Yoshida Kohsuke, Nakai Ayako, Kaneshiro Kenta, Hashimoto Naonori, Suzuki Kohjin, Uchida Koto, Hashimoto Teppei, Kawasaki Yoshiko, Tateishi Koji, Nakagawa Natsuko, Shibanuma Nao, Sakai Yoshitada, Hashiramoto Akira
Department of Biophysics, Kobe University Graduate School of Health Sciences, Kobe 654-0142, Japan.
Department of Rheumatology, Kobe Kaisei Hospital, Kobe 657-0068, Japan.
Biochem Biophys Res Commun. 2018 Jan 8;495(2):1675-1680. doi: 10.1016/j.bbrc.2017.12.015. Epub 2017 Dec 5.
Tumor necrosis factor (TNF)-α is responsible for expressions of several clock genes and affects joint symptoms of rheumatoid arthritis (RA) with diurnal fluctuation. We tried to determine the mechanism involved in over-expression of Bmal1, induced by TNF-α, in primary cultured rheumatoid synovial cells. Cells were incubated with intra-cellular Ca chelator BAPTA-AM, calcineurin inhibitor FK506 and p300/CBP (CREB binding protein) inhibitor C646, respectively, or transfected with p300 and CBP small interfering RNA (siRNA) before stimulation with TNF-α. Oscillation phase and amplitude of Bmal1, transcriptional activator Rorα, transcriptional repressor Rev-erbα, and histone acetyltransferases (p300 and Cbp) were evaluated by quantitative real-time PCR. As results, TNF-α did not influence the oscillation phase of Rev-erbα, while enhanced those of Rorα, resulting in over-expression of Bmal1. When Ca influx was inhibited by BAPTA-AM, TNF-α-mediated up-regulation of Rorα was cancelled, however, that of Bmal1 was still apparent. When we further explored another pathway between TNF-α and Bmal1, TNF-α suppressed the expression of Rev-erbα in the absence of Ca influx, as well as those of p300 and Cbp genes. Finally, actions of TNF-α, in increasing Bmal1/Rorα and decreasing Rev-erbα, were cancelled by C646 treatment or silencing of both p300 and Cbp. In conclusion, we determined a novel role of TNF-α in inducing Bmal1 via dual calcium dependent pathways; Rorα was up-regulated in the presence of Ca influx and Rev-erbα was down-regulated in the absence of that. Results proposed that inhibition of p300/CBP could be new therapeutic targets for RA.
肿瘤坏死因子（TNF）-α负责多种生物钟基因的表达，并通过昼夜波动影响类风湿关节炎（RA）的关节症状。我们试图确定在原代培养的类风湿滑膜细胞中，TNF-α诱导的Bmal1过表达所涉及的机制。分别用细胞内钙螯合剂BAPTA-AM、钙调神经磷酸酶抑制剂FK506和p300/CBP（CREB结合蛋白）抑制剂C646孵育细胞，或在用TNF-α刺激之前用p300和CBP小干扰RNA（siRNA）转染细胞。通过定量实时PCR评估Bmal1、转录激活因子Rorα、转录抑制因子Rev-erbα和组蛋白乙酰转移酶（p300和Cbp）的振荡相位和幅度。结果显示，TNF-α不影响Rev-erbα的振荡相位，但增强了Rorα的振荡相位，导致Bmal1过表达。当BAPTA-AM抑制钙内流时，TNF-α介导的Rorα上调被消除，但Bmal1的上调仍然明显。当我们进一步探索TNF-α和Bmal1之间的另一条途径时，TNF-α在无钙内流的情况下抑制Rev-erbα的表达，以及p300和Cbp基因的表达。最后，C646处理或p300和Cbp的沉默消除了TNF-α增加Bmal1/Rorα和降低Rev-erbα的作用。总之，我们确定了TNF-α通过双钙依赖途径诱导Bmal1的新作用；在有钙内流时Rorα上调，在无钙内流时Rev-erbα下调。结果表明，抑制p300/CBP可能是RA的新治疗靶点。
