RESUMEN
Spermatid production is a complex regulatory process in which coordination between hormonal control and apoptosis plays a pivotal role in maintaining a balanced number of sperm cells. Apoptosis in spermatogenesis is controlled by pro-apoptotic and anti-apoptotic molecules. Hormones involved in the apoptotic process during spermatogenesis include gonadotrophins, sex hormones, and glucocorticoid (GC). GC acts broadly as an apoptosis inducer by binding to its receptor (glucocorticoid receptor: GR) during organ development processes, such as spermatogenesis. However, the downstream pathway induced in GC-GR signaling and the apoptotic process during spermatogenesis remains poorly understood. We reported previously that GC induces full-length glucocorticoid-induced transcript 1 (GLCCI1-long), which functions as an anti-apoptotic mediator in thymic T cell development. Here, we demonstrate that mature murine testis expresses a novel isoform of GLCCI1 protein (GLCCI1-short) in addition to GLCCI1-long. We demonstrate that GLCCI1-long is expressed in spermatocytes along with GR. In contrast, GLCCI1-short is primarily expressed in spermatids where GR is absent; instead, the estrogen receptor is expressed. GLCCI1-short also binds to LC8, which is a known mediator of the anti-apoptotic effect of GLCCI1-long. A luciferase reporter assay revealed that ß-estradiol treatment synergistically increased Glcci1-short promotor-driven luciferase activity in Erα-overexpressing cells. Together with the evidence that the conversion of testosterone to estrogen is preceded by aromatase expression in spermatids, we hypothesize that estrogen induces GLCCI1-short, which, in turn, may function as a novel anti-apoptotic mediator in mature murine testis.
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Glucocorticoides , Semen , Masculino , Ratones , Animales , Espermatogénesis , Espermátides , EstrógenosRESUMEN
Vascular endothelial growth factor (VEGF) signaling plays a central role in vascular development and maintenance of vascular homeostasis. In endothelial cells (ECs), VEGF activates the gene expression of angiogenic transcription factors (TFs), followed by induction of downstream angiogenic responsive genes. Recent findings support that histone modification dynamics contribute to the transcriptional control of genes that are important for EC functions. Lysine demethylase 2B (KDM2B) demethylates histone H3K4me3 and H3K36me2/3 and mediates the monoubiquitination of histone H2AK119. KDM2B functions as a transcriptional repressor in somatic cell reprogramming and tumor development. However, the role of KDM2B in VEGF signaling remains to be elucidated. Here, we show that KDM2B knockdown enhances VEGF-induced angiogenesis in cultured human ECs via increased migration and proliferation. In contrast, ectopic expression of KDM2B inhibits angiogenesis. The function of KDM2B may depend on its catalytic Jumonji C domain. Genome-wide analysis further reveals that KDM2B selectively controls the transcription of VEGF-induced angiogenic TFs that are associated with increased H3K4me3/H3K36me3 and decreased H2AK119ub. These findings suggest an essential role of KDM2B in VEGF signaling in ECs. As dysregulation of VEGF signaling in ECs is involved in various diseases, including cancer, KDM2B may be a potential therapeutic target in VEGF-mediated vasculopathic diseases.
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Proteínas F-Box , Histonas , Proliferación Celular , Células Endoteliales/metabolismo , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Histonas/metabolismo , Humanos , Histona Demetilasas con Dominio de Jumonji/genética , Histona Demetilasas con Dominio de Jumonji/metabolismo , Lisina/metabolismo , Factores de Transcripción/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
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.
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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
Altered expression of nephrin underlies the pathophysiology of proteinuria in both congenital and acquired nephrotic syndrome. However, the epigenetic mechanisms of nephrin gene regulation remain elusive. Here, we show that Wolf-Hirschhorn syndrome candidate 1-like 1 long form (WHSC1L1-L) is a novel epigenetic modifier of nephrin gene regulation. WHSC1L1-L was associated with histone H3K4 and H3K36 in human embryonic kidney cells. WHSC1L1-L gene was expressed in the podocytes, and functional protein product was detected in these cells. WHSC1L1-L was found to bind nephrin but not other podocyte-specific gene promoters, leading to its inhibition/suppression, abrogating the stimulatory effect of WT1 and NF-κB. Gene knockdown of WHSC1L1-L in primary cultured podocytes accelerated the transcription of nephrin but not CD2AP. An in vivo zebrafish study involving the injection of Whsc1l1 mRNA into embryos demonstrated an apparent reduction of nephrin mRNA but not podocin and CD2AP mRNA. Immunohistochemistry showed that both WHSC1L1-L and nephrin emerged at the S-shaped body stage in glomeruli. Immunofluorescence and confocal microscopy displayed WHSC1L1 to colocalize with trimethylated H3K4 in the glomerular podocytes. Chromatin immunoprecipitation assay revealed the reduction of the association of trimethylated H3K4 at the nephrin promoter regions. Finally, nephrin mRNA was upregulated in the glomerulus at the early proteinuric stage of mouse nephrosis, which was associated with the reduction of WHSC1L1. In conclusion, our results demonstrate that WHSC1L1-L acts as a histone methyltransferase in podocytes and regulates nephrin gene expression, which may in turn contribute to the integrity of the slit diaphragm of the glomerular filtration barrier.
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Epigénesis Genética , N-Metiltransferasa de Histona-Lisina/genética , Proteínas de la Membrana/genética , Síndrome Nefrótico/genética , Proteínas Nucleares/genética , Podocitos/enzimología , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Sitios de Unión , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Modelos Animales de Enfermedad , Doxorrubicina , Regulación de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Células HEK293 , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Proteínas de la Membrana/metabolismo , Metilación , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Síndrome Nefrótico/inducido químicamente , Síndrome Nefrótico/enzimología , Síndrome Nefrótico/patología , Proteínas Nucleares/metabolismo , Podocitos/patología , Regiones Promotoras Genéticas , Interferencia de ARN , Transfección , Pez Cebra/genética , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
BACKGROUND: Leucine stimulates cancer cell proliferation through the mTOR pathway, therefore, inhibiting leucine transporters may be a novel therapeutic target for cancer. L-type amino acid transporter (LAT) 1, a Na+ -independent amino acid transporter, is highly expressed in many tumor cells. However, leucine transporter(s) in different stages of prostate cancer, particularly in the stages of castration resistance with androgen receptor (AR) expression, is unclear. METHODS: LNCaP and DU145 and PC-3 cell lines were used as a model of androgen dependent, and metastatic prostate cancer. A new "LN-cr" cell line was established after culturing LNCaP cells for 6 months under androgen-free conditions, which is considered a model of castration resistant prostate cancer (CRPC) with androgen AR expression. The expression of leucine transporters was investigated with quantitative PCR and immunofluorescence. Uptake of 14 C Leucine was examined in the presence or absence of BCH (a pan-LAT inhibitor), JPH203 (an LAT1-specific inhibitor), or Na+ . Cell growth was assessed with MTT assay. siRNA studies were performed to evaluate the indispensability of y+ LAT2 on leucine uptake and cell viability in LN-cr. RESULTS: Cell viability showed a 90% decrease in the absence of leucine in all four cell lines. LNCaP cells principally expressed LAT3, and their leucine uptake was more than 90% Na+ -independent. BCH, but not JPH203, inhibited leucine uptake, and cell proliferation (IC50BCH :15 mM). DU145 and PC-3 cells predominantly expressed LAT1. Leucine uptake and cell growth were suppressed by BCH or JPH203 in a dose-dependent manner (IC50BCH : â¼20 mM, IC50JPH203 : â¼5 µM). In LN-cr cells, Na+ -dependent uptake of leucine was 3.8 pmol/mgprotein/min, while, Na+ -independent uptake was only 0.52 (P < 0.05). Leucine uptake of LN-cr was largely (â¼85%) Na+ -dependent. y+ LAT2 expression was confirmed in LN-cr. Knockdown of y+ LAT2 lead to significant leucine uptake inhibition (40%) and cell growth inhibition (20%). CONCLUSIONS: New CRPC cell line with increased expression of y+ LAT2 as a leucine transporter was established in vitro. Anti-leucine transporter therapy could be an important option against prostate cancer. Prostate 77:222-233, 2017. © 2016 Wiley Periodicals, Inc.
Asunto(s)
Sistemas de Transporte de Aminoácidos/metabolismo , Leucina/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Receptores Androgénicos/metabolismo , Línea Celular Tumoral , Supervivencia Celular/fisiología , Humanos , Masculino , Neoplasias de la Próstata Resistentes a la Castración/patologíaRESUMEN
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.
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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.
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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
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
Some endothelial cells in the tumor vasculature expressed a system L amino acid transporter LAT1. To elucidate the role of LAT1 in tumor related endothelial cells, tumor cells were injected into endothelial specific LAT1 conditional knockout mice (Slc7a5flox/flox; Cdh5-Cre-ERT2) and found that the shape of the tumor vasculature was normalized and that the size and numbers of lung metastasis was reduced. TNFα-induced expression of VCAM1 and E-selectin at the surface of HUVEC, both of which are responsible for enhanced monocyte attachment and pre-metastatic niche formation, was reduced in the presence of LAT1 inhibitor, nanvuranlat. Deprivation of tryptophan, an LAT1 substrate, mimicked LAT1 inhibition, which led to activation of MEK1/2-ERK1/2 pathway and subsequent cystathionine γ lyase (CTH) induction. Increased production of hydrogen sulfide (H2S) by CTH was at least partially responsible for tumor vascular normalization, leading to decreased leakiness and enhanced delivery of chemotherapeutic agents to the tumor.
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
The endothelial layers of the microvasculature regulate the transport of solutes to the surrounding tissues. It remains unclear how this barrier function is affected by blood flow-induced intraluminal pressure. Using a 3D microvessel model, we compare the transport of macromolecules through endothelial tissues at mechanical rest or with intraluminal pressure, and correlate these data with electron microscopy of endothelial junctions. On application of an intraluminal pressure of 100 Pa, we demonstrate that the flow through the tissue increases by 2.35 times. This increase is associated with a 25% expansion of microvessel diameter, which leads to tissue remodeling and thinning of the paracellular junctions. We recapitulate these data with the deformable monopore model, in which the increase in paracellular transport is explained by the augmentation of the diffusion rate across thinned junctions under mechanical stress. We therefore suggest that the deformation of microvasculatures contributes to regulate their barrier function.
RESUMEN
Endothelial cell activation and dysfunction underlie many vascular disorders, including atherosclerosis, tumor growth, and sepsis. Endothelial cell activation, in turn, is mediated primarily at the level of gene transcription. Here, we show that in response to several activation agonists, including vascular endothelial growth factor (VEGF), tumor necrosis factor-alpha, and thrombin, endothelial cells demonstrate rapid and profound induction of the early growth response (Egr) genes egr-1 and egr-3. In VEGF-treated endothelial cells, induction of Egr-3 was far greater and more prolonged compared with Egr-1. VEGF-mediated stimulation of Egr-3 involved the inducible binding of NFATc, serum response factor, and CREB to their respective consensus motifs in the upstream promoter region of Egr-3. Knockdown of Egr-3 markedly impaired VEGF-mediated proliferation, migration, and tube formation of endothelial cells and blocked VEGF-induced monocyte adhesion. Egr-3 knockdown abrogated VEGF-mediated vascular outgrowth from ex vivo aortic rings and attenuated Matrigel plug vascularization and melanoma tumor growth in vivo. Together, these findings suggest that Egr-3 is a critical determinant of VEGF signaling in activated endothelial cells. Thus, Egr-3 represents a potential therapeutic target in VEGF-mediated vasculopathic diseases.
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Proteína 3 de la Respuesta de Crecimiento Precoz/genética , Células Endoteliales/citología , Células Endoteliales/metabolismo , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Animales , División Celular/efectos de los fármacos , División Celular/fisiología , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Células Cultivadas , Vasos Coronarios/citología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Proteína 3 de la Respuesta de Crecimiento Precoz/metabolismo , Humanos , Melanoma/irrigación sanguínea , Melanoma/metabolismo , Ratones , Ratones Endogámicos C57BL , Factores de Transcripción NFATC/metabolismo , Arteria Pulmonar/citología , Factor de Respuesta Sérica/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Neoplasias Cutáneas/irrigación sanguínea , Neoplasias Cutáneas/metabolismo , Trombina/metabolismo , Trombina/farmacología , Activación Transcripcional/efectos de los fármacos , Activación Transcripcional/fisiología , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Venas Umbilicales/citología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/farmacologíaRESUMEN
L type amino acid transporter 1 (LAT1) is an attractive molecular target for cancer therapy because of its overexpression in many cancer cells. JPH203, a selective LAT1 inhibitor, causes amino acid deprivation and suppresses cancer cell proliferation. However, several cancer cells showed resistance to amino acid deprivation. In this study, we aimed to elucidate the molecular mechanism of different sensitivity between 2 breast cancer cells to anti-LAT1 therapy. MDA-MB-231 cells were more resistant to growth suppression effect of JPH203 than T-47D cells (IC50 was 200 ± 12.5 µM for MDA-MB-231, and 5 ± 1.1 µM for T-47D cells; p < 0.05). Transcriptome and biochemical analysis were done in these cells in the presence/absence of JPH203. JPH203 induced intracellular amino acid deprivation stress in both cells, but it upregulated cystathionine γ lyase (CTH), an enzyme for synthesis of antioxidants, only in MDA-MB-231 cells. Moreover, siRNA-mediated CTH knockdown induced oxidative stress in response to JPH203 leading to decreased cell viability in MDA-MB-231 cells. These results suggest that activation of anti-oxidation pathways in response to amino acid deprivation confers resistance to anti-LAT1 therapy.
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Aminoácidos/efectos de los fármacos , Benzoxazoles/farmacología , Cistationina gamma-Liasa/metabolismo , Transportador de Aminoácidos Neutros Grandes 1/efectos de los fármacos , Tirosina/análogos & derivados , Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cistationina gamma-Liasa/genética , Femenino , Técnicas de Silenciamiento del Gen/métodos , Humanos , ARN Interferente Pequeño , Tirosina/farmacologíaRESUMEN
Endothelial cells (ECs) are phenotypically heterogeneous, mainly due to their dynamic response to the tissue microenvironment. Vascular endothelial cell growth factor (VEGF), the best-known angiogenic factor, activates calcium-nuclear factor of activated T cells (NFAT) signaling following acute angiogenic gene transcription. Here, we evaluate the global mapping of VEGF-mediated dynamic transcriptional events, focusing on major histone-code profiles using chromatin immunoprecipitation sequencing (ChIP-seq). Remarkably, the gene loci of immediate-early angiogenic transcription factors (TFs) exclusively acquire bivalent H3K4me3-H3K27me3 double-positive histone marks after the VEGF stimulus. Moreover, NFAT-associated Pax transactivation domain-interacting protein (PTIP) directs bivalently marked TF genes transcription through a limited polymerase II running. The non-canonical polycomb1 variant PRC1.3 specifically binds to and allows the transactivation of PRC2-enriched bivalent angiogenic TFs until conventional PRC1-mediated gene silencing is achieved. Knockdown of these genes abrogates post-natal aberrant neovessel formation via the selective inhibition of indispensable bivalent angiogenic TF gene transcription. Collectively, the reported dynamic histone mark landscape may uncover the importance of immediate-early genes and the development of advanced anti-angiogenic strategies.
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Inductores de la Angiogénesis/metabolismo , Genes Inmediatos-Precoces/genética , Histonas/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Inmunoprecipitación de Cromatina , Secuenciación de Inmunoprecipitación de Cromatina , Células Endoteliales/metabolismo , Epigénesis Genética/genética , Silenciador del Gen/fisiología , Humanos , Ratones , Neovascularización Patológica/genética , Regiones Promotoras Genéticas/genéticaRESUMEN
Vascular endothelial growth factor receptor 1 (VEGFR1) is a marker for endothelial-specific gene expression. We previously reported that the human VEGFR1 promoter (between -748 and +284) contains information for expression in the intact endothelium of transgenic mice. The objective of this study was to dissect the cis-regulatory elements underlying VEGFR1 promoter activity in vitro and in vivo. In primary endothelial cells, binding sites for E74-like factor 1 (ELF-1; between -49 and -52), cyclic adenosine monophosphate response element binding (CREB; between -74 and -81), and early growth response factor 1/3 (EGR-1/3; between -16 to -25) were shown to play a positive role in gene transcription, whereas a putative E26 transformation-specificsequence (ETS) motif between -36 and -39 had a net negative effect on promoter activity. When targeted to the Hprt locus of mice, mutations of the ELF-1 binding site and the CRE element reduced promoter activity in the embryonic vasculature and resulted in a virtual loss of expression in adult endothelium. Postnatally, the EGR binding site mutant displayed significantly reduced promoter activity in a subset of vascular beds. In contrast, mutation of the -39 ETS site resulted in increased LacZ staining in multiple vascular beds. Together, these results provide new insights into the transcriptional regulatory mechanisms of VEGFR1.
Asunto(s)
Proteína de Unión a CREB/metabolismo , Factores de Transcripción de la Respuesta de Crecimiento Precoz/metabolismo , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas/genética , Factores de Transcripción/metabolismo , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genética , Animales , Secuencia de Bases , Sitios de Unión/genética , Sitios de Unión/fisiología , Células Cultivadas , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Proteína 3 de la Respuesta de Crecimiento Precoz/metabolismo , Células Endoteliales/citología , Células Endoteliales/metabolismo , Femenino , Regulación de la Expresión Génica/fisiología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Datos de Secuencia Molecular , Mutación , Unión Proteica , Homología de Secuencia de Ácido NucleicoRESUMEN
Endothelial phenotypes are highly regulated in space and time by both transcriptional and post-transcriptional mechanisms. There is increasing evidence that the GATA family of transcription factors function as signal transducers, coupling changes in the extracellular environment to changes in downstream target gene expression. Here we show that human primary endothelial cells derived from large blood vessels express GATA2, -3, and -6. Of these factors, GATA3 was expressed at the highest levels. In DNA microarrays of human umbilical vein endothelial cells (HUVEC), small interfering RNA-mediated knockdown of GATA3 resulted in reduced expression of genes associated with angiogenesis, including Tie2. At a functional level, GATA3 knockdown inhibited angiopoietin (Ang)-1-mediated but not vascular endothelial cell growth factor (VEGF)-mediated AKT signaling, cell migration, survival, and tube formation. In electrophoretic gel mobility shift assays and chromatin immunoprecipitation, GATA3 was shown to bind to regulatory regions within the 5'-untranslated region of the Tie2 gene. In co-immunoprecipitation and co-transfection assays, GATA3 and the Ets transcription factor, ELF1, physically interacted and synergized to transactivate the Tie2 promoter. GATA3 knockdown blocked the ability of Ang-1 to attenuate vascular endothelial cell growth factor stimulation of vascular cell adhesion molecule-1 expression and monocytic cell adhesion. Moreover, exposure of human umbilical vein endothelial cells to tumor necrosis factor-alpha resulted in marked down-regulation of GATA3 expression and reduction in Tie2 expression. Together, these findings suggest that GATA3 is indispensable for Ang-1-Tie2-mediated signaling in large vessel endothelial cells.
Asunto(s)
Factor de Transcripción GATA3/fisiología , Regulación de la Expresión Génica , Receptor TIE-2/biosíntesis , Angiopoyetina 1/metabolismo , Células Endoteliales/citología , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Factor de Transcripción GATA3/metabolismo , Humanos , Inmunoprecipitación , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Regiones Promotoras Genéticas , ARN Interferente Pequeño/metabolismo , Receptor TIE-2/química , Factor de Necrosis Tumoral alfa/metabolismo , Venas Umbilicales/citología , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
TNF-α is a pleiotropic cytokine that has the potential to induce apoptosis under inflammation. How endothelial cells (ECs) are spared from this fate in inflammatory environments where TNF-α is present is not known. Here, we show that TGF-ß-activated kinase 1 (TAK1) ensures EC survival and maintains vascular integrity upon TNF-α stimulation. Endothelial-specific TAK1 knockout mice exhibit intestinal and liver hemorrhage due to EC apoptosis, leading to vascular destruction and rapid death. This EC apoptosis was induced by TNF-α from myeloid cells responding to intestinal microbiota. TNF-α secretion associated with inflammation also induced vascular defects in inflamed organs. Additionally, we determined that TAK1 deletion in tumor ECs resulted in blood vessel and hence tumor regression. Our results illuminate mechanisms ensuring survival of intestinal and liver ECs under physiological conditions and ECs of other organs under inflammatory conditions that could be exploited for anti-angiogenic therapy to treat cancer.
Asunto(s)
Células Endoteliales/patología , Hepatocitos/citología , Inflamación/patología , Quinasas Quinasa Quinasa PAM/metabolismo , Animales , Apoptosis/fisiología , Ratones Transgénicos , Transducción de Señal/fisiologíaRESUMEN
The generation of new blood vessels via angiogenesis is critical for meeting tissue oxygen demands. A role for adult stem cells in this process remains unclear. Here, we identified CD157 (bst1, bone marrow stromal antigen 1) as a marker of tissue-resident vascular endothelial stem cells (VESCs) in large arteries and veins of numerous mouse organs. Single CD157+ VESCs form colonies in vitro and generate donor-derived portal vein, sinusoids, and central vein endothelial cells upon transplantation in the liver. In response to injury, VESCs expand and regenerate entire vasculature structures, supporting the existence of an endothelial hierarchy within blood vessels. Genetic lineage tracing revealed that VESCs maintain large vessels and sinusoids in the normal liver for more than a year, and transplantation of VESCs rescued bleeding phenotypes in a mouse model of hemophilia. Our findings show that tissue-resident VESCs display self-renewal capacity and that vascular regeneration potential exists in peripheral blood vessels.
Asunto(s)
ADP-Ribosil Ciclasa/metabolismo , Antígenos CD/metabolismo , Células Progenitoras Endoteliales/metabolismo , Homeostasis , Regeneración , Animales , Biomarcadores/metabolismo , Vasos Sanguíneos/metabolismo , Linaje de la Célula , Ensayo de Unidades Formadoras de Colonias , Células Progenitoras Endoteliales/citología , Células Progenitoras Endoteliales/trasplante , Células Progenitoras Endoteliales/ultraestructura , Factor VIII/metabolismo , Proteínas Ligadas a GPI/metabolismo , Hígado/citología , Hígado/fisiología , Ratones Endogámicos C57BLRESUMEN
Three-dimensional (3D) in vitro microvasculature in a polydimethylsiloxane-based microdevice was developed as a physiologically relevant model of angiogenesis. The angiogenic process is monitored using stage-top optical coherence tomography (OCT). OCT allows non-invasive monitoring of the 3D structures of the prepared host microvasculature and sprouted neovasculature without fluorescence staining. OCT monitoring takes only a few minutes to scan through the several-millimetre scale range, which provides the advantage of rapid observation of living samples. The obtained OCT cross-sectional images capture 3D features of the angiogenic sprouting process and provide information on the dynamics of luminal formation. The stage-top system used in this study enables the observer to visualize the in vitro dynamics of 3D cultured cells simply and conveniently, offering an alternative monitoring method for studies on angiogenesis and providing quantitative information about vascular morphological changes.
Asunto(s)
Microvasos/diagnóstico por imagen , Neovascularización Patológica/diagnóstico por imagen , Neovascularización Fisiológica , Tomografía de Coherencia Óptica , Humanos , Imagenología Tridimensional/métodos , Neovascularización Patológica/metabolismo , Tomografía de Coherencia Óptica/métodos , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
The vascular barrier is an important function of the endothelium and its dysfunction is involved in several diseases. The barrier function of the endothelial cell monolayer is governed by cell-cell, cell-extracellular matrix (cell-ECM) contacts, and inflammatory factors such as thrombin, histamine or vascular endothelial growth factor. Several in vivo and in vitro assays that measure the vascular permeability induced by these factors have been developed. However, they suffer limitations such as being challenging for assessing details of biological processes at a cellular level or lacking the architecture of a vessel, that raise the need for new methods. In vitro 3D model-based assays have thus been developed but assays for investigating compounds that protects the barrier function are lacking. Here we describe the development of an in vitro three-dimensional (3D) vascular endothelium model in which we can manipulate the endothelial barrier function and permeability to molecules, which have a molecular weight similar to human serum albumin, allowing to assess the protective effect of compounds. A microvessel was prepared by culturing human umbilical vein endothelial cells (HUVECs) within a collagen gel on a polydimethylsiloxane (PDMS) chip. Using fluorescein isothiocyanate (FITC)-conjugated dextran (70 kDa, FITC-dextran) and confocal fluorescence microscopy, we showed that the microvessel presented an effective barrier function. We were then able to induce the loss of this barrier function by treatment with the inflammatory factor thrombin. The loss of barrier function was quantified by the extravasation of FITC-dextran into collagen matrix. Furthermore, we were able to analyze the protective effect on the endothelial barrier function of the cyclic adenosine monophosphate (cAMP) analog, 8-pCPT-2'-O-Me-cAMP (also called 007). In an attempt to understand the effects of thrombin and 007 in our model, we analyzed the adherens junctions and cytoskeleton through immunostaining of the vascular endothelial cadherin and actin, respectively. Our assay method could be used to screen for compounds modulating the barrier function of endothelial cells, as well as investigating mechanistic aspects of barrier dysfunction.