Iannetti Alessio, Ledoux Adeline C, Tudhope Susan J, Sellier Hélène, Zhao Bo, Mowla Sophia, Moore Adam, Hummerich Holger, Gewurz Benjamin E, Cockell Simon J, Jat Parmjit S, Willmore Elaine, Perkins Neil D
Institute for Cell and Molecular Biosciences, Faculty of Medical Sciences, Newcastle University, Newcastle Upon Tyne, United Kingdom.
Northern Institute for Cancer Research, Faculty of Medical Sciences, Newcastle University, Newcastle Upon Tyne, United Kingdom.
PLoS Genet. 2014 Sep 25;10(9):e1004642. doi: 10.1371/journal.pgen.1004642. eCollection 2014 Sep.
There are two major pathways leading to induction of NF-κB subunits. The classical (or canonical) pathway typically leads to the induction of RelA or c-Rel containing complexes, and involves the degradation of IκBα in a manner dependent on IκB kinase (IKK) β and the IKK regulatory subunit NEMO. The alternative (or non-canonical) pathway, involves the inducible processing of p100 to p52, leading to the induction of NF-κB2(p52)/RelB containing complexes, and is dependent on IKKα and NF-κB inducing kinase (NIK). Here we demonstrate that in primary human fibroblasts, the alternative NF-κB pathway subunits NF-κB2 and RelB have multiple, but distinct, effects on the expression of key regulators of the cell cycle, reactive oxygen species (ROS) generation and protein stability. Specifically, following siRNA knockdown, quantitative PCR, western blot analyses and chromatin immunoprecipitation (ChIP) show that NF-κB2 regulates the expression of CDK4 and CDK6, while RelB, through the regulation of genes such as PSMA5 and ANAPC1, regulates the stability of p21WAF1 and the tumour suppressor p53. These combine to regulate the activity of the retinoblastoma protein, Rb, leading to induction of polycomb protein EZH2 expression. Moreover, our ChIP analysis demonstrates that EZH2 is also a direct NF-κB target gene. Microarray analysis revealed that in fibroblasts, EZH2 antagonizes a subset of p53 target genes previously associated with the senescent cell phenotype, including DEK and RacGAP1. We show that this pathway provides the major route of crosstalk between the alternative NF-κB pathway and p53, a consequence of which is to suppress cell senescence. Importantly, we find that activation of NF-κB also induces EZH2 expression in CD40L stimulated cells from Chronic Lymphocytic Leukemia patients. We therefore propose that this pathway provides a mechanism through which microenvironment induced NF-κB can inhibit tumor suppressor function and promote tumorigenesis.
有两条主要途径可导致NF-κB亚基的诱导。经典（或标准）途径通常导致含有RelA或c-Rel的复合物的诱导，并涉及以依赖IκB激酶（IKK）β和IKK调节亚基NEMO的方式降解IκBα。替代（或非经典）途径涉及p100向p52的诱导性加工，导致含有NF-κB2(p52)/RelB的复合物的诱导，并且依赖于IKKα和NF-κB诱导激酶（NIK）。在这里，我们证明在原代人成纤维细胞中，替代NF-κB途径亚基NF-κB2和RelB对细胞周期关键调节因子的表达、活性氧（ROS）生成和蛋白质稳定性具有多种但不同的影响。具体而言，在siRNA敲低、定量PCR、蛋白质印迹分析和染色质免疫沉淀（ChIP）之后表明，NF-κB2调节CDK4和CDK6的表达，而RelB通过调节诸如PSMA5和ANAPC1等基因，调节p21WAF1和肿瘤抑制因子p53的稳定性。这些共同调节视网膜母细胞瘤蛋白Rb的活性，导致多梳蛋白EZH2表达的诱导。此外，我们的ChIP分析表明EZH2也是一个直接的NF-κB靶基因。微阵列分析显示，在成纤维细胞中，EZH2拮抗先前与衰老细胞表型相关的一部分p53靶基因，包括DEK和RacGAP1。我们表明，该途径提供了替代NF-κB途径与p53之间相互作用的主要途径，其结果是抑制细胞衰老。重要的是，我们发现NF-κB的激活也在慢性淋巴细胞白血病患者的CD40L刺激细胞中诱导EZH2表达。因此，我们提出该途径提供了一种机制，通过该机制微环境诱导的NF-κB可以抑制肿瘤抑制功能并促进肿瘤发生。
