RESUMO
Thrombopoietin (TPO) regulates megakaryocytopoiesis and platelet production in vivo and in vitro. Exogenous overexpression of TPO in vivo by viral-mediated gene transfer induced bone marrow (BM) fibrosis and osteosclerosis. On the other hand, transgenic mice (Tg) overexpressing TPO using a liver-specific apolipoprotein E (Apo-E) promoter did not exhibit myelofibrosis or osteosclerosis. These discrepancies in phenotype are not fully understood. Then we have investigated the consequences of long-term in vivo overexpression of TPO in a mouse model. Murine TPO Tg mice driven by the IgH promoter were generated. The number of platelets and neutrophils in peripheral blood, and the number of megakaryocytes and granulocytic immature cells in the BM was elevated, together with the number of progenitor cells for megakaryocyte and myeloid cells. TPO Tg mice demonstrated anemia but the number of progenitor cells for the erythrocyte was increased. TPO Tg mice developed myelofibrosis and osteosclerosis as they aged with extramedullary hematopoiesis in the spleen. As plasma transforming growth factors (TGF)-beta1 and osteoprotegerin (OPG) levels were higher in TPO Tg mice than in wild-type mice, the development of myelofibrosis and osteosclerosis depends on local TPO levels in BM and might be due to elevated TGF-beta1 and OPG.
Assuntos
Células da Medula Óssea/fisiologia , Osteosclerose/genética , Mielofibrose Primária/genética , Trombopoetina/genética , Animais , Apolipoproteínas E/genética , Células da Medula Óssea/citologia , Clonagem Molecular , Primers do DNA , Eritropoetina/sangue , Rearranjo Gênico , Globinas/genética , Cadeias Pesadas de Imunoglobulinas/genética , Camundongos , Camundongos Transgênicos , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Coelhos , Valores de Referência , Trombopoetina/fisiologiaRESUMO
G-CSF specifically stimulates the proliferation and differentiation of cells that are committed to the neutrophil-granulocyte lineage. Although Stat3 was thought to be essential for the transduction of G-CSF-induced cell proliferation and differentiation signals, mice deficient for Stat3 in hematopoietic cells show neutrocytosis and infiltration of cells into the digestive tract. The number of progenitor cells in the neutrophil lineage is not changed, and G-CSF-induced proliferation of progenitor cells and prolonged neutrophil survival were observed in Stat3-deficient mice. In hematopoietic cells from Stat3-deficient mice, trace levels of SOCS3, a negative regulator of granulopoiesis, were observed, and SOCS3 expression was not induced by G-CSF stimulation. Stat3-null bone marrow cells displayed a significant activation of extra-cellular regulated kinase 1 (ERK1)/ERK2 under basal conditions, and the activation of ERK was enhanced and sustained by G-CSF stimulation. Furthermore, the augmented proliferation of Stat3-deficient bone marrow cells in response to G-CSF was dramatically decreased by addition of a MEK1 inhibitor. These results indicate that Stat3 functions as a negative regulator of G-CSF signaling by inducing SOCS3 expression and that ERK activation is the major factor responsible for inducing the proliferation of hematopoietic cells in response to G-CSF.
Assuntos
Proteínas de Ligação a DNA/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fator Estimulador de Colônias de Granulócitos/administração & dosagem , Transdução de Sinais/efeitos dos fármacos , Transativadores/metabolismo , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/fisiologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Proteínas de Ligação a DNA/deficiência , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/fisiologia , Camundongos , Camundongos Knockout , Neutrófilos/citologia , Neutrófilos/fisiologia , Fator de Transcrição STAT3 , Transdução de Sinais/fisiologia , Transativadores/deficiênciaRESUMO
The Janus kinase (Jak)-Stat pathway plays an essential role in cytokine signaling. Granulocyte colony-stimulating factor (G-CSF) promotes granulopoiesis and granulocytic differentiation, and Stat3 is the principle Stat protein activated by G-CSF. Upon treatment with G-CSF, the interleukin-3-dependent cell line 32D clone 3(32Dcl3) differentiates into neutrophils, and 32Dcl3 cells expressing dominant-negative Stat3 (32Dcl3/DNStat3) proliferate in G-CSF without differentiation. Gene expression profile and quantitative PCR analysis of G-CSF-stimulated cell lines revealed that the expression of C/EBPalpha was up-regulated by the activation of Stat3. In addition, activated Stat3 bound to CCAAT/enhancer-binding protein (C/EBP)alpha, leading to the enhancement of the transcription activity of C/EBPalpha. Conditional expression of C/EBPalpha in 32Dcl3/DNStat3 cells after G-CSF stimulation abolishes the G-CSF-dependent cell proliferation and induces granulocytic differentiation. Although granulocyte-specific genes, such as the G-CSF receptor, lysozyme M, and neutrophil gelatinase-associated lipocalin precursor (NGAL) are regulated by Stat3, only NGAL was induced by the restoration of C/EBPalpha after stimulation with G-CSF in 32Dcl3/DNStat3 cells. These results show that one of the major roles of Stat3 in the G-CSF signaling pathway is to augment the function of C/EBPalpha, which is essential for myeloid differentiation. Additionally, cooperation of C/EBPalpha with other Stat3-activated proteins are required for the induction of some G-CSF responsive genes including lysozyme M and the G-CSF receptor.
Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Fator Estimulador de Colônias de Granulócitos/metabolismo , Células Mieloides/metabolismo , Transdução de Sinais , Transativadores/metabolismo , Transativadores/fisiologia , Transcrição Gênica , Proteínas de Fase Aguda/metabolismo , Animais , Western Blotting , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Citocinas/metabolismo , Citometria de Fluxo , Regulação da Expressão Gênica , Genes Dominantes , Granulócitos/citologia , Granulócitos/metabolismo , Humanos , Immunoblotting , Imunoprecipitação , Interferon-alfa/metabolismo , Interleucina-3/metabolismo , Lipocalina-2 , Lipocalinas , Camundongos , Muramidase/química , Muramidase/metabolismo , Neutrófilos/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Oncogênicas/metabolismo , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT3 , Fatores de TempoRESUMO
Receptor for activated C kinase (Rack)-1 is a protein kinase C-interacting protein, and contains a WD repeat but has no enzymatic activity. In addition to protein kinase C, Rack-1 also binds to Src, phospholipase Cgamma, and ras-GTPase-activating proteins. Thus, Rack-1 is thought to function as a scaffold protein that recruits specific signaling elements. In a cytokine signaling cascade, Rack-1 has been reported to interact with the IFN-alphabeta receptor and Stat1. In addition, we show here that Rack-1 associates with a member of Jak, tyrosine kinase 2 (Tyk2). Rack-1 interacts weakly with the kinase domain and interacts strongly with the pseudokinase domain of Tyk2. Rack-1 associates with Tyk2 via two regions, one in the N terminus and one in the middle portion (aa 138-203) of Rack-1. Jak activation causes the phosphorylation of tyrosine 194 on Rack-1. After phosphorylation, Rack-1 is translocated toward the perinuclear region. In addition to functioning as a scaffolding protein, these results raise the possibility that Rack-1 functions as a signaling molecule in cytokine signaling cascades.
Assuntos
Proteínas Tirosina Quinases/metabolismo , Receptores de Superfície Celular/metabolismo , Motivos de Aminoácidos , Humanos , Fosforilação , Estrutura Terciária de Proteína , Receptores de Quinase C Ativada , TYK2 Quinase , Tirosina/metabolismoRESUMO
OBJECTIVE: Limitin, an interferon-like cytokine, suppresses B lymphopoiesis through ligation of the interferon-alpha/beta (IFN-alpha/beta) receptor. The aim of this study was to examine the intracellular signal transduction pathways activated by limitin. MATERIALS AND METHODS: The effects of limitin on cell growth, the activation of Jak kinase and Stat proteins, and the induction of interferon regulatory factor-1 (IRF-1) and Daxx were examined using the mouse pre-B-cell line 18.81, wild-type, and Tyk2-deficient mouse bone marrow cells. In addition, the change of localization of the Daxx protein after limitin treatment in wild-type and Tyk2-deficient mice was examined. RESULTS: Limitin phosphorylates Tyk2, Jak1, Stat1, and Stat2 and rapidly induces IRF-1 mRNA production. Phosphorylation of Stat1 by limitin is partially dependent on Tyk2. Suppression of B-cell growth by limitin, however, is severely impaired in the absence of Tyk2, whereas it is unaffected by the absence of Stat1. Limitin also induces the expression and nuclear translocation of Daxx, which is essential for IFN-alpha-induced inhibition of B-lymphocyte development. The absence of Tyk2 abrogates this induction of Daxx expression and nuclear translocation. CONCLUSIONS: Limitin suppresses B-cell growth through activation of Tyk2, resulting in the up-regulation and nuclear translocation of Daxx. This limitin-mediated signaling pathway does not require Stat1.
Assuntos
Linfócitos B/citologia , Proteínas de Transporte/metabolismo , Imunoglobulinas/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana/farmacologia , Proteínas Nucleares/metabolismo , Proteínas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Animais , Linfócitos B/efeitos dos fármacos , Células da Medula Óssea , Proteínas de Transporte/biossíntese , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Proteínas Correpressoras , Proteínas de Ligação a DNA/metabolismo , Janus Quinase 1 , Camundongos , Camundongos Knockout , Chaperonas Moleculares , Proteínas Nucleares/biossíntese , Fosforilação/efeitos dos fármacos , Proteínas Tirosina Quinases/metabolismo , Proteínas/genética , Fator de Transcrição STAT1 , TYK2 Quinase , Transativadores/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
Interferon (IFN)-alpha and IFN-gamma suppress the growth of haematopoietic progenitor cells. IFN-alpha activates Janus kinase-1 (Jak1) and Tyrosine kinase-2 (Tyk2), followed by the phosphorylation of the signal transducers and activators of transcription, Stat1 and Stat2. IFN-gamma activates Jak1 and Jak2, followed by the activation of Stat1. Activated Stats bind the promoter regions of IFN-inducible genes. We evaluated the role of Tyk2 and Stat1 in the IFN-mediated inhibition of haematopoietic progenitor cell growth. While IFN-alpha (1000 U/ml) suppressed the number of granulocyte-macrophage colony-forming units (CFU-GM) or erythroid burst-forming units (BFU-E) from wild-type mouse bone marrow cells, this suppression was partially inhibited by a deficiency in Tyk2 and completely inhibited by a deficiency in Stat1. High levels of IFN-alpha (10,000 U/ml) suppressed the CFU-GM or BFU-E obtained from Stat1-deficient mice, but did not suppress this growth in cells from Tyk2-deficient mice. Stat1 was phosphorylated by IFN-alpha in Tyk2-deficient cells, although the level of phosphorylation was weaker than that observed in wild type mice. Thus, the inhibitory signal on haematopoietic progenitor cells mediated by IFN-alpha may be transduced by two signalling pathways, one regulated by Tyk2 and the other dependent on Stat1. IFN-gamma also suppressed the number of CFU-GM or BFU-E, and this pathway was mediated by IFN-gamma in a Stat1-dependent manner, independently of Tyk2.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Células-Tronco Hematopoéticas/fisiologia , Interferons/farmacologia , Líquido Intracelular/imunologia , Proteínas Tirosina Quinases , Proteínas/metabolismo , Transdução de Sinais/fisiologia , Transativadores/metabolismo , Animais , Divisão Celular/fisiologia , Ensaio de Unidades Formadoras de Colônias , Proteínas de Ligação a DNA/genética , Depressão Química , Interferon-alfa/farmacologia , Interferon gama/farmacologia , Camundongos , Camundongos Knockout , Fosforilação , Proteínas/genética , Fator de Transcrição STAT1 , TYK2 Quinase , Transativadores/genéticaRESUMO
Many hematopoietic cells undergo apoptosis when deprived of specific cytokines. Lipocalin 24p3, reported to be induced in hematopoietic cells by interleukin 3 (IL-3) depletion, induces hematopoietic cell apoptosis despite the presence of IL-3. As granulocyte colony stimulating factor (G-CSF) depletion also induces the apoptosis of G-CSF-dependent cell line cells, we examined the effect of 24p3 on the apoptotic function induced by G-CSF depletion. 24p3 was induced by the depletion of IL-3, but not G-CSF, in cytokine-dependent cell lines. Although 24p3 suppressed growth induced by IL-3, it did not influence G-CSF-dependent cell growth. These observations show that 24p3 is not involved in the G-CSF withdrawal-induced apoptosis, although it is essential in IL-3 withdrawal-induced apoptosis.