Analysis Of The TGFb-Induced Program In Primary Airway Epithelial Cells Shows Essential Role Of NF-kB/RelA Signaling Network In Type II Epithelial Mesenchymal Transition

The airway epithelial cell plays a central role in coordinating pulmonary response to injury and inflammation. Here, transforming growth factor-b (TGFb) activates gene expression programs to induce stem cell-like properties, inhibit expression of differentiated epithelial adhesion proteins and express mesenchymal contractile proteins. This process is known as epithelial mesenchymal transition (EMT); although much is known about the role of EMT in cellular metastasis in an oncogene-transformed cell, less is known about Type II EMT, that occurring in normal epithelial cells. In this study, we applied next generation sequencing (RNA-seq) in primary human airway epithelial cells to understand the gene program controlling Type II EMT and how cytokine-induced inflammation modifies it. Generalized linear modeling was performed on a two-factor RNA-seq experiment of 6 treatments of telomerase immortalized human small airway epithelial cells (3 replicates). Using a stringent cut-off, we identified 3,478 differentially expressed genes (DEGs) in response to EMT. Unbiased transcription factor enrichment analysis identified three clusters of EMT regulators, one including SMADs/TP63 and another NF-kB/RelA. Surprisingly, we also observed 527 of the EMT DEGs were also regulated by the TNF-NF-kB/RelA pathway. This Type II EMT program was compared to Type III EMT in TGFb stimulated A549 alveolar lung cancer cells, revealing significant functional differences. Moreover, we observe that Type II EMT modifies the outcome of the TNF program, reducing IFN signaling and enhancing integrin signaling. We confirmed experimentally that TGFb-induced the NF-kB/RelA pathway by observing a 2-fold change in NF-kB/RelA nuclear translocation. A small molecule IKK inhibitor blocked TGFb-induced core transcription factor (SNAIL1, ZEB1 and Twist1) and mesenchymal gene (FN1 and VIM) expression. These data indicate that NF-kB/RelA controls a SMAD-independent gene network whose regulation is required for initiation of Type II EMT. Type II EMT dramatically affects the induction and kinetics of TNF-dependent gene networks.

气道上皮细胞（airway epithelial cell）在协调肺部对损伤与炎症的应答过程中发挥核心作用。本研究中，转化生长因子-β（transforming growth factor-b，TGFb）可激活基因表达程序，诱导干细胞样特性、抑制分化型上皮黏附蛋白的表达，并表达间质收缩蛋白。该过程即为上皮间质转化（epithelial mesenchymal transition，EMT）；尽管目前学界已明确EMT在癌基因转化细胞的细胞转移中发挥的作用，但对于正常上皮细胞中发生的II型EMT的了解仍相对匮乏。本研究中，我们通过对原代人气道上皮细胞开展下一代测序（next generation sequencing，RNA-seq），以解析调控II型EMT的基因程序，以及细胞因子诱导的炎症如何对其进行修饰。我们针对端粒酶永生化人小气道上皮细胞设置6种处理条件并设置3次生物学重复，基于双因素RNA-seq实验开展广义线性建模分析。通过严格的筛选阈值，我们共鉴定出3478个响应EMT的差异表达基因（differentially expressed genes，DEGs）。无偏转录因子富集分析鉴定出3类EMT调控因子簇，其中一类包含SMAD家族/TP63，另一类包含核因子κB/RelA（NF-kB/RelA）。令人意外的是，我们还发现527个EMT相关DEGs同时受肿瘤坏死因子（TNF）-核因子κB/RelA通路调控。我们将该II型EMT程序与TGFb刺激的A549肺泡肺癌细胞中的III型EMT进行比较，发现二者存在显著的功能差异。此外，我们观察到II型EMT可改变TNF应答程序的结局：降低干扰素（IFN）信号通路的活性，同时增强整合素信号通路（integrin signaling）的传导。我们通过实验证实，TGFb可激活核因子κB/RelA通路：观察到该因子的核转位（nuclear translocation）水平上调2倍。小分子IKK抑制剂（small molecule IKK inhibitor）可阻断TGFb诱导的核心转录因子（SNAIL1、ZEB1和Twist1）以及间质基因（FN1和VIM）的表达。上述数据表明，核因子κB/RelA可调控一条不依赖SMAD的基因网络，该网络的调控是启动II型EMT的必要条件。II型EMT可显著影响TNF依赖型基因网络的诱导过程及其动力学特征。