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

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都起着关键作用。在静息细胞中，NF-κB与i -κB蛋白抑制家族成员结合，导致这些复合物在细胞质中保留。在适当的刺激下，IκB蛋白在两个特定的nh_2末端丝氨酸残基上被IκB激酶（IKK）的一个催化亚基磷酸化。磷酸化的i -κB随后被蛋白酶体泛素化和降解，使NF-κB自由地转运到细胞核，在那里它与靶基因群中的同源增强子/启动子元件结合。然而，我们和其他人已经证明，除了i -κ b的调控降解外，核NF-κ b p65的磷酸化也是NF-κ b有效转录的必要步骤。已经确定了几种不同的蛋白激酶和p65上的推定磷酸化位点，一般认为这些磷酸化事件与ikk介导的i - κ b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NF-κB响应适当的诱导信号从细胞质进入细胞核才能触发基因表达。为了研究p65、Ser 276和Ser 534上可能的磷酸化位点的生物学重要性，我们产生了表达丝氨酸到丙氨酸突变的敲入小鼠。与预期的在缺乏NF-κB活性时肝细胞凋亡导致的胚胎死亡不同，S276A敲入胚胎在不同的胚胎天数由于不同的发育异常而死亡。我们证明，这种多样化的表型是由于NF-κB的非磷酸化形式将组蛋白去乙酰化酶募集到碰巧位于NF-κB结合位点附近的基因附近所导致的表观遗传抑制。因此，未磷酸化的核NF-κB可以通过表观遗传机制影响正常情况下不受NF-κB调控的基因的表达。另一方面，S534A敲入小鼠发育正常，体重略有增加。与这些结果一致，我们观察到与对照组相比，S534A小鼠皮下脂肪增加。为了进一步确定将Ser 276改变为类似磷酸化的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，其中Ser 276被改变为天冬氨酸（S276D）。携带p65 S276D突变的小鼠以正常的孟德尔比率出生，但很快就开始表现出进行性、全身性的高炎症状态，导致严重的跑步，通常在出生后8-20天死亡。我们证明了大量NF-κB靶基因在这些小鼠中上调，从而解释了高炎症表型。值得注意的是，将敲入菌株与缺乏TNF受体1 （TNFR1）的小鼠杂交可以完全挽救全身炎症表型，但在某些局部炎症条件下却不能，包括类似于人类干燥性角膜结膜炎（KCS）或“干眼症”的炎症。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在全身性炎症和某些局部炎症性疾病中的独特作用。少

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