RESUMEN
During the perinatal period, the immune system sets the threshold to select either response or tolerance to environmental Ags, which leads to the potential to provide a lifetime of protection and health. B-1a B cells have been demonstrated to develop during this perinatal time window, showing a unique and restricted BCR repertoire, and these cells play a major role in natural Ab secretion and immune regulation. In the current study, we developed a highly efficient temporally controllable RAG2-based lymphoid lineage cell labeling and tracking system and applied this system to understand the biological properties and contribution of B-1a cells generated at distinct developmental periods to the adult B-1a compartments. This approach revealed that B-1a cells with a history of RAG2 expression during the embryonic and neonatal periods dominate the adult B-1a compartment, including those in the bone marrow (BM), peritoneal cavity, and spleen. Moreover, the BCR repertoire of B-1a cells with a history of RAG2 expression during the embryonic period was restricted, becoming gradually more diverse during the neonatal period, and then heterogeneous at the adult stage. Furthermore, more than half of plasmablasts/plasma cells in the adult BM had embryonic and neonatal RAG2 expression histories. Moreover, BCR analysis revealed a high relatedness between BM plasmablasts/plasma cells and B-1a cells derived from embryonic and neonatal periods, suggesting that these cell types have a common origin. Taken together, these findings define, under native hematopoietic conditions, the importance in adulthood of B-1a cells generated during the perinatal period.
Asunto(s)
Linaje de la Célula , Proteínas de Unión al ADN , Animales , Ratones , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Linaje de la Célula/inmunología , Linfocitos B/inmunología , Rastreo Celular/métodos , Receptores de Antígenos de Linfocitos B/inmunología , Subgrupos de Linfocitos B/inmunología , Ratones Endogámicos C57BL , HematopoyesisRESUMEN
Thymocytes having diverse Ag specificities are selected in response to self-MHC-peptide expressed in thymic epithelial cells, which contributes to the formation of a T cell repertoire. However, it is not well understood whether additional signals from epithelial cells are required to drive positive selection. In this study, we found that one of the TNFR superfamily members, herpes virus entry mediator (HVEM), when expressed on thymocytes provides signals for positive selection. HVEM deficiency in double-positive (DP) thymocytes impaired positive selection of CD8 thymocytes. HVEM-deficient thymocytes in OT-1 TCR transgenic mice exhibited significant defects in positive selection and impaired CD69 upregulation of selected thymocytes. HVEM ligands (lymphotoxin-like, exhibits inducible expression, and competes with HSV glycoprotein D for HVEM, a receptor expressed by T lymphocytes, and B and T lymphocyte attenuator) were expressed in cortical thymic epithelial cells. Weak TCR ligation combined with HVEM signals enhanced ERK activation in DP thymocytes developed in vitro. Insufficient signals for positive selection in HVEM-deficient DP thymocytes led to the development of innate memory-like CD8 T cells expressing high levels of CD122, along with the increased development of PLZF+ NKT cells. These results suggest that thymocytes receive activation signals through HVEM during positive selection. Thus, our findings provide evidence that the threshold of thymocyte positive selection is set by signals from TCR in association with HVEM.
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Miembro 14 de Receptores del Factor de Necrosis Tumoral , Timocitos , Animales , Ratones , Linfocitos T CD8-positivos/metabolismo , Diferenciación Celular , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptores de Antígenos de Linfocitos T/metabolismo , Timo , Miembro 14 de Receptores del Factor de Necrosis Tumoral/metabolismoRESUMEN
Intracellular signaling pathways that promote axon regeneration are closely linked to the mechanism of neurite outgrowth. TC10, a signaling molecule that acts on neurite outgrowth through membrane transport, is a member of the Rho family G proteins. Axon injury increases the TC10 levels in motor neurons, suggesting that TC10 may be involved in axon regeneration. In this study, we tried to understand the roles of TC10 in the nervous system using TC10 knock-out mice. In cultured hippocampal neurons, TC10 ablation significantly reduced axon elongation without affecting ordinary polarization. We determined a role of TC10 in microtubule stabilization at the growth cone neck; therefore, we assume that TC10 limits axon retraction and promotes in vitro axon outgrowth. In addition, there were no notable differences in the size and structure of brains during prenatal and postnatal development between wild-type and TC10 knock-out mice. In motor neurons, axon regeneration after injury was strongly suppressed in mice lacking TC10 (both in conventional and injured nerve specific deletion). In retinal ganglion cells, TC10 ablation suppressed the axon regeneration stimulated by intraocular inflammation and cAMP after optic nerve crush. These results show that TC10 plays an important role in axon regeneration in both the peripheral and central nervous systems, and the role of TC10 in peripheral axon regeneration is neuron-intrinsic.
Asunto(s)
Axones/metabolismo , Regeneración Nerviosa/fisiología , Proteínas de Unión al GTP rho/metabolismo , Animales , Hipocampo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proyección Neuronal/fisiología , Neuronas/metabolismo , Transducción de Señal/fisiologíaRESUMEN
BLNK (BASH/SLP-65) encodes an adaptor protein that plays an important role in B-cell receptor (BCR) signaling. Loss-of-function mutations in this gene are observed in human pre-B acute lymphoblastic leukemia (ALL), and a subset of Blnk knock-out (KO) mice develop pre-B-ALL. To understand the molecular mechanism of the Blnk mutation-associated pre-B-ALL development, retroviral tagging was applied to KO mice using the Moloney murine leukemia virus (MoMLV). The Blnk mutation that significantly accelerated the onset of MoMLV-induced leukemia and increased the incidence of pre-B-ALL Cebpb was identified as a frequent site of retroviral integration, suggesting that its upregulation cooperates with Blnk mutations. Transgenic expression of the liver-enriched activator protein (LAP) isoform of Cebpb reduced the number of mature B-lymphocytes in the bone marrow and inhibited differentiation at the pre-BI stage. Furthermore, LAP expression significantly accelerated leukemogenesis in Blnk KO mice and alone acted as a B-cell oncogene. Furthermore, an inverse relationship between BLNK and C/EBPß expression was also noted in human pre-B-ALL cases, and the high level of CEBPB expression was associated with short survival periods in patients with BLNK-downregulated pre-B-ALL. These results indicate the association between the C/EBPß transcriptional network and BCR signaling in pre-B-ALL development and leukemogenesis. This study gives insight into ALL progression and suggests that the BCR/C/EBPß pathway can be a therapeutic target.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Proteína beta Potenciadora de Unión a CCAAT/genética , Virus de la Leucemia Murina de Moloney/fisiología , Mutación , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Animales , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Perfilación de la Expresión Génica , Regulación Leucémica de la Expresión Génica , Humanos , Ratones , Ratones Noqueados , Ratones Transgénicos , Análisis de Secuencia por Matrices de Oligonucleótidos , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/virología , Regulación hacia Arriba , Integración ViralRESUMEN
Cerebellar granule cell precursors (GCPs) and granule cells (GCs) represent good models to study neuronal development. Here, we report that the transcription factor myeloid ectopic viral integration site 1 homolog (Meis1) plays pivotal roles in the regulation of mouse GC development. We found that Meis1 is expressed in GC lineage cells and astrocytes in the cerebellum during development. Targeted disruption of the Meis1 gene specifically in the GC lineage resulted in smaller cerebella with disorganized lobules. Knock-down/knock-out (KO) experiments for Meis1 and in vitro assays showed that Meis1 binds to an upstream sequence of Pax6 to enhance its transcription in GCPs/GCs and also suggested that the Meis1-Pax6 cascade regulates morphology of GCPs/GCs during development. In the conditional KO (cKO) cerebella, many Atoh1-positive GCPs were observed ectopically in the inner external granule layer (EGL) and a similar phenomenon was observed in cultured cerebellar slices treated with a bone morphogenic protein (BMP) inhibitor. Furthermore, expression of Smad proteins and Smad phosphorylation were severely reduced in the cKO cerebella and Meis1-knock-down GCPs cerebella. Reduction of phosphorylated Smad was also observed in cerebellar slices electroporated with a Pax6 knock-down vector. Because it is known that BMP signaling induces Atoh1 degradation in GCPs, these findings suggest that the Meis1-Pax6 pathway increases the expression of Smad proteins to upregulate BMP signaling, leading to degradation of Atoh1 in the inner EGL, which contributes to differentiation from GCPs to GCs. Therefore, this work reveals crucial functions of Meis1 in GC development and gives insights into the general understanding of the molecular machinery underlying neural differentiation from neural progenitors.SIGNIFICANCE STATEMENT We report that myeloid ectopic viral integration site 1 homolog (Meis1) plays pivotal roles in the regulation of mouse granule cell (GC) development. Here, we show Meis1 is expressed in GC precursors (GCPs) and GCs during development. Our knock-down and conditional knock-out (cKO) experiments and in vitro assays revealed that Meis1 is required for proper cerebellar structure formation and for Pax6 transcription in GCPs and GCs. The Meis1-Pax6 cascade regulates the morphology of GCs. In the cKO cerebella, Smad proteins and bone morphogenic protein (BMP) signaling are severely reduced and Atoh1-expressing GCPs are ectopically detected in the inner external granule layer. These findings suggest that Meis1 regulates degradation of Atoh1 via BMP signaling, contributing to GC differentiation in the inner EGL, and should provide understanding into GC development.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Proteínas Morfogenéticas Óseas/biosíntesis , Proteínas Morfogenéticas Óseas/genética , Cerebelo/citología , Cerebelo/crecimiento & desarrollo , Proteína 1 del Sitio de Integración Viral Ecotrópica Mieloide/fisiología , Factor de Transcripción PAX6/biosíntesis , Factor de Transcripción PAX6/genética , Transducción de Señal/genética , Transducción de Señal/fisiología , Animales , Astrocitos/metabolismo , Ciclo Celular/genética , Ciclo Celular/fisiología , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Gránulos Citoplasmáticos , Femenino , Masculino , Ratones , Ratones Endogámicos ICR , Ratones Noqueados , Fosforilación , Embarazo , Proteínas Smad/metabolismoRESUMEN
The dermal papilla (DP) is a key mesenchymal compartment of hair follicles that orchestrates mesenchymal-epithelial interaction regulating hair growth cycles. In the present study, we demonstrate that a TALE-family transcription factor, Meis1, is selectively localized in the nucleus of the DP in the anagen phase of the hair cycle. By using an ex vivo organ culture of vibrissae follicles, conditional Meis1 loss causes retardation in hair growth, accompanied by defects in cell proliferation of hair matrix cells. This cell proliferation defect is partly rescued by the addition of culture supernatants derived from Meis1-sufficient but not -deficient DP cells. These findings indicate that nuclear Meis1 in DP activate genes involved in secretion of some unknown factors, which promote proliferation of hair matrix cells in the anagen phase of the hair cycle.
Asunto(s)
Proliferación Celular , Dermis/metabolismo , Folículo Piloso/metabolismo , Cabello/metabolismo , Proteína 1 del Sitio de Integración Viral Ecotrópica Mieloide/metabolismo , Animales , Núcleo Celular/metabolismo , Células Cultivadas , Dermis/citología , Regulación de la Expresión Génica , Cabello/citología , Cabello/crecimiento & desarrollo , Folículo Piloso/citología , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Proteína 1 del Sitio de Integración Viral Ecotrópica Mieloide/genética , Técnicas de Cultivo de Órganos , Vibrisas/citología , Vibrisas/metabolismoRESUMEN
MAIN CONCLUSION: Enzymatic activities of Oryza sativa expansins, which were heterologously overexpressed in Escherichia coli , were analyzed. Results suggested that expansins promote degradation of cellulose by cellulase in a synergistic manner. Sustainable production of future biofuels is dependent on efficient saccharification of lignocelluloses. Expansins have received a lot of attention as proteins promoting biological degradation of cellulose using cellulase. The expansins are a class of plant cell wall proteins that induce cell wall loosening without hydrolysis. In this study, the expansins from Oryza sativa were classified using phylogenetic analysis and five proteins were selected for functional evaluation. At low cellulose loading, the cellulase in expansin mixtures was up to 2.4 times more active than in mixtures containing only cellulase, but at high cellulose loading the activity of cellulase in expansin mixtures and cellulase only mixtures did not differ. Furthermore, expansin activity was greater in cellulase mixtures compared with cellulase-deficient mixtures. Therefore, the expansins showed significant synergistic activity with cellulase. Expansin may play an important role in efficient saccharification of cellulose.
Asunto(s)
Celulasa/metabolismo , Celulosa/metabolismo , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Pared Celular/metabolismo , Celulosa/química , Cristalización , Electroforesis en Gel de Poliacrilamida , Hidrólisis , Modelos Biológicos , Oryza/genética , Filogenia , Proteínas de Plantas/clasificación , Proteínas de Plantas/genética , Unión Proteica , Difracción de Rayos XRESUMEN
The spleen is a lymphoid organ that serves as a unique niche for immune reactions, extramedullary hematopoiesis, and the removal of aged erythrocytes from the circulation. While much is known about the immunological functions of the spleen, the mechanisms governing the development and organization of its stromal microenvironment remain poorly understood. Here we report the generation and analysis of a Tlx1(Cre) (ER) (-Venus) knock-in mouse strain engineered to simultaneously express tamoxifen-inducible CreER(T2) and Venus fluorescent protein under the control of regulatory elements of the Tlx1 gene, which encodes a transcription factor essential for spleen development. We demonstrated that Venus as well as CreER expression recapitulates endogenous Tlx1 transcription within the spleen microenvironment. When Tlx1(Cre) (ER) (-Venus) mice were crossed with the Cre-inducible reporter strain, Tlx1-expressing cells as well as their descendants were specifically labeled following tamoxifen administration. We also showed by cell lineage tracing that asplenia caused by Tlx1 deficiency is attributable to altered contribution of mesenchymal cells in the spleen anlage to the pancreatic mesenchyme. Thus, Tlx1(Cre) (ER) (-Venus) mice represent a new tool for lineage tracing and conditional gene manipulation of spleen mesenchymal cells, essential approaches for understanding the molecular mechanisms of spleen development.
Asunto(s)
Técnicas de Sustitución del Gen/métodos , Proteínas de Homeodominio/genética , Células Madre Mesenquimatosas/metabolismo , Modelos Animales , Morfogénesis/fisiología , Bazo/embriología , Animales , Proteínas Bacterianas/metabolismo , Linaje de la Célula/fisiología , Cruzamientos Genéticos , Cartilla de ADN/genética , Citometría de Flujo , Proteínas de Homeodominio/metabolismo , Inmunohistoquímica , Integrasas/metabolismo , Proteínas Luminiscentes/metabolismo , Ratones , Microscopía Fluorescente , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Bazo/citología , TamoxifenoRESUMEN
Sphingosine 1-phosphate (S1P) regulates lymphocyte trafficking through the type 1 sphingosine 1-phosphate receptor (S1P(1)) and participates in many pathological conditions, including autoimmune diseases. We developed a novel S1P(1)-selective antagonist, TASP0277308, which is structurally unrelated to S1P. This antagonist competitively inhibited S1P-induced cellular responses, such as chemotaxis and receptor internalization. Furthermore, differing from previously reported S1P(1) antagonists, TASP0277308 demonstrated in vivo activities to induce lymphopenia, a block in T cell egress from the thymus, displacement of marginal zone B cells, and upregulation of CD69 expression on both T and B cells, all of which recapitulate phenotypes of S1P(1)-deficient lymphocytes. In a mouse collagen-induced arthritis model, TASP0277308 significantly suppressed the development of arthritis, even after the onset of disease. These findings provide the first chemical evidence to our knowledge that S1P(1) antagonism is responsible for immunosuppression in the treatment of autoimmune diseases and also resolve the discrepancies between genetic and chemical studies on the functions of S1P(1) in lymphocytes.
Asunto(s)
Artritis Experimental/tratamiento farmacológico , Linfocitos B/inmunología , Tolerancia Inmunológica/efectos de los fármacos , Inmunosupresores/farmacología , Lisofosfolípidos/antagonistas & inhibidores , Esfingosina/análogos & derivados , Sulfonas/farmacología , Linfocitos T/inmunología , Triazoles/farmacología , Animales , Artritis Experimental/genética , Artritis Experimental/inmunología , Artritis Experimental/patología , Linfocitos B/patología , Cricetinae , Cricetulus , Células HEK293 , Humanos , Tolerancia Inmunológica/genética , Tolerancia Inmunológica/inmunología , Inmunosupresores/química , Linfopenia/inducido químicamente , Linfopenia/genética , Linfopenia/inmunología , Linfopenia/patología , Lisofosfolípidos/genética , Lisofosfolípidos/inmunología , Masculino , Ratones , Esfingosina/antagonistas & inhibidores , Esfingosina/genética , Esfingosina/inmunología , Sulfonas/toxicidad , Linfocitos T/patología , Timo/inmunología , Timo/patología , Triazoles/toxicidadRESUMEN
G protein-coupled receptors (GPCRs) regulate many cellular processes in response to various stimuli, including light, hormones, neurotransmitters, and odorants, some of which play critical roles in innate and adaptive immune responses. However, the physiological functions of many GPCRs and the involvement of them in autoimmune diseases of the central nervous system remain unclear. Here, we demonstrate that GPR141, an orphan GPCR belonging to the class A receptor family, suppresses immune responses. High GPR141 messenger RNA levels were expressed in myeloid-lineage cells, including neutrophils (CD11b + Gr1+), monocytes (CD11b + Gr1-Ly6C+ and CD11b + Gr1-Ly6C-), macrophages (F4/80+), and dendritic cells (CD11c+). Gpr141 -/- mice, which we independently generated, displayed almost no abnormalities in myeloid cell differentiation and compartmentalization in the spleen and bone marrow under steady-state conditions. However, Gpr141 deficiency exacerbated disease conditions of experimental autoimmune encephalomyelitis, an autoimmune disease model for multiple sclerosis, with increased inflammation in the spinal cord. Gpr141 -/- mice showed increased CD11b + Gr1+ neutrophils, CD11b + Gr1- monocytes, CD11c+ dendritic cells, and CD4+ T cell infiltration into the experimental autoimmune encephalomyelitis-induced spinal cord compared with littermate control mice. Lymphocytes enriched from Gpr141 -/- mice immunized with myelin oligodendrocyte glycoprotein 35-55 produced high amounts of interferon-γ, interleukin-17A, and interleukin-6 compared with those from wild-type mice. Moreover, CD11c+ dendritic cells (DCs) purified from Gpr141 -/- mice increased cytokine production of myelin oligodendrocyte glycoprotein 35-55-specific T cells. These findings suggest that GPR141 functions as a negative regulator of immune responses by controlling the functions of monocytes and dendritic cells and that targeting GPR141 may be a possible therapeutic intervention for modulating chronic inflammatory diseases.
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Encefalomielitis Autoinmune Experimental , Inflamación , Ratones Noqueados , Células Mieloides , Receptores Acoplados a Proteínas G , Animales , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/deficiencia , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/patología , Encefalomielitis Autoinmune Experimental/metabolismo , Células Mieloides/metabolismo , Células Mieloides/inmunología , Inflamación/inmunología , Inflamación/metabolismo , Ratones , Ratones Endogámicos C57BL , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Glicoproteína Mielina-Oligodendrócito/inmunología , Fragmentos de PéptidosRESUMEN
Sphingosine 1-phosphate (S1P) regulates lymphocyte trafficking via type-1 S1P receptor (S1P(1)) and participates in many pathological conditions. We developed a novel type S1P(1)-selective antagonist, TASP0251078, which is structurally unrelated to S1P. This competitive antagonist inhibited binding of S1P to S1P(1) resulting in reduced signaling downstream of S1P(1), including GTPγS-binding and cAMP formation. TASP0251078 also inhibited S1P-induced cellular responses such as chemotaxis and receptor-internalization. Furthermore, when administered in vivo, TASP0251078 induced lymphopenia in blood, which is different from previously reported effects of other S1P(1)-antagonists. In a mouse contact hypersensitivity model, TASP0251078 effectively suppressed ear swelling, leukocyte infiltration, and hyperplasia. These findings provide the chemical evidence that S1P(1) antagonism is responsible for lymphocyte sequestration from the blood, and suggest that the effect of S1P(1) agonists on lymphocyte sequestration results from their functional antagonism.
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Linfopenia/metabolismo , Lisofosfolípidos/metabolismo , Receptores de Lisoesfingolípidos/antagonistas & inhibidores , Esfingosina/análogos & derivados , Sulfonamidas/farmacología , Triazoles/farmacología , Animales , Células CHO , Quimiotaxis/efectos de los fármacos , Cricetinae , Cricetulus , AMP Cíclico/metabolismo , Dermatitis por Contacto/metabolismo , Dermatitis por Contacto/patología , Dermatitis por Contacto/prevención & control , Oído/patología , Edema/prevención & control , Femenino , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Células HEK293 , Humanos , Hiperplasia/prevención & control , Leucocitos/efectos de los fármacos , Leucocitos/patología , Linfopenia/inducido químicamente , Lisofosfolípidos/química , Lisofosfolípidos/farmacología , Masculino , Ratones , Ratones Endogámicos BALB C , Estructura Molecular , Unión Proteica/efectos de los fármacos , Ratas , Ratas Endogámicas Lew , Receptores de Lisoesfingolípidos/genética , Receptores de Lisoesfingolípidos/metabolismo , Esfingosina/química , Esfingosina/metabolismo , Esfingosina/farmacología , Sulfonamidas/química , Sulfonamidas/toxicidad , Triazoles/química , Triazoles/toxicidadRESUMEN
Basophils have been reported to play a critical role in allergic inflammation by secreting IL-4 in response to IL-3 or high-affinity IgE receptor (FcεRI)-cross-linking. However, the signaling pathways downstream of FcεRI and the IL-3 receptor in basophils have yet to be determined. In the present study, we used mice deficient in SLP-76 (Src homology 2 domain-containing leukocyte phosphoprotein of 76kDa) to demonstrate critical functions of this adaptor molecule in transducing FcεRI- and IL-3 receptor-mediated signals that induce basophil activation. Although SLP-76 was dispensable for in vivo differentiation, as well as IL-3-induced in vitro proliferation of basophils, IL-4 production induced by both stimuli was completely ablated by SLP-76 deficiency. Biochemical analyses revealed that IL-3-induced phosphorylation of phospholipase C (PLC) γ2 and Akt, but not STAT5, was severely reduced in SLP-76-deficient basophils, whereas FcεRI cross-linking phosphorylation of PLCγ2, but not Akt, was abrogated by SLP-76 deficiency, suggesting important differences in the requirement of SLP-76 for Akt activation between FcεRI- and IL-3 receptor-mediated signaling pathways in basophils. Because IL-3-induced IL-4 production was sensitive to calcineurin inhibitors and an intracellular calcium chelator, in addition to PI3K inhibitors, SLP-76 appears to regulate FcεRI- and IL-3 receptor-induced IL-4 production via mediating PLCγ2 activation in basophils. Taken together, these findings indicate that SLP-76 is an essential signaling component for basophil activation downstream of both FcεRI and the IL-3 receptor.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/inmunología , Basófilos/inmunología , Fosfoproteínas/inmunología , Receptores de IgE/inmunología , Receptores de Interleucina-3/inmunología , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Basófilos/efectos de los fármacos , Basófilos/metabolismo , Células Cultivadas , Relación Dosis-Respuesta a Droga , Femenino , Citometría de Flujo , Immunoblotting , Interleucina-3/inmunología , Interleucina-3/farmacología , Interleucina-4/inmunología , Interleucina-4/metabolismo , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos BALB C , Ratones Noqueados , Fosfolipasa C gamma/inmunología , Fosfolipasa C gamma/metabolismo , Fosfoproteínas/deficiencia , Fosfoproteínas/genética , Fosforilación/inmunología , Receptores de IgE/genética , Receptores de IgE/metabolismo , Receptores de Interleucina-3/genética , Receptores de Interleucina-3/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Bazo/citología , Bazo/inmunología , Bazo/metabolismo , Tirosina/inmunología , Tirosina/metabolismoRESUMEN
BACKGROUND: Hemophilia A (HA) is a hereditary bleeding disorder caused by defects in endogenous factor (F)VIII. Approximately 30 % of patients with severe HA treated with FVIII develop neutralizing antibodies (inhibitors) against FVIII, which render the therapy ineffective. The managements of HA patients with high-titter inhibitors are especially challenging. Therefore, it is important to understand the mechanism(s) of high-titer inhibitor development and dynamics of FVIII-specific plasma cells (FVIII-PCs). AIMS: To identify the dynamics of FVIII-PCs and the lymphoid organs in which FVIII-PCs are localized during high-titer inhibitor formation. METHODS AND RESULTS: When FVIII-KO mice were intravenously injected with recombinant (r)FVIII in combination with lipopolysaccharide (LPS), a marked enhancement of anti-FVIII antibody induction was observed with increasing FVIII-PCs, especially in the spleen. When splenectomized or congenitally asplenic FVIII-KO mice were treated with LPS + rFVIII, the serum inhibitor levels decreased by approximately 80 %. Furthermore, when splenocytes or bone marrow (BM) cells from inhibitor+ FVIII-KO mice treated with LPS + rFVIII were grafted into immune-deficient mice, anti-FVIII IgG was detected only in the serum of splenocyte-administered mice and FVIII-PCs were detected in the spleen but not in the BM. In addition, when splenocytes from inhibitor+ FVIII-KO mice were grafted into splenectomized immuno-deficient mice, inhibitor levels were significantly reduced in the serum. CONCLUSION: The spleen is the major site responsible for the expansion and retention of FVIII-PCs in the presence of high-titer inhibitors.
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Hemofilia A , Humanos , Animales , Ratones , Hemofilia A/tratamiento farmacológico , Bazo , Lipopolisacáridos , Factor VIII/farmacología , Anticuerpos NeutralizantesRESUMEN
Sphingosine 1-phosphate receptor type 1 (S1P(1)) was shown to be essential for vascular maturation during embryonic development and it has been demonstrated that substantial crosstalk exists between S1P(1) and other pro-angiogenic growth factors, such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor. We developed a novel S1P(1)-selective antagonist, TASP0277308, which is structurally unrelated to S1P as well as previously described S1P(1) antagonists. TASP0277308 inhibited S1P- as well as VEGF-induced cellular responses, including migration and proliferation of human umbilical vein endothelial cells. Furthermore, TASP0277308 effectively blocked a VEGF-induced tube formation in vitro and significantly suppressed tumor cell-induced angiogenesis in vivo. These findings revealed that S1P(1) is a critical component of VEGF-related angiogenic responses and also provide evidence for the efficacy of TASP0277308 for anti-cancer therapies.
Asunto(s)
Neovascularización Fisiológica/efectos de los fármacos , Receptores de Lisoesfingolípidos/antagonistas & inhibidores , Sulfonas/farmacología , Triazoles/farmacología , Línea Celular Tumoral , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/fisiología , Humanos , Lisofosfolípidos/metabolismo , Lisofosfolípidos/farmacología , Neoplasias/irrigación sanguínea , Neovascularización Patológica/metabolismo , Receptores de Lisoesfingolípidos/metabolismo , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Esfingosina/farmacología , Receptores de Esfingosina-1-Fosfato , Factor A de Crecimiento Endotelial Vascular/farmacologíaRESUMEN
Monocytic leukemia zinc finger protein (MOZ, MYST3, or KAT6A) is a MYST-type acetyltransferase involved in chromosomal translocation in acute myelogenous leukemia (AML) and myelodysplastic syndrome. MOZ is established as essential for hematopoiesis; however, the role of MOZ in AML has not been addressed. We propose that MOZ is critical for AML development induced by MLL-AF9, MLL-AF10, or MOZ-TIF2 fusions. Moz-deficient hematopoietic stem/progenitor cells (HSPCs) transduced with an MLL-AF10 fusion gene neither formed colonies in methylcellulose nor induced AML in mice. Moz-deficient HSPCs bearing MLL-AF9 also generated significantly reduced colony and cell numbers. Moz-deficient HSPCs expressing MOZ-TIF2 could form colonies in vitro but could not induce AML in mice. By contrast, Moz was dispensable for colony formation by HOXA9-transduced cells and AML development caused by HOXA9 and MEIS1, suggesting a specific requirement for MOZ in AML induced by MOZ/MLL fusions. Expression of the Hoxa9 and Meis1 genes was decreased in Moz-deficient MLL fusion-expressing cells, while expression of Meis1, but not Hoxa9, was reduced in Moz-deficient MOZ-TIF2 AML cells. AML development induced by MOZ-TIF2 was rescued by introducing Meis1 into Moz-deficient cells carrying MOZ-TIF2. Meis1 deletion impaired MOZ-TIF2-mediated AML development. Active histone modifications were also severely reduced at the Meis1 locus in Moz-deficient MOZ-TIF2 and MLL-AF9 AML cells. These results suggest that endogenous MOZ is critical for MOZ/MLL fusion-induced AML development and maintains active chromatin signatures at target gene loci.
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Leucemia Mieloide Aguda , Animales , Cromatina , Hematopoyesis , Histona Acetiltransferasas/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Metilcelulosa , RatonesRESUMEN
The type 2 Ca2+-dependent activator protein for secretion (CAPS2/CADPS2) regulates dense-core vesicle trafficking and exocytosis and is involved in the regulated release of catecholamines, peptidergic hormones, and neuromodulators. CAPS2 is expressed in the pancreatic exocrine acinar cells that produce and secrete digestive enzymes. However, the functional role of CAPS2 in vesicular trafficking and/or exocytosis of non-regulatory proteins in the exocrine pancreas remains to be determined. Here, we analyzed the morpho-pathological indicators of the pancreatic exocrine pathway in Cadps2-deficient mouse models using histochemistry, biochemistry, and electron microscopy. We used whole exosome sequencing to identify CADPS2 variants in patients with chronic pancreatitis (CP). Caps2/Cadps2-knockout (KO) mice exhibited morphophysiological abnormalities in the exocrine pancreas, including excessive accumulation of secretory granules (zymogen granules) and their amylase content in the cytoplasm, deterioration of the fine intracellular membrane structures (disorganized rough endoplasmic reticulum, dilated Golgi cisternae, and the appearance of empty vesicles and autophagic-like vacuoles), as well as exocrine pancreatic cell injury, including acinar cell atrophy, increased fibrosis, and inflammatory cell infiltration. Pancreas-specific Cadps2 conditional KO mice exhibited pathological abnormalities in the exocrine pancreas similar to the global Cadps2 KO mice, indicating that these phenotypes were caused either directly or indirectly by CAPS2 deficiency in the pancreas. Furthermore, we identified a rare variant in the exon3 coding region of CADPS2 in a non-alcoholic patient with CP and showed that Cadps2-dex3 mice lacking CAPS2 exon3 exhibited symptoms similar to those exhibited by the Cadps2 KO and cKO mice. These results suggest that CAPS2 is critical for the proper functioning of the pancreatic exocrine pathway, and its deficiency is associated with a risk of pancreatic acinar cell pathology.
RESUMEN
Pre-B-cell leukemia spontaneously develops in BLNK-deficient mice, and pre-B-cell acute lymphoblastic leukemia cells in children often lack BLNK protein expression, demonstrating that BLNK functions as a tumor suppressor. However, the mechanism by which BLNK suppresses pre-B-cell leukemia, as well as the identification of other genetic alterations that collaborate with BLNK deficiency to cause leukemogenesis, are still unknown. Here, we demonstrate that the JAK3/STAT5 signaling pathway is constitutively activated in pre-B leukemia cells derived from BLNK(-/-) mice, mostly due to autocrine production of IL-7. Inhibition of IL-7R signaling or JAK3/STAT5 activity resulted in the induction of p27(kip1) expression and cell-cycle arrest, accompanied by apoptosis in the leukemia cells. Transgene-derived constitutively active STAT5 (STAT5b-CA) strongly synergized with the loss of BLNK to initiate leukemia in vivo. In the leukemia cells, exogenously expressed BLNK inhibited autocrine JAK3/STAT5 signaling, resulting in p27(kip1) induction, cell-cycle arrest, and apoptosis. BLNK-inhibition of JAK3 was dependent on the binding of BLNK to JAK3. These data indicate that BLNK normally regulates IL-7-dependent proliferation and survival of pre-B cells through direct inhibition of JAK3. Thus, somatic loss of BLNK and concomitant mutations leading to constitutive activation of Jak/STAT5 pathway result in the generation of pre-B-cell leukemia.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Janus Quinasa 3/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Células Precursoras de Linfocitos B/citología , Células Precursoras de Linfocitos B/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Agammaglobulinemia Tirosina Quinasa , Animales , Apoptosis/inmunología , Ciclo Celular/inmunología , Línea Celular Tumoral , Supervivencia Celular/inmunología , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Regulación hacia Abajo/inmunología , Fase G1/inmunología , Regulación Leucémica de la Expresión Génica , Interleucina-7/genética , Interleucina-7/metabolismo , Ratones , Ratones Transgénicos , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patología , Proteínas Tirosina Quinasas/metabolismo , Factor de Transcripción STAT5/genética , Transducción de Señal/inmunologíaRESUMEN
The aggregation of high affinity IgE receptors (Fcepsilon receptor I [FcepsilonRI]) on mast cells is potent stimulus for the release of inflammatory and allergic mediators from cytoplasmic granules. However, the molecular mechanism of degranulation has not yet been established. It is still unclear how FcepsilonRI-mediated signal transduction ultimately regulates the reorganization of the cytoskeleton and how these events lead to degranulation. Here, we show that FcepsilonRI stimulation triggers the formation of microtubules in a manner independent of calcium. Drugs affecting microtubule dynamics effectively suppressed the FcepsilonRI-mediated translocation of granules to the plasma membrane and degranulation. Furthermore, the translocation of granules to the plasma membrane occurred in a calcium-independent manner, but the release of mediators and granule-plasma membrane fusion were completely dependent on calcium. Thus, the degranulation process can be dissected into two events: the calcium-independent microtubule-dependent translocation of granules to the plasma membrane and calcium-dependent membrane fusion and exocytosis. Finally, we show that the Fyn/Gab2/RhoA (but not Lyn/SLP-76) signaling pathway plays a critical role in the calcium-independent microtubule-dependent pathway.
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Membrana Celular/inmunología , Mastocitos/inmunología , Mastocitos/metabolismo , Microtúbulos/metabolismo , Receptores de IgE/inmunología , Vesículas Secretoras/inmunología , Proteínas Adaptadoras Transductoras de Señales , Animales , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/inmunología , Degranulación de la Célula/fisiología , Células Cultivadas , Exocitosis/efectos de los fármacos , Exocitosis/inmunología , Mediadores de Inflamación/inmunología , Mediadores de Inflamación/metabolismo , Mastocitos/citología , Fusión de Membrana/efectos de los fármacos , Fusión de Membrana/inmunología , Ratones , Ratones Endogámicos C57BL , Fosfoproteínas/metabolismo , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/inmunología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-fyn , Agregación de Receptores/inmunología , Vesículas Secretoras/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Proteína de Unión al GTP rhoA/metabolismo , Familia-src Quinasas/metabolismoRESUMEN
Pre-B cell receptor (pre-BCR) signals promote pre-B cell differentiation, in which the adaptor protein B-cell linker (BLNK) plays a crucial role. However, the molecular pathways downstream of BLNK are currently unclear. Utilizing pre-B leukemia cell lines (BKO84 and others) derived from BLNK-deficient mice as in vitro models of the pre-B cell differentiation, we have demonstrated that reconstitution of BLNK as well as an active form of protein kinase C (PKC)eta induces the differentiation events, such as pre-BCR down-regulation and kappa gene rearrangement. Here we show that the same events are induced by cross-linking of pre-BCR with anti-mu antibody in these pre-B cell lines, as well as in ex vivo pre-B cells from BLNK-deficient mice, suggesting a function of BLNK as an internal cross-linker of pre-BCR. Anti-mu treatment of BKO84 cells up-regulated membrane recruitment of PKC eta and the expression of IRF-4, a transcription factor known to promote light chain gene rearrangements. Anti-mu induction of surface kappa chain on BKO84 cells was blocked by reagents that inhibit phospholipase C or PKC. Enforced expression of the active PKC eta in BKO84 cells resulted in up-regulation of IRF-4 expression. Conversely, siRNA-mediated silencing of PKC eta expression strikingly attenuated the anti-mu-induced IRF-4 expression and kappa gene rearrangement, which were restored by PKC eta reconstitution. Finally, enforced expression of IRF-4, but not of BLNK, in the PKC eta-silenced BKO84 cells resulted in kappa gene rearrangement. These results indicate that PKC eta directs the induction of IRF-4 expression downstream of BLNK in the pre-BCR signaling pathway promoting kappa gene rearrangement.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Reordenamiento Génico de Cadena Ligera de Linfocito B , Factores Reguladores del Interferón/metabolismo , Proteína Quinasa C/metabolismo , Activación Transcripcional , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Diferenciación Celular , Línea Celular Tumoral , Regulación hacia Abajo , Cadenas mu de Inmunoglobulina/metabolismo , Factores Reguladores del Interferón/genética , Ratones , Células Precursoras de Linfocitos B/inmunología , Células Precursoras de Linfocitos B/metabolismo , Proteína Quinasa C/genética , Agregación de Receptores , Receptores de Antígenos de Linfocitos B/genética , Receptores de Antígenos de Linfocitos B/metabolismo , Transducción de Señal/inmunología , Activación Transcripcional/inmunologíaRESUMEN
Activation of NK cells is triggered by multiple receptors. We demonstrate here that SLP-76 is required for CD16- and NKG2D-mediated NK cell cytotoxicity, while MIST negatively regulates these responses in an SLP-76-dependent manner. Exceptionally, MIST acts as a positive regulator of cytotoxicity against YAC-1 cells, although SLP-76 plays a more key role. SLP-76 acts as a dominant positive regulator for both NKG2D-mediated and YAC-1 cell-triggered IFN-gamma production. Although NKG2D-mediated IFN-gamma production depends on phospholipase C (PLC) gamma 2, YAC-1 cell-triggered IFN-gamma production is PLC gamma 2- and Syk/ZAP-70 independent and nuclear factor-kappa B mediated. SLP-76 is required for this process in the presence of MIST but is dispensable in the absence of MIST. Thus, YAC-1 cell-triggered NKG2D-independent IFN-gamma production appears to be regulated by SLP-76-dependent and -independent pathways, in which the latter is negatively regulated by MIST. Taken together, these results suggest that SLP-76 and MIST distinctly but interactively regulate NK cell cytotoxicity and IFN-gamma production.