Pang Shu-Jie, Jiang Tian-Yi, Wang Nai-Guo, Cui Xiao-Wen, Wang Hui, Pan Yu-Fei, Yang Ning, Dong Li-Wei
Department of Surgery, Eastern Hepatobiliary Surgery Hospital, the Naval Medical University, 700 Moyu Road, Shanghai, 201805, Shanghai, China.
International Cooperation Laboratory on Signal Transduction, Eastern Hepatobiliary Surgery Institute, the Naval Medical University, Shanghai, 200438, China.
Cell Commun Signal. 2025 Jun 10;23(1):274. doi: 10.1186/s12964-025-02278-w.
Nuclear factor κB (NF-κB) activity is a central component of inflammatory and innate immune responses, which plays a crucial role in sepsis. The inhibition of NF-κB signaling and the IκB kinase (IKK) complex is important for understanding the control of innate immunity and regulating the progress of sepsis.
We constructed transgenic mouse strains (Rmp; Lyz2-Cre), and then established lipopolysaccharide (LPS), cecal ligation and perforation (CLP)-induced sepsis models. Hematoxylin-eosin (HE) staining, ELISA, and flow cytometry assay were employed to evaluate the sepsis-related damage and the activation of the inflammatory-related signaling pathway. In vitro, differential expression of RMP cell lines and primary macrophage isolated from transgenic mice were utilized to assess the activation of the NF-κB signaling pathway by Western blot (WB), reverse transcription-polymerase chain reaction (RT-PCR), and ELISA tests. Co‑immunoprecipitation (Co-IP), WB, GST-pulldown, phosphorylation mass spectrometry, surface plasmon resonance (SPR), and IKK activity detection assay were employed to investigate the underlying molecular mechanism by which RMP restrains IKK-NF-κB pathway.
We identified RNA polymerase II subunit 5 (RPB5)-mediating protein (RMP) as an inhibitor of the IKK complex, which thus inhibited NF-κB signaling in macrophages. In resting macrophages, RMP was directly bound to the kinase domain of IKKβ and inhibited its activity by recruiting protein phosphatase 2 A (PP2A) to the IKK complex. When mouse macrophages were treated with LPS, a Toll-like receptor 4 (TLR4) agonist that stimulates NF-κB signaling, RMP was phosphorylated by IKKβ at Ser and dissociated from the IKK complex, which further activated NF-κB signaling. Macrophage-specific deletion of Rmp reduced survival in mice due to an increased inflammatory response in experimental models of sepsis.
RMP inhibits TLR4-induced NF-κB activation and exerts homeostatic control of innate immunity, and may be promising as a therapeutic target in the limiting of NF-κB signaling and attenuating sepsis-related damage.
核因子κB（NF-κB）活性是炎症和固有免疫反应的核心组成部分，在脓毒症中起关键作用。抑制NF-κB信号通路和IκB激酶（IKK）复合物对于理解固有免疫的调控以及脓毒症进展的调节至关重要。
我们构建了转基因小鼠品系（Rmp；Lyz2-Cre），然后建立脂多糖（LPS）、盲肠结扎穿孔（CLP）诱导的脓毒症模型。采用苏木精-伊红（HE）染色、酶联免疫吸附测定（ELISA）和流式细胞术分析来评估脓毒症相关损伤以及炎症相关信号通路的激活。在体外，利用从转基因小鼠分离的RMP细胞系和原代巨噬细胞的差异表达，通过蛋白质免疫印迹（WB）、逆转录-聚合酶链反应（RT-PCR）和ELISA检测来评估NF-κB信号通路的激活。采用免疫共沉淀（Co-IP）、WB、谷胱甘肽-S-转移酶下拉实验（GST-pulldown）、磷酸化质谱分析、表面等离子体共振（SPR）和IKK活性检测分析来研究RMP抑制IKK-NF-κB通路的潜在分子机制。
我们鉴定出RNA聚合酶II亚基5（RPB5）介导蛋白（RMP）是IKK复合物的抑制剂，从而在巨噬细胞中抑制NF-κB信号。在静息巨噬细胞中，RMP直接与IKKβ的激酶结构域结合，并通过将蛋白磷酸酶2A（PP2A）募集到IKK复合物来抑制其活性。当用LPS（一种刺激NF-κB信号的Toll样受体4（TLR4）激动剂）处理小鼠巨噬细胞时，RMP在丝氨酸位点被IKKβ磷酸化并从IKK复合物解离，进而激活NF-κB信号。在脓毒症实验模型中，巨噬细胞特异性缺失Rmp会因炎症反应增加而降低小鼠存活率。
RMP抑制TLR4诱导的NF-κB激活并对固有免疫发挥稳态控制作用，在限制NF-κB信号和减轻脓毒症相关损伤方面有望成为治疗靶点。
