ATF6 Aggravates Angiogenesis-Osteogenesis Coupling During Ankylosing Spondylitis by Mediating FGF2 Expression in Chondrocytes

Abstract

Background: Although angiogenesis-osteogenesis coupling is important in ankylosing spondylitis (AS), therapeutic agents targeting the vasculature remain elusive. Here, we identified activating transcription factor 6 (ATF6) as an important regulator of angiogenesis. Methods: ATF6 and fibroblast growth factor 2 (FGF2) levels were quantified in SKG mice and AS patient cartilage by immunohistochemistry. Next, we isolated and cultured human chondrocytes and stimulated them with TNF-α, IFN-γ, and IL-17. ATF6 and FGF2 expression was measured by quantitative real-time PCR and western blotting. The angiogenic ability of chondrocytes was assessed with human umbilical vein endothelial cell tube formation and transwell migration assays. Theendoplasmic reticulum stress alleviator 4-phenyl butyric acid and the ATF6 inhibitor Ceapin-A7 were used to indicate the relationship between ATF6 and FGF2. We also performed luciferase reporter and chromatin immunoprecipitation assays to test whether ATF6 was directly responsible for FGF2 transcription, treated SKG mice with Ceapin-A7 to evaluate the role of ATF6 in AS and performed micro-CT and immunofluorescence analyses. Findings: ATF6 and FGF2 expression was elevated in AS cartilage following long-term stimulation with inflammatory factors. Among the three pathways involved in the unfolded protein response, the ATF6 pathway was activated mostly in chondrocytes stimulated long-term with proinflammatory cytokines. In chondrocytes, ATF6 knockdown decreased FGF2 expression and inhibited angiogenesis, whereas ATF6 overexpression had the opposite effects. Mechanistically, ATF6 interacted with the FGF2 promotor and promoted its transcription. Treatment with the ATF6 inhibitor Ceapin-A7 inhibited angiogenesis in vitro and angiogenesis-osteogenesis coupling in vivo. Interpretation: ATF6 may aggravate angiogenesis-osteogenesis coupling during AS by mediating FGF2 transcription in chondrocytes, implying that ATF6 represents a promising therapeutic target for AS. Funding Statement: This study was financially supported by grants from the National Natural Science Foundation of China (81971518), the National Natural Science Foundation of China (81871750), the Natural Science Foundation of Guangdong Province (2018A030313232), the Shenzhen Key Medical Discipline Construction Fund (ZDSYS20190902092851024) and the Health Welfare Fund Project of Futian District (FTWS2020078) Declaration of Interests: All authors declare no conflict of interest. Ethics Approval Statement: This study was approved by the Ethics Committee of The Eighth Affiliated Hospital, Sun Yat-sen University, Shenzhen, China. Written informed consent was obtained from all participants.