RESUMEN
Interleukin (IL)-3 has long been known for its hematopoietic properties. However, recent evidence has expanded our understanding of IL-3 function by identifying IL-3 as a critical orchestrator of inflammation in a wide array of diseases. Depending on the type of disease, the course of inflammation, the cell or the tissue involved, IL-3 promotes either pathologic inflammation or its resolution. Here, we describe the cell-specific functions of IL-3 and summarize its role in diseases. We discuss the current treatments targeting IL-3 or its receptor, and highlight the potential and the limitations of targeting IL-3 in clinics.
Asunto(s)
Inflamación , Interleucina-3 , Humanos , Inflamación/inmunología , Inflamación/metabolismo , Interleucina-3/metabolismo , Animales , Transducción de Señal , Receptores de Interleucina-3/metabolismo , Receptores de Interleucina-3/inmunologíaRESUMEN
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
IL-3 has been reported to be involved in various inflammatory disorders, but its role in inflammatory bowel disease (IBD) has not been addressed so far. Here, we determined IL-3 expression in samples from patients with IBD and studied the impact of Il3 or Il3r deficiency on T cell-dependent experimental colitis. We explored the mechanical, cytoskeletal and migratory properties of Il3r -/- and Il3r +/+ T cells using real-time deformability cytometry, atomic force microscopy, scanning electron microscopy, fluorescence recovery after photobleaching and in vitro and in vivo cell trafficking assays. We observed that, in patients with IBD, the levels of IL-3 in the inflamed mucosa were increased. In vivo, experimental chronic colitis on T cell transfer was exacerbated in the absence of Il-3 or Il-3r signalling. This was attributable to Il-3r signalling-induced changes in kinase phosphorylation and actin cytoskeleton structure, resulting in increased mechanical deformability and enhanced egress of Tregs from the inflamed colon mucosa. Similarly, IL-3 controlled mechanobiology in human Tregs and was associated with increased mucosal Treg abundance in patients with IBD. Collectively, our data reveal that IL-3 signaling exerts an important regulatory role at the interface of biophysical and migratory T cell features in intestinal inflammation and suggest that this might be an interesting target for future intervention.
Asunto(s)
Colitis , Enfermedades Inflamatorias del Intestino , Humanos , Linfocitos T Reguladores , Receptores de Interleucina-3/metabolismo , Interleucina-3/metabolismo , Inflamación/metabolismo , Colitis/metabolismo , Enfermedades Inflamatorias del Intestino/metabolismo , Mucosa Intestinal/metabolismoRESUMEN
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
IL-3, a cytokine secreted by activated T lymphocytes, is known to regulate the proliferation, survival, and differentiation of hematopoietic cells. However, the role of IL-3 in regulation of T cell functions is not fully delineated. Previously, we have reported that IL-3 plays an important role in development of regulatory T cells in mice. In this study, we investigated the regulation of IL-3R expression on human Th cells and also examined the role of IL-3 in effector functions of these cells. We found that human peripheral blood Th cells in resting state do not show surface expression of IL-3R; however, its expression was observed at transcript and intracellular protein levels. The functional IL-3R expression on the surface was seen only after antigenic stimulation. When naive Th cells were activated in the presence of various cytokines, we found that IL-4 significantly increases the surface expression of IL-3R and also increases the number of IL-3R+ Th cells. Interestingly, IL-3R+ cells exhibit a Th2 cell-like phenotype and show high GATA-3 expression. Moreover, Th2 cells in presence of IL-3 show increased expression of type 2 effector cytokines, such as IL-4, IL-5, and IL-13. Furthermore, IL-3R expressing and IL-3-secreting Th cells were high in house dust mite-allergic patients. Thus, to our knowledge, we provide the first evidence that the expression of IL-3R on activated human Th cells is modulated by IL-4, and IL-3 regulates the effector functions of Th2 cells. Our results suggest that IL-3 may play an important role in regulating allergic immune responses.
Asunto(s)
Diferenciación Celular/inmunología , Interleucina-3/inmunología , Interleucina-4/inmunología , Receptores de Interleucina-3/inmunología , Células Th2/inmunología , Humanos , Hipersensibilidad/inmunología , Interleucina-3/metabolismo , Interleucina-4/metabolismo , Activación de Linfocitos/inmunología , Receptores de Interleucina-3/metabolismoRESUMEN
Sepsis is defined as a life-threatening organ dysfunction caused by a dysregulated host response to infection. There are multiple cytokines involved in the process of sepsis. As an important upstream cytokine in inflammation, Interleukin-3 (IL-3) plays a crucial role during sepsis, however, its exact role is unclear. The purpose of this study is to discuss the role of IL-3 and its receptor in cecal ligation and puncture (CLP)-induced sepsis in a rat model. The Cluster of Differentiation 123 (CD123, IL-3 receptor alpha chain, IL-3Rac) antibody (anti-CD123) was used to directly target IL-3's receptor and alleviate the effect of IL-3 in the CLP + anti-CD123 group during the early stage of sepsis. CLP was performed in the CLP and CLP + anti-CD123 groups. The time points of observation included 12 h, 24 h, and 5d after the operation. The results showed that the rats in the CLP + anti-CD123 group had lower levels of lactate, serum tumor necrosis factor-α (TNF-α), Interleukin-1ß (IL-1ß), and Interleukin-6 (IL-6), and also exhibited a higher core temperature, mean arterial pressure (MAP), Oxygenation Index (PO2/FiO2), and end-tidal carbon dioxide (ETCO2) and serum Interleukin-10 (IL-10) levels after CLP than those in the CLP group. Additionally, administration of anti-CD123 led to a stable down-regulation of tyrosine phosphorylation of the IL-3 receptor, a decline in phosphorylation of the Janus kinase 2 (JAK2) protein, and the signal transduction and activation of transcription 5 (STAT5) proteins in lung tissues. Meanwhile, the study revealed that treatment of anti-CD123 can markedly attenuate histological damages in lung and kidney tissues, improve sublingual microcirculation, and prolong survival post sepsis. In conclusion, anti-CD123 reduces mortality and alleviates organ dysfunction by restraining the JAK2-STAT5 signaling pathway and reduces serum cytokines in the development of early sepsis in a rat model induced by CLP.
Asunto(s)
Ciego/patología , Receptores de Interleucina-3/antagonistas & inhibidores , Sepsis/patología , Sepsis/prevención & control , Animales , Anticuerpos/farmacología , Modelos Animales de Enfermedad , Mediadores de Inflamación/metabolismo , Interleucina-3/metabolismo , Subunidad alfa del Receptor de Interleucina-3/metabolismo , Riñón/efectos de los fármacos , Riñón/patología , Ligadura , Pulmón/metabolismo , Pulmón/patología , Masculino , Microcirculación/efectos de los fármacos , Punciones , Ratas Sprague-Dawley , Receptores de Interleucina-3/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Despite Imatinib (IM), a selective inhibitor of Bcr-Abl, having led to improved prognosis in Chronic Myeloid Leukemia (CML) patients, acquired resistance and long-term adverse effects is still being encountered. There is, therefore, urgent need to develop alternative strategies to overcome drug resistance. According to the molecules expressed on their surface, exosomes can target specific cells. Exosomes can also be loaded with a variety of molecules, thereby acting as a vehicle for the delivery of therapeutic agents. In this study, we engineered HEK293T cells to express the exosomal protein Lamp2b, fused to a fragment of Interleukin 3 (IL3). The IL3 receptor (IL3-R) is overexpressed in CML blasts compared to normal hematopoietic cells and thus is able to act as a receptor target in a cancer drug delivery system. Here we show that IL3L exosomes, loaded with Imatinib or with BCR-ABL siRNA, are able to target CML cells and inhibit in vitro and in vivo cancer cell growth.
Asunto(s)
Antineoplásicos/farmacocinética , Proliferación Celular/efectos de los fármacos , Portadores de Fármacos/metabolismo , Exosomas/metabolismo , Mesilato de Imatinib/farmacocinética , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Receptores de Interleucina-3/metabolismo , Animales , Antineoplásicos/administración & dosificación , Línea Celular Tumoral , Modelos Animales de Enfermedad , Células HEK293 , Xenoinjertos , Humanos , Mesilato de Imatinib/administración & dosificación , Ratones , Resultado del TratamientoRESUMEN
Acute myeloid leukemia (AML) is the most common type of acute leukemia in adults and is associated with high relapse rates. It is known that leukemia stem cells (LSCs), a very small subpopulation of the total number of leukemic cells, maintain the leukemia phenotype (â¼80-90% of AML remain the same as at first diagnosis), display chemotherapy resistance, and contribute to disease regeneration. Therefore, targeting LSCs could control the relapse of AML. Small interfering RNA (siRNA), an effector of the RNA interference (RNAi) pathway, can selectively downregulate any gene implicated in the pathology of disease, presenting great potential for treatment of AML. In this study an antibody targeted cyclodextrin-based nanoparticle (NP) (CD.DSPE-PEG-Fab) was developed for siRNA delivery specifically to AML LSCs. The targeted CD.siRNA.DSPE-PEG-Fab formulation, where Fab specifically targets the IL-3 receptor α-chain (IL-3Rα, also known as CD123, a cell surface antigen for human AML LSCs), achieved antigen-mediated cellular uptake in KG1 cells (an AML leukemia stem and progenitor cell line). Efficient delivery of bromodomain-containing protein 4 (BRD4) siRNA using the targeted formulation resulted in downregulation of the corresponding mRNA and protein in KG1 cells and in ex vivo primary AML patient derived samples. The resulting silencing of BRD4 induced myeloid differentiation and triggered leukemia apoptosis. In addition, a synergistic therapeutic effect was detected when administered in combination with the chemotherapeutic, cytarabine (Ara-C). These results indicate the clinical potential of the antibody-tagged cyclodextrin NP for targeted delivery of therapeutic siRNA in the treatment of AML.
Asunto(s)
Anticuerpos/administración & dosificación , Ciclodextrinas/administración & dosificación , Leucemia Mieloide Aguda/tratamiento farmacológico , Nanopartículas/administración & dosificación , ARN Interferente Pequeño/administración & dosificación , Apoptosis/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Citarabina/farmacología , Regulación hacia Abajo/efectos de los fármacos , Humanos , Células K562 , Leucemia Mieloide Aguda/metabolismo , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Receptores de Interleucina-3/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Cytokines of the GM-CSF family signal via the same receptor subunit (ßc) and, thus, have overlapping effects on cells that express all cytokine-specific α-chains (IL-3Rα, IL-5Rα, GM-CSFRα), such as human basophils, whose rapid effector functions are similarly enhanced by IL-3, IL-5, and GM-CSF. However, previous work has shown that IL-3, but not IL-5 and GM-CSF, supports and induces allergy-associated functions of human basophils at later time points. This includes induction of Th2 cytokine and chemokine secretion, high-affinity IgE receptor-independent leukotriene C4 (LTC4) formation, expression of enzymes (e.g., RALDH2, granzyme B), and kinases (e.g., Pim1). Here, we address the question of why IL-3, but not IL-5 or GM-CSF, is capable of inducing these late responses in human basophils, and we investigate the mechanism that underlies the unique regulatory capacity of IL-3. We find that IL-3, IL-5, and GM-CSF rapidly activate the same canonical signaling cascades in a qualitatively identical manner with comparable strength, but we identify signaling duration as major discriminating factor. IL-5 and GM-CSF rapidly down-regulate surface levels of their receptors within minutes, concomitant with a rapid decay in signaling molecule activation and time-dependent loss of ability of these cytokines to prime basophils for functional responses. By contrast, IL-3 hardly down-regulates the α-chain of its receptor without depleting the common ß-chain, which enables extraordinarily sustained signaling events, predominantly the activation of Stat5. Of interest, acute IL-3 signaling is not sufficient to induce persistent phenotypical and functional changes in human basophils. Induction of these functional late responses depends on continuous IL-3 receptor activation and signaling.
Asunto(s)
Basófilos/metabolismo , Interleucina-3/metabolismo , Receptores de Interleucina-3/metabolismo , Transducción de Señal , Regulación hacia Abajo/efectos de los fármacos , Eosinófilos/efectos de los fármacos , Eosinófilos/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Humanos , Interleucina-5/metabolismo , Cinética , Ligandos , Fenotipo , Fosforilación/efectos de los fármacos , Factor de Transcripción STAT5/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Myeloproliferative neoplasms (MPN), which overproduce blood cells in the bone marrow, have recently been linked with a genetically determined decrease in expression of the MYB transcription factor. Here, we use a mouse MYB knockdown model with an MPN-like phenotype to show how lower levels of MYB lead to stem cell characteristics in myeloid progenitors. The altered progenitor properties feature elevated cytokine responsiveness, especially to interleukin-3, which results from increased receptor expression and increased MAPK activity leading to enhanced phosphorylation of a key regulator of protein synthesis, ribosomal protein S6. MYB acts on MAPK signaling by directly regulating transcription of the gene encoding the negative modulator SPRY2. This mechanistic insight points to pathways that might be targeted therapeutically in MPN.
Asunto(s)
Regulación de la Expresión Génica , Interleucina-3/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de la Membrana/genética , Células Mieloides/metabolismo , Células Progenitoras Mieloides/citología , Células Progenitoras Mieloides/metabolismo , Proteínas Proto-Oncogénicas c-myb/metabolismo , Transcripción Genética , Animales , Biomarcadores , Línea Celular , Proliferación Celular , Sangre Fetal/citología , Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Inmunofenotipificación , Interleucina-3/farmacología , Modelos Moleculares , Células Progenitoras Mieloides/efectos de los fármacos , Fenotipo , Receptores de Interleucina-3/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Physical and chemical insult-induced bone marrow (BM) damage often leads to lethality resulting from the depletion of hematopoietic stem and progenitor cells (HSPCs) and/or a deteriorated BM stroma. Notch signaling plays an important role in hematopoiesis, but whether it is involved in BM damage remains unclear. In this study, we found that conditional disruption of RBP-J, the transcription factor of canonical Notch signaling, increased irradiation sensitivity in mice. Activation of Notch signaling with the endothelial cell (EC)-targeted soluble Dll1 Notch ligand mD1R promoted BM recovery after irradiation. mD1R treatment resulted in a significant increase in myeloid progenitors and monocytes in the BM, spleen and peripheral blood after irradiation. mD1R also enhanced hematopoiesis in mice treated with cyclophosphamide, a chemotherapeutic drug that induces BM suppression. Mechanistically, mD1R increased the proliferation and reduced the apoptosis of myeloid cells in the BM after irradiation. The ß chain cytokine receptor Csf2rb2 was identified as a downstream molecule of Notch signaling in hematopoietic cells. mD1R improved hematopoietic recovery through up-regulation of the hematopoietic expression of Csf2rb2. Our findings reveal the role of Notch signaling in irradiation- and drug-induced BM suppression and establish a new potential therapy of BM- and myelo-suppression induced by radiotherapy and chemotherapy.
Asunto(s)
Médula Ósea/fisiología , Predisposición Genética a la Enfermedad , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/metabolismo , Péptidos y Proteínas de Señalización Intercelular/administración & dosificación , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Traumatismos Experimentales por Radiación/fisiopatología , Receptores de Interleucina-3/metabolismo , Animales , Células Sanguíneas , Médula Ósea/efectos de la radiación , Proteínas de Unión al Calcio , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/deficiencia , Masculino , Ratones Endogámicos C57BL , Células Progenitoras Mieloides/fisiología , Regeneración , Transducción de Señal , Bazo/citología , Regulación hacia ArribaRESUMEN
Th2 type immune responses are essential for protective immunity against parasites and play crucial roles in allergic disorders. Helminth parasites secrete a variety of proteases for their infectious cycles including for host entry, tissue migration, and suppression of host immune effector cell function. Furthermore, a number of pathogen-derived antigens, as well as allergens such as papain, belong to the family of cysteine proteases. Although the link between protease activity and Th2 type immunity is well documented, the mechanisms by which proteases regulate host immune responses are largely unknown. Here, we demonstrate that the cysteine proteases papain and bromelain selectively cleave the α subunit of the IL-3 receptor (IL-3Rα/CD123) on the surface of murine basophils. The decrease in CD123 expression on the cell surface, and the degradation of the extracellular domain of recombinant CD123 were dependent on the protease activity of papain and bromelain. Pre-treatment of murine basophils with papain resulted in inhibition of IL-3-IL-3R signaling and suppressed IL-3- but not thymic stromal lymphopoietin-induced expansion of basophils in vitro. Our unexpected findings illuminate a novel mechanism for the regulation of basophil functions by protease antigens. Because IL-3 plays pivotal roles in the activation and proliferation of basophils and in protective immunity against helminth parasites, pathogen-derived proteases might contribute to the pathogenesis of infections by regulating IL-3-mediated functions in basophils.
Asunto(s)
Basófilos/metabolismo , Proteasas de Cisteína/inmunología , Subunidad alfa del Receptor de Interleucina-3/inmunología , Interleucina-3/metabolismo , Receptores de Interleucina-3/metabolismo , Secuencia de Aminoácidos , Animales , Basófilos/citología , Basófilos/inmunología , Western Blotting , Ensayo de Inmunoadsorción Enzimática , Hidrólisis , Subunidad alfa del Receptor de Interleucina-3/química , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Receptores de Interleucina-3/químicaRESUMEN
Mast cell, basophil, and eosinophil lineages all derive from CD34(+) hemopoietic stem cells; however, mast cells are derived from a distinct, nonmyeloid progenitor, while eosinophils and basophils share a common myeloid progenitor. These progenitors likely evolved from an ancestral leukocyte population involved in innate immunity and currently play a central role in the pathology of allergic disease. Advances in isolation and analysis of mast cell and basophil/eosinophil progenitor populations have been critical to understanding lineage commitment, differentiation, function, and transcriptional regulation of these cells and have provided a way of monitoring the effect of novel investigational therapies on these cell populations in samples of blood, bone marrow, and airway secretions.
Asunto(s)
Basófilos/citología , Eosinófilos/citología , Mastocitos/citología , Células Madre/citología , Células de la Médula Ósea/citología , Sangre Fetal/citología , Citometría de Flujo , Humanos , Metilcelulosa/química , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Receptores de Interleucina-3/metabolismo , Receptores de Interleucina-5/metabolismo , Esputo/inmunología , Células Madre/metabolismoRESUMEN
The corepressor Rcor1 has been linked biochemically to hematopoiesis, but its function in vivo remains unknown. We show that mice deleted for Rcor1 are profoundly anemic and die in late gestation. Definitive erythroid cells from mutant mice arrest at the transition from proerythroblast to basophilic erythroblast. Remarkably, Rcor1 null erythroid progenitors cultured in vitro form myeloid colonies instead of erythroid colonies. The mutant proerythroblasts also aberrantly express genes of the myeloid lineage as well as genes typical of hematopoietic stem cells (HSCs) and/or progenitor cells. The colony-stimulating factor 2 receptor ß subunit (Csf2rb), which codes for a receptor implicated in myeloid cytokine signaling, is a direct target for both Rcor1 and the transcription repressor Gfi1b in erythroid cells. In the absence of Rcor1, the Csf2rb gene is highly induced, and Rcor1(-/-) progenitors exhibit CSF2-dependent phospho-Stat5 hypersensitivity. Blocking this pathway can partially reduce myeloid colony formation by Rcor1-deficient erythroid progenitors. Thus, Rcor1 promotes erythropoiesis by repressing HSC and/or progenitor genes, as well as the genes and signaling pathways that lead to myeloid cell fate.
Asunto(s)
Proteínas Co-Represoras/metabolismo , Eritropoyesis , Animales , Células Cultivadas , Proteínas Co-Represoras/genética , Subunidad beta Común de los Receptores de Citocinas/metabolismo , Embrión de Mamíferos/metabolismo , Embrión de Mamíferos/patología , Eritroblastos/citología , Eritroblastos/metabolismo , Células Precursoras Eritroides/citología , Células Precursoras Eritroides/metabolismo , Células Precursoras Eritroides/patología , Eliminación de Gen , Regulación del Desarrollo de la Expresión Génica , Ratones , Ratones Noqueados , Células Mieloides/citología , Receptores de Interleucina-3/metabolismo , Transducción de SeñalRESUMEN
The activation of the Akt signalling in response to cytokine receptor signalling promotes protein synthesis, cellular growth and proliferation. To determine the role of Akt in interleukin-3 (IL-3) signalling, we generated IL-3-dependent myeloid cell lines from mice lacking Akt1, Akt2 or Akt3. Akt1 deletion resulted in accelerated apoptosis at low concentrations of IL-3. Expression of constitutively active Akt1 was sufficient to delay apoptosis in response to IL-3 withdrawal, but not sufficient to induce proliferation in the absence of IL-3. Akt1 prolonged survival of Bim- or Bad-deficient cells, but not cells lacking Puma, indicating that Akt1-dependent repression of apoptosis was in part dependent on Puma and independent of Bim or Bad. Our data show that a key role of Akt1 during IL-3 signalling is to repress p53-dependent apoptosis pathways, including transcriptional upregulation of Puma. Moreover, our data indicate that regulation of BH3-only proteins by Akt is dispensable for Akt-dependent cell survival.
Asunto(s)
Apoptosis/fisiología , Citocinas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Animales , Procesos de Crecimiento Celular/fisiología , Células HEK293 , Humanos , Interleucina-3/metabolismo , Isoenzimas , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Células Mieloides/citología , Células Mieloides/enzimología , Receptores de Interleucina-3/metabolismo , Transducción de SeñalRESUMEN
Granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 are members of a discrete family of cytokines that regulates the growth, differentiation, migration and effector function activities of many hematopoietic cells and immunocytes. These cytokines are involved in normal responses to infectious agents, bridging innate and adaptive immunity. However, in certain cases, the overexpression of these cytokines or their receptors can lead to excessive or aberrant initiation of signaling resulting in pathological conditions, with chronic inflammatory diseases and myeloid leukemias the most notable examples. Recent crystal structures of the GM-CSF receptor ternary complex and the IL-5 binary complex have revealed new paradigms of cytokine receptor activation. Together with a wealth of associated structure-function studies, they have significantly enhanced our understanding of how these receptors recognize cytokines and initiate signals across cell membranes. Importantly, these structures provide opportunities for structure-based approaches for the discovery of novel and disease-specific therapeutics. In addition, recent biochemical evidence has suggested that the GM-CSF/IL-3/IL-5 receptor family is capable of interacting productively with other membrane proteins at the cell surface. Such interactions may afford additional or unique biological activities and might be harnessed for selective modulation of the function of these receptors in disease.
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Factor Estimulante de Colonias de Granulocitos y Macrófagos/química , Interleucina-3/química , Interleucina-5/química , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/química , Receptores de Interleucina-3/química , Receptores de Interleucina-5/química , Cristalografía por Rayos X , Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Humanos , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/patología , Interleucina-3/inmunología , Interleucina-3/metabolismo , Interleucina-5/inmunología , Interleucina-5/metabolismo , Leucemia Mieloide/inmunología , Leucemia Mieloide/metabolismo , Leucemia Mieloide/patología , Linfocitos/inmunología , Linfocitos/metabolismo , Linfocitos/patología , Modelos Moleculares , Unión Proteica , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Receptores de Interleucina-3/inmunología , Receptores de Interleucina-3/metabolismo , Receptores de Interleucina-5/inmunología , Receptores de Interleucina-5/metabolismo , Transducción de Señal , Relación Estructura-ActividadRESUMEN
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.
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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
Lymphatic endothelial cells (LECs) are differentiated from blood vascular endothelial cells (BECs) during embryogenesis and this physiological cell fate specification is controlled by PROX1, the master regulator for lymphatic development. When Kaposi sarcoma herpes virus (KSHV) infects host cells, it activates the otherwise silenced embryonic endothelial differentiation program and reprograms their cell fates. Interestingly, previous studies demonstrated that KSHV drives BECs to acquire a partial lymphatic phenotype by upregulating PROX1 (forward reprogramming), but stimulates LECs to regain some BEC-signature genes by downregulating PROX1 (reverse reprogramming). Despite the significance of this KSHV-induced bidirectional cell fate reprogramming in KS pathogenesis, its underlying molecular mechanism remains undefined. Here, we report that IL3 receptor alpha (IL3Rα) and NOTCH play integral roles in the host cell type-specific regulation of PROX1 by KSHV. In BECs, KSHV upregulates IL3Rα and phosphorylates STAT5, which binds and activates the PROX1 promoter. In LECs, however, PROX1 was rather downregulated by KSHV-induced NOTCH signal via HEY1, which binds and represses the PROX1 promoter. Moreover, PROX1 was found to be required to maintain HEY1 expression in LECs, establishing a reciprocal regulation between PROX1 and HEY1. Upon co-activation of IL3Rα and NOTCH, PROX1 was upregulated in BECs, but downregulated in LECs. Together, our study provides the molecular mechanism underlying the cell type-specific endothelial fate reprogramming by KSHV.
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Células Endoteliales/virología , Infecciones por Herpesviridae/metabolismo , Proteínas de Homeodominio/metabolismo , Receptores de Interleucina-3/metabolismo , Receptores Notch/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Diferenciación Celular/fisiología , Linaje de la Célula , Células Cultivadas , Ensayo de Cambio de Movilidad Electroforética , Células Endoteliales/metabolismo , Herpesvirus Humano 8/metabolismo , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de SeñalRESUMEN
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.
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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.