ABSTRACT

Aim: 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.

Materials and methods: 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.

Results: 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.

Conclusions: 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.

摘要

目的：成纤维细胞的病理性增生是导致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，被发现在各种纤维化组织中过表达。然而，LAMA4 在膝关节纤维化中的作用仍然难以捉摸。因此，本研究旨在探讨LAMA4对成纤维细胞增殖和迁移的影响及其机制。

材料与方法：膝关节手术后，分别在第2周和第4周检测到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 测定评估细胞迁移，并通过 qRT-PCR、蛋白质印迹和免疫荧光分析。此外，环巴胺在 LAMA4 过表达的成纤维细胞中进一步阻断了经典的 Shh 级联，并研究了细胞增殖和迁移的变化。

结果：LAMA4表达与膝关节纤维化严重程度呈正相关。功能研究表明，LAMA4 过表达促进了成纤维细胞的增殖、细胞周期进程和迁移。机械地，LAMA4 激活典型的 Shh/Gli1 信号并促进 Gli1 的核转位以上调与细胞增殖和迁移相关的基因的表达。有趣的是，环巴胺阻断 Shh/Gli1 信号通路逆转了 LAMA4 对成纤维细胞增殖和迁移的促进作用。

结论：LAMA4通过激活Shh/Gli1信号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引起的关节内纤维化的潜在治疗靶点。