Ankylosis progressive homolog upregulation inhibits cell viability and mineralization during fibroblast ossification by regulating the Wnt/β‑catenin signaling pathway

Abstract

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.

Identification of Immune Infiltration-Related ceRNAs as Novel Biomarkers for Prognosis of Patients With Primary Open-Angle Glaucoma

“…It has been demonstrated that the loss of ANKH transporter function in mice led to excessive mineralization because of reduced PPi transport (Ho et al, 2000). Other studies indicated that ANKH took part in chondrocalcinosis articularis (Kumar et al, 2019), fibroblast ossification (He and Dong, 2020), and vascular smooth muscle cell calcification (Chen et al, 2019a), suggesting that ANKH may play a crucial role in the hardening and remodeling of TM cells in POAG patients. This view has been established in studies on POAG identifying novel risk loci (Choquet et al, 2018).…”

miR‑148a‑3p facilitates osteogenic differentiation of fibroblasts in ankylosing spondylitis by activating the Wnt pathway and targeting DKK1

“…However, apoptosis-related changes were not detected in this study. ANKH, a multichannel transmembrane protein, has been reported to affect the metabolism of AS fibroblasts and inhibit fibroblast viability, ossification and mineralization ( 50 ). Recent findings have elucidated that ANKH plays a regulatory role in fibroblast viability, ossification and mineralization ( 50 ).…”

“…ANKH, a multichannel transmembrane protein, has been reported to affect the metabolism of AS fibroblasts and inhibit fibroblast viability, ossification and mineralization ( 50 ). Recent findings have elucidated that ANKH plays a regulatory role in fibroblast viability, ossification and mineralization ( 50 ). In the current study, the Starbase database predicted ANKH as a downstream target gene of miR-148a-3p.…”

Tail suspension delays ectopic ossification in proteoglycan‑induced ankylosing spondylitis in mice via miR‑103/DKK1

“…In addition, a significant increase in β-catenin was observed in the PG + FLB group compared with the PG model group. The level of β-catenin is positively associated with abnormal bone formation in AS (35). The expression levels of ROCK1 and p-Erk1/2 in the ligaments of the sacroiliac joint were significantly decreased in the groups with suspension with a reduced mechanical load compared with the PG model group, and the opposite trend was observed in the upright group.…”