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. Recently, a functional role of the BET bromodomain proteins BRD2, BRD3, and BRD4 in inflammation was described (Nicodeme et al., Nature. 2010) and we have shown that the BET protein inhibitor I-BET151 suppressed the inflammatory, matrix degrading, proliferating and chemoattraction properties of rheumatoid arthritis synovial fibroblasts (RASF).
Objectives To evaluate the expression of the BET proteins BRD2, BRD3 and BRD4 in synovial tissues and to investigate their individual roles in the regulation of gene expression in RASF.
Methods Synovial tissues from RA patients were stained with anti-BRD2, anti-BRD3 and anti-BRD4 antibodies and double stained with anti-CD68 or anti-prolyl 4 hydroxylase beta antibodies as markers for macrophages and fibroblasts, respectively. RASF (n=6-8) were transfected with siRNAs targeting BRD2, BRD3, and BRD4, or a pool of siRNAs targeting all three BRDs (triple transfection), or scrambled siRNAs as a control. 24 hours after transfection cells were stimulated with the cytokines TNFα (10 ng/ml), IL1β (1 ng/ml), or the TLR2 agonist Pam3 (100 ng/ml), the TLR3 agonist pIC (10 μg/ml), and the TLR4 agonist LPS (100 ng/ml) for another 24h. Knockdown of BRD2, BRD3, and BRD4 was verified by quantitative RT-PCR and Western blotting. The secretion rates into cell culture supernatants of MMP1, MMP3, IL6, and IL8 were evaluated by ELISA.
Results Nuclear staining of BRD2, BRD3 and BRD4 was detected in 50-100% of synovial cells and was confirmed in both RASF and macrophages. Silencing of one BET protein in RASF did not affect expression levels of the other two BET proteins. Silencing of BRD2 increased the TNFα and LPS-induced secretion rates of MMP1 (p<0.001) and MMP3 (TNFα p<0.05, LPS p<0.01), while basal IL6 (p<0.05) secretion levels were decreased. Silencing of BRD3 reduced the IL1β -induced levels of MMP1 (p<0.01) as well as basal (p<0.01), IL1β (p<0.05) and pIC-induced levels (p<0.08) of IL6. pIC-induced levels of MMP1 (p<0.05), MMP3 (p<0.05) and IL8 (p<0.05) were suppressed by silencing of BRD4. Furthermore, IL1β–induced levels of MMP1 (p<0.01) were decreased by silencing of BRD4. Triple transfections had the same effect on MMP1, MMP3 and IL8 secretion rates as silencing of BRD4 alone. In addition, basal (p<0.05) and pIC-induced (p<0.08) levels of IL6 were reduced.
Conclusions Our results demonstrate that each BET protein regulates the effect of distinct inflammatory pathways. BRD4 mostly mediates pIC-induced pathways and mimicks most effects obtained in triple transfections.
Acknowledgements IMI-BT Cure (Pfizer), IAR Epalinges, euroTEAM
Disclosure of Interest K. Klein Grant/research support: IMI-BT Cure, IAR Epalinges, euroTEAM, R. Gay Grant/research support: IMI-BT Cure, IAR Epalinges, euroTEAM, C. Kolling: None declared, L.-L. Lin Employee of: Pfizer, S. Gay Grant/research support: IMI-BT Cure, IAR Epalinges, euroTEAM, C. Ospelt Grant/research support: IMI-BT Cure, IAR Epalinges, euroTEAM