Comprehensive Pneumology Center, Ludwig-Maximilians-Universität and Helmholtz Zentrum Munich, Max-Lebsche-Platz 31, 81377, Munich, Germany.
Member of the German Center of Lung Research (DZL), Munich, Germany.
Respir Res. 2018 Apr 19;19(1):67. doi: 10.1186/s12931-018-0768-1.
In idiopathic pulmonary fibrosis (IPF), fibroblasts gain a more migratory phenotype and excessively secrete extracellular matrix (ECM), ultimately leading to alveolar scarring and progressive dyspnea. Here, we analyzed the effects of deficiency of FK506-binding protein 10 (FKBP10), a potential IPF drug target, on primary human lung fibroblast (phLF) adhesion and migration.
Using siRNA, FKBP10 expression was inhibited in phLF in absence or presence of 2ng/ml transforming growth factor-β1 (TGF-β1) and 0.1mM 2-phosphoascorbate. Effects on cell adhesion and migration were monitored by an immunofluorescence (IF)-based attachment assay, a conventional scratch assay, and single cell tracking by time-lapse microscopy. Effects on expression of key players in adhesion dynamics and migration were analyzed by qPCR and Western Blot. Colocalization was evaluated by IF microscopy and by proximity ligation assays.
FKBP10 knockdown significantly attenuated adhesion and migration of phLF. Expression of collagen VI was decreased, while expression of key components of the focal adhesion complex was mostly upregulated. The effects on migration were 2-phosphoascorbate-dependent, suggesting collagen synthesis as the underlying mechanism. FKBP10 colocalized with collagen VI and coating culture dishes with collagen VI, and to a lesser extent with collagen I, abolished the effect of FKBP10 deficiency on migration.
These findings show, to our knowledge for the first time, that FKBP10 interacts with collagen VI and that deficiency of FKBP10 reduces phLF migration mainly by downregulation of collagen VI synthesis. The results strengthen FKBP10 as an important intracellular regulator of ECM remodeling and support the concept of FKBP10 as drug target in IPF.
在特发性肺纤维化（IPF）中，成纤维细胞获得更具迁移能力的表型，并过度分泌细胞外基质（ECM），最终导致肺泡瘢痕形成和进行性呼吸困难。在这里，我们分析了 FK506 结合蛋白 10（FKBP10）缺失对原代人肺成纤维细胞（phLF）黏附和迁移的影响，FKBP10 是潜在的 IPF 药物靶点。
使用 siRNA，在不存在或存在 2ng/ml 转化生长因子-β1（TGF-β1）和 0.1mM 2-磷酸抗坏血酸的情况下，抑制 phLF 中的 FKBP10 表达。通过免疫荧光（IF）基础附着测定、传统划痕测定和延时显微镜下的单细胞跟踪监测细胞粘附和迁移的影响。通过 qPCR 和 Western Blot 分析对粘附动力学和迁移关键参与者表达的影响。通过 IF 显微镜和邻近连接测定评估共定位。
FKBP10 敲低显着减弱了 phLF 的粘附和迁移。胶原 VI 的表达减少，而焦点粘附复合物的关键组成部分的表达大多上调。迁移的影响依赖于 2-磷酸抗坏血酸，表明胶原合成是潜在机制。FKBP10 与胶原 VI 共定位，并与胶原 VI 包被培养皿，以及在较小程度上与胶原 I 共定位，消除了 FKBP10 缺乏对迁移的影响。
这些发现表明，FKBP10 与胶原 VI 相互作用，并且 FKBP10 缺乏会降低 phLF 迁移，主要是通过下调胶原 VI 的合成。结果加强了 FKBP10 作为 ECM 重塑的重要细胞内调节剂的作用，并支持 FKBP10 作为 IPF 药物靶点的概念。
Zotero 插件
