RESUMO
OBJECTIVE: During megakaryopoiesis, hematopoietic progenitor cells in the bone marrow proliferate and ultimately differentiate in mature megakaryocytes (MK). We and others have recently described a role for the mammalian target of Rapamycin (mTOR) in proliferation and differentiation of MK cells. Two non-redundant complexes of mTOR have been described; mTORC1 containing rapamycin-associated TOR protein (Raptor) and mTORC2 containing Rapamycin-insensitive companion of mTOR (Rictor). The individual roles of these complexes in MK development have so far not been elucidated, and were investigated in this study. METHODS: We have used an siRNA approach to selectively knock down either Rictor or Raptor expression in MO7e megakaryoblastic cells. Using flow cytometry, nuclear ploidity, and cell cycling as assessed by BrdU incorporation were investigated. Electron microscopy and cotransductions with GFP-LC3 were used to quantify autophagy. Activation of intracellular signal transduction pathways was studied by Western blot analysis. RESULTS: We observed a reduced cell cycling upon Rictor siRNA transduction, resulting in decreased numbers of polypoid cells. Knocking down Raptor expression resulted in a reduced expansion and a reduced cell size. In addition, increased autophagy was observed in Raptor siRNA-transduced cells, in correspondence with an attenuation of activation of the p70S6K/S6, and 4E-BP pathways. CONCLUSIONS: The current study shows that the mTORC1 and mTORC2 complexes have distinct, non-redundant functions in MO7e MK cell proliferation, and development. The mTOR/Rictor complex affects megakaryopoiesis by regulating nuclear division and subsequent cell cycle progression, whereas Raptor signaling protects MK cells from autophagic cell death, enabling normal megakaryopoiesis to take place.
Assuntos
Proteínas de Transporte/metabolismo , Regulação da Expressão Gênica , Megacariócitos/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Células da Medula Óssea/citologia , Proliferação de Células , Citometria de Fluxo/métodos , Vetores Genéticos , Proteínas de Fluorescência Verde/metabolismo , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Complexos Multiproteicos , RNA Interferente Pequeno/metabolismo , Proteína Companheira de mTOR Insensível à Rapamicina , Proteína Regulatória Associada a mTOR , Transdução de Sinais , Serina-Treonina Quinases TOR , Fatores de Transcrição/metabolismoRESUMO
Patients with myelodysplasia (MDS) show a differentiation defect in the multipotent stem-cell compartment. An important factor in stem-cell differentiation is their proper localization within the bone marrow microenvironment, which is regulated by stromal cell-derived factor (SDF-1). We now show that SDF-1-induced migration of CD34(+) progenitor cells from MDS patients is severely impaired. In addition, these cells show a reduced capacity to polymerize F-actin in response to SDF-1. We demonstrate a major role for Rac and phosphatidylinositol 3-kinase (PI3K) and a minor role for the extracellular signal-regulated kinase (ERK)1/2 signaling pathway in SDF-1-induced migration of normal CD34(+) cells. Furthermore, SDF-1-stimulated activation of Rac and the PI3K target protein kinase B is impaired in CD34(+) cells from MDS patients. Lentiviral transduction of MDS CD34(+) cells with constitutive active Rac1V12 results in a partial restoration of F-actin polymerization in response to SDF-1. In addition, expression of constitutive active Rac increases the motility of MDS CD34(+) cells in the absence of SDF-1, although the directional migration of cells toward this chemoattractant is not affected. Taken together, our results show a reduced migration of MDS CD34(+) cells toward SDF-1, as a result of impaired activation of the PI3K and Rac pathways and a decreased F-actin polymerization.
Assuntos
Actinas/metabolismo , Movimento Celular/fisiologia , Quimiocina CXCL12/metabolismo , Defeitos do Tubo Neural/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos CD34/metabolismo , Movimento Celular/efeitos dos fármacos , Quimiocina CXCL12/farmacologia , Células HL-60 , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Pessoa de Meia-Idade , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Defeitos do Tubo Neural/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Polímeros/metabolismo , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
Biallelic germline mutations of MUTYH-a gene encoding a base excision repair protein-are associated with an increased susceptibility of colorectal cancer. Whether monoallelic MUTYH mutations also increase cancer risk is not yet clear, although there is some evidence suggesting a slight increase of risk. As the MUTYH protein interacts with the mismatch repair (MMR) system, we hypothesised that the combination of a monoallelic MUTYH mutation with an MMR gene mutation increases cancer risk. We therefore investigated the prevalence of monoallelic MUTYH mutations in carriers of a germline MMR mutation: 40 carriers of a truncating mutation (group I) and 36 of a missense mutation (group II). These patients had been diagnosed with either colorectal or endometrial cancer. We compared their MUTYH mutation frequencies with those observed in a group of 134 Dutch colorectal and endometrial cancer patients without an MMR gene mutation (0.7%) and those reported for Caucasian controls (1.5%). In group I one monoallelic MUTYH mutation was found (2.5%). In group II five monoallelic germline MUTYH mutations were found (14%), four of them in MSH6 missense mutation carriers (20%). Of all patients with an MMR gene mutation, only those with a missense mutation showed a significantly higher frequency of (monoallelic) MUTYH mutations than the Dutch cancer patients without MMR gene mutations (P = 0.002) and the published controls (P = 0.001). These results warrant further study to test the hypothesis of mutations in MMR genes (in particular MSH6) and MUTYH acting together to increase cancer risk.
Assuntos
Neoplasias Colorretais Hereditárias sem Polipose/genética , DNA Glicosilases/genética , Reparo de Erro de Pareamento de DNA , Mutação , Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte/análise , Proteínas de Transporte/genética , Neoplasias Colorretais Hereditárias sem Polipose/patologia , Análise Mutacional de DNA , Reparo do DNA/genética , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/genética , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/patologia , Feminino , Mutação em Linhagem Germinativa , Humanos , Imuno-Histoquímica , Instabilidade de Microssatélites , Proteína 1 Homóloga a MutL , Proteína 2 Homóloga a MutS/análise , Proteína 2 Homóloga a MutS/genética , Mutação de Sentido Incorreto , Proteínas Nucleares/análise , Proteínas Nucleares/genéticaRESUMO
Thrombopoietin (TPO) is a potent regulator of megakaryopoiesis and stimulates megakaryocyte (MK) progenitor expansion and MK differentiation. In this study, we show that TPO induces activation of the mammalian target of rapamycin (mTOR) signaling pathway, which plays a central role in translational regulation and is required for proliferation of MO7e cells and primary human MK progenitors. Treatment of MO7e cells, human CD34+, and primary MK cells with the mTOR inhibitor rapamycin inhibits TPO-induced cell cycling by reducing cells in S phase and blocking cells in G0/G1. Rapamycin markedly inhibits the clonogenic growth of MK progenitors with high proliferative capacity but does not reduce the formation of small MK colonies. Addition of rapamycin to MK suspension cultures reduces the number of MK cells, but inhibition of mTOR does not significantly affect expression of glycoproteins IIb/IIIa (CD41) and glycoprotein Ib (CD42), nuclear polyploidization levels, cell size, or cell survival. The downstream effectors of mTOR, p70 S6 kinase (S6K) and 4E-binding protein 1 (4E-BP1), are phosphorylated by TPO in a rapamycin- and LY294002-sensitive manner. Part of the effect of the phosphatidyl inositol 3-kinase pathway in regulating megakaryopoiesis may be mediated by the mTOR/S6K/4E-BP1 pathway. In conclusion, these data demonstrate that the mTOR pathway is activated by TPO and plays a critical role in regulating proliferation of MK progenitors, without affecting differentiation or cell survival.