Aoki Tsukasa, Gao Jing, Li Aonan, Huang Fei, Tu Yiran, Wu Wei, Matsuda Miho, Kiyoshima Tamotsu, Nishimura Fusanori, Jimi Eijiro
Laboratory of Molecular and Cellular Biochemistry, Division of Oral Biological Sciences, Faculty of Dental Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan.
Department of Periodontology, Division of Oral Rehabilitation, Faculty of Dental Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan.
Inflammation. 2024 Sep 8. doi: 10.1007/s10753-024-02140-0.
Nuclear factor-κB (NF-κB) is a transcription factor that regulates the expression of various genes involved in inflammatory diseases and immune responses. Recently, a novel transcriptional regulatory mechanism of NF-κB involving the phosphorylation of serine 536 (534 in mice; S534) of its p65 subunit was reported; however, further research is required to elucidate the physiological role of S534 phosphorylation. Therefore, we generated S534A knock-in (KI) mice, in which the S534 of p65 was substituted with alanine. Similar to the wild-type (WT) mice, S534A KI mice developed normally. After stimulation with tumor necrosis factor α (TNFα), mouse embryonic fibroblasts (MEFs) derived from S534A KI mice exhibited increased target gene expression compared with that in the WT MEFs, which was induced by long-term binding of p65 to DNA. According to comprehensive gene expression analysis after stimulation with TNFα, the expression of genes p65ted to inflammatory and immune responses was increased, and the expression of genes p65ted to lipolysis was decreased in S534A KI MEFs. Analyses of a periodontal disease model established using WT and S534A KI mice revealed that alveolar bone resorption was enhanced in S534A KI mice owing to an increase in the number of osteoclasts, which was not attributed to the differentiation of osteoclast precursor cells but to an increased expression of interleukin-1β and receptor activator of NF-κB ligand in the periodontal tissue. Hence, phosphorylation of S536 negatively regulates inflammatory responses in vitro and in vivo.
核因子-κB（NF-κB）是一种转录因子，可调节参与炎症性疾病和免疫反应的各种基因的表达。最近，有报道称NF-κB存在一种新的转录调节机制，涉及其p65亚基丝氨酸536（小鼠中为534；S534）的磷酸化；然而，需要进一步研究以阐明S534磷酸化的生理作用。因此，我们构建了S534A基因敲入（KI）小鼠，其中p65的S534被丙氨酸取代。与野生型（WT）小鼠相似，S534A KI小鼠正常发育。用肿瘤坏死因子α（TNFα）刺激后，与WT小鼠胚胎成纤维细胞（MEF）相比，来自S534A KI小鼠的MEF表现出靶基因表达增加，这是由p65与DNA的长期结合诱导的。根据TNFα刺激后的综合基因表达分析，在S534A KI MEF中，与炎症和免疫反应相关的基因表达增加，与脂肪分解相关的基因表达减少。对使用WT和S534A KI小鼠建立的牙周病模型的分析表明，由于破骨细胞数量增加，S534A KI小鼠的牙槽骨吸收增强，这并非归因于破骨细胞前体细胞的分化，而是由于牙周组织中白细胞介素-1β和NF-κB受体激活剂配体的表达增加。因此，S536的磷酸化在体外和体内均对炎症反应起负调节作用。
