RESUMEN

MicroRNAs (miRNA) are small noncoding RNAs that regulate gene expression by targeting mRNAs in a sequence specific manner, thereby determining their degradation or inhibiting translation. They are involved in processes such as proliferation, differentiation and apoptosis by fine-tuning the expression of genes underlying such events. The expression of specific miRNAs is involved in hematopoietic differentiation and their deregulation contributes to the development of hematopoietic malignancies such as acute myeloid leukemia (AML). miR-130a is over-expressed in AML. Here we show that miR-130a is physiologically expressed in myeloblasts and down-regulated during monocyte differentiation. Gain- and loss-of-function experiments performed on CD34+ human hematopoietic stem cells confirmed that expression of miR-130a inhibits monocyte differentiation by interfering with the expression of key transcription factors HOXA10, IRF8, KLF4, MAFB and PU-1. The data obtained in this study highlight that the correct modulation of miR-130a is necessary for normal differentiation to occur and confirming that deregulation of this miRNA might underlie the differentiation block occurring in AML.

Asunto(s)

Regulación de la Expresión Génica , Células Precursoras de Granulocitos/metabolismo , Células Madre Hematopoyéticas/metabolismo , MicroARNs/fisiología , Monocitos/citología , Mielopoyesis/fisiología , Proteínas de Neoplasias/fisiología , Antígenos CD34/análisis , Línea Celular Tumoral , Linaje de la Célula , Células Cultivadas , Ensayo de Unidades Formadoras de Colonias , Mutación con Ganancia de Función , Células Precursoras de Granulocitos/citología , Células Madre Hematopoyéticas/citología , Humanos , Factor 4 Similar a Kruppel , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Mutación con Pérdida de Función , MicroARNs/antagonistas & inhibidores , MicroARNs/biosíntesis , MicroARNs/genética , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Ácidos Nucleicos de Péptidos/farmacología , ARN Neoplásico/genética , ARN Neoplásico/fisiología , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética

RESUMEN

BACKGROUND/AIMS: Many potentially toxic molecules accumulate in the blood during hepatic dysfunction. In clinical practice, it is very difficult to remove bilirubin, the most widely studied toxin, and particularly the unconjugated form, strongly albumin-bound. The aim of this in vitro study was to assess irreversible bilirubin adsorption as a protein-bound compound marker, using Cytosorb® (Cytosorbents Corp.), a new hemoadsorption device designed to remove cytokines. METHODS: We performed 4 in vitro experiments, dynamic and static, with different albumin-bilirubin solutions. RESULTS: All experiments showed the resin's ability to break the albumin-bilirubin complex (Experiment 1, 2), leading to efficient bilirubin removal for 24 h (Removal Rate: 90% Experiment 3) with minimal albumin loss. No sign of bilirubin release from the charged resin was detected (Experiment 4). CONCLUSION: Cytosorb® seems a promising artificial liver support, thanks to its ability to adsorb bilirubin and its proven ability to modulate the cytokines involved in hepatic dysfunction.

Asunto(s)

Bilirrubina/sangre , Fallo Hepático/sangre , Fallo Hepático/terapia , Desintoxicación por Sorción/instrumentación , Desintoxicación por Sorción/métodos , Humanos , Albúmina Sérica Humana

RESUMEN

Mesalazine (5-ASA) is an aminosalicylate anti-inflammatory drug capable of inducing µ-protocadherin, a protein expressed by colorectal epithelial cells that is downregulated upon malignant transformation. Treatment with 5-ASA restores µ-protocadherin expression and promotes the sequestration of ß-catenin to the plasma membrane. Here, we show that 5-ASA-induced µ-protocadherin expression is directly regulated by the KLF4 transcription factor. In addition, we suggest the existence of a dual mechanism whereby 5-ASA-mediated ß-catenin inhibition is caused by µ-protocadherin-dependent sequestration of ß-catenin to the plasma membrane and by the direct binding of KLF4 to ß-catenin. In addition, we found that 5-ASA treatment suppresses the expression of miR-130a and miR-135b, which target KLF4 mRNA, raising the possibility that this mechanism is involved in the increased expression of KLF4 induced by 5-ASA. Cancer Prev Res; 11(8); 503-10. ©2018 AACR.

Asunto(s)

Antiinflamatorios no Esteroideos/farmacología , Neoplasias del Colon/prevención & control , Factores de Transcripción de Tipo Kruppel/metabolismo , Mesalamina/farmacología , Vía de Señalización Wnt/efectos de los fármacos , Antiinflamatorios no Esteroideos/uso terapéutico , Células CACO-2 , Proteínas Relacionadas con las Cadherinas , Cadherinas/metabolismo , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Regulación hacia Abajo/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HEK293 , Células HT29 , Humanos , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Mesalamina/uso terapéutico , MicroARNs/metabolismo , Unión Proteica/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Vía de Señalización Wnt/genética , beta Catenina/metabolismo

RESUMEN

A human bone marrow-derived mesenchymal stromal cell (MSCs) and cord blood-derived CD34+ stem cell co-culture system was set up in order to evaluate the proliferative and differentiative effects induced by MSCs on CD34+ stem cells, and the reciprocal influences on gene expression profiles. After 10 days of co-culture, non-adherent (SN-fraction) and adherent (AD-fraction) CD34+ stem cells were collected and analysed separately. In the presence of MSCs, a significant increase in CD34+ cell number was observed (fold increase = 14.68), mostly in the SN-fraction (fold increase = 13.20). This was combined with a significant increase in CD34+ cell differentiation towards the BFU-E colonies and with a decrease in the CFU-GM. These observations were confirmed by microarray analysis. Through gene set enrichment analysis (GSEA), we noted a significant enrichment in genes involved in heme metabolism (e.g. LAMP2, CLCN3, BMP2K), mitotic spindle formation and proliferation (e.g. PALLD, SOS1, CCNA1) and TGF-beta signalling (e.g. ID1) and a down-modulation of genes participating in myeloid and lymphoid differentiation (e.g. PCGF2) in the co-cultured CD34+ stem cells. On the other hand, a significant enrichment in genes involved in oxygen-level response (e.g. TNFAIP3, SLC2A3, KLF6) and angiogenesis (e.g. VEGFA, IGF1, ID1) was found in the co-cultured MSCs. Taken together, our results suggest that MSCs can exert a priming effect on CD34+ stem cells, regulating their proliferation and erythroid differentiation. In turn, CD34+ stem cells seem to be able to polarise the BM-niche towards the vascular compartment by modulating molecular pathways related to hypoxia and angiogenesis.

Asunto(s)

Células Eritroides/citología , Sangre Fetal/citología , Células Madre Hematopoyéticas/citología , Células Madre Mesenquimatosas/citología , Adulto , Células Madre Adultas/citología , Células Madre Adultas/metabolismo , Antígenos CD34/análisis , Proliferación Celular , Separación Celular , Células Cultivadas , Técnicas de Cocultivo , Células Eritroides/metabolismo , Femenino , Células Madre Hematopoyéticas/metabolismo , Humanos , Masculino , Células Madre Mesenquimatosas/metabolismo , Transcriptoma , Adulto Joven

RESUMEN

CD271 is the low-affinity neurotrophin (p75NTR) receptor that belongs to the tumor necrosis factor receptor superfamily. Because in human epidermis, CD271 is predominantly expressed in transit-amplifying (TA) cells, we evaluated the role of this receptor in keratinocyte differentiation and in the transition from keratinocyte stem cells (KSCs) to progeny. Calcium induced an upregulation of CD271 in subconfluent keratinocytes, which was prevented by CD271 small interfering RNA. Furthermore, CD271 overexpression provoked the switch of KSCs to TA cells, whereas silencing CD271 induced TA cells to revert to a KSC phenotype, as shown by the expression of ß1-integrin and by the increased clonogenic ability. CD271(+) keratinocytes sorted from freshly isolated TA cells expressed more survivin and keratin 15 (K15) compared with CD271(-) cells and displayed a higher proliferative capacity. Early differentiation markers and K15 were more expressed in the skin equivalent generated from CD271(+) TA than from those derived from CD271(-) TA cells. By contrast, late differentiation markers were more expressed in skin equivalents from CD271(-) than in reconstructs from CD271(+) TA cells. Finally, skin equivalents originated from CD271(-) TA cells displayed a psoriatic phenotype. These results indicate that CD271 is critical for keratinocyte differentiation and regulates the transition from KSCs to TA cells.

Asunto(s)

Diferenciación Celular/fisiología , Células Epidérmicas , Queratinocitos/citología , Proteínas del Tejido Nervioso/metabolismo , Receptores de Factor de Crecimiento Nervioso/metabolismo , Células Madre/citología , Calcio/farmacología , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Epidermis/efectos de los fármacos , Epidermis/metabolismo , Humanos , Técnicas In Vitro , Proteínas Inhibidoras de la Apoptosis/metabolismo , Queratina-15/metabolismo , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/genética , Fenotipo , Psoriasis/patología , ARN Interferente Pequeño/farmacología , Receptores de Factor de Crecimiento Nervioso/deficiencia , Receptores de Factor de Crecimiento Nervioso/genética , Células Madre/efectos de los fármacos , Células Madre/metabolismo , Survivin

RESUMEN

AIMS: The aim of this study is to investigate the effect of hemin in colonic epithelial cells (Caco-2) cell proliferation and if this effect was due to a direct modulation of 18-kDa translocator protein (TSPO) and/or heme oxygenase type 1 (HO-1). MAIN METHODS: The main methods are as follow: cell proliferation and cell cytotoxic assays on Caco-2 cell lines treated with hemin in the presence or not of 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide (PK 11195) and Sn-protoporphyrin IX (Sn-PPIX), and immunoblotting for TSPO and HO-1 protein analysis, siRNA directed against TSPO. KEY FINDINGS: Hemin was shown to be toxic for the Caco-2 cell line in a concentration and time dependent manner. Although hemin was able to induce HO-1 in a dose dependent manner, a specific HO-1 inhibitor, Sn-PPIX, was unable to interfere with the effect of hemin on Caco-2 cells. Instead, PK 11195, a specific TSPO ligand, was able to counteract the effect of hemin, suggesting an important role of TSPO in the hemin activity. Cell viability assay further confirms the high cytotoxic effects exerted by hemin on Caco-2 cells expressing TSPO compared to the siRNA-TSPO targeted cells. In addition, hemin was able to decrease significantly the TSPO protein density in a dose dependent manner after 24h of incubation. SIGNIFICANCE: The interaction and the consecutive down regulation of TSPO by hemin played an important role in the control of Caco-2 cell viability. The presented data suggest that TSPO might contribute to protect cells from potential toxic compounds such as free tetrapyrroles, candidating this receptor as a survival receptor protein.

Asunto(s)

Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Hemina/farmacología , Receptores de GABA/metabolismo , Antineoplásicos/farmacología , Western Blotting , Células CACO-2/efectos de los fármacos , Células CACO-2/metabolismo , Inhibidores Enzimáticos/farmacología , Hemo-Oxigenasa 1/antagonistas & inhibidores , Hemo-Oxigenasa 1/metabolismo , Humanos , Isoquinolinas/farmacología , Metaloporfirinas/farmacología , Protoporfirinas/farmacología , ARN Interferente Pequeño/genética , Receptores de GABA/química , Receptores de GABA/genética

RESUMEN

Monocytes/macrophages are key players in all phases of physiological and pathological inflammation. To understanding the regulation of macrophage functional differentiation during inflammation, we designed an in vitro model that recapitulates the different phases of the reaction (recruitment, initiation, development, and resolution), based on human primary blood monocytes exposed to sequential changes in microenvironmental conditions. All reaction phases were profiled by transcriptomic microarray analysis. Distinct clusters of genes were identified that are differentially regulated through the different phases of inflammation. The gene sets defined by GSEA analysis revealed that the inflammatory phase was enriched in inflammatory pathways, while the resolution phase comprised pathways related to metabolism and gene rearrangement. By comparing gene clusters differentially expressed in monocytes vs. M1 and vs. M2 macrophages extracted from an in-house created meta-database, it was shown that cells in the model resemble M1 during the inflammatory phase and M2 during resolution. The validation of inflammatory and transcriptional factors by qPCR and ELISA confirmed the transcriptomic profiles in the different phases of inflammation. The accurate description of the development of the human inflammatory reaction provided by this in vitro kinetic model can help in identifying regulatory mechanisms in physiological conditions and during pathological derangements.

Asunto(s)

Diferenciación Celular/genética , Perfilación de la Expresión Génica , Macrófagos/metabolismo , Monocitos/metabolismo , Adulto , Células Cultivadas , Microambiente Celular/genética , Quimiocina CCL5/genética , Quimiocina CCL5/metabolismo , Análisis por Conglomerados , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Inflamación/genética , Inflamación/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Macrófagos/citología , Masculino , Persona de Mediana Edad , Monocitos/citología , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/genética , Adulto Joven

RESUMEN

In spite of the numerous reports implicating MafB transcription factor in the molecular control of monocyte-macrophage differentiation, the precise genetic program underlying this activity has been, to date, poorly understood. To clarify this issue, we planned a number of experiments that were mainly conducted on human primary macrophages. In this regard, a preliminary gene function study, based on MafB inactivation and over-expression, indicated MMP9 and IL-7R genes as possible targets of the investigated transcription factor. Bioinformatics analysis of their promoter regions disclosed the presence of several putative MARE elements and a combined approach of EMSA and luciferase assay subsequently demonstrated that expression of both genes is indeed activated by MafB through a direct transcription mechanism. Additional investigation, performed with similar procedures to elucidate the biological relevance of our observation, revealed that MafB is a downstream target of the IL-10/STAT3 signaling pathway, normally inducing the macrophage de-activation process. Taken together our data support the existence of a signaling cascade by which stimulation of macrophages with the IL-10 cytokine determines a sequential activation of STAT3 and MafB transcription factors, in turn leading to an up-regulated expression of MMP9 and IL-7R genes.

Asunto(s)

Interleucina-10/metabolismo , Activación de Macrófagos , Factor de Transcripción MafB/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Secuencia de Bases , Línea Celular , Sondas de ADN , Silenciador del Gen , Humanos , Factor de Transcripción MafB/genética , Metaloproteinasa 7 de la Matriz/genética , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Receptores de Interleucina-7/genética

RESUMEN

Orosomucoid 1 (ORM1), also named Alpha 1 acid glycoprotein A (AGP-A), is an abundant plasma protein characterized by anti-inflammatory and immune-modulating properties. The present study was designed to identify a possible correlation between ORM1 and Vitamin D3 (1,25(OH)2D3), a hormone exerting a widespread effect on cell proliferation, differentiation and regulation of the immune system. In particular, the data described here indicated that ORM1 is a 1,25(OH)2D3 primary response gene, characterized by the presence of a VDRE element inside the 1kb sequence of its proximal promoter region. This finding was demonstrated with gene expression studies, Chromatin Immunoprecipitation and luciferase transactivation experiments and confirmed by VDR full length and dominant negative over-expression. In addition, several experiments carried out in human normal monocytes demonstrated that the 1,25(OH)2D3--VDR--ORM1 pathway plays a functional role inside the macrophage de-activation process and that ORM1 may be considered as a signaling molecule involved in the maintenance of tissue homeostasis and remodeling.

Asunto(s)

Activación de Macrófagos/efectos de los fármacos , Macrófagos/efectos de los fármacos , Orosomucoide/metabolismo , Vitamina D/farmacología , Perfilación de la Expresión Génica , Células HL-60 , Humanos , Macrófagos/metabolismo , Orosomucoide/genética , Orosomucoide/aislamiento & purificación , Células U937

RESUMEN

RNA binding proteins belonging to the TIS11/TTP gene family regulate the stability of multiple targets. Their inactivation or deregulated expression has recently been related to cancer, and it has been suggested that they are capable of displaying tumor suppressor activities. Here we describe three new targets of ZFP36 (PIM-1, PIM-3 and XIAP) and show by different approaches that its ectopic expression is capable of impairing glioblastoma cell lines viability and invasiveness by interfering with different transduction pathways. Moreover, we provide evidence that compounds capable of inducing the expression of TIS11/TTP genes determine a comparable biological effect on the same cell contexts.

Asunto(s)

Transducción de Señal , Tristetraprolina/metabolismo , Regiones no Traducidas 3' , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular , Supervivencia Celular , Proteínas Fúngicas , Glioblastoma/metabolismo , Glioblastoma/patología , Células HEK293 , Humanos , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Polifenoles/farmacología , Unión Proteica , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Tristetraprolina/genética , Proteína Inhibidora de la Apoptosis Ligada a X/antagonistas & inhibidores , Proteína Inhibidora de la Apoptosis Ligada a X/genética , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismo

RESUMEN

ZFP36L1 is a member of a family of CCCH tandem zinc finger proteins (TTP family) able to bind to AU-rich elements in the 3'-untranslated region of mRNAs, thereby triggering their degradation. The present study suggests that such mechanism is used during hematopoiesis to regulate differentiation by posttranscriptionally modulating the expression of specific target genes. In particular, it demonstrates that ZFP36L1 negatively regulates erythroid differentiation by directly binding the 3' untranslated region of Stat5b encoding mRNA. Stat5b down-regulation obtained by ZFP36L1 overexpression results, in human hematopoietic progenitors, in a drastic decrease of erythroid colonies formation. These observations have been confirmed by silencing experiments targeting Stat5b and by treating hematopoietic stem/progenitor cells with drugs able to induce ZFP36L1 expression. Moreover, this study shows that different members of ZFP36L1 family act redundantly, because cooverexpression of ZFP36L1 and family member ZFP36 determines a cumulative effect on Stat5b down-regulation. This work describes a mechanism underlying ZFP36L1 capability to regulate hematopoietic differentiation and suggests a new target for the therapy of hematopoietic diseases involving Stat5b/JAK2 pathway, such as chronic myeloproliferative disorders.

Asunto(s)

Antígenos CD34/metabolismo , Factor 1 de Respuesta al Butirato/metabolismo , Diferenciación Celular , Células Eritroides/citología , Células Madre Hematopoyéticas/citología , Factor de Transcripción STAT5/metabolismo , Transducción de Señal , Regiones no Traducidas 3'/genética , Biomarcadores , Diferenciación Celular/efectos de los fármacos , Cinnamomum zeylanicum/química , Regulación hacia Abajo/efectos de los fármacos , Células Eritroides/efectos de los fármacos , Células Eritroides/metabolismo , Sangre Fetal/citología , Flavonoides/farmacología , Silenciador del Gen/efectos de los fármacos , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Humanos , Factores Reguladores del Interferón/genética , Factores Reguladores del Interferón/metabolismo , Fenoles/farmacología , Extractos Vegetales/farmacología , Polifenoles , Unión Proteica/efectos de los fármacos , Estabilidad del ARN/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Tristetraprolina/metabolismo , Regulación hacia Arriba/efectos de los fármacos

RESUMEN

Integrins regulate adhesive cell-matrix interactions and mediate survival signals. On the other hand, unligated or free cytoplasmic fragments of integrins induce apoptosis in many cell types (integrin-mediated death). We have previously shown that ß(1) integrin expression protects keratinocyte stem cells from anoikis, whereas the role of the ß(1)B integrin isoform has not been clarified. In this study we report that suspended keratinocytes undergo apoptosis through the activation of caspase-8, independently of the Fas/Fas ligand system. Indeed, anti-ß(1) integrin-neutralizing antibodies induced apoptosis in short hairpin RNA Fas-associated death domain-treated cells. Moreover, before and during suspension, caspase-8 directly associated with ß(1) integrin, which in turn internalized and progressively degraded, shedding the cytoplasmic domain. ß(1)B was expressed only in the cytoplasm in a perinuclear manner and remained unaltered during suspension. At 24 hours, as ß(1)A was located close to the nucleus, ß(1)B colocalized with ß(1)A and coimmunoprecipitated with caspase-8. Caspase-8 was activated earlier in ß(1)B integrin-transfected keratinocytes, and these cells underwent a higher rate of apoptosis than mock cells. In contrast, caspase-8 was not activated in small interfering RNA (siRNA) ß(1)B-transfected cells. These results indicate that when ß(1)A is unligated, ß(1)B is responsible for "integrin-mediated death" in human keratinocytes.

Asunto(s)

Anoicis/fisiología , Caspasa 8/metabolismo , Integrina beta1/metabolismo , Queratinocitos/citología , Queratinocitos/fisiología , Apoptosis/fisiología , Línea Celular Tumoral , Endocitosis/fisiología , Matriz Extracelular/metabolismo , Proteína Ligando Fas/metabolismo , Proteína de Dominio de Muerte Asociada a Fas/metabolismo , Humanos , Integrina beta1/química , Integrina beta1/genética , Isomerismo , Masculino , ARN Interferente Pequeño/farmacología , Receptor fas/metabolismo

RESUMEN

Transcription Factor for Immunoglobulin Heavy-Chain Enhancer 3 (Tfe3) is a transactivator of metabolic genes that are regulated through an EBox located in their promoters. It is involved in physiological processes such as osteoclast and macrophage differentiation, as well as in pathological processes such as translocations underlying different cancer diseases. MAFB is a basic region/leucine zipper transcription factor that affects transcription by binding specific DNA regions known as MARE. It plays a pivotal role in regulating lineage-specific hematopoiesis by repressing transcription of erythroid specific genes in myeloid cells and enhancing expression of macrophage and megakaryocytic genes. Here we have shown MAFB to be highly induced in human hematopoietic cells undergoing macrophage differentiation following Tfe3 ectopic expression, and to be down regulated, compared to the controls, in the same cell population following Phorbol Esters (PMA) dependent differentiation coupled to Tfe3 gene silencing. Electrophoretic mobility shift assays identified a Tfe3-binding site (EBox) in the MAFB promoter region that is conserved in different mammalian species. MAFB promoter was transactivated by co-expression of Tfe3 in reporter gene assays while deletion or mutation of the MAFB EBox prevented transactivation by Tfe3. Both of these genes were previously included in the group of transcription factors able to drive macrophage differentiation. The observation that MAFB belongs to the Tfe3 regulon suggests the existence of a pathway where these two gene families act synergistically to determine differentiation.

Asunto(s)

Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Macrófagos/citología , Macrófagos/metabolismo , Factor de Transcripción MafB/genética , Factor de Transcripción MafB/metabolismo , Animales , Secuencia de Bases , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Sitios de Unión/genética , Diferenciación Celular/efectos de los fármacos , Línea Celular , Cartilla de ADN/genética , Expresión Génica , Humanos , Macrófagos/efectos de los fármacos , Ratones , Datos de Secuencia Molecular , Mutación , Células 3T3 NIH , Regiones Promotoras Genéticas , Interferencia de ARN , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Ácido Nucleico , Acetato de Tetradecanoilforbol/farmacología , Activación Transcripcional , Células U937

RESUMEN

A number of reports indicate that peroxisome proliferator-activated receptor (PPAR) delta is involved in the molecular control of monocyte-macrophage differentiation. In this regard, the recent demonstration that PPARdelta is a primary response gene of 1alpha,25-dihydroxyvitamin D3 (VD), i.e. a powerful inducer of such process, allowed us to hypothesize the existence of a cross talk between PPARdelta and VD receptor pathways. To address this issue, we analyzed the effects promoted by stimulation with PPARdelta ligands and by overexpression of this nuclear receptor in monoblastic cell lines undergoing exposure to VD. The results obtained evidenced that, although promoting a weak differentiation effect by themselves, PPARdelta ligands efficiently co-operated with VD treatment. In spite of this, PPARdelta overexpression exerted a remarkable inhibitory effect on monocyte-macrophage differentiation induced by VD that was, at least partly, reverted by stimulation with a highly specific PPARdelta ligand. These data indicate that, although acting through a ligand-dependent modality, PPARdelta is a negative regulator of VD-mediated monocyte differentiation, allowing us to hypothesize a role of the investigated nuclear receptor in the differentiation block of M5 type (monoblastic) acute myeloid leukemias (AMLs). Bioinformatic analysis of a microarray database, containing the expression profiles of 285 AML cases, further supported this hypothesis demonstrating the existence of a subset of M5 type (monoblastic) AMLs that overexpress PPARdelta gene.

Asunto(s)

Diferenciación Celular/fisiología , Colecalciferol/farmacología , Monocitos/citología , PPAR delta/fisiología , Antígenos CD34/metabolismo , Antígenos de Diferenciación/metabolismo , Ácido Araquidónico/farmacología , Diferenciación Celular/efectos de los fármacos , Línea Celular , Biología Computacional , Bases de Datos Factuales , Epoprostenol/análogos & derivados , Epoprostenol/farmacología , Perfilación de la Expresión Génica , Hematopoyesis , Humanos , Leucemia Mieloide Aguda/metabolismo , Ligandos , Monocitos/efectos de los fármacos , Monocitos/fisiología , PPAR delta/biosíntesis , Tiazoles/farmacología , Regulación hacia Arriba

RESUMEN

Although a considerable number of reports indicate an involvement of the Hox-A10 gene in the molecular control of hemopoiesis, the conclusions of such studies are quite controversial given that they support, in some cases, a role in the stimulation of stem cell self-renewal and myeloid progenitor expansion, whereas in others they implicate this transcription factor in the induction of monocyte-macrophage differentiation. To clarify this issue, we analyzed the biological effects and the transcriptome changes determined in human primary CD34(+) hemopoietic progenitors by retroviral transduction of a full-length Hox-A10 cDNA. The results obtained clearly indicated that this homeogene is an inducer of monocyte differentiation, at least partly acting through the up-regulation of the MafB gene, recently identified as the master regulator of such a maturation pathway. By using a combined approach based on computational analysis, EMSA experiments, and luciferase assays, we were able to demonstrate the presence of a Hox-A10-binding site in the promoter region of the MafB gene, which suggested the likely molecular mechanism underlying the observed effect. Stimulation of the same cells with the vitamin D(3) monocyte differentiation inducer resulted in a clear increase of Hox-A10 and MafB transcripts, indicating the existence of a precise transactivation cascade involving vitamin D(3) receptor, Hox-A10, and MafB transcription factors. Altogether, these data allow one to conclude that the vitamin D(3)/Hox-A10 pathway supports MafB function during the induction of monocyte differentiation.

Asunto(s)

Antígenos CD34 , Diferenciación Celular/inmunología , Colecalciferol/farmacología , Proteínas de Homeodominio/inmunología , Factor de Transcripción MafB/inmunología , Monocitos/inmunología , Células Progenitoras Mieloides/inmunología , Vitaminas/farmacología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Células HL-60 , Hematopoyesis/efectos de los fármacos , Hematopoyesis/genética , Hematopoyesis/inmunología , Proteínas Homeobox A10 , Proteínas de Homeodominio/biosíntesis , Proteínas de Homeodominio/genética , Humanos , Células K562 , Factor de Transcripción MafB/biosíntesis , Factor de Transcripción MafB/genética , Monocitos/metabolismo , Células Progenitoras Mieloides/metabolismo , Elementos de Respuesta/genética , Elementos de Respuesta/inmunología , Retroviridae , Transducción Genética , Células U937 , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genética , Regulación hacia Arriba/inmunología

RESUMEN

BACKGROUND: Human myelopoiesis is an exciting biological model for cellular differentiation since it represents a plastic process where multipotent stem cells gradually limit their differentiation potential, generating different precursor cells which finally evolve into distinct terminally differentiated cells. This study aimed at investigating the genomic expression during myeloid differentiation through a computational approach that integrates gene expression profiles with functional information and genome organization. RESULTS: Gene expression data from 24 experiments for 8 different cell types of the human myelopoietic lineage were used to generate an integrated myelopoiesis dataset of 9,425 genes, each reliably associated to a unique genomic position and chromosomal coordinate. Lists of genes constitutively expressed or silent during myelopoiesis and of genes differentially expressed in commitment phase of myelopoiesis were first identified using a classical data analysis procedure. Then, the genomic distribution of myelopoiesis genes was investigated integrating transcriptional and functional characteristics of genes. This approach allowed identifying specific chromosomal regions significantly highly or weakly expressed, and clusters of differentially expressed genes and of transcripts related to specific functional modules. CONCLUSION: The analysis of genomic expression during human myelopoiesis using an integrative computational approach allowed discovering important relationships between genomic position, biological function and expression patterns and highlighting chromatin domains, including genes with coordinated expression and lineage-specific functions.

Asunto(s)

Expresión Génica , Genoma Humano , Genómica , Células Mieloides/metabolismo , Mielopoyesis/genética , Antígenos CD34/genética , Antígenos CD34/metabolismo , Diferenciación Celular , Linaje de la Célula , Cromosomas Humanos , Análisis por Conglomerados , Biología Computacional , Eosinófilos/citología , Eosinófilos/metabolismo , Eritroblastos/citología , Eritroblastos/metabolismo , Sangre Fetal/citología , Sangre Fetal/metabolismo , Perfilación de la Expresión Génica , Células Precursoras de Granulocitos/citología , Células Precursoras de Granulocitos/metabolismo , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Humanos , Modelos Biológicos , Monocitos/citología , Monocitos/metabolismo , Células Mieloides/citología , Neutrófilos/citología , Neutrófilos/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Programas Informáticos

RESUMEN

The MItf-Tfe family of basic helix-loop-helix leucine zipper (bHLH-Zip) transcription factors encodes four family members: MItf, Tfe3, TfeB and TfeC. In vitro, each protein of the family binds DNA in a homo- or heterodimeric form with other family members. Tfe3 is involved in chromosomal translocations recurrent in different tumors and it has been demonstrated, by in vivo studies, that it plays, redundantly with MItf, an important role in the process of osteoclast formation, in particular during the transition from mono-nucleated to multi-nucleated osteoclasts. Since mono-nucleated osteoclasts derive from macrophages we investigated whether Tfe3 might play a role upstream during hematopoietic differentiation. Here we show that Tfe3 is able to induce mono-macrophagic differentiation of U937 cells, in association with a decrease of cell proliferation and an increase of apoptosis. We also show that Tfe3 does not act physiologically during commitment of CD34+ hematopoietic stem cells (HSCs), since it is not able to direct HSCs toward a specific lineage as observed by clonogenic assay, but is a strong actor of terminal differentiation since it allows human primary myeloblasts' maturation toward the macrophage lineage.

Asunto(s)

Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Macrófagos/metabolismo , Células Progenitoras Mieloides/fisiología , Antígenos de Diferenciación/análisis , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Ciclo Celular , Diferenciación Celular , Línea Celular Tumoral , Células Cultivadas , Sangre Fetal/metabolismo , Sangre Fetal/fisiología , Silenciador del Gen , Células Precursoras de Granulocitos/metabolismo , Células HL-60 , Humanos , Receptores de Lipopolisacáridos/metabolismo , Acetato de Tetradecanoilforbol/farmacocinética , Acetato de Tetradecanoilforbol/farmacología , Transducción Genética

RESUMEN

The gene expression profile of CD34(-) hematopoietic stem cells (HSCs) and the correlations with their biological properties are still poorly understood. To address this issue, we used the DNA microarray technology to compare the expression profiles of different peripheral blood hemopoietic stem/progenitor cell subsets, lineage-negative (Lin(-)) CD34(-), Lin(-)CD34(+), and Lin(+)CD34(+) cells. The analysis of gene categories differentially expressed shows that the expression of CD34 is associated with cell cycle entry and metabolic activation, such as DNA, RNA, and protein synthesis. Moreover, the significant upregulation in CD34(-) cells of pathways inhibiting HSC proliferation induces a strong differential expression of cyclins, cyclin-dependent kinases (CDKs), CDK inhibitors, and growth-arrest genes. According to the expression of their receptors and transducers, interleukin (IL)-10 and IL-17 showed an inhibitory effect on the clonogenic activity of CD34(-) cells. Conversely, CD34(+) cells were sensitive to the mitogenic stimulus of thrombopoietin. Furthermore, CD34(-) cells express preferentially genes related to neural, epithelial, and muscle differentiation. The analysis of transcription factor expression shows that the CD34 induction results in the upregulation of genes related to self-renewal and lineage commitment. The preferential expression in CD34(+) cells of genes supporting the HSC mobilization and homing to the bone marrow, such as chemokine receptors and integrins, gives the molecular basis for the higher engraftment capacity of CD34(+) cells. Thus, the different kinetic status of CD34(-) and CD34(+) cells, detailed by molecular and functional analysis, significantly influences their biological behavior.

Asunto(s)

Antígenos CD34/metabolismo , Perfilación de la Expresión Génica , Células Madre Hematopoyéticas/metabolismo , Biomarcadores/metabolismo , Diferenciación Celular , Linaje de la Célula , Proliferación Celular , Células Cultivadas , Análisis por Conglomerados , Técnicas de Cultivo , Quinasas Ciclina-Dependientes/biosíntesis , Quinasas Ciclina-Dependientes/genética , Ciclinas/biosíntesis , Ciclinas/genética , Regulación de la Expresión Génica , Células Madre Hematopoyéticas/citología , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Transducción de Señal , Trombopoyetina/metabolismo

RESUMEN

Interleukin-6 (IL-6) is a pleiotropic cytokine involved in the regulation of proliferation and differentiation of hematopoietic cells and in the pathogenesis of many diseases, including multiple myeloma. This study pursues a way to interfere with IL-6 pathway in an attempt to modulate its biological activity. Here we describe the rational design and biological evaluation of peptides able to antagonize the murine IL-6 activity by interfering with IL-6 Receptor alpha in 7TD1 cells, a IL-6-dependent B-cell line. Of the peptide tested, only Guess 4a is capable of interfering with IL-6 transducing pathway, therefore inducing growth arrest and apoptosis of 7TD1 cells.

Asunto(s)

Apoptosis/efectos de los fármacos , Linfocitos B/efectos de los fármacos , Interleucina-6/antagonistas & inhibidores , Péptidos/farmacología , Secuencia de Aminoácidos , Animales , Apoptosis/fisiología , Linfocitos B/metabolismo , Biología Computacional , Simulación por Computador , Interleucina-6/metabolismo , Ratones , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Alineación de Secuencia

RESUMEN

A wide difference in the susceptibility to undergo in vitro apoptosis exists among individuals, and this fact has potential implications in predicting the in vivo response to apoptotic agents, such as those employed in chemotherapy. In this report, we addressed the question whether the natural variability at p53 locus (the proline-arginine substitution at codon 72) affects the capacity of peripheral-blood mononuclear cells from healthy subjects to undergo in vitro apoptosis in response to the cytotoxic drug cytosine arabinoside. We found that cells from subjects carrying the arginine/arginine genotype undergo in vitro apoptosis at a higher extent in comparison to those from arginine/proline subjects. This finding suggests that naturally occurring genetic variability at p53 gene explains part of the inter-individual difference in the in vitro susceptibility to a chemotherapeutic drug, thus resulting as an eligible predictor marker of in vivo response to chemotherapy and its adverse effects.

Asunto(s)

Antimetabolitos Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Citarabina/farmacología , Leucocitos/efectos de los fármacos , Proteína p53 Supresora de Tumor/genética , Adulto , Análisis de Varianza , Apoptosis/fisiología , Codón , Exones , Variación Genética , Genotipo , Humanos , Leucocitos/metabolismo