RESUMO
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.
Assuntos
Inflamação , Interleucina-3 , Humanos , Inflamação/imunologia , Inflamação/metabolismo , Interleucina-3/metabolismo , Animais , Transdução de Sinais , Receptores de Interleucina-3/metabolismo , Receptores de Interleucina-3/imunologiaRESUMO
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.
Assuntos
Linfócitos B/metabolismo , Células da Medula Óssea/metabolismo , Macrófagos/metabolismo , Mastócitos/metabolismo , Peritônio/metabolismo , Transcriptoma , Proteína ADAMTS9/genética , Proteína ADAMTS9/metabolismo , Animais , Linfócitos B/citologia , Biomarcadores/metabolismo , Células da Medula Óssea/citologia , Complemento C2/genética , Complemento C2/metabolismo , Feminino , Regulação da Expressão Gênica , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Histidina Descarboxilase/genética , Histidina Descarboxilase/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Macrófagos/citologia , Mastócitos/citologia , Camundongos , Camundongos Endogâmicos BALB C , Especificidade de Órgãos , Peritônio/citologia , Proteínas Proto-Oncogênicas c-myb/genética , Proteínas Proto-Oncogênicas c-myb/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Receptores de Interleucina-3/genética , Receptores de Interleucina-3/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismoRESUMO
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.
Assuntos
Diferenciação Celular/imunologia , Interleucina-3/imunologia , Interleucina-4/imunologia , Receptores de Interleucina-3/imunologia , Células Th2/imunologia , Humanos , Hipersensibilidade/imunologia , Interleucina-3/metabolismo , Interleucina-4/metabolismo , Ativação Linfocitária/imunologia , Receptores de Interleucina-3/metabolismoRESUMO
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.
Assuntos
Ceco/patologia , Receptores de Interleucina-3/antagonistas & inibidores , Sepse/patologia , Sepse/prevenção & controle , Animais , Anticorpos/farmacologia , Modelos Animais de Doenças , Mediadores da Inflamação/metabolismo , Interleucina-3/metabolismo , Subunidade alfa de Receptor de Interleucina-3/metabolismo , Rim/efeitos dos fármacos , Rim/patologia , Ligadura , Pulmão/metabolismo , Pulmão/patologia , Masculino , Microcirculação/efeitos dos fármacos , Punções , Ratos Sprague-Dawley , Receptores de Interleucina-3/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
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.
Assuntos
Antineoplásicos/farmacocinética , Proliferação de Células/efeitos dos fármacos , Portadores de Fármacos/metabolismo , Exossomos/metabolismo , Mesilato de Imatinib/farmacocinética , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Receptores de Interleucina-3/metabolismo , Animais , Antineoplásicos/administração & dosagem , Linhagem Celular Tumoral , Modelos Animais de Doenças , Células HEK293 , Xenoenxertos , Humanos , Mesilato de Imatinib/administração & dosagem , Camundongos , Resultado do TratamentoRESUMO
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.
Assuntos
Anticorpos/administração & dosagem , Ciclodextrinas/administração & dosagem , Leucemia Mieloide Aguda/tratamento farmacológico , Nanopartículas/administração & dosagem , RNA Interferente Pequeno/administração & dosagem , Apoptose/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Citarabina/farmacologia , Regulação para Baixo/efeitos dos fármacos , Humanos , Células K562 , Leucemia Mieloide Aguda/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Receptores de Interleucina-3/metabolismo , Fatores de Transcrição/metabolismoRESUMO
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.
Assuntos
Basófilos/metabolismo , Interleucina-3/metabolismo , Receptores de Interleucina-3/metabolismo , Transdução de Sinais , Regulação para Baixo/efeitos dos fármacos , Eosinófilos/efeitos dos fármacos , Eosinófilos/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Interleucina-5/metabolismo , Cinética , Ligantes , Fenótipo , Fosforilação/efeitos dos fármacos , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
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.
Assuntos
Regulação da Expressão Gênica , Interleucina-3/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Células Mieloides/metabolismo , Células Progenitoras Mieloides/citologia , Células Progenitoras Mieloides/metabolismo , Proteínas Proto-Oncogênicas c-myb/metabolismo , Transcrição Gênica , Animais , Biomarcadores , Linhagem Celular , Proliferação de Células , Sangue Fetal/citologia , Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Imunofenotipagem , Interleucina-3/farmacologia , Modelos Moleculares , Células Progenitoras Mieloides/efeitos dos fármacos , Fenótipo , Receptores de Interleucina-3/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
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.
Assuntos
Medula Óssea/fisiologia , Predisposição Genética para Doença , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/administração & dosagem , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Lesões Experimentais por Radiação/fisiopatologia , Receptores de Interleucina-3/metabolismo , Animais , Células Sanguíneas , Medula Óssea/efeitos da radiação , Proteínas de Ligação ao Cálcio , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/deficiência , Masculino , Camundongos Endogâmicos C57BL , Células Progenitoras Mieloides/fisiologia , Regeneração , Transdução de Sinais , Baço/citologia , Regulação para CimaRESUMO
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.
Assuntos
Basófilos/metabolismo , Cisteína Proteases/imunologia , Subunidade alfa de Receptor de Interleucina-3/imunologia , Interleucina-3/metabolismo , Receptores de Interleucina-3/metabolismo , Sequência de Aminoácidos , Animais , Basófilos/citologia , Basófilos/imunologia , Western Blotting , Ensaio de Imunoadsorção Enzimática , Hidrólise , Subunidade alfa de Receptor de Interleucina-3/química , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Receptores de Interleucina-3/químicaRESUMO
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.
Assuntos
Basófilos/citologia , Eosinófilos/citologia , Mastócitos/citologia , Células-Tronco/citologia , Células da Medula Óssea/citologia , Sangue Fetal/citologia , Citometria de Fluxo , Humanos , Metilcelulose/química , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Receptores de Interleucina-3/metabolismo , Receptores de Interleucina-5/metabolismo , Escarro/imunologia , Células-Tronco/metabolismoRESUMO
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.
Assuntos
Proteínas Correpressoras/metabolismo , Eritropoese , Animais , Células Cultivadas , Proteínas Correpressoras/genética , Subunidade beta Comum dos Receptores de Citocinas/metabolismo , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/patologia , Eritroblastos/citologia , Eritroblastos/metabolismo , Células Precursoras Eritroides/citologia , Células Precursoras Eritroides/metabolismo , Células Precursoras Eritroides/patologia , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Knockout , Células Mieloides/citologia , Receptores de Interleucina-3/metabolismo , Transdução de SinaisRESUMO
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.
Assuntos
Apoptose/fisiologia , Citocinas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Processos de Crescimento Celular/fisiologia , Células HEK293 , Humanos , Interleucina-3/metabolismo , Isoenzimas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células Mieloides/citologia , Células Mieloides/enzimologia , Receptores de Interleucina-3/metabolismo , Transdução de SinaisRESUMO
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.
Assuntos
Fator Estimulador de Colônias de Granulócitos e Macrófagos/química , Interleucina-3/química , Interleucina-5/química , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/química , Receptores de Interleucina-3/química , Receptores de Interleucina-5/química , Cristalografia por Raios X , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , Interleucina-3/imunologia , Interleucina-3/metabolismo , Interleucina-5/imunologia , Interleucina-5/metabolismo , Leucemia Mieloide/imunologia , Leucemia Mieloide/metabolismo , Leucemia Mieloide/patologia , Linfócitos/imunologia , Linfócitos/metabolismo , Linfócitos/patologia , Modelos Moleculares , Ligação Proteica , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/imunologia , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Receptores de Interleucina-3/imunologia , Receptores de Interleucina-3/metabolismo , Receptores de Interleucina-5/imunologia , Receptores de Interleucina-5/metabolismo , Transdução de Sinais , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Células Endoteliais/virologia , Infecções por Herpesviridae/metabolismo , Proteínas de Homeodomínio/metabolismo , Receptores de Interleucina-3/metabolismo , Receptores Notch/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Diferenciação Celular/fisiologia , Linhagem da Célula , Células Cultivadas , Ensaio de Desvio de Mobilidade Eletroforética , Células Endoteliais/metabolismo , Herpesvirus Humano 8/metabolismo , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de SinaisRESUMO
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.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Quinase I-kappa B/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Proteínas Proto-Oncogênicas/metabolismo , Receptores de Interleucina-3/metabolismo , Transdução de Sinais/fisiologia , Proteínas Supressoras de Tumor/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Morte Celular/fisiologia , Linhagem Celular , Células-Tronco Hematopoéticas/citologia , Humanos , Quinase I-kappa B/genética , Interleucina-3/genética , Interleucina-3/metabolismo , Camundongos , Camundongos Knockout , NF-kappa B/genética , NF-kappa B/metabolismo , Fosforilação/fisiologia , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Proteínas Proto-Oncogênicas/genética , Receptores de Interleucina-3/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/genéticaRESUMO
Erythroid myeloid lymphoid (EML) cells are an established multipotent hematopoietic precursor cell line that can be maintained in medium including stem cell factor (SCF). EML cultures contain a heterogeneous mixture of cells, including a lineage-negative, CD34+ subset of cells that propagate rapidly in SCF and can clonally regenerate the mixed population. A second major subset of EML cells consists of lineage-negative. CD34- cells that can be propagated in IL-3 but grow slowly, if at all, in SCF, although they express the SCF receptor (c-kit). The response of these cells to IL-3 is stimulated synergistically by SCF, and we present evidence that both the synergy and the inhibition of c-kit responses may be mediated by direct interaction with IL-3 receptor. Further, the relative level of tyrosine phosphorylation of various substrates by either cytokine alone differs from that produced by the combination of the two cytokines, suggesting that cell signaling by the combination of the two cytokines differs from that produced by either alone.
Assuntos
Antígenos CD34 , Células-Tronco Hematopoéticas/citologia , Interleucina-3/farmacologia , Fator de Células-Tronco/farmacologia , Animais , Linhagem Celular , Células-Tronco Hematopoéticas/metabolismo , Humanos , Interleucina-3/metabolismo , Camundongos , Proteínas Proto-Oncogênicas c-kit/metabolismo , Receptores de Interleucina-3/metabolismo , Fator de Células-Tronco/metabolismoRESUMO
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.
Assuntos
Subunidade beta Comum dos Receptores de Citocinas/metabolismo , Células Endoteliais/enzimologia , Eritropoetina/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Receptores de Interleucina-3/metabolismo , Transdução de Sinais , Animais , Anticorpos Neutralizantes/farmacologia , Bovinos , Movimento Celular , Proliferação de Células , Células Cultivadas , Subunidade beta Comum dos Receptores de Citocinas/genética , Subunidade beta Comum dos Receptores de Citocinas/imunologia , Células Endoteliais/efeitos dos fármacos , Ativação Enzimática , Eritropoetina/genética , Humanos , Imunoprecipitação , Janus Quinase 2/antagonistas & inibidores , Janus Quinase 2/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Confocal , Neovascularização Fisiológica , Óxido Nítrico/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , Receptores da Eritropoetina/genética , Receptores da Eritropoetina/imunologia , Receptores da Eritropoetina/metabolismo , Receptores de Interleucina-3/genética , Receptores de Interleucina-3/imunologia , Proteínas Recombinantes , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Transfecção , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/metabolismoRESUMO
BACKGROUND: RhoH is a constitutively active member of the family of Rho GTPases. Its expression is restricted to the haematopoietic lineage, where it serves as a positive regulator for T cell selection and mast cell function and as a negative regulator for growth-related functions in other lineages. Here, we examined the activation of signal transducer and activator of transcription (STAT) proteins in response to stimulation with interleukin 3 (IL3). RESULTS: Using the murine IL3-dependent cell line BaF3 we investigated the influence of RhoH protein expression levels on IL3-mediated cellular responses. RhoH overexpressing cells showed lower sensitivity to IL3 and decreased STAT5 activation. SiRNA-mediated repression of RhoH gene expression led to an increase in proliferation and STAT5 activity which correlated with an increased number of IL3 receptor α chain molecules, also known as CD123, expressed at the cell surface. Interestingly, these findings could be reproduced using human THP-1 cells as a model system for acute myeloid leukaemia, where low RhoH levels are known to be an unfavourable prognostic marker. Overexpression of RhoH on the other hand caused an induction of STAT1 activity and western blot analysis revealed that activated STAT1 is phosphorylated on Tyr701. STAT1 is known to induce apoptosis or cell cycle arrest and we detected an upregulation of cyclin-dependent kinase inhibitors (CDKI) p21Cip1 and p27Kip1 in RhoH overexpressing BaF3 cells. CONCLUSIONS: We propose that RhoH functions as a negative regulator for IL3-induced signals through modulation of the JAK-STAT pathway. High levels of RhoH allow the IL3-dependent activation of STAT1 causing decreased proliferation through upregulation of p21Cip1 and p27Kip1. Low RhoH levels on the other hand led to an upregulation of IL3-dependent cell growth, STAT5 activity and an increase of CD123 surface expression, linking RhoH to a CD123/STAT5 phenotype that has been described in AML patients.
Assuntos
Interleucina-3/metabolismo , Receptores de Interleucina-3/metabolismo , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais , Fatores de Transcrição/fisiologia , Proteínas rho de Ligação ao GTP/fisiologia , Animais , Linhagem Celular , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Camundongos , Regulação para CimaRESUMO
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.