RESUMO
THOC5 is a nuclear/cytoplasmic protein member of the spliceosome complex which potentiates C/EBP expression in adipocyte differentiation. As C/EBP family members are important regulators of myelopoiesis and THOC5 is highly expressed in neutrophil/macrophage progenitor cells we assessed the role of THOC5 in cytokine-stimulated monocytic development. M-CSF stimulated maturation of the NFS60 cell line was associated with enhanced THOC5 expression and phosphorylation. THOC5 was also shown to form a complex with C/EBPbeta. Ectopic expression of THOC5 mimicked M-CSF mediated cell maturation and enhanced protein expression of the myeloid transcription factors C/EBPbeta, C/EBPalpha, Pu-1 and also GAB2 (a PI-3 Kinase and macrophage development regulator). Increased THOC5 expression also mimicked M-CSF stimulated increases in the lipid second messenger PtdInsP(3). Inhibition of THOC5-induced increases in PtdInsP(3) levels abrogated the elevated levels of C/EBPbeta. Thus THOC5 expression can potentiate receptor signalling to transcription factor expression and monocyte differentiation.
Assuntos
Fator Estimulador de Colônias de Macrófagos/farmacologia , Proteínas Nucleares/metabolismo , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Linhagem Celular , Humanos , Fator Estimulador de Colônias de Macrófagos/metabolismo , Camundongos , Monócitos/citologia , Monócitos/metabolismo , Proteínas Nucleares/imunologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Transdução de Sinais , Fatores de Transcrição/genética , TransfecçãoRESUMO
The chemokine, stromal cell-derived factor-1 (SDF-1) is a crucial regulator of stem cell homing and tethering, and potentiation of this pathway in leukaemias may contribute to the pathogenesis of the disease. A key second messenger in SDF-1 signal/response coupling is phosphatidylinositol 3,4,5-triphosphate [PtdIns(3,4,5)P3]. SDF-1 elevated PtdIns(3,4,5)P3 levels markedly in the multipotent FDCP-mix stem cell line. Similarly, transfection with BCR/ABL or TEL/PDGFRbeta leukaemogenic tyrosine kinases chronically elevated PtdIns(3,4,5)P3 levels. However, whilst an SDF-1 chemotactic response was observed in TEL/PDGFRbeta-transfected cells, in BCR/ABL cells this was markedly decreased, which was not due to Ras-pathway activation. Thus, multipotent cells can respond to SDF-1, despite chronic increases in this second messenger indicating that a discrete pool of SDF-1-stimulated PtdIns(3,4,5)P3 production drives the chemotactic response. To discern the mechanism for the differential effects of these oncogenes we considered subcellular localisation. As TEL/PDGFRbeta has a cytosolic location whilst BCR/ABL associates with actin, we removed the actin-binding domain from BCR/ABL. We observed relocation of BCR/ABL to the cytosol and increased SDF-1 responses. We conclude that the localisation of BCR/ABL to the cytoskeleton is essential for effects on motility and moderating SDF-1 responses is not essential in tyrosine kinase-mediated leukaemic transformation.
Assuntos
Transformação Celular Neoplásica/metabolismo , Quimiocinas CXC/farmacologia , Quimiotaxia/fisiologia , Proteínas de Fusão bcr-abl/fisiologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Actinas/metabolismo , Animais , Linhagem Celular , Quimiocina CXCL12 , Quimiotaxia/efeitos dos fármacos , Citoesqueleto/metabolismo , Relação Dose-Resposta a Droga , Proteínas de Fusão bcr-abl/análise , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/fisiologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Camundongos , Proteínas de Fusão Oncogênica/metabolismo , Fosfatidilinositol 3-Quinases/fisiologia , Fosfatos de Fosfatidilinositol/biossíntese , Transdução de Sinais , TransfecçãoRESUMO
Lineage-marker depleted (Lin(-)) murine bone marrow cells expressing stem cell antigen 1 (Sca-1) were sorted on the basis of stem cell factor receptor (c-kit) expression to obtain Lin(-)Sca(+)Kit(+) or Lin(-)Sca(+)Kit(-) cells. Lin(-)Sca(+)Kit(-) cells have a markedly greater chemotactic response to stromal derived factor-1 (SDF-1). Using a novel fluorescent stain, we show that both populations generate similar levels of a key messenger, phosphatidylinositol 3,4,5 trisphosphate (PIP(3)), in response to SDF-1. Differences in motile behavior may therefore lie downstream of phosphatidylinositol 3-kinase (PI3-kinase) activation at the level of cytoskeleton regulation. The 2 cell populations were compared using 2-dimensional difference gel electrophoresis (2D-DIGE), with a maleimide CyDye fluorescent protein labeling technique that has enhanced sensitivity for low abundance samples. Comparative proteomic analysis of Cy3- and Cy5-labeled protein samples allows relative quantification of protein spots present in both cell populations; of these, 73% were common. Key protein differences were adseverin and gelsolin, actin micro-filament splicing proteins, regulated by Rac, downstream of PI3-kinase activation. Adseverin was shown to be acetylated, a novel modification for this protein. Differences in major regulators of cell shape and motility between the 2 populations can explain the differential response to SDF-1.
