Department of Orthopedics, Affiliated Hospital of North Sichuan Medical College, 637000, Nanchong, China.
Department of Orthopedics, West China Hospital, Sichuan University, 610044, Chengdu, China.
Tissue Cell. 2022 Jun;76:101760. doi: 10.1016/j.tice.2022.101760. Epub 2022 Feb 21.
This study aimed to investigate the role of connexin 43 (CX43) in thoracic ossification of ligamentum flavum (TOLF) based on the p38 mitogen-activated protein kinase (p38MAPK)-runt-related transcription factor 2 (RUNX2) pathway. Immunohistochemistry was used to detect CX43 expression in TOLF and non-TOLF patients, fibroblasts of TOLF were isolated and induced osteogenic differentiation, and CX43 expression was detected by western blot analysis (WB). In addition, si-CX43 was used to intervene CX43, and SB203580 was used to inhibit the p38MAPK. The expressions of bone differentiation marker protein were detected by WB, and the ossification ability was analyzed by alizarin red staining. The interaction between RUNX2 and CX43 was identified by dual-luciferase reporter assay. Results found that CX43 was highly expressed during TOLF, and si-CX43 could inhibit the expression of alkaline phosphatase (ALP) and osteopontin (OPN), as well as inhibit TOLF and the p38MAPK-RUNX2 pathway. In addition, SB203580 showed a synergistic effect with si-CX43 to further inhibit TOLF and the expression of RUNX2. The dual-luciferase reporter assay confirmed that RUNX2 could bind to the CX43 promoter. In conclusion, CX43 promotes TOLF, which may be mediated by p38MAPK-RUNX2, and RUNX2 binds to the CX43 promoter to form a positive feedback regulatory loop during TOLF.
本研究旨在探讨缝隙连接蛋白 43（Connexin 43，CX43）在胸韧带骨化（Thoracic Ossification of Ligamentum Flavum，TOLF）中的作用，其作用机制可能与丝裂原活化蛋白激酶 p38（p38 mitogen-activated protein kinase，p38MAPK）- runt 相关转录因子 2（Runt-related transcription factor 2，RUNX2）通路有关。采用免疫组化法检测 TOLF 患者与非 TOLF 患者韧带中的 CX43 表达，分离 TOLF 成纤维细胞并诱导其成骨分化，采用 Western blot 分析（Western blot analysis，WB）检测 CX43 表达。转染 si-CX43 干扰 CX43 表达，用 p38MAPK 抑制剂 SB203580 抑制 p38MAPK，WB 检测骨分化标记蛋白的表达，茜素红染色分析成骨能力，双荧光素酶报告基因检测 RUNX2 与 CX43 的相互作用。结果显示，TOLF 中 CX43 高表达，si-CX43 可抑制碱性磷酸酶（Alkaline phosphatase，ALP）和骨桥蛋白（Osteopontin，OPN）表达，抑制 TOLF 形成及其 p38MAPK-RUNX2 通路。SB203580 与 si-CX43 具有协同作用，进一步抑制 TOLF 形成及其 RUNX2 表达。双荧光素酶报告基因检测证实 RUNX2 可与 CX43 启动子结合。综上所述，CX43 通过 p38MAPK-RUNX2 促进 TOLF 形成，RUNX2 与 CX43 启动子结合形成正反馈调节环，可能在 TOLF 中发挥作用。
