The endoplasmic reticulum stress response participates in connexin 43-mediated ossification of the posterior longitudinal ligament.

Ossification of the posterior longitudinal ligament (OPLL) manifests as ectopic bone formation in spinal ligament tissue. As revealed by in vitro 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.