RESUMEN
In cytotoxic T cells (CTL), Akt, also known as protein kinase B, is activated by the T cell antigen receptor (TCR) and the cytokine interleukin 2 (IL-2). Akt can control cell metabolism in many cell types but whether this role is important for CTL function has not been determined. Here we have shown that Akt does not mediate IL-2- or TCR-induced cell metabolic responses; rather, this role is assumed by other Akt-related kinases. There is, however, a nonredundant role for sustained and strong activation of Akt in CTL to coordinate the TCR- and IL-2-induced transcriptional programs that control expression of key cytolytic effector molecules, adhesion molecules, and cytokine and chemokine receptors that distinguish effector versus memory and naive T cells. Akt is thus dispensable for metabolism, but the strength and duration of Akt activity dictates the CTL transcriptional program and determines CTL fate.
Asunto(s)
Regulación de la Expresión Génica , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Proto-Oncogénicas c-akt/fisiología , Linfocitos T Citotóxicos/inmunología , Transcripción Genética , Proteínas Quinasas Dependientes de 3-Fosfoinosítido , Adenina/análogos & derivados , Adenina/farmacología , Animales , División Celular , Movimiento Celular , Fosfatidilinositol 3-Quinasa Clase I , Citotoxicidad Inmunológica , Glucosa/metabolismo , Interferón gamma/biosíntesis , Interferón gamma/genética , Interleucina-2/farmacología , Interleucina-2/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fenilalanina/metabolismo , Fosfatidilinositol 3-Quinasas/fisiología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Citotóxicas Formadoras de Poros/biosíntesis , Proteínas Citotóxicas Formadoras de Poros/genética , Quinazolinas/farmacología , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Citocinas/biosíntesis , Receptores de Citocinas/genética , Linfocitos T Citotóxicos/metabolismoRESUMEN
In normal T cell progenitors, phosphoinositide-dependent kinase l (PDK1)-mediated phosphorylation and activation of protein kinase B (PKB) is essential for the phosphorylation and inactivation of Foxo family transcription factors, and also controls T cell growth and proliferation. The current study has characterized the role of PDK1 in the pathology caused by deletion of the tumor suppressor phosphatase and tensin homologue deleted on chromosome 10 (PTEN). PDK1 is shown to be essential for lymphomagenesis caused by deletion of PTEN in T cell progenitors. However, PTEN deletion bypasses the normal PDK1-controlled signaling pathways that determine thymocyte growth and proliferation. PDK1 does have important functions in PTEN-null thymocytes, notably to control the PKB-Foxo signaling axis and to direct the repertoire of adhesion and chemokine receptors expressed by PTEN-null T cells. The results thus provide two novel insights concerning pathological signaling caused by PTEN loss in lymphocytes. First, PTEN deletion bypasses the normal PDK1-controlled metabolic checkpoints that determine cell growth and proliferation. Second, PDK1 determines the cohort of chemokine and adhesion receptors expressed by PTEN-null cells, thereby controlling their migratory capacity.
Asunto(s)
Movimiento Celular , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Fosfohidrolasa PTEN/deficiencia , Proteínas Serina-Treonina Quinasas/metabolismo , Linfocitos T/citología , Linfocitos T/enzimología , Proteínas Quinasas Dependientes de 3-Fosfoinosítido , Animales , Moléculas de Adhesión Celular/metabolismo , Proliferación Celular , Factores de Transcripción Forkhead/metabolismo , Eliminación de Gen , Integrasas/metabolismo , Tejido Linfoide/citología , Ratones , Fosfohidrolasa PTEN/metabolismo , Fosforilación , Receptores de Quimiocina/metabolismo , Transducción de Señal , Células Madre/citología , Células Madre/enzimología , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
We have developed hematopoietic cells resistant to the cytotoxic effects of oxysterols. Oxysterol-resistant HL60 cells were generated by continuous exposure to three different oxysterols-25-hydroxycholesterol (25-OHC), 7-beta-hydroxycholesterol (7beta-OHC) and 7-keto-cholesterol (7kappa-C). We investigated the effects of 25-OHC, 7beta-OHC, 7kappa-C and the apoptotic agent staurosporine on these cells. The effect of the calcium channel blocker nifedipine on oxysterol cytotoxicity was also investigated. Differential display and real-time PCR were used to quantitate gene expression of oxysterol-sensitive and -resistant cells. Our results demonstrate that resistance to the cytotoxic effects of oxysterols is relatively specific to the type of oxysterol, and that the cytotoxicity of 25-OHC but not that of 7beta-OHC and 7kappa-C, appears to occur by a calcium dependent mechanism. Oxysterol-resistant cells demonstrated no significant difference in the expression of several genes previously implicated in oxysterol resistance, but expressed the bcl-2 gene at significantly lower levels than those observed in control cells. We identified three novel genes differentially expressed in resistant cells when compared to HL60 control cells. Taken together, the results of this study reveal potentially novel mechanisms of oxysterol cytotoxicity and resistance, and indicate that cytotoxicity of 25-OHC, 7beta-OHC and 7kappa-C occur by independent, yet overlapping mechanisms.
Asunto(s)
Leucemia/metabolismo , Esteroles/farmacología , Secuencia de Aminoácidos , Secuencia de Bases , Cartilla de ADN , Células HL-60 , Humanos , Datos de Secuencia Molecular , Nifedipino/farmacología , Homología de Secuencia de AminoácidoRESUMEN
UNLABELLED: Human osteoclasts can be efficiently generated in vitro from cord blood mononuclear cells and derived CFU-GM colonies. However, CFU-M colonies are poorly osteoclastogenic. Short-term (2-48 h) treatment with GM-CSF stimulates osteoclast formation by proliferating precursors, whereas longer exposure favors dendritic cell formation. INTRODUCTION: Osteoclasts (OC) differentiate from cells of the myelomonocytic lineage under the influence of macrophage-colony stimulating factor (M-CSF) and RANKL. However, cells of this lineage can also differentiate to macrophages and dendritic cells (DC) depending on the cytokine environment. The aims of this study were to develop an efficient human osteoclastogenesis model and to investigate the roles of granulocyte macrophage-colony stimulating factor (GM-CSF) and M-CSF in human OC differentiation. MATERIALS AND METHODS: A human osteoclastogenesis model, using as precursors colony forming unit-granulocyte macrophage (CFU-GM) colonies generated from umbilical cord mononuclear cells cultured in methylcellulose with GM-CSF, interleukin (IL)-3 and stem cell factor (SCF), has been developed. CFU-GM, colony forming unit-macrophage (CFU-M), or mixed colonies were cultured on dentine with soluble RANKL (sRANKL) and human M-CSF with and without GM-CSF. Major endpoints were OC number, dentine resorption, and CD1a+ DC clusters. RESULTS: Osteoclast generation from CFU-GM and mixed colonies treated with M-CSF and sRANKL for 7-14 days was highly efficient, but CFU-M colonies were poorly osteoclastogenic under these conditions. Pretreatment of precursors with M-CSF for 7 or 14 days maintained the precursor pool, but OCs were smaller and resorption was reduced. The effect of GM-CSF treatment was biphasic, depending on the timing and duration of exposure. Short-term treatment (2-48 h) at the beginning of the culture stimulated cell proliferation and enhanced OC formation up to 100%, independent of sRANKL. Longer-term GM-CSF treatment in the presence of sRANKL, however, inhibited OC generation with the formation of extensive CD1a+ DC clusters, accompanied by downregulation of c-Fos mRNA. Delaying the addition of GM-CSF resulted in progressively less inhibition of osteoclastogenesis. CONCLUSIONS: Human CFU-GM, but not CFU-M, progenitors have high osteoclastogenic potential. GM-CSF plays an important role in osteoclastogenesis and has a biphasic effect: Short-term treatment potentiates OC differentiation by proliferating precursors, but persistent exposure favors DC formation.
Asunto(s)
Factor Estimulante de Colonias de Granulocitos y Macrófagos/fisiología , Osteoclastos/citología , Secuencia de Bases , Diferenciación Celular/fisiología , Cartilla de ADN , Humanos , Inmunohistoquímica , Factor Estimulante de Colonias de Macrófagos/fisiologíaRESUMEN
BACKGROUND: The aim of this study was to determine the influence of mode of birth and umbilical cord blood (CB) collection before (in utero) or after delivery of the placenta (ex utero) on total number of WBCs and CD34+ cells in CB units. STUDY DESIGN AND METHODS: Consecutively donated, banked CB units were assessed for net volume, WBC concentration, total number of WBCs, proportion of CD34+ cells, and total number of CD34+ cells. These parameters were then correlated with the mode of birth and the mode of CB collection relative to the delivery of the placenta. RESULTS: A significantly higher CB volume was seen following cesarean section (n = 61) than following vaginal delivery (n = 157; median volume, 76 vs. 63 mL, respectively; p < 0.0001). In contrast, CB from vaginal delivery had a significantly higher WBC concentration compared with CB from cesarean section (medians, 17.1 x 10(9) and 13.6 x 10(9) WBCs/L, respectively; p < 0.0001). The mode of birth did not influence the proportion of CD34+ cells. A correlation was demonstrated between the total number of CD34+ cells and the total number of WBCs. As a consequence of the opposing effects on volume and WBC counts by cesarean section and vaginal delivery, there were no significant differences in the total number of WBCs or CD34+ cells for the CB units with mode of delivery in this study. No significant differences were found in CB with mode of CB collection (in utero [n = 58] or ex utero [n = 99]) following vaginal delivery. CONCLUSIONS: The mode of birth influences the CB WBC concentration and volume collected and should be taken into consideration for establishing any acceptance limits for CB units to be banked. There were no differences in CB with in utero or ex utero collections.