Background/Purpose: Nitric oxide (NO), a proinflammatory mediator responsible for various physiological processes, plays a central role in the pathogenesis of rheumatoid arthritis (RA). As a heme-based sensor for NO, a transcription factor neuronal PAS domain protein 2 (NPAS2) forms a heterodimer with clock gene BMAL1. Using RA fibroblast-like synoviocytes (-FLS), we previously reported the interaction of clock genes and Bcl-2-interacting killer (BIK) which was known to be an inducer of mitochondrial apoptosis, and the promoter site of BIK has the NPAS2/BMAL1 heterodimer binding enhanced box sites (E-box sites). In this study, we evaluated the role of NPAS2 in RA synovium in views of relationship between NO and BIK.

Methods: The synovium were obtained during total knee replacement surgery from patients. Immunohistochemistry was performed to determine NPAS2 expression in synovial tissue. Cell proliferation was assessed with WST-8 assay in RA-FLS, using varying concentrations of the NO donor, S-Nitroso-N-acetyl-DL-penicillamine (SNAP) (0-20 nM). To evaluate the relationship between NPAS2 and BIK, NPAS2-siRNA was used to measure BIK mRNA expression in the absence and presence (5, 20 nM) of SNAP in RA-FLS. Moreover, we cloned the three BIK promoters (see Figure1), made luciferase assay model, and measured the luciferase activity in the absence and presence of SNAP in RA-FLS.

Results: NPAS2 expression was observed in RA synovium. Cell proliferation was significantly increased by SNAP. In the presence of SNAP, BIK mRNA expression was significantly reduced in the control-siRNA group, but not in the NPAS2-siRNA group (Figure 2, *; P < 0.05). In the presence of SNAP, the luciferase activity was significantly reduced in the Bik-E2-promoter group, but not in the Bik-E0-promoter group (Figure 3, *; P < 0.05).

Conclusion: NPAS2 enhance Bik expression, and NO inhibited Bik expression by NPAS2. These results suggests that NO may inhibit NPAS2 to bind E-box of Bik promoter. We propose a novel action of NO that inhibits mitochondrial apoptosis of RA-FLS by preventing the binding between NPAS2 and BIK. Therefore, we suggested that the inhibition of NO and NPAS2 may clinically be potential of anti-hyperproliferation and anti-bone destruction in RA synovium.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/bik-plays-an-important-role-of-cell-proliferation-caused-by-nitric-oxide-in-rheumatoid-arthritis-synovium/