RESUMEN
Down syndrome critical region (DSCR)-1 functions as a feedback modulator for calcineurin-nuclear factor for activated T cell (NFAT) signals, which are crucial for cell proliferation and inflammation. Stable expression of DSCR-1 inhibits pathological angiogenesis and septic inflammation. DSCR-1 also plays a critical role in vascular wall remodeling associated with aneurysm development that occurs primarily in smooth muscle cells. Besides, Dscr-1 deficiency promotes the M1-to M2-like phenotypic switch in macrophages, which correlates to the reduction of denatured cholesterol uptakes. However, the distinct roles of DSCR-1 in cholesterol and lipid metabolism are not well understood. Here, we show that loss of apolipoprotein (Apo) E in mice with chronic hypercholesterolemia induced Dscr-1 expression in the liver and aortic atheroma. In Dscr-1-null mice fed a high-fat diet, oxidative- and endoplasmic reticulum (ER) stress was induced, and sterol regulatory element-binding protein (SREBP) 2 production in hepatocytes was stimulated. This exaggerated ApoE-/--mediated nonalcoholic fatty liver disease (NAFLD) and subsequent hypercholesterolemia. Genome-wide screening revealed that loss of both ApoE and Dscr-1 resulted in the induction of immune- and leukocyte activation-related genes in the liver compared with ApoE deficiency alone. However, expressions of inflammation-activated markers and levels of monocyte adhesion were suspended upon induction of the Dscr-1 null background in the aortic endothelium. Collectively, our study shows that the combined loss of Dscr-1 and ApoE causes metabolic dysfunction in the liver but reduces atherosclerotic plaques, thereby leading to a dramatic increase in serum cholesterol and the formation of sporadic vasculopathy.
Asunto(s)
Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Proteínas de Unión al Calcio/deficiencia , Colesterol/metabolismo , Eliminación de Gen , Hipercolesterolemia/genética , Proteínas Musculares/deficiencia , Animales , Proteínas de Unión al Calcio/genética , Regulación de la Expresión Génica , Hepatocitos/metabolismo , Hipercolesterolemia/metabolismo , Ratones , Proteínas Musculares/genética , FenotipoRESUMEN
Cell signaling and the following gene regulation are tightly regulated to keep homeostasis. NF-κB is a famous key transcription factor for inflammatory cell regulations that obtain a closed feedback loop with IκB. Similarly, we show here, NFAT is also tightly regulated via its downstream target, down syndrome critical region (DSCR)-1. In primary cultured endothelium, either shear stress or VEGF treatment revealed quick NFAT1 nuclear localization following the DSCR-1 transactivation, which in turn induced NFAT1 cytoplasm sequestration. Interestingly, both NFAT and DSCR-1 can be competitive substrates for calcineurin phosphatase and DSCR-1 is known to unstable protein, which caused NFAT1-nucleocytoplasmic damped oscillation via sustained shear stress or VEGF stimulation in endothelial cell (EC)s. To understand the molecular mechanism underlying the NFAT1 oscillation, we built a mathematical model of spatiotemporal regulation of NFAT1 combined with calcineurin and DSCR-1. Theoretically, manipulation of DSCR-1 expression in simulation predicted that DSCR-1 reduction would cause nuclear retention of dephosphorylated NFAT1 and disappearance of NFAT1 oscillation. To confirm this in ECs, DSCR-1 knockdown analysis was performed. DSCR-1 reduction indeed increased dephosphorylated NFAT1 in both the nucleus and cytoplasm, which eventually led to nuclear retention of NFAT1. Taken together, these studies suggest that DSCR-1 is a responsible critical factor for NFAT1 nucleocytoplasmic oscillation in shear stress or VEGF treated ECs. Our mathematical model successfully reproduced the experimental observations of NFAT1 dynamics. Combined mathematical and experimental approaches would provide a quantitative understanding way for the spatiotemporal NFAT1 feedback system.
Asunto(s)
Calcineurina/metabolismo , Proteínas de Unión al ADN/metabolismo , Células Endoteliales/metabolismo , Proteínas Musculares/metabolismo , Factores de Transcripción NFATC/metabolismo , Transporte Activo de Núcleo Celular , Línea Celular , Humanos , Transducción de SeñalRESUMEN
OBJECTIVE: The calcineurin-NFAT (nuclear factor for activated T cells)-DSCR (Down syndrome critical region)-1 pathway plays a crucial role as the downstream effector of VEGF (vascular endothelial growth factor)-mediated tumor angiogenesis in endothelial cells. A role for DSCR-1 in different organ microenvironment such as the cornea and its role in ocular diseases is not well understood. Corneal changes can be indicators of various disease states and are easily detected through ocular examinations. Approach and Results: The presentation of a corneal arcus or a corneal opacity due to lipid deposition in the cornea often indicates hyperlipidemia and in most cases, hypercholesterolemia. Although the loss of Apo (apolipoprotein) E has been well characterized and is known to lead to elevated serum cholesterol levels, there are few corneal changes observed in ApoE-/- mice. In this study, we show that the combined loss of ApoE and DSCR-1 leads to a dramatic increase in serum cholesterol levels and severe corneal opacity with complete penetrance. The cornea is normally maintained in an avascular state; however, loss of Dscr-1 is sufficient to induce hyper-inflammatory and -oxidative condition, increased corneal neovascularization, and lymphangiogenesis. Furthermore, immunohistological analysis and genome-wide screening revealed that loss of Dscr-1 in mice triggers increased immune cell infiltration and upregulation of SDF (stromal derived factor)-1 and its receptor, CXCR4 (C-X-C motif chemokine ligand receptor-4), potentiating this signaling axis in the cornea, thereby contributing to pathological corneal angiogenesis and opacity. CONCLUSIONS: This study is the first demonstration of the critical role for the endogenous inhibitor of calcineurin, DSCR-1, and pathological corneal angiogenesis in hypercholesterolemia induced corneal opacity.
Asunto(s)
Proteínas de Unión al Calcio/deficiencia , Neovascularización de la Córnea/etiología , Opacidad de la Córnea/etiología , Células Endoteliales/metabolismo , Endotelio Corneal/metabolismo , Hipercolesterolemia/complicaciones , Proteínas Musculares/deficiencia , Animales , Proteínas de Unión al Calcio/genética , Quimiocina CXCL12/metabolismo , Quimiotaxis de Leucocito , Neovascularización de la Córnea/genética , Neovascularización de la Córnea/metabolismo , Neovascularización de la Córnea/patología , Opacidad de la Córnea/genética , Opacidad de la Córnea/metabolismo , Opacidad de la Córnea/patología , Proteínas de Unión al ADN/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Células Endoteliales/patología , Endotelio Corneal/patología , Infecciones Fúngicas del Ojo/metabolismo , Infecciones Fúngicas del Ojo/patología , Células HEK293 , Humanos , Hipercolesterolemia/genética , Hipercolesterolemia/metabolismo , Linfangiogénesis , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados para ApoE , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Estrés Oxidativo , Receptores CXCR4/metabolismo , Transducción de Señal , Síndrome de Stevens-Johnson/metabolismo , Síndrome de Stevens-Johnson/patología , Factores de Tiempo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Endothelial cell (EC) plasticity in pathological settings has recently been recognized as a driver of disease progression. Endothelial-to-mesenchymal transition (EndMT), in which ECs acquire mesenchymal properties, has been described for a wide range of pathologies, including cancer. However, the mechanism regulating EndMT in the tumor microenvironment and the contribution of EndMT in tumor progression are not fully understood. Here, we found that combined knockdown of two ETS family transcription factors, ERG and FLI1, induces EndMT coupled with dynamic epigenetic changes in ECs. Genome-wide analyses revealed that ERG and FLI1 are critical transcriptional activators for EC-specific genes, among which microRNA-126 partially contributes to blocking the induction of EndMT. Moreover, we demonstrated that ERG and FLI1 expression is downregulated in ECs within tumors by soluble factors enriched in the tumor microenvironment. These data provide new insight into the mechanism of EndMT, functions of ERG and FLI1 in ECs, and EC behavior in pathological conditions.
Asunto(s)
Transición Epitelial-Mesenquimal/genética , Proteína Proto-Oncogénica c-fli-1/genética , Animales , Regulación hacia Abajo , Células Endoteliales/metabolismo , Células Endoteliales/patología , Epigénesis Genética , Transición Epitelial-Mesenquimal/fisiología , Femenino , Técnicas de Silenciamiento del Gen , Estudio de Asociación del Genoma Completo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ratones , Ratones Endogámicos C57BL , MicroARNs/genética , MicroARNs/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Pronóstico , Proteína Proto-Oncogénica c-fli-1/antagonistas & inhibidores , Proteína Proto-Oncogénica c-fli-1/metabolismo , Regulador Transcripcional ERG/antagonistas & inhibidores , Regulador Transcripcional ERG/genética , Regulador Transcripcional ERG/metabolismo , Microambiente Tumoral/genéticaRESUMEN
BACKGROUND: The rapid increase in the number of heart failure (HF) patients in parallel with the increase in the number of older people is receiving attention worldwide. HF not only increases mortality but decreases quality of life, creating medical and social problems. Thus, it is necessary to define molecular mechanisms underlying HF development and progression. HMGB2 is a member of the high-mobility group superfamily characterized as nuclear proteins that bind DNA to stabilize nucleosomes and promote transcription. A recent in vitro study revealed that HMGB2 loss in cardiomyocytes causes hypertrophy and increases HF-associated gene expression. However, it's in vivo function in the heart has not been assessed. MethodsâandâResults: Western blotting analysis revealed increased HMGB2 expression in heart tissues undergoing pressure overload by transverse aorta constriction (TAC) in mice. Hmgb2 homozygous knockout (Hmgb2-/-) mice showed cardiac dysfunction due to AKT inactivation and decreased sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)2a activity. Compared to wild-type mice, Hmgb2-/- mice had worsened cardiac dysfunction after TAC surgery, predisposing mice to HF development and progression. CONCLUSIONS: This study demonstrates that upregulation of cardiac HMGB2 is an adaptive response to cardiac stress, and that loss of this response could accelerate cardiac dysfunction, suggesting that HMGB2 plays a cardioprotective role.
Asunto(s)
Proteína HMGB2/análisis , Insuficiencia Cardíaca/etiología , Animales , Western Blotting , Cardiotónicos/análisis , Cardiotónicos/farmacología , Constricción Patológica/complicaciones , Proteína HMGB2/genética , Proteína HMGB2/farmacología , Insuficiencia Cardíaca/prevención & control , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Proto-Oncogénicas c-akt/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismoRESUMEN
The vascular system forms the largest surface in our body, serving as a critical interface between blood circulation and our diverse organ/tissue environments. Thus, the vascular system performs a gatekeeper function for organ/tissue homeostasis and the body's adjustment to pathological challenges. The endothelium, as the most inner layer of the vasculature, regulates the tissue microenvironment, which is critical for development, hemostatic balance, inflammation, and angiogenesis, with a role as well in tumor malignancy and metastasis. These multitudinous functions are primarily mediated by organ/tissue-specifically differentiated endothelial cells, in which heterogeneity has long been recognized at the molecular and histological level. Based on these general principles of vascular-bed heterogeneity and characterization, this review largely covers landmark discoveries regarding organ/tissue microenvironment-governed endothelial cell phenotypic changes. These involve the physical features of continuous, discontinuous, fenestrated, and sinusoidal endothelial cells, in addition to the more specialized endothelial cell layers of the lymphatic system, glomerulus, tumors, and the blood brain barrier (BBB). Major signal pathways of endothelial specification are outlined, including Notch as a key factor of tip/stalk- and arterial-endothelial cell differentiation. We also denote the shear stress sensing machinery used to convey blood flow-mediated biophysical forces that are indispensable to maintaining inert and mature endothelial phenotypes. Since our circulatory system is among the most fundamental and emergent targets of study in pharmacology from the viewpoint of drug metabolism and delivery, a better molecular understanding of organ vasculature-bed heterogeneity may lead to better strategies for novel vascular-targeted treatments to fight against hitherto intractable diseases.
Asunto(s)
Células Endoteliales , Especificidad de Órganos , Animales , Enfermedad , Endotelio Vascular , Salud , HumanosRESUMEN
Although studies of the differentiation from mouse embryonic stem (ES) cells to vascular endothelial cells (ECs) provide an excellent model for investigating the molecular mechanisms underlying vascular development, temporal dynamics of gene expression and chromatin modifications have not been well studied. Herein, using transcriptomic and epigenomic analyses based on H3K4me3 and H3K27me3 modifications at a genome-wide scale, we analysed the EC differentiation steps from ES cells and crucial epigenetic modifications unique to ECs. We determined that Gata2, Fli1, Sox7 and Sox18 are master regulators of EC that are induced following expression of the haemangioblast commitment pioneer factor, Etv2. These master regulator gene loci were repressed by H3K27me3 throughout the mesoderm period but rapidly transitioned to histone modification switching from H3K27me3 to H3K4me3 after treatment with vascular endothelial growth factor. SiRNA knockdown experiments indicated that these regulators are indispensable not only for proper EC differentiation but also for blocking the commitment to other closely aligned lineages. Collectively, our detailed epigenetic analysis may provide an advanced model for understanding temporal regulation of chromatin signatures and resulting gene expression profiles during EC commitment. These studies may inform the future development of methods to stimulate the vascular endothelium for regenerative medicine.
Asunto(s)
Células Endoteliales/metabolismo , Epigénesis Genética , Factor de Transcripción GATA2/genética , Histonas/genética , Células Madre Embrionarias de Ratones/metabolismo , Proteína Proto-Oncogénica c-ets-1/genética , Factores de Transcripción SOXF/genética , Animales , Diferenciación Celular , Linaje de la Célula/genética , Células Endoteliales/citología , Factor de Transcripción GATA2/antagonistas & inhibidores , Factor de Transcripción GATA2/metabolismo , Histonas/metabolismo , Ratones , Células Madre Embrionarias de Ratones/citología , Análisis de Secuencia por Matrices de Oligonucleótidos , Cultivo Primario de Células , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteína Proto-Oncogénica c-ets-1/antagonistas & inhibidores , Proteína Proto-Oncogénica c-ets-1/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Factores de Transcripción SOXF/antagonistas & inhibidores , Factores de Transcripción SOXF/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The androgen receptor (AR) is a key driver of prostate cancer (PC), even in the state of castration-resistant PC (CRPC) and frequently even after treatment with second-line hormonal therapies such as abiraterone and enzalutamide. The persistence of AR activity via both ligand-dependent and ligand-independent mechanisms (including constitutively active AR splice variants) highlights the unmet need for alternative approaches to block AR signaling in CRPC. We investigated the transcription factor GATA-binding protein 2 (GATA2) as a regulator of AR signaling and an actionable therapeutic target in PC. We demonstrate that GATA2 directly promotes expression of both full-length and splice-variant AR, resulting in a strong positive correlation between GATA2 and AR expression in both PC cell lines and patient specimens. Conversely, GATA2 expression is repressed by androgen and AR, suggesting a negative feedback regulatory loop that, upon androgen deprivation, derepresses GATA2 to contribute to AR overexpression in CRPC. Simultaneously, GATA2 is necessary for optimal transcriptional activity of both full-length and splice-variant AR. GATA2 colocalizes with AR and Forkhead box protein A1 on chromatin to enhance recruitment of steroid receptor coactivators and formation of the transcriptional holocomplex. In agreement with these important functions, high GATA2 expression and transcriptional activity predicted worse clinical outcome in PC patients. A GATA2 small molecule inhibitor suppressed the expression and transcriptional function of both full-length and splice-variant AR and exerted potent anticancer activity against PC cell lines. We propose pharmacological inhibition of GATA2 as a first-in-field approach to target AR expression and function and improve outcomes in CRPC.
Asunto(s)
Factor de Transcripción GATA2/fisiología , Coactivadores de Receptor Nuclear/metabolismo , Receptores Androgénicos/metabolismo , Proliferación Celular , Cromatina/metabolismo , Elementos de Facilitación Genéticos , Factor Nuclear 3-alfa del Hepatocito/metabolismo , Humanos , Masculino , Pronóstico , Receptores Androgénicos/fisiología , Transducción de Señal , Transcripción Genética/fisiologíaRESUMEN
Slit proteins and their receptors, the Roundabout (Robo) family, are known to have a pivotal role in the vascular system. Slit2/Robo1 regulates the migration of human umbilical vein endothelial cells (HUVECs) and tumor-associated endothelial cells. Robo4, the endothelial-specific Robo, is also considered to be involved in vascular cell migration. However, the Slit/Robo signaling pathway is still unclear. Using a Boyden chamber assay, we found that Slit2 induces the migration of HUVECs under a Robo4 knockdown condition. This effect disappeared in Robo1 knockdown cells. The co-existence of the N-terminal extracellular portion of Robo1 blocked the Slit2-evoked migration of HUVECs, while that of Robo4 caused no effect. These results show that the Slit2 signal is transduced through Robo1, while the negative regulation of Robo4 is an intracellular event. Targeted proteomics using an anti-Robo1 monoclonal antibody identified CdGAP, an adhesion-localized Rac1-and Cdc42-specific GTPase activating protein, as a candidate for Slit2/Robo1 signaling. Robo1 and CdGAP were co-immunoprecipitated from CHO cells co-transfected with Robo1 and CdGAP genes. These results suggest that Slit2/Robo1 binding exerts an effect on cell migration, which is negatively regulated by Robo4, and Robo1 may function by interacting with CdGAP in HUVECs.
Asunto(s)
Movimiento Celular/fisiología , Células Endoteliales/fisiología , Proteínas Activadoras de GTPasa/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Fosfoproteínas/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores Inmunológicos/metabolismo , Línea Celular , Células Endoteliales/citología , Humanos , Péptidos y Proteínas de Señalización Intercelular , Transducción de Señal/fisiología , Proteínas RoundaboutRESUMEN
VEGF is a key regulator of endothelial cell migration, proliferation, and inflammation, which leads to activation of several signaling cascades, including the calcineurin-nuclear factor of activated T cells (NFAT) pathway. NFAT is not only important for immune responses but also for cardiovascular development and the pathogenesis of Down syndrome. By using Down syndrome model mice and clinical patient samples, we showed recently that the VEGF-calcineurin-NFAT signaling axis regulates tumor angiogenesis and tumor metastasis. However, the connection between genome-wide views of NFAT-mediated gene regulation and downstream gene function in the endothelium has not been studied extensively. Here we performed comprehensive mapping of genome-wide NFATc1 binding in VEGF-stimulated primary cultured endothelial cells and elucidated the functional consequences of VEGF-NFATc1-mediated phenotypic changes. A comparison of the NFATc1 ChIP sequence profile and epigenetic histone marks revealed that predominant NFATc1-occupied peaks overlapped with promoter-associated histone marks. Moreover, we identified two novel NFATc1 regulated genes, CXCR7 and RND1. CXCR7 knockdown abrogated SDF-1- and VEGF-mediated cell migration and tube formation. siRNA treatment of RND1 impaired vascular barrier function, caused RhoA hyperactivation, and further stimulated VEGF-mediated vascular outgrowth from aortic rings. Taken together, these findings suggest that dynamic NFATc1 binding to target genes is critical for VEGF-mediated endothelial cell activation. CXCR7 and RND1 are NFATc1 target genes with multiple functions, including regulation of cell migration, tube formation, and barrier formation in endothelial cells.
Asunto(s)
Endotelio Vascular/metabolismo , Factores de Transcripción NFATC/metabolismo , Neovascularización Patológica , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Células COS , Movimiento Celular , Chlorocebus aethiops , Técnicas de Cocultivo , Células Endoteliales/citología , Epigénesis Genética , Fibroblastos/metabolismo , Estudio de Asociación del Genoma Completo , Células HEK293 , Homeostasis , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Receptores CXCR4/metabolismo , Transducción de Señal , Activación Transcripcional , Proteínas de Unión al GTP rho/metabolismoRESUMEN
GATA2 is well recognized as a key transcription factor and regulator of cell-type specificity and differentiation. Here, we carried out comparative chromatin immunoprecipitation with comprehensive sequencing (ChIP-seq) to determine genome-wide occupancy of GATA2 in endothelial cells and erythroids, and compared the occupancy to the respective gene expression profile in each cell type. Although GATA2 was commonly expressed in both cell types, different GATA2 bindings and distinct cell-specific gene expressions were observed. By using the ChIP-seq with epigenetic histone modifications and chromatin conformation capture assays; we elucidated the mechanistic regulation of endothelial-specific GATA2-mediated endomucin gene expression, that was regulated by the endothelial-specific chromatin loop with a GATA2-associated distal enhancer and core promoter. Knockdown of endomucin markedly attenuated endothelial cell growth, migration and tube formation. Moreover, abrogation of GATA2 in endothelium demonstrated not only a reduction of endothelial-specific markers, but also induction of mesenchymal transition promoting gene expression. Our findings provide new insights into the correlation of endothelial-expressed GATA2 binding, epigenetic modification, and the determination of endothelial cell specificity.
Asunto(s)
Endotelio Vascular/metabolismo , Epigénesis Genética/fisiología , Factor de Transcripción GATA2/metabolismo , Sialoglicoproteínas/genética , Animales , Secuencia de Bases , Células COS , Células Cultivadas , Chlorocebus aethiops , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Endotelio Vascular/efectos de los fármacos , Factor de Transcripción GATA2/genética , Factor de Transcripción GATA2/fisiología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Técnicas de Silenciamiento del Gen , Humanos , Células K562 , Análisis por Micromatrices , Modelos Biológicos , Especificidad de Órganos/efectos de los fármacos , Especificidad de Órganos/genética , Unión Proteica/genética , Unión Proteica/fisiología , ARN Interferente Pequeño/farmacología , Sialoglicoproteínas/metabolismoRESUMEN
OBJECTIVE: Cardiovascular disease (CVD), the most common morbidity resulting from atherosclerosis, remains a frequent cause of death. Efforts to develop effective therapeutic strategies have focused on vascular inflammation as a critical pathology driving atherosclerosis progression. Nonetheless, molecular mechanisms underlying this activity remain unclear. Here, we ask whether angiopoietin-like protein 2 (Angptl2), a proinflammatory protein, contributes to vascular inflammation that promotes atherosclerosis progression. APPROACH AND RESULTS: Histological analysis revealed abundant Angptl2 expression in endothelial cells and macrophages infiltrating atheromatous plaques in patients with cardiovascular disease. Angptl2 knockout in apolipoprotein E-deficient mice (ApoE(-/-)/Angptl2(-/-)) attenuated atherosclerosis progression by decreasing the number of macrophages infiltrating atheromatous plaques, reducing vascular inflammation. Bone marrow transplantation experiments showed that Angptl2 deficiency in endothelial cells attenuated atherosclerosis development. Conversely, ApoE(-/-) mice crossed with transgenic mice expressing Angptl2 driven by the Tie2 promoter (ApoE(-/-)/Tie2-Angptl2 Tg), which drives Angptl2 expression in endothelial cells but not monocytes/macrophages, showed accelerated plaque formation and vascular inflammation because of increased numbers of infiltrated macrophages in atheromatous plaques. Tie2-Angptl2 Tg mice alone did not develop plaques but exhibited endothelium-dependent vasodilatory dysfunction, likely because of decreased production of endothelial cell-derived nitric oxide. Conversely, Angptl2(-/-) mice exhibited less severe endothelial dysfunction than did wild-type mice when fed a high-fat diet. In vitro, Angptl2 activated proinflammatory nuclear factor-κB signaling in endothelial cells and increased monocyte/macrophage chemotaxis. CONCLUSIONS: Endothelial cell-derived Angptl2 accelerates vascular inflammation by activating proinflammatory signaling in endothelial cells and increasing macrophage infiltration, leading to endothelial dysfunction and atherosclerosis progression.
Asunto(s)
Angiopoyetinas/metabolismo , Aterosclerosis/metabolismo , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Mediadores de Inflamación/metabolismo , Vasculitis/metabolismo , Anciano de 80 o más Años , Proteína 2 Similar a la Angiopoyetina , Proteínas Similares a la Angiopoyetina , Angiopoyetinas/deficiencia , Angiopoyetinas/genética , Animales , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Aterosclerosis/genética , Aterosclerosis/inmunología , Aterosclerosis/patología , Aterosclerosis/prevención & control , Trasplante de Médula Ósea , Células Cultivadas , Quimiotaxis de Leucocito , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Dislipidemias/metabolismo , Dislipidemias/fisiopatología , Células Endoteliales/inmunología , Células Endoteliales/patología , Endotelio Vascular/inmunología , Endotelio Vascular/patología , Endotelio Vascular/fisiopatología , Femenino , Humanos , Integrina alfa5beta1/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Monocitos/inmunología , Monocitos/metabolismo , FN-kappa B/metabolismo , Factores de Transcripción NFATC/metabolismo , Óxido Nítrico/metabolismo , Obesidad/metabolismo , Obesidad/fisiopatología , Placa Aterosclerótica , Transducción de Señal , Factores de Tiempo , Vasculitis/genética , Vasculitis/inmunología , Vasculitis/patología , Vasculitis/prevención & control , VasodilataciónRESUMEN
The incidence of many cancer types is significantly reduced in individuals with Down's syndrome, and it is thought that this broad cancer protection is conferred by the increased expression of one or more of the 231 supernumerary genes on the extra copy of chromosome 21. One such gene is Down's syndrome candidate region-1 (DSCR1, also known as RCAN1), which encodes a protein that suppresses vascular endothelial growth factor (VEGF)-mediated angiogenic signalling by the calcineurin pathway. Here we show that DSCR1 is increased in Down's syndrome tissues and in a mouse model of Down's syndrome. Furthermore, we show that the modest increase in expression afforded by a single extra transgenic copy of Dscr1 is sufficient to confer significant suppression of tumour growth in mice, and that such resistance is a consequence of a deficit in tumour angiogenesis arising from suppression of the calcineurin pathway. We also provide evidence that attenuation of calcineurin activity by DSCR1, together with another chromosome 21 gene Dyrk1a, may be sufficient to markedly diminish angiogenesis. These data provide a mechanism for the reduced cancer incidence in Down's syndrome and identify the calcineurin signalling pathway, and its regulators DSCR1 and DYRK1A, as potential therapeutic targets in cancers arising in all individuals.
Asunto(s)
Síndrome de Down/genética , Inositol/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Animales , Calcineurina/metabolismo , Proteínas de Unión al Calcio , Catecoles , Células Cultivadas , Proteínas de Unión al ADN , Modelos Animales de Enfermedad , Síndrome de Down/metabolismo , Células Endoteliales/metabolismo , Dosificación de Gen/genética , Humanos , Ratones , Ratones Transgénicos , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Quinasas DyrKRESUMEN
The COUP-TFII (chicken ovalbumin upstream promoter-transcription factor II) nuclear receptor, which is composed of a DNA-binding domain and a ligand-binding domain, exerts pleiotropic effects on development and cell differentiation by regulating the transcription of its target genes, including Cyp7a1 (cytochrome P450, family 7, subfamily a, polypeptide 1), which plays important roles in catabolism of cholesterol in the liver. Although multiple variants of COUP-TFII exist, their roles in the regulation of Cyp7a1 expression have not been elucidated. In the present study, we investigated the roles of COUP-TFII-V2 (variant 2), which lacks a DNA-binding domain, in the regulation of the transcriptional control of the Cyp7a1 gene by COUP-TFII in hepatocellular carcinoma cells. We found that COUP-TFII-V2 was significantly expressed in Huh7 cells, in which Cyp7a1 was not expressed. Furthermore, knockdown of COUP-TFII-V2 enhanced endogenous Cyp7a1 expression in Huh7 cells. Although COUP-TFII activates the Cyp7a1 promoter through direct binding to DNA, this activation was affected by COUP-TFII-V2, which physically interacted with COUP-TFII and inhibited its DNA-binding ability. Chromatin immunoprecipitation assays showed that COUP-TFII-V2 inhibited the binding of endogenous COUP-TFII to the intact Cyp7a1 promoter. The results of the present study suggest that COUP-TFII-V2 negatively regulates the function of COUP-TFII by inhibiting its binding to DNA to decrease Cyp7a1 expression.
Asunto(s)
Factor de Transcripción COUP II/química , Factor de Transcripción COUP II/genética , Colesterol 7-alfa-Hidroxilasa/antagonistas & inhibidores , Colesterol 7-alfa-Hidroxilasa/genética , Proteínas de Unión al ADN/antagonistas & inhibidores , Variación Genética , Regiones Promotoras Genéticas , Factor de Transcripción COUP II/metabolismo , Línea Celular Tumoral , Colesterol 7-alfa-Hidroxilasa/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación Enzimológica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Células Hep G2 , Humanos , Unión Proteica/genética , Estructura Terciaria de Proteína/genéticaRESUMEN
Histone demethylase JHDM1D (also known as KDM7A) modifies the level of methylation in histone and participates in epigenetic gene regulation; however, the role of JHDM1D in tumor progression is unknown. Here, we show that JHDM1D plays a tumor-suppressive role by regulating angiogenesis. Expression of JHDM1D was increased in mouse and human cancer cells under long-term nutrient starvation in vitro. Expression of JHDM1D mRNA was increased within avascular tumor tissue at the preangiogenic switch, along with increased expression of angiogenesis-regulating genes such as Vegf-A. Stable expression of JHDM1D cDNA or siRNA silencing of JHDM1D in cancer cells did not affect cell proliferation, anchorage-independent cell growth, or cell cycle progression in vitro. Notably, JHDM1D-expressing mouse melanoma (B16) and human cervical carcinoma (HeLa) cells exhibited significantly slower tumor growth in vivo compared with the original cells. This reduction in tumor growth was associated with decreased formation of CD31(+) blood vessels and reduced infiltration of CD11b(+) macrophage linage cells into tumor tissues. Expression of multiple angiogenic factors such as VEGF-B and angiopoietins was decreased in tumor xenografts of JHDM1D-expressing B16 and HeLa cells. Our results provide evidence that increased JHDM1D expression suppressed tumor growth by down-regulating angiogenesis under nutrient starvation.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Histona Demetilasas con Dominio de Jumonji/genética , Neovascularización Patológica , Animales , Proliferación Celular , Medios de Cultivo/química , Progresión de la Enfermedad , Células HeLa , Humanos , Histona Demetilasas con Dominio de Jumonji/metabolismo , Melanoma Experimental , Ratones , Ratones Endogámicos C57BL , Trasplante de Neoplasias , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Forkhead box O (FOXO) family proteins are expressed in various cells, and play crucial roles in cellular metabolism, apoptosis, and aging. FOXO1-null mice exhibit embryonic lethality due to impaired endothelial cell (EC) maturation and vascular remodeling. However, FOXO1-mediated genome-wide regulation in ECs remains unclear. Here, we demonstrate that VEGF dynamically regulates FOXO1 cytosol-nucleus translocation. FOXO1 re-localizes to the nucleus via PP2A phosphatase. RNA-seq combined with FOXO1 overexpression/knockdown in ECs demonstrated that FOXO1 governs the VEGF-responsive tip cell-enriched genes, and further inhibits DLL4-NOTCH signaling. Endogenous FOXO1 ChIP-seq revealed that FOXO1 binds to the EC-unique tip-enriched genes with co-enrichment of EC master regulators, and the condensed chromatin region as a pioneer factor. We identified new promoter/enhancer regions of the VEGF-responsive tip cell genes regulated by FOXO1: ESM1 and ANGPT2. This is the first study to identify cell type-specific FOXO1 functions, including VEGF-mediated tip cell definition in primary cultured ECs.
RESUMEN
BACKGROUND/AIM: Individuals with Down syndrome (DS), attributed to triplication of human chromosome 21 (Hsa21), exhibit a reduced incidence of solid tumors. However, the prevalence of glioblastoma among individuals with DS remains a contentious issue in epidemiological studies. Therefore, this study examined the gliomagenicity in Ts1Cje mice, a murine model of DS. MATERIALS AND METHODS: We employed the Sleeping Beauty transposon system for the integration of human oncogenes into cells of the subventricular zone of neonatal mice. RESULTS: Notably, Sleeping Beauty-mediated de novo murine gliomagenesis was significantly suppressed in Ts1Cje mice compared to wild-type mice. In glioblastomas of Ts1je mice, we observed an augmented presence of M1-polarized tumor-associated macrophages and microglia, known for their anti-tumor efficacy in the early stage of tumor development. CONCLUSION: Our findings in a mouse model of DS offer novel perspectives on the diminished gliomagenicity observed in individuals with DS.
Asunto(s)
Síndrome de Down , Ratones , Animales , Humanos , Síndrome de Down/genética , Síndrome de Down/patología , Modelos Animales de EnfermedadRESUMEN
The genetic lesions that drive acute megakaryoblastic leukemia (AMKL) have not been fully elucidated. To search for genetic alterations in AMKL, we performed targeted deep sequencing in 34 AMKL patient samples and 8 AMKL cell lines and detected frequent genetic mutations in the NOTCH pathway in addition to previously reported alterations in GATA-1 and the JAK-STAT pathway. Pharmacological and genetic NOTCH activation, but not inhibition, significantly suppressed AMKL cell proliferation in both in vitro and in vivo assays employing a patient-derived xenograft model. These results suggest that NOTCH inactivation underlies AMKL leukemogenesis. and NOTCH activation holds the potential for therapeutic application in AMKL.
RESUMEN
Prox1 plays pivotal roles during embryonic lymphatic development and maintenance of adult lymphatic systems by modulating the expression of various lymphatic endothelial cell (LEC) markers, such as vascular endothelial growth factor receptor 3 (VEGFR3). However, the molecular mechanisms by which Prox1 transactivates its target genes remain largely unknown. Here, we identified Ets-2 as a candidate molecule that regulates the functions of Prox1. Whereas Ets-2 has been implicated in angiogenesis, its roles during lymphangiogenesis have not yet been elucidated. We found that endogenous Ets-2 interacts with Prox1 in LECs. Using an in vivo model of chronic aseptic peritonitis, we found that Ets-2 enhanced inflammatory lymphangiogenesis, whereas a dominant-negative mutant of Ets-1 suppressed it. Ets-2 also enhanced endothelial migration towards VEGF-C through induction of expression of VEGFR3 in collaboration with Prox1. Furthermore, we found that both Prox1 and Ets-2 bind to the VEGFR3 promoter in intact chromatin. These findings suggest that Ets family members function as transcriptional cofactors that enhance Prox1-induced lymphangiogenesis.
Asunto(s)
Células Endoteliales/metabolismo , Proteínas de Homeodominio/metabolismo , Peritonitis/metabolismo , Proteína Proto-Oncogénica c-ets-1/metabolismo , Proteína Proto-Oncogénica c-ets-2/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Células Cultivadas , Inmunoprecipitación de Cromatina , Células Endoteliales/inmunología , Células Endoteliales/patología , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Humanos , Inflamación , Linfangiogénesis/genética , Linfangiogénesis/inmunología , Ratones , Ratones Endogámicos BALB C , Mutación/genética , Peritonitis/inducido químicamente , Peritonitis/genética , Peritonitis/fisiopatología , Proteína Proto-Oncogénica c-ets-1/genética , Proteína Proto-Oncogénica c-ets-2/genética , ARN Interferente Pequeño/genética , Tioglicolatos/administración & dosificación , Proteínas Supresoras de Tumor/genética , Receptor 3 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 3 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
A region of the human von Willebrand factor (VWF) gene between -2812 and the end of the first intron (termed vWF2) was previously shown to direct expression in the endothelium of capillaries and a subset of larger blood vessels in the heart and skeletal muscle. Here, our goal was to delineate the DNA sequences responsible for this effect. A series of constructs containing deletions or mutations of vWF2 coupled to LacZ were targeted to the Hprt locus of mice, and the resulting animals were analyzed for reporter gene expression. The findings demonstrate that DNA sequences between -843 and -620 are necessary for expression in capillary but not large vessel endothelium in heart and skeletal muscle. Further, expression of VWF in capillaries and larger vessels of both tissues required the presence of a native or heterologous intron. In vitro assays implicated a role for ERG-binding ETS motif at -56 in mediating basal expression of VWF. In Hprt-targeted mice, mutation of the ETS consensus motif resulted in loss of LacZ expression in the endothelium of the heart and skeletal muscle. Together, these data indicate that distinct DNA modules regulate vascular bed-specific expression of VWF.