RESUMEN
Maturation of newly formed vessels is a multistep phenomenon during which functional endothelial barriers are established. Disruption of vessel integrity is an important feature in many physiological and pathological processes. We previously reported that lymphoblastic leukemia-derived sequence 1 (LYL1) is required for the late stages of postnatal angiogenesis to limit the formation of new blood vessels, notably by regulating the activity of the small GTPase Rap1. In this study, we show that LYL1 is also required during the formation of the mature endothelial barrier in the lungs of adult mice. Specifically, LYL1 knockdown in human endothelial cells downregulated the expression of ARHGAP21 and ARHGAP24, which encode two Rho GTPase-activating proteins, and this was correlated with increased RhoA activity and reorganization of the actin cytoskeleton into stress fibers. Importantly, in lungs of Lyl1-deficient mice, both vascular endothelial (VE)-cadherin and p120-catenin were poorly recruited to endothelial adherens junctions, indicative of defective cell-cell junctions. Consistent with this, higher Evans blue dye extravasation, edema, and leukocyte infiltration in the lung parenchyma of Lyl1-/- mice than in wild-type littermates confirmed that lung vascular permeability is constitutively elevated in Lyl1-/- adult mice. Our data show that LYL1 acts as a stabilizing signal for adherens junction formation by operating upstream of VE-cadherin and of the two GTPases Rap1 and RhoA. As increased vascular permeability is a key feature and a major mechanism of acute respiratory distress syndrome, molecules that regulate LYL1 activity could represent additional tools to modify the endothelial barrier permeability.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Permeabilidad de la Membrana Celular , Endotelio Vascular/patología , Células Endoteliales de la Vena Umbilical Humana/patología , Pulmón/patología , Proteínas de Neoplasias/fisiología , Actinas/genética , Actinas/metabolismo , Animales , Western Blotting , Células Cultivadas , Endotelio Vascular/metabolismo , Técnica del Anticuerpo Fluorescente , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Técnicas para Inmunoenzimas , Pulmón/irrigación sanguínea , Pulmón/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Fibras de Estrés , Proteínas de Unión al GTP rap1/genética , Proteínas de Unión al GTP rap1/metabolismo , Proteína de Unión al GTP rhoA/genética , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
The two related basic helix-loop-helix, TAL1 and LYL1, and their cofactor LIM-only-2 protein (LMO2) are present in blood and endothelial cells. While their crucial role in early hematopoiesis is well established, their function in endothelial cells and especially in angiogenesis is less understood. Here, we identified ANGIOPOIETIN-2 (ANG-2), which encodes a major regulator of angiogenesis, as a direct transcriptional target of TAL1, LYL1 and LMO2. Knockdown of any of the three transcription factors in human blood and lymphatic endothelial cells caused ANG-2 mRNA and protein down-regulation. Transient transfections showed that the full activity of the ANG-2 promoter required the integrity of a highly conserved Ebox-GATA composite element. Accordingly, chromatin immunoprecipitation assays demonstrated that TAL1, LYL1, LMO2 and GATA2 occupied this region of ANG-2 promoter in human endothelial cells. Furthermore, we showed that LMO2 played a central role in assembling TAL1-E47, LYL1-LYL1 or/and LYL1-TAL1 dimers with GATA2. The resulting complexes were able to activate endogenous ANG-2 expression in endothelial cells as well as in non-endothelial cells. Finally, we showed that ANG-2 gene activation during angiogenesis concurred with the up-regulation of TAL1 and LMO2. Altogether, we identified ANG-2 as a bona fide target gene of LMO2-complexes with TAL1 and/or LYL1, highlighting a new function of the three hematopoietic factors in the endothelial lineage.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Angiopoyetina 2/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Regulación de la Expresión Génica , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Proteínas con Dominio LIM/fisiología , Proteínas de Neoplasias/fisiología , Proteínas Proto-Oncogénicas/fisiología , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Angiopoyetina 2/metabolismo , Secuencia de Bases , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Secuencia Conservada , Factor de Transcripción GATA2/fisiología , Técnicas de Silenciamiento del Gen , Genes Reporteros , Células HEK293 , Humanos , Proteínas con Dominio LIM/genética , Proteínas con Dominio LIM/metabolismo , Luciferasas de Luciérnaga/biosíntesis , Luciferasas de Luciérnaga/genética , Microvasos/citología , Datos de Secuencia Molecular , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Multimerización de Proteína , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , ARN Interferente Pequeño/genética , Proteína 1 de la Leucemia Linfocítica T Aguda , Transcripción GenéticaRESUMEN
The 2 related basic helix loop helix genes, LYL1 and TAL-1 are active in hematopoietic and endothelial lineages. While Tal-1 is essential for both hematopoietic and vascular development, the role of Lyl1 appears to be distinct as deficient mice are viable and display modest hematopoietic defects. Here, we reveal a role for Lyl1 as a major regulator of adult neovascularization. Tumors implanted into Lyl1-deficient mice showed higher proliferation and angiogenesis, as evidenced by enlarged lumens, reduced pericyte coverage and increased permeability, compared with wild type littermates. Of note, Lyl1-deficient tumor vessels exhibited an up-regulation of Tal-1, the VE-Cadherin target gene, as well as Angiopoietin-2, 3 major actors in angiogenesis. Hematopoietic reconstitution experiments demonstrated that this sustained tumor angiogenesis was of endothelial origin. Moreover, the angiogenic phenotype observed in the absence of Lyl1 function was not tumor-restricted as microvessels forming in Matrigel or originating from aortic explants were also more numerous and larger than their wild-type counterparts. Finally, LYL1 depletion in human endothelial cells revealed that LYL1 controls the expression of molecules involved in the stabilization of vascular structures. Together, our data show a role for LYL1 in the postnatal maturation of newly formed blood vessels.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Experimentales/irrigación sanguínea , Neoplasias Experimentales/metabolismo , Neovascularización Patológica/metabolismo , Angiopoyetina 2/biosíntesis , Angiopoyetina 2/genética , Animales , Antígenos CD/biosíntesis , Antígenos CD/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Cadherinas/biosíntesis , Cadherinas/genética , Regulación Neoplásica de la Expresión Génica/genética , Hematopoyesis/genética , Humanos , Ratones , Ratones Mutantes , Proteínas de Neoplasias/genética , Trasplante de Neoplasias , Neoplasias Experimentales/genética , Neovascularización Patológica/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteína 1 de la Leucemia Linfocítica T Aguda , Regulación hacia Arriba/genéticaRESUMEN
In this paper, we describe how, in a model embryonic system, cyclin-dependent kinase (Cdk) activity controls the efficiency of DNA replication by determining the frequency of origin activation. Using independent approaches of protein depletion and selective chemical inhibition of a single Cdk, we find that both Cdk1 and Cdk2 are necessary for efficient DNA replication in Xenopus egg extracts. Eliminating Cdk1, Cdk2 or their associated cyclins changes replication origin spacing, mainly by decreasing frequency of activation of origin clusters. Although there is no absolute requirement for a specific Cdk or cyclin, Cdk2 and cyclin E contribute more to origin cluster efficiency than Cdk1 and cyclin A. Relative Cdk activity required for DNA replication is very low, and even when both Cdk1 and Cdk2 are strongly inhibited, some origins are activated. However, at low levels, Cdk activity is limiting for the pre-replication complex to pre-initiation complex transition, origin activation and replication efficiency. As such, unlike mitosis, initiation of DNA replication responds progressively to changes in Cdk activity at low activity levels.
Asunto(s)
Proteína Quinasa CDC2/metabolismo , Quinasa 2 Dependiente de la Ciclina/metabolismo , Replicación del ADN , ADN/metabolismo , Animales , Extractos Celulares/aislamiento & purificación , Cromatina/metabolismo , Oocitos/química , XenopusRESUMEN
The basic helix-loop-helix TAL-1/SCL essential for hematopoietic development is also required during vascular development for embryonic angiogenesis. We reported that TAL-1 acts positively on postnatal angiogenesis by stimulating endothelial morphogenesis. Here, we investigated the functional consequences of TAL-1 silencing in human primary endothelial cells. We found that TAL-1 knockdown caused the inhibition of in vitro tubulomorphogenesis, which was associated with a dramatic reduction in vascular endothelial cadherin (VE-cadherin) at intercellular junctions. Consistently, silencing of TAL-1 as well as of its cofactors E47 and LMO2 down-regulated VE-cadherin at both the mRNA and the protein level. Endogenous VE-cadherin transcription could be activated in nonendothelial HEK-293 cells by the sole concomitant ectopic expression of TAL-1, E47, and LMO2. Transient transfections in human primary endothelial cells derived from umbilical vein (HUVECs) demonstrated that VE-cadherin promoter activity was dependent on the integrity of a specialized E-box associated with a GATA motif and was maximal with the coexpression of the different components of the TAL-1 complex. Finally, chromatin immunoprecipitation assays showed that TAL-1 and its cofactors occupied the VE-cadherin promoter in HUVECs. Together, these data identify VE-cadherin as a bona fide target gene of the TAL-1 complex in the endothelial lineage, providing a first clue to TAL-1 function in angiogenesis.
Asunto(s)
Antígenos CD/biosíntesis , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Cadherinas/biosíntesis , Proteínas de Unión al ADN/fisiología , Células Endoteliales/metabolismo , Metaloproteínas/fisiología , Proteínas Proto-Oncogénicas/fisiología , Factores de Transcripción TCF/fisiología , Regulación hacia Arriba , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Antígenos CD/genética , Secuencia de Bases , Cadherinas/genética , Células Cultivadas , Colágeno , Combinación de Medicamentos , Células Endoteliales/fisiología , Endotelio Vascular/citología , Humanos , Proteínas con Dominio LIM , Laminina , Datos de Secuencia Molecular , Neovascularización Fisiológica , Regiones Promotoras Genéticas , Unión Proteica , Proteoglicanos , Proteína 1 de la Leucemia Linfocítica T Aguda , Proteína 1 Similar al Factor de Transcripción 7RESUMEN
The TAL-1/SCL gene encodes a basic helix-loop-helix (bHLH) transcription factor essential for primitive hematopoiesis and for adult erythroid and megakaryocytic development. Activated transcription of TAL-1 as a consequence of chromosomal rearrangements is associated with a high proportion of human T cell acute leukemias, showing that appropriate control of TAL-1 is crucial for the formation and subsequent fate of hematopoietic cells. Hence, the knowledge of the mechanisms, which govern the pattern of TAL-1 expression in hematopoiesis, is of great interest. We previously described a silencer in the 3'-untranslated region of human TAL-1, the activity of which is mediated through binding of a tissue-specific 40 kDa nuclear protein to a new DNA recognition motif, named tal-RE. Here, we show that tal-RE-binding activity, high in immature human hematopoietic progenitors is down regulated upon erythroid and megakaryocytic differentiation. This expression profile helped us to identify that PU.1/Spi-1 binds to the tal-RE sequences in vitro and occupies the TAL-1 silencer in vivo. By expressing a mutant protein containing only the ETS domain of PU.1 in human erythroleukemic HEL cells, we demonstrated that PU.1 mediates the transcriptional repression activity of the silencer. We found that ectopic PU.1 is not able to induce silencing activity in PU.1-negative Jurkat T cells, indicating that PU.1 activity, although necessary, is not sufficient to confer transcriptional repression activity to the TAL-1 silencer. Finally, we showed that the silencer is also active in TAL-1-negative myeloid HL60 cells that express PU.1 at high levels. In summary, our study shows that PU.1, in addition to its positive role in TAL-1 expression in early hematopoietic progenitors, may also act as a mediator of TAL-1 silencing in some hematopoietic lineages.
Asunto(s)
Silenciador del Gen , Proteínas Proto-Oncogénicas/fisiología , Transactivadores/fisiología , Ubiquitina-Proteína Ligasas/genética , Línea Celular Tumoral , Regulación hacia Abajo , Hematopoyesis/genética , Humanos , Leucemia/patología , Unión Proteica , Proteínas Proto-Oncogénicas/metabolismo , Elementos de Respuesta , Transactivadores/metabolismo , Factores de Transcripción , Activación Transcripcional , TransfecciónRESUMEN
The basic helix-loop-helix tal-1 gene (or scl), known for its fundamental role in embryonic and adult hematopoiesis in vertebrates, is also required for embryonic vascular remodeling. In adults, TAL-1 protein is undetectable in quiescent endothelium but it is present in newly formed vessels including tumoral vasculature, indicating its involvement in angiogenesis. Here, we demonstrate that TAL-1 expression is tightly regulated during in vitro angiogenesis: it is low during the initial step of migration and is upregulated during formation of capillary-like structures. We investigated whether ectopic expression of either wild-type TAL-1 or a dominant-negative mutant lacking the DNA-binding domain (Delta-bas) modulates the activity of human primary endothelial cells in the angiogenic processes of migration, proliferation and cell morphogenesis. Overexpression of either wild-type or Delta-bas TAL-1 affected chemotactic migration of primary endothelial cells without modifying their proliferative properties. Ectopic expression of wild-type TAL-1 accelerated the formation of capillary-like structures in vitro and, in vivo, enhanced vascularisation in mice (Matrigel implants) associated with a general enlargement of capillary lumens. Importantly, transduction of the mutant Delta-bas completely impaired in vitro angiogenesis and strongly inhibited vascularisation in mice. Taken together, our data show that TAL-1 modulates the angiogenic response of endothelial cells by stimulating cell morphogenesis and by influencing their behavior in migration. This study highlights the importance of TAL-1 regulation in postnatal vascular remodeling and provides the first physiological evidence that links TAL-1 activity to endothelial cell morphogenic processes.