Thus, these results show that although p65 can bind to the NF-B site within this region of the ICAM-1 promoter, when presented as an oligonucleotide, such as in the EMSA (Fig. Stockman DL, Hornick JL, Deavers MT, Lev DC, Lazar AJ, Wang WL. provided reagents and contributed to scientific discussion; J.C.M. Accordingly, the expression of ERG in 22Rv1 and C4-2B cells co-cultured with HUVEC was determined, respectively. Immunoprecipitated DNA was analyzed by qPCR for primers covering the NF-B site and EBS in cIAP2 (D, panel iii), or IL-8 (panel iv) (see supplemental Fig. ERG transcription factor as an immunohistochemical marker for vascular endothelial tumors and prostatic carcinoma. Endothelial-to-mesenchymal transition (EndMT), in which ECs acquire mesenchymal properties, has been described for a wide range of pathologies, including cancer. Target Gene TF Knock-Method Tissue Type Biosample Name Fold Change Log2FC P_Value Corrected _P Dataset ID; GPR128: HNF1A: shRNA: Liver: HuH7: .26791-1.90017--DataSet_01_002 ERG knockdown causes spontaneously cardiac fibrosis and dysfunction. Cross sectioning through neovessels (right) shows localization of the tracers. Cooperation of liver cells in health and disease. National Library of Medicine Han E, Kim J, Jung MJ, Chin S, Lee JH, Won KY, Moon A. Int J Clin Exp Pathol. government site. provided funding, conceived, designed, and supervised the study, interpreted results, and wrote the manuscript. https://doi.org/10.1038/s44161-022-00128-3, Cooperative ETS transcription factors enforce adult endothelial cell fate and cardiovascular homeostasis. All graphical data are SEM, To investigate the role of ERG in physiological and pathological postnatal angiogenesis, floxed. Mod Pathol. (2006), The principal eosinophil peroxidase product, HOSCN, is a uniquely potent phagocyte oxidant inducer of endothelial cell tissue factor activity. Numerous studies have shown that the new vasculature induced by VEGF invivo, to promote revascularization in ischemic diseases (. We evaluated 57 CNS tumors, including glioblastomas (GBMs) and hemangioblastomas (HBs), as well as two arteriovenous malformations and four samples of normal brain tissue with immunohistochemistry using a specific ERG rabbit monoclonal antibody. Embryonic Pericytes Promote Microglial Homeostasis and Their Effects on Neural Progenitors in the Developing Cerebral Cortex. To confirm that Erg repression of NF-B activation is a common mechanism in resting EC, we focused on two genes: cellular inhibitor of apoptosis (cIAP)-2 and IL-8. For normalization, tubulin was used as a cytoplasmic control and HDAC1 as a nuclear marker (n= 3). Nature Cardiovascular Research This article describes the possibility of direct reprogramming of non-vascular cells into endothelial cells using ETV2, ERG and FLI1 transcription factors. 2022 Sep 16;23(18):10848. doi: 10.3390/ijms231810848. These data indicate that Erg binds to the ICAM-1 promoter in a region detected by the R4 primers, 188 to 103 bp upstream of the transcription start site. Tight control of NF-B activation is crucial to cellular homeostasis, and several mechanisms, both at the transcriptional and non-transcriptional levels, have been identified. 5B). https://doi.org/10.1182/blood-2007-08-105346, https://doi.org/10.1038/s41569-019-0176-3, https://doi.org/10.1038/s41467-017-01169-0, 81470516 and 81530012/National Natural Science Foundation of China, 81700254/National Natural Science Foundation of China, 2018YFC1311300/National Key R&D Program of China, 2042018kf1032/Fundamental Research Funds for the Central Universities, 2042017kf0085/Fundamental Research Funds for the Central Universities, 2016ZX-008-01/Development Center for Medical Science and Technology National Health and Family Planning Commission of the People's Republic of China (The prevention and control project of cardiovascular disease). This is likely to be the result of endothelial activation by proinflammatory stimuli, because Erg levels have been shown to decrease upon LPS or TNF- stimulation (9, 13). Overlapping and divergent signaling pathways of N-cadherin and VE-cadherin in endothelial cells. Statistical significance was determined by using unpaired two-tailed Students t test. The role of EBS 118 in Erg repression of ICAM-1 activity, however, is less clear. The role of Erg as a repressor of inflammation is in contrast to that of other ETS factors, which have previously been shown to act synergistically with NF-B in promoting inflammatory gene expression. Endothelial cell (EC) plasticity in pathological settings has recently been recognized as a driver of disease progression. C, ICAM-1 promoter activity of EBS mutants after AdErg treatment, expressed as luciferase activity relative to AdLacZ (n = 37). Gene set enrichment analysis (GSEA) overlap studies were carried out using GSEA software version 2.0 (Broad Institute). 2C). Here, we applied CoBATCH for single-cell epigenomic tracing of dynamic EC lineage histories in five mouse organs from development to ageing. Addition of an anti-Erg antibody resulted in the appearance of a supershift (Fig. Chimeric decoy oligonucleotides containing a combination of EBS and NF-B consensus sequences were able to inhibit inflammation in abdominal aortic aneurysms to a greater level than either site alone (53). This site needs JavaScript to work properly. Results show additional HLA-DPB1 polymorphism in exons 1, 3, 4 and 5 and the 5' and 3'-UTR. NF-B activity was inhibited using an adenovirus expressing a mutant super repressor version of IB (AdIBSR), which cannot be phosphorylated and degraded, resulting in sequestration of NF-B in the cytoplasm (14). Takeuchi H, Hashimoto N, Kitai R, Kubota T, Kikuta K. J Neurosurg. FEV contains an ETS DNA binding domain, which is highly homologous to Erg and Fli-1 (48); however, its restricted pattern of expression suggests that FEV is not likely to play a role in transcriptional repression in endothelial cells. Epub 2010 May 24. This smooth anticoagulant surface functions as a selective filter to regulate the passage of gases, fluid, immune cells, and various molecules. There was no recurrence at the 1-year follow up. Interestingly, ERG overexpression in the invivo Matrigel plug model resulted in increased pericyte recruitment to vessels. Please enable it to take advantage of the complete set of features! Mice were administered Tamoxifen (50g per mouse; Sigma) by intraperitoneal injection (IP) at postnatal (P) day 1, P2 and P3. official website and that any information you provide is encrypted Immunoprecipitated DNA was then used as template for quantitative PCR using primers specific for the ICAM-1 promoter, cIAP2 promoter, IL-8 promoter, and the negative control gene GAPDH. 3A, solid arrow), indicating that Erg binds to the EBS 118 site. The first study, in pancreatic cancer cells, investigated genes up-regulated (at least 1.5-fold) by TNF- treatment that were inhibited by an IKK inhibitor, therefore regulated by NF-B (17). Interestingly, similar angiogenic defects are observed in the retinas from. Erg interacts with the histone-3 lysine-9 specific-methyltranferase Erg-associated protein with a SET domain (ESET), and ESET binds co-repressors HDAC-1 and -2, and mSin3A and -B (42, 43). Constitutive Wnt signaling activation caused by mutations in -catenin or genes that control -catenin stability has been associated with aberrant cell proliferation and subsequent cancer progression (reviewed in. All graphical data are SEM, Pericyte recruitment is a critical step in vascular stability and maturation, and lack of pericytes has been shown to cause increased permeability (. A potential mechanism for thrombosis in eosinophilic inflammatory states, Regulation of the human P-selectin promoter by Bcl-3 and specific homodimeric members of the NF-B/Rel family, Xue J., Thippegowda P. B., Hu G., Bachmaier K., Christman J. W., Malik A. Because EMSA studies have shown that Erg can bind to this site, we investigated the possibility that, in resting EC, Erg might block NF-B p65 binding to the ICAM-1 promoter at EBS 181. (1995), Regulation of cell type-specific interleukin-2 receptor -chain gene expression. Clipboard, Search History, and several other advanced features are temporarily unavailable. Primers for a downstream region within the Fzd4 gene were used as a negative control. 3A, lane 4, arrowhead) and a decrease in the intensity of a complex (Fig. Professor Nancy Hogg (Cancer Research UK) for the kind gift of the anti-ICAM-1 antibody, clone 15.5. Conversely, -SMA immunoreactivity was identified in the abluminal cells of these hyperplastic vessels. Figure 7.. EVI of different CNS lesions, plotted with SD. A., Taborsky G. J., Jr., Chansky H. A. ERG knockdown causes spontaneously cardiac fibrosis and dysfunction. ERG silence in HUVECs promotes the secretion of endothelin-1, which in turn activates cardiac fibroblasts in a paracrine manner. The query dataset were the 1138 genes identified as being up-regulated following a 48-h Erg inhibition in HUVEC (16), which were then compared against all the studies in the C2 curated gene sets. Taken together, these data suggest that Erg repression involves two EBS, 118 and 181 bp upstream of the transcription start site and either of these sites is sufficient for Erg-mediated repression. C57BL/6 mice received a subcutaneous injection of Matrigel (BD Biosciences), as described (, Primary mouse lung EC were isolated from control, Primary HUVEC were harvested from umbilical cords (. The supershift was specific, as it was competed off by an excess of unlabeled probe, but not by unlabeled probe containing a mutation of the EBS 118 (Fig. a: H & E demonstrates glomeruloid microvascular proliferation. In the ICAM-1 promoter, the EBS 118 and the NF-B site are 70 bp apart, which suggest they are approximately half a nucleosome apart. S1S3. In conclusion, this study provides evidence of the crucial role Erg plays in maintaining a quiescent state in endothelial cells. FOIA Accessibility is a recipient of a DOC-fFORTE fellowship of the Austrian Academy of Sciences at the London Research Institute. We show that in quiescent EC Erg prevents NF-B p65 binding to DNA, suggesting that Erg may compete with p65 for DNA binding. Inactivation of AR/TMPRSS2-ERG/Wnt signaling networks attenuates the aggressive behavior of prostate cancer cells. FEV expression is restricted to prostate and small intestine tissue (47) and Dami megakaryocytic cells (27). The new PMC design is here! 3C); however, NF-B p65 was enriched in R4 after treatment with TNF-, as expected (34) (Fig. 1 The transcriptional mechanisms regulating EC lineage identity and maintenance of endothelial homeostasis are also areas of immense interest for vascular regenerative therapies but remain poorly understood. 2014, Received: 2010 Aug;113(2):218-24. doi: 10.3171/2009.10.JNS08631. ERG is abundant in murine hearts, especially in cardiac ECs, but decreased during fibrotic remodeling. Analysis of oligonucleotide screening and ChIP sequencing (ChIP Seq) data has suggested a possible specific Erg consensus motif of (A/C)GGAA(G/A) (28) or AGGA(A/T)(G/A) (29). and JavaScript. Values are presented as means SEM. In support of the specificity of the interaction of Erg with this site, addition of control IgG antibodies had no effect on the band pattern (Fig. Location of qPCR amplicon covering region R1 is indicated. 2001;161:III-XIII, 1-151. doi: 10.1007/978-3-642-56553-3. 2007; 114: 97109. Immunohistochemistry analysis revealed endothelial cells stained positive for ERG, D2-40 (podoplanin) and negative for WT1, calretinin. Deletion of either VEGF-A or VEGFR2 in endothelial cells regulates levels of Dll4 in tip cells and, in turn, Notch inhibition provides a feedback loop that reinforces expression of VEGF-A and C-X-C chemokine receptor type 4 (CXCR4), which stimulate endothelial sprouting and proliferation in the expanding vascular plexus [13 ]. To further define the role of ERG in regulating EC function, we evaluated the effect of ERG knockdown on EC lumen formation in 3D collagen matrices. 3C). 3A, lane 1, arrows), two of which were competed by excess unlabeled probe but not by a probe containing a mutation in the EBS 118 site, indicating specificity for this site. Values are represented as the fold change in relative luciferase activity over the empty pGL4 vector alone. volume1,pages 880881 (2022)Cite this article. 2B). Angiogenesis of glioma: evaluation of ultrastructural characteristics of microvessels and tubular bodies (Weibel-Palade) in endothelial cells and immunohistochemical findings with VEGF and p53 protein. Optimized fibrin gel bead assay for the study of angiogenesis. You are using a browser version with limited support for CSS. Also, analysis of the genome-wide binding sites of 10 key regulators of blood stem/progenitor cells identified a combinatorial interaction between a heptad of transcription factors including Erg (29). Dysfunctional ERG signaling drives pulmonary vascular aging and persistent fibrosis. Circ. In this study we investigate the mechanisms used by Erg to repress inflammatory gene expression in quiescent EC, focusing on ICAM-1 as a model gene. Previously, using an ICAM-1 promoter luciferase construct containing the first 1.3 kb upstream of the ICAM-1 transcription start site, we have shown that Erg represses ICAM-1 promoter activity in resting EC (12). -. They constitute a heterogeneous cell population in the human body. (F) Representative images of B16F0 tumors which were grown for 14days on adult. Federal government websites often end in .gov or .mil. (1990), The ETS domain. This showed that Erg binds to the promoter of cIAP2. CD31 demonstrated variable and sometimes weak immunoreactivity for endothelial cells. This study aims at investigating the potential role and molecular basis of ERG in fibrotic remodeling within the adult heart. -, Semenza GL Targeting HIF-1 for cancer therapy. (1997), ETS1, NFB, and AP1 synergistically transactivate the human GM-CSF promoter, Gri G., Savio D., Trinchieri G., Ma X. Miettinen M, Wang ZF, Paetau A, Tan SH, Dobi A, Srivastava S, Sesterhenn I. 2019;16(8):491502. Dev. Essential roles of a variant NF-B site and p65 homodimers, Birdsey G. M., Dryden N. H., Shah A. V., Hannah R., Hall M. D., Haskard D. O., Parsons M., Mason J. C., Zvelebil M., Gottgens B., Ridley A. J., Randi A. M. (2012), The transcription factor Erg regulates expression of histone deacetylase 6 and multiple pathways involved in endothelial cell migration and angiogenesis, Zhang Y., Gavriil M., Lucas J., Mandiyan S., Follettie M., Diesl V., Sum F. W., Powell D., Haney S., Abraham R., Arndt K. (2008), IB kinase inhibitor IKI-1 conferred tumor necrosis factor sensitivity to pancreatic cancer cells and a xenograft tumor model, Sana T. R., Janatpour M. J., Sathe M., McEvoy L. M., McClanahan T. K. (2005), Microarray analysis of primary endothelial cells challenged with different inflammatory and immune cytokines, Barish G. D., Yu R. T., Karunasiri M., Ocampo C. B., Dixon J., Benner C., Dent A. L., Tangirala R. K., Evans R. M. (2010), Bcl-6 and NF-B cistromes mediate opposing regulation of the innate immune response, Prasad D. D., Rao V. N., Reddy E. S. (1992), Jeong B. C., Kim M. Y., Lee J. H., Kee H. J., Kho D. H., Han K. E., Qian Y. R., Kim J. K., Kim K. K. (2006), Brain-specific angiogenesis inhibitor 2 regulates VEGF through GABP that acts as a transcriptional repressor, Okada Y., Yano K., Jin E., Funahashi N., Kitayama M., Doi T., Spokes K., Beeler D. L., Shih S. C., Okada H., Danilov T. A., Maynard E., Minami T., Oettgen P., Aird W. C. (2007), A 3-kb fragment of the human Robo4 promoter directs cell type-specific expression in endothelium, Voraberger G., Schfer R., Stratowa C. (1991), Cloning of the human gene for intercellular adhesion molecule 1 and analysis of its 5-regulatory region. This article demonstrates the role of ERG in the control of vascular gene expression through epigenetic regulation of super-enhancers. Using EMSA, we investigated whether, in resting HUVEC, p65 interacts with the NF-B site at 188, which overlaps with EBS 181. Careers. The identification of key mediators to regain control of EC homeostasis has great potential for the development of novel therapeutics. Images are a single representation of at least 3 separate experiments. Disclaimer, National Library of Medicine Here we describe a novel mechanism that controls the activation of NF-B in EC. The https:// ensures that you are connecting to the 2022 Dec;58:102513. doi: 10.1016/j.redox.2022.102513. Endothelial Fli1 deficiency impairs vascular homeostasis: a role in scleroderma vasculopathy. (2002), Molecular cloning of ESET, a novel histone H3-specific methyltransferase that interacts with ERG transcription factor, Yang L., Mei Q., Zielinska-Kwiatkowska A., Matsui Y., Blackburn M. L., Benedetti D., Krumm A. From 2008 to 2016, a total of $36 946 764.00 USD has . EBS 834 and 907, suggested to have a role in H2O2-mediated activation of the ICAM-1 promoter (24), do not appear to have a role in Erg-mediated repression. B, ICAM-1 promoter activity of EBS mutants after Erg Genebloc treatment. MicroRNA-147a Targets SLC40A1 to Induce Ferroptosis in Human Glioblastoma. By inhibiting the activity of constitutive low levels of nuclear NF-B, Erg represses the transactivation of a set of proinflammatory NF-B target genes. Before Rafii, S., Butler, J. M. & Ding, B.-S. Angiocrine functions of organ-specific endothelial cells. Erg is a crucial regulator of endocardial-mesenchymal transformation during cardiac valve morphogenesis. Published by Elsevier Inc. We use cookies to help provide and enhance our service and tailor content. Accepted: Internet Explorer). An official website of the United States government. 1E). is a recipient of a National Lung and Heart Institute Foundation Studentship. If you don't remember your password, you can reset it by entering your email address and clicking the Reset Password button. (A) 3D rendering of confocal microscopy images of whole-mount Matrigel plugs perfused with the dextran tracers. To confirm the specificity for Erg binding at this site, ChIP was carried out on chromatin from HUVEC treated with Erg siRNA. Erg enrichment at R4, which includes EBS 118 and EBS 181, was greater than at other regions containing EBS (Fig. Copyright 2022 Elsevier Inc. except certain content provided by third parties. 124, 13371349 (2019). Chen R, Cao C, Liu H, Jiang W, Pan R, He H, Ding K, Meng Q. Redox Biol. Specificity was measured by addition of saturating amounts of competing oligonucleotide (competitor) or competing oligonucleotide with a mutation in the EBS 118 (A) or 181 (B) (M-competitor). The endothelial transcription factor ERG promotes vascular stability and growth through Wnt/-catenin signaling. (D and E) (D) qPCR and (E) western blot analysis of Fzd4 expression in control and ERG-deficient cells (n= 3). Loss of endothelial CFTR drives barrier failure and edema formation in lung infection and can be targeted by CFTR potentiation. These data suggest that Erg may be important in maintaining endothelial quiescence and in the termination of the inflammatory response, by preventing the induction of proinflammatory gene expression. Immunoprecipitated DNA was analyzed by qPCR for primers covering ICAM-1 promoter regions (R) 35 (C) or 4 (D) and negative control region. 2008;111(7):3498506. Endothelial cell-derived endothelin-1 promotes cardiac fibrosis in diabetic hearts through stimulation of endothelial-to-mesenchymal transition. In the presence of bFGF (, Matrigel containing bFGF or VEGF with adenovirus expressing either Lacz (Ad.Lacz) or ERG (Ad.ERG) was injected into C57BL6 mice. 21 in view of the widespread expression of the gm-csf receptor in the cerebral parenchyma, including the microglia, ependymal cells, choroid plexus cells, neurons, and endothelial cells, 18, 3D Reconstruction of Neovessels inside Matrigel Plugs Supplemented with VEGF and Adenovirus Expressing ERG, Related to Figure6. Immunoprecipitated DNA was analyzed by qPCR for primers covering ICAM-1 promoter regions 15 (D) or region 4 only (E) and negative control region. Blood. Cell 151, 559575 (2012). A role for Erg is identified as a gatekeeper in quiescent endothelial cells to inhibit basal NF-B activity, thus providing an important barrier to protect against inappropriate endothelial activation. E ndothelial cells (EC) have many functions and play a central role in the control of coagulation, thrombolysis, vascular tone, permeability, inflammation, tissue repair, and angiogenesis. Efficient direct reprogramming of mature amniotic cells into endothelial cells by ETS factors and TGF suppression. We selected the following ETS factors, all expressed in resting HUVEC, although at lower levels than Erg (supplemental Fig. Accessibility Intriguingly, ERG knockdown in human umbilical vein endothelial cells (HUVECs) promoted the secretion of endothelin-1 (ET-1), which subsequently accelerated the proliferation, phenotypic transition, and collagen synthesis of cardiac fibroblasts in a paracrine manner. The .gov means its official. siRNA treatment to inhibit Erg, Fli1, and Ets2 expression was carried out using Hs_ERG_7, Hs_FLI1_7, Hs_ETS2_7, and Hs_GAPB_10 FlexiTube siRNA (Qiagen), respectively, and AllStars Negative Control siRNA (Qiagen). HHS Vulnerability Disclosure, Help Thus, the ETS and GATA factors . Our results show that ERG is a novel, reliable, and specific marker for endothelial cells within CNS tumors that can be used to better study the process of neovascularization. The role of wnt signaling in physiological and pathological angiogenesis. contributed to scientific discussion; H.G. A. Notably, stimulation of ECs with Toll-like receptor 3 ligand poly(I:C) suppressed ERG expression and induced ERG dissociation from the IFNB1 promoter, while promoting signal transducers and activators . a: H & E demonstrates a vascular lumen. -, Louis DN Ohgaki H Wiestler OD Cavenee WK Burger PC Jouvet A Scheithauer BW Kleihues P The 2007 WHO classification of tumours of the central nervous system. Macrophage Sprouty4 deficiency diminishes sepsis-induced acute lung injury in mice. To obtain FZD4 as a mediator of ERG oncogene-induced WNT signaling and epithelial-to-mesenchymal transition in human prostate cancer cells. Interestingly, inhibition of Erg expression in quiescent EC results in increased NF-B-dependent ICAM-1 expression, indicating that Erg represses basal NF-B activity. September 24, Data are shown as fold change over IgG (n= 3). Nat Commun. (BD) (B) Representative images of EC sprouts on fibrin gel beads using siCtrl or siERG-treated HUVEC in the presence or absence of LiCl; (C) quantification of numbers of sprouts; and (D) tube length (n= 20). Acute myeloid leukemia with complex karyotypes and abnormal chromosome 21: amplification discloses overexpression of APP, ETS2, and ERG genes. and transmitted securely. The following day, cells were transduced with 100 multiplicity of infection of IB Super Repressor Adenovirus (AdIBSR) (14) or AdLacZ in serum-free M199 medium for 2 h before replacing with complete M199 medium. Oligonucleotides used in this study are described in supplemental Table S1. provided advice and contributed to scientific discussion; R.H.A. The LNCaP-androgen dependent cell line was cultured and passaged 60 times over 16 months. ETS-related gene (ERG) is a transcription factor that has been linked to angiogenesis. 8600 Rockville Pike 2020 Aug 1;319(2):H443-H455. (2003), An ERG (ets-related gene)-associated histone methyltransferase interacts with histone deacetylases 1/2 and transcription co-repressors mSin3A/B, De Val S., Chi N. C., Meadows S. M., Minovitsky S., Anderson J. P., Harris I. S., Ehlers M. L., Agarwal P., Visel A., Xu S. M., Pennacchio L. A., Dubchak I., Krieg P. A., Stainier D. Y., Black B. L. (2008), Combinatorial regulation of endothelial gene expression by Ets and Forkhead transcription factors, Buchwalter G., Gross C., Wasylyk B. The double mutant EBS 96/118, shown previously to have a role in both transactivation and repression of ICAM-1 by ETS factors in rabbit kidney RK13 cells and HEK 293 cells (2527), also showed no increase in promoter activity after Erg inhibition; however, this is likely to occur through EBS 118, as mutation of EBS 96 alone was responsive to Erg inhibition. Nuclear lysates from a confluent monolayer of HUVEC were extracted using the Nuclear extract kit (Active Motif) following the manufacturer's instructions. We then carried out ChIP to test whether Erg binds to the promoter of cIAP2 in resting HUVEC, using primers designed around the Erg consensus sequence near NF-B sites (Fig. Cell sorting and proliferation assays performed after sustained ERG knockdown indicate that ERG drives proliferation and blocks the differentiation of prostate cells to both NE and luminal cell types. EIF5 EIF6 ELF2 ELK1 ELK3 EMC8 EME1 EMG1 ENKD1 ENO3 ENTR1 ENY2 EOGT EP400P1 EPB41 EPB41L1 EPB41L2 EPB41L4A EPB41L5 EPHA2 EPHB4 EPS8L2 ERC1 ERCC1 ERG ERH ERMN ERVFRD-1 ESRRG ETFB ETHE1 ETNPPL ETS1 ETV3 ETV4 ETV5 EVI2A EXOC3 EXOC6 EXOSC1 EXOSC3 EXOSC7 F12 FAAH2 FAAP100 . Alternatively, after 42 h following adenovirus transduction, cells were treated with 10 ng/ml of TNF- for 6 h. ICAM-1 mRNA levels were assessed by quantitative RT-PCR, normalized to GAPDH. Liu J, Zhuang T, Pi J, Chen X, Zhang Q, Li Y, Wang H, Shen Y, Tomlinson B, Chan P, Yu Z, Cheng Y, Zheng X, Reilly M, Morrisey E, Zhang L, Liu Z, Zhang Y. ERG is a novel and reliable marker for endothelial cells in central nervous system tumors Authors Matthew A Haber , Amir Iranmahboob , Cheddhi Thomas , Mengling Liu , Amanda Najjar , David Zagzag PMID: 25881913 PMCID: PMC4542182 DOI: 10.5414/NP300817 Abstract ETS-related gene (ERG) is a transcription factor that has been linked to angiogenesis. The site is secure. (D and E) (D) Western blot and (E) qPCR analysis of -catenin expression in control (siCtrl) and ERG-deficient (siERG) HUVEC (n= 4). Interestingly, this resulted in a significant increase in NF-B p65 binding to R4 of the ICAM-1 promoter (Fig. Perfused vessels are labeled with FITC-dextran (green) and vessel leakage is visualized with TRITC-dextran (red). [Cancer Letters] Full Article Published In Prostate Cell News December 2, 2022 Zhou N, Ye Y, Wang X, Ma B, Wu J, Li L, Wang L, Wang DW, Zou Y. J Mol Med (Berl). We then carried out a separate GSEA between the whole Erg dataset and these two studies. Cell 32, 8296 (2015). ERG can serve as a reliable marker of immature myeloid cells in cases of extramedullary hematopoiesis, bone marrow trephine biopsies and in adrenal myelolipomas ( Am J Surg Pathol 2011;35:432, Hum Pathol 2022;124:1 ) Reliably identifies the presence of ERG fusions in Ewing sarcomas ( Mod Pathol 2012;25:1378, Genes Chromosomes Cancer 2016;55:340 ) PGA-PTX-E-[c (RGDfK)2] inhibited the growth of avb3-expressing endothelial cells and several cancer cells. First, we showed that Erg inhibition in HUVEC significantly increased the mRNA levels of cIAP2 (Fig. Epub 2017 Jan 13. Hypoxia is implicated in loss of retinal ganglion cells (RGCs) occurring in such conditions. It has been recently shown that the ETS factor ERG interacts with endothelial-specific genes, including vWF . This pro-fibrotic effect was also negated by RGD (Arg-Gly-Asp)-peptide magnetic nanoparticles target delivery of ET-1 small interfering RNA to ECs in mice. Epub 2021 Nov 13. To confirm that ERG controls angiogenesis and vascular development in a Wnt/-catenin-dependent manner invivo, we carried out a rescue experiment by pharmacological stabilization of Wnt/-catenin signaling (. Embryos were harvested at E10.5 and yolk sac vasculature was analyzed by light microscopy and immunostaining. FOIA B., Tiruppathi C. (2009), NF-B regulates thrombin-induced ICAM-1 gene expression in cooperation with NFAT by binding to the intronic NF-B site in the ICAM-1 gene, Pierce J. W., Schoenleber R., Jesmok G., Best J., Moore S. A., Collins T., Gerritsen M. E. (1997), Novel inhibitors of cytokine-induced IB phosphorylation and endothelial cell adhesion molecule expression show anti-inflammatory effects, Subramanian A., Tamayo P., Mootha V. K., Mukherjee S., Ebert B. L., Gillette M. A., Paulovich A., Pomeroy S. L., Golub T. R., Lander E. S., Mesirov J. P. (2005), Gene set enrichment analysis. (A) Invitro Brdu incorporation in control and ERG-deficient HUVEC treated in presence or absence of LiCl (n= 4). 2022 Jan 19;42(3):362-376. doi: 10.1523/JNEUROSCI.1201-21.2021. 3A, lane 7), confirming that Erg specifically interacts with the oligonucleotide containing EBS 118. Dustri-Verlag Dr. Karl Feistle GmbH & Co. KG. Little is known about the maintenance of endothelial cell fate in adults. a: H & E demonstrates pseudopalisading cells surrounding areas of central necrosis,, Figure 3.. GBM, glomeruloid type. Scale bar, 20m. . Using bioinformatic analysis of transcriptome profiling datasets and validation by ChIP, we show that this mechanism is common to other proinflammatory genes and we identify a specific subset of NF-B target genes repressed by Erg in quiescent endothelial cells. 8600 Rockville Pike Accessibility Advances knowledge of these transcription factors, in terms of structure, function . n = 5. No supershifted band was visible after the addition of the anti NF-B p65 antibody. D and E, ChIP was carried out using an anti-Erg or control IgG antibody on sheared chromatin from confluent resting HUVEC (D) or HUVEC treated with Erg or control siRNA (E). Epub 2014 Oct 28. Briefly, pGL4 ICAM-1 1.3 plasmid was amplified using a primer designed to mutate a specific EBS from GGAA to CCAA or the NF-B site from TTGGAAATTCC to TTCTAGATTAG. Cell proliferation was determined invitro using a BrdU proliferation ELISA kit (Roche) according to the manufacturers instructions. The data presented so far show that the transcription factor ERG controls angiogenesis through pathways mediating vascular stability and growth. 7): Fli-1, the ETS factor with the closest homology to Erg; Ets-2, a more distantly related ETS factor known to interact with Erg (20); and GABP, which can act as an activator or repressor of transcription (21, 22). Circulation. A, and B, biotinylated oligonucleotides containing sequences from the ICAM-1 promoter EBS 118 (A) or the EBS 181 (B) were incubated with nuclear lysate from resting HUVEC. and K.H.D. Hypoxia-ischemia induces the expression of hypoxia inducible factor-1 and its target genes such as vascular endothelial growth factor (VEGF) and nitric oxide synthase (NOS). Data are expressed as fold-change compared with IgG normalized to input and control region (n = 8). This modality has been successfully applied to destruct ERG, a TF overexpressed in 50% of both primary and metastatic prostate cancer , and LEF1, another cancer-related TF involved in migration and invasion, with potent efficacy in cultured cells. Healthy EC maintain homeostasis through a dynamic balance between expression of protective genes and repression of proinflammatory genes. This article underscores the importance of ERG. (H) mRNA expression of Erg, -catenin, and its target genes Cyclin D1, Axin-2, and TCF-1 in primary, (I) qPCR analysis of total brain mRNA from control and, (J) GSEA shows enrichment and significant correlation (normalized enrichment score, 2.46; p< 0.001) between genes downregulated in -catenin siRNA-treated HPAEC (green curve) (. 1C). Res. In this study, we investigated the miRNA expression changes . 3B), in fact p65 does not bind to this same sequence in native chromatin from unstimulated HUVEC. 2022 Feb;102(2):204-211. doi: 10.1038/s41374-021-00698-z. Disruption of PPAR/-catenin-mediated regulation of apelin impairs BMP-induced mouse and human pulmonary arterial EC survival. Virchows Archiv. Get the most important science stories of the day, free in your inbox. ( C) Endothelial cells ERG positive. Please enter a term before submitting your search. and ancillary testing results (attenuation of rods, and later cones, on the full-field electroretinogram (ERG) mainstay of diagnosis and constriction of the visual field on Goldmann perimetry (see image) . The ETS transcription factor family is implicated in vascular development and angiogenesis (reviewed in. 12 was mutated within ETS binding sites (EBS), or within the NF-B binding site as previously shown (15), using the QuikChange lightning multi site-directed mutagenesis kit (Agilent), all primers were designed using the QuikChange Primer Design Program (Agilent). Learn more Combined genomic and antisense analysis reveals that the transcription factor Erg is implicated in endothelial cell differentiation. designed and carried out invitro experiments, analyzed, interpreted, and conceptualized results, and wrote the manuscript. These cells capture visual information and ultimately respond to light . We demonstrate that Erg binds to two ETS binding sites (EBS) in the ICAM-1 promoter and we show that both EBS and NF-B consensus sites are required for the repressive activity of Erg. In the retinal vasculature of littermate controls ( Ergfl/fl ), ERG was strongly expressed in endothelial cells (EC) from all regions of the vascular plexus, including tip and stalk cells, arteries, veins, and capillaries ( Figure S1 K), in line with previous studies ( Korn et al., 2014 ). ERG is a critical regulator of Wnt/LEF1 signaling in prostate cancer. ERG knockdown within murine hearts caused spontaneously cardiac fibrosis and dysfunction, accompanied by the activation of multiple Smad-dependent and independent pathways. (2010), Genome-wide analysis of ETS family DNA-binding, Wilson N. K., Foster S. D., Wang X., Knezevic K., Schtte J., Kaimakis P., Chilarska P. M., Kinston S., Ouwehand W. H., Dzierzak E., Pimanda J. E., de Bruijn M. F., Gttgens B. 2014 Apr;27(4):496-501. doi: 10.1038/modpathol.2013.161. granulocyte-macrophage colony-stimulating factor (gm-csf) is produced in diverse cns cells under pathological conditions, 18 - 20 and it can cross the bbb. (G and H) Panels show endomucin staining of blood vessels in B16F0 tumors and the quantification of the number of endomucin-positive vessels, (n= 6), scale bar, 50m. Differences were considered significant with a p value< 0.05. (G) qPCR of downstream -catenin target gene expression in control and ERG-deficient HUVEC: Cyclin D1, Axin-2, and TCF-1 (n= 4). The https:// ensures that you are connecting to the C and D, ChIP was carried out on sheared chromatin from confluent resting HUVEC TNF (10 ng/ml for 30 min) (C), or HUVEC treated with Erg or control siRNA (D) using an anti-NF-B p65 or control IgG antibody. Provided by the Springer Nature SharedIt content-sharing initiative, Nature Cardiovascular Research (Nat Cardiovasc Res) (D) Vascular density of isolectin B4 stained branches in the central plexus, scale bar, 50m; quantification (n= 6). B, Erg or control Genebloc-transfected HUVEC were treated with BAY-11-7085 or dimethyl sulfoxide (DMSO) control. Gene Regulatory Network of ETS Domain Transcription Factors in Different Stages of Glioma. A knowledge-based approach for interpreting genome-wide expression profiles, Subramanian A., Kuehn H., Gould J., Tamayo P., Mesirov J. P. (2007), GSEA-P, a desktop application for Gene Set Enrichment Analysis, Mootha V. K., Lindgren C. M., Eriksson K. F., Subramanian A., Sihag S., Lehar J., Puigserver P., Carlsson E., Ridderstrle M., Laurila E., Houstis N., Daly M. J., Patterson N., Mesirov J. P., Golub T. R., Tamayo P., Spiegelman B., Lander E. S., Hirschhorn J. N., Altshuler D., Groop L. C. (2003), PGC-1-responsive genes involved in oxidative phosphorylation are coordinately down-regulated in human diabetes, Furusu A., Nakayama K., Xu Q., Konta T., Sugiyama H., Kitamura M. (2001), Expression, regulation, and function of inhibitor of apoptosis family genes in rat mesangial cells, Strieter R. M., Kunkel S. L., Showell H. J., Remick D. G., Phan S. H., Ward P. A., Marks R. M. (1989), Endothelial cell gene expression of a neutrophil chemotactic factor by TNF-, LPS, and IL-1, Payankaulam S., Li L. M., Arnosti D. N. (2010), Transcriptional repression. Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project. B. However, the direct silence of ERG in cardiac fibroblasts did not affect the expression of fibrotic markers. Therefore both genes are candidates for regulation by both Erg and NF-B. This indicates a correlation between NF-B pathway target genes in endothelial cells and genes up-regulated by Erg inhibition in HUVEC. INTRODUCTION HISTORY Results are expressed as fold-change compared with IgG normalized to input and negative control region. ERG and FLI1, induces EndMT coupled with dynamic . endothelial junctions are crucial for the maintenance and regulation of vascular homeostasis and function and mediate a complex signaling network. ISSN 2731-0590 (online). 2021 Jan 1;14(1):116-125. eCollection 2021. The second study investigated the response of microvascular and macrovascular endothelial cells to TNF-, interferon , or IL-4 (18). Federal government websites often end in .gov or .mil. An official website of the United States government. Comparative genomic analysis of the Fzd4 promoter revealed the presence of three highly conserved ERG DNA binding motifs in the 800 base pair (bp) region upstream of the Fzd4 transcription start site (, Wnt/-catenin signaling can promote EC proliferation (. (2006), Endoglin expression in the endothelium is regulated by Fli-1, Erg, and Elf-1 acting on the promoter and a 8-kb enhancer, McLaughlin F., Ludbrook V. J., Kola I., Campbell C. J., Randi A. M. (1999), Characterization of the tumor necrosis factor (TNF)- response elements in the human ICAM-2 promoter, Birdsey G. M., Dryden N. H., Amsellem V., Gebhardt F., Sahnan K., Haskard D. O., Dejana E., Mason J. C., Randi A. M. (2008), Transcription factor Erg regulates angiogenesis and endothelial apoptosis through VE-cadherin, Deramaudt B. M., Remy P., Abraham N. G. (1999), Up-regulation of human heme oxygenase gene expression by Ets family proteins, Sperone A., Dryden N. H., Birdsey G. M., Madden L., Johns M., Evans P. C., Mason J. C., Haskard D. O., Boyle J. J., Paleolog E. M., Randi A. M. (2011), The transcription factor Erg inhibits vascular inflammation by repressing NF-B activation and proinflammatory gene expression in endothelial cells, Yuan L., Nikolova-Krstevski V., Zhan Y., Kondo M., Bhasin M., Varghese L., Yano K., Carman C. V., Aird W. C., Oettgen P. (2009), Anti-inflammatory effects of the ETS factor ERG in endothelial cells are mediated through transcriptional repression of the interleukin-8 gene, Brockman J. Over the last decade, major progress has been made in understanding the molecular mechanisms that regulate angiogenesis. official website and that any information you provide is encrypted A number of these EBS have been shown to play a role in ICAM-1 expression, including two AP1-EBS repeats involved in ICAM-1 induction after H2O2 stimulation (24), and two EBS that are involved in ICAM-1 expression in non-endothelial cells (2527). Bookshelf (C) Western blot (left) and quantification (right) of -catenin expression in nuclear/cytoplasmic fractions of ERG-deficient HUVEC transduced with GFP or VEC-GFP adenovirus in presence or absence of LiCl. There were two labeled dextran molecules of different molecular weights, 210. Suppressing ET-1 with either a neutralizing antibody or a receptor blocker abolished ERG knockdown-mediated deleterious effect in vivo and in vitro. Chromatin was enriched with an NF-B p65 antibody and binding sites were analyzed by quantitative PCR, using the ICAM-1 promoter primers described above (see Fig. Am J Surg Pathol. 3B, lanes 46, arrows), suggesting that the anti-NF-B p65 antibody binds to the protein-oligonucleotide complex at this site. The endothelial transcription factor ERG promotes vascular stability and growth through Wnt/-catenin signaling. This site is also responsible for the repression of ICAM-1 by the ETS factor FEV. 15 a major regulator of adherent junctions is vascular endothelial (ve)-cadherin, a ca 2+ -dependent cell-surface adhesion molecule that forms homophilic interactions and is required for the integrity Smad-dependent and Smad-independent pathways in TGF-beta family signalling. HUVEC were isolated and cultured in supplemented M199 media as previously described (10). 3D). Epub 2013 Sep 27. 43 the absence of. (F) Quantification of yolk sac vitelline vessel diameter. One of these sites (EBS 181) is located within the consensus binding site for NF-B. [BMC Molecular and Cell Biology] Full Article Published In Prostate Cell News Transcription factor Erg regulates angiogenesis and endothelial apoptosis through VE-cadherin. Additionally, we mutated the NF-B site at 188, previously identified as important for cytokine-mediated up-regulation of ICAM-1 (15) (Fig. Proc Natl Acad Sci U S A. We observed significantly higher quantitative expression of ERG in HBs than in other CNS tumors. Interestingly, not all the genes identified by the GSEA comparison of Erg and NF-B-dependent datasets were repressed through the mechanisms identified here (data not shown), suggesting that other pathways may be involved in Erg-mediated repression. These results indicate that, as with ICAM-1, Erg inhibits constitutive binding of nuclear NF-B p65 to the promoters of IL-8 and cIAP2. Document S1. Birdsey, G. M. et al. See this image and copyright information in PMC. Background: In quiescent endothelial cells, the transcription factor Erg regulates cell homeostasis by repressing expression of proinflammatory genes. Sequencing was carried out to confirm mutation of the desired EBS. In this study, Erg was shown to interact with two EBS in the ICAM-1 promoter, and both were found to be required for Erg repression of ICAM-1 expression. EBS 181 is part of an NF-B consensus sequence that is bound by NF-B p65 after TNF- stimulation in HUVEC (15, 30). Dev. 2010 Jun 8;121(22):2407-18. doi: 10.1161/CIRCULATIONAHA.110.938217. The transcription factor Erg, the most highly expressed ETS member in resting EC, controls quiescence by repressing proinflammatory gene expression. 2 erg has been recently studied immunohisto-chemically in prostate carcinoma, subsets of which have oncogenic tmprss2-erg gene fusions Article This consensus sequence appears in the ICAM-1 promoter five times, at 1239, 1136, 773, 118, and 75 relative to the transcription start site (Fig. . Abstract:Endothelial cells (ECs) across ages and tissues are highly heterogeneous in developmental origins, structures, functions, and cellular plasticity. Thus Erg provides a checkpoint to protect endothelial cells against inappropriate activation, and may prevent the onset of chronic inflammatory vascular diseases, such as atherosclerosis. A recent example is provided by BCL6, which was shown to repress a subset of NF-B target genes in macrophages by binding the promoters at a distance equivalent to approximately one nucleosome or 200 bp away from NF-B binding sites (19). In resting endothelial cells (EC),2 NF-B is sequestered in the cytoplasm by proteins of the inhibitor of NF-B (IB) family. The different expression levels and activities of ETS factors highlights their important role in regulating inflammation. Loss of ERG expression is associated with diseases including atherosclerosis. The recent years have witnessed an increased activity in biocompatibility research aimed at limiting biomaterial-induced blood coagulation. The following day, siRNA (10 nm) was mixed with AtuFect01 lipid (1 g/ml, Silence Therapeutics) at 5 times concentration in Opti-MEM (Invitrogen), then added to cells for 24 or 48 h. Erg overexpression was carried out using a V5-tagged Erg-3 adenovirus (AdErg), as described previously (12). ChIP on resting HUVEC treated with control or Erg siRNA showed that NF-B p65 binding to the IL-8 and cIAP2 promoters increases significantly after Erg inhibition (Fig. Results are expressed as luciferase activity relative to control Genebloc-treated cells. This may allow Erg-mediated repression through modification of the nucleosomal structure of the ICAM-1 promoter. (D, panels iii and iv) ChIP was carried out on sheared chromatin from HUVEC treated with control or Erg siRNA, using an anti NF-B p65 or control IgG antibody. After 24 h, cells were transfected with Erg or control siRNA as described above. Epub 2020 Jul 3. MB), Help with a: H & E demonstrates a vascular lumen. G.M.B. performed experiments and analyzed results. We show that constitutive deletion of endothelial ERG in the mouse embryo causes embryonic lethality with severe vascular disruption. Would you like email updates of new search results? Acta Neuropathol. (F) There are three putative ERG binding sites (black bars) located within the Fzd4 locus upstream of the transcription start site (arrow); sequence conservation between 100 vertebrates is shown across this region. Previous studies have demonstrated that Erg binds to an EBS in the IL-8 promoter (13). This is a preview of subscription content, access via your institution, Get immediate online access to Nature and 55 other Nature journal. 2022 Jul 25;13(1):4170. doi: 10.1038/s41467-022-31890-4. Epub 2019 Jun 10. Typically only the nuclei are visible, at the boundary between the lumen and the wall of a vessel. Showing cell line RNA expression of ERG (erg-3, p55). Endothelial ERG overexpression prevents pressure overload-induced cardiac fibrosis. We first tested whether NF-B p65 could bind to the region containing the NF-B site and EBS 181 within the native chromatin structure of resting HUVEC, using ChIP. Transcription cofactor HES-6 is a protein that in humans is encoded by the HES6 gene. OMIM: 610331 MGI: 1859852 HomoloGene: 23111 GeneCards: HES6. It is possible that some of the genes may be indirect targets of Erg; genomic ChIP sequencing of Erg DNA binding profiles will address this question. To update your cookie settings, please visit the. Androgen receptor levels identification were detected in qRT-PCR and Western blot assay. Am J Physiol Heart Circ Physiol. Depending on the tissue and organ, endothelial cells can vary from each other. ERG deficiency results in vessel regression and reduced pericyte recruitment, demonstrating that ERG controls vascular stability. LiCl or NaCl (400mg/kg, dissolved in water) was injected IP into pregnant female mice at E8.5 and E9.5. Erg is the most highly expressed ETS factor in resting EC (7), where it acts as an activator of genes involved in homeostasis, including endoglin (8), ICAM-2 (9), vascular endothelial-cadherin (10), and heme oxygenase-1 (11). This review highlights the adaptability of endothelial cells to support the function of each organ, while maintaining their endothelial cell fate. (F) Quantification of pericyte coverage, pixel intensity (n= 8). 1C). pdf files, http://creativecommons.org/licenses/by/3.0/, Redistribute or republish the final article, Reuse portions or extracts from the article in other works. Results: Erg represses NF-B p65 . Briefly, biotinylated double-stranded oligonucleotides containing the ICAM-1 promoter sequence surrounding and including EBS 118 and 181 were incubated with 2 g of HUVEC nuclear lysate with or without excess unbiotinylated oligonucleotides. The The transcription factor Erg is essential for definitive hematopoiesis and the function of adult hematopoietic stem cells. Single cell type. Image, Download Hi-res Sullivan HC, Edgar MA, Cohen C, Kovach CK, HooKim K, Reid MD. Am. In quiescent EC, nuclear-localized NF-B p65 appears to have a role in constitutive expression of genes, such as ICAM-2 and P-selectin, as mutation of the NF-B binding sites within the promoters of these genes results in a decrease in basal activity (9, 33). 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