p100 deficiency alone is insufficient for full activation of the alternative NF-κB pathway: TNF signaling cooperates with p52-RelB activation in the transcriptional regulation of the enpp2/autotaxin promoter. Mus musculus

Background: Constitutive activation of the alternative NF-κB pathway leads to marginal zone B cell expansion and disorganized spleen microarchitecture. Furthermore, uncontrolled alternative NF-κB signaling results in the development and progression of cancer. We aimed here to learn about the mechanisms how does the constitutively active alternative NF-κB pathway exert its effects in these malignant processes. Methodology/Principal Findings: To explore the consequences of constitutive alternative NF-κB activation on genome-wide transcription, we compared gene expression profiles of wild-type and NF-kB2/p100-deficient (p100-/-) primary mouse embryonic fibroblasts (MEFs) and spleens. Microarray experiments revealed 73 differentially regulated genes in p100-/- vs. wild-type MEFs. Chromatin immunoprecipitation (ChIP) assays showed in p100-/- MEFs direct binding of RelB and p52 to the promoter of the enpp2 gene encoding Enpp2/Autotaxin, a protein with an important role in lymphocyte homing and cell migration. Gene ontology analysis revealed upregulation of genes with anti-apoptotic/proliferative activity (enpp2, serpina3g, traf1, rrad), chemotactic/locomotory activity (enpp2, ccl8), and lymphocyte homing activity (enpp2, cd34). Most importantly, biochemical analyses of MEFs and gene expression analyses of mice indicated a crosstalk between classical and alternative NF-κB pathways. Conclusions/Significance: The present results show that uncontrolled alternative NF-κB signaling is further enhanced by classical NF-κB activation, indicating that p100 deficiency alone is insufficient for full induction of a subset of genes by the alternative NF-κB pathway. Overall design: cell type comparison (wt vs p100-/-) after genetic modification

Background: 替代性NF-κB通路（alternative NF-κB pathway）的组成型激活可导致边缘区B细胞扩增以及脾脏微结构紊乱。此外，不受调控的替代性NF-κB信号转导会促进癌症的发生与进展。本研究旨在阐明组成型激活的替代性NF-κB通路在上述恶性进程中发挥调控作用的具体分子机制。
Methodology/Principal Findings: 为探究组成型激活的替代性NF-κB通路对全基因组转录的影响，本研究对比了野生型与NF-κB2/p100敲除（p100-/-）原代小鼠胚胎成纤维细胞（primary mouse embryonic fibroblasts, MEFs）及脾脏的基因表达谱。基因芯片实验结果显示，p100-/-与野生型MEFs之间存在73个差异表达基因。染色质免疫沉淀（chromatin immunoprecipitation, ChIP）实验证实，在p100-/- MEFs中，RelB与p52可直接结合至enpp2基因的启动子区域；该基因编码的Enpp2/自分泌运动因子（Autotaxin）在淋巴细胞归巢与细胞迁移过程中发挥关键作用。基因本体（Gene Ontology, GO）富集分析显示，具有抗凋亡/增殖活性的基因（enpp2、serpina3g、traf1、rrad）、具备趋化/运动活性的基因（enpp2、ccl8）以及参与淋巴细胞归巢的基因（enpp2、cd34）均呈现上调表达。最为关键的是，对MEFs的生化分析以及对小鼠的基因表达分析结果证实，经典NF-κB通路与替代性NF-κB通路之间存在串扰调控。
Conclusions/Significance: 本研究结果显示，经典NF-κB激活可进一步增强不受调控的替代性NF-κB信号转导，这表明仅p100缺失不足以通过替代性NF-κB通路完全诱导特定基因子集的表达。
Overall design: 遗传修饰后的细胞类型对比（野生型 vs p100-/-）