/- mice. Mechanistic analysis revealed that the mTOR-Raptor-S6K1 axis regulates Runx2 expression through phosphorylation of estrogen receptor α, which binds to Distal-less homeobox 5 (DLX5) and augments the activity of Runx2 enhancer. Moreover, heterozygous mutation of raptor in osteoblasts aggravates the bone defects observed in Runx2+/- mice, indicating a genetic interaction between Raptor and Runx2. Collectively, these findings reveal that mTOR/Raptor signaling is essential for bone formation in vivo through the regulation of Runx2 expression. These results also suggest that a selective mTOR/Raptor antagonist, which has been developed for treatment of many diseases, may have the side effect of causing bone loss.


Assuntos
Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Osteogênese , Proteína Regulatória Associada a mTOR/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Animais , Osso e Ossos/patologia , Diferenciação Celular , Displasia Cleidocraniana/metabolismo , Displasia Cleidocraniana/patologia , Elementos Facilitadores Genéticos/genética , Epistasia Genética , Camundongos , Tamanho do Órgão , Osteoblastos/metabolismo , Fenótipo , Proteína Regulatória Associada a mTOR/deficiência , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Serina-Treonina Quinases TOR/deficiência
14.
J Bone Miner Res ; 32(9): 1829-1840, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28520214

RESUMO

The mechanistic target of rapamycin complex 1 (mTORC1) is a critical sensor for bone homeostasis and bone formation; however, the role of mTORC1 in osteoclast development and the underlying mechanisms have not yet been fully established. Here, we found that mTORC1 activity declined during osteoclast precursors differentiation in vitro and in vivo. We further targeted deletion of Raptor (mTORC1 key component) or Tsc1 (mTORC1 negative regulator) to constitutively inhibit or activate mTORC1 in osteoclast precursors (monocytes/macrophages), using LyzM-cre mice. Osteoclastic formation was drastically increased in cultures of Raptor deficient bone marrow monocytes/macrophages (BMMs), and Raptor-deficient mice displayed osteopenia with enhanced osteoclastogenesis. Conversely, BMMs lacking Tsc1 exhibited a severe defect in osteoclast-like differentiation and absorptive function, both of which were restored following rapamycin treatment. Importantly, expression of nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) and nuclear factor of activated T cells, cytoplasmic 1 (NFATc1), transcription factors that are essential for osteoclast differentiation was negatively regulated by mTORC1 in osteoclast lineages. These results provide evidence that mTORC1 plays as a critical role as an osteoclastic differentiation-limiting signal and suggest a potential drawback in treating bone loss-related diseases with mTOR inhibitors clinically. © 2017 American Society for Bone and Mineral Research.


Assuntos
Diferenciação Celular , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , NF-kappa B/metabolismo , Fatores de Transcrição NFATC/metabolismo , Osteoclastos/metabolismo , Transdução de Sinais , Animais , Doenças Ósseas Metabólicas/genética , Doenças Ósseas Metabólicas/metabolismo , Doenças Ósseas Metabólicas/patologia , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Camundongos , Camundongos Knockout , NF-kappa B/genética , Fatores de Transcrição NFATC/genética , Osteoclastos/patologia , Células RAW 264.7 , Proteína Regulatória Associada a mTOR/deficiência , Proteína 1 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/deficiência
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