The role of phosphorylated NF-κB, p65 subunit on physiological and inflammatory responses

磷酸化 NF-κB、p65 亚基对生理和炎症反应的作用

• 批准号: 20592179
• 负责人: MATSUO Kou
• 金额: $ 3万
• 依托单位: Kyushu Dental College
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2008
• 资助国家: 日本
• 起止时间: 2008 至 2010
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-20592179/
• 关键词: 歯学; NF-κB; リン酸化; 炎症反応; NF-кB

The transcription factor NF-κB regulates the expression of a wide range of genes, including various proinflammatory cytokines, cell adhesion proteins, and several anti-apoptotic molecules that together play pivotal roles in almost all aspects of immune and inflammatory responses. In resting cells, NF-κB associates with members of the inhibitory family of IκB proteins, resulting in retention of these complexes in the cytoplasm. Following appropriate stimulation, IκB proteins are phosphorylated on two specific NH_2-terminal serine residues by one of the catalytic subunits of the IκB kinase (IKK). Phosphorylated IκBs are subsequently ubiquitinated and degraded by the proteasome, leaving NF-κB free to translocate to the nucleus, where it binds to cognate enhancer/promoter elements in its cohort of target genes. However, we and others have shown that, besides the regulated degradation of IκBs, phosphorylation of nuclear NF-κB p65 is also an obligatory step for efficient transcription of NF- … More κB-dependent genes. Several different protein kinases and putative sites of phosphorylation on p65 have been identified, and, in general, it is believed that these phosphorylation events occur concomitantly with IKK-mediated phosphorylation of IκB proteins. We suggest that this additional step in the NF-κB activation pathway helps ensure that only NF-κB that enters the nucleus from the cytoplasm in response to appropriate inducing signals is able to trigger gene expression.To address the biological importance of the putative sites of phosphorylation on p65, Ser 276 and Ser 534, we generated knock-in mice expressing a serine-to-alanine mutation. Instead of the expected embryonic lethality from hepatocyte apoptosis seen in the absence of NF-κB activity, the S276A knock-in embryos die at different embryonic days due to variegated developmental abnormalities. We demonstrate that this variegated phenotype is due to epigenetic repression resulting from the recruitment of histone deacetylases by the nonphosphorylatable form of NF-κB into the vicinity of genes positioned fortuitously near NF-κB-binding sites. Therefore, unphosphorylated nuclear NF-κB can affect expression of genes not normally regulated by NF-κB through epigenetic mechanisms. On the other hand, S534A knock-in mice demonstrated normal development with slight increasing body weight. Consistent with these results, we observed increased subcutaneous fat in S534A mice compared with control littermate.To further determine whether changing the Ser 276 to a phospyhomimetic amino acid could mimic phosphorylation, and to examine the biological consequences of such a modification, we knocked in a mutant form of p65, where Ser 276 was changed to aspartic acid (S276D), into the genome. Mice bearing the p65 S276D mutation are born at normal Mendelian ratios, but soon begin to display a progressive, systemic hyperinflammatory condition that results in severe runting and, typically, death 8-20 d after birth. We demonstrated that a significant number of NF-κB target genes are up-regulated in these mice, thereby explaining the hyperinflammatory phenotype. Remarkably, crossing the knock-in strain with mice lacking TNF receptor 1 (TNFR1) leads to a complete rescue of the systemic inflammatory phenotype, but not in certain local inflammatory conditions-including one that resembles human keratoconjunctivitis sicca (KCS) or "dry eye". Therefore, the p65 S276D knock-in mice provide a unique model system, demonstrating the distinct roles of NF-κB in systemic inflammation and certain localized inflammatory diseases. Less

转录因子 NF-κB 调节多种基因的表达，包括各种促炎细胞因子、细胞粘附蛋白和几种抗凋亡分子，它们在免疫和炎症反应的几乎所有方面共同发挥着关键作用。在静息细胞中，NF-κB 与 IκB 蛋白抑制家族的成员结合，导致这些复合物保留在细胞质中。经过适当的刺激后，IκB 蛋白在两个特定的 NH_2 末端丝氨酸残基上被 IκB 激酶 (IKK) 的催化亚基之一磷酸化。磷酸化的 IκB 随后被蛋白酶体泛素化和降解，使 NF-κB 自由易位到细胞核，在细胞核中与其靶基因组中的同源增强子/启动子元件结合。然而，我们和其他人已经证明，除了 IκB 的受调节降解之外，核 NF-κB p65 的磷酸化也是 NF-κB 依赖性基因有效转录的必要步骤。已鉴定出几种不同的蛋白激酶和 p65 上磷酸化的推定位点，并且通常认为这些磷酸化事件与 IKK 介导的 IκB 蛋白磷酸化同时发生。我们认为，NF-κB 激活途径中的这一额外步骤有助于确保只有响应适当的诱导信号从细胞质进入细胞核的 NF-κB 才能够触发基因表达。为了解决 p65、Ser 276 和 Ser 534 磷酸化位点的生物学重要性，我们生成了表达丝氨酸至丙氨酸突变的敲入小鼠。与在缺乏 NF-κB 活性的情况下观察到的肝细胞凋亡导致的预期胚胎致死不同，S276A 敲入胚胎由于多种发育异常而在不同的胚胎日死亡。我们证明，这种多样化的表型是由于非磷酸化形式的 NF-κB 将组蛋白脱乙酰酶募集到偶然位于 NF-κB 结合位点附近的基因附近而引起的表观遗传抑制所致。因此，未磷酸化的核 NF-κB 可以通过表观遗传机制影响通常不受 NF-κB 调节的基因的表达。另一方面，S534A敲入小鼠表现出正常发育，体重略有增加。与这些结果一致，我们观察到与对照同窝小鼠相比，S534A 小鼠的皮下脂肪增加。为了进一步确定将 Ser 276 更改为拟磷氨基酸是否可以模拟磷酸化，并检查这种修饰的生物学后果，我们将 p65 的突变形式敲入基因组中，其中 Ser 276 更改为天冬氨酸 (S276D)。携带 p65 S276D 突变的小鼠以正常孟德尔比率出生，但很快开始表现出进行性、全身性高炎症状况，导致严重矮小，通常在出生后 8-20 天死亡。我们证明这些小鼠中大量 NF-κB 靶基因上调，从而解释了高炎症表型。值得注意的是，将敲入品系与缺乏 TNF 受体 1 (TNFR1) 的小鼠杂交可以完全挽救全身炎症表型，但在某些局部炎症状况下却不能，包括类似于人类干燥性角结膜炎 (KCS) 或“干眼症”的炎症状况。因此，p65 S276D 敲入小鼠提供了一个独特的模型系统，证明了 NF-κB 在全身炎症和某些局部炎症性疾病中的独特作用。较少的

项目成果

Promoting effects of thymosin.4 on granulation tissue and new bone formation after extraction in rats.

胸腺肽4对大鼠拔牙后肉芽组织和新骨形成的促进作用。

• 发表时间: 2011
• 期刊: Oral Surg Oral Med Oral Pathol Oral Radiol Endod (accepted)
• 作者: Matsuo K;Akasaki Y;Adachi K;Zhang M;Ishikawa A;Jimi E;Nishihara T;Hosokawa R.
• 通讯作者: Hosokawa R.

Regulation of gene expression by phosphorylated NF-.B, p65

磷酸化 NF-.B、p65 对基因表达的调节

• 发表时间: 2008
• 作者: N.Yonehara;M.Takemura (7人中5番目);Jimi E
• 通讯作者: Jimi E

Ornithine decarboxylase antizyme induces hypomethylation of genome DNA and histone H3 lysine 9 dimethylation (H3K9me2) in human oral cancer cell line.

• DOI: 10.1371/journal.pone.0012554
• 发表时间: 2010-09-03
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Yamamoto D;Shima K;Matsuo K;Nishioka T;Chen CY;Hu GF;Sasaki A;Tsuji T
• 通讯作者: Tsuji T

-thymosinsの歯科口腔外科治療応用に向けた基礎的研究

-胸腺肽在牙科和口腔外科治疗中应用的基础研究

• 发表时间: 2011
• 作者: A.Yano;S.Kikuchi;Y.Yamashita;Y.Sakamoto;Y.Nakagawa;Y.Yoshida;松尾拡
• 通讯作者: 松尾拡

Responses of immunocompetent cells in dentin bridge formation after pulpotomy.

活髓切断术后牙本质桥形成中免疫活性细胞的反应。

• 发表时间: 2010
• 作者: M.Zhang;E.Jimi;K.Matsuo
• 通讯作者: K.Matsuo