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Mammalian target of rapamycin (mTOR) is an important mediator of phosphoinositol-3-kinase (PI3K) signaling. PI3K signaling regulates B cell development, homeostasis, and immune responses. However, the function and molecular mechanism of mTOR-mediated PI3K signaling in B cells has not been fully elucidated. Here we show that Sin1, an essential component of mTOR complex 2 (mTORC2), regulates B cell development. Sin1 deficiency results in increased IL-7 receptor (il7r) and RAG recombinase (rag1 and rag2) gene expression, leading to enhanced pro-B cell survival and augmented V(D)J recombinase activity. We further show that Akt2 specifically mediates the Sin1-mTORC2 dependent suppression of il7r and rag gene expression in B cells by regulating FoxO1 phosphorylation. Finally, we demonstrate that the mTOR inhibitor rapamycin induces rag expression and promotes V(D)J recombination in B cells. Our study reveals that the Sin1/mTORC2-Akt2 signaling axis is a key regulator of FoxO1 transcriptional activity in B cells.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Linfocitos B/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica/fisiología , Proteínas de Homeodominio/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores de Interleucina-7/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Linfocitos B/citología , Línea Celular Transformada , Proteínas de Unión al ADN/genética , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Reordenamiento Génico de Linfocito B/fisiología , Proteínas de Homeodominio/genética , Ratones , Ratones Noqueados , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Receptores de Interleucina-7/genética , Transducción de Señal/fisiología , Factores de TranscripciónRESUMEN
Mesenchymal stem/stromal cells (MSCs) have great clinical potential in modulating inflammation and promoting tissue repair. Human embryonic stem cells (hESCs) have recently emerged as a potentially superior cell source for MSCs. However, the generation methods reported so far vary greatly in quality and efficiency. Here, we describe a novel method to rapidly and efficiently produce MSCs from hESCs via a trophoblast-like intermediate stage in approximately 11-16 days. We term these cells "T-MSCs" and show that T-MSCs express a phenotype and differentiation potential minimally required to define MSCs. T-MSCs exhibit potent immunomodulatory activity in vitro as they can remarkably inhibit proliferation of cocultured T and B lymphocytes. Unlike bone marrow MSCs, T-MSCs do not have increased expression of inflammatory mediators in response to IFNγ. Moreover, T-MSCs constitutively express a high level of the immune inhibitory ligand PD-L1 and elicit strong and durable efficacy in two distinct animal models of autoimmune disease, dextran sulfate sodium induced colitis, and experimental autoimmune encephalomyelitis, at doses near those approved for clinical trials. Together, we present a simple and fast derivation method to generate MSCs from hESCs, which possess potent immunomodulatory properties in vitro and in vivo and may serve as a novel and ideal candidate for MSC-based therapies.
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Diferenciación Celular/inmunología , Proliferación Celular , Células Madre Embrionarias Humanas/inmunología , Inmunomodulación , Células Madre Mesenquimatosas/inmunología , Trofoblastos/inmunología , Células Madre Embrionarias Humanas/citología , Humanos , Células Madre Mesenquimatosas/citología , Trofoblastos/citologíaRESUMEN
Constitutive activation of the kinases Akt or protein kinase C (PKC) in blood cancers promotes tumor-cell proliferation and survival and is associated with poor patient survival. The mammalian target of rapamycin (mTOR) complex 2 (mTORC2) regulates the stability of Akt and conventional PKC (cPKC; PKCα and PKCß) proteins by phosphorylating the highly conserved turn motif of these proteins. In cells that lack mTORC2 function, the turn motif phosphorylation of Akt and cPKC is abolished and therefore Akt and cPKC protein stability is impaired. However, the chaperone protein HSP90 can stabilize Akt and cPKC, partially rescuing the expression of these proteins. In the present study, we investigated the antitumor effects of inhibiting mTORC2 plus HSP90 in mouse and human leukemia cell models and show that the HSP90 inhibitor 17-allylaminogeldanamycin (17-AAG) preferentially inhibits Akt and cPKC expression and promotes cell death in mTORC2 deficient pre-B leukemia cells. Furthermore, we show that 17-AAG selectively inhibits mTORC2 deficient leukemia cell growth in vivo. Finally, we show that the mTOR inhibitors rapamycin and pp242 work together with 17-AAG to inhibit leukemia cell growth to a greater extent than either drug alone. These studies provide a mechanistic and clinical rationale to combine mTOR inhibitors with chaperone protein inhibitors to treat human blood cancers.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Leucemia/tratamiento farmacológico , Chaperonas Moleculares/antagonistas & inhibidores , Transactivadores/antagonistas & inhibidores , Animales , Benzoquinonas/administración & dosificación , Células Cultivadas , Evaluación Preclínica de Medicamentos , Células HEK293 , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Humanos , Indoles/administración & dosificación , Células Jurkat , Lactamas Macrocíclicas/administración & dosificación , Ratones , Ratones Transgénicos , Chaperonas Moleculares/metabolismo , Purinas/administración & dosificación , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Sirolimus/administración & dosificación , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Transactivadores/metabolismo , Factores de TranscripciónRESUMEN
B cells are an attractive platform for engineering to produce protein-based biologics absent in genetic disorders, and potentially for the treatment of metabolic diseases and cancer. As part of pre-clinical development of B cell medicines, we demonstrate a method to collect, ex vivo expand, differentiate, radioactively label, and track adoptively transferred non-human primate (NHP) B cells. These cells underwent 10- to 15-fold expansion, initiated IgG class switching, and differentiated into antibody secreting cells. Zirconium-89-oxine labeled cells were infused into autologous donors without any preconditioning and tracked by PET/CT imaging. Within 24 hours of infusion, 20% of the initial dose homed to the bone marrow and spleen and distributed stably and equally between the two. Interestingly, approximately half of the dose homed to the liver. Image analysis of the bone marrow demonstrated inhomogeneous distribution of the cells. The subjects experienced no clinically significant side effects or laboratory abnormalities. A second infusion of B cells into one of the subjects resulted in an almost identical distribution of cells, suggesting a non-limiting engraftment niche and feasibility of repeated infusions. This work supports the NHP as a valuable model to assess the potential of B cell medicines as potential treatment for human diseases.
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Mammalian Sin1 plays key roles in the regulation of mitogen-activated protein kinase (MAPK) and mammalian target of rapamycin (mTOR) signaling. Sin1 is an essential component of mTOR complex 2 (mTORC2). The functions of Sin1 and mTORC2 remain largely unknown in T cells. Here, we investigate Sin1 function in T cells using mice that lack Sin1 in the hematopoietic system. Sin1 deficiency blocks the mTORC2-dependent Akt phosphorylation in T cells during development and activation. Sin1-deficient T cells exhibit normal thymic cellularity and percentages of double-negative, double-positive, and single-positive CD4(+) and CD8(+) thymocytes. Sin1 deficiency does not impair T-cell receptor (TCR) induced growth and proliferation. Sin1 appears dispensable for in vitro CD4(+) helper cell differentiation. However, Sin1 deficiency results in an increased proportion of Foxp3(+) natural T-regulatory (nTreg) cells in the thymus. The TGF-ß-dependent differentiation of CD4(+) T cells in vitro is enhanced by the inhibition of mTOR but not by loss of Sin1 function. Our results reveal that Sin1 and mTORC2 are dispensable for the development and activation of T cells but play a role in nTreg-cell differentiation.
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Proteínas Portadoras/fisiología , Diferenciación Celular , Proliferación Celular , Subgrupos de Linfocitos T/fisiología , Linfocitos T Reguladores/fisiología , Animales , Activación de Linfocitos , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Complejos Multiproteicos , Proteínas/fisiología , Proteínas Proto-Oncogénicas c-akt/fisiología , Receptores de Antígenos de Linfocitos T/fisiología , Linfocitos T Reguladores/citología , Serina-Treonina Quinasas TOR/fisiología , Transactivadores/fisiología , Factores de Transcripción , Factor de Crecimiento Transformador beta/fisiologíaRESUMEN
The protein kinase Akt (also known as protein kinase B) is a critical signaling hub downstream of various cellular stimuli such as growth factors that control cell survival, growth, and proliferation. The activity of Akt is tightly regulated, and the aberrant activation of Akt is associated with diverse human diseases including cancer. Although it is well documented that the mammalian target of rapamycin complex 2 (mTORC2)-dependent phosphorylation of the Akt hydrophobic motif (Ser-473 in Akt1) is essential for full Akt activation, it remains unclear whether this phosphorylation has additional roles in regulating Akt activity. In this study, we found that abolishing Akt Ser-473 phosphorylation stabilizes Akt following agonist stimulation. The Akt Ser-473 phosphorylation promotes a Lys-48-linked polyubiquitination of Akt, resulting in its rapid proteasomal degradation. Moreover, blockade of this proteasomal degradation pathway prolongs agonist-induced Akt activation. These data reveal that mTORC2 plays a central role in regulating the Akt protein life cycle by first stabilizing Akt protein folding through the turn motif phosphorylation and then by promoting Akt protein degradation through the hydrophobic motif phosphorylation. Taken together, this study reveals that the Akt Ser-473 phosphorylation-dependent ubiquitination and degradation is an important negative feedback regulation that specifically terminates Akt activation.
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Regulación Enzimológica de la Expresión Génica , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Transactivadores/metabolismo , Secuencias de Aminoácidos , Animales , Línea Celular , Humanos , Ratones , Modelos Biológicos , Fosforilación , Unión Proteica , Serina/química , Transducción de SeñalRESUMEN
The target of rapamycin (TOR), as part of the rapamycin-sensitive TOR complex 1 (TORC1), regulates various aspects of protein synthesis. Whether TOR functions in this process as part of TORC2 remains to be elucidated. Here, we demonstrate that mTOR, SIN1 and rictor, components of mammalian (m)TORC2, are required for phosphorylation of Akt and conventional protein kinase C (PKC) at the turn motif (TM) site. This TORC2 function is growth factor independent and conserved from yeast to mammals. TM site phosphorylation facilitates carboxyl-terminal folding and stabilizes newly synthesized Akt and PKC by interacting with conserved basic residues in the kinase domain. Without TM site phosphorylation, Akt becomes protected by the molecular chaperone Hsp90 from ubiquitination-mediated proteasome degradation. Finally, we demonstrate that mTORC2 independently controls the Akt TM and HM sites in vivo and can directly phosphorylate both sites in vitro. Our studies uncover a novel function of the TOR pathway in regulating protein folding and stability, processes that are most likely linked to the functions of TOR in protein synthesis.
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Proteína Quinasa C/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Secuencias de Aminoácidos , Animales , Proteínas Portadoras/metabolismo , Línea Celular , Embrión de Mamíferos/citología , Fibroblastos/metabolismo , Humanos , Ratones , Modelos Moleculares , Complejos Multiproteicos/metabolismo , Pliegue de Proteína , Proteína Quinasa C/química , Proteínas Proto-Oncogénicas c-akt/química , Serina-Treonina Quinasas TORRESUMEN
Radiotherapy is the most widely used cancer treatment and improvements in its efficacy and safety are highly sought-after. Peposertib (also known as M3814), a potent and selective DNA-dependent protein kinase (DNA-PK) inhibitor, effectively suppresses the repair of radiation-induced DNA double-strand breaks (DSB) and regresses human xenograft tumors in preclinical models. Irradiated cancer cells devoid of p53 activity are especially sensitive to the DNA-PK inhibitor, as they lose a key cell-cycle checkpoint circuit and enter mitosis with unrepaired DSBs, leading to catastrophic consequences. Here, we show that inhibiting the repair of DSBs induced by ionizing radiation with peposertib offers a powerful new way for improving radiotherapy by simultaneously enhancing cancer cell killing and response to a bifunctional TGFß "trap"/anti-PD-L1 cancer immunotherapy. By promoting chromosome misalignment and missegregation in p53-deficient cancer cells with unrepaired DSBs, DNA-PK inhibitor accelerated micronuclei formation, a key generator of cytosolic DNA and activator of cGAS/STING-dependent inflammatory signaling as it elevated PD-L1 expression in irradiated cancer cells. Triple combination of radiation, peposertib, and bintrafusp alfa, a fusion protein simultaneously inhibiting the profibrotic TGFß and immunosuppressive PD-L1 pathways was superior to dual combinations and suggested a novel approach to more efficacious radioimmunotherapy of cancer. IMPLICATIONS: Selective inhibition of DNA-PK in irradiated cancer cells enhances inflammatory signaling and activity of dual TGFß/PD-L1 targeted therapy and may offer a more efficacious combination option for the treatment of locally advanced solid tumors.
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Neoplasias , Inhibidores de Proteínas Quinasas , Antígeno B7-H1/metabolismo , ADN , Humanos , Inmunoterapia , Neoplasias/tratamiento farmacológico , Neoplasias/radioterapia , Inhibidores de Proteínas Quinasas/farmacología , Piridazinas , Quinazolinas , Factor de Crecimiento Transformador betaRESUMEN
BACKGROUND: Bintrafusp alfa (BA) is a bifunctional fusion protein designed for colocalized, simultaneous inhibition of two immunosuppressive pathways, transforming growth factor-ß (TGF-ß) and programmed death-ligand 1 (PD-L1), within the tumor microenvironment (TME). We hypothesized that targeting PD-L1 to the tumor by BA colocalizes the TGF-ß trap (TGF-ßRII) to the TME, enabling it to sequester TGF-ß in the tumor more effectively than systemic TGF-ß blockade, thereby enhancing antitumor activity. METHODS: Multiple technologies were used to characterize the TGF-ß trap binding avidity. BA versus combinations of anti-PD-L1 and TGF-ß trap or the pan-TGF-ß antibody fresolimumab were compared in proliferation and two-way mixed lymphocyte reaction assays. Immunophenotyping of tumor-infiltrating lymphocytes (TILs) and RNA sequencing (RNAseq) analysis assessing stromal and immune landscape following BA or the combination therapy were performed in MC38 tumors. TGF-ß and PD-L1 co-expression and their associated gene signatures in MC38 tumors and human lung carcinoma tissue were studied with single-cell RNAseq (scRNAseq) and immunostaining. BA-induced internalization, degradation, and depletion of TGF-ß were investigated in vitro. RESULTS: BA and fresolimumab had comparable intrinsic binding to TGF-ß1, but there was an ~80× avidity-based increase in binding affinity with BA. BA inhibited cell proliferation in TGF-ß-dependent and PD-L1-expressing cells more potently than TGF-ß trap or fresolimumab. Compared with the combination of anti-PD-L1 and TGF-ß trap or fresolimumab, BA enhanced T cell activation in vitro and increased TILs in MC38 tumors, which correlated with efficacy. BA induced distinct gene expression in the TME compared with the combination therapy, including upregulation of immune-related gene signatures and reduced activities in TGF-ß-regulated pathways, such as epithelial-mesenchymal transition, extracellular matrix deposition, and fibrosis. Regulatory T cells, macrophages, immune cells of myeloid lineage, and fibroblasts were key PD-L1/TGF-ß1 co-expressing cells in the TME. scRNAseq analysis suggested BA modulation of the macrophage phenotype, which was confirmed by histological assessment. PD-L1/TGF-ß1 co-expression was also seen in human tumors. Finally, BA induced TGF-ß1 internalization and degradation in the lysosomes. CONCLUSION: BA more effectively blocks TGF-ß by targeting TGF-ß trap to the tumor via PD-L1 binding. Such colocalized targeting elicits distinct and superior antitumor responses relative to single agent combination therapy.
Asunto(s)
Neoplasias Pulmonares , Factor de Crecimiento Transformador beta , Antígeno B7-H1 , Humanos , Factores Inmunológicos , Receptor de Muerte Celular Programada 1 , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta1 , Microambiente TumoralRESUMEN
Localized radiotherapy (RT) induces an immunogenic antitumor response that is in part counterbalanced by activation of immune evasive and tissue remodeling processes, e.g., via upregulation of programmed cell death-ligand 1 (PD-L1) and transforming growth factor ß (TGF-ß). We report that a bifunctional fusion protein that simultaneously inhibits TGF-ß and PD-L1, bintrafusp alfa (BA), effectively synergizes with radiotherapy, leading to superior survival in multiple therapy-resistant murine tumor models with poor immune infiltration. The BA + RT (BART) combination increases tumor-infiltrating leukocytes, reprograms the tumor microenvironment, and attenuates RT-induced fibrosis, leading to reconstitution of tumor immunity and regression of spontaneous lung metastases. Consistently, the beneficial effects of BART are in part reversed by depletion of cytotoxic CD8+ T cells. Intriguingly, targeting of the TGF-ß trap to PD-L1+ endothelium and the M2/lipofibroblast-like cell compartment by BA attenuated late-stage RT-induced lung fibrosis. Together, the results suggest that the BART combination has the potential to eradicate therapy-resistant tumors while sparing normal tissue, further supporting its clinical translation.
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Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Evasión Inmune/inmunología , Neoplasias/tratamiento farmacológico , Neoplasias/radioterapia , Factor de Crecimiento Transformador beta/metabolismo , Animales , Humanos , Inhibidores de Puntos de Control Inmunológico/farmacología , Ratones , Microambiente TumoralRESUMEN
The immunoglobulin kappa light chain (Igkappa) locus is regulated in a lineage- and stage-specific manner during B-cell development. The highly restricted timing of V to J gene recombination at the pre-B-cell stage is under the control of two enhancers, the intronic enhancer (kappaEi) and the 3' enhancer (kappaE3'), flanking the constant exon. E2A transcription factors have been indicated to be directly involved in the regulation of Igkappa locus activation. In this study, we utilize E2A-deficient pre-B cells to directly investigate the mechanism of E2A-mediated Igkappa activation. We demonstrate that Igkappa germ line transcription is severely impaired and recombination is blocked in the absence of E2A. Reconstitution of E2A-/- pre-B cells with inducible human E2A (E47R) is sufficient to promote chromatin modification of Igkappa and rescue Igkappa germ line transcription and Jkappa gene recombinase accessibility. Furthermore, we show that increased E2A recruitment to kappaEi and kappaE3' correlates with activation of Igkappa in pre-B cells and that recruitment of E2A to kappaE3' is in part dependent on the transcription factor IRF-4. Inhibition of IRF-4 expression in pre-B cells leads to a significant reduction of Igkappa germ line transcription and enhancer acetylation. In the absence of E2A, increased IRF-4 expression is not sufficient to promote Igkappa enhancer chromatin modification or transcription, suggesting that the sequential involvement of IRF-4 and E2A is necessary for the activation of the Igkappa locus. Finally, we provide genetic evidence in the mouse that E2A gene dosage can influence the development of pre-B cells during the phase of Igkappa gene activation.
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Linfocitos B/inmunología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Cromatina/metabolismo , Reordenamiento Génico de Cadena Ligera de Linfocito B , Cadenas kappa de Inmunoglobulina/genética , Factores Reguladores del Interferón/metabolismo , Acetilación , Animales , Linfocitos B/citología , Linfocitos B/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Benzamidas , Diferenciación Celular , Elementos de Facilitación Genéticos , Dosificación de Gen , Células Germinativas/metabolismo , Histonas/metabolismo , Humanos , Mesilato de Imatinib , Factores Reguladores del Interferón/antagonistas & inhibidores , Ratones , Ratones Mutantes , Mutación , Piperazinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , Recombinación Genética , Transcripción Genética , Activación TranscripcionalRESUMEN
Proper control of B cell growth and metabolism is crucial for B-cell-mediated immunity, but the underlying molecular mechanisms remain incompletely understood. In this study, Sin1, a key component of mTOR complex 2 (mTORC2), specifically regulates B cell growth and metabolism. Genetic ablation of Sin1 in B cells reduces the cell size at either the transitional stage or upon antigen stimulation and severely impairs metabolism. Sin1 deficiency also severely impairs B-cell proliferation, antibody responses, and anti-viral immunity. At the molecular level, Sin1 controls the expression and stability of the c-Myc protein and maintains the activity of mTORC1 through the Akt-dependent inactivation of GSK3 and TSC1/2, respectively. Therefore, our study reveals a novel and specific role for Sin1 in coordinating the activation of mTORC2 and mTORC1 to control B cell growth and metabolism.
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Linfocitos B/citología , Linfocitos B/metabolismo , Proteínas Portadoras/fisiología , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Animales , Linfocitos B/inmunología , Proliferación Celular , Células Cultivadas , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Diana Mecanicista del Complejo 2 de la Rapamicina/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosforilación , Proteínas Proto-Oncogénicas c-myc/genética , Transducción de SeñalRESUMEN
In the version of this Article originally published, the labels for Rictor and mTOR in the whole cell lysate (WCL) blots were swapped in Fig. 3b and the mTOR blot was placed upside down. Unprocessed blots of mTOR were also missing from Supplementary Fig. 9. The corrected Figs are shown below. In addition, control blots for the mTOR antibody (Cell Signalling Technology #2972) were also missing. These are now provided below, as Fig. 9, and show that the lower band is likely non-specific.
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Current therapies for multiple sclerosis (MS) are largely palliative, not curative. Mesenchymal stem cells (MSCs) harbor regenerative and immunosuppressive functions, indicating a potential therapy for MS, yet the variability and low potency of MSCs from adult sources hinder their therapeutic potential. MSCs derived from human embryonic stem cells (hES-MSCs) may be better suited for clinical treatment of MS because of their unlimited and stable supply. Here, we show that hES-MSCs significantly reduce clinical symptoms and prevent neuronal demyelination in a mouse experimental autoimmune encephalitis (EAE) model of MS, and that the EAE disease-modifying effect of hES-MSCs is significantly greater than that of human bone-marrow-derived MSCs (BM-MSCs). Our evidence also suggests that increased IL-6 expression by BM-MSCs contributes to the reduced anti-EAE therapeutic activity of these cells. A distinct ability to extravasate and migrate into inflamed CNS tissues may also be associated with the robust therapeutic effects of hES-MSCs on EAE.
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Células de la Médula Ósea/citología , Células Madre Embrionarias/citología , Encefalomielitis Autoinmune Experimental/terapia , Células Madre Mesenquimatosas/citología , Esclerosis Múltiple/patología , Esclerosis Múltiple/terapia , Animales , Sistema Nervioso Central/patología , Modelos Animales de Enfermedad , Humanos , Trasplante de Células Madre Mesenquimatosas , RatonesRESUMEN
The mechanistic target of rapamycin (mTOR) functions as a critical regulator of cellular growth and metabolism by forming multi-component, yet functionally distinct complexes mTORC1 and mTORC2. Although mTORC2 has been implicated in mTORC1 activation, little is known about how mTORC2 is regulated. Here we report that phosphorylation of Sin1 at Thr 86 and Thr 398 suppresses mTORC2 kinase activity by dissociating Sin1 from mTORC2. Importantly, Sin1 phosphorylation, triggered by S6K or Akt, in a cellular context-dependent manner, inhibits not only insulin- or IGF-1-mediated, but also PDGF- or EGF-induced Akt phosphorylation by mTORC2, demonstrating a negative regulation of mTORC2 independent of IRS-1 and Grb10. Finally, a cancer-patient-derived Sin1-R81T mutation impairs Sin1 phosphorylation, leading to hyper-activation of mTORC2 by bypassing this negative regulation. Together, our results reveal a Sin1-phosphorylation-dependent mTORC2 regulation, providing a potential molecular mechanism by which mutations in the mTORC1-S6K-Sin1 signalling axis might cause aberrant hyper-activation of the mTORC2-Akt pathway, which facilitates tumorigenesis.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Carcinogénesis , Complejos Multiproteicos/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Humanos , Diana Mecanicista del Complejo 2 de la Rapamicina , Mutación , FosforilaciónRESUMEN
Mammalian target of rapamycin complex 2 (mTORC2) is a key downstream mediator of phosphoinositol-3-kinase (PI3K) dependent growth factor signaling. In lymphocytes, mTORC2 has emerged as an important regulator of cell development, homeostasis and immune responses. However, our current understanding of mTORC2 functions and the molecular mechanisms regulating mTORC2 signaling in B and T cells are still largely incomplete. Recent studies have begun to shed light on this important pathway. We have previously reported that mTORC2 mediates growth factor dependent phosphorylation of Akt and facilitates Akt dependent phosphorylation and inactivation of transcription factors FoxO1 and FoxO3a. We have recently explored the functions of mTORC2 in B cells and show that mTORC2 plays a key role in regulating survival and immunoglobulin (Ig) gene recombination of bone marrow B cells through an Akt2-FoxO1 dependent mechanism. Ig recombination is suppressed in proliferating B cells to ensure that DNA double strand breaks are not generated in actively dividing cells. Our results raise the possibility that genetic or pharmacologic inhibition of mTORC2 may promote B cell tumor development as a result of inefficient suppression of Ig recombination in dividing B cells. We also propose a novel strategy to treat cancers based on our recent discovery that mTORC2 regulates Akt protein stability.
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Linfocitos B , Diferenciación Celular , Transformación Celular Neoplásica , Factores de Transcripción/metabolismo , Linfocitos B/citología , Linfocitos B/inmunología , HumanosRESUMEN
The basic helix-loop-helix transcription factor E2A is an essential regulator of B lymphocyte lineage commitment and is required to activate the expression of numerous B lineage-specific genes. Studies involving ectopic expression of Id proteins, which inhibit E2A as well as other basic helix-loop-helix proteins such as HEB, suggest additional roles of E2A at later stages of B cell development. We use E2A-deficient and E2A and HEB double-deficient pre-B cell lines to directly assess the function of E2A and HEB in B cell development after lineage commitment. We show that, in contrast to the established role of E2A in lineage commitment, elimination of E2A and HEB in pre-B cell lines has only a modest negative impact on B lineage gene expression. However, E2A single and E2A and HEB double-deficient but not HEB single-deficient cell lines show dramatically enhanced apoptosis upon growth arrest. To address the possible role of E2A in the regulation of B cell survival in vivo, we crossed IFN-inducible Cre-transgenic mice to E2A conditional mice. Cre-mediated E2A deletion resulted in a block in bone marrow B cell development and a significant reduction in the proportion and total number of splenic B cells in these mice. We show that Cre-mediated deletion of E2A in adoptively transferred mature B cells results in the rapid depletion of the transferred population within 24 h of Cre induction. These results reveal that E2A is not required to maintain B cell fate but is essential in promoting pre-B and B cell survival.
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Subgrupos de Linfocitos B/citología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Diferenciación Celular/inmunología , Células Madre Hematopoyéticas/citología , Traslado Adoptivo , Animales , Subgrupos de Linfocitos B/inmunología , Subgrupos de Linfocitos B/trasplante , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/deficiencia , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Línea Celular , Linaje de la Célula/genética , Linaje de la Célula/inmunología , Supervivencia Celular/inmunología , Regulación del Desarrollo de la Expresión Génica/inmunología , Células Madre Hematopoyéticas/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones TransgénicosRESUMEN
Lymphocyte development has long served as an experimental paradigm, revealing fundamental mechanisms of gene regulation and cellular differentiation in mammals. The study of E-protein-mediated transcriptional regulation in lymphocyte development provides a means to address these mechanistic issues. Both genetic and biochemical studies have defined many important regulatory events during lymphocyte development that are mediated by E-proteins. The E2A gene, one of the three known E-protein genes in mammals, has a particularly important role in B-lymphocyte development. Major progress has been made in recent years towards understanding the physiological targets of E2A during B-lymphocyte development. Most notably, new insights have been gained regarding the role of E2A in controlling lineage commitment and V(D)J recombination. This Review focuses primarily on E2A-mediated gene regulation during B-lymphocyte development.
Asunto(s)
Diferenciación Celular , Proteínas de Unión al ADN/metabolismo , Linfocitos/citología , Linfocitos/metabolismo , Factores de Transcripción/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Linaje de la Célula , Proteínas de Unión al ADN/clasificación , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Humanos , Cadenas kappa de Inmunoglobulina/inmunología , Linfocitos/inmunología , Factores de Transcripción/clasificación , Factores de Transcripción/genéticaRESUMEN
The transcription factors encoded by the E2A gene have been shown to play essential roles in the initiation and progression of lymphocyte development. However, there is still a lack of comprehensive understanding of E2A downstream genes in B-cell development. We previously developed a gene tagging-based chromatin immunoprecipitation (ChIP) system to directly evaluate E2A target genes in B-cell development. Here, we have improved this ChIP strategy and used it in conjunction with microarray analysis on E2A-deficient pre-B-cell lines to determine E2A target genes in lymphocyte development. Both microarray data and ChIP studies confirmed that E2A directly controls IgH gene expression. The microarray assay also revealed genes that were significantly up-regulated after E2A disruption. ChIP analysis showed that E2A was most likely to be directly involved in repression of some of these target genes such as Nfil3 and FGFR2. An inducible E2A reconstitution system further demonstrated that E2A-mediated repression of Nfil3 and FGFR2 was reversible. Collectively, these findings indicate that E2A is a positive regulator for one set of genes and a negative regulator for another set of genes in developing B lymphocytes.