Yoshihide Asano

Simultaneous downregulation of KLF5 and Fli1 is a key feature underlying systemic sclerosis

Systemic sclerosis (SSc) is manifested by fibrosis, vasculopathy and immune dysregulation. So far, a unifying hypothesis underpinning these pathological events remains unknown. Given that SSc is a multifactorial disease caused by both genetic and environmental factors, we focus on the two transcription factors, which modulate the fibrotic reaction and are epigenetically suppressed in SSc dermal fibroblasts, Friend leukemia integration 1 (Fli1) and Krüppel-like factor 5 (KLF5). In addition to Fli1 silencing-dependent collagen induction, simultaneous knockdown of Fli1 and KLF5 synergistically enhances expression of connective tissue growth factor. Notably, mice with double heterozygous deficiency of Klf5 and Fli1 mimicking the epigenetic phenotype of SSc skin spontaneously recapitulate all the three features of SSc, including fibrosis and vasculopathy of the skin and lung, B cell activation, and autoantibody production. These studies implicate the epigenetic downregulation of Fli1 and KLF5 as a central event triggering the pathogenic triad of SSc.

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

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.

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

AbstractnCXCL5 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 (<1xa0year) and positively correlated with disease duration in patients with disease duration of <6xa0years. In non-early stage dcSSc (≥1xa0year), 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.

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.

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.

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

OBJECTIVESnChemerin 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.nnnMETHODSnChemerin 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.nnnRESULTSnIn 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.nnnCONCLUSIONnChemerin 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.

Endothelin Receptor Blockade Ameliorates Vascular Fragility in Endothelial Cell–Specific Fli‐1–Knockout Mice by Increasing Fli‐1 DNA Binding Ability

It is generally accepted that blockade of endothelin receptors has potentially beneficial effects on vasculopathy associated with systemic sclerosis (SSc). The aim of this study was to clarify the molecular mechanism underlying these effects using endothelial cell–specific Fli‐1–knockout (Fli‐1 ECKO) mice, an animal model of SSc vasculopathy.

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

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).

Progranulin Overproduction Due to Fli-1 Deficiency Contributes to the Resistance of Dermal Fibroblasts to Tumor Necrosis Factor in Systemic Sclerosis.

Progranulin is a growth factor that is active in wound repair and is an antagonist of tumor necrosis factor (TNF) receptors, regulating fibroblast activation, angiogenesis, and inflammation. Because long‐standing activation of gene programs related to wound healing is a hallmark of systemic sclerosis (SSc), we sought to investigate the role of progranulin in SSc.

A potential contribution of altered cathepsin L expression to the development of dermal fibrosis and vasculopathy in systemic sclerosis.

Cathepsin L (CTSL) is a lysosomal proteolytic enzyme involved in inflammation and vascular and extracellular matrix remodelling, which are the three cardinal pathological events associated with systemic sclerosis (SSc). To elucidate the potential role of CTSL in the development of SSc, we here investigated CTSL expression in the lesional skin of patients with SSc and SSc animal models and the clinical correlation of serum CTSL levels. CTSL expression was elevated in dermal small vessels of SSc patients compared with those of healthy controls. Consistently, CTSL mRNA levels were increased in SSc lesional skin samples, but not in cultivated SSc dermal fibroblasts, compared with corresponding control samples from healthy individuals. Serum CTSL levels were significantly higher in SSc patients than in healthy controls and inversely correlated with skin score. Furthermore, the elevation of serum CTSL levels was linked to SSc vasculopathy. Supporting these results, Ctsl mRNA levels were decreased in the skin of bleomycin‐treated mice, an SSc animal model recapitulating its fibrotic aspect, and CTSL expression was enhanced in dermal small vessels of endothelial cell‐specific Fli1 knockout mice, reminiscent of SSc vasculopathy. Importantly, gene silencing of FLI1 induced CTSL mRNA expression and Fli1 occupied the CTSL promoter in human dermal microvascular endothelial cells. Collectively, these results suggest that endothelial CTSL up‐regulation partially due to Fli1 deficiency may contribute to the development of vasculopathy, while the decrease in dermal CTSL expression is likely associated with dermal fibrosis in SSc.