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

转录因子NF-κB调控多种基因的表达，包括多种致炎细胞因子、细胞黏附蛋白和几种抗凋亡分子，这些分子共同在免疫和炎症反应的几乎所有方面发挥关键作用。在静息细胞中，NF-κB与IκB抑制蛋白家族的成员结合，导致这些复合体保留在细胞质中。在适当的刺激下，IκB蛋白被IκB激酶(IKK)的一个催化亚基磷酸化到两个特定的NH_2末端丝氨酸残基上。磷酸化的IκB随后被蛋白酶体泛素化和降解，使NF-κB自由移位到核，在那里它与其靶基因队列中的同源增强子/启动子元件结合。然而，我们和其他人已经证明，除了IκB的受控降解外，核NF-κB p65的磷酸化也是有效转录NF-…的必备步骤更多依赖于κB的基因。已经确定了几种不同的蛋白激酶和p65上的磷酸化位点，通常认为这些磷酸化事件伴随着IKK介导的IκB蛋白的磷酸化而发生。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在适当的诱导信号下从细胞质进入细胞核的NF-κB能够触发基因表达。为了解决p65、Ser276和Ser534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。在缺乏NF-κB活性的情况下，S276A敲入胚胎不同于预期的肝细胞凋亡导致的胚胎死亡，而是由于各种发育异常而在不同的胚胎天数死亡。我们证明，这种多样化的表型是由于表观遗传抑制引起的，组蛋白脱乙酰酶被非磷酸化形式的NF-κB招募到恰好位于NF-κB结合位点附近的基因附近。因此，核内非磷酸化的NF-κB可以通过表观遗传机制影响非正常调控基因的表达。另一方面，S534A基因敲除小鼠发育正常，体重略有增加。与这些结果一致，我们观察到S534A小鼠的皮下脂肪比对照小鼠增加。为了进一步确定将Ser276改变为磷酸同源氨基酸是否可以模拟磷酸化，并检查这种修改的生物学后果，我们在基因组中敲入了P65的突变形式，其中Ser 276改变为天冬氨酸(S276D)。携带p65 S276D突变的小鼠出生时孟德尔比例正常，但很快就开始表现出进行性的全身性炎症状态，导致严重的跑动，通常情况下，出生后8-20天就会死亡。我们证明，在这些小鼠中，大量的NF-κB靶基因上调，从而解释了高炎症表型。值得注意的是，将敲入菌株与缺乏肿瘤坏死因子受体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结膜炎(KCS)或“干眼”的炎症。因此，p65 S276D敲入小鼠提供了一个独特的模型系统，展示了NF-κB在全身性炎症和某些局限性炎症性疾病中的独特作用。较少

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