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

ABSTRACT: BACKGROUND AND PURPOSE:Celastrol exhibits anti-arthritic effects in rheumatoid arthritis (RA), but the role of celastrol-mediated Ca2+ mobilization in treatment of RA remains undefined. Here, we describe a regulatory role for celastrol-induced Ca2+ signalling in synovial fibroblasts of RA patients and adjuvant-induced arthritis (AIA) in rats. EXPERIMENTAL APPROACH:We used computational docking, Ca2+ dynamics and functional assays to study the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase pump (SERCA). In rheumatoid arthritis synovial fibroblasts (RASFs)/rheumatoid arthritis fibroblast-like synoviocytes (RAFLS), mechanisms of Ca2+ -mediated autophagy were analysed by histological, immunohistochemical and flow cytometric techniques. Anti-arthritic effects 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, using siRNA methods to study calmodulin, calpains, and calcineurin. KEY RESULTS:Celastrol inhibited SERCA to induce autophagy-dependent cytotoxicity in RASFs/RAFLS via Ca2+ /calmodulin-dependent kinase kinase-?-AMP-activated protein kinase-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 down-regulated 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 Ca2+ in celastrol-regulated gene expression. CONCLUSION AND IMPLICATIONS:Celastrol triggered Ca2+ 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 Ca2+ signalling.

SUBMITTER: Wong VKW

PROVIDER: S-EPMC6637043 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Ca<sup>2+</sup> signalling plays a role in celastrol-mediated suppression of synovial fibroblasts of rheumatoid arthritis patients and experimental arthritis in rats.

Wong Vincent Kam Wai VKW Qiu Congling C Xu Su-Wei SW Law Betty Yuen Kwan BYK Zeng Wu W Wang Hui H Michelangeli Francesco F Dias Ivo Ricardo De Seabra Rodrigues IRSR Qu Yuan Qing YQ Chan Tsz Wai TW Han Yu Y Zhang Ni N Mok Simon Wing Fai SWF Chen Xi X Yu Lu L Pan Hudan H Hamdoun Sami S Efferth Thomas T Yu Wen Jing WJ Zhang Wei W Li Zheng Z Xie Yuesheng Y Luo Riqiang R Jiang Quan Q Liu Liang L

British journal of pharmacology 20190704 16

<h4>Background and purpose</h4>Celastrol exhibits anti-arthritic effects in rheumatoid arthritis (RA), but the role of celastrol-mediated Ca<sup>2+</sup> mobilization in treatment of RA remains undefined. Here, we describe a regulatory role for celastrol-induced Ca<sup>2+</sup> signalling in synovial fibroblasts of RA patients and adjuvant-induced arthritis (AIA) in rats.<h4>Experimental approach</h4>We used computational docking, Ca<sup>2+</sup> dynamics and functional assays to study the sarco ...[more]

PMID: 31124139

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Project description:Cannabidiol (CBD) is a non-intoxicating phytocannabinoid from cannabis sativa that has demonstrated anti-inflammatory effects in several inflammatory conditions including arthritis. However, CBD binds to several receptors and enzymes and, therefore, its mode of action remains elusive. In this study, we show that CBD increases intracellular calcium levels, reduces cell viability and IL-6/IL-8/MMP-3 production of rheumatoid arthritis synovial fibroblasts (RASF). These effects were pronounced under inflammatory conditions by activating transient receptor potential ankyrin (TRPA1), and by opening of the mitochondrial permeability transition pore. Changes in intracellular calcium and cell viability were determined by using the fluorescent dyes Cal-520/PoPo3 together with cell titer blue and the luminescent dye RealTime-glo. Cell-based impedance measurements were conducted with the XCELLigence system and TRPA1 protein was detected by flow cytometry. Cytokine production was evaluated by ELISA. CBD reduced cell viability, proliferation, and IL-6/IL-8 production of RASF. Moreover, CBD increased intracellular calcium and uptake of the cationic viability dye PoPo3 in RASF, which was enhanced by pre-treatment with TNF. Concomitant incubation of CBD with the TRPA1 antagonist A967079 but not the TRPV1 antagonist capsazepine reduced the effects of CBD on calcium and PoPo3 uptake. In addition, an inhibitor of the mitochondrial permeability transition pore, cyclosporin A, also blocked the effects of CBD on cell viability and IL-8 production. PoPo3 uptake was inhibited by the voltage-dependent anion-selective channel inhibitor DIDS and Decynium-22, an inhibitor for all organic cation transporter isoforms. CBD increases intracellular calcium levels, reduces cell viability, and IL-6/IL-8/MMP-3 production of RASF by activating TRPA1 and mitochondrial targets. This effect was enhanced by pre-treatment with TNF suggesting that CBD preferentially targets activated, pro-inflammatory RASF. Thus, CBD possesses anti-arthritic activity and might ameliorate arthritis via targeting synovial fibroblasts under inflammatory conditions.

Dataset Information

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

Publications

Ca<sup>2+</sup> signalling plays a role in celastrol-mediated suppression of synovial fibroblasts of rheumatoid arthritis patients and experimental arthritis in rats.

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