RESUMEN

Stem cell identity depends on the integration of extrinsic and intrinsic signals, which directly influence the maintenance of their epigenetic state. Although Myc transcription factors play a major role in stem cell self-renewal and pluripotency, their integration with signalling pathways and epigenetic regulators remains poorly defined. We addressed this point by profiling the gene expression and epigenetic pattern in ESCs whose growth depends on conditional Myc activity. Here we show that Myc potentiates the Wnt/ß-catenin signalling pathway, which cooperates with the transcriptional regulatory network in sustaining ESC self-renewal. Myc activation results in the transcriptional repression of Wnt antagonists through the direct recruitment of PRC2 on these targets. The consequent potentiation of the autocrine Wnt/ß-catenin signalling induces the transcriptional activation of the endogenous Myc family members, which in turn activates a Myc-driven self-reinforcing circuit. Thus, our data unravel a Myc-dependent self-propagating epigenetic memory in the maintenance of ESC self-renewal capacity.

Asunto(s)

Redes Reguladoras de Genes/genética , Células Madre Embrionarias de Ratones/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Animales , Autorrenovación de las Células/efectos de los fármacos , Epigénesis Genética/efectos de los fármacos , Retroalimentación Fisiológica/efectos de los fármacos , Redes Reguladoras de Genes/efectos de los fármacos , Factor Inhibidor de Leucemia/farmacología , Ratones , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/efectos de los fármacos , Proteínas del Grupo Polycomb/metabolismo , Transcripción Genética/efectos de los fármacos , Vía de Señalización Wnt/efectos de los fármacos , Vía de Señalización Wnt/genética

RESUMEN

The inhibition of tumor angiogenesis is one of the main challenges in cancer therapy. With the aim of developing monoclonal antibodies able to inhibit angiogenesis, we immunized mice with proliferating human umbilical vein endothelial cells. We generated a library of monoclonal antibodies able to recognize antigens expressed on endothelial cells and screened the antibodies for their ability to inhibit endothelial cell proliferation, migration, and sprouting in vitro. Here, we show that the antibody, designated as 4E1, is able to neutralize the formation of new vessels both in vitro and in vivo without affecting endothelial cell survival. By mass spectrometry we identified CD93 as the antigen bound by 4E1 and mapped the recognized epitope. CD93 is a transmembrane protein heavily glycosylated preferentially expressed in the vascular endothelium. CD93 silencing by lentiviral-mediated small hairpin RNA expression impairs human endothelial cell proliferation, migration, and sprouting. Altogether these findings reveal 4E1 as a novel antiangiogenic antibody and identify CD93 as a new target suitable for antiangiogenic therapy.

Asunto(s)

Inhibidores de la Angiogénesis/farmacología , Anticuerpos Monoclonales/farmacología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Glicoproteínas de Membrana/antagonistas & inhibidores , Neovascularización Patológica/tratamiento farmacológico , Receptores de Complemento/antagonistas & inhibidores , Animales , Apoptosis , Western Blotting , Adhesión Celular , Movimiento Celular , Proliferación Celular , Células Cultivadas , Femenino , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/patología , Humanos , Inmunoprecipitación , Glicoproteínas de Membrana/inmunología , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Microscopía Fluorescente , Receptores de Complemento/inmunología , Receptores de Complemento/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción

RESUMEN

Vascular Endothelial Growth Factor-A (VEGF-A) is a key molecule in normal and tumor angiogenesis. This study addresses the role of c-ABL as a novel downstream target of VEGF-A in primary Human Umbilical Vein Endothelial Cells (HUVEC). On the basis of immunoprecipitation experiments, in vitro kinase assay and RNA interference, we demonstrate that VEGF-A induces the c-ABL kinase activity through the VEGF Receptor-2/Phosphatidylinositol-3-Kinase pathway. By treating HUVEC with the specific tyrosine kinase inhibitor STI571 and over-expressing a dominant negative c-ABL mutant, we show that the VEGF-A-activated c-ABL reduces the amplitude of Mitogen-Activated Protein Kinases (ERK1/2, JNKs and p38) activation in a dose-dependent manner by a negative feedback mechanism. By analysis of the adaptor proteins NCK1 and GRB2 mutants we further show that the negative loop on p38 is mediated by c-ABL phosphorylation at tyrosine 105 of the adaptor protein NCK1, while the phosphorylation at tyrosine 209 of GRB2 down-modulates ERK1/2 and JNKs signaling. These findings suggest that c-ABL function is to establish a correct and tightly controlled response of endothelial cells to VEGF-A during the angiogenic process.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales/inmunología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Proteína Adaptadora GRB2/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Neovascularización Fisiológica/fisiología , Proteínas Oncogénicas/inmunología , Proteínas Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-abl/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Benzamidas , Células Cultivadas , Células Endoteliales/citología , Células Endoteliales/metabolismo , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Quinasas MAP Reguladas por Señal Extracelular/genética , Proteína Adaptadora GRB2/genética , Humanos , Mesilato de Imatinib , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Mutación , Neovascularización Fisiológica/efectos de los fármacos , Proteínas Oncogénicas/genética , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación/efectos de los fármacos , Fosforilación/fisiología , Piperazinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-abl/genética , Pirimidinas/farmacología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética

RESUMEN

Myc family members are critical to maintain embryonic stem cells (ESC) in the undifferentiated state. However, the mechanism by which they perform this task has not yet been elucidated. Here we show that Myc directly upregulates the transcription of all core components of the Polycomb repressive complex 2 (PRC2) as well as the ESC-specific PRC2-associated factors. By expressing Myc protein fused with the estrogen receptor (Myc-ER) in fibroblasts, we observed that Myc, binding to the regulatory elements of Suz12, Ezh2, and Eed, induces the acetylation of histones H3 and H4 and the recruitment of elongating RNA polymerase II at their promoters. The silencing of both c-Myc and N-Myc in ESC results in reduced expression of PRC2 and H3K27me3 at Polycomb target developmental regulators and upregulation of genes involved in primitive endoderm differentiation. The ectopic expression of PRC2 in ESC, either silenced for c-Myc and N-Myc or induced to differentiate by leukemia inhibitory factor (LIF) withdrawal, is sufficient to maintain the H3K27me3 mark at genes with bivalent histone modifications and keep repressed the genes involved in ESC differentiation. Thus, Myc proteins control the expression of developmental regulators via the upregulation of the Polycomb PRC2 complex.

Asunto(s)

Células Madre Embrionarias/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Represoras/genética , Células 3T3 , Animales , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Cromatina/genética , Cromatina/metabolismo , Elementos E-Box , Células Madre Embrionarias/citología , Regulación del Desarrollo de la Expresión Génica , Histonas/metabolismo , Metilación , Ratones , Proteínas del Grupo Polycomb , Proteínas Proto-Oncogénicas c-myc/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-myc/genética , ARN Interferente Pequeño/genética , Transcripción Genética

RESUMEN

RATIONALE: Integrins cooperate with growth factor receptors to promote downstream signaling for cell proliferation and migration. However, the mechanism of receptor activation is still unknown. OBJECTIVE: To analyze the mechanism of phosphorylation of the vascular endothelial growth factor receptor (VEGFR)-3 by cell adhesion. METHODS AND RESULTS: We show that VEGFR-3 phosphorylation, induced by cell attachment to the extracellular matrix, is independent from the intrinsic kinase activity of the receptor, as evidenced from phosphorylation cell adhesion experiments with a mutant kinase dead receptor or in the presence of the specific kinase inhibitor MAZ 51. Cell adhesion experiments in the presence of the c-Src inhibitor PP2 or in fibroblast triple knockout for c-Src, Yes, and Fyn (SYF) demonstrate that VEGFR-3 phosphorylation, induced by extracellular matrix, is mediated by c-Src. Kinase assays in vitro with recombinant c-Src show that VEGFR-3 is a direct c-Src target and mass spectrometry analysis identified the sites phosphorylated by c-Src as tyrosine 830, 833, 853, 1063, 1333, and 1337, demonstrating that integrin-mediated receptor phosphorylation induces a phosphorylation pattern that is distinct from that induced by growth factors. Furthermore, pull-down assays show that integrin-mediated VEGFR-3 phosphorylation activates the recruitment to the receptor of the adaptor proteins CRKI/II and SHC inducing activation of JNK. CONCLUSIONS: These data suggest that cell adhesion to extracellular matrix induces a downstream signaling using the tyrosine kinase receptor VEGFR-3 as scaffold.

Asunto(s)

Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Matriz Extracelular/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Receptor 3 de Factores de Crecimiento Endotelial Vascular/metabolismo , Animales , Proteína Tirosina Quinasa CSK , Adhesión Celular , Línea Celular , Células Cultivadas , Colágeno Tipo I/metabolismo , Humanos , MAP Quinasa Quinasa 4/metabolismo , Ratones , Ratones Noqueados , Modelos Animales , Fosforilación , Proteínas Proto-Oncogénicas c-crk/metabolismo , Proteínas Proto-Oncogénicas c-fyn/genética , Proteínas Proto-Oncogénicas c-fyn/metabolismo , Proteínas Proto-Oncogénicas c-yes/genética , Proteínas Proto-Oncogénicas c-yes/metabolismo , Proteínas Adaptadoras de la Señalización Shc/metabolismo , Familia-src Quinasas

RESUMEN

The phosphorylation of the serine 10 at histone H3 has been shown to be important for transcriptional activation. Here, we report the molecular mechanism through which H3S10ph triggers transcript elongation of the FOSL1 gene. Serum stimulation induces the PIM1 kinase to phosphorylate the preacetylated histone H3 at the FOSL1 enhancer. The adaptor protein 14-3-3 binds the phosphorylated nucleosome and recruits the histone acetyltransferase MOF, which triggers the acetylation of histone H4 at lysine 16 (H4K16ac). This histone crosstalk generates the nucleosomal recognition code composed of H3K9acS10ph/H4K16ac determining a nucleosome platform for the bromodomain protein BRD4 binding. The recruitment of the positive transcription elongation factor b (P-TEFb) via BRD4 induces the release of the promoter-proximal paused RNA polymerase II and the increase of its processivity. Thus, the single phosphorylation H3S10ph at the FOSL1 enhancer triggers a cascade of events which activate transcriptional elongation.

Asunto(s)

Código de Histonas , Histonas/metabolismo , Transcripción Genética , Animales , Línea Celular , Quinasa 9 Dependiente de la Ciclina/metabolismo , Drosophila , Recuperación de Fluorescencia tras Fotoblanqueo , Transferencia Resonante de Energía de Fluorescencia , Humanos , Ratones , Nucleosomas/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Factores de Transcripción/metabolismo , Levaduras

RESUMEN

Disabled-2 (DAB2) is an adaptor protein implicated in signal transduction pathways and in protein traffic regulation. Here, we show that DAB2 is highly expressed in human endothelial cells. DAB2 silencing in endothelial cells by lentiviral-mediated small hairpin RNA expression affects cell migration and differentiation into capillary-like structures while increasing cell proliferation and viability. DAB2 knockdown causes activation of the Src-FAK signal pathway, extracellular-signal regulated kinase and c-Jun NH2-terminal kinase activation, and inhibition of p38 phosphorylation. In DAB2 silenced endothelial cells, pharmacological inhibition of Src with its specific inhibitor PP2 abolishes focal adhesion kinase activation and restores differentiation of endothelial cells. These results suggest that DAB2, via Src and focal adhesion signaling, plays a role in human endothelial cell function.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales/fisiología , Diferenciación Celular , Endotelio Vascular/crecimiento & desarrollo , Neovascularización Fisiológica , Familia-src Quinasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Proteínas Reguladoras de la Apoptosis , Diferenciación Celular/genética , Movimiento Celular/genética , Células Cultivadas , Endotelio Vascular/metabolismo , Quinasa 1 de Adhesión Focal/genética , Quinasa 1 de Adhesión Focal/metabolismo , Silenciador del Gen , Humanos , Ratones , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Morfogénesis , Neovascularización Fisiológica/genética , ARN Interferente Pequeño/genética , Proteínas Supresoras de Tumor , Familia-src Quinasas/antagonistas & inhibidores

RESUMEN

Peroxynitrite, via post-translational modifications to target proteins, contributes to cardiovascular injury and cancer. Since tissue inhibitor of metalloproteinase-4 (TIMP-4), the activity of which is impaired in both pathological conditions, has several amino acid residues susceptible to peroxynitrite, we investigated its role as a potential target of peroxynitrite. Peroxynitrite-induced nitration and oligomerization of TIMP-4 attenuated its inhibitory activity against MMP-2 activity and endothelial or tumor cell invasiveness. Moreover, cell treatment with peroxynitrite promoted the nitration of endogenous TIMP-4. HPLC/ESI-MS/MS analysis of peroxynitrite-treated TIMP-4 showed modifications at Y114, Y195, Y188 and Y190. In conclusion, TIMP-4 nitration might be a potential mechanism contributing to cardiovascular disease and cancer.

Asunto(s)

Ácido Peroxinitroso/farmacología , Inhibidores Tisulares de Metaloproteinasas/química , Animales , Bovinos , Línea Celular Tumoral , Movimiento Celular , Células Cultivadas , Endotelio Vascular/citología , Humanos , Inhibidores Tisulares de Metaloproteinasas/antagonistas & inhibidores , Inhibidores Tisulares de Metaloproteinasas/metabolismo , Tirosina/análogos & derivados , Tirosina/análisis , Inhibidor Tisular de Metaloproteinasa-4

RESUMEN

Vascular Endothelial Growth Factor (VEGF)-D is a member of the VEGF family of angiogenic growth factors that activate the Vascular Endothelial Growth Factor Receptor (VEGFR)-2 and VEGFR-3, which are mainly expressed in blood and lymphatic vessels. Here we have analyzed by using monoclonal antibodies, the expression of VEGF-D and its cognate receptor VEGFR-3 in normal and pathologic bone marrow and lymph node biopsies. This analysis revealed that VEGF-D is expressed in B cells of the germinal centers, scattered B and T blasts, myeloid progenitors, acute leukemia, several types of non Hodgkin lymphoma, and classical Hodgkin's lymphoma. In normal tissues VEGFR-3 was only expressed in fenestrated capillaries of bone marrow and in lymphatic vessels of lymph nodes, while in VEGF-D expressing tumors newly formed vessels, but not malignant cells, showed high VEGFR-3 expression. These data suggest that VEGF-D could contribute to leukemia and lymphoma growth via the induction of angiogenesis in bone marrow and lymphoid tissues.

Asunto(s)

Regulación Leucémica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Células Madre Hematopoyéticas/metabolismo , Linfocitos/metabolismo , Factor D de Crecimiento Endotelial Vascular/biosíntesis , Anticuerpos Monoclonales/química , Biopsia , Células de la Médula Ósea/metabolismo , Línea Celular Tumoral , Células HL-60 , Humanos , Células K562 , Ganglios Linfáticos/patología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Receptor 3 de Factores de Crecimiento Endotelial Vascular/biosíntesis , Receptor 3 de Factores de Crecimiento Endotelial Vascular/metabolismo

RESUMEN

Vascular endothelial growth factor receptor-3 (VEGFR-3) is constitutively expressed in lymphatic vessels and transiently in endothelial cells of blood vessels during angiogenesis. Here we report that VEGFR-3 localizes in the caveolae membrane of endothelial cells and co-immunoprecipitates with caveolin-1. Caveolin-1 silencing or its depletion from the cell membrane by cholesterol increases VEGFR-3 autophosphorylation, suggesting that caveolin acts as a negative regulator of VEGFR-3 activity. Receptor activation induces caveolin-1 phosphorylation on tyrosine residues including tyrosine 14. Cell treatment with Src or Abl inhibitors PP2 or STI571, prior to receptor stimulation, affects caveolin-1 phosphorylation without affecting receptor autophosphorylation, suggesting that both Src and Abl are involved in VEGFR-3-dependent caveolin-1 phosphorylation. Caveolin-1 phosphorylation in Src/Fyn/Yes knockout cells demonstrated that Abl phosphorylates caveolin-1 independently from Src family members. These results suggest a functional interaction between VEGFR-3 and caveolin-1 to modulate endothelial cell activation during angiogenesis.

Asunto(s)

Caveolina 1/metabolismo , Endotelio Vascular/citología , Receptor 3 de Factores de Crecimiento Endotelial Vascular/efectos de los fármacos , Receptor 3 de Factores de Crecimiento Endotelial Vascular/metabolismo , Animales , Benzamidas , Caveolas/química , Membrana Celular/fisiología , Células Clonales , Humanos , Mesilato de Imatinib , Tejido Linfoide/química , Ratones , Fosforilación , Piperazinas/farmacología , Proteínas Proto-Oncogénicas c-abl/fisiología , Proteínas Proto-Oncogénicas pp60(c-src)/fisiología , Pirimidinas/farmacología , Sus scrofa

RESUMEN

Vascular endothelial growth factor (VEGF)-D is a member of the VEGF family of angiogenic growth factors that recognizes and activates the vascular endothelial growth factor receptor (VEGFR)-2 and VEGFR-3 on blood and/or lymphatic vessels. We show that in the long bones of newborn mice, VEGF-D and VEGFR-3 are expressed in the osteoblasts of the growing plate. The treatment of primary human osteoblasts with recombinant VEGF-D induces the expression of osteocalcin and the formation of mineralized nodules in a dose-dependent manner. A monoclonal neutralizing antibody, anti-VEGF-D, or silencing of VEGFR-3 by lentiviral-mediated expression of VEGFR-3 small hairpin RNA affects VEGF-D-dependent osteocalcin expression and nodule formation. Moreover, in primary human osteoblasts, VEGF-D expression is under the control of VEGF, and inhibition of VEGF-D/VEGFR-3 signaling, by monoclonal antibodies or VEGFR-3 silencing, affects VEGF-dependent osteoblast differentiation. These experiments establish that VEGF-D/VEGFR-3 signaling plays a critical role in osteoblast maturation and suggest that VEGF-D is a downstream effector of VEGF in osteogenesis.

Asunto(s)

Osteoblastos/citología , Osteoblastos/fisiología , Osteogénesis/fisiología , Factor D de Crecimiento Endotelial Vascular/metabolismo , Receptor 3 de Factores de Crecimiento Endotelial Vascular/metabolismo , Anciano , Animales , Animales Recién Nacidos , Diferenciación Celular/fisiología , Células Cultivadas , Endotelio Vascular/citología , Femenino , Humanos , Masculino , Ratones , Persona de Mediana Edad , Osteocalcina/genética , ARN Interferente Pequeño , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Venas Umbilicales/citología , Factor D de Crecimiento Endotelial Vascular/genética , Factor D de Crecimiento Endotelial Vascular/farmacología , Receptor 3 de Factores de Crecimiento Endotelial Vascular/genética

RESUMEN

Nucleotide metabolism was studied in erythrocytes of a mentally retarded child and family members. Partial hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency was found in the propositus and an asymptomatic maternal uncle. Studies in crude lysates demonstrated decreased apparent V(max) and slightly decreased apparent K(m) for hypoxanthine in both HPRT-deficient subjects. Genomic DNA analysis revealed a single nucleotide change with leucine-147 to phenylalanine substitution in both subjects; mother and grandmother were heterozygous carriers of the same defect. This new variant has been termed HPRT(Potenza). Increased erythrocyte concentration of NAD and rate of synthesis by intact erythrocytes were found in the patient; increased activities of nicotinic acid phosphoribosyltransferase (NAPRT) and NAD synthetase (NADs) were demonstrated in erythrocyte lysates, with normal apparent K(m) for their substrates and increased V(max). These alterations were not found in any member of the family, including the HPRT-deficient uncle. These findings show multiple derangement of nucleotide metabolism associated with partial HPRT deficiency. The enzyme alteration was presumably not the cause of neurological impairment since no neurological symptoms were found in the HPRT-deficient uncle, whereas they were present in the propositus' elder brother who had normal HPRT activity.

Asunto(s)

Eritrocitos/metabolismo , Hipoxantina Fosforribosiltransferasa/deficiencia , Discapacidad Intelectual/genética , Niño , Preescolar , Estabilidad de Enzimas , Eritrocitos/enzimología , Humanos , Hipoxantina Fosforribosiltransferasa/química , Hipoxantina Fosforribosiltransferasa/genética , Lactante , Discapacidad Intelectual/enzimología , Discapacidad Intelectual/metabolismo , Masculino , Mutación , Linaje , Polimorfismo Conformacional Retorcido-Simple , Purinas/metabolismo , Piridinas/metabolismo