Ca2+ signalling plays a role in celastrol-mediated suppression of synovial fibroblasts of rheumatoid arthritis patients and experimental arthritis in rats

Abstract

Celastrol exhibits anti-arthritic effect in rheumatoid arthritis (RA), but the role of celastrol-mediated Ca mobilization in treatment of RA remains unelucidated. Here, we illustrate the regulatory role of celastrol-induced Ca signalling in synovial fibroblasts of RA patients and adjuvant-induced arthritis (AIA) in rats. Molecular target of celastrol was determined by computational docking, Ca dynamic and functional assays on SERCA. Ca -mediated autophagy in RASFs/RAFLS and the underlying mechanism were verified by quantification of endogenous LC3-II puncta, immunoblotting, and flow cytometry with the Ca chelator (BAPTA/AM) or suitable inhibitors. The anti-arthritic effect of celastrol, autophagy induction and growth rate of synovial fibroblasts in AIA rats were monitored by microCT and immunofluorescence staining. mRNA from joint tissues of AIA rats was isolated for transcriptional analysis of inflammatory genes. The role of Ca in regulating the identified genes was investigated by knockdown of calmodulin, calpains, and calcineurin. Celastrol inhibited SERCA to induce autophagy-dependent cytotoxicity in RASFs/RAFLS via CaMKKβ-AMPK-mTOR pathway and repressed arthritis symptoms in AIA rats. BAPTA/AM hampered the in vitro and in vivo effectiveness of celastrol. Inflammatory- and autoimmunity-associated genes downregulated by celastrol in joint tissues of AIA rat were restored by BAPTA/AM. Knockdown of calmodulin, calpains, and calcineurin in RAFLS confirmed the role of Ca in celastrol-regulated gene expression. Celastrol triggered Ca signalling to induce autophagic cell death in RASFs/RAFLS and ameliorated arthritis in AIA rats mediated by calcium-dependent/-binding proteins facilitating the exploitation of anti-arthritic drugs based on manipulation of Ca signalling. [Abstract copyright: This article is protected by copyright. All rights reserved.]