Endothelial CCR6 expression due to FLI1 deficiency contributes to vasculopathy associated with systemic sclerosis.

BACKGROUND: We have recently demonstrated that serum CCL20 levels positively correlate with mean pulmonary arterial pressure in patients with systemic sclerosis (SSc). Considering a proangiogenic effect of CCL20 on endothelial cells via CCR6, the CCL20/CCR6 axis may contribute to the development of SSc vasculopathy. Therefore, we explored this hypothesis using clinical samples, cultured cells, and murine SSc models. METHODS: The expression levels of CCL20 and CCR6 in the skin, mRNA levels of target genes, and the binding of transcription factor FLI1 to the target gene promoter were evaluated by immunostaining, quantitative reverse transcription PCR, and chromatin immunoprecipitation, respectively. Vascular permeability was evaluated by Evans blue dye injection in bleomycin-treated mice. Angiogenic activity of endothelial cells was assessed by in vitro angiogenesis assay. RESULTS: CCL20 expression was significantly elevated in dermal fibroblasts of patients with early diffuse cutaneous SSc, while CCR6 was significantly up-regulated in dermal small vessels of SSc patients irrespective of disease subtypes and disease duration. In human dermal microvascular endothelial cells, FLI1 siRNA induced the expression of CCR6, but not CCL20, and FLI1 bound to the CCR6 promoter. Importantly, vascular permeability, a representative SSc-like vascular feature of bleomycin-treated mice, was attenuated by Ccr6 siRNA treatment, and CCR6 siRNA suppressed the angiogenic activity of human dermal microvascular endothelial cells assayed by in vitro tube formation. CONCLUSIONS: The increased expression of endothelial CCR6 due to FLI1 deficiency may contribute to the development of SSc vasculopathy.

Progranulin overproduction due to constitutively activated c-Abl/PKC-δ/Fli1 pathway contributes to the resistance of dermal fibroblasts to the anti-fibrotic effect of tumor necrosis factor-α in localized scleroderma.

BACKGROUND: Dermal fibroblasts derived from patients with systemic sclerosis (SSc) overproduce progranulin (PGRN), an endogenous antagonist of tumor necrosis factor (TNF) receptors, due to the deficiency of transcription factor Fli1. Fli1 expression is also decreased in dermal fibroblasts derived from patients with localized scleroderma (LSc). OBJECTIVE: To investigate the expression levels of PGRN and its contribution to the induction of pro-fibrotic phenotype in LSc dermal fibroblasts. METHODS: PGRN expression levels were determined by immunohistochemistry and quantitative reverse transcription PCR in the skin of human subjects. The role of PGRN in fibroblast activation was examined with gene silencing technique. The involvement of c-Abl/protein kinase C (PKC)-δ/Fli1 pathway in the regulation of PGRN expression was investigated by immunoblotting. RESULTS: The expression levels of PGRN and TNF-α were elevated in LSc skin lesions compared with healthy control skin. LSc dermal fibroblasts were less responsive to the anti-fibrotic effect of TNF-α than normal dermal fibroblasts. Importantly, gene silencing of PGRN reversed the response to TNF-α in LSc dermal fibroblasts. Similar to SSc dermal fibroblasts, the inhibition of c-Abl/PKC-δ/Fli1 pathway by gene silencing of ABL1 or PRKCD significantly suppressed PGRN expression in LSc dermal fibroblasts. CONCLUSION: PGRN overproduction due to constitutively activated c-Abl/PKC-δ/Fli1 pathway may contribute to the resistance of LSc dermal fibroblasts to the anti-fibrotic effect of TNF-α, which may be involved in maintaining their pro-fibrotic phenotype under the pro-inflammatory condition, as is the case with SSc.

CXCL13 produced by macrophages due to Fli1 deficiency may contribute to the development of tissue fibrosis, vasculopathy and immune activation in systemic sclerosis.

CXCL13, a chemokine for B cells, follicular T cells, T helper 17 cells, and regulatory T cells, is reported to contribute to the development of systemic sclerosis (SSc), reflecting aberrant activation of immune system. To better understand the role of CXCL13 in SSc, we investigated the influence of Fli1 deficiency, a potential predisposing factor of this disease, on CXCL13 expression and assessed the clinical correlation of serum CXCL13 levels by multivariate regression analysis. Haploinsufficient loss of Fli1 remarkably induced CXCL13 expression in murine peritoneal macrophages, while gene silencing of FLI1 did not affect the expression of CXCL13 in human dermal fibroblasts and human dermal microvascular endothelial cells. Serum CXCL13 levels were elevated in SSc patients compared with healthy controls and correlated positively with skin score and negatively with pulmonary function test results. SSc patients with elevated serum CXCL13 levels had longer disease duration, diffuse cutaneous involvement, interstitial lung disease (ILD), heart involvement, pulmonary arterial hypertension, Raynaud's phenomenon, pitting scars, digital ulcers, telangiectasia, and high serum IgG levels more frequently than the other patients. In particular, serum CXCL13 levels were associated with ILD and digital ulcers by multivariate regression analysis. Taken together, these results indicate that CXCL13 expression is upregulated by Fli1 deficiency in macrophages, potentially contributing to the development of tissue fibrosis, vasculopathy and immune activation in SSc, especially ILD and digital ulcers.

Systemic Sclerosis Dermal Fibroblasts Suppress Th1 Cytokine Production via Galectin-9 Overproduction due to Fli1 Deficiency.

Dermal fibroblasts promote skin-localized transdifferentiation of regulatory T cells to T helper (Th) type 2-like cells in systemic sclerosis (SSc). However, the entire effect of SSc dermal fibroblasts on immune cells still remains unknown. Because galectin-9 induces Th2 cytokine-predominant immune imbalance by negatively regulating Th1/Th17 cells in inflammatory diseases, we investigated the contribution of galectin-9 to Th immune balance in SSc lesional skin. We used human clinical samples and Fli1+/- mice because Fli1 deficiency induces SSc-like phenotypes in various cell types. Galectin-9 was overexpressed in SSc dermal fibroblasts in vivo and in vitro. Serum galectin-9 levels were significantly elevated in SSc patients and positively correlated with skin score. Galectin-9 was up-regulated by autocrine endothelin stimulation and Fli1 deficiency, and Fli1 occupied the LGALS9 promoter in dermal fibroblasts. Co-culture of splenic CD4+ T cells with Fli1+/- dermal fibroblasts significantly increased IL-4-producing cell proportion, and this effect was cancelled in parallel with the increased interferon-γ production when Fli1+/- dermal fibroblasts were transfected with Lgals9 small interfering RNA. Furthermore, Lgals9 small interfering RNA suppressed dermal collagen deposition by increasing interferon-γ production of skin-infiltrating CD4+ T cells in bleomycin-treated mice. These results suggest that SSc dermal fibroblasts suppress interferon-γ expression of skin-infiltrating CD4+ T cells through galectin-9 overproduction, promoting skin fibrosis development.

The impact of transcription factor Fli1 deficiency on the regulation of angiogenesis.

The insufficiency of Friend leukaemia virus integration 1 (Fli1), a member of the Ets family transcription factors, is implicated in the pathogenesis of vasculopathy associated with systemic sclerosis (SSc). Fli1 deficiency accelerates early steps of angiogenesis, including detachment of pre-existing pericytes and extracellular matrix degradation by endothelial proteinases, but the impact of Fli1 deficiency on the other steps of angiogenesis has not been investigated. Therefore, we evaluated the effect of Fli1 deficiency on migration, proliferation, cell survival and tube formation of human dermal microvascular endothelial cells (HDMECs). HDMECs transfected with FLI1 siRNA exhibited a greater migratory property in scratch assay and transwell migration assay and a higher proliferation rate in BrdU assay than HDMECs transfected with non-silencing scrambled RNA. In flow cytometry-based apoptosis assay, FLI1 siRNA-transduced HDMECs revealed the decreased number of annexin and propidium iodide-double-positive apoptotic cells compared with control cells, reflecting the promotion of cell survival. On the other hand, tubulogenic activity on Matrigel was remarkably suppressed in Fli1-deficient HDMECs relative to control cells. These results indicate that Fli1 deficiency promotes migration, proliferation and cell survival, while abating tube formation of endothelial cells, suggesting that Fli1 deficiency is potentially attributable to the development of both proliferative obliterative vasculopathy (occlusion of arterioles and small arteries) and destructive vasculopathy (loss of small vessels) characteristic of SSc vasculopathy.

A potential contribution of antimicrobial peptide LL-37 to tissue fibrosis and vasculopathy in systemic sclerosis.

BACKGROUND: LL-37 is an antimicrobial peptide with pleiotropic effects on the immune system, angiogenesis and tissue remodelling. These are cardinal pathological events in systemic sclerosis (SSc). OBJECTIVES: To elucidate the potential role of LL-37 in SSc. METHODS: The expression of target molecules was evaluated by immunostaining and quantitative reverse-transcription real-time polymerase chain reaction in human and murine skin. The mechanisms regulating LL-37 expression in endothelial cells were examined by gene silencing and chromatin immunoprecipitation. Serum LL-37 levels were determined by enzyme-linked immunosorbent assay. RESULTS: In SSc lesional skin, LL-37 expression was increased in dermal fibroblasts, perivascular inflammatory cells, keratinocytes and, particularly, dermal small vessels. Expression positively correlated with interferon-α expression, possibly reflecting LL-37-dependent induction of interferon-α. In SSc animal models, bleomycin-treated skin exhibited the expression pattern of CRAMP, a murine homologue of LL-37, similar to that of LL-37 in SSc lesional skin. Furthermore, Fli1+/- mice showed upregulated expression of CRAMP in dermal small vessels. Fli1 binding to the CAMP (LL-37 gene) promoter and Fli1 deficiency-dependent induction of LL-37 were also confirmed in human dermal microvascular endothelial cells. In the analysis of sera, patients with SSc had serum LL-37 levels significantly higher than in healthy controls. Furthermore, serum LL-37 levels positively correlated with skin score and the activity of alveolitis and were significantly elevated in patients with digital ulcers compared with those without. CONCLUSIONS: LL-37 upregulation, induced by Fli1 deficiency at least in endothelial cells, potentially contributes to the development of skin sclerosis, interstitial lung disease and digital ulcers in SSc.

Decreased expression of neuropilin-1 as a novel key factor contributing to peripheral microvasculopathy and defective angiogenesis in systemic sclerosis.

OBJECTIVES: In systemic sclerosis (SSc), vascular involvement is characterised by vascular endothelial growth factor (VEGF)-A/VEGF receptor (VEGFR) system disturbances. Neuropilin-1 (NRP1), a receptor for both class-3 semaphorins (Sema3s) and VEGF-A, is required for optimal VEGF-A/VEGFR-2 signalling. Here, we investigated the possible involvement of Sema3A/NRP1 axis in SSc. METHODS: Circulating Sema3A and soluble NRP1 (sNRP1) were measured in patients with SSc and controls. NRP1 and Sema3A expression in skin biopsies was evaluated by immunofluorescence and western blotting. NRP1 expression was assessed in SSc and healthy dermal microvascular endothelial cells (SSc-MVECs and H-MVECs), and in SSc and control endothelial progenitor cell (EPC)-derived endothelial cells (ECs). The possible impact of transcription factor Friend leukaemia integration 1 (Fli1) deficiency on endothelial NRP1 expression was investigated by gene silencing. The binding of Fli1 to NRP1 gene promoter was evaluated using chromatin immunoprecipitation. Capillary morphogenesis was performed on Matrigel. RESULTS: Decreased sNRP1 levels in SSc were associated with active and late nailfold videocapillaroscopy patterns and digital ulcers. No difference in Sema3A was found between patients and controls. NRP1 was significantly decreased in SSc-MVECs both ex vivo and in vitro. NRP1 and Fli1 significantly decreased in H-MVECs challenged with SSc sera, while they were not different in SSc and control EPC-derived ECs. Fli1 occupied the NRP1 gene promoter and Fli1 gene silencing reduced NRP1 expression in H-MVECs. NRP1 gene silencing in H-MVECs resulted in a significantly impaired angiogenic capacity comparable to that of cells treated with SSc sera. CONCLUSION: In SSc, NRP1 deficiency may be an additional factor in the perturbed VEGF-A/VEGFR-2 system contributing to peripheral microvasculopathy and defective angiogenesis.

Fli1 deficiency contributes to the downregulation of endothelial protein C receptor in systemic sclerosis: a possible role in prothrombotic conditions.

BACKGROUND: Endothelial protein C receptor (EPCR), expressed predominantly on endothelial cells, plays a critical role in the regulation of the coagulation system and also mediates various cytoprotective effects by binding and activating protein C. So far, the role of EPCR has not been studied in systemic sclerosis (SSc). OBJECTIVES: To investigate the potential contribution of EPCR to the development of SSc. METHODS: EPCR expression was examined in skin samples and cultivated dermal microvascular endothelial cells by immunostaining, immunoblotting and/or quantitative reverse-transcription polymerase chain reaction. Fli1, binding to the PROCR promoter, was assessed by chromatin immunoprecipitation. Serum EPCR levels were determined by enzyme-linked immunosorbent assay in 65 patients with SSc and 20 healthy subjects. RESULTS: EPCR expression was decreased in dermal small vessels of SSc lesional skin compared with those of healthy control skin. Transcription factor Fli1, deficiency of which is implicated in SSc vasculopathy, occupied the PROCR promoter, and EPCR expression was suppressed in Fli1 small interfering RNA-treated endothelial cells and dermal small vessels of Fli1(+/-) mice. In patients with SSc, decreased serum EPCR levels were associated with diffuse skin involvement, interstitial lung disease and digital ulcers. Furthermore, serum EPCR levels inversely correlated with plasma levels of plasmin-α2-plasmin inhibitor complex (PIC). Importantly, bosentan significantly reversed circulating EPCR and PIC levels in patients with SSc, and the expression of Fli1 and EPCR in dermal small vessels was elevated in patients treated with bosentan compared with untreated patients. CONCLUSIONS: Endothelial EPCR downregulation due to Fli1 deficiency may contribute to hypercoagulation status leading to tissue fibrosis and impaired peripheral circulation in SSc.

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.

Fibrosis, vascular activation, and immune abnormalities resembling systemic sclerosis in bleomycin-treated Fli-1-haploinsufficient mice.

OBJECTIVE: Fli-1, a potential predisposing factor for systemic sclerosis (SSc), is constitutively down-regulated in the lesional skin of patients with SSc by an epigenetic mechanism. To investigate the impact of Fli-1 deficiency on the induction of an SSc phenotype in various cell types, we generated bleomycin-induced skin fibrosis in Fli-1(+/-) mice and investigated the molecular mechanisms underlying its phenotypic alterations. METHODS: Messenger RNA (mRNA) levels and protein expression of target molecules were examined by quantitative reverse transcription-polymerase chain reaction and immunostaining. Transforming growth factor ß (TGFß) bioassay was used to evaluate the activation of latent TGFß. The binding of Fli-1 to the target gene promoters was assessed with chromatin immunoprecipitation. RESULTS: Bleomycin induced more severe dermal fibrosis in Fli-1(+/-) mice than in wild-type mice. Fli-1 haploinsufficiency activated dermal fibroblasts via the up-regulation of αvß3 and αvß5 integrins and activation of latent TGFß. Dermal fibrosis in Fli-1(+/-) mice was also attributable to endothelial-to-mesenchymal transition, which is directly induced by Fli-1 deficiency and amplified by bleomycin. Th2/Th17-skewed inflammation and increased infiltration of mast cells and macrophages were seen, partly due to the altered expression of cell adhesion molecules in endothelial cells as well as the induction of the skin chemokines. Fli-1(+/-) mouse macrophages preferentially differentiated into an M2 phenotype upon stimulation with interleukin-4 (IL-4) or IL-13. CONCLUSION: Our findings provide strong evidence for the fundamental role of Fli-1 deficiency in inducing SSc-like phenotypic alterations in dermal fibroblasts, endothelial cells, and macrophages in a manner consistent with human disease.

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

OBJECTIVES: 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. METHODS: 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. RESULTS: 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-ß1 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. CONCLUSION: Chemerin is down-regulated in SSc dermal fibroblasts by autocrine TGF-ß, 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.

Fli1 acts downstream of Etv2 to govern cell survival and vascular homeostasis via positive autoregulation.

RATIONALE: Cardiovascular health depends on proper development and integrity of blood vessels. Ets variant 2 (Etv2), a member of the E26 transforming-specific family of transcription factors, is essential to initiate a transcriptional program leading to vascular morphogenesis in early mouse embryos. However, endothelial expression of the Etv2 gene ceases at midgestation; therefore, vascular development past this stage must continue independent of Etv2. OBJECTIVE: To identify molecular mechanisms underlying transcriptional regulation of vascular morphogenesis and homeostasis in the absence of Etv2. METHODS AND RESULTS: Using loss- and gain-of-function strategies and a series of molecular techniques, we identify Friend leukemia integration 1 (Fli1), another E26 transforming-specific family transcription factor, as a downstream target of Etv2. We demonstrate that Etv2 binds to conserved Ets-binding sites within the promoter region of the Fli1 gene and governs Fli1 expression. Importantly, in the absence of Etv2 at midgestation, binding of Etv2 at Ets-binding sites in the Fli1 promoter is replaced by Fli1 protein itself, sustaining expression of Fli1 as well as selective Etv2-regulated endothelial genes to promote endothelial cell survival and vascular integrity. Consistent with this, we report that Fli1 binds to the conserved Ets-binding sites within promoter and enhancer regions of other Etv2-regulated endothelial genes, including Tie2, to control their expression at and beyond midgestation. CONCLUSIONS: We have identified a novel positive feed-forward regulatory loop in which Etv2 activates expression of genes involved in vasculogenesis, including Fli1. Once the program is activated in early embryos, Fli1 then takes over to sustain the process in the absence of Etv2.

Fli1 deficiency contributes to the suppression of endothelial CXCL5 expression in systemic sclerosis.

CXCL5 is a member of CXC chemokines with neutrophilic chemoattractant and pro-angiogenic properties, which has been implicated in the pathological angiogenesis of rheumatoid arthritis and inflammatory bowel diseases. Since aberrant angiogenesis is also involved in the developmental process of systemic sclerosis (SSc), we herein measured serum CXCL5 levels in 63 SSc and 18 healthy subjects and investigated their clinical significance and the mechanism explaining altered expression of CXCL5 in SSc. Serum CXCL5 levels were significantly lower in SSc patients than in healthy subjects. In diffuse cutaneous SSc (dcSSc), serum CXCL5 levels were uniformly decreased in early stage (<1 year) and positively correlated with disease duration in patients with disease duration of <6 years. In non-early stage dcSSc (≥1 year), decreased serum CXCL5 levels were linked to the development of digital ulcers. Consistently, the expression levels of CXCL5 proteins were decreased in dermal blood vessels of early stage dcSSc. Importantly, Fli1 bound to the CXCL5 promoter and its gene silencing significantly suppressed the CXCL5 mRNA expression in human dermal microvascular endothelial cells. Furthermore, endothelial cell-specific Fli1 knockout mice, an animal model of SSc vasculopathy, exhibited decreased CXCL5 expression in dermal blood vessels. Collectively, these results indicate that CXCL5 is a member of angiogenesis-related genes, whose expression is suppressed at least partially due to Fli1 deficiency in SSc endothelial cells. Since Fli1 deficiency is deeply related to aberrant angiogenesis in SSc, it is plausible that serum CXCL5 levels inversely reflect the severity of SSc vasculopathy.

[A Uuifying hypothesis for the pathogenesis of systemic sclerosis based on the deficiency of transcription factor Fli1 - the development of a new animal model of systemic sclerosis -].

Systemic sclerosis (SSc) is a multisystem connective tissue disease featured by immune abnormalities, vasculopathy and resultant fibrosis of the skin and various internal organs. Although the pathogenesis of SSc remains incompletely elucidated, it is currently accepted that this disease is caused by the complex interplay between hereditary and environmental factors. The deficiency of transcription factor Fli1, which is epigenetically suppressed in SSc dermal fibroblasts, potentially causes SSc-like phenotypical alteration in various cell types such as fibroblasts, endothelial cells, and macrophages, suggesting that Fli1 is a predisposing factor of SSc. KLF5 is another transcription factor which is suppressed in SSc dermal fibroblasts through an epigenetic mechanism. Importantly, double heterozygous mice for Fli1 and KLF5 develop three cardinal features of SSc, including immune abnormalities, vasculopathy and fibrosis. Therefore, these two transcription factors are likely to be critical predisposing factors regulating the development of SSc. Given that potential disease modifying drugs, bosentan and imatinib, reverse the expression and transcriptional activity of Fli1, the studies on the pathological process of double heterozygous mice and the impact of these transcription factors on various cell types may provide a new clue to further understand the pathogenesis of SSc leading to the development of new therapies.

Flightless I over-expression impairs skin barrier development, function and recovery following skin blistering.

Development of an intact epidermis is critical for maintaining the integrity of the skin. Patients with epidermolysis bullosa (EB) experience multiple erosions, which breach the epidermal barrier and lead to increased microbial colocalization of wounds, infections and sepsis. The cytoskeletal protein Flightless I (Flii) is a known regulator of both development and wound healing. Using Flii(+/-), WT and Flii(Tg/Tg) mice, we investigated the effect of altering Flii levels in embryos and adult mice on the development of the epidermal barrier and, consequently, how this affects the integrity of the skin in EB. Flii over-expression resulted in delayed formation of the epidermal barrier in embryos and decreased expression of tight junction (TJ) proteins Claudin-1 and ZO-2. Increased intercellular space and transepidermal water loss was observed in Flii(Tg)(/Tg) adult mouse skin, while Flii(Tg/Tg) keratinocytes showed altered TJ protein localization and reduced transepithelial resistance. Flii is increased in the blistered skin of patients with EB, and over-expression of Flii in experimental EBA showed impaired Claudin-1 and -4 TJ protein expression and delayed recovery of functional barrier post-blistering. Immunoprecipitation confirmed Flii associated with TJ proteins and in vivo actin assays showed that the effect of Flii on actin polymerization underpinned the impaired barrier function observed in Flii(Tg/Tg) mice. These results therefore demonstrate an important role for Flii in the development and regulation of the epidermal barrier, which may contribute to the impaired healing and skin fragility of EB patients.

Reducing FLI1 levels in the MRL/lpr lupus mouse model impacts T cell function by modulating glycosphingolipid metabolism.

Systemic Lupus erythematosus (SLE) is an autoimmune disease caused, in part, by abnormalities in cells of the immune system including B and T cells. Genetically reducing globally the expression of the ETS transcription factor FLI1 by 50% in two lupus mouse models significantly improves disease measures and survival through an unknown mechanism. In this study we analyze the effects of reducing FLI1 in the MRL/lpr lupus prone model on T cell function. We demonstrate that adoptive transfer of MRL/lpr Fli1(+/+) or Fli1(+/-) T cells and B cells into Rag1-deficient mice results in significantly decreased serum immunoglobulin levels in animals receiving Fli1(+/-) lupus T cells compared to animals receiving Fli1(+/+) lupus T cells regardless of the genotype of co-transferred lupus B cells. Ex vivo analyses of MRL/lpr T cells demonstrated that Fli1(+/-) T cells produce significantly less IL-4 during early and late disease and exhibited significantly decreased TCR-specific activation during early disease compared to Fli1(+/+) T cells. Moreover, the Fli1(+/-) T cells expressed significantly less neuraminidase 1 (Neu1) message and decreased NEU activity during early disease and significantly decreased levels of glycosphingolipids during late disease compared to Fli1(+/+) T cells. FLI1 dose-dependently activated the Neu1 promoter in mouse and human T cell lines. Together, our results suggest reducing FLI1 in lupus decreases the pathogenicity of T cells by decreasing TCR-specific activation and IL-4 production in part through the modulation of glycosphingolipid metabolism. Reducing the expression of FLI1 or targeting the glycosphingolipid metabolic pathway in lupus may serve as a therapeutic approach to treating lupus.

Decreased cathepsin V expression due to Fli1 deficiency contributes to the development of dermal fibrosis and proliferative vasculopathy in systemic sclerosis.

OBJECTIVES: Cathepsin V (CTSV) is a proteolytic enzyme potentially modulating angiogenic processes, collagen degradation and keratinocyte differentiation. We aimed to investigate the clinical association of serum CTSV levels and the mechanism by which CTSV expression is altered in SSc. METHODS: Serum CTSV levels were determined by ELISA in 51 SSc and 18 healthy subjects. CTSV expression was evaluated by immunostaining in SSc and normal skin and by RT-real-time PCR in normal and SSc dermal fibroblasts, normal dermal fibroblasts treated with TGF-ß1 or Fli1 siRNA and human dermal microvascular endothelial cells (ECs) treated with Fli1 siRNA. RESULTS: Serum CTSV levels were significantly lower in dcSSc and lcSSc patients than in healthy controls. In early-stage dcSSc, serum CTSV levels were remarkably and uniformly decreased compared with healthy controls. The decrease in serum CTSV levels in mid- and late-stage dcSSc and in lcSSc was linked to the development of proliferative vasculopathy. CTSV expression was decreased in microvascular ECs, pericytes/vascular smooth muscle cells and keratinocytes of dcSSc and lcSSc skin and in dermal fibroblasts of dcSSc skin compared with control skin. Consistently, CTSV expression was decreased in cultured dermal fibroblasts from early-stage dcSSc. Furthermore, mRNA levels of the CTSV gene were significantly decreased in normal fibroblasts treated with TGF-ß1 or Fli1 siRNA and in human dermal microvascular ECs treated with Fli1 siRNA. CONCLUSION: Loss of CTSV expression may contribute to the development of fibrosis, vasculopathy and the altered phenotype of keratinocytes in SSc.

Fli-1 transcription factor affects glomerulonephritis development by regulating expression of monocyte chemoattractant protein-1 in endothelial cells in the kidney.

Expression of transcription factor Fli-1 is implicated in the development of glomerulonephritis. Fli-1 heterozygous knockout (Fli1(+/-)) NZM2410 mice, a murine model of lupus, had significantly improved survival and reduced glomerulonephritis. In this study, we found that infiltrated inflammatory cells were significantly decreased in the kidneys from Fli-1(+/-) NZM2410 mice. The expression of monocyte chemoattractant protein-1 (MCP-1) was significantly decreased in kidneys from Fli-1(+/-) NZM2410 mice. The primary endothelial cells isolated from the kidneys of Fli-1(+/-) NZM2410 mice produced significantly less MCP-1. In endothelial cells transfected with specific Fli-1 siRNA the production of MCP-1 was significantly reduced compared to cells transfected with negative control siRNA. By Chromatin Immunoprecipitation (ChIP) assay, we further demonstrated that Fli-1 directly binds to the promoter of the MCP-1 gene. Our data indicate that Fli-1 impacts glomerulonephritis development by regulating expression of inflammatory chemokine MCP-1 and inflammatory cell infiltration in the kidneys in the NZM2410 mice.

A novel oncogenic mechanism in Ewing sarcoma involving IGF pathway targeting by EWS/Fli1-regulated microRNAs.

MicroRNAs (miRs) are a novel class of cellular bioactive molecules with critical functions in the regulation of gene expression in normal biology and disease. MiRs are frequently misexpressed in cancer, with potent biological consequences. However, relatively little is known about miRs in pediatric cancers, including sarcomas. Moreover, the mechanisms behind aberrant miR expression in cancer are poorly understood. Ewing sarcoma is an aggressive pediatric malignancy driven by EWS/Ets fusion oncoproteins, which are gain-of-function transcriptional regulators. We employed stable silencing of EWS/Fli1, the most common of the oncogenic fusions, and global miR profiling to identify EWS/Fli1-regulated miRs with oncogenesis-modifying roles in Ewing sarcoma. In this report, we characterize a group of miRs (100, 125b, 22, 221/222, 27a and 29a) strongly repressed by EWS/Fli1. Strikingly, all of these miRs have predicted targets in the insulin-like growth factor (IGF) signaling pathway, a pivotal driver of Ewing sarcoma oncogenesis. We demonstrate that miRs in this group negatively regulate the expression of multiple pro-oncogenic components of the IGF pathway, namely IGF-1, IGF-1 receptor, mammalian/mechanistic target of rapamycin and ribosomal protein S6 kinase A1. Consistent with tumor-suppressive functions, these miRs manifest growth inhibitory properties in Ewing sarcoma cells. Our studies thus uncover a novel oncogenic mechanism in Ewing sarcoma, involving post-transcriptional derepression of IGF signaling by the EWS/Fli1 fusion oncoprotein via miRs. This novel pathway may be amenable to innovative therapeutic targeting in Ewing sarcoma and other malignancies with activated IGF signaling.