Abstract
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.
Highlights
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
ET-1 rapidly induced the expression of type I collagen via the non-Smad signaling pathway As an initial experiment, to determine the optimal dose of ET-1 to induce the expression of type I collagen in normal dermal fibroblasts, cells were treated with ET-1 at the concentration of 0, 50, 100, 200, or 400 nM for 24 hours and mRNA levels of the human α2 collagen (COL1A2) gene were determined by reverse transcription (RT) real-time PCR
As Smad3 has a big impact on the transforming growth factor (TGF)-β-dependent regulation of type I collagen expression, we investigated the effect of Smad3 inhibitor SIS3 on the ET-1-induced mRNA expression of the COL1A2 gene
Summary
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. A wealth of evidence suggests that, once activated, SSc dermal fibroblasts establish a selfactivation system by autocrine transforming growth factor (TGF)-β stimulation at least partially via upregulating cell surface receptors for latent-form TGF-β, such as integrin αVβ3, integrin αVβ5, and thrombospondin-1 [2,3,4,5,6]. In animal models, forced expression of ET-1 accelerates dermal wound healing as well as TGF-β1, while blockade of endothelin signaling with bosentan, a dual endothelin receptor antagonist, significantly inhibits the effect of TGF-β1 on dermal wound healing [12]. Endothelins are potentially implicated in the pathogenesis of fibrotic disorders
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