RESUMO
We recently demonstrated that STAT5 can induce a variety of biological functions in mouse IL-3-dependent Ba/F3 cells; STAT5-induced expression of pim-1, p21(WAF/Cip1), and suppressor of cytokine signaling-1/STAT-induced STAT inhibitor-1/Janus kinase binding protein is responsible for induction of proliferation, differentiation, and apoptosis, respectively. In the present study, using a constitutively active STAT5A (STAT5A1*6), we show that STAT5 induces macrophage differentiation of mouse leukemic M1 cells through a distinct mechanism, autocrine production of IL-6. The supernatant of STAT5A1*6-transduced cells contained sufficient concentrations of IL-6 to induce macrophage differentiation of parental M1 cells, and STAT3 was phosphorylated on their tyrosine residues in these cells. Treatment of the cells with anti-IL-6 blocking Abs profoundly inhibited the differentiation. We also found that the STAT5A1*6 transactivated the IL-6 promoter, which was mediated by the enhanced binding of NF-kappaB p65 (RelA) to the promoter region of IL-6. These findings indicate that STAT5A cooperates with Rel/NF-kappaB to induce production of IL-6, thereby inducing macrophage differentiation of M1 cells in an autocrine manner. In summary, we have shown a novel mechanism by which STAT5 induces its pleiotropic functions. Cytokines
Assuntos
Proteínas de Ligação a DNA/fisiologia , Interleucina-6/genética , Macrófagos/imunologia , Proteínas do Leite , NF-kappa B/fisiologia , Transativadores/fisiologia , Animais , Anticorpos/imunologia , Apoptose , Comunicação Autócrina , Células COS , Diferenciação Celular , Linhagem Celular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Interleucina-6/biossíntese , Interleucina-6/imunologia , Leucemia Mieloide Aguda , Macrófagos/citologia , Camundongos , Mutação , Fosforilação , Regiões Promotoras Genéticas , Fator de Transcrição STAT3 , Fator de Transcrição STAT5 , Transativadores/genética , Transativadores/metabolismo , Fator de Transcrição RelA , Ativação Transcricional , Células Tumorais CultivadasRESUMO
In a search for a human sequence related to a recently identified type I cytokine receptor delta1, which turned out to be a receptor subunit for a cytokine called TSLP, we have now identified a novel human type I cytokine receptor from a human T lymphocyte cDNA library. The deduced amino acid sequence of 371 residues has a typical signal sequence and a membrane-spanning region. The mature protein is predicted to have a molecular mass of 39,698 Da. The N-terminal extracellular region contains two fibronectin type III-like domains, four conserved cysteine residues, and a WSXWS box-like motif. The C-terminal intracellular region contains box 1 and box 2-like motifs. Thus, it has common characteristics of type I cytokine receptor family members, and we tentatively termed this protein CRLF2, which stands for cytokine receptor-like factor 2. Northern blot analysis revealed CRLF2 mRNA in liver, kidney, heart, and skeletal muscle. The fetal liver also expresses CRLF2 transcripts. The gene for CRLF2 was mapped to the pseudoautosomal region, Xp22.3 and Yp11.3 by FISH analysis, a region where genes encoding the IL-3 receptor alpha and the GM-CSF receptor alpha chains are also located. The biological function of this newly identified receptor is now under investigation.
Assuntos
Receptores de Citocinas/química , Receptores de Citocinas/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Sequência Conservada , Perfilação da Expressão Gênica , Humanos , Imunoglobulinas , Hibridização in Situ Fluorescente , Camundongos , Dados de Sequência Molecular , Mapeamento Físico do Cromossomo , Estrutura Terciária de Proteína , Subunidades Proteicas , RNA Mensageiro/análise , RNA Mensageiro/genética , Receptores de Citocinas/classificação , Alinhamento de Sequência , Cromossomo X/genéticaRESUMO
To identify the key molecules that regulate differentiation of hematopoietic cells, we carried out retrovirus-mediated functional screening for cDNAs whose expression suppresses IL-6-induced differentiation of mouse myeloid leukemic M1 cells. From this screening, we obtained a full length cDNA encoding a mouse homologue of human MgcRacGAP. Overexpression of the anti-sense MgcRacGAP profoundly inhibited IL-6-induced macrophage-differentiation of M1 cells. On the other hand, overexpression of the full-length form of MgcRacGAP alone enhanced macrophage differentiation of M1 cells in response to IL-6, and induced macrophage differentiation of HL-60 leukemic cells. To determine how this protein regulates differentiation and proliferation, an antibody against MgcRacGAP was prepared. Immunohistochemical studies revealed that MgcRacGAP mainly localizes in the nucleus in interphase, accumulates on the mitotic spindle in metaphase, and is condensed in the midbody during cytokinesis. Overexpression of an N-terminal domain deletion mutant, which lacks the ability to localize to the midbody through association with tubulins, or a GAP-inactive mutant resulted in the formation of multinucleated cells in HeLa cells as well as in hemopoietic cells. Interestingly, MgcRacGAP in the midbody was phosphorylated probably on serine and threonine residues. These results indicate that MgcRacGAP regulates cytokinesis and cellular differentiation as a regulator of Rho family of GTPase and suggest that this process is controlled by some serine/threonine kinases.
Assuntos
Diferenciação Celular , Divisão Celular , Proteínas Ativadoras de GTPase/metabolismo , Proteínas do Leite , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Proteínas de Ligação a DNA/metabolismo , Ativadores de GTP Fosfo-Hidrolase/metabolismo , Proteínas Ativadoras de GTPase/genética , Células HeLa , Humanos , Imuno-Histoquímica , Interleucina-6/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Modelos Biológicos , NF-kappa B/metabolismo , Fator de Transcrição STAT5 , Transativadores/metabolismoRESUMO
We analyzed a megakaryocyte-derived protein immunoreactive with an antiserum to cartilage oligomeric matrix protein (COMP) in the developing rat liver. Staining with the anti-COMP antiserum in the developing rat liver increased during embryogenesis, and was strongest in the livers of 17-day-old embryos. However, staining in the liver was not detected at eight days after birth or thereafter. The stained cells were found to be megakaryocytes. We partially purified the protein showing cross-reaction with the antiserum to COMP from a megakaryocyte-rich cells fraction in 17-day-old embrionic rat livers. The molecular weight of this protein (approximately 95 kDa) was close to the molecular weight of COMP (105 kDa). Amplification of an RT-PCR fragment (225 bp) corresponding to part of COMP mRNA was detected in cartilage, but not in megakaryocytes of fetal liver or bone marrow. Based on these results, the fetal rat liver megakaryocyte-derived protein that reacted with the antiserum against COMP was thought to contain a common epitope with COMP from cartilage, but to be a different protein from COMP.
