RESUMEN
IL-3/STAT5 signaling pathway is crucial for the development and activation of immune cells, contributing to the cellular response to infections and inflammatory stimuli. Dysregulation of the IL-3/STAT5 signaling have been associated with inflammatory and autoimmune diseases characterized by inflammatory cell infiltration and organ damage. IL-3 receptor α (IL-3Rα) specifically binds to IL-3 and initiates intracellular signaling, resulting in the phosphorylation of STAT5. However, the regulatory mechanisms of IL-3Rα remain unclear. Here, we identified the E3 ubiquitin ligase RNF128 as a negative regulator of IL-3/STAT5 signaling by targeting IL-3Rα for lysosomal degradation. RNF128 was shown to selectively bind to IL-3Rα, without interacting with the common beta chain IL-3Rß, which shares the subunit with GM-CSF. The deficiency of Rnf128 had no effect on GM-CSF-induced phosphorylation of Stat5, but it resulted in heightened Il-3-triggered activation of Stat5 and increased transcription of the Id1, Pim1, and Cd69 genes. Furthermore, we found that RNF128 promoted the K27-linked polyubiquitination of IL-3Rα in a ligase activity-dependent manner, ultimately facilitating its degradation through the lysosomal pathway. RNF128 inhibited the activation and chemotaxis of macrophages in response to LPS stimulation, thereby attenuating excessive inflammatory responses. Collectively, these results reveal that RNF128 negatively regulates the IL-3/STAT5 signaling pathway by facilitating K27-linked polyubiquitination of IL-3Rα. This study uncovers E3 ubiquitin ligase RNF128 as a novel regulator of the IL-3/STAT5 signaling pathway, providing potential molecular targets for the treatment of inflammatory diseases.
Asunto(s)
Interleucina-3 , Factor de Transcripción STAT5 , Transducción de Señal , Ubiquitina-Proteína Ligasas , Ubiquitinación , Factor de Transcripción STAT5/metabolismo , Factor de Transcripción STAT5/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Humanos , Animales , Interleucina-3/metabolismo , Ratones , Lisosomas/metabolismo , Células HEK293 , Fosforilación , Receptores de Interleucina-3/metabolismo , Receptores de Interleucina-3/genéticaRESUMEN
Interleukin-3 (IL-3) is a cytokine belonging to the family of common ß (ßc) and is involved in various biological systems. Its activity is mediated by the interaction with its receptor (IL-3R), a heterodimer composed of two distinct subunits: IL-3Rα and ßc. IL-3 and its receptor, especially IL-3Rα, play a crucial role in pathologies like inflammatory diseases and therefore are interesting therapeutic targets. Here, we have performed an analysis of these proteins and their interaction based on structural and evolutionary information. We highlighted that IL-3 and IL-3Rα structural architectures are conserved across evolution and shared with other proteins belonging to the same ßc family interleukin-5 (IL-5) and granulocyte-macrophage colony-stimulating factor (GM-CSF). The IL-3Rα/IL-3 interaction is mediated by a large interface in which most residues are surprisingly not conserved during evolution and across family members. In spite of this high variability, we suggested small regions constituted by few residues conserved during the evolution in both proteins that could be important for the binding affinity.
Asunto(s)
Evolución Molecular , Interleucina-3/química , Receptores de Interleucina-3/química , Secuencia de Aminoácidos , Animales , Humanos , Interleucina-3/genética , Conformación Proteica , Receptores de Interleucina-3/genética , Homología de Secuencia de AminoácidoRESUMEN
Bone marrow-derived mast cells (BMMCs) are often used as a model system for studies of the role of MCs in health and disease. These cells are relatively easy to obtain from total bone marrow cells by culturing under the influence of IL-3 or stem cell factor (SCF). After 3 to 4 weeks in culture, a nearly homogenous cell population of toluidine blue-positive cells are often obtained. However, the question is how relevant equivalents these cells are to normal tissue MCs. By comparing the total transcriptome of purified peritoneal MCs with BMMCs, here we obtained a comparative view of these cells. We found several important transcripts that were expressed at very high levels in peritoneal MCs, but were almost totally absent from the BMMCs, including the major chymotryptic granule protease Mcpt4, the neurotrophin receptor Gfra2, the substance P receptor Mrgprb2, the metalloprotease Adamts9 and the complement factor 2 (C2). In addition, there were a number of other molecules that were expressed at much higher levels in peritoneal MCs than in BMMCs, including the transcription factors Myb and Meis2, the MilR1 (Allergin), Hdc (Histidine decarboxylase), Tarm1 and the IL-3 receptor alpha chain. We also found many transcripts that were highly expressed in BMMCs but were absent or expressed at low levels in the peritoneal MCs. However, there were also numerous MC-related transcripts that were expressed at similar levels in the two populations of cells, but almost absent in peritoneal macrophages and B cells. These results reveal that the transcriptome of BMMCs shows many similarities, but also many differences to that of tissue MCs. BMMCs can thereby serve as suitable models in many settings concerning the biology of MCs, but our findings also emphasize that great care should be taken when extrapolating findings from BMMCs to the in vivo function of tissue-resident MCs.
Asunto(s)
Linfocitos B/metabolismo , Células de la Médula Ósea/metabolismo , Macrófagos/metabolismo , Mastocitos/metabolismo , Peritoneo/metabolismo , Transcriptoma , Proteína ADAMTS9/genética , Proteína ADAMTS9/metabolismo , Animales , Linfocitos B/citología , Biomarcadores/metabolismo , Células de la Médula Ósea/citología , Complemento C2/genética , Complemento C2/metabolismo , Femenino , Regulación de la Expresión Génica , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial/genética , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Histidina Descarboxilasa/genética , Histidina Descarboxilasa/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Macrófagos/citología , Mastocitos/citología , Ratones , Ratones Endogámicos BALB C , Especificidad de Órganos , Peritoneo/citología , Proteínas Proto-Oncogénicas c-myb/genética , Proteínas Proto-Oncogénicas c-myb/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Receptores de Interleucina-3/genética , Receptores de Interleucina-3/metabolismo , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismoRESUMEN
Signaling by Kit has been extensively studied in hematopoietic cells and is essential for the survival, proliferation and maintenance of hematopoietic stem and progenitor cells. In addition to the activation of intrinsic signaling pathways, Kit has been shown to interact with lineage-restricted type I cytokine receptors and produce cross signals, e.g. erythropoietin receptor, interleukin-7 receptor (IL-7R), IL-3R. Based on the earlier studies, we hypothesize that Kit activate other type I cytokine receptors in a cell-specific manner and execute cell-specific function. To investigate other Kit-activated receptors, we tested Kit and IL-4R cross-receptor activation in murine bone-marrow-derived mast cells, which express both Kit and IL-4R at the surface level. Kit upon activation by Kit ligand (KL), activated IL-4Rα, γC , and signal transducer and activator of transcription 6 independent of its cognate ligand IL-4. Though KL and IL-4 are individually mitogenic, combinations of KL and IL-4 synergistically promoted mast cell proliferation. Furthermore, inhibition of lipid raft formation by methyl-ß-cyclodextrin resulted in loss of synergistic proliferation. Together the data suggest IL-4R as a novel Kit-activated receptor. Such cross-receptor activations are likely to be a universal mechanism of Kit signaling in hematopoiesis.
Asunto(s)
Interleucina-4/farmacología , Mastocitos/efectos de los fármacos , Proteínas Proto-Oncogénicas c-kit/genética , Receptores de Interleucina-4/genética , Factor de Transcripción STAT6/genética , Factor de Células Madre/farmacología , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/inmunología , Proliferación Celular/efectos de los fármacos , Regulación de la Expresión Génica , Hematopoyesis/efectos de los fármacos , Hematopoyesis/genética , Hematopoyesis/inmunología , Interleucina-4/genética , Interleucina-4/inmunología , Mastocitos/citología , Mastocitos/inmunología , Microdominios de Membrana/efectos de los fármacos , Microdominios de Membrana/inmunología , Microdominios de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Cultivo Primario de Células , Proteínas Proto-Oncogénicas c-kit/inmunología , Receptores de Eritropoyetina/genética , Receptores de Eritropoyetina/inmunología , Receptores de Interleucina-3/genética , Receptores de Interleucina-3/inmunología , Receptores de Interleucina-4/inmunología , Receptores de Interleucina-7/genética , Receptores de Interleucina-7/inmunología , Factor de Transcripción STAT6/inmunología , Transducción de Señal , Factor de Células Madre/genética , Factor de Células Madre/inmunología , beta-Ciclodextrinas/farmacologíaRESUMEN
The ß common chain cytokines GM-CSF, IL-3, and IL-5 regulate varied inflammatory responses that promote the rapid clearance of pathogens but also contribute to pathology in chronic inflammation. Therapeutic interventions manipulating these cytokines are approved for use in some cancers as well as allergic and autoimmune disease, and others show promising early clinical activity. These approaches are based on our understanding of the inflammatory roles of these cytokines; however, GM-CSF also participates in the resolution of inflammation, and IL-3 and IL-5 may also have such properties. Here, we review the functions of the ß common cytokines in health and disease. We discuss preclinical and clinical data, highlighting the potential inherent in targeting these cytokine pathways, the limitations, and the important gaps in understanding of the basic biology of this cytokine family.
Asunto(s)
Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Inflamación/inmunología , Interleucina-3/inmunología , Interleucina-5/inmunología , Animales , Enfermedades Autoinmunes/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/deficiencia , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Factor Estimulante de Colonias de Granulocitos y Macrófagos/uso terapéutico , Hematopoyesis/inmunología , Humanos , Inflamación/terapia , Interleucina-3/antagonistas & inhibidores , Interleucina-3/deficiencia , Interleucina-3/genética , Interleucina-5/antagonistas & inhibidores , Interleucina-5/deficiencia , Interleucina-5/genética , Ratones , Ratones Noqueados , Familia de Multigenes , Neoplasias/inmunología , Neoplasias/terapia , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Receptores de Interleucina-3/genética , Receptores de Interleucina-3/inmunología , Receptores de Interleucina-5/genética , Receptores de Interleucina-5/inmunología , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/uso terapéutico , Transducción de Señal , Relación Estructura-Actividad , Vacunación , Cicatrización de Heridas/inmunologíaAsunto(s)
Regulación Neoplásica de la Expresión Génica , Neoplasias Hematológicas/inmunología , Interleucina-3/inmunología , Janus Quinasa 2/inmunología , Trastornos Mieloproliferativos/inmunología , Receptores de Interleucina-3/inmunología , Animales , Médula Ósea/efectos de los fármacos , Médula Ósea/inmunología , Médula Ósea/patología , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/patología , Humanos , Interleucina-3/genética , Janus Quinasa 2/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación , Trastornos Mieloproliferativos/genética , Trastornos Mieloproliferativos/patología , Receptores de Interleucina-3/genética , Factor de Transcripción STAT5/genética , Factor de Transcripción STAT5/inmunología , Transducción de Señal , Trombopoyetina/farmacologíaRESUMEN
OBJECTIVE: To investigate the effects of total saponins from Sanguisorba officinalis (DYS) on hematopoietic cell proliferation, differentiation and the expression level of IL-3R and c-kit. METHOD: Baf3 and 32D cells were cultured with or without IL-3, then the cells were exposed to DYS in different concentrations of 5, 10, 20, 30 and 40 mg x L(-1) for 24, 48, 72 and 96 hours separately. After that, the cell proliferation and differentiation capacity were determinated by the methods of CCK8 and Giemsa staining separately. The effects of DYS on the expression level of IL-3 receptor in Baf3 cells and the expression level of c-kit in 32D cells were determinated using RT-PCR. RESULT: DYS promotes alone proliferation of Baf3 cells and 32D cells after 48 h. In contrast to control cells, 32D cells containing DYS without IL-3 form many large clusters. DYS also increases the proliferation when cultured with IL-3. High concentration of DYS induce alone the differentiation of 32D cells and increase alone the number of the polyploidy megakaryocyte. Moreover, DYS increases alone the expression level of IL-3R in Baf3 cells and the expression level of c-kit in 32D cells separately. CONCLUSION: Our data shows DYS can promote alone proliferation and differentiation of megakaryocyte progenitor cells. The proliferative and differentiative effect of DYS on megakaryocyte progenitor cells is correlated to the up-regulation of IL-3 receptor and c-kit expression.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Progenitoras de Megacariocitos/efectos de los fármacos , Proteínas Proto-Oncogénicas c-kit/genética , Receptores de Interleucina-3/genética , Sanguisorba/química , Saponinas/farmacología , Animales , Diferenciación Celular/genética , Línea Celular , Relación Dosis-Respuesta a Droga , Expresión Génica/efectos de los fármacos , Interleucina-3/farmacología , Células Progenitoras de Megacariocitos/metabolismo , Ratones , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de TiempoRESUMEN
Chronic myeloid leukemia (CML) stem cells are not dependent on BCR-ABL kinase for their survival, suggesting that kinase-independent mechanisms must contribute to their persistence. We observed that CML stem/progenitor cells (SPCs) produce tumor necrosis factor-α (TNF-α) in a kinase-independent fashion and at higher levels relative to their normal counterparts. We therefore investigated the role of TNF-α and found that it supports survival of CML SPCs by promoting nuclear factor κB/p65 pathway activity and expression of the interleukin 3 and granulocyte/macrophage-colony stimulating factor common ß-chain receptor. Furthermore, we demonstrate that in CML SPCs, inhibition of autocrine TNF-α signaling via a small-molecule TNF-α inhibitor induces apoptosis. Moreover TNF-α inhibition combined with nilotinib induces significantly more apoptosis relative to either treatment alone and a reduction in the absolute number of primitive quiescent CML stem cells. These results highlight a novel survival mechanism of CML SPCs and suggest a new putative therapeutic target for their eradication.
Asunto(s)
Cromonas/farmacología , Indoles/farmacología , Células Madre Neoplásicas/efectos de los fármacos , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Regulación Leucémica de la Expresión Génica , Humanos , Interleucina-3/antagonistas & inhibidores , Interleucina-3/genética , Interleucina-3/inmunología , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , FN-kappa B/antagonistas & inhibidores , FN-kappa B/genética , FN-kappa B/inmunología , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Cultivo Primario de Células , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , Receptores de Interleucina-3/antagonistas & inhibidores , Receptores de Interleucina-3/genética , Receptores de Interleucina-3/inmunología , Transducción de Señal , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
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
P53-upregulated modifier of apoptosis (PUMA), a pro-apoptotic member of the Bcl-2 family, is transcriptionally activated by p53 and is a key effector of p53-dependent apoptosis. We show that PUMA protein is subject to rapid post-translational regulation by phosphorylation at a conserved residue, serine 10, following serum or interleukin-3 (IL-3) stimulation. Serine 10 is not within the Bcl-2 homology (BH3) domain, and PUMA phosphorylated at serine 10 retained the ability to co-immunoprecipitate with antiapoptotic Bcl-2 family members. However, phosphorylated PUMA was targeted for proteasomal degradation indicating that it is less stable than unphosphorylated PUMA. Importantly, we identified IKK1/IKK2/Nemo as the kinase complex that interacts with and phosphorylates PUMA, thereby also demonstrating that IL-3 activates NFκB signaling. The identification and characterization of this novel survival pathway has important implications for IL-3 signaling and hematopoietic cell development.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Células Madre Hematopoyéticas/metabolismo , Quinasa I-kappa B/metabolismo , Procesamiento Proteico-Postraduccional/fisiología , Proteínas Proto-Oncogénicas/metabolismo , Receptores de Interleucina-3/metabolismo , Transducción de Señal/fisiología , Proteínas Supresoras de Tumor/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/genética , Muerte Celular/fisiología , Línea Celular , Células Madre Hematopoyéticas/citología , Humanos , Quinasa I-kappa B/genética , Interleucina-3/genética , Interleucina-3/metabolismo , Ratones , Ratones Noqueados , FN-kappa B/genética , FN-kappa B/metabolismo , Fosforilación/fisiología , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Proteínas Proto-Oncogénicas/genética , Receptores de Interleucina-3/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/genéticaRESUMEN
CD4(+) T-helper type 2 (T(H)2) cells, characterized by their expression of interleukin (IL)-4, IL-5, IL-9 and IL-13, are required for immunity to helminth parasites and promote the pathological inflammation associated with asthma and allergic diseases. Polymorphisms in the gene encoding the cytokine thymic stromal lymphopoietin (TSLP) are associated with the development of multiple allergic disorders in humans, indicating that TSLP is a critical regulator of T(H)2 cytokine-associated inflammatory diseases. In support of genetic analyses, exaggerated TSLP production is associated with asthma, atopic dermatitis and food allergies in patients, and studies in murine systems demonstrated that TSLP promotes T(H)2 cytokine-mediated immunity and inflammation. However, the mechanisms through which TSLP induces T(H)2 cytokine responses remain poorly defined. Here we demonstrate that TSLP promotes systemic basophilia, that disruption of TSLP-TSLPR interactions results in defective basophil responses, and that TSLPR-sufficient basophils can restore T(H)2-cell-dependent immunity in vivo. TSLP acted directly on bone-marrow-resident progenitors to promote basophil responses selectively. Critically, TSLP could elicit basophil responses in both IL-3-IL-3R-sufficient and -deficient environments, and genome-wide transcriptional profiling and functional analyses identified heterogeneity between TSLP-elicited versus IL-3-elicited basophils. Furthermore, activated human basophils expressed TSLPR, and basophils isolated from eosinophilic oesophagitis patients were distinct from classical basophils. Collectively, these studies identify previously unrecognized heterogeneity within the basophil cell lineage and indicate that expression of TSLP may influence susceptibility to multiple allergic diseases by regulating basophil haematopoiesis and eliciting a population of functionally distinct basophils that promote T(H)2 cytokine-mediated inflammation.
Asunto(s)
Basófilos/citología , Citocinas/metabolismo , Hematopoyesis , Hipersensibilidad Inmediata/inmunología , Inflamación/inmunología , Inflamación/metabolismo , Interleucina-3 , Animales , Asma/inmunología , Basófilos/metabolismo , Citocinas/genética , Citocinas/inmunología , Dermatitis Atópica/inmunología , Hipersensibilidad a los Alimentos/inmunología , Humanos , Interleucina-3/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Fenotipo , Receptores de Citocinas/metabolismo , Receptores de Interleucina-3/deficiencia , Receptores de Interleucina-3/genética , Receptores de Interleucina-3/metabolismo , Células Th2/inmunología , Linfopoyetina del Estroma TímicoRESUMEN
Erythropoietin (EPO), the key hormone for erythropoiesis, also increases nitric oxide (NO) bioavailability in endothelial cells (ECs), yet the definitive mechanisms are not fully understood. Increasing evidence has demonstrated that ß common receptor (ßCR) plays a crucial role in EPO-mediated non-hematopoietic effects. We investigated the role of ßCR in EPO-induced endothelial NO synthase (eNOS) activation in bovine aortic ECs (BAECs) and the molecular mechanisms involved. Results of confocal microscopy and immunoprecipitation analyses revealed that ßCR was colocalized and interacted with EPO receptor (EPOR) in ECs. Inhibition of ßCR or EPOR by neutralizing antibodies or small interfering RNA abolished the EPO-induced NO production. Additionally, blockage of ßCR abrogated the EPO-induced increase in the phosphorylation of eNOS, Akt, Src, or Janus kinase 2 (JAK2). Immunoprecipitation analysis revealed that treatment with EPO increased the interaction between ßCR and eNOS, which was suppressed by inhibition of Src, JAK2, or Akt signaling with specific pharmacological inhibitors. Furthermore, EPO-induced EC proliferation, migration, and tube formation were blocked by pretreatment with ßCR antibody and Src, JAK2, or PI3K/Akt inhibitors. Moreover, in vivo experiments showed that EPO increased the level of phosphorylated eNOS, Src, JAK2, and Akt, as well as ßCR-eNOS association in aortas and promoted the angiogenesis in Matrigel plug, which was diminished by ßCR or EPOR neutralizing antibodies. Our findings suggest that ßCR may play an integrative role in the EPO signaling-mediated activation of eNOS in ECs.
Asunto(s)
Subunidad beta Común de los Receptores de Citocinas/metabolismo , Células Endoteliales/enzimología , Eritropoyetina/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Receptores de Interleucina-3/metabolismo , Transducción de Señal , Animales , Anticuerpos Neutralizantes/farmacología , Bovinos , Movimiento Celular , Proliferación Celular , Células Cultivadas , Subunidad beta Común de los Receptores de Citocinas/genética , Subunidad beta Común de los Receptores de Citocinas/inmunología , Células Endoteliales/efectos de los fármacos , Activación Enzimática , Eritropoyetina/genética , Humanos , Inmunoprecipitación , Janus Quinasa 2/antagonistas & inhibidores , Janus Quinasa 2/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Confocal , Neovascularización Fisiológica , Óxido Nítrico/metabolismo , Fosfatidilinositol 3-Quinasa/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/metabolismo , Interferencia de ARN , Receptores de Eritropoyetina/genética , Receptores de Eritropoyetina/inmunología , Receptores de Eritropoyetina/metabolismo , Receptores de Interleucina-3/genética , Receptores de Interleucina-3/inmunología , Proteínas Recombinantes , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Transfección , Familia-src Quinasas/antagonistas & inhibidores , Familia-src Quinasas/metabolismoRESUMEN
Transmembrane domains (TMD) connect the inner with the outer world of a living cell. Single TMD containing (bitopic) receptors are of particular interest, because their oligomerization seems to be a common activation mechanism in cell signaling. We analyzed the composition of TMDs in bitopic proteins within the proteomes of 12 model organisms. The average number of strongly polar and charged residues decreases during evolution, while the occurrence of a dimerization motif, GxxxG, remains unchanged. This may reflect the avoidance of unspecific binding within a growing receptor interaction network. In addition, we propose a new experimental approach for studying helix-helix interactions in giant plasma membrane vesicles using scanning fluorescence cross-correlation spectroscopy. Measuring eGFP/mRFP tagged versions of cytokine receptors confirms the homotypic interactions of the erythropoietin receptor in contrast to the Interleukin-4 receptor chains. As a proof of principle, by swapping the TMDs, the interaction potential of erythropoietin receptor was partially transferred to Interleukin-4 receptor α and vice versa. Non-interacting receptors can therefore serve as host molecules for TMDs whose oligomerization capability must be assessed. Computational analysis of the free energy gain resulting from TMD dimer formation strongly corroborates the experimental findings, potentially allowing in silico pre-screening of interacting pairs.
Asunto(s)
Proteínas de la Membrana/química , Secuencia de Aminoácidos , Simulación por Computador , Humanos , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Mapeo de Interacción de Proteínas/métodos , Estructura Secundaria de Proteína , Proteoma/química , Receptores de Eritropoyetina/química , Receptores de Eritropoyetina/genética , Receptores de Interleucina-2/química , Receptores de Interleucina-2/genética , Receptores de Interleucina-3/química , Receptores de Interleucina-3/genética , Receptores de Interleucina-4/química , Receptores de Interleucina-4/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas de Saccharomyces cerevisiae/química , Espectrometría de FluorescenciaRESUMEN
IL-3 is an important cytokine that regulates hematopoiesis and functions as a link between the immune and the hematopoietic system. In this study, we investigated the role and mechanism of IL-3 action on human osteoclast formation and bone resorption using PBMCs. PBMCs differentiate into functional osteoclasts in the presence of M-CSF and receptor activator of NF-kappaB ligand as evaluated by 23c6 expression and bone resorption. We found that IL-3 dose-dependently inhibited formation of 23c6-positive osteoclasts, bone resorption and C-terminal telopeptide of type I collagen, a collagen degradation product. The inhibitory effect of IL-3 on bone resorption was irreversible. To investigate the mechanism of IL-3 action, we analyzed the effect of IL-3 on the receptor activator of NF-kappaB and c-Fms receptors and c-Fos, PU.1, NFAT cytoplasmic 1, and RelB transcription factors essential for osteoclastogenesis. IL-3 significantly inhibited c-Fms and downregulated both PU.1 and c-Fos at both mRNA and protein level. Furthermore, IL-3-treated cells showed increased expression of dendritic cell markers CD1a and CD80 and decreased expression of monocyte/macrophage marker CD14. Interestingly, IL-3 inhibited formation of human osteoclasts derived from blood monocytes and bone marrow cells of osteoporotic individuals. Thus, IL-3 may have therapeutic potential as an antiosteolytic agent in treatment of osteoporosis.
Asunto(s)
Células Dendríticas/efectos de los fármacos , Interleucina-3/farmacología , Osteoclastos/efectos de los fármacos , Receptor de Factor Estimulante de Colonias de Macrófagos/metabolismo , Adulto , Anciano , Resorción Ósea/prevención & control , Diferenciación Celular/efectos de los fármacos , Linaje de la Célula/efectos de los fármacos , Células Cultivadas , Células Dendríticas/citología , Células Dendríticas/metabolismo , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo/efectos de los fármacos , Citometría de Flujo , Expresión Génica/efectos de los fármacos , Humanos , Persona de Mediana Edad , Osteoclastos/citología , Osteoclastos/metabolismo , Osteoprotegerina/genética , Osteoprotegerina/metabolismo , Ligando RANK/farmacología , Receptor de Factor Estimulante de Colonias de Macrófagos/genética , Receptores de Interleucina-3/genética , Receptores de Interleucina-3/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Adulto JovenRESUMEN
Human plasmacytoid predendritic cells (pDCs) can be activated during microbial infection through Toll-like receptor engagement. They are also involved in nonmicrobial inflammatory diseases, but their activation pathways in this context remain elusive. To identify Toll-like receptor-independent pDC activators, we performed a systematic analysis of cytokine receptors on primary human pDCs. Six receptors were expressed both at mRNA and protein levels: interleukin-3 receptor (IL-3R), IL-6R, IL-10R, IL-18R, interferon-gamma receptor, and granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor. Only GM-CSF and IL-3 were able to efficiently promote pDC survival and induce their differentiation into dendritic cells. Allogeneic naive CD4 T cells primed with GM-CSF-activated pDCs produced more interferon-gamma and less IL-4 and IL-10 compared with IL-3-activated pDCs, indicating a shift in the Th1/Th2 balance. Our data point at a novel function of GM-CSF, which may serve as a link between a pathologic inflammatory environment, pDC activation, and the modulation of CD4 T-cell responses.
Asunto(s)
Células Dendríticas/fisiología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Receptores Toll-Like/metabolismo , Adulto , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/fisiología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/inmunología , Células Cultivadas , Células Dendríticas/citología , Células Dendríticas/efectos de los fármacos , Perfilación de la Expresión Génica , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Humanos , ARN Mensajero/metabolismo , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Receptores de Interferón/genética , Receptores de Interferón/metabolismo , Receptores de Interleucina-10/genética , Receptores de Interleucina-10/metabolismo , Receptores de Interleucina-18/genética , Receptores de Interleucina-18/metabolismo , Receptores de Interleucina-3/genética , Receptores de Interleucina-3/metabolismo , Receptores de Interleucina-6/genética , Receptores de Interleucina-6/metabolismo , Receptor de Interferón gammaRESUMEN
Promyelocytic leukemia zinc finger is a negative regulator of cell cycle progression. In this study, we showed that PLZF inhibits endothelial cell angiogenesis using a human umbilical vein endothelial cell system. We also focused on characterizing the specific function of the BTB domain of PLZF as a novel apoptotic and anti-angiogenic protein via deletion mapping analysis. The BTB domain directly inhibited tube formation, as well as the biological functions of angiostatic activity in vivo, and reduced the expression of p-Akt and p-eNOS, which play a significant role in angiogenesis when stimulated by VEGF. These results strongly suggest that the BTB domain could potentially modulate the apoptotic and anti-angiogenic effects of PLZF.
Asunto(s)
Leucemia Promielocítica Aguda/genética , Dedos de Zinc/fisiología , Animales , Ciclo Celular , Movimiento Celular , Pollos , Membrana Corioalantoides/fisiología , Clonación Molecular , Ciclina A2/genética , Cartilla de ADN , Endotelio Vascular/citología , Endotelio Vascular/fisiología , Sangre Fetal , Eliminación de Gen , Humanos , Factores de Transcripción de Tipo Kruppel/genética , Leucemia Promielocítica Aguda/patología , Invasividad Neoplásica , Óxido Nítrico Sintasa de Tipo III/metabolismo , Reacción en Cadena de la Polimerasa , Proteína de la Leucemia Promielocítica con Dedos de Zinc , Proteínas Proto-Oncogénicas c-akt/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/genética , Receptores de Interleucina-3/genética , Timidina/metabolismo , Transcripción Genética , Cordón Umbilical , Dedos de Zinc/genéticaRESUMEN
Basophils are recognized as immune modulators through their ability to produce IL-4, a key cytokine required for Th2 immunity. It has also recently been reported that basophils are transiently recruited into the draining lymph node (LN) after allergen immunization and that the recruited basophils promote the differentiation of naive CD4 T cells into Th2 effector cells. Using IL-3(-/-) and IL-3Rbeta(-/-) mice, we report in this study that the IL-3/IL-3R system is absolutely required to recruit circulating basophils into the draining LN following helminth infection. Unexpectedly, the absence of IL-3 or of basophil LN recruitment played little role in helminth-induced Th2 immune responses. Moreover, basophil depletion in infected mice did not diminish the development of IL-4-producing CD4 T cells. Our results reveal a previously unknown role of IL-3 in recruiting basophils to the LN and demonstrate that basophils are not necessarily associated with the development of Th2 immunity during parasite infection.
Asunto(s)
Basófilos/inmunología , Basófilos/patología , Movimiento Celular/inmunología , Interleucina-3/fisiología , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/patología , Infecciones por Strongylida/inmunología , Células Th2/inmunología , Animales , Basófilos/parasitología , Movimiento Celular/genética , Técnicas de Sustitución del Gen , Interleucina-3/deficiencia , Interleucina-3/genética , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Nippostrongylus/inmunología , Receptores de Interleucina-3/deficiencia , Receptores de Interleucina-3/genética , Receptores de Interleucina-3/fisiología , Infecciones por Strongylida/parasitología , Infecciones por Strongylida/patología , Células Th2/parasitologíaRESUMEN
Recent work has established important roles for basophils in regulating immune responses. To exert their biological functions, basophils need to be expanded to critical numbers. However, the mechanisms underlying basophil expansion remain unclear. In this study, we established that IL-3 played an important role in the rapid and specific expansion of basophils. We found that the IL-3 complex (IL-3 plus anti-IL-3 Ab) greatly facilitated the differentiation of GMPs into basophil lineage-restricted progenitors (BaPs) but not into eosinophil lineage-restricted progenitors or mast cells in the bone marrow. We also found that the IL-3 complex treatment resulted in approximately 4-fold increase in the number of basophil/mast cell progenitors (BMCPs) in the spleen. IL-3-driven basophil expansion depended on STAT5 signaling. We showed that GMPs but not common myeloid progenitors expressed low levels of IL-3 receptor. IL-3 receptor expression was dramatically up-regulated in BaPs but not eosinophil lineage-restricted progenitors. Approximately 38% of BMCPs expressed the IL-3R alpha-chain. The up-regulated IL-3 receptor expression was not affected by IL-3 or STAT5. Our findings demonstrate that IL-3 induced specific expansion of basophils by directing GMPs to differentiate into BaPs in the bone marrow and by increasing the number of BMCPs in the spleen.
Asunto(s)
Basófilos/inmunología , Diferenciación Celular/inmunología , Linaje de la Célula/inmunología , Células Precursoras de Granulocitos/inmunología , Células Progenitoras de Granulocitos y Macrófagos/inmunología , Interleucina-3/fisiología , Bazo/inmunología , Regulación hacia Arriba/inmunología , Animales , Basófilos/citología , Basófilos/metabolismo , Regulación de la Expresión Génica/inmunología , Células Precursoras de Granulocitos/citología , Células Precursoras de Granulocitos/metabolismo , Células Progenitoras de Granulocitos y Macrófagos/citología , Células Progenitoras de Granulocitos y Macrófagos/metabolismo , Interleucina-3/administración & dosificación , Interleucina-3/deficiencia , Interleucina-3/genética , Recuento de Leucocitos , Mastocitos/citología , Mastocitos/inmunología , Mastocitos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Células Progenitoras Mieloides/citología , Células Progenitoras Mieloides/inmunología , Células Progenitoras Mieloides/metabolismo , Receptores de Interleucina-3/biosíntesis , Receptores de Interleucina-3/genética , Receptores de Interleucina-3/fisiología , Bazo/citología , Bazo/metabolismo , Regulación hacia Arriba/genéticaRESUMEN
Interleukin-3 (IL-3) promotes both self-renewal and differentiation of early multipotential progenitors and is involved in inducible hematopoiesis in response to infections. Here we report new insights into these processes with the identification of a new isoform (SP2) of IL-3 receptor alpha (IL-3Ralpha), present in mouse and human hematopoietic cells, which lacks domain 1 of the full-length receptor (SP1). Binding assays with beta(IL-3) mutants showed that mouse SP2 uses a different high affinity binding mode to SP1, although both mouse and human SP2 and SP1 can stimulate IL-3-dependent growth. In IL-3-dependent differentiation models, human SP2 and SP1 gave differential effects on lineage commitment or self-renewal dependent on the cellular context, suggesting that different modes of ectodomain binding may modulate intracellular signaling. In a multipotential factor dependent cell-Paterson mix, the transcription factors C/EBPalpha and PU.1 and microRNAs miRNA-15a, -223, and -181a were up-regulated in cells undergoing SP2-supported differentiation compared with SP1-supported self-renewal. Similarly in M1 cells, SP2 promoted differentiation compared with SP1 and gave up-regulation of PU.1 and miRNA-155 and -223. These findings suggest that IL-3-promoted lineage commitment uses similar mechanisms to those of steady-state hematopoiesis. Both the SP1 and SP2 isoforms activated the Jak2/STAT5, Akt, and Erk1/2 signaling pathways in M1 cells, although the activation was more prolonged for the SP2 isoform.
Asunto(s)
Empalme Alternativo , Diferenciación Celular , Subunidad alfa del Receptor de Interleucina-3/metabolismo , Receptores de Interleucina-3/metabolismo , Transducción de Señal , Animales , Western Blotting , Células COS , Proliferación Celular , Células Cultivadas , Chlorocebus aethiops , Citometría de Flujo , Glicosilación , Humanos , Subunidad alfa del Receptor de Interleucina-3/genética , Janus Quinasa 2/metabolismo , Ratones , Ratones Transgénicos , MicroARNs/farmacología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Isoformas de Proteínas , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores de Interleucina-3/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción STAT5/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Schizophrenia has been observed to be associated with various abnormalities in cytokines and cytokine receptors. Three very recent reports showed the evidence that the IL3 gene, colony stimulating factor 2 receptor alpha (CSF2RA), beta (CSF2RB) and IL-3 receptor alpha (IL3RA), the IL-specific receptor subunits for CSF2 and IL3, respectively, are associated with schizophrenia. To examine the association of the IL3RA polymorphism (rs6603272) with schizophrenia in a Chinese population, 310 physically healthy patients with schizophrenia were compared with 330 age-, sex- matched normal controls. Statistically significant differences were observed in both allelic and genotypic frequencies of the rs6603272 polymorphism (Allele, chi2=6.24, d.f.=1, p=0.013, odds ratio (OR)=1.35, 95% CI 1.07-1.71; Genotype, chi2=6.85, d.f.=2, p=0.033). Our results indicate a small but significant contribution of the IL3RA polymorphism to susceptibility to schizophrenia, suggesting that the IL3 pathway may be involved in schizophrenia.