Estrogen Modulates NFκB Signaling by Enhancing IκBα Levels and Blocking p65 Binding at the Promoters of Inflammatory Genes via Estrogen Receptor-β

BackgroundNFκB signaling is critical for expression of genes involved in the vascular injury response. We have shown that estrogen (17β-estradiol, E2) inhibits expression of these genes in an estrogen receptor (ER)-dependent manner in injured rat carotid arteries and in tumor necrosis factor (TNF)-α treated rat aortic smooth muscle cells (RASMCs). This study tested whether E2 inhibits NFκB signaling in RASMCs and defined the mechanisms. Methodology/Principal FindingsTNF-α treated RASMCs demonstrated rapid degradation of IκBα (10–30 min), followed by dramatic increases in IκBα mRNA and protein synthesis (40–60 min). E2 enhanced TNF-α induced IκBα synthesis without affecting IκBα degradation. Chromatin immunoprecipitation (ChIP) assays revealed that E2 pretreatment both enhanced TNF-α induced binding of NFκB p65 to the IκBα promoter and suppressed TNF-α induced binding of NFκB p65 to and reduced the levels of acetylated histone 3 at promoters of monocyte chemotactic protein (MCP)-1 and cytokine-induced neutrophil chemoattractant (CINC)-2β genes. ChIP analyses also demonstrated that ERβ can be recruited to the promoters of MCP-1 and CINC-2β during co-treatment with TNF-α and E2. ConclusionsThese data demonstrate that E2 inhibits inflammation in RASMCs by two distinct mechanisms: promoting new synthesis of IκBα, thus accelerating a negative feedback loop in NFκB signaling, and directly inhibiting binding of NFκB to the promoters of inflammatory genes. This first demonstration of multifaceted modulation of NFκB signaling by E2 may represent a novel mechanism by which E2 protects the vasculature against inflammatory injury.研究背景 核因子κB（NFκB）信号通路对于参与血管损伤应答的基因表达至关重要。本团队前期研究证实，雌激素（17β-雌二醇，E2）可通过雌激素受体（ER）依赖的方式，在损伤大鼠颈动脉以及经肿瘤坏死因子（TNF）-α处理的大鼠主动脉平滑肌细胞（RASMCs）中抑制此类基因的表达。本研究旨在验证E2是否可在RASMCs中抑制NFκB信号通路，并阐明其具体分子机制。 材料与方法/主要研究结果 经TNF-α处理的RASMCs可出现核因子κB抑制蛋白α（IκBα）的快速降解（10~30分钟），随后IκBα的mRNA及蛋白合成水平显著升高（40~60分钟）。E2可增强TNF-α诱导的IκBα合成，但不影响IκBα的降解过程。染色质免疫沉淀（ChIP）实验结果显示，E2预处理既可增强TNF-α诱导的NFκB p65与IκBα启动子的结合，又可抑制TNF-α诱导的NFκB p65与单核细胞趋化蛋白（MCP）-1及细胞因子诱导中性粒细胞趋化因子（CINC）-2β基因启动子的结合，并降低这些位点的乙酰化组蛋白3水平。ChIP分析还证实，在TNF-α与E2联合处理过程中，雌激素受体β（ERβ）可被招募至MCP-1及CINC-2β基因的启动子区域。 研究结论 上述结果表明，E2可通过两种截然不同的机制抑制RASMCs中的炎症反应：一是促进IκBα的新合成，从而加速NFκB信号通路的负反馈环路；二是直接抑制NFκB与炎症基因启动子的结合。本研究首次证实E2可多维度调控NFκB信号通路，这或许代表了E2保护血管免受炎症性损伤的全新分子机制。