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Background <p>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.</p> Methods <p>We constructed transgenic mouse strains (<i>Rmp</i><sup><i>f/f</i></sup>; <i>Lyz2-Cre</i><sup><i>+</i></sup>), 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.</p> Results <p>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&#xa0;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<sup>439</sup> and dissociated from the IKK complex, which further activated NF-κB signaling. Macrophage-specific deletion of <i>Rmp</i> reduced survival in mice due to an increased inflammatory response in experimental models of sepsis.</p> Conclusions <p>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.</p>

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RNA polymerase II subunit 5-mediating protein limits TLR4-induced innate immune activation in macrophages by inhibiting IKKβ/NF-κB signaling during sepsis

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年6月10日

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  • Shu-jie Pang,
  • Tian-yi Jiang,
  • Nai-guo Wang,
  • Xiao-wen Cui,
  • Hui Wang,
  • Yu-fei Pan,
  • Ning Yang,
  • Li-wei Dong

摘要

Background

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.

Methods

We constructed transgenic mouse strains (Rmpf/f; 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.

Results

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 Ser439 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.

Conclusions

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.