Bosentan reverses the pro-fibrotic phenotype of systemic sclerosis dermal fibroblasts via increasing DNA binding ability of transcription factor Fli1.
Bosentan reverses the pro-fibrotic phenotype of systemic sclerosis dermal fibroblasts via increasing DNA binding ability of transcription factor Fli1.
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DOI：
10.1186/ar4529
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发表时间：
2014-04-03
影响因子：
4.9
通讯作者：
Sato S
中科院分区：
文献类型：
--
作者：
Akamata K;Asano Y;Aozasa N;Noda S;Taniguchi T;Takahashi T;Ichimura Y;Toyama T;Sato S
尽管系统性硬化症（SSc）的发病机制仍不清楚，但最近的研究表明，内皮素深深参与了与SSc相关的纤维化和血管病变的发展过程，而双重内皮素受体拮抗剂波生坦有可能作为这种疾病的疾病缓解药物。重要的是，内皮素-1（ET-1）对正常真皮成纤维细胞发挥促纤维化作用，波生坦逆转SSc真皮成纤维细胞的促纤维化表型。本研究的目的是阐明ET-1和波生坦对真皮成纤维细胞影响的分子机制的细节，这些机制尚未得到充分研究。采用逆转录实时荧光定量PCR和免疫印迹法分别检测靶基因的mRNA水平和靶蛋白的表达及磷酸化水平。使用人α 2（I）胶原（COL1A2）启动子的顺序缺失进行启动子测定。DNA亲和沉淀和染色质免疫沉淀法检测Fli1与DNA的结合能力。通过免疫染色评估小鼠皮肤中的Fli1蛋白水平。在正常成纤维细胞中，ET-1激活c-Abl和蛋白激酶C（PKC）-δ，并诱导Fli1在苏氨酸312处磷酸化，导致Fli1（COL1A2基因的有效阻遏物）的DNA结合减少，以及I型胶原表达增加。另一方面，波生坦降低了c-Abl和PKC-δ的表达、PKC-δ的核定位和Fli1磷酸化，导致SSc成纤维细胞中Fli1的DNA结合增加和I型胶原表达抑制。在博来霉素处理的小鼠中，波生坦可预防真皮纤维化并增加皮损真皮成纤维细胞中Fli1的表达。ET-1通过激活"c-Abl-PKC-δ-Fli1"通路对正常成纤维细胞发挥强有力的促纤维化作用。波生坦逆转SSc成纤维细胞的促纤维化表型，并通过阻断这一信号传导途径防止博来霉素处理小鼠中真皮纤维化的发展。虽然波生坦对SSc皮肤和肺纤维化的疗效有限，但目前的观察结果无疑为我们提供了一个有用的线索，以进一步探索即将推出的新型双重内皮素受体拮抗剂作为SSc疾病改善药物的潜力。
Although 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. The 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. In 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. ET-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.