Shenzhen Key Laboratory of Ophthalmology, Shenzhen Eye Hospital, Shenzhen University School of Medicine, No. 18, Zetian Road, Futian District, Shenzhen, 518040, China.
In Vitro Cell Dev Biol Anim. 2020 Mar;56(3):234-242. doi: 10.1007/s11626-019-00424-6. Epub 2020 Mar 18.
Granular corneal dystrophy (GCD) is featured by corneal deposits of transforming growth factor beta-induced gene (TGFBI) mediated by the TGF-β (transforming growth factor-β)/Smad signaling. However, the roles of c-Jun amino-terminal kinase (JNK) pathway in GCD pathogenesis remains unexplored, which was investigated in this study. JNK signaling activation and inhibition in primary corneal fibroblasts were obtained by treatments with anisomycin and SP600125, respectively. Protein abundance and phosphorylation were detected by immunoblotting. Cell viability and apoptosis were analyzed by CCK-8 and flow cytometry respectively. TGFBI deposit and autophagy progression were assessed by immunofluorescence. The results found that JNK1 expression and phosphorylation were greatly increased in corneal tissues from GCD2 patients. JNK signaling activation impaired the viability and promoted apoptosis and autophagy processes in primary corneal fibroblasts, along with Smad2/3 phosphorylation, TGFBI accumulation and Bcl-2 suppression. Autophagy related proteins, such as ATG5 (autophagy related 5), ATG12 (autophagy related 12) and LC3B (microtubule-associated protein 1 light chain 3 beta), were also increased in anisomycin or TGF-β1 treated corneal fibroblasts. However, SP600125 effectively reversed the above effect induced by TGF-β1 treatment in corneal fibroblasts, including the TGF-β-induced autophagy progression. The results suggested that JNK signaling was activated in GCD2 corneal tissues, and it mediated the TGF-β-induced TGFBI protein accumulation and apoptosis of corneal fibroblasts during GCD2 pathogenesis.
颗粒状角膜营养不良（GCD）的特征是转化生长因子β诱导基因（TGFBI）介导的转化生长因子-β（TGF-β）/Smad 信号转导在角膜中的沉积。然而，c-Jun 氨基末端激酶（JNK）通路在 GCD 发病机制中的作用仍未被探索，本研究对此进行了研究。通过用anisomycin 和 SP600125 处理分别获得原代角膜成纤维细胞中 JNK 信号通路的激活和抑制。通过免疫印迹检测蛋白质丰度和磷酸化。通过 CCK-8 和流式细胞术分别分析细胞活力和凋亡。通过免疫荧光评估 TGFBI 沉积和自噬进展。结果发现，GCD2 患者角膜组织中 JNK1 表达和磷酸化显著增加。JNK 信号通路的激活损害了原代角膜成纤维细胞的活力，并促进了凋亡和自噬过程，同时还伴随着 Smad2/3 磷酸化、TGFBI 积累和 Bcl-2 抑制。自噬相关蛋白，如 ATG5（自噬相关 5）、ATG12（自噬相关 12）和 LC3B（微管相关蛋白 1 轻链 3β），也在 anisomycin 或 TGF-β1 处理的角膜成纤维细胞中增加。然而，SP600125 可有效逆转 TGF-β1 处理诱导的角膜成纤维细胞中的上述效应，包括 TGF-β 诱导的自噬进展。结果表明，JNK 信号在 GCD2 角膜组织中被激活，它介导了 TGF-β 诱导的 TGFBI 蛋白积累和 GCD2 发病过程中角膜成纤维细胞的凋亡。
