Department of Spinal Surgery, The People's Hospital of Xinchang, Xinchang, Zhejiang 312500, P.R. China.
Mol Med Rep. 2020 Dec;22(6):4551-4560. doi: 10.3892/mmr.2020.11576. Epub 2020 Oct 11.
Ankylosis progressive homolog (ANKH) is associated with fibroblast ossification in ankylosing spondylitis (AS). As the human ANKH gene is poorly characterized relative to its murine counterpart, the aim of the present study was to examine ANKH expression in ligament tissue isolated from patients with AS and the role played by this gene in AS‑associated fibroblast ossification. Fibroblasts were isolated from ligament tissue collected from patients with AS and ligament tissue from individuals with spinal cord fractures, then cultured. Fibroblasts from patients with AS were subsequently transfected with an ANKH overexpression vector, while those collected from individuals with spinal cord fractures were transfected with small interfering RNA specific for ANKH. Cell viability, apoptosis and mineralization were analyzed using MTT assays, flow cytometry and Alizarin Red staining, respectively. Furthermore, ANKH mRNA and protein expression levels were analyzed using reverse transcription‑quantitative PCR and western blotting analysis, respectively. The expression levels of osteogenesis markers, including alkaline phosphatase, osteocalcin, Runt‑related transcription factor 2, c‑Myc, as well as the β‑catenin signaling protein, were also determined using western blotting. The results of the present study revealed that ANKH protein expression levels were downregulated in AS total ligament tissue extract, compared with spinal fracture ligament. Moreover, the fibroblasts derived from patients with AS exhibited an increased viability and reduced apoptosis rates, compared with the fibroblasts from patients with spinal fracture. Notably, ANKH overexpression inhibited viability, mineralization and ossification, increased the phosphorylation of β‑catenin and downregulated β‑catenin and c‑Myc protein expression levels in fibroblasts from patients with AS. In addition, ANKH overexpression increased the ratio of p‑β‑catenin/β‑catenin in fibroblasts from patients with AS. By contrast, ANKH silencing in fibroblasts from patients with spinal fracture resulted in the opposite effect. In conclusion, the findings of the present study suggested that ANKH may inhibit fibroblast viability, mineralization and ossification, possibly by regulating the Wnt/β‑catenin signaling pathway.
骨钙素（ANKH）与强直性脊柱炎（AS）中的成纤维细胞骨化有关。由于人类 ANKH 基因相对于其鼠类对应物的特征描述较差，因此本研究的目的是检测 AS 患者韧带组织中 ANKH 的表达及其在 AS 相关成纤维细胞骨化中的作用。从 AS 患者的韧带组织和脊髓骨折患者的韧带组织中分离出成纤维细胞，然后进行培养。随后，用 ANKH 过表达载体转染 AS 患者的成纤维细胞，而用针对 ANKH 的小干扰 RNA 转染脊髓骨折患者的成纤维细胞。使用 MTT 测定法、流式细胞术和茜素红染色分别分析细胞活力、细胞凋亡和矿化。此外，使用逆转录-定量 PCR 和 Western blot 分析分别分析 ANKH mRNA 和蛋白表达水平。还使用 Western blot 测定骨形成标志物碱性磷酸酶、骨钙素、Runt 相关转录因子 2、c-Myc 以及 β-连环蛋白信号蛋白的表达水平。本研究的结果表明，与脊髓骨折韧带相比，AS 总韧带组织提取物中 ANKH 蛋白表达水平下调。此外，与脊髓骨折患者的成纤维细胞相比，AS 患者的成纤维细胞活力增加，凋亡率降低。值得注意的是，ANKH 过表达抑制了 AS 患者成纤维细胞的活力、矿化和骨化，增加了 AS 患者成纤维细胞中 β-连环蛋白的磷酸化，并下调了 β-连环蛋白和 c-Myc 蛋白的表达水平。此外，ANKH 过表达增加了 AS 患者成纤维细胞中 p-β-连环蛋白/β-连环蛋白的比值。相比之下，在脊髓骨折患者的成纤维细胞中沉默 ANKH 则产生相反的效果。综上所述，本研究的结果表明，ANKH 可能通过调节 Wnt/β-连环蛋白信号通路来抑制成纤维细胞的活力、矿化和骨化。
