Overexpression Of ETS2 Research Articles

Discovering therapeutic possibilities for polycystic ovary syndrome by targeting XIST and its associated ceRNA network through the analysis of transcriptome data

Long non-coding RNA (lncRNA) regulates many physiological processes by acting as competitive endogenous RNA (ceRNA). The dysregulation of lncRNA X-inactive specific transcript (XIST) has been shown in various human disorders. However, its role in the pathogenesis of polycystic ovary syndrome (PCOS) is yet to be explored. This study aimed to explore the underlying mechanism of XIST in the pathogenesis of PCOS, specifically through dataset functional analysis. GEO PCOS datasets including RNA-seq, microarray, and miRNA-seq in granulosa cells (GCs) and blood, were examined and comprehensively analyzed. Enrichment analysis, ROC curve constructions, lncRNA-miRNA-mRNA interaction network analyses, and qRT-PCR validation were performed followed by a series of drug signature screenings. Our results revealed significant dysregulation in the expression of 1131 mRNAs, 30 miRNAs, and XIST in GCs of PCOS patients compared to healthy individuals. Of the120 XIST-correlated upregulated genes, 25 were enriched in inflammation-related pathways. Additionally, 5 miRNAs were identified as negative regulators of XIST-correlated genes. Accordingly, a ceRNA network containing XIST-miRNAs-mRNAs interactions was constructed. Furthermore, 6 genes, including AQP9, ETS2, PLAU, PLEK, SOCS3, and TNFRSF1B served as both GCs and blood-based biomarkers. By analyzing the number of interactions among XIST, miRNAs, and mRNAs, we pinpointed ETS2 as the pivotal gene within the ceRNA network. Our findings reveal a novel XIST- hsa-miR-146a-5p, hsa-miR-144-3p, and hsa-miR-1271-5p-ETS2 axis that comprehensively elucidates the XIST-associated mechanism underlying PCOS onset. qRT-PCR analysis further confirmed the, overexpression of both XIST and ETS2 . Furthermore, our results demonstrated that XIST and ETS2 were correlated with some assisted reproductive technologies outcomes. Finally, we identified two novel compounds including, methotrexate/folate and threonine using drug–gene interaction databases for PCOS management. These findings provide novel insights into the molecular etiology, diagnosis, and potential therapeutic interventions for PCOS.

ETS2 overexpression ameliorates cartilage injury in osteoarthritis by the ETS2/miR-155/STAT1/DNMT1 feedback loop pathway

Osteoarthritis (OA) is the most common irreversible chronic joint dysfunction disease, which is pathologically characterized by disturbance of articular cartilage homeostasis leading to subsequent inflammatory response and cartilage extracellular matrix (ECM) degradation. Increasing evidence has demonstrated the dysregulation of transcription factors play crucial roles in the occurrence and development of osteoarthritis (OA), but the potential functions and mechanism of most transcription factors in OA has not been completely illuminated. In this study, we identified that transcription factor V-ets erythroblastosis virus E26 oncogene homolog 2 (ETS2) was significantly down-regulated in OA cartilage and IL-1β-induced OA chondrocytes. Functional experiments in vitro demonstrated that the overexpressed ETS2 strikingly enhanced proliferation, outstandingly suppressed apoptosis, and dramatically reduced inflammation and ECM degradation in IL-1β-induced OA chondrocytes, whereas the knockdown of ETS2 led to the opposite effects. Further in vivo studies have shown that up-regulated ETS2 dramatically ameliorates cartilage injury in DMM-induced OA mice. Mechanical studies have disclosed that DNMT1-mediated downregulation of ETS2 dramatically promotes STAT1 by inhibiting miR-155 transcription, and increased STAT1 initiates a feedback loop that may enhance DNMT1-mediated hypermethylation of ETS2 to inhibit ETS2 expression, thus forming a DNMT1/ETS2/miR-155/STAT1 feedback loop that inhibits MAPK signaling pathways and aggravates OA cartilage injury. In all, our results revealed that overexpression of ETS2 markedly ameliorated OA cartilage injury through the ETS2/miR-155/STAT1/DNMT1 feedback loop, providing a new perspective on the pathogenesis and therapeutic strategies for OA.

LncRNA DLEU2 promotes gastric cancer progression through ETS2 via targeting miR-30a-5p

BackgroundGastric cancer (GC) remains an important cancer worldwide. Further understanding of the molecular mechanisms of gastric carcinogenesis will enhance the diagnosis and treatment of GC.MethodsThe expression of DLEU2 and ETS2 was analyzed in several GC cell lines using GEPIA online analyze, qRT-PCR and immunohistochemistry. The biological behavior of GC cells was detected by CCK8, clone formation, transwell, wound healing, western blot, and flow cytometry assay. More in-depth mechanisms were studied.ResultsDLEU2 was significantly up-regulated in GC tissues and cell lines. The expression of DLEU2 was significantly associated with pathological grading and TNM stage of GC patients. Furthermore, knockdown of DLEU2 inhibited the proliferation, migration, and invasion of AGS and MKN-45 cells, while overexpression of DLEU2 promoted the proliferation, migration, and invasion of HGC-27 cells. MiR-30a-5p could directly bind to the 3’ UTR region of ETS2. Moreover, DLEU2 bound to miR-30a-5p through the same binding site, which facilitated the expression of ETS2. Knockdown of DLEU2 reduced the protein level of intracellular ETS2 and inhibited AKT phosphorylation, while overexpression of DLEU2 induced the expression of ETS2 and the phosphorylation of AKT. ETS2 was highly expressed in GC tissues. The expression of ETS2 was significantly associated with age, pathological grading, and TNM stage. ETS2 overexpression promoted cell proliferation and migration of AGS and MKN-45 cells. Furthermore, ETS2 overexpression rescued cell proliferation and migration inhibition induced by DLEU2 down-regulation and miR-30a-5p up-regulation in AGS and MKN-45 cells.ConclusionsDLEU2 is a potential molecular target for GC treatment.

A Genomic Signature Predicting Venetoclax Treatment Response in Acute Myeloid Leukemia (AML) Identified By Protein Network Mapping and Validated By Ex Vivo Ddrug Sensitivity Testing

A Genomic Signature Predicting Venetoclax Treatment Response in Acute Myeloid Leukemia (AML) Identified By Protein Network Mapping and Validated By Ex Vivo Ddrug Sensitivity Testing

Transcription factor Ets-2 regulates the expression of key lymphotropic factors.

Transcription factor Ets-2 downregulates the expression of cytokine genes and HIV-1 in resting T-cells. Herein, we studied whether Ets-2 regulates the expression of lymphotropic factors (LFs) NFAT2, NF-κΒ/p65, c-Jun, c-Fos, which regulate the activation/differentiation of T-cells, and kinase CDK10, which controls Ets-2 degradation and repression activity. In silico analysis revealed Ets-2 binding sites on the promoters of NFAT2, c-Jun, c-Fos. The T-cell lines Jurkat (models T-cell signaling/activation) and H938 (contains the HIV-1-LTR) were transfected with an Ets-2 overexpressing vector, in the presence/absence of mitogens. mRNA and protein levels were assessed by qPCR and Western immunoblotting, respectively. Ets-2 overexpression in unstimulated Jurkat increased NFAT2 and c-Jun mRNA/protein, c-Fos mRNA and NF-κΒ/p65 protein, and decreased CDK10 protein. In unstimulated H938, Ets-2 upregulated NFAT2, c-Jun and CDK10 mRNA/protein and NF-κΒ/p65 protein. In stimulated Jurkat, Ets-2 increased NFAT2, c-Jun and c-Fos mRNA/protein and decreased CDK10 mRNA/protein. In stimulated H938 Ets-2 increased NFAT2, c-Jun and c-Fos protein and reduced CDK10 protein levels. Furthermore, Ets-2 overexpression modulated the expression of pro- and anti-apoptotic genes in both cell lines. Ets-2 upregulates the expression of key LFs involved in the activation of cytokine genes or HIV-1 in T-cells, either through its physical interaction with gene promoters or through its involvement in signaling pathways that directly impact their expression. The effect of Ets-2 on CDK10 expression in H938 vs Jurkat cells dictates that, additionally to Ets-2 degradation, CDK10 may facilitate Ets-2 repression activity in cells carrying the HIV-1-LTR, contributing thus to the regulation of HIV latency in virus-infected T-cells.

RNA-binding protein CELF1 enhances cell migration, invasion, and chemoresistance by targeting ETS2 in colorectal cancer.

Colorectal cancer (CRC) is often diagnosed at later stages after it has metastasized to other organs. The development of chemoresistance also contributes to a poor prognosis. Therefore, an increased understanding of the metastatic properties of CRC and chemoresistance could improve patient survival. CUGBP elav-like family member 1 (CELF1) is an RNA-binding protein, which is overexpressed in many human malignant tumors. However, the influence of CELF1 in CRC is unclear. V-ets erythroblastosis virus E26 oncogene homologue 2 (ETS2) is an evolutionarily conserved proto-oncogene known to be overexpressed in a variety of human cancers including CRC. In thespresent tudy, we investigated the association between CELF1 and ETS2 in CRC tumorigenesis and oxaliplatin (L-OHP) resistance. We found a positive correlation between the elevated expression of CELF1 and ETS2 in human CRC tissues. Overexpression of CELF1 increased CRC cell proliferation, migration, and invasion in vitro and in a xenograft tumor growth model in vivo, and induced resistance to L-OHP. In contrast, CELF1 knockdown improved the response of CRC cells to L-OHP. Overexpression of ETS2 increased the malignant behavior of CRC cells (growth, migration, and invasion) and L-OHP resistance in vitro. Moreover, L-OHP resistance induced by CELF1 overexpression was reversed by ETS2 knockdown. The results of luciferase reporter and ribonucleoprotein immunoprecipitation assays indicated that CELF1 up-regulates ETS2 by binding to its 3'-UTR. Taken together, our findings have identified that CELF1 regulates ETS2 in a mechanism that results in CRC tumorigenesis and L-OHP resistance, and CELF1 may be a promising target for overcoming chemoresistance in CRC.

Choice of factors and medium impinge on success of ESC to TSC conversion

IntroductionThe first lineage separation in mammalian development occurs when totipotent cells of the zygote give rise to the inner cell mass and the trophectoderm. The lineages are strictly separated by an epigenetic barrier. In vitro derivatives of these lineages embryonic stem cells (ESC) and trophoblast stem cells (TSC) are used to study the requirements needed to overcome the barrier in ESC to TSC conversion approaches. MethodsDifferent combinations of TSC transcription factors were induced in ESC for three days. Cells were kept in TS medium with fetal bovine serum (FBS) or the chemically defined TX medium. Obtained cells were analysed for OCT4 levels, TSC surface marker levels, expression of TSC markers and methylation status of Elf5, Oct4 and Nanog promoters. Further, long-term culture stability and in vitro and in vivo differentiation was tested. ResultsOverexpression of Gata3, Eomes, Tfap2c, Ets2 and Cdx2 in ESC resulted in induction of TSC fate. Overexpression of Cdx2 or four factors (Gata3, Eomes, Tfap2c and Ets2) resulted in complete conversion only when cells were cultured in TX medium. The obtained induced TSC (iTSC) display characteristics of bona fide TSC in terms of marker expression and promoter methylation patterns. The generated converted cells were shown to display self-renewal and to be capable to differentiate into TSC derivatives in vitro and in vivo. ConclusionGata3, Eomes, Tfap2c, Ets2 and Cdx2 overexpression in ESC resulted in stable iTSC fate independent of culture conditions. For four factors or Cdx2 alone, TX medium is required for complete TSC conversion.

EGF suppresses the expression of miR-124a in pancreatic β cell lines via ETS2 activation through the MEK and PI3K signaling pathways.

Diabetes mellitus is characterized by pancreatic β cell dysfunction. Previous studies have indicated that epidermal growth factor (EGF) and microRNA-124a (miR-124a) play opposite roles in insulin biosynthesis and secretion by beta cells. However, the underlying mechanisms remain poorly understood. In the present study, we demonstrated that EGF could inhibit miR-124a expression in beta cell lines through downstream signaling pathways, including mitogen-activated protein kinase kinase (MEK) and phosphatidylinositol 3-kinase (PI3K) cascades. Further, the transcription factor ETS2, a member of the ETS (E26 transformation-specific) family, was identified to be responsible for the EGF-mediated suppression of miR-124a expression, which was dependent on ETS2 phosphorylation at threonine 72. Activation of ETS2 decreased miR-124a promoter transcriptional activity through the putative conserved binding sites AGGAANA/TN in three miR-124a promoters located in different chromosomes. Of note, ETS2 played a positive role in regulating beta cell function-related genes, including miR-124a targets, Forkhead box a2 (FOXA2) and Neurogenic differentiation 1 (NEUROD1), which may have partly been through the inhibition of miR-124 expression. Knockdown and overexpression of ETS2 led to the prevention and promotion of insulin biosynthesis respectively, while barely affecting the secretion ability. These results suggest that EGF may induce the activation of ETS2 to inhibit miR-124a expression to maintain proper beta cell functions and that ETS2, as a novel regulator of insulin production, is a potential therapeutic target for diabetes mellitus treatment.

Ets-2 Acts As a Transcriptional Repressor of the Human Immunodeficiency Virus Type 1 through Binding to a Repressor-Activator Target Sequence of 5'-LTR.

HIV-1 is transcriptionally active in activated T helper (Th)-cells and inactive in naive or resting memory Th-cells. Ets-2 is a preinduction transcriptional repressor of the IL-2 gene in naive Th-cells and a candidate transcriptional repressor of HIV-1 in the same cells, because the −279 to −250 upstream region of HIV-1-LTR [repressor–activator target sequence (RATS)], that participates in HIV-1-LTR transcriptional silencing, encompasses the AAGGAG Ets-2 binding site. In this proof of concept study, we investigated whether Ets-2 represses the expression of HIV-1. To assess whether Ets-2 can repress HIV-1 transcriptional activation acting through RATS, we transfected Jurkat cells with an Ets-2 overexpression plasmid (pCDNA3-ets-2) or Ets-2 silencing plasmids (ets-2-shRNA) and, as target genes, plasmids carrying the whole HIV-1-LTR sequence (HIV-1-LTR-CAT) or two copies of the RATS sequence (2× RATS-CAT) or a point mutation in the Ets-2 binding site (2× mutantRATS-CAT) or CMV-CAT (control). Ets-2 overexpression resulted in a significant reduction of HIV-1-LTR-CAT and 2× RATS-CAT activities in stimulated cells, but not of the 2× mutantRATS-CAT or CMV-CAT. Ets-2 silencing led to increased activities of HIV-1-LTR-CAT and 2× RATS-CAT in unstimulated cells, but had no effect on the activities of 2× mutantRATS-CAT and CMV-CAT. To assess Ets-2 binding to HIV-1-LTR–RATS in naive Th-cells, we isolated naive Th-cell nuclear proteins and passed them through an Ets-2 antibody column; electrophoretic mobility shift assays were performed using an RATS probe mixed with consecutive protein eluates. Ets-2 bound to the HIV-1-LTR–RATS in a dose-dependent manner. To assess Ets-2 binding to RATS in vivo, Jurkat cells were transfected with 2× RATS-CAT and stained for the Ets-2 protein and the RATS sequence by combining immunofluorescence and fluorescence in situ hybridization techniques. In unstimulated cells, Ets-2 bound to RATS, whereas no binding was observed in stimulated cells. To test for RATS specificity, the same experiments were performed with 2× mutantRATS-CAT, and no binding of Ets-2 was observed. The results were corroborated by chromatin immunoprecipitation assays performed with the same cells. Our results show that Ets-2 is a transcriptional repressor of HIV-1. Repression of HIV-LTR-RATS mediated by Ets-2 may account for the low-level transcription and replication of HIV-1 in naive Th-cells, and contribute to the viral latency and maintenance of viral reservoirs in patients, despite long-term therapy.

Protocol for the Direct Conversion of Murine Embryonic Fibroblasts into Trophoblast Stem Cells

Trophoblast stem cells (TSCs) arise as a consequence of the first cell fate decision in mammalian development. They can be cultured in vitro, retaining the ability to self-renew and to differentiate into all subtypes of the trophoblast lineage, equivalent to the in vivo stem cell population giving rise to the fetal portion of the placenta. Therefore, TSCs offer a unique model to study placental development and embryonic versus extra-embryonic cell fate decision in vitro. From the blastocyst stage onwards, a distinct epigenetic barrier consisting of DNA methylation and histone modifications tightly separates both lineages. Here, we describe a protocol to fully overcome this lineage barrier by transient over-expression of trophoblast key regulators Tfap2c, Gata3, Eomes and Ets2 in murine embryonic fibroblasts. The induced trophoblast stem cells are able to self-renew and are almost identical to blastocyst derived trophoblast stem cells in terms of morphology, marker gene expression and methylation pattern. Functional in vitro and in vivo assays confirm that these cells are able to differentiate along the trophoblast lineage generating polyploid trophoblast giant cells and chimerizing the placenta when injected into blastocysts. The induction of trophoblast stem cells from somatic tissue opens new avenues to study genetic and epigenetic characteristics of this extra-embryonic lineage and offers the possibility to generate trophoblast stem cell lines without destroying the respective embryo.

Protocol for the Direct Conversion of Murine Embryonic Fibroblasts into Trophoblast Stem Cells.

Trophoblast stem cells (TSCs) arise as a consequence of the first cell fate decision in mammalian development. They can be cultured in vitro, retaining the ability to self-renew and to differentiate into all subtypes of the trophoblast lineage, equivalent to the in vivo stem cell population giving rise to the fetal portion of the placenta. Therefore, TSCs offer a unique model to study placental development and embryonic versus extra-embryonic cell fate decision in vitro. From the blastocyst stage onwards, a distinct epigenetic barrier consisting of DNA methylation and histone modifications tightly separates both lineages. Here, we describe a protocol to fully overcome this lineage barrier by transient over-expression of trophoblast key regulators Tfap2c, Gata3, Eomes and Ets2 in murine embryonic fibroblasts. The induced trophoblast stem cells are able to self-renew and are almost identical to blastocyst derived trophoblast stem cells in terms of morphology, marker gene expression and methylation pattern. Functional in vitro and in vivo assays confirm that these cells are able to differentiate along the trophoblast lineage generating polyploid trophoblast giant cells and chimerizing the placenta when injected into blastocysts. The induction of trophoblast stem cells from somatic tissue opens new avenues to study genetic and epigenetic characteristics of this extra-embryonic lineage and offers the possibility to generate trophoblast stem cell lines without destroying the respective embryo.

Ets-2 Protein Is a Transcriptional Repressor Of The HIV-1 Virus and Acts Through Binding To The HIV-LTR-RATS Element

HIV-1 is transcriptionally active in activated CD4+ T (Th) cells, and inactive in naive Th cells. Our previous work has shown that Ets-2 is a candidate transcriptional repressor of HIV-1 in naive Th cells, because the -279 to -250 upstream region of HIV-1-LTR (RATS, Repressor Activator Target Sequence), that participates in HIV-1-LTR transcriptional silencing, encompasses the AAGGAG Ets-2 binding site. In this proof of concept study, we investigated whether Ets-2 represses the expression of HIV-1.We measured the levels of ets-2 mRNA in peripheral blood T-cells, Jurkat cells and 10 other human leukemic T, B and monocytic cell lines, before and after activation of the cells with the mitogens PMA and ionomycin (P/I). Ets-2 mRNA was synthesized in T-cells, Jurkat cells and in most cell lines tested when cultured in plain culture medium (CM), but its synthesis was severely reduced upon cell activation.To directly assess whether Ets-2 can silence HIV-1 activation acting through the RATS sequence, we transfected Jurkat cells with (i) the plasmids HIV-1-LTR-CAT (carries the whole LTR sequence of the HIV-1 virus) or 2xRATS-CAT (carries 2 copies of the RATS sequence) or 2xmutantRATS-CAT (carries a point mutation in the Ets-2 binding site) or CMV-CAT (control), (ii) pCDNA3-ets-2 (for ets-2 overexpression) and (iii) ets-2 shRNA (to silence ets-2 expression in the cells). When Jurkat cells were transfected with HIV-1-LTR-CAT, 2xRATS-CAT or CMV-CAT, there was no transcriptional activity of the genes in CM. After activation of the cells, the expression of the transfected genes increased, except of CMV-CAT. Co-transfection of Jurkat cells with increasing amounts of pCDNA3-ets-2, led to a gradual reduction of HIV-1-LTR-CAT and 2xRATS-CAT activities upon cell stimulation, but not of CMV-CAT. No transcriptional response was observed for the 2xmutantRATS-CAT gene. Co-transfection experiments with HIV-1-LTR-CAT, 2xRATS-CAT and ets-2 shRNA, led to an increase in the activity of both reporter genes, but had no effect on the activities of 2xmutantRATS-CAT and CMV-CAT.To assess whether Ets-2 binds to HIV-1 LTR directly in vivo, Jurkat cells were transfected with the plasmid HIV-1-LTR-CAT, left in CM for 24h, and then in CM±P/I for a further 12h. The cells were then attached to slides and stained for the Ets-2 protein and the HIV-1-LTR sequence by combining immunofluorescence and FISH techniques respectively. In CM condition, Ets-2 protein bound to the HIV-1-LTR sequence (Fig. 1A), whereas after P/I activation of the cells [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Telomerase activity and apoptosis genes as parameters of lymphocyte aging in Down syndrome patients

It is hypothesized that Down syndrome (DS) patients are associated with abnormalities of the immune system. Accordingly, this study was conducted to measure replicative aging and apoptosis in lymphocytes, which play an important role in the immune system, before and after being biostimulated with He:Ne laser. Replicative aging was measured in terms of telomerase activity, and ETS-2 gene relative expression. Apoptosis was measured in terms of DNA fragmentation and apoptosis genes (Fas, FasL and Bax) and antiapoptotic Bcl-2 protein. Results showed that Telomerase activity, ETS-2 mRNA expression, plasma DNA fragmentation, Fas and FasL were significantly higher among DS patients compared to controls: Telomerase activity (1.5±0.5 vs. 0.9±0.4, p<0.001); ETS2 mRNA expression (0.6±0.1 vs. 0.43±0.04, p<0.0001); plasma DNA fragmentation (0.45%±0.12 vs. 0.2%±0.1, p<0.0001); Fas protein (5.3±1.2 vs. 2.3±0.2, p<0.0001); FasL mRNA relative expression (0.37±0.05 vs. 0.24±0.01, p<0.001); Bax mRNA relative expression (0.9±0.1 vs. 0.5±0.1, p<0.00001). Bcl-2 protein was significantly low in DS patients compared to controls (8.6±1.3 vs. 10±2.1, p<0.01). He:Ne laser biostimulation applied to evaluate lymphocytes’ response significantly increased the former parameters in DS patients compared to their level before irradiation, except for Bcl-2, which was significantly decreased. In conclusion: increased telomerase activity associated with increased activity and overexpression of ETS-2 on chromosome 21 in DS patients may contribute to the increased rate of early senescence in circulating lymphocytes, which consequently contributes to the abnormalities of the immune system observed in DS. Increased apoptosis is due to increased oxidative stress, which induces an increase in the apoptotic genes Bax, Fas and FasL accompanied by a decrease in the antiapoptotic gene Bcl-2.

Linkage of cardiac gene expression profiles and ETS2 with lifespan variability in rats

Longevity variability is a common feature of aging in mammals, but the mechanisms responsible for this remain largely unknown. Using microarray datasets coupled with prediction analysis of microarrays (PAM), we identified a set of 252 cardiac transcripts predictive of relative lifespan in Wistar and Fisher 344 rats. Prediction analysis of microarrays 'tests' of rat heart transcriptomes from a third longer lived Fisher × Norway Brown rat strain validated the predictive value of this gene subset. The expression patterns of these genes were highly conserved, and corresponding promoter regions were employed to identify common cis-elements and trans-activating factors implicated in their control. Specifically, four transcription factors (Max, Ets2, Erg, and Msx2) present in heart displayed longevity-dependent, strain-independent changes in abundance, but only ETS2 had an expression profile that directly correlated with the relative lifespan gene set. In heart, ETS2 was prevalent in cardiomyocytes (CMs) and showed a high degree of myocyte-to-myocyte variability predominantly in adult rat hearts prior to the exponential increase in the rate of mortality. Exclusively in this group, elevated ETS2 significantly overlapped with TUNEL staining in heart myocytes. In response to sympathetic stimuli, ETS2 is also up-regulated, and functionally, adenovirus-mediated over-expression of ETS2 promotes apoptosis-inducing factor-mediated, caspase-independent programmed necrosis exclusively in CMs that can be fully inhibited by the PARP-1 inhibitor DPQ. We conclude that variations in ETS2 abundance in hearts of adult rodents and the associated loss of CMs contribute at least partially, to the longevity variability observed during normal aging of rats through activation of programmed necrosis.

The manipulation of miRNA-gene regulatory networks by KSHV induces endothelial cell motility

AbstractmiRNAs have emerged as master regulators of cancer-related events. miRNA dysregulation also occurs in Kaposi sarcoma (KS). Exploring the roles of KS-associated miRNAs should help to identify novel angiogenesis and lymphangiogenesis pathways. In the present study, we show that Kaposi sarcoma-associated herpesvirus (KSHV), the etiological agent of KS, induces global miRNA changes in lymphatic endothelial cells (LECs). Specifically, the miR-221/miR-222 cluster is down-regulated, whereas miR-31 is up-regulated. Both latent nuclear antigen (LANA) and Kaposin B repress the expression of the miR-221/miR-222 cluster, which results in an increase of endothelial cell (EC) migration. In contrast, miR-31 stimulates EC migration, so depletion of miR-31 in KSHV-transformed ECs reduces cell motility. Analysis of the putative miRNA targets among KSHV-affected genes showed that ETS2 and ETS1 are the downstream targets of miR-221 and miR-222, respectively. FAT4 is one of the direct targets of miR-31. Overexpression of ETS1 or ETS2 alone is sufficient to induce EC migration, whereas a reduction in FAT4 enhances EC motility. Our results show that KSHV regulates multiple miRNA-mRNA networks to enhance EC motility, which eventually contributes to KS progression by promoting the spread of malignant KS progenitor cells. Targeting KSHV-regulated miRNAs or genes might allow the development of novel therapeutic strategies that induce angiogenesis or allow the treatment of pathogenic (lymph)angiogenesis.

Ets2 Determines the Inflammatory State of Endothelial Cells in Advanced Atherosclerotic Lesions

Neovascularization is required for embryonic development and plays a central role in diseases in adults. In atherosclerosis, the role of neovascularization remains to be elucidated. In a genome-wide microarray-screen of Flk1+ angioblasts during murine embryogenesis, the v-ets erythroblastosis virus E26 oncogene homolog 2 (Ets2) transcription factor was identified as a potential angiogenic factor. We assessed the role of Ets2 in endothelial cells during atherosclerotic lesion progression toward plaque instability. In 91 patients treated for carotid artery disease, Ets2 levels showed modest correlations with capillary growth, thrombogenicity, and rising levels of tumor necrosis factor-α (TNFα), monocyte chemoattractant protein 1, and interleukin-6 in the atherosclerotic lesions. Experiments in ApoE(-/-) mice, using a vulnerable plaque model, showed that Ets2 expression was increased under atherogenic conditions and was augmented specifically in the vulnerable versus stable lesions. In endothelial cell cultures, Ets2 expression and activation was responsive to the atherogenic cytokine TNFα. In the murine vulnerable plaque model, overexpression of Ets2 promoted lesion growth with neovessel formation, hemorrhaging, and plaque destabilization. In contrast, Ets2 silencing, using a lentiviral shRNA construct, promoted lesion stabilization. In vitro studies showed that Ets2 was crucial for TNFα-induced expression of monocyte chemoattractant protein 1, interleukin-6, and vascular cell adhesion molecule 1 in endothelial cells. In addition, Ets2 promoted tube formation and amplified TNFα-induced loss of vascular endothelial integrity. Evaluation in a murine retina model further validated the role of Ets2 in regulating vessel inflammation and endothelial leakage. We provide the first evidence for the plaque-destabilizing role of Ets2 in atherosclerosis development by induction of an intraplaque proinflammatory phenotype in endothelial cells.

Abstract 5611: ETS2 Contributes to a Pro-atherogenic Endothelial Cell Phenotype, Causing Plaque Destabilization in Advanced Atherosclerotic Lesions

Neovascularization is a requirement for embryonic development and plays an important role in the pathogenesis of various diseases in adults. In atherosclerosis, the role of neovascularization remains to be elucidated. Previously, we conducted a genome-wide microarray analysis for vasculogenesis during murine embryonic development. The E 26 T ransformation-specific S equence 2 (ETS2) transcription factor was upregulated in Flk1+ angioblasts and vascular expression of ETS2 was validated by whole mount in situ hybridization in the developing zebrafish. We sought to assess the role of ETS2 on neovascularization during atherosclerotic lesion progression toward plaque instability. We conducted experiments in ApoE−/− mice using a vulnerable plaque (VP) model that creates vulnerable and stable plaques in the common carotid artery by flow alteration. QPCR analysis showed that ETS2 expression is augmented in vulnerable versus stable lesions (P&lt;0,05, N=3). Stimulation with the pro-atherogenic cytokine TNF α increased ETS2 expression and induced ETS2 translocation from the golgi area to the nucleus. Western Diet increased ETS2 expression on the endothelium of the aorta of ApoE−/− animals compared with chow-fed controls, indicated by whole mount staining (P&lt;0,05, N=4). In vitro knockdown of ETS2 by SiRNA in HUVECs diminished the expression of the cytokines MCP1, IL6, and the adhesion molecule VCAM1, under basal conditions and in response to TNF α (P&lt;0,05, N=4). ETS2 knockdown by SiRNA impeded new vessel formation in a matrigel tube-formation assay (P&lt;0,05, N=8). In vivo , adenoviral transduction of ETS2 in the adventitia of the VP region significantly augmented the vascular network of the adventitia compared with shamvirus treated controls. ETS2 overexpression increased both lesion size and necrotic core area, and promoted plaque destabilization by increasing intraplaque lipid and macrophage accumulation, while vascular smooth muscle cell accumulation was diminished. In conclusion, ETS2 expression correlates with plaque instability and responds to pro-atherogenic stimuli. Subsequently, ETS2 function is crucial for induction of the pro-atherogenic EC phenotype and promotes destabilization in advanced atherosclerotic lesions.

Transcriptional Regulation of the Novel Toll-like Receptor Tlr13

Little has been known about Tlr13 (Toll-like receptor 13), a novel member of the Toll-like receptor family. To elucidate the molecular basis of murine Tlr13 gene expression, the activity of the Tlr13 gene promoter was characterized. Reporter gene analysis and electrophoretic mobility shift assays demonstrated that Tlr13 gene transcription was regulated through three cis-acting elements that interacted with the Ets2, Sp1, and PU.1 transcription factors. Furthermore, our work suggests that these transcription factors may cooperate, culminating in maximal transcription of the Tlr13 gene. In contrast, NF-kappaB appeared to act as an inhibitor of Tlr13 transcription. Overexpression of Ets2 caused a strong increase in the transcriptional activity of the Tlr13 promoter; however, overexpression of NF-kappaB p65 dramatically inhibited it. Additionally, interferon-beta is capable of acting Tlr13 transcription, but the activated signaling of lipopolysaccharide/TLR4 and peptidoglycan/TLR2 strongly inhibited the Tlr13 gene promoter. Thus, these findings reveal the mechanism of Tlr13 gene regulation, thereby providing insight into the function of Tlr13 in the immune response to pathogen.

Ets2 Maintains hTERT Gene Expression and Breast Cancer Cell Proliferation by Interacting with c-Myc

Human telomerase reverse transcriptase (hTERT) underlies cancer cell immortalization, and the expression of hTERT is regulated strictly at the gene transcription. Here, we report that transcription factor Ets2 is required for hTERT gene expression and breast cancer cell proliferation. Silencing Ets2 induces a decrease of hTERT gene expression and increase in human breast cancer cell death. Reconstitution with recombinant hTERT rescues the apoptosis induced by Ets2 depression. In vitro and in vivo analyses show that Ets2 binds to the EtsA and EtsB DNA motifs on the hTERT gene promoter. Mutation of either Ets2 binding site reduces the hTERT promoter transcriptional activity. Moreover, Ets2 forms a complex with c-Myc as demonstrated by co-immunoprecipitation and glutathione S-transferase pulldown assays. Immunological depletion of Ets2, or mutation of the EtsA DNA motif, disables c-Myc binding to the E-box, whereas removal of c-Myc or mutation of the E-box also compromises Ets2 binding to EtsA. Thus, hTERT gene expression is maintained by a mechanism involving Ets2 interactions with the c-Myc transcription factor and the hTERT gene promoter, a protein-DNA complex critical for hTERT gene expression and breast cancer cell proliferation.

ETS2 and ERG1 Promote Megakaryopoiesis and Immortalize GATA1 Knockdown (ΔneoΔHS) Fetal Liver Progenitors: Implications for Leukemogenesis in Down Syndrome.

ETS2 and ERG are two ETS family transcription factors encoded on chromosome 21 that have been implicated in leukemia and other types of cancer. In the setting of trisomy 21 (e.g. in individuals with Down syndrome) there is a significant increase in the propensity for leukemia, including AMKL (acute megakaryoblastic leukemia). It has been theorized that genes located on chromosome 21 may contribute to the predisposition of children with Down syndrome for pediatric AMKL, possibly by collaborating with mutations in the transcriptional regulator GATA1. To test the oncogenic potential of these genes in the hematopoietic system, ETS2 and the ERG isoform, ERG1, were over-expressed in both wild-type and GATA1 knockdown (ΔneoΔHS) fetal liver hematopoietic progenitors and the effects on growth and differentiation were studied. We discovered that over-expression of either ETS2 or ERG1 in wild-type progenitors increased the formation of CD41 positive megakaryocytes by 2–3 fold in liquid culture. However, whereas ETS2 appeared to primarily promote the early stages of megakaryopoiesis, ERG1 over-expression led to a dramatic rise in the percentage of cells expressing CD42, a marker of more mature megakaryocytes. Ectopic ERG1 expression also affected the ploidy profile, with nearly all cells containing ≥4N DNA content. Quantitative real time RT-PCR analysis of lineage specific genes confirmed that expression of megakaryocyte genes, including GPIIb (CD41), platelet factor 4 (PF4), and ß1-tubulin, was significantly increased in undifferentiated cells over-expressing either ETS2 or ERG1. These results show that over-expression of ETS2 or ERG1 rapidly initiates the megakaryocyte program. Over-expression of either ETS2 or ERG1 in GATA1 knockdown fetal liver cells similarly led to a 2.5–5 fold increase in the formation of CD41 positive megakaryocytes in liquid culture; however, neither ETS2 or ERG1 could circumvent the differentiation block that is characteristic of the GATA1 knockdown strain, including the inability to form proplatelet extensions. We next assessed the capacity of ETS2 or ERG1 over-expression to alter the colony forming ability of wild-type and GATA1 knockdown fetal liver progenitors. Both ETS2 and ERG1 expression led to a significant increase in CFU-MK derived from wild-type progenitors, as evidenced by an increase in the number of acetylcholinesterase positive colonies. In contrast, neither gene altered the formation of CFU-MK when over-expressed in the GATA1 knockdown progenitors, which already show a prominent enhancement in megakaryocyte colony formation over wild-type fetal liver cells. Finally, serial re-plating assays performed with GATA1 knockdown fetal liver cells showed that ectopic expression of either ETS2 or ERG1 could immortalize hematopoietic progenitors, whereas only ERG1 could enhance colony re-plating of wild-type cells. These findings provide evidence for a specific synergy between GATA-1 deficiency and ETS2 over-expression and suggest a distinct oncogenic potential for each factor. Overall, these results in primary murine fetal liver cells demonstrate that ETS2 and ERG1 promote the megakaryocyte lineage and facilitate immortalization of GATA1 knockdown cells, consistent with a potential oncogenic role in Down syndrome AMKL for these two ETS family genes located on chromosome 21.