Wan Qi, Liu Fang, Zhang Jie, Chen Hui, Yan Lianqi, Li Xiaolei, Sun Yu, Wang Jingcheng
Department of Orthopedics, The Second Xiangya Hospital, Central South University, Changsha, Hunan, China.
Department of Orthopedics, Yueyang Second People's Hospital, Yueyang Hospital Affiliated to Hunan Normal University, Yueyang, Hunan, China.
Connect Tissue Res. 2021 Jul;62(4):464-474. doi: 10.1080/03008207.2020.1773451. Epub 2020 Jun 10.
: Pathologic hyperplasia of fibroblast is responsible for the progression of intraarticular fibrosis. Laminin α4 (LAMA4), a subunit of laminin macromolecule family, was found to be overexpressed in various fibrotic tissues. However, the role of LAMA4 in knee arthrofibrosis remains elusive. Therefore, the aim of this study was to investigate the effect and mechanism of LAMA4 on fibroblast proliferation and migration. : Following knee surgery, LAMA4 expression was detected in intraarticular fibrous tissues in rabbits at week 2 and week 4, respectively. In lentivirus-mediated LAMA4-overexpressed fibroblasts, cellular proliferation was assessed by EdU labeling and cell cycle analysis, cellular migration was evaluated using Transwell assay, and the expressions of key components in Shh/Gli1 signaling were detected by qRT-PCR, western blot and immunofluorescence analysis. Additionally, canonical Shh cascade was further blocked in LAMA4-overexpressed fibroblasts by cyclopamine, and the changes in cellular proliferation and migration were investigated. : LAMA4 expression was positively correlated with the severity of knee arthrofibrosis. Functional studies demonstrated that LAMA4 overexpression facilitated proliferation, cell cycle progression and migration in fibroblasts. Mechanically, LAMA4 activated the canonical Shh/Gli1 signaling and promoted the nuclear translocation of Gli1 to upregulate expression of genes associated with cellular proliferation and migration. Intriguingly, blockage of Shh/Gli1 signaling with cyclopamine reversed the promoting effects of LAMA4 on proliferation and migration of fibroblasts. : LAMA4 positively regulated cellular proliferation and migration in fibroblasts via activating the Shh/Gli1 signaling. LAMA4/Shh/Gli1 signaling axis might be a potential therapeutic target for the prevention of surgery-induced intraarticular fibrosis.
成纤维细胞的病理性增生是关节内纤维化进展的原因。层粘连蛋白α4（LAMA4）是层粘连蛋白大分子家族的一个亚基，在各种纤维化组织中被发现过度表达。然而，LAMA4在膝关节纤维性关节病中的作用仍不清楚。因此，本研究的目的是探讨LAMA4对成纤维细胞增殖和迁移的影响及机制。：膝关节手术后，分别在第2周和第4周检测兔关节内纤维组织中LAMA4的表达。在慢病毒介导的LAMA4过表达的成纤维细胞中，通过EdU标记和细胞周期分析评估细胞增殖，使用Transwell实验评估细胞迁移，并通过qRT-PCR、蛋白质免疫印迹和免疫荧光分析检测Shh/Gli1信号通路关键成分的表达。此外，用环杷明进一步阻断LAMA4过表达的成纤维细胞中的经典Shh信号级联反应，并研究细胞增殖和迁移的变化。：LAMA4表达与膝关节纤维性关节病的严重程度呈正相关。功能研究表明，LAMA4过表达促进成纤维细胞的增殖、细胞周期进程和迁移。机制上，LAMA4激活经典的Shh/Gli1信号通路，促进Gli1的核转位，上调与细胞增殖和迁移相关的基因表达。有趣的是，用环杷明阻断Shh/Gli1信号通路可逆转LAMA4对成纤维细胞增殖和迁移的促进作用。：LAMA4通过激活Shh/Gli1信号通路正向调节成纤维细胞的增殖和迁移。LAMA4/Shh/Gli1信号轴可能是预防手术诱导的关节内纤维化的潜在治疗靶点。
