Background/Purpose: The mammalian clock genes including Clock (circadian locomotor output cycles kaput), Bmal1 (brain and muscle Arnt-like protein 1), Per (Period) and Cry (Cryptochrome) regulate the circadian rhythm. We previously showed that arthritis was significantly accelerated in Cry knockout mice due to the activation of TNF-α (tumor necrosis factor a) transcription, and TNF-α inhibited the expression of Per2 gene via D-box binding protein, such as Dbp (D site of albumin promoter binding protein), in rheumatoid synovial cells. Since the effect of TNF-α on expression of Dbp gene has been reported to be dependent on intra-cellular calcium signaling, we tried to elucidate the contribution of the calcium signaling on TNF-α-induced Per2 inhibition in rheumatoid synovial cells.

Methods: Primary cultured rheumatoid synovial cells were synchronized upon incubation with 50% horse serum for 2 hours. Cells were treated with an intra-cellular [Ca²⁺] chelator BAPTA-AM (25μg/mL) or a calcineurin inhibitor FK-506 (Tacrolimus; 0.25 to 25 μg/mL) for 60 min, and then stimulated with or without TNF-α (10 ng/mL) for 24 hours. Total RNA was extracted from synovial cells, and mRNA expression of D-box binding protein genes, including Dbp, Hlf (hepatic leukemia factor), Tef (thyrotroph embryonic factor) and E4BP4 (E4-binding protein 4), were analyzed by real-time PCR. The viability of the synovial cells was determined using Cell Counting Kit-8.

Results: The mRNA expression of Per2 was inhibited upon incubation with TNF-α in rheumatoid synovial cells, which was cancelled by BAPTA-AM treatment (P < 0.05), but not FK-506, suggesting that Per2 inhibition by TNF-α could be induced via calcium signaling. Since Dbp, Hlf, Tef and E4bp4 genes could transactivate and suppress the expression of Per2 gene, respectively, we next examined the effect of BAPTA-AM on expression of these genes. As well as the result of Per2, the inhibition of Dbp, Hlf and Tef mRNA expression upon incubation with TNF-α were cancelled by BAPTA-AM treatment (P < 0.05). However, BAPTA-AM treatment did not affect the increase of E4bp4 expression by TNF-α. In addition, the viability of the synovial cells was increased by stimulation with TNF-α, and this was reversed by treatment with BAPTA-AM (P < 0.01).

Conclusion:  In rheumatoid synovial cells, TNF-α modulates the expression of Dbp, Hlf, Tef genes and then inhibits Per2 gene via calcium signaling. Since Per2 knockout mice exhibit increased resistance to apoptosis in thymocytes, our results suggest a novel role of TNF-α in the relationship between clock gene expression via calcium signaling and the anti-apoptotic character of rheumatoid synovial cells.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/tnf-a-modulates-the-expression-of-circadian-clock-genes-via-calcium-signaling-in-rheumatoid-synovial-cells/