Dataset Information

BRD3 Regulates the Inflammatory and Stress Response in Rheumatoid Arthritis Synovial Fibroblasts.

ABSTRACT:

Background

Individual functions of members of the bromodomain (BRD) and extra-terminal (BET) protein family underlying the anti-inflammatory effects of BET inhibitors in rheumatoid arthritis (RA) are incompletely understood. Here, we aimed to analyze the regulatory functions of BRD3, an understudied member of the BET protein family, in RA synovial fibroblasts (FLS).

Methods

BRD3 was silenced in FLS prior to stimulation with TNF. Alternatively, FLS were treated with I-BET. Transcriptomes were analyzed by RNA sequencing (RNAseq), followed by pathway enrichment analysis. We confirmed results for selective target genes by real-time PCR, ELISA, and Western blotting.

Results

BRD3 regulates the expression of several cytokines and chemokines in FLS, and positively correlates with inflammatory scores in the RA synovium. In addition, RNAseq pointed to a profound role of BRD3 in regulating FLS proliferation, metabolic adaption, and response to stress, including oxidative stress, and autophagy.

Conclusions

BRD3 acts as an upstream regulatory factor that integrates the response to inflammatory stimuli and stress conditions in FLS and executes many functions of BET proteins that have previously been identified using pan-BET inhibitors.

SUBMITTER: Seifritz T

PROVIDER: S-EPMC10741099 | biostudies-literature | 2023 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

BRD3 Regulates the Inflammatory and Stress Response in Rheumatoid Arthritis Synovial Fibroblasts.

Seifritz Tanja T Brunner Matthias M Camarillo Retamosa Eva E Maciukiewicz Malgorzata M Krošel Monika M Moser Larissa L Züllig Thomas T Tomšič Matija M Distler Oliver O Ospelt Caroline C Klein Kerstin K

Biomedicines 20231130 12

<h4>Background</h4>Individual functions of members of the bromodomain (BRD) and extra-terminal (BET) protein family underlying the anti-inflammatory effects of BET inhibitors in rheumatoid arthritis (RA) are incompletely understood. Here, we aimed to analyze the regulatory functions of BRD3, an understudied member of the BET protein family, in RA synovial fibroblasts (FLS).<h4>Methods</h4>BRD3 was silenced in FLS prior to stimulation with TNF. Alternatively, FLS were treated with I-BET. Transcri ...[more]

PMID: 38137409

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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.

Project description:The nonresolving character of synovial inflammation in rheumatoid arthritis (RA) is a conundrum. To identify the contribution of fibroblast-like synoviocytes (FLS) to the perpetuation of synovitis, we investigated the molecular mechanisms that govern the tumor necrosis factor ? (TNF?)-driven inflammatory program in human FLS.FLS obtained from the synovial tissues of patients with RA or osteoarthritis were stimulated with TNF? and assayed for gene expression and cytokine production by real-time quantitative reverse transcription-polymerase chain reaction analysis and enzyme-linked immunosorbent assay. NF-?B signaling was evaluated by Western blotting. Histone acetylation, chromatin accessibility, and NF-?B p65 and RNA polymerase II (Pol II) occupancy at the interleukin-6 (IL-6) promoter were measured by chromatin immunoprecipitation and restriction enzyme accessibility assays.In FLS, TNF? induced prolonged transcription of messenger RNA (mRNA) for IL-6 and progressive accumulation of IL-6 protein over 4 days. Similarly, induction of mRNA for CXCL8/IL-8, CCL5/RANTES, matrix metalloproteinase 1 (MMP-1), and MMP-3 after TNF? stimulation was sustained for several days. This contrasted with the macrophage response to TNF?, which characteristically involved a transient increase in the expression of proinflammatory genes. In FLS, TNF? induced prolonged activation of NF-?B signaling and sustained transcriptional activity, as indicated by increased histone acetylation, chromatin accessibility, and p65 and Pol II occupancy at the IL-6 promoter. Furthermore, FLS expressed low levels of the feedback inhibitors A20-binding inhibitor of NF-?B activation 3 (ABIN-3), IL-1 receptor-associated kinase M (IRAK-M), suppressor of cytokine signaling 3 (SOCS-3), and activating transcription factor 3 (ATF-3), which terminate inflammatory responses in macrophages.TNF? signaling is not effectively terminated in FLS, which leads to an uncontrolled inflammatory response. The results suggest that prolonged and sustained inflammatory responses by FLS in response to synovial TNF? contribute to the persistence of synovial inflammation in RA.

Dataset Information

BRD3 Regulates the Inflammatory and Stress Response in Rheumatoid Arthritis Synovial Fibroblasts.

Background

Methods

Results

Conclusions

Publications

BRD3 Regulates the Inflammatory and Stress Response in Rheumatoid Arthritis Synovial Fibroblasts.

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