Transcription Factor Fli-1 Research Articles

Direct conversion of human amniotic cells into endothelial cells without transitioning through a pluripotent state.

Endothelial cells (ECs) have essential roles in organ development and regeneration, and therefore they could be used for regenerative therapies. However, generation of abundant functional endothelium from pluripotent stem cells has been difficult because ECs generated by many existing strategies have limited proliferative potential and display vascular instability. The latter difficulty is of particular importance because cells that lose their identity over time could be unsuitable for therapeutic use. Here, we describe a 3-week platform for directly converting human mid-gestation lineage-committed amniotic fluid-derived cells (ACs) into a stable and expandable population of vascular ECs (rAC-VECs) without using pluripotency factors. By transient expression of the ETS transcription factor ETV2 for 2 weeks and constitutive expression the ETS transcription factors FLI1 and ERG1, concomitant with TGF-β inhibition for 3 weeks, epithelial and mesenchymal ACs are converted, with high efficiency, into functional rAC-VECs. These rAC-VECs maintain their vascular repertoire and morphology over numerous passages in vitro, and they form functional vessels when implanted in vivo. rAC-VECs can be detected in recipient mice months after implantation. Thus, rAC-VECs can be used to establish a cellular platform to uncover the molecular determinants of vascular development and heterogeneity and potentially represent ideal ECs for the treatment of regenerative disorders.

Abstract 1654: The small molecule YK-4-279 shows anti-lymphoma activity in pre-clinical models

Abstract We have reported that up to 20% of diffuse large B cell lymphomas (DLBCL) present recurrent 11q23 gains leading to deregulated expression of the two transcription factors ETS1 and FLI1 and silencing of FLI1 was shown to lead to cell death in 6/6 DLBCL cell lines (Blood 2013). YK-4-279 is a small molecule that inhibits the binding of EWS1-FLI1 to RHA, leading to growth arrest and apoptosis of pre-clinical models of Ewing Sarcoma (Nat Med 2009). RHA is transcriptional co-activator, part of the EWS1-FLI1 transcription complex, but also involved in the activity of NFKB and STAT transcription complexes. Indeed, the compound also presented anti-cancer activity in a few additional cancer cell lines perhaps targeting not EWS1-FLI1 or FLI1 but other ETS factors, which are very similar to FLI1 in terms of DNA binding site (PLoS One 2011). Here, we assessed the anti-proliferative activity of YK-4-279 in a panel of human cell lines derived from B- and T-cell lymphomas. Methods. 48 cell lines [27 derived from DLBCL, 10 from mantle cell lymphoma (MCL), 3 from splenic marginal zone lymphoma (SMZL), 8 from anaplastic large cell lymphoma (ALCL)] were treated with increasing doses of YK-4-279) and IC50s were calculated with the MTT assays after 72 hrs exposure. Baseline gene expression profiling (GEP) was obtained on the cell lines with the Illumina HumanHT-12 Expression BeadChips and integrated with the anti-proliferative effect. Results. YK-4-279 showed potent dose-dependent anti-proliferative activity in most of the cell lines tested, with the majority of IC50 values being below 1000 nM. The median IC50 value was 393 nM (95% C.I., 272 - 3622 nM). There were no apparent differences among the different lymphoma subtypes: DLBCL (median IC50 = 386 nM; 95% C.I., 223-497), MCL (596 nM; 95% C.I., 230-1305), SMZL (217 nM; 95% C.I., 183-524) and ALCL (403 nM; 95% C.I., 170-3387). Results were confirmed by performing further MTT experiments using the R-YK-4-279 and S-YK-4-279, showing activity only when cells were exposed to the latter compound. The comparison of baseline GEP between 14 less sensitive (IC50 > 500 nM) and 9 highly sensitive (IC < 200 nM) B-cell lines highlighted that the expression signature of the latter group was enriched of E2F1 and PAX5 targets (including FLI1), genes involved in germinal center, cell cycle, sequence-specific DNA binding, and RNA processing. Conversely, the low sensitivity was associated with NFKB-related genes. Genes such as BCL6, BACH2, LMO2, CXCR4 and FAK were among the 50 most up-regulated transcripts in the sensitive cells. Conclusion. YK-4-279 shows strong anti-proliferative activity in lymphomas. Its activity appears positively correlated with the presence of germinal center B cell-related expression signatures and negatively with NFKB activity. The compound appears worth of further investigations in the lymphoma setting. Citation Format: Elain YL Chung, Valdemar Priebe, Eugenio Gaudio, Ivo Kwee, Chiara Tarantelli, Andrea Rinaldi, Laura Carrassa, Monica Testoni, Luciano Cascione, Massimo Broggini, Jeffrey A. Toretsky, Emanuele Zucca, Francesco Bertoni. The small molecule YK-4-279 shows anti-lymphoma activity in pre-clinical models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1654. doi:10.1158/1538-7445.AM2015-1654

A possible contribution of lipocalin-2 to the development of dermal fibrosis, pulmonary vascular involvement and renal dysfunction in systemic sclerosis.

Lipocalin-2 is an adipocytokine implicated in apoptosis, innate immunity, angiogenesis, and the development of chronic kidney disease. To investigate the role of lipocalin-2 in systemic sclerosis (SSc). Serum lipocalin-2 levels were determined by enzyme-linked immunosorbent assay in 50 patients with SSc and 19 healthy subjects. Lipocalin-2 expression was evaluated in the skin of patients with SSc and bleomycin (BLM)-treated mice and in Fli1-deficient endothelial cells by reverse transcriptase-real time polymerase chain reaction, immunoblotting and/or immunohistochemistry. Although serum lipocalin-2 levels were comparable between patients with SSc and healthy controls, the prevalence of scleroderma renal crisis was significantly higher in patients with SSc with elevated serum lipocalin-2 levels than in those with normal levels. Furthermore, serum lipocalin-2 levels inversely correlated with estimated glomerular filtration rate in patients with SSc with renal dysfunction. Among patients with SSc with normal renal function, serum lipocalin-2 levels positively correlated with skin score in patients with diffuse cutaneous SSc with disease duration of <3years and inversely correlated with estimated right ventricular systolic pressure in total patients with SSc. Importantly, in SSc lesional skin, lipocalin-2 expression was increased in dermal fibroblasts and endothelial cells. In BLM-treated mice, lipocalin-2 was highly expressed in dermal fibroblasts, but not in endothelial cells. On the other hand, the deficiency of transcription factor Fli1, which is implicated in SSc vasculopathy, induced lipocalin-2 expression in cultivated endothelial cells. Lipocalin-2 may be involved in renal dysfunction and dermal fibrosis of SSc. Dysregulated matrix metalloproteinase-9/lipocalin-2-dependent angiogenesis due to Fli1 deficiency may contribute to the development of pulmonary arterial hypertension associated with SSc.

Identifying novel mechanisms regulating expression of inflammatory chemokine CXCL2 (CCR3P.206)

Abstract Mammalian cells produce inflammatory mediators in response to the innate immune signal and their expression is tightly regulated. Chemokine (C-X-C motif) ligand 2 (CXCL2), also known as MIP2-alpha, belongs to the CXC chemokine family. CXCL2 is chemotactic for neutrophils. Elevated expression of CXCL2 is associated with many inflammatory and autoimmune diseases. Recently we have found that the Fli-1 transcription factor is a novel regulator in modulating the expression of many inflammatory mediators. including MCP-1,CCL5) and IL-6. Since we found that lupus-prone mice with reduced expression of Fli-1 had significantly decreased neutrophil infiltration into the kidneys, we investigated if Fli-1 regulates the expression of CXCL2. We found that Fli-1 protein expression in endothelial cells transfected with Fli-1 specific siRNA was significantly decreased compared to the expression of Fli-1 in cells transfected with control siRNA after stimulation by Toll-like receptor (TLR) 4 ligands, and tumor necrosis factor (TNF) alpha. The production of CXCL2 in endothelial cells with LPS stimulation is dose-dependent. We found that Fli-1 binds to the CXCL2 promoter by Chromatin immunoprecipitation‎ (ChIP) assay. Transient transfection assays show that Fli-1 drives transcription from the CXCL2 promoter in a dose-dependent manner. These results indicate that Fli-1 is a novel, critical transcription factor in regulating the expression of the inflammatory chemokine CXCL2.

Regulation of granulocyte colony stimulating factor by Fli-1, a novel transcriptional activator of inflammatory mediators (CCR3P.213)

Abstract The Fli-1 transcription factor plays a significant role in the pathogenesis of systemic lupus erythematosus (SLE). Fli-1 expression is significantly increased in patients with lupus nephritis and lupus-prone mice with reduced Fli-1 expression (Fli-1+/-) live longer and have significantly less nephritis compared to wild type controls. Recently, Fli-1 has emerged as a critical regulator of inflammatory mediators, including MCP-1, CCL5, and IL-6. The cytokine, granulocyte colony stimulating factor (G-CSF) regulates neutrophil precursor maturation and survival, and activates mature neutrophils. In clinical settings, G-CSF increases the inflammatory response and worsens tissue damage. Previously, a significant decrease in neutrophil infiltration into the kidneys of Fli-1+/- lupus-prone mice was observed. In this study, a significant decrease in G-CSF protein expression was detected in stimulated endothelial cells transfected with Fli-1 specific siRNA. The murine G-CSF promoter contains numerous putative Fli-1 binding sites and five regions within the proximal promoter were found to be significantly enriched for Fli-1 binding. Transient transfection assays indicate that Fli-1 drives transcription from the G-CSF promoter and mutation of the Fli-1 DNA binding domain resulted in a 94% loss of transcriptional activation. In conclusion, we have discovered that Fli-1 plays a major role in the regulation of inflammatory cytokines and chemokines and inflammatory disease pathogenesis.

Fli1 deficiency and beyond: a unique pathway linking peripheral vasculopathy and dermal fibrosis in systemic sclerosis

Fli1 deficiency and beyond: a unique pathway linking peripheral vasculopathy and dermal fibrosis in systemic sclerosis

A possible contribution of endothelial CCN1 downregulation due to Fli1 deficiency to the development of digital ulcers in systemic sclerosis.

CCN1 is a pleiotropic molecule involved in angiogenesis and postnatal vasculogenesis, both of which are impaired in systemic sclerosis (SSc). To elucidate the potential role of CCN1 in the development of SSc, we investigated CCN1 expression in the lesional skin of SSc patients and SSc animal models and the clinical correlation of serum CCN1 levels. CCN1 expression was markedly decreased in dermal small blood vessels of SSc patients compared with those of healthy controls, while comparable between normal and SSc dermal fibroblasts. Transcription factor Fli1, whose deficiency due to epigenetic suppression is implicated in the pathogenesis of SSc, occupied the CCN1 promoter and gene silencing of Fli1 resulted in the reduction of CCN1 expression in human dermal microvascular endothelial cells. Consistently, CCN1 expression was suppressed uniformly and remarkably in dermal blood vessels of Fli1(+/-) mice and partially in those of endothelial cell-specific Fli1 knockout mice. Furthermore, serum CCN1 levels were significantly decreased in SSc patients with previous and current history of digital ulcers as compared to those without. Collectively, these results suggest that endothelial CCN1 downregulation at least partially due to Fli1 deficiency may contribute to the development of digital ulcers in SSc patients. This study further supports the idea that epigenetic downregulation of Fli1 is a potential predisposing factor in the pathogenesis of SSc.

Transdifferentiation of erythroblasts to megakaryocytes using FLI1 and ERG transcription factors.

Platelet transfusion has been widely used to prevent and treat life-threatening thrombocytopenia; however, preparation of a unit of concentrated platelet for transfusion requires at least 4-6 units of whole blood. At present, a platelet unit from a single donor can be prepared using apheresis, but lack of donors is still a major problem. Several approaches to produce platelets from other sources, such as haematopoietic stem cells and pluripotent stem cells, have been attempted but the system is extremely complicated, time-consuming and expensive. We now report a novel and simpler technology to obtain platelets using transdifferentiation of human bone marrow erythroblasts to megakaryocytes with overexpression of the FLI1 and ERG genes. The obtained transdifferentiated erythroblasts (both from CD71+ and GPA+ erythroblast subpopulations) exhibit typical features of megakaryocytes including morphology, expression of specific genes (cMPL and TUBB1) and a marker protein (CD41). They also have the ability to generate megakaryocytic CFU in culture and produce functional platelets, which aggregate with normal human platelets to form a normal-looking clot. Overexpression of FLI1 and ERG genes is sufficient to transdifferentiate erythroblasts to megakaryocytes that can produce functional platelets.

Increased expression of chemerin in endothelial cells due to Fli1 deficiency may contribute to the development of digital ulcers in systemic sclerosis.

Chemerin is a member of adipocytokines with a chemoattractant effect on plasmacytoid dendritic cells and macrophages and pro-angiogenic properties. We investigated the potential role of chemerin in the development of SSc. Chemerin expression was evaluated by immunostaining and/or real-time quantitative RT-PCR in human and murine skin. The mechanisms regulating chemerin expression in dermal fibroblasts and endothelial cells were examined using the gene silencing technique and chromatin immunoprecipitation. Serum chemerin levels were determined by ELISA in 64 SSc patients and 19 healthy subjects. In SSc lesional skin, chemerin was up-regulated in small blood vessels, while it was down-regulated in fibroblasts surrounded with thickened collagen bundles. The decreased expression of chemerin was significantly reversed by TGF-β1 antisense oligonucleotide in cultured SSc dermal fibroblasts and chemerin expression was markedly decreased in dermal fibroblasts of bleomycin-treated mice. Gene silencing of transcription factor Fli1, which binds to the chemerin promoter, induced chemerin expression in human dermal microvascular endothelial cells and Fli1(+/-) mice exhibited elevated chemerin expression in dermal blood vessels. Serum chemerin levels inversely correlated with estimated glomerular filtration rate in SSc patients with renal dysfunction. In SSc patients with normal renal function, patients with digital ulcers had higher serum chemerin levels than those without. Chemerin is down-regulated in SSc dermal fibroblasts by autocrine TGF-β, while it is up-regulated in SSc dermal blood vessels through endothelial Fli1 deficiency. Increased chemerin expression in dermal blood vessels may be associated with the development of digital ulcers in SSc.

A critical role of the transcription factor fli-1 in murine lupus development by regulation of interleukin-6 expression.

The Fli-1 transcription factor is implicated in the pathogenesis of systemic lupus erythematosus (SLE), both in humans and in animal models. Dysregulation of interleukin-6 (IL-6) is also associated with SLE. The purpose of this study was to investigate whether Fli-1 directly regulates the expression of IL-6. Sera were collected from wild-type and Fli-1-heterozygous (Fli-1(+/-) ) MRL/lpr mice, and the concentration of IL-6 was measured by enzyme-linked immunosorbent assay (ELISA). Expression of IL-6 in the kidney was measured by real-time polymerase chain reaction analysis. T cells were isolated from wild-type and Fli-1(+/-) MRL/lpr mice and stimulated with CD3/CD28 beads, and the concentration of IL-6 in the supernatants was measured by ELISA. MS1 endothelial cells were transfected with Fli-1 and control small interfering RNA, and the production of IL-6 was compared after lipopolysaccharide stimulation. A chromatin immunoprecipitation (ChIP) assay was performed to determine whether Fli-1 binds to the IL-6 promoter region. Transient transfections with the NIH3T3 cell line were performed to examine whether Fli-1 regulates the expression of IL-6. Fli-1(+/-) MRL/lpr mice had significantly decreased IL-6 levels in sera and reduced expression of IL-6 in kidneys as compared to their wild-type littermates. T cells isolated from Fli-1(+/-) MRL/lpr mice produced less IL-6 than did those from wild-type mice. Inhibiting the expression of Fli-1 in endothelial cells resulted in reduced production of IL-6. The ChIP assay revealed direct binding of Fli-1 to 3 regions within the IL-6 promoter. Fli-1 activated transcription from the IL-6 promoter in a dose-dependent manner. The direct regulation of IL-6 expression by Fli-1 represents one possible mechanism for the protective effect of decreased Fli-1 expression in lupus.

Long noncoding RNA EWSAT1-mediated gene repression facilitates Ewing sarcoma oncogenesis.

Chromosomal translocation that results in fusion of the genes encoding RNA-binding protein EWS and transcription factor FLI1 (EWS-FLI1) is pathognomonic for Ewing sarcoma. EWS-FLI1 alters gene expression through mechanisms that are not completely understood. We performed RNA sequencing (RNAseq) analysis on primary pediatric human mesenchymal progenitor cells (pMPCs) expressing EWS-FLI1 in order to identify gene targets of this oncoprotein. We determined that long noncoding RNA-277 (Ewing sarcoma-associated transcript 1 [EWSAT1]) is upregulated by EWS-FLI1 in pMPCs. Inhibition of EWSAT1 expression diminished the ability of Ewing sarcoma cell lines to proliferate and form colonies in soft agar, whereas EWSAT1 inhibition had no effect on other cell types tested. Expression of EWS-FLI1 and EWSAT1 repressed gene expression, and a substantial fraction of targets that were repressed by EWS-FLI1 were also repressed by EWSAT1. Analysis of RNAseq data from primary human Ewing sarcoma further supported a role for EWSAT1 in mediating gene repression. We identified heterogeneous nuclear ribonucleoprotein (HNRNPK) as an RNA-binding protein that interacts with EWSAT1 and found a marked overlap in HNRNPK-repressed genes and those repressed by EWS-FLI1 and EWSAT1, suggesting that HNRNPK participates in EWSAT1-mediated gene repression. Together, our data reveal that EWSAT1 is a downstream target of EWS-FLI1 that facilitates the development of Ewing sarcoma via the repression of target genes.

NLS-tagging: an alternative strategy to tag nuclear proteins.

The characterization of transcription factor complexes and their binding sites in the genome by affinity purification has yielded tremendous new insights into how genes are regulated. The affinity purification requires either the use of antibodies raised against the factor of interest itself or by high-affinity binding of a C- or N-terminally added tag sequence to the factor. Unfortunately, fusing extra amino acids to the termini of a factor can interfere with its biological function or the tag may be inaccessible inside the protein. Here, we describe an effective solution to that problem by integrating the ‘tag’ close to the nuclear localization sequence domain of the factor. We demonstrate the effectiveness of this approach with the transcription factors Fli-1 and Irf2bp2, which cannot be tagged at their extremities without loss of function. This resulted in the identification of novel proteins partners and a new hypothesis on the contribution of Fli-1 to hematopoiesis.

The Fli-1 transcription factor regulates the expression of CCL5/RANTES.

The friend leukemia insertion site 1 (Fli-1) transcription factor, an Ets family member, is implicated in the pathogenesis of systemic lupus erythematosus in human patients and murine models of lupus. Lupus-prone mice with reduced Fli-1 expression have significantly less nephritis, prolonged survival, and decreased infiltrating inflammatory cells into the kidney. Inflammatory chemokines, including CCL5, are critical for attracting inflammatory cells. In this study, decreased CCL5 mRNA expression was observed in kidneys of lupus-prone NZM2410 mice with reduced Fli-1 expression. CCL5 protein expression was significantly decreased in endothelial cells transfected with Fli-1-specific small interfering RNA compared with controls. Fli-1 binds to endogenous Ets binding sites in the distal region of the CCL5 promoter. Transient transfection assays demonstrate that Fli-1 drives transcription from the CCL5 promoter in a dose-dependent manner. Both Ets1, another Ets family member, and Fli-1 drive transcription from the CCL5 promoter, although Fli-1 transactivation was significantly stronger. Ets1 acts as a dominant-negative transcription factor for Fli-1, indicating that they may have at least one DNA binding site in common. Systematic deletion of DNA binding sites demonstrates the importance of the sites located within a 225-bp region of the promoter. Mutation of the Fli-1 DNA binding domain significantly reduces transactivation of the CCL5 promoter by Fli-1. We identified a novel regulator of transcription for CCL5. These results suggest that Fli-1 is a novel and critical regulator of proinflammatory chemokines and affects the pathogenesis of disease through the regulation of factors that recruit inflammatory cells to sites of inflammation.

Fli-1 controls transcription from the MCP-1 gene promoter, which may provide a novel mechanism for chemokine and cytokine activation

Regulation of proinflammatory cytokines and chemokines is a primary role of the innate immune response. MCP-1 is a chemokine that recruits immune cells to sites of inflammation. Expression of MCP-1 is reduced in primary kidney endothelial cells from mice with a heterozygous knockout of the Fli-1 transcription factor. Fli-1 is a member of the Ets family of transcription factors, which are evolutionarily conserved across several organisms including Drosophilla, Xenopus, mouse and human. Ets family members bind DNA through a consensus sequence GGAA/T, or Ets binding site (EBS). Fli-1 binds to EBSs within the endogenous MCP-1 promoter by ChIP assay. In this study, transient transfection assays indicate that the Fli-1 gene actively promotes transcription from the MCP-1 gene promoter in a dose-dependent manner. Mutation of the DNA binding domain of Fli-1 demonstrated that Fli-1 activates transcription of MCP-1 both directly, by binding to the promoter, and indirectly, likely through interactions with other transcription factors.Another Ets transcription factor, Ets-1, was also tested, but failed to promote transcription. While Ets-1 failed to drive transcription independently, a weak synergistic activation of the MCP-1 promoter was observed between Ets-1 and Fli-1. In addition, Fli-1 and the NFκB family member p65 were found to interact synergistically to activate transcription from the MCP-1 promoter, while Sp1 and p50 inhibit this interaction. Deletion studies identified that EBSs in the distal and proximal MCP-1 promoter are critical for Fli-1 activation from the MCP-1 promoter. Together, these results demonstrate that Fli-1 is a novel regulator of the proinflammatory chemokine MCP-1, that interacts with other transcription factors to form a complex transcriptional mechanism for the activation of MCP-1 and mediation of the inflammatory response.

The ets transcription factor Fli-1 in development, cancer and disease.

Friend Leukemia Virus Induced erythroleukemia-1 (Fli-1), an ETS transcription factor, was isolated a quarter century ago through a retrovirus mutagenesis screen. Fli-1 has since been recognized to play critical roles in normal development and homeostasis. For example, it transcriptionally regulates genes that drive normal hematopoiesis and vasculogenesis. Indeed, Fli-1 is one of 10 key regulators of hematopoietic stem/progenitor cell maintenance and differentiation. Aberrant expression of Fli-1 also underlies a number of virally induced leukemias, including Friend virus-induced erythroleukemia and various types of human cancers, and it is the target of chromosomal translocations in childhood Ewing’s sarcoma. Abnormal expression of Fli-1 is important in the aetiology of auto-immune diseases such as Systemic Lupus Erythematosus (SLE) and Systemic Sclerosis (SSc). These studies establish Fli-1 as a strong candidate for drug development. Despite difficulties in targeting transcription factors, recent studies identified small molecule inhibitors for Fli-1. Here we review past and ongoing research on Fli-1 with emphasis on its mechanistic function in autoimmune disease and malignant transformation. The significance of identifying Fli-1 inhibitors and their clinical applications for treatment of disease and cancer with deregulated Fli-1 expression are discussed.

The Friend leukaemia virus integration 1 (Fli-1) transcription factor affects lupus nephritis development by regulating inflammatory cell infiltration into the kidney

The transcription factor Friend leukaemia virus integration 1 (Fli-1) is implicated in the pathogenesis of systemic lupus erythematosus in both human patients and murine models of lupus. Murphy Roths large (MRL)/lpr mice and New Zealand mixed (NZM)2410 mice, murine models of lupus, with decreased expression of Fli-1 had significantly prolonged survival and reduced nephritis. Lupus nephritis is a major cause of mortality and morbidity in patients, and inflammatory cell infiltration plays a key role in the development of the disease. To study how the expression of Fli-1 affects the infiltration of inflammatory cells into the kidneys, we generated congenic enhanced green fluorescent protein (GFP) transgenic MRL/lpr mice. A significantly increased number of GFP-expressing inflammatory cells infiltrated the kidneys of wild-type MRL/lpr mice compared to Fli-1 heterozygous (Fli-1(+/-)) MRL/lpr mice after injection of GFP(+) cells. Expression of inflammatory chemokine mRNA, including chemokine (C-C motif) ligand (CCL)2, CCL3, CCL4 and CCL5, was significantly lower in the kidneys from Fli-1(+/-) MRL/lpr mice compared to wild-type littermates. Numbers of infiltrated cells into the kidneys correlate with expression levels of CCL2, CCL4 and CCL5, but not the titres of anti-dsDNA autoantibodies in these mice. Significantly increased inflammatory cells from wild-type MRL/lpr mice infiltrated into kidneys compared to the cells from Fli-1(+/-) MRL/lpr mice. The chemotaxis of inflammatory cells from Fli-1(+/-) MRL/lpr mice towards each chemokine was decreased significantly compared to inflammatory cells from wild-type MRL/lpr mice in the transwell migration assay in vitro. Our results indicate that Fli-1 affects lupus nephritis development by regulating the expression of chemokines in the kidney and the migration of inflammatory cells.

Transcription factor Fli-1 directly regulates the expression of IL-6 (BA2P.114)

Abstract Fli-1, a member of the Ets family of transcription factors, is highly expressed in immune cells and endothelial cells and is implicated in the development of systemic lupus erythematosus (SLE) in both human patients and mouse models. Increased expression of IL-6 is also involved in the SLE and other autoimmune disease development. We examined the role of Fli-1 on production of IL-6 in this study. We found expression of IL-6 was significantly reduced in sera from Fli-1 heterozygous knockout MRL/lpr mice (Fli1+/-; Fli-1 homozygous knockout is embryonic lethal), a murine model of lupus, compared to wild-type littermates. The production of IL-6 were significantly decreased in endothelial cells transfected with specific Fli-1 siRNA compared to cells transfected with negative control siRNA after LPS stimulation. We demonstrated that Fli-1 directly binds to the IL-6 promoter by Chromatin Immunoprecipitation (ChIP) assay and transient transfection assays show that Fli-1 drives transcription from the IL-6 promoter in a dose-dependent manner. Our data indicate that transcription factor Fli-1 binds to the promoter of IL-6 and directly regulates the expression of IL-6.

Identification of Fli-1 as a novel regulator of the inflammatory chemokine CCL5/RANTES (CCR5P.253)

Abstract The Fli-1 transcription factor is implicated in the pathogenesis of systemic lupus erythematosus (SLE) in both human patients and murine models of lupus. Lupus-prone mice with reduced Fli-1 expression have significantly less nephritis, prolonged survival and significantly decreased infiltrating inflammatory cells into the kidney. The chemokine, CCL5, attracts inflammatory cells and expression is increased in patients with SLE. In this study, decreased expression of CCL5 mRNA was observed in kidneys of NZM2410 mice with reduced Fli-1 expression. CCL5 protein expression was significantly impaired in endothelial cells treated with Fli-1 specific siRNA compared to controls. Fli-1 binds to Ets binding sites in the CCL5 promoter and transfection assays show that Fli-1 drives transcription in a dose-dependent manner. Mutation of the Fli-1 DNA binding domain demonstrates that activation of the CCL5 promoter is through direct binding by Fli-1. Ets1 also drives transcription from the CCL5 promoter, although Fli-1 driven transactivation is significantly stronger. The Ets1 transcription factor acts as a dominant negative to Fli-1, indicating that the genes may share a DNA binding site. Fli-1 is a novel regulator of CCL5 gene transcription. Combined with previous results, demonstrating that Fli-1 regulates MCP-1, we conclude that Fli-1 is a critical regulator of proinflammatory chemokines and affects disease pathogenesis by regulating factors involved inflammatory cell recruitment.

Proteome-wide Analysis and CXCL4 as a Biomarker in Systemic Sclerosis

BackgroundPlasmacytoid dendritic cells have been implicated in the pathogenesis of systemic sclerosis through mechanisms beyond the previously suggested production of type I interferon.MethodsWe isolated plasmacytoid dendritic cells from healthy persons and from patients with systemic sclerosis who had distinct clinical phenotypes. We then performed proteome-wide analysis and validated these observations in five large cohorts of patients with systemic sclerosis. Next, we compared the results with those in patients with systemic lupus erythematosus, ankylosing spondylitis, and hepatic fibrosis. We correlated plasma levels of CXCL4 protein with features of systemic sclerosis and studied the direct effects of CXCL4 in vitro and in vivo.ResultsProteome-wide analysis and validation showed that CXCL4 is the predominant protein secreted by plasmacytoid dendritic cells in systemic sclerosis, both in circulation and in skin. The mean (±SD) level of CXCL4 in patients with systemic sclerosis was 25,624±2652 pg per milliliter, which was significantly higher than the level in controls (92.5±77.9 pg per milliliter) and than the level in patients with systemic lupus erythematosus (1346±1011 pg per milliliter), ankylosing spondylitis (1368±1162 pg per milliliter), or liver fibrosis (1668±1263 pg per milliliter). CXCL4 levels correlated with skin and lung fibrosis and with pulmonary arterial hypertension. Among chemokines, only CXCL4 predicted the risk and progression of systemic sclerosis. In vitro, CXCL4 down-regulated expression of transcription factor FLI1, induced markers of endothelial-cell activation, and potentiated responses of toll-like receptors. In vivo, CXCL4 induced the influx of inflammatory cells and skin transcriptome changes, as in systemic sclerosis.ConclusionsLevels of CXCL4 were elevated in patients with systemic sclerosis and correlated with the presence and progression of complications, such as lung fibrosis and pulmonary arterial hypertension. (Funded by the Dutch Arthritis Association and others.)

Bosentan reverses the pro-fibrotic phenotype of systemic sclerosis dermal fibroblasts via increasing DNA binding ability of transcription factor Fli1

IntroductionAlthough the pathogenesis of systemic sclerosis (SSc) still remains unknown, recent studies have demonstrated that endothelins are deeply involved in the developmental process of fibrosis and vasculopathy associated with SSc, and a dual endothelin receptor antagonist, bosentan, has a potential to serve as a disease modifying drug for this disorder. Importantly, endothelin-1 (ET-1) exerts a pro-fibrotic effect on normal dermal fibroblasts and bosentan reverses the pro-fibrotic phenotype of SSc dermal fibroblasts. The purpose of this study was to clarify the details of molecular mechanisms underlying the effects of ET-1 and bosentan on dermal fibroblasts, which have not been well studied.MethodsThe mRNA levels of target genes and the expression and phosphorylation levels of target proteins were determined by reverse transcription real-time PCR and immunoblotting, respectively. Promoter assays were performed using a sequential deletion of human α2 (I) collagen (COL1A2) promoter. DNA affinity precipitation and chromatin immunoprecipitation were employed to evaluate the DNA binding ability of Fli1. Fli1 protein levels in murine skin were evaluated by immunostaining.ResultsIn normal fibroblasts, ET-1 activated c-Abl and protein kinase C (PKC)-δ and induced Fli1 phosphorylation at threonine 312, leading to the decreased DNA binding of Fli1, a potent repressor of the COL1A2 gene, and the increase in type I collagen expression. On the other hand, bosentan reduced the expression of c-Abl and PKC-δ, the nuclear localization of PKC-δ, and Fli1 phosphorylation, resulting in the increased DNA binding of Fli1 and the suppression of type I collagen expression in SSc fibroblasts. In bleomycin-treated mice, bosentan prevented dermal fibrosis and increased Fli1 expression in lesional dermal fibroblasts.ConclusionsET-1 exerts a potent pro-fibrotic effect on normal fibroblasts by activating “c-Abl - PKC-δ - Fli1” pathway. Bosentan reverses the pro-fibrotic phenotype of SSc fibroblasts and prevents the development of dermal fibrosis in bleomycin-treated mice by blocking this signaling pathway. Although the efficacy of bosentan for dermal and pulmonary fibrosis is limited in SSc, the present observation definitely provides us with a useful clue to further explore the potential of the upcoming new dual endothelin receptor antagonists as disease modifying drugs for SSc.