Shi Lei, Shi Guodong, Li Tiefeng, Luo Yibin, Chen Deyu, Miao Jinhao, Chen Yu
Spine Center, Department of Orthopaedics, Changzheng Hospital, Second Military Medical University Shanghai 200003, China.
Am J Transl Res. 2019 Jul 15;11(7):4113-4125. eCollection 2019.
Ossification of the posterior longitudinal ligament (OPLL) manifests as ectopic bone formation in spinal ligament tissue. As revealed by studies, fibroblasts from patients with OPLL or healthy ligament fibroblasts undergo mechanical stress (MS). We previously demonstrated that a cell-cell junction protein, connexin 43 (Cx43), is significantly up-regulated in OPLL cells and previous data indicated that some proteins related to the endoplasmic reticulum (ER) stress response are elevated during the development of OPLL. The present study utilized gain- and loss-of-function tools to delineate the contribution of the ER stress response within ligament fibroblasts under OPLL-inducing stimuli and the crosstalk between Cx43 signaling and the ER stress response. The ER stress process was augmented by the induction of Cx43 expression in OPLL cells or cells under MS. Cx43 over-expression also promoted ER stress and ossification in OPLL cells. Moreover, the activation of ER stress was accompanied with increased oxidative stress, which was inhibited by Cx43 gene silencing. Cx43 knockdown also improved ER stress-related ossification in OPLL cells. The blockage of ER stress using a chemical compound or small interfering RNA was sufficient to overcome MS-induced ossification in OPLL cells. These findings were further validated in patients with OPLL, as the mRNA levels of Cx43 and PKR-like endoplasmic reticulum kinase (a single-pass type I ER membrane protein kinase), a major transducer of ER stress, were significantly increased compared with non-OPLL subjects. In conclusion, this study demonstrates that ER stress participates in Cx43-related OPLL.
后纵韧带骨化（OPLL）表现为脊柱韧带组织中的异位骨形成。研究表明，OPLL患者的成纤维细胞或健康韧带成纤维细胞会受到机械应力（MS）作用。我们之前证明，一种细胞间连接蛋白，连接蛋白43（Cx43），在OPLL细胞中显著上调，并且之前的数据表明，一些与内质网（ER）应激反应相关的蛋白质在OPLL发展过程中升高。本研究利用功能获得和功能丧失工具来描述在OPLL诱导刺激下韧带成纤维细胞内ER应激反应的作用以及Cx43信号与ER应激反应之间的相互作用。在OPLL细胞或受到MS作用的细胞中诱导Cx43表达可增强ER应激过程。Cx43过表达还促进了OPLL细胞中的ER应激和骨化。此外，ER应激的激活伴随着氧化应激增加，而Cx43基因沉默可抑制氧化应激。Cx43敲低也改善了OPLL细胞中与ER应激相关的骨化。使用化合物或小干扰RNA阻断ER应激足以克服OPLL细胞中MS诱导的骨化。这些发现在OPLL患者中得到进一步验证，因为与非OPLL受试者相比，Cx43和PKR样内质网激酶（一种单次跨膜I型内质网膜蛋白激酶，是ER应激的主要转导因子）的mRNA水平显著升高。总之，本研究表明ER应激参与了与Cx43相关的OPLL。
