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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.
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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.
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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.
Assuntos
Glucocorticoides , Sêmen , Masculino , Camundongos , Animais , Espermatogênese , Espermátides , EstrogêniosRESUMO
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.
Assuntos
Proteínas F-Box , Histonas , Proliferação de Células , Células Endoteliais/metabolismo , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Histonas/metabolismo , Humanos , Histona Desmetilases com o Domínio Jumonji/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Lisina/metabolismo , Fatores de Transcrição/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
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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Indutores da Angiogênese/metabolismo , Genes Precoces/genética , Histonas/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Imunoprecipitação da Cromatina , Sequenciamento de Cromatina por Imunoprecipitação , Células Endoteliais/metabolismo , Epigênese Genética/genética , Inativação Gênica/fisiologia , Humanos , Camundongos , Neovascularização Patológica/genética , Regiões Promotoras Genéticas/genéticaRESUMO
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.
Assuntos
Células Endoteliais/patologia , Hepatócitos/citologia , Inflamação/patologia , MAP Quinase Quinase Quinases/metabolismo , Animais , Apoptose/fisiologia , Camundongos Transgênicos , Transdução de Sinais/fisiologiaRESUMO
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.
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ADP-Ribosil Ciclase/metabolismo , Antígenos CD/metabolismo , Células Progenitoras Endoteliais/metabolismo , Homeostase , Regeneração , Animais , Biomarcadores/metabolismo , Vasos Sanguíneos/metabolismo , Linhagem da Célula , Ensaio de Unidades Formadoras de Colônias , Células Progenitoras Endoteliais/citologia , Células Progenitoras Endoteliais/transplante , Células Progenitoras Endoteliais/ultraestrutura , Fator VIII/metabolismo , Proteínas Ligadas por GPI/metabolismo , Fígado/citologia , Fígado/fisiologia , Camundongos Endogâmicos C57BLRESUMO
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.
Assuntos
Células Endoteliais/metabolismo , Epigênese Genética , Fator de Transcrição GATA2/genética , Histonas/genética , Células-Tronco Embrionárias Murinas/metabolismo , Proteína Proto-Oncogênica c-ets-1/genética , Fatores de Transcrição SOXF/genética , Animais , Diferenciação Celular , Linhagem da Célula/genética , Células Endoteliais/citologia , Fator de Transcrição GATA2/antagonistas & inibidores , Fator de Transcrição GATA2/metabolismo , Histonas/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Análise de Sequência com Séries de Oligonucleotídeos , Cultura Primária de Células , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteína Proto-Oncogênica c-ets-1/antagonistas & inibidores , Proteína Proto-Oncogênica c-ets-1/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Fatores de Transcrição SOXF/antagonistas & inibidores , Fatores de Transcrição SOXF/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
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.
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Microvasos/diagnóstico por imagem , Neovascularização Patológica/diagnóstico por imagem , Neovascularização Fisiológica , Tomografia de Coerência Óptica , Humanos , Imageamento Tridimensional/métodos , Neovascularização Patológica/metabolismo , Tomografia de Coerência Óptica/métodos , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
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.
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Sistemas de Transporte de Aminoácidos/metabolismo , Leucina/metabolismo , Neoplasias de Próstata Resistentes à Castração/metabolismo , Receptores Androgênicos/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Humanos , Masculino , Neoplasias de Próstata Resistentes à Castração/patologiaRESUMO
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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Endotélio Vascular/metabolismo , Fatores de Transcrição NFATC/metabolismo , Neovascularização Patológica , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Células COS , Movimento Celular , Chlorocebus aethiops , Técnicas de Cocultura , Células Endoteliais/citologia , Epigênese Genética , Fibroblastos/metabolismo , Estudo de Associação Genômica Ampla , Células HEK293 , Homeostase , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Receptores CXCR4/metabolismo , Transdução de Sinais , Ativação Transcricional , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
Statins exert atheroprotective effects through the induction of specific transcriptional factors in multiple organs. In endothelial cells, statin-dependent atheroprotective gene up-regulation is mediated by Kruppel-like factor (KLF) family transcription factors. To dissect the mechanism of gene regulation, we sought to determine molecular targets by performing microarray analyses of human umbilical vein endothelial cells (HUVECs) treated with pitavastatin, and KLF4 was determined to be the most highly induced gene. In addition, it was revealed that the atheroprotective genes induced with pitavastatin, such as nitric oxide synthase 3 (NOS3) and thrombomodulin (THBD), were suppressed by KLF4 knockdown. Myocyte enhancer factor-2 (MEF2) family activation is reported to be involved in pitavastatin-dependent KLF4 induction. We focused on MEF2C among the MEF2 family members and identified a novel functional MEF2C binding site 148 kb upstream of the KLF4 gene by chromatin immunoprecipitation along with deep sequencing (ChIP-seq) followed by luciferase assay. By applying whole genome and quantitative chromatin conformation analysis {chromatin interaction analysis with paired end tag sequencing (ChIA-PET), and real time chromosome conformation capture (3C) assay}, we observed that the MEF2C-bound enhancer and transcription start site (TSS) of KLF4 came into closer spatial proximity by pitavastatin treatment. 3D-Fluorescence in situ hybridization (FISH) imaging supported the conformational change in individual cells. Taken together, dynamic chromatin conformation change was shown to mediate pitavastatin-responsive gene induction in endothelial cells.
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Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Cromatina/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Fatores de Transcrição Kruppel-Like/biossíntese , Quinolinas/farmacologia , Cromatina/genética , Regulação da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição MEF2/genética , Fatores de Transcrição MEF2/metabolismo , Óxido Nítrico Sintase Tipo III/biossíntese , Óxido Nítrico Sintase Tipo III/genética , Elementos de Resposta , Trombomodulina/biossíntese , Trombomodulina/genéticaRESUMO
BACKGROUND: Synergistic transcriptional activation by different stimuli has been reported along with a diverse array of mechanisms, but the full scope of these mechanisms has yet to be elucidated. RESULTS: We present a detailed investigation of hypoxia-inducible factor (HIF) 1 dependent gene expression in endothelial cells which suggests the importance of crosstalk between the peroxisome proliferator-activated receptor (PPAR) ß/δ and HIF signaling axes. A migration assay shows a synergistic interaction between these two stimuli, and we identify angiopoietin-like 4 (ANGPTL4) as a common target gene by using a combination of microarray and ChIP-seq analysis. We profile changes of histone marks at enhancers under hypoxia, PPARß/δ agonist and dual stimulations and these suggest that the spatial proximity of two response elements is the principal cause of the synergistic transcription induction. A newly developed quantitative chromosome conformation capture assay shows the quantitative change of the frequency of proximity of the two response elements. CONCLUSIONS: To the best of our knowledge, this is the first report that two different transcription factors cooperate in transcriptional regulation in a synergistic fashion through conformational change of their common target genes.
Assuntos
Angiopoietinas/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , PPAR delta/metabolismo , PPAR beta/metabolismo , Elementos de Resposta , Proteína 4 Semelhante a Angiopoietina , Angiopoietinas/genética , Hipóxia Celular , Cromatina/química , Cromatina/genética , Montagem e Desmontagem da Cromatina , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , PPAR delta/genética , PPAR beta/genéticaRESUMO
The premetastatic niche is a predetermined site of metastases, awaiting the influx of tumor cells. However, the regulation of the angiogenic switch at these sites has not been examined. Here, we demonstrate that the calcineurin and nuclear factor of activated T cells (NFAT) pathway is activated specifically in lung endothelium prior to the detection of tumor cells that preferentially metastasize to the lung. Upregulation of the calcineurin pathway via deletion of its endogenous inhibitor Dscr1 leads to a significant increase in lung metastases due to increased expression of a newly identified NFAT target, Angiopoietin-2 (ANG2). Increased VEGF levels specifically in the lung, and not other organ microenvironments, trigger a threshold of calcineurin-NFAT signaling that transactivates Ang2 in lung endothelium. Further, we demonstrate that overexpression of DSCR1 or the ANG2 receptor, soluble TIE2, prevents the activation of lung endothelium, inhibiting lung metastases in our mouse models. Our studies provide insights into mechanisms underlying angiogenesis in the premetastatic niche and offer targets for lung metastases.
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Calcineurina/metabolismo , Neoplasias Pulmonares/metabolismo , Pulmão/metabolismo , Fatores de Transcrição NFATC/metabolismo , Ribonuclease Pancreático/metabolismo , Animais , Proteínas de Ligação ao Cálcio , Linhagem Celular Tumoral , Células Cultivadas , Endotélio/metabolismo , Endotélio/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Pulmão/patologia , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Receptor TIE-2/genética , Receptor TIE-2/metabolismo , Ribonuclease Pancreático/genética , Transdução de Sinais , Ativação Transcricional , Microambiente TumoralRESUMO
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.
Assuntos
Fator II de Transcrição COUP/química , Fator II de Transcrição COUP/genética , Colesterol 7-alfa-Hidroxilase/antagonistas & inibidores , Colesterol 7-alfa-Hidroxilase/genética , Proteínas de Ligação a DNA/antagonistas & inibidores , Variação Genética , Regiões Promotoras Genéticas , Fator II de Transcrição COUP/metabolismo , Linhagem Celular Tumoral , Colesterol 7-alfa-Hidroxilase/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação Enzimológica da Expressão Gênica , Técnicas de Silenciamento de Genes , Células Hep G2 , Humanos , Ligação Proteica/genética , Estrutura Terciária de Proteína/genéticaRESUMO
Antiangiogenic strategies can be effective for cancer therapy, but like all therapies resistance poses a major clinical challenge. Hypoxia and nutrient starvation select for aggressive qualities that may render tumors resistant to antiangiogenic attack. Here, we show that hypoxia and nutrient starvation cooperate to drive tumor aggressiveness through epigenetic regulation of the histone demethylase JMJD1A (JHDM2A; KDM3A). In cancer cells rendered resistant to long-term hypoxia and nutrient starvation, we documented a stimulation of AKT phosphorylation, cell morphologic changes, cell migration, invasion, and anchorage-independent growth in culture. These qualities associated in vivo with increased angiogenesis and infiltration of macrophages into tumor tissues. Through expression microarray analysis, we identified a cluster of functional drivers such as VEGFA, FGF18, and JMJD1A, the latter which was upregulated in vitro under conditions of hypoxia and nutrient starvation and in vivo before activation of the angiogenic switch or the prerefractory phase of antiangiogenic therapy. JMJD1A inhibition suppressed tumor growth by downregulating angiogenesis and macrophage infiltration, by suppressing expression of FGF2, HGF, and ANG2. Notably, JMJD1A inhibition enhanced the antitumor effects of the anti-VEGF compound bevacizumab and the VEGFR/KDR inhibitor sunitinib. Our results form the foundation of a strategy to attack hypoxia- and nutrient starvation-resistant cancer cells as an approach to leverage antiangiogenic treatments and limit resistance to them.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Histona Desmetilases com o Domínio Jumonji/antagonistas & inibidores , Macrófagos/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Animais , Hipóxia Celular , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Humanos , Histona Desmetilases com o Domínio Jumonji/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/irrigação sanguínea , Neoplasias/patologia , Neovascularização Patológica/etiologia , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidoresRESUMO
Hypoxia-inducible factor 1 (HIF1) is a master regulator of adaptive gene expression under hypoxia. However, a role for HIF1 in the epigenetic regulation remains unknown. Genome-wide analysis of HIF1 binding sites (chromatin immunoprecipitation [ChIP] with deep sequencing) of endothelial cells clarified that HIF1 mainly binds to the intergenic regions distal from transcriptional starting sites under both normoxia and hypoxia. Next, we examined the temporal profile of gene expression under hypoxic conditions by using DNA microarrays. We clarified that early hypoxia-responsive genes are functionally associated with glycolysis, including GLUT3 (SLC2A3). Acetylated lysine 27 of histone 3 covered the HIF1 binding sites, and HIF1 functioned as an enhancer of SLC2A3 by interaction with lysine (K)-specific demethylase 3A (KDM3A). Knockdown of HIF1α and KDM3A showed that glycolytic genes are regulated by both HIF1 and KDM3A and respond to hypoxia in a manner independent of cell type specificity. We elucidated that both the chromatin conformational structure and histone modification change under hypoxic conditions and enhance the expression of SLC2A3 based on the combined results of chromatin conformation capture (3C) and ChIP assays. KDM3A is recruited to the SLC2A3 locus in an HIF1-dependent manner and demethylates H3K9me2 so as to upregulate its expression. These findings provide novel insights into the interaction between HIF1 and KDM3A and also the epigenetic regulation of HIF1.
Assuntos
Hipóxia Celular , Cromatina/metabolismo , Transportador de Glucose Tipo 3/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Sítios de Ligação , Linhagem Celular , Transportador de Glucose Tipo 3/biossíntese , Histonas/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Histona Desmetilases com o Domínio Jumonji/genética , Conformação Proteica , Interferência de RNA , RNA Interferente PequenoRESUMO
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.
Assuntos
Endotélio Vascular/metabolismo , Epigênese Genética/fisiologia , Fator de Transcrição GATA2/metabolismo , Sialoglicoproteínas/genética , Animais , Sequência de Bases , Células COS , Células Cultivadas , Chlorocebus aethiops , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Endotélio Vascular/efeitos dos fármacos , Fator de Transcrição GATA2/genética , Fator de Transcrição GATA2/fisiologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Humanos , Células K562 , Análise em Microsséries , Modelos Biológicos , Especificidade de Órgãos/efeitos dos fármacos , Especificidade de Órgãos/genética , Ligação Proteica/genética , Ligação Proteica/fisiologia , RNA Interferente Pequeno/farmacologia , Sialoglicoproteínas/metabolismoRESUMO
Endothelial cell activation and dysfunction underlie many vascular disorders, including atherosclerosis and inflammation. Here, we show that interleukin-4 (IL-4) markedly induced vascular cell adhesion molecule 1 (VCAM-1), both in cultured endothelial cells and in the intact endothelium in mice. Combined treatment with IL-4 and tumor necrosis factor alpha (TNF-α) resulted in further, sustained induction of VCAM-1 expression. IL-4-mediated induction of VCAM-1 and secondary monocyte adhesion was predominantly regulated by the transcription factor STAT6. Genome-wide survey of IL-4-mediated STAT6 binding from sequential chromatin-immunoprecipitation with deep sequencing (chromatin immunoprecipitation sequencing [ChIP-seq]) in endothelial cells revealed regions of transient and sustained transcription factor binding. Through the combination of DNA microarrays and ChIP-seq at the same time points, the majority of IL-4-responsive genes were shown to be STAT6 dependent and associated with direct STAT6 binding to their promoter. IL-4-mediated stable binding of STAT6 led to sustained target gene expression. Moreover, our strategy led to the identification of a novel functionally important STAT6 binding site within 16 kb upstream of the VCAM-1 gene. Taken together, these findings support a critical role for STAT6 in mediating IL-4 signal transduction in endothelial cells. Identification of a novel IL-4-mediated VCAM-1 enhancer may provide a foundation for targeted therapy in vascular disease.
Assuntos
Interleucina-4/metabolismo , Regiões Promotoras Genéticas , Sequências Reguladoras de Ácido Nucleico , Fator de Transcrição STAT6/metabolismo , Molécula 1 de Adesão de Célula Vascular/genética , Animais , Sítios de Ligação , Adesão Celular , Imunoprecipitação da Cromatina , Células Endoteliais/metabolismo , Endotélio/metabolismo , Expressão Gênica , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Transdução de Sinais , Transcrição Gênica , Fator de Necrose Tumoral alfa/metabolismo , Molécula 1 de Adesão de Célula Vascular/biossíntese , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
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.