Session Type: Abstract Submissions (ACR)
Bromodomain proteins (BRD) contain conserved acetyl-lysine binding domains that specifically recognize ε-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 and BET inhibitors were shown to inhibit the expression of pro-inflammatory cytokines and chemokines in LPS-stimulated macrophages (Nicodeme et al., Nature. 2010). Our objectives were to evaluate the expression of the BET proteins BRD2, BRD3 and BRD4 in rheumatoid arthritis synovial fibroblasts (RASF) and to investigate the effects of the BET protein inhibitor I-BET151 on expression levels and functional properties of RASF.
The mRNA expression levels of BRD2, BRD3 and BRD4 in RASF and osteoarthritis synovial fibroblasts (OASF) were verified by quantitative Real-time PCR. RASF were treated with the inflammatory cytokines TNF-α (10 ng/ml), IL1β (1 ng/ml) and the Toll-like receptor (TLR) ligands Pam3 (100 ng/ml), pIC (10 µg/ml), and LPS (100 ng/ml) in presence or absence of I-BET151 (1 µM, R&D Systems) for 24h. The mRNA expression levels and secretion rates into cell culture supernatants of MMP1, MMP3, IL6, IL8 and CCL5 were evaluated by quantitative Real-time PCR and ELISA. Rates of proliferation and migratory properties of RASF before and after I-BET151 treatment were investigated using a BrdU-based proliferation assay and scratch assays, respectively.
BRD2 (delta Ct: OASF: 5.93 ± 0.22; RASF: 5.47 ± 0.47; p<0.01) and BRD3 (delta Ct: OASF: 7.94 ± 0.67; RASF: 7.27 ± 0.56; p<0.01) mRNA levels were slightly increased in RASF (n=20) compared to OASF (n=10). On the other hand, BRD4 mRNA levels were similar in OASF and RASF (delta Ct: OASF: 4.94 ± 0.26; RASF: 4.95 ± 0.36). Treatment of RASF (n=12) with I-BET151 significantly reduced basal IL6 mRNA levels (p<0.001), as well as the mRNA expression levels of IL6, IL8, MMP1, MMP3 and CCL5 after stimulation with cytokines and TLR ligands (p<0.05). Furthermore, basal IL6 and IL8 secretion rates were decreased after I-BET151 treatment of RASF (n=10-12) by 75.9% (p<0.01) and 39.4% (p<0.01), respectively. Both, IL6 and IL8 secretion rates remained decreased after I-BET151 treatment of RASF (n=9-12, p<0.01) after stimulation with cytokines and TLR ligands between 53.6 and 79%. Whereas I-BET151 significantly reduced MMP1 secretion rates only after TNF-α (71.5%, p<0.01), IL1β (75.6%, p<0.01) and pIC (32.8%, p<0.05) (n=12), MMP3 levels in supernatants (n=12) were reduced after stimulation with all cytokines and TLR ligands by 29.5–82.2% (p<0.05). CCL5 secretion was reduced in I-BET151 treated RASF (n=6) by 51.3–67.2%, depending on the stimuli. Furthermore, rates of proliferation were reduced by 61.4 ± 14.3 % (p=0.0625) in RASF (n=5) treated with I-BET151, whereas migratory properties of RASF (n=2) were not affected.
Our results demonstrate that I-BET151 is a strong suppressor of the inflammatory, matrix degrading and proliferating properties of RASF and suggest a therapeutic potential of blocking the recruitment of BET proteins to promoters of target genes in RA.
R. E. Gay,
L. L. Lin,
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/the-bromodomain-protein-inhibitor-i-bet151-suppresses-inflammatory-and-matrix-degrading-properties-of-rheumatoid-arthritis-synovial-fibroblasts/