Background Bromodomain proteins (BRD) contain conserved acetyl-lysine binding domains that specifically recognize ɛ-N-lysine acetylation motifs, a key event in the reading process of epigenetic marks. We have recently identified a single nucleotide polymorphism in the BRD1 locus that is associated with the progression of joint destruction in rheumatoid arthritis (RA) in stage-I of a genome-wide association study (Knevel et al., Ann Rheum Dis. 2014).
Objectives To evaluate the expression of BRD1 in synovial tissues and in rheumatoid arthritis synovial fibroblasts (RASF) and to investigate its role in the regulation of gene expression levels in RASF.
Methods Synovial tissues from RA patients were stained with anti-BRD1 antibodies and double stained with anti-CD68 or anti-prolyl 4 hydroxylase beta antibodies as markers for macrophages and fibroblasts, respectively. RASF (n=4) were stimulated with the cytokines TNFα (10 ng/ml), IL1β (1 ng/ml), or the Toll-like receptor (TLR) 2 agonist Pam3 (100 ng/ml), the TLR3 agonist pIC (10 μg/ml) and the TLR4 agonist LPS (100 ng/ml) for 24h. The expression levels of BRD1 were evaluated by Western blotting. RASF (n=8-10) were transfected with siRNAs targeting BRD1, or scrambled siRNAs as a control. 24 hours later cells were stimulated with cytokines or TLR ligands. Knockdown of BRD1 was verified by quantitative RT-PCR and Western blotting. Expression levels and secretion rates into cell culture supernatants of MMP1, MMP3, IL6 and IL8 were evaluated by RT-PCR and ELISA, respectively.
Results Nuclear staining of BRD1 was detected in RASF and in macrophages in synovial tissues of RA patients. BRD1 protein levels in RASF were not regulated by stimulation with inflammatory cytokines or TLR ligands. Silencing of BRD1 increased the TNFα and LPS-induced secretion rates of MMP3 (TNFα p<0.01, LPS p=0.056), IL8 (TNFα p<0.01, LPS p<0.01) as well as LPS-induced levels of IL6 (p<0.01). In contrast, basal MMP1 (p<0.05) secretion levels were decreased by BRD1 silencing. Similar results were obtained on mRNA levels. Silencing of BRD1 had no effect on secretion rates of MMP1, MMP3, IL6 and IL8 after stimulation with other inflammatory stimuli in RASF.
Conclusions Our results demonstrate that BRD1 is a specific mediator of TNFα and LPS-induced pathways in RASF that limits expression levels of matrix degrading enzymes and cytokines under inflammatory conditions. The anti-inflammatory role of BRD1 should be kept in mind in the drug development of bromodomain inhibitors.
Knevel et al., Ann Rheum Dis. 2014
Acknowledgements IMI-BT Cure (Pfizer), IAR Epalinges, euroTEAM
Disclosure of Interest K. Klein Grant/research support from: IMI-BT Cure (Pfizer), IAR Epalinges, euroTEAM, R. Gay Grant/research support from: IMI-BT Cure (Pfizer), IAR Epalinges, euroTEAM, C. Kolling: None declared, M. Kato: None declared, B. Michel: None declared, S. Gay Grant/research support from: IMI-BT Cure (Pfizer), IAR Epalinges, euroTEAM, C. Ospelt Grant/research support from: IMI-BT Cure (Pfizer), IAR Epalinges, euroTEAM