Cao Hua-Li, Zhou Jiong, Chen Xi-Bei, Landeck Lilla, Yang Jian-Qiang, Chen Jia-Qi, Li Wei, Cai Sui-Qing, Zheng Min, Man Xiao-Yong
Department of Dermatology, Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310009, China.
These authors contributed equally to this article.
Discov Med. 2016 Nov;22(122):311-318.
Dermal fibrosis is characterized by the activation of the matrix-producing 'positive' myofibroblasts, and the relentless production and deposition of extracellular matrix. The hedgehog pathway has recently been demonstrated to work in a pro-fibrotic manner in systemic sclerosis (SSc). A negative regulator of the hedgehog pathway (Hh), the suppressor of fused (Sufu), was shown to be involved in the activation of fibrotic diseases. However, the exact role of Sufu in fibrosis has not been investigated so far. In our study, we aimed to define the role of sufu in the process of fibrosis using dermal fibroblasts of healthy donors that were cultured in vitro. Cyclopamine, a Smo antagonist, and Sufu lentivector were used to treat or transfect cells. The expression of fibrosis markers and ERK1/2, Smad2, and GSK3β at the protein level was determined by Western blot. Fibroblast migration was measured by in vitro wound healing assay. Bleomycin-induced dermal fibrosis mouse model was introduced to assess the effect of cyclopamine on dermal fibrosis in vivo. We found that cyclopamine significantly upregulated the expression of Sufu. Both cyclopamine and Sufu lentivector reduced migration and myofibroblast differentiation of human dermal fibroblasts at a statistically significant level. Furthermore, cyclopamine reversed dermal fibrosis induced by TGF-β1. Cyclopamine and the overexpression of Sufu inhibited the phosphorylation of GSK-3β and restrained the migration of fibroblasts. Dermal fibrosis was inhibited by intraperitoneal injection of cyclopamine in a mouse model of scleroderma. Our findings suggest that cyclopamine and Sufu-overexpression may effectively inhibit the endogenous as well as the TGF-β1-induced activation of fibroblasts through subsequent activation of GSK-3β. Sufu agonists may be a promising approach in the development of antifibrotic medications for dermal fibrosis and systemic sclerosis.
皮肤纤维化的特征是产生基质的“阳性”肌成纤维细胞被激活，以及细胞外基质持续产生和沉积。最近已证明刺猬信号通路在系统性硬化症（SSc）中以促纤维化方式发挥作用。刺猬信号通路（Hh）的负调节因子，即融合抑制因子（Sufu），被证明与纤维化疾病的激活有关。然而，迄今为止，Sufu在纤维化中的确切作用尚未得到研究。在我们的研究中，我们旨在利用体外培养的健康供体的皮肤成纤维细胞来确定Sufu在纤维化过程中的作用。使用环杷明（一种Smo拮抗剂）和Sufu慢病毒载体来处理或转染细胞。通过蛋白质印迹法测定纤维化标志物以及ERK1/2、Smad2和GSK3β在蛋白质水平的表达。通过体外伤口愈合试验测量成纤维细胞迁移。引入博来霉素诱导的皮肤纤维化小鼠模型以评估环杷明对体内皮肤纤维化的影响。我们发现环杷明显着上调Sufu的表达。环杷明和Sufu慢病毒载体均在统计学显著水平上降低了人皮肤成纤维细胞的迁移和肌成纤维细胞分化。此外，环杷明逆转了TGF-β1诱导的皮肤纤维化。环杷明和Sufu的过表达抑制了GSK-3β的磷酸化并抑制了成纤维细胞的迁移。在硬皮病小鼠模型中，腹腔注射环杷明可抑制皮肤纤维化。我们的研究结果表明，环杷明和Sufu过表达可能通过随后激活GSK-3β有效抑制内源性以及TGF-β1诱导的成纤维细胞激活。Sufu激动剂可能是开发用于皮肤纤维化和系统性硬化症的抗纤维化药物的一种有前景的方法。
