The SUMO1/ESR1 Pathway Upregulates ANKH Level to Inhibit Fibroblast Ossification in Ankylosing Spondylitis

Abstract

Background: The chronic progression of ankylosing spondylitis is accompanied by inflammatory bone erosion, vertebral bone loss, and abnormal bone overgrowth. It is known that targeting osteogenic differentiation of fibroblasts can prevent new bone formation, representing an effective approach to alleviating the burden caused by ankylosing spondylitis. The upregulation of ankylosis progressive homolog (ANKH) has been found to inhibit ankylosing spondylitis ligament-derived fibroblast ossification. This study was designed to decipher the mechanism behind this effect mediated by ANKH. Methods: The interplay between ANKH and estrogen receptor 1 (ESR1) gene was assessed by bioinformatics/chromatin-immunoprecipitation/dual-luciferase reporter assay. Ankylosing spondylitis-derived fibroblasts were isolated from human ankylosing spondylitis ligaments, where ESR1 expression was assessed with Western blotting. Co-immunoprecipitation assay was utilized to investigate the affinity between ESR1 and small ubiquitin-like modifier 1 (SUMO1). After inducing ESR1 overexpression and SUMO1 knockdown, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and Alizarin red staining were performed to evaluate the viability and mineralization of fibroblasts. Real-time quantitative polymerase chain reaction and Western blotting were carried out to determine the gene and protein expression of ANKH, ESR1, SUMO1, osteogenesis-related markers, and Wnt/β-catenin pathway-related markers. Results: ESR1 overexpression upregulated ANKH and p-β-catenin/β-catenin levels, decreased the viability, inhibited the mineralization, and downregulated the expression levels of osteogenesis-related genes, c-Myc and β-catenin in ankylosing spondylitis-derived fibroblasts. It was found that SUMO1 could bind to ESR1, and the SUMO1 knockdown downregulated ESR1 level, exerting effects contrary to those after ESR1 overexpression. This finding indicates that the effects of SUMO1 knockdown and ESR1 overexpression on ankylosing spondylitis-derived fibroblasts are opposite to each other. Conclusions: In sum, the SUMO1/ESR1 pathway upregulates ANKH level to inhibit ankylosing spondylitis-associated fibroblast ossification.