Department of Rheumatology and Immunology, Shanghai Changzheng Hospital, Naval Medical University, Shanghai, 200003, China.
Department of General Surgery, 72nd Group Army Hospital, Huzhou University, Huzhou, 313000, Zhejiang, China.
Arthritis Res Ther. 2023 Feb 21;25(1):28. doi: 10.1186/s13075-023-03011-z.
Ectopic ossification is an important cause of disability in patients with ankylosing spondylitis (AS). Whether fibroblasts can transdifferentiate into osteoblasts and contribute to ossification remains unknown. This study aims to investigate the role of stem cell transcription factors (POU5F1, SOX2, KLF4, MYC, etc.) of fibroblasts in ectopic ossification in patients with AS.
Primary fibroblasts were isolated from the ligaments of patients with AS or osteoarthritis (OA). In an in vitro study, primary fibroblasts were cultured in osteogenic differentiation medium (ODM) to induce ossification. The level of mineralization was assessed by mineralization assay. The mRNA and protein levels of stem cell transcription factors were measured by real-time quantitative PCR (q-PCR) and western blotting. MYC was knocked down by infecting primary fibroblasts with lentivirus. The interactions between stem cell transcription factors and osteogenic genes were analysed by chromatin immunoprecipitation (ChIP). Recombinant human cytokines were added to the osteogenic model in vitro to evaluate their role in ossification.
We found that MYC was elevated significantly in the process of inducing primary fibroblasts to differentiate into osteoblasts. In addition, the level of MYC was remarkably higher in AS ligaments than in OA ligaments. When MYC was knocked down, the expression of the osteogenic genes alkaline phosphatase (ALP) and bone morphogenic protein 2 (BMP2) was decreased, and the level of mineralization was reduced significantly. In addition, the ALP and BMP2 were confirmed to be the direct target genes of MYC. Furthermore, interferon-γ (IFN-γ), which showed high expression in AS ligaments, was found to promote the expression of MYC in fibroblasts in the process of ossification in vitro.
This study demonstrates the role of MYC in ectopic ossification. MYC may act as the critical bridge that links inflammation with ossification in AS, thus providing new insights into the molecular mechanisms of ectopic ossification in AS.
异位骨化是强直性脊柱炎（AS）患者致残的重要原因。成纤维细胞是否可以转分化为成骨细胞并促进骨化尚不清楚。本研究旨在探讨 AS 患者成纤维细胞中的干细胞转录因子（POU5F1、SOX2、KLF4、MYC 等）在异位骨化中的作用。
从 AS 或骨关节炎（OA）患者的韧带中分离原代成纤维细胞。在体外研究中，将原代成纤维细胞在成骨分化培养基（ODM）中培养以诱导骨化。通过矿化测定评估矿化水平。通过实时定量 PCR（q-PCR）和 Western blot 测定干细胞转录因子的 mRNA 和蛋白水平。通过慢病毒感染原代成纤维细胞敲低 MYC。通过染色质免疫沉淀（ChIP）分析干细胞转录因子与成骨基因之间的相互作用。在体外成骨模型中添加重组人细胞因子以评估其在骨化中的作用。
我们发现，在诱导原代成纤维细胞分化为成骨细胞的过程中，MYC 显著升高。此外，AS 韧带中的 MYC 水平明显高于 OA 韧带。当 MYC 被敲低时，碱性磷酸酶（ALP）和骨形态发生蛋白 2（BMP2）的成骨基因表达降低，矿化水平显著降低。此外，ALP 和 BMP2 被确认为 MYC 的直接靶基因。此外，在体外骨化过程中，AS 韧带中高表达的干扰素-γ（IFN-γ）被发现可促进成纤维细胞中 MYC 的表达。
本研究表明 MYC 在异位骨化中的作用。MYC 可能作为炎症与 AS 中骨化之间的关键桥梁，从而为 AS 中异位骨化的分子机制提供新的见解。
