Background/Purpose: Transcription factors belonging to the family of nuclear factors of activated T cells (NFAT) play an essential role in diverse biological processes, such as inflammation, immune response and cell proliferation. Known NFAT5 target genes are involved in the pathogenesis of rheumatoid arthritis (RA), including inflammation promotion and joint destruction. The TLR4 ligands was shown to trigger the activity of NFAT5 in macrophages through the IKK-NF-κB pathway. Some reports indicated that there is an interaction between NF-κB p65 and NFAT5 for the induction of the expression of the target genes of these transcription factors. These findings suggested that NFAT5 might play an important role in not only innate immune but also autoimmune rheumatic disorders. However, it is still not clear whether the TLR4 pathway induces NFAT5-p65 complex enhanceosome activation in RA synovial fibroblasts (RASFs). The aim of this study to investigate the role of NFAT5-p65 enhanceosome in the inflammatory responses of RASFs via TLR4 signaling.

Methods: Human neutrophil-derived lactoferrin (LTF) was used as TLR4 ligand. RASFs were stimulated with LTF, and the expressions of inflammatory cytokines in RASFs were measured. The expression of IL-6, CCL20 and IL-8 mRNA in RASFs was measured using real-time quantitative PCR. The protein levels of IL-6, CCL20 and IL-8 in culture medium was measured by ELISA. To clarify the TLR4 signalling pathway associated with LTF stimulation, a small molecular inhibitor of TLR4 (TAK242) and NF-κB inhibitor were used. The role of nuclear factor of activated T cells 5 (NFAT5) was identified using small interfering RNA. Cerulenin is an inhibitor of fatty acid synthase, which is isolated from Cephalosporium caerulens as an antibiotic agent. Cerulenin was reported as one of agents to disrupt the p65-NFAT5-p300 interaction and inhibited the activity of NF-κB. Therefore, to reveal the interaction between NF-κB and NFAT5, cerulenin, which disrupts their interaction, was used.

Results: Stimulation of RASFs with LTF significantly increased the expressions of inflammatory cytokines and chemokines, such as IL-6, CCL20 and IL-8, in RASFs. LTF enhanced the mRNA expressions of these cytokines in RASFs stimulated by TNF-α. TAK242 almost completely inhibited the expressions of inflammatory cytokines and chemokines in RASFs stimulated by LTF. The NF-κB inhibitor partially repressed the expressions of IL-6 and IL-8 mRNAs induced by LTF, but not CCL20 mRNA expression. On the other hand, NFAT5 silencing decreased the expressions of CCL20 and IL-8 mRNAs induced by LTF, but not IL-6 mRNA expression. Cerulenin repressed the expressions of IL-6, CCL20 and IL-8 mRNAs in RASFs treated with LTF or LPS.

Conclusion: NFAT5-p65 enhanceosome might regulate the expressions of LTF-TLR4-responsive genes in RASFs. NFAT5-p65 enhanceosome has potential as novel therapeutic target in the pathogenesis of RA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/the-role-of-nfat5-p65-complex-enhanceosome-in-the-inflammatory-responses-of-rheumatoid-arthritis-synovial-fibroblasts-via-tlr4-signaling/