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

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

doi:10.1186/s12964-025-02278-w

. 2025 Jun 10;23:274. doi: 10.1186/s12964-025-02278-w

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

Shu-jie Pang ^1,^#, Tian-yi Jiang ^2,^3,^✉,^#, Nai-guo Wang ^4,^#, Xiao-wen Cui ^3,^5,^#, Hui Wang ⁶, Yu-fei Pan ^2,³, Ning Yang ^1,^✉, Li-wei Dong ^2,^3,^7,^✉

PMCID: PMC12150503 PMID: 40495190

Abstract

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 (Rmp^f/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.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12964-025-02278-w.

Keywords: IκB kinase β (IKKβ), RNA polymerase II subunit 5-mediating protein (RMP), NF-κB, Sepsis, Macrophage, Protein phosphatase 2A (PP2A)

Background

The innate immune response can be activated by pathogen-associated molecular patterns (PAMPs) through a family of Toll-like receptors (TLRs) [1–3], which maintain immune homeostasis. The hyper- or hypoactivation of TLR signaling causes human diseases [4, 5]. TLRs are primarily expressed in macrophages and neutrophils and play a crucial role in modulating the innate immune-inflammatory response and septic outcome. Sepsis is a complex syndrome characterized by infection and systemic inflammatory response, and it is attributed to over 14 million fatalities annually [6]; understanding the molecular switches underlying sepsis is, therefore, a high priority [7, 8].

Upon activation of TLRs, the cytoplasmic domains recruit signaling adaptor molecules such as myeloid differential protein-88 (MYD88) and TIR-domain-containing adapter-inducing interferon-β (TRIF), which then trigger a cascade of signaling events that lead to the activation of mitogen-activated protein kinases (MAPKs) and IκB kinases (IKKs) as well as downstream transcription factors AP-1, NF-κB, and interferon regulatory factor 3 (IRF3) [9]. The NF-κB signaling pathway is central to regulating of inflammatory and immune responses. In the canonical NF-κB pathway, proinflammatory cytokines, such as tumor necrosis factor α (TNFα) and interleukin (IL)-1β, induce the activation of the canonical IKK complex [10]. The activated IKK complex phosphorylates the NF-κB inhibitor IκBα and thereby promotes its ubiquitination and proteasomal degradation [11, 12]. Both positive and negative feedback mechanisms maintain appropriate NF-κB activity during an inflammatory response [13–15].

RNA polymerase II subunit 5 (RPB5)-mediating protein (RMP) is also known as an unconventional prefoldin RBP5 interactor (URI) [16]. URI/RMP is an important chaperone member of Particle for Arrangement of Quaternary Structure (PAQosome), formerly known as the R2TP/PFDL complex [17]. URI/RMP is involved in essential cellular processes as a functional chaperone, including protein synthesis, gene transcription, and mRNA splicing [18]. RMP was also identified as a downstream effector of the mammalian target of rapamycin (mTOR) /ribosomal protein S6 kinase (S6K1) signaling, beyond its chaperone function [19, 20]. In mammalian cells, RMP could be phosphorylated by mTOR to reach the hyperphosphorylated state, an effect induced by insulin or insulin-like growth factor 1 (IGF1) and blocked by rapamycin [21]. Additionally, RMP may be important for tumor progression in various cancers, including hepatocellular carcinoma [22, 23], prostate cancer [24], and ovarian cancer [25]. For example, RMP increases the transcription of IL-6 by interacting with NF-κB, P65, and RPB5 in liver tumor cells [23], indicating a role for innate inflammatory responses. Here, we identified RMP as an inflammatory regulator and an inhibitor of NF-κB signaling in macrophages through direct interactions with IKKβ and inhibition of IKKβ kinase activity. In vivo, evidence further showed that myeloid-specific deletion of the Rmp gene impaired survival in mice with a dysregulated inflammatory response in an experimental model of sepsis.

Methods

Cell culture and treatment

Monocyte cell lines RAW264.7 and THP-1 were purchased from the Shanghai Cell Resource Center of the Chinese Academy of Sciences and cultured in RPMI 1640 medium supplemented with 10% Fetal Bovine Serum(FBS)(Biological Industries), 100 IU/mL penicillin (Gibco), and 100 µg/mL streptomycin (Gibco). Human embryonic kidney cell line HEK-293T was obtained from the American Type Culture Collection (ATCC) and cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS (Biological Industries). All cell lines were maintained at 37 °C and 5% CO₂. RAW264.7 and THP-1 cells were activated by LPS (100 ng/mL) or TNFα (20 ng/mL) for the indicated period. The concentrations of small molecular inhibitors or agonists used were as follows: IKK-16 (3 µM), TPCA-1 (5 µM), PMA (150 nM), CHX (50 ng/mL), SP600125 (10 µM), SCH772984 (10 µM), BAY (10 µM), Rapamycin (10 µM), PP2A inhibitor LB100 (10 µM), Resiquimod (5 µg/mL), and Pam3csk4 (100 ng/mL).

Isolation of pMφs and spleen cells

Thioglycollate broth (3% in phosphate-buffered saline; Sigma-Aldrich) was steam-sterilized before use. Each mouse was injected intraperitoneally with 2.5 mL of broth. On the third day, mice were euthanized and enriched peritoneal macrophages were obtained by peritoneal lavage using 8 mL phosphate buffer. Peritoneal macrophages were activated by LPS (100 ng/mL) or TNFα (20 ng/mL). Spleen was collected aseptically, immediately after the euthanasia of mice. Then, the spleen was placed on a sterile 60-mesh stainless-steel sieve above a collector tube, perfused with culture medium, teased apart using micro-dissecting scissors, and gently pushed through the sieve using a syringe plunger. The splenic cell suspension was counted under the microscope by employing a blood-counting cell plate.

Animals

Rmp^flox/flox was constructed by the Shanghai Southern Model Biology Research Center, as previously described [26]. Lyz-Cre mice were purchased from the Jackson Laboratory. All mice were on the genetic background of C57BL/6j and inhabited in a standard condition meeting the criteria of the guidelines of the Naval Medical University Animal Care Facility. In this study, to conveniently record, KO mice mean a mice-specific deficiency of Rmp in myeloid cells (Rmp^f/f, Lyz2-Cre⁺). All animal operating procedures complied with the ethical standards of the Institutional Animal Care and Use Committee of the Naval Medical University. Animal experiments were approved by the Ethics Board of the Eastern Hepatobiliary Surgery Hospital.

Plasmids, DNA transfection, and lentivirus infection

Plasmids pCDNA3.1 A, pCDNA3.1 A-mouse RMP, mouse RMP truncated mutants, mouse RMP S439A mutant, mouse RMP S439E mutant, mouse RMP S369A mutant, human RMP S372A mutant, pCDNA3.1 A-mouse TLR4, pCDNA3.1 A-mouse IKKβ, mouse IKKβ truncated mutants, mouse IKK-SSEE mutant, pCDNA3.1 A-mouse PP1γ, pCDNA3.1 A-mouse PP2Aa, pCDNA3.1 A-mouse PP2Ab, pCDNA3.1 A-mouse PP2Ac were constructed by Biochat Co., Ltd, Shanghai. Plasmids were transfected into cell lines using jetPEI DNA Transfection Reagent (Polyplus), according to the manufacturer’s instructions. The lentivirus-shRNA targeting human RMP was constructed by Genechem Co., Ltd, Shanghai. Viral solutions were added to a cell culture medium containing 4 µg/mL polybrene. After 48 h of infection, cells were selected using 2 µg/mL puromycin. After that, the RMP expression of cells was detected by RT-PCR or immunoblotting.

Antibodies and reagents

Anti-RMP (11277-1-AP, RRID: AB_10596929), anti-beta-actin (66009-1-Ig, RRID: AB_2687938), and anti-GAPDH (60004-1-Ig, RRID: AB_2107436) were purchased from Proteintech Group, Inc. Anti-HA (AE008, RRID: AB_2770404), anti-His (AE003, RRID: AB_2728734), anti-Myc (AE010, RRID: AB_2770408), and anti-Flag (AE005, RRID: AB_2770401) were purchased from ABclonal, Inc. Anti-P65 (8242, RRID: AB_10859369), anti-p-P65 (Ser⁵³⁶)(3033, RRID: AB_331284), anti-IKKβ (8943, RRID: AB_11024092), anti-p-IKKα/β (Ser^176/180)(2697, RRID: AB_2079382), anti-IKKα (2682, RRID: AB_331626), anti-IKKγ (2695, RRID: AB_2124826), anti-IκBα (4814, RRID: AB_390781), anti-p-IκBα (Ser³²) (2859, RRID: AB_561111), anti-P38 (9212, RRID: AB_330713), anti-p-p38 (Thr^180/182) (9211, RRID: AB_331641), anti-S6 (2217, RRID: AB_331355), anti-p-S6 (Ser^235/236) (4858, RRID: AB_916156), anti-ERK1/2 (9102, RRID: AB_330744), anti-p-ERK1/2 (Thr^202/204) (9101, RRID: AB_331646), anti-p-AKT (Ser473) (4060, RRID: AB_2315049), and-pan-AKT (4691, RRID: AB_915783), anti-SAPK/JNK (9252, RRID: AB_2250373), anti-p-SAPK/JNK (Thr^183/185) (9251, RRID: AB_331659), anti-PP2Aa (2041, RRID: AB_2168121), anti-PP2Ab (2290, RRID: AB_659890), and anti-PP2Ac (2259, RRID: AB_561239) were purchased from Cell Signaling Technology. Mouse IgG (sc-2025, RRID: AB_737182) and PP1γ (sc-515943, RRID: AB_2909495) were purchased from Santa Cruz Biotechnology, Inc. LPS (S1732) was purchased from Beyotime. TNFα (315–01 A) was purchased from PeproTech. IKK inhibitors including TPCA-1 (S2824), IKK-16 (S2882), PP2A inhibitor, SP600125 (a JNK inhibitor) (S1460), SCH772984 (an ERK inhibitor) (S7101), MK2206 (an AKT inhibitor) (S1078), rapamycin (an mTOR inhibitor) (S1039), LB100 (a PP2A inhibitor) (S7537) and BAY (a P65 inhibitor) (S2913) were purchased from Selleck. Pam3csk4 (an agonist of TLR1/2) (TP1068) and Resiquimod (an agonist of TLR7/8) (T6965) were purchased from Target Molecule Corp.

Immunoblot analysis and co-immunoprecipitation

Whole-cell extracts were collected and lysed in RIPA buffer (Beyotime, 50 mM Tris pH 7.4, 150 mM NaCl, 1% NP40, 0.1% SDS, and 0.5% sodium deoxycholate, 150 mM NaCl, 2 mM EDTA, and 50 mM NaF) supplemented with a cocktail of protease inhibitor (Roche) and phosphatase inhibitors (Sigma-Aldrich). The lysed products were centrifuged at 12,000 rpm (Microfuge 20R, BECKMAN COULTER) for 15 min at 4 °C to remove precipitates. Primary antibodies used for immunoblot analysis were diluted at 1:1000 with 5% BSA buffer. Immunoblots were performed using specific primary antibodies, followed by a fluorescein-conjugated secondary antibody, and then detected using an Odyssey fluorescence scanner (Li-Cor). Immunoprecipitation was determined according to the instructions of MAg25K/Protein A/G beads (Enriching Biotechnology LTD).

Luciferase reporter gene assay

Cells (20,000 cells/well) were plated in 300 µL/well in a 48-well plate. The Renilla (pRL-TK) 10 ng, and pNF-κB-luc 200 ng were co-transfected into cells. Cells were lysed by positive lysis buffer (Promega) and assayed employing the Dual-Luciferase Reporter Assay System (Promega, E1910) after 36 h according to the manufacturer’s instructions. Luciferase activities were detected by Single Tube Luminometer LB 9507 (Lumat).

RNA extraction and RT-PCR

Total RNA was extracted from cells using TRIzol reagent (Invitrogen) and then reverse-transcribed into cDNA using iScript cDNA Synthesis Kit (Bio-Rad). The resulting cDNA was diluted (1:20) for quantitative PCR using SYBR Green SuperMix (Roche) with gene-specific primers. The results were normalized to 18 S control. The sequences of the gene-specific primers involved in this study (forward and reverse, respectively) are as follows:

Rmp, 5’-CAAGCAGGCTGTGGGTTTAGT-3’ and 5’-GTGAGCAATTCGTTGTTTTCCTT-3’; Il-6, 5’-TAGTCCTTCCTACCCCAATTTCC-3’ and 5’-TTGGTCCTTAGCCACTCCTTC − 3’; Il-1β, 5’-GCAACTGTTCCTGAACTCAACT-3’and 5’-ATCTTTTGGGGTCCGTCAACT − 3’; Tnfα, 5’-CCCTCACACTCAGATCATCTTCT-3’and 5’-GCTACGACGTGGGCTACAG − 3’; Ifnγ, 5’-TCTGGAGGAACTGGCAAAAG-3’and 5’-AGTGACAGGCTGGGATGG − 3’; Ccl2, 5’-TAAAAACCTGGATCGGAACCAAA-3’and 5’-GCATTAGCTTCAGATTTACGGGT-3’; Ccl5, 5’-GCTGCTTTGCCTACCTCTCC-3’and 5’-TCGAGTGACAAACACGACTGC-3’; Trail, 5’-ATGATGGTGATTTGCATAGTGCT − 3’ and 5’-AGCTGCTTCATCTCGTTGGTG − 3’; 18S, 5’-CGGCTACCACATCCAAGGAA-3’and 5’-GCTGGAATTACCGCGGCT-3’.

Cytokine measurement

The concentrations of TNFα, IL-1β, and IL-6 in cellular supernatants or serum were measured using ELISA kits (eBioscience) according to the manufacturer’s instructions. Cellular supernatants were collected and centrifuged at 10,000 rpm (Microfuge 20R, BECKMAN COULTER) for 5 min to remove precipitates. Standard concentrations of IL-1β, TNFα, and IL-6 were prepared by serial dilutions of the peak concentration. 20 µL of each sample was added to 80 µL dilution buffer and transferred to the 96-well plate in the ELISA kit. After incubation and washing, 50 µL TMB One-Step Substrate Reagent was added to each well and incubated for 30 min in the dark at room temperature. The absolute absorbance value of each sample was obtained by subtracting the optical density readings at 570 nm from readings at 450 nm using a microplate reader (Synergy H1, BioTek).

Mass spectrometry of phosphoproteins

RAW264.7 cells were treated with LPS (100 ng/mL) for 30 min. Then, whole cell extracts were collected and lysed to obtain protein samples as described above. After that, each protein sample was incubated with 3 µg anti-RMP antibody and 50 µL MAg25K/Protein A/G beads overnight according to the manufacturer’s instructions. Western blot and Coomassie brilliant blue staining procedures were performed to stain the targeted bands (between 72 and 95 kDa). Further LC-MS/MS analysis was performed on a Q Exactive Mass Spectrometer (ThermoFisher Scientific) that was coupled to Easy nLC (ThermoFisher Scientific) for 60/120/240 min. The digested peptides were loaded onto a reverse phase trap column (ThermoFisher Scientific, Acclaim PepMap100, 100 μm*2 cm, nanoViper C18) connected to the C18-reversed-phase analytical column (ThermoFisher Scientific Easy Column, 10 cm long, 75 μm inner diameter, 3 μm resin) in buffer A (0.1% Formic acid) and separated with a linear gradient of buffer B (84% acetonitrile and 0.1% Formic acid) at a flow rate of 300 nL/min controlled by IntelliFlow technology. The mass spectrometer was operated in positive ion mode. MS data were acquired using a data-dependent top10 method. The MS raw data for each sample were combined and searched using the MaxQuant 1.5.3.17 software for further identification and quantitative analysis.

Phos-tag SDS-PAGE

Phosphorylated proteins were separated using conventional Phos-tag SDS-PAGE, as previously reported [27]. Phos-tag is a functional molecule that can specifically bind phosphate ions. In Phos-tag SDS-PAGE, a separation gel was prepared using acrylamide containing Mn²⁺ Phos-tag (FUJIFILM Wako Pure Chemical), according to the manufacturer’s instructions. By electrophoresis, phosphorylated and non-phosphorylated proteins can be separated according to their phosphorylation levels. The separation gel can be used for Coomassie blue staining, Western blotting, and mass spectrometry (MS) experiments.

LPS-induced lethal septic shock

For the LPS-induced endotoxemia model, male mice (8–12 weeks old) were injected intraperitoneally with LPS (Escherichia coli 0111:B4, Sigma-Aldrich) at a dose of 25 mg/kg. Control animals were administered with an equal amount of saline. Mice grouping was blind to the model maker and data collector. The survival was monitored and recorded for up to 72 h. Mice were grouped and euthanized for determination of inflammatory factors in plasma (6 h), or collection of lung tissues for histological evaluation (24 h), respectively. Mouse blood samples were collected and left to set for at least 10 h without anticoagulants at room temperature, and then centrifuged at 3000 rpm/min for 10 min. Serum samples were used to measure the concentrations of corresponding cytokines using ELISA kits (eBioscience). Littermates of myeloid-specific Rmp-KO and Rmp-WT mice were grouped and tested.

Establishment of CLP-induced sepsis and bacterial load measurement

Male mice, 8–12 weeks old, were completely anesthetized with 1% pentobarbital sodium (Sigma) according to weight (50 µg/g) by intraperitoneal injection before the surgery. After anesthetized, the central abdominal laparotomy was performed with an incision of 1–1.5 cm. 50% of the cecum was ligated with 4 − 0 silk suture, and one penetrable hole was made at the ligated cecum with a 21-G needle. Sham-operated animals underwent neither ligation nor puncture. Littermates of myeloid-specific Rmp-KO and Rmp-WT mice were grouped and tested. Mice grouping was blind to the model maker and data collector. Mice were resuscitated with 500 µL pre-warmed sterile physiological saline by subcutaneous injection after surgery. All mice had free access to food and water after recovery from anesthesia. Mice were checked for their survival state every 6 h after model construction until the 10th day. Test contents included the presence of heartbeat and mobility. Ascites obtained from tested mice were diluted at 1:1000, and then infiltrated cells in ascites were counted under the microscope by employing a blood cell counting plate. All blood, liver, and lung tissue collection and tissue homogenates were performed using sterile instruments. Serial dilutions were plated onto tryptic soy agar pre-poured petri dishes and incubated aerobically overnight at 37 °C.

Flow cytometry

Primary BMDM or peritoneal lavage fluids, and spleen homogenates were prepared at 1 × 10⁷/mL in 1.5 mL Eppendorf tubes (final volume of 100 µL). After washing with flow cytometry buffer (PBS with 2% FCS, 2 mM EDTA, and 25 mM HEPES, pH 7.4), cells were incubated with blocking buffer (5% goat serum in flow cytometry buffer) for 15 min at 4 °C. 0.5 µg/mL propidium iodide was added to each plate to identify and exclude dead cells. Then, cells were stained with the indicated antibody combinations for 30 min at room temperature. The antibodies used for flow cytometry are purchased from eBioscience and listed as follows: live/dead AmCyan, CD45.2-BV605, Cd11c-APC, Cd11b-PE, F4/80-FITC, Ly6C-pacific blue and Ly6G -Pe-Cy7 (all diluted at 1:100). Then, cells were washed twice with Hanks’ Balanced Salt Solution/Ca/Mg and then transferred into flow testing tubes (FALCON). Unstained cells and single-stained cells were used as control groups. The cells were gated using forward and side scatters on a BD FACS Aria II (BD Biosciences). Raw FCS files were analyzed with the FlowJo software.

Histology and injury evaluation of lung

Lung tissues were fixed in situ using 4% paraformaldehyde and then paraffin-embedded. Lung Sect. (4 μm thick) were stained with H&E and imaged using the Aperio ImageScope Viewer. Assessment of lung injury was previously described [28]. In brief, lung injury was assessed in four aspects, namely alveolar congestion, edema, cellular infiltrates, and widening of alveolar septa. Each aspect was given a score of 0 to 2 on the scale of severity. As a result, the total score of lung injury ranged from 0 to 8. The evaluation of lung injury was blindly conducted by two independent pathologists.

GST pull-down assay

The proteins including GST, GST-mouse RMP-WT, GST-mouse RMP-S439A, and His-mouse IKKβ were constructed and purified by AtaGenix. 5 µg GST fusion proteins plus 50 µL glutathione beads (Boppard) were added to a purified protein of IKKβ in vitro. After incubation with gentle rotation overnight at 4 °C, beads were washed three times with cell lysis buffer, and proteins were eluted with 2 × SDS loading buffer and detected by immunoblot.

ADP-Glo kinase assay

ADP-Glo Kinase kit was purchased from Promega. ADP-Glo Kinase Assay was conducted according to the manufacturer’s instructions. Purified His-tagged IKKβ (0.5 µg), GST-RMP WT (1 µg) or GST-RMP S439A (1 µg), ATP (25 µM), and protease inhibitors were mixed with 50 µL kinase buffer. Mixtures were added to 96-well plates and incubated for 60 min at room temperature. Then, 50 µL of ADP-Glo Reagent was added into each well and incubated for 40 min at room temperature. Subsequently, 100 µL of Kinase Detection Reagent was added to each well. After incubation for 30 min at room temperature, samples underwent luminescence detection using a microplate reader (Synergy H1, BioTek).

Immunofluorescence staining

RAW264.7 cell was treated with DMSO or LPS (30 min, 3 h). Then, the cells for immunostaining were fixed in 4% methanol-free formaldehyde at room temperature for 20 min and penetrated with 0.2% Triton X-100 for 20 min at 4 °C. After rinsing with PBS three times, cells were blocked with 10% goat serum for 1 h at room temperature and incubated with primary antibodies (RMP, 1:100; IKKβ, 1:100) overnight at 4 °C. After washing three times with PBS, the cells were incubated with secondary antibodies conjugated with Alexa 488 or Alexa 568 (1:100) at room temperature for 1 h. After immunostaining, the cells were briefly stained with Hoechst to display the cell nuclei. Finally, images were visualized and photographed using a Leica confocal microscope.

Surface plasmon resonance analysis

Interactions between the GST-RMP WT, GST-RMP S439A, Flag-PP2Ab, and His-IKKβ proteins were analyzed using the OpenSPR system (Nicoya) at 25 °C. Recombinant His-IKKβ protein was immobilized on a sensor chip (NTA, Nicoya) using an amine coupling kit (Nicoya). Final His-IKKβ immobilized levels were typically ∼ 16,000 RU. Subsequently, GST, GST-RMP WT, GST-RMP S439A, and Flag-PP2Ab were injected as analytes at various concentrations. PBS-P (10 mM phosphate buffer with 2.7 mM KCl and 137 mM NaCl, 0.05% Surfactant P20, pH 4.5) was used as the running buffer. For binding studies, analytes were applied at corresponding concentrations in the running buffer at a flow rate of 20 µL/min with a contact time of 240 s and a dissociation time of 480 s. Chip platforms were washed with the running buffer and 50% DMSO. Data were analyzed with TraceDrawer (Ridgeview Instruments ab, Sweden) by curve fitting using a 1:1 binding model.

Statistical analysis

All grouped data were statistically analyzed by GraphPad Prism software (version 7.0) and presented as bar plots in the figures. Data were presented as mean ± SEM. An unpaired Student’s t-test (two-tailed) was used to define the statistical significance between the two groups. One-way ANOVA was used to define the statistical significance in multiple groups of variables with more than three different managements. Kaplan-Meier survival curves were generated using GraphPad Prism software (version 7.0), and a log-rank test was performed to assess statistical significance. Asterisks presented P values in figures and P < 0.05 was considered statistically significant.

Results

RMP is a negative regulator of LPS-induced NF-κB signaling activation

To determine whether RMP affects innate immune activation of macrophages, we first constructed RAW264.7 cells stably transfected with RMP or GFP (Fig. 1A). When these cells were triggered by LPS, a TLR4 ligand, phosphorylation of IKK, P65, and IκBα was enhanced in GFP-overexpressing cells, compared with RMP-overexpressing cells (Fig. 1B, C). Notably, the most marked disparities manifested within the 15–30 min timeframe, coinciding temporally with the kinetic profile of NF-κB/IKKβ pathway activation. Given that IKK is the vital kinase in the NF-κB signaling pathway, we focused on its inhibition by RMP. NF-κB signaling is pivotal in modulating innate immune responses; thus, we investigated whether RMP affects cytokine production in macrophages under LPS treatment. GFP- and RMP-overexpressing cells were stimulated with LPS for 12 to 24 h and the mRNA and protein expressions of cytokines were assayed. RMP overexpression inhibited the transcription of Il-1β and Tnfα (Fig. 1D), two NF-κB-responsive cytokine genes, under 12 h of LPS treatment. Consistent with these results, the secretion of IL-1β and TNFα decreased in RMP-overexpressing cells, compared with control cells, under LPS treatment (Fig. 1E).

Fig. 1 — RMP is a negative regulator of LPS-induced NF-κB signaling activation. (A) Immunoblot analysis and quantification of RMP in lysates of RAW264.7 cells stably transfected with His-tagged mouse RMP or GFP plasmid. Images are representative of three independent experiments. n = 3 over three independent experiments. (B) Immunoblot analysis of phosphorylated (p-) and total IKK, P65, IκBα in lysates of RAW264.7 cells stably transfected with His-tagged mouse RMP or GFP plasmid and treated with LPS. Images are representative of three independent experiments. (C) Quantification of p-IKK, p-P65, p-IκBα levels normalized to their total protein levels in (B). n = 3 over three independent experiments. (D) RT-PCR analysis of *Il-1β* and *Tnfα* mRNA expression in RAW264.7 cells stably transfected with His-tagged mouse RMP or GFP plasmid and treated with LPS. n = 3 over three independent experiments. (E) ELISA analysis of IL-1β and TNFα concentrations in supernatants of RAW264.7 cells stably transfected with His-tagged mouse RMP or GFP plasmid and treated with LPS. n = 3 over three independent experiments. (F) Immunoblot analysis of RMP in lysates of THP-1 cells stably transfected with human RMP plasmids or shRNA. Images are representative of three independent experiments. (G) Immunoblot analysis of phosphorylated (p-) and total IKK, P65, IκBα in lysates of THP-1 cells stably transfected with human RMP plasmids or shRNA. Cells were treated with PMA and then with LPS. Images are representative of three independent experiments. (H) Quantification of p-IKK, p-P65, and p-IκBα levels normalized to their total protein levels in (G). n = 3 over three independent experiments. (I, J) NF-κB-responsive luciferase reporter analysis of THP-1 cells transfected with human RMP plasmids or shRNA. Cells were treated with PMA and then stimulated with LPS. n = 3 over three independent experiments. (K, L) RT-PCR analysis of *Il-1β*, *IL-6*, *Tnfα*, *Ccl2*, and *Ccl5* mRNA expression in THP-1 cells transfected with human RMP plasmids or shRNA. Cells were treated with PMA and then stimulated with LPS. n = 3 over three independent experiments. Data are presented with mean ± SEM and analyzed by unpaired two-tailed t-test. Data in (D), (K) and (L) are presented as the fold change in mRNA abundance relative to *18 S*. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant

THP-1, a human monocyte cell line, was used to validate the negative regulation of NF-κB signaling by RMP. We constructed a cell line with stable knockdown of RMP (shRMP) and a cell line of RMP overexpressing THP-1 cells (Fig. 1F). The LPS treatment induced strong activation of IKK and P65 in shRMP cells, whereas the phosphorylation of IKK and P65 were much lower in RMP-overexpressing THP-1 cells than in GFP-overexpressing cells (Fig. 1G, H). Then, we performed NF-κB-luc reporter assays in THP-1 cells and found that LPS strongly induced NF-κB-Luciferases(luc) activity and this activity, which was increased in shRMP cells and reduced in RMP-overexpressing cells (Fig. 1I, J). Furthermore, real-time PCR (RT-PCR) analysis revealed that the knockdown of RMP in THP-1 cells enhanced the mRNA abundance of LPS-induced NF-κB-targeting genes, including Il-1β, Il-6, Tnfα, Ccl2, and Ccl5, whereas RMP overexpression decreased them (Fig. 1K, L). Taken together, these results suggested that RMP inhibited TLR-induced NF-κB activation in macrophages.

Genetic knockout of Rmp enhances LPS-induced NF-κB signaling in macrophages

To explore the role of RMP in macrophage activation, floxed Rmp (Rmp^f/f) mice were constructed. The loxp sites were knocked into the RMP gene spanning the third exon, which led to a premature stop codon, as previously described [26]. We crossed Rmp^f/f mice with the Lysozyme 2-Cre (Lyz2-Cre⁺) line to create myeloid-specific RMP-knockout (KO) mice (Rmp^f/f; Lyz2-Cre⁺) (Fig. 2A), in which the expression of RMP was abolished in peritoneal macrophages (pMφs) but intact in liver cells (Fig. 2B, C). Relative to their Rmp^f/f; Lyz2-Cre⁻ (Rmp-Wild type,Rmp-WT) counterparts, Rmp^f/f; Lyz2-Cre⁺ (Rmp-KO) mice did not exhibit defective developmental phenotypes or differences in body weight, organ weight, hemogram, or blood biochemistry (Fig. S1A-D), as well as the total cell number of leukocytes in the bone marrow or spleen (Fig. S2A). Moreover, flow cytometry analysis revealed that the percentages of monocytes, macrophages, and neutrophils from bone marrow or spleen did not differ between myeloid-specific Rmp-KO and -WT mice (Fig. S2B-I), indicating that myeloid-specific Rmp-KO had a limited influence on the maturation and differentiation of mononuclear macrophages.

Fig. 2 — Genetic knockout of RMP enhances LPS-induced NF-κB signaling in macrophages. (A) Map of *Rmp*^flox/flox mice crossed with *Lyz2-Cre*⁺ mice. (B) RT-PCR analysis of *Rmp* mRNA expression in primary peritoneal macrophages (pMφs) and liver cells derived from *Lyz-Cre*⁺ or *Lyz-Cre*⁻, *Rmp*^flox/flox mice. n = 3 over three independent experiments. (C) Immunoblot analysis of RMP expression in pMφs and liver cells derived from *Lyz-Cre*⁺ or *Lyz-Cre*⁻, *Rmp*^flox/flox (*Rmp-*KO or *Rmp-*WT) mice. Images are representative of three independent experiments. (D) Immunoblot analysis of phosphorylated (p-) and total IKK, P65, and IκBα in lysates of primary pMφs derived from myeloid *Rmp-*KO or *Rmp-*WT mice and treated with LPS. Images are representative of three independent experiments. (E) Quantification of p-IKK, p-P65, and p-IκBα levels normalized to their total protein levels in (D). n = 3 over three independent experiments. (F) ELISA analysis of IL-1β, IL-6, and TNFα concentrations in supernatants of pMφs derived from myeloid *Rmp-*KO or *Rmp-*WT mice. Cells were treated with LPS. n = 3 over three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant

We then evaluated the activation of IKK and P65 stimulated by LPS in pMφs derived from genetically engineered mice. Rmp-knockout (Rmp-KO) macrophages exhibited much higher IKK and P65 phosphorylation activities than the floxed control (Rmp-WT) cells (Fig. 2D, E). Nevertheless, no alterations were observed in the phosphorylation status of AKT (Ser473), S6 (Ser235/236), JNK (Thr183/185), or ERK1/2 (Ser202/204) within various innate immunity-associated signaling pathways, such as PI3K/AKT/mTOR/MAPKs (Fig. S1E). Quantitative analysis revealed that Rmp knockout significantly augmented LPS-induced secretion of these proinflammatory cytokines into the cellular supernatant. (Fig. 2F).

Myeloid cell-specific deletion of Rmp promotes susceptibility to lethal LPS shock in mice

To investigate the function of myeloid-derived RMP in the innate immune response in vivo, endotoxin shock was induced by intraperitoneal injection of LPS. After administration of LPS, 40% of Rmp-WT mice survived within 48 h, whereas 80-90% of Rmp-KO mice died during the same period (Fig. 3A). Histopathological analysis revealed more severe lung injury in Rmp-KO mice than in Rmp-WT mice (Fig. 3B, C). Analyses of inflammatory cytokines in serum revealed an exacerbated cytokine storm in Rmp-KO mice. Enhanced LPS toxicity in Rmp-KO mice was correlated with increased serum abundance of TNFα, IL-1β, and IL-6 (Fig. 3D-F). Furthermore, by examining the peritoneal lavage fluid, we found a much higher cell count in Rmp-KO mice than in RMP-WT mice under LPS treatment (Fig. 3G). In addition, the percentage of neutrophils was greater in the Rmp-KO group under LPS treatment, although there was no significant difference in the percentage of macrophages and monocytes (Fig. 3H and Fig. S3A). Moreover, quantitative RT-PCR analysis demonstrated that genetic ablation of Rmp significantly upregulated transcriptional levels of Il-1β, Il-6 and Tnfα in pMφs isolated from lavage fluid of mice subjected to 12-hour LPS challenge (Fig. 3I). Taken together, these results suggested that RMP in myeloid cells was crucial for limiting the hyperactivation of the innate immune response and protecting hosts from lethal LPS shock.

Fig. 3 — Myeloid cell-specific deletion of RMP increases the susceptibility of mice to lethal LPS shock. (A) Survival curves of *Rmp-*WT (n = 16) and *Rmp-*KO (n = 17) mice after injection of LPS. Data are representative of three independent experiments. (B) Representative histopathologic images of lung tissues of *Rmp-*WT and *Rmp-*KO mice 12 h after LPS injection. Scale bar, 500 μm. (C) Lung damage score of *Rmp-*KO or *Rmp-*WT mice that underwent LPS-induced endotoxin shock. n = 6 over three independent experiments. (**D-F**) ELISA analysis of TNFα, IL-1β, and IL-6 concentrations in the serum of myeloid *Rmp-*KO or *Rmp-*WT mice undergoing LPS-induced endotoxin shock. n = 6 over three independent experiments. (G) Cell number of leukocytes in peritoneal lavage fluids derived from myeloid *Rmp-*KO or *Rmp-*WT mice that experienced LPS-induced endotoxin shock. n = 6 over three independent experiments. (H) Flow cytometry analysis of the percentages of neutrophils, macrophages, and monocytes in peritoneal lavage fluids of mice in the LPS-induced endotoxic shock model. n = 6 over three independent experiments. (I) RT-PCR analysis of *Il-1β*, *IL-6* and *Tnfα* mRNA expression in pMφs derived from myeloid *Rmp-*KO or *Rmp-*WT mice under LPS injection for 12 h. n = 3 over three independent experiments. Data in (A) are analyzed with the log-rank test. Data in (**C-H**) are presented with mean ± SEM and analyzed by unpaired two-tailed t-test. Data in (I) are presented as the fold change in mRNA abundance relative to *18 S*.*P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant

Myeloid cell-specific deletion of Rmp augments mortality in polymicrobial sepsis

The LPS shock model has been extensively employed in mechanistic investigations of sepsis pathogenesis. However, its pathophysiological relevance is fundamentally constrained by its exclusive dependence on LPS/TLR4 signaling pathway activation, representing a sterile inflammatory paradigm. In contrast, the CLP model induces a polymicrobial septic state characterized by substantial intestinal microbiota translocation and subsequent systemic dissemination. This polymicrobial infection model more faithfully recapitulates the complex immunoregulatory mechanisms involving multiple cellular components and signaling pathways observed in clinical human sepsis, particularly those associated with host-pathogen interactions and bacterial toxin-mediated pathogenesis. Thus, to validate whether Rmp disruption in myeloid cells aggravated mortality after sepsis, myeloid-specific Rmp-KO and Rmp-WT mice were placed in a moderate grade of CLP model and the survival was monitored. After CLP treatment, the death of Rmp-KO mice was observed within 1 day. Within 2 days, 50% of Rmp-KO mice died, whereas Rmp-WT mice reached the same mortality rate (50%) on the fourth day. The survival of Rmp-KO mice was shorter compared to Rmp-WT mice (Fig. 4A). Sham surgery caused no death in mice of any genotypes. These data indicate that RMP plays a critical role in myeloid cells in regulating mortality after polymicrobial sepsis. The lethality in sepsis is associated with the failure of vital organs. Therefore, we next examined histopathological differences between Rmp-KO and Rmp-WT mice and showed that lung injury was more severe in Rmp-KO mice (Fig. 4B, C).

Fig. 4 — Myeloid cell-specific deletion of RMP augments mortality in polymicrobial sepsis. (A) Survival curves of *Rmp-*WT (n = 22) and *Rmp-*KO (n = 22) mice in the CLP-induced septic shock model. Data are representative of three independent experiments. (**B) R**epresentative histopathologic images of three independent experiments of lung tissues of *Rmp-*WT and *Rmp-*KO mice in the CLP model. Scale bar, 500 μm. (C) Lung damage score of *Rmp-*KO or *Rmp-*WT mice in CLP model. n = 6 over three independent experiments. (**D-G**) Bacterial colonies are generated from peritoneal lavage fluids, blood, lung, and liver tissues after CLP. Quantitative analyses of bacterial CFUs are shown in histograms. Images are representative of three independent experiments. n = 6 over three independent experiments. (**H-J**) ELISA analysis of TNFα, IL-1β, and IL-6 concentrations in the serum of myeloid *Rmp-*KO and *Rmp-*WT mice that experienced CLP-induced septic shock. n = 6 over three independent experiments. (K) Cell number of leukocytes in peritoneal lavage fluids derived from myeloid *Rmp-*KO or *Rmp-*WT mice in CLP model. n = 6 over three independent experiments. (L) Flow cytometry analysis of the percentages of monocytes, macrophages, and neutrophils in peritoneal lavage fluids. n = 6 over three independent experiments. CLP was conducted in male littermates. Data in (A) are analyzed with the log-rank test. Data in (**C-L**) are presented with mean ± SEM and analyzed by unpaired two-tailed t-test. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant

Next, we collected peritoneal lavage fluid, blood samples, and liver and lung homogenates from the sham-operated, CLP Rmp-KO, and CLP Rmp-WT groups. Microflora was cultured on agarose plates and there was no obvious colony formation from the sham-operated mice, whereas the colony numbers from CLP-treated Rmp-KO mice were much higher than those from CLP-treated Rmp-WT mice (Fig. 4D-G). ELISA analysis showed that the serum abundance of TNFα, IL-1β, and IL-6 was greater in Rmp-KO mice than in Rmp-WT mice 24 h after CLP (Fig. 4H-J). Similarly to the LPS-induced endotoxin shock model, the number of cells and the percentage of macrophages in the peritoneal lavage fluid of Rmp-KO mice increased after 24 h of CLP, whereas the percentages of neutrophils and monocytes were not significantly different (Fig. 4K, L and Fig. S3B). Collectively, these results indicate that RMP in myeloid cells protected hosts from CLP-induced sepsis.

Inflammatory stimulation induces RMP phosphorylation in macrophages

To understand the cellular mechanisms by which RMP mediates LPS-induced NF-κB activation, we analyzed the expressions of RMP protein and mRNA after initial LPS treatment in RAW264.7 cell lines. The results showed a rapid downregulation of RMP protein after LPS stimulation for 3 to 24 h (Fig. 5A). RT-PCR analysis further showed that the Rmp mRNA expression reduced after 24 h of LPS treatment (Fig. 5B). The LPS-induced reduction of RMP suggested its role as a regulator of TLR4 signaling.

When endogenous RMP protein was detected in RAW264.7 cells, an upward band shift was observed after 15 to 60 min of stimulation with LPS (Fig. 5C). However, neither protein stability nor the transcriptional expressions of RMP were altered in LPS-treated macrophages within a short period (Fig. S4A-D). It has also been demonstrated that RMP can be hyperphosphorylated by mTOR and S6K1, and this hyperphosphorylation is efficiently blocked by rapamycin [21]. We postulated that this upward band shift may be a phosphorylation modification of the RMP protein. As expected, the slowly upshifting bands of RMP were sensitive to treatment with γPPase(Fig. 5D), representing it as one or more phosphorylated forms of RMP. To identify the exact phosphorylation site, we performed phosphorylation mass spectrometry identification of immunoprecipitation products from LPS-triggered macrophages and revealed that RMP was phosphorylated at Ser⁴³⁹ (Fig. 5E and Fig. S4E).

Then, we pretreated cells with several signaling pathway inhibitors, including rapamycin, and then stimulated these cells with LPS. We found that SP600125 (JNK inhibitor), SCH772984 (ERK inhibitor), BAY (P65 inhibitor), and rapamycin, did not abrogate LPS-induced phosphorylation of RMP (Fig. S4F-I). However, treatment with the IKK inhibitors IKK-16 and TPCA-1 slowed the upward migration of RMP bands (Fig. 5F, G), a phenomenon that was consistently recapitulated in primary peritoneal macrophages under identical experimental conditions (Fig. S4L). In addition, activation by other ligands of TLRs, including Resiquimod (TLR7/8) and Pam3csk4 (TLR1/2), caused a similar upward band shift of RMP in RAW264.7 cells (Fig. S4J, K). Together, these data suggested that RMP may participate in inflammatory stimulation and serve as a downstream substrate of IKK kinase.

IKKβ physically associates with and phosphorylates RMP

To determine the molecular mechanisms by which RMP negatively regulates NF-κB signaling, we detected whether RMP directly interacts with the IKK complex and P65 protein, as we found that IKK may phosphorylate RMP at Ser⁴³⁹. We transfected IKKα, IKKβ, IKKγ, and P65 plasmids into HEK-293T cells and performed co-immunoprecipitation experiments with specific antibodies. Overexpressed RMP could interact with all these proteins, whereas IKKβ showed the strongest interaction with RMP protein (Fig. S5A, B). The same result was also observed in RAW264.7 macrophages by endogenous co-immunoprecipitation assays, in which RMP interacted with IKKβ (Fig. 6A, B). To determine whether IKKβ directly binds to RMP, a GST pull-down assay was performed in vitro, which showed that purified GST-tagged RMP pulled down IKKβ protein (Fig. 6C). Given that IKKβ binds to RMP and may phosphorylate RMP at Ser⁴³⁹ in response to LPS stimulation, we constructed and purified the GST-tagged RMP S439A mutant protein for the GST-pulldown assay. Compared to its wild type, RMP S439A pulled down more IKKβ protein (Fig. 6D), suggesting that phosphorylation of the RMP Ser⁴³⁹ site affects its binding to IKKβ. Moreover, we constructed constitutively active IKKβ-SSEE, in which the Ser¹⁷⁷ and Ser¹⁸¹ sites were replaced by glutamic acids [12, 29]. The co-transfection of plasmids and immunoprecipitation experiments were performed in HEK-293T cells. Compared to RMP-WT, the RMP-S439A mutant immunoprecipitated more IKKβ-WT protein. Likewise, compared to active IKKβ-SSEE mutant, the IKKβ-WT immunoprecipitated more RMP-WT protein (Fig. S5C).

Fig. 6 — IKKβ physically associates with and phosphorylates RMP. (A) Immunoblot analysis of P65, IKKα, IKKβ, and IKKγ after immunoprecipitation with RMP from whole cell lysates (input) from RAW264.7 cells. Images are representative of three independent experiments. (B) Immunoblot analysis of endogenous RMP after immunoprecipitation with IKKα, IKKβ, and IKKγ antibodies from whole cell lysates (input) from RAW264.7 cells. Images are representative of three independent experiments. (C) Immunoblot analysis of bound proteins in a GST pulldown assay. Purified GST or GST-tagged mouse RMP (GST-RMP) was incubated with His-tagged IKKβ (His-IKKβ) and then pulled down with glutathione beads. Images are representative of three independent experiments. (D) Immunoblot analysis and quantification of bound proteins in a GST pulldown assay. Purified GST, GST-tagged mouse RMP-WT, or GST-tagged mouse RMP S439A was incubated with His-IKKβ and then pulled down with glutathione beads. Ratios of IKKβ/RMP were calculated. Images are representative of three independent experiments. n = 3 over three independent experiments. (E) Immunoblot analysis and quantification of endogenous RMP after immunoprecipitation with IKKβ from whole cell lysates (input) from RAW264.7 cells treatment with LPS. Ratios of RMP/IKKβ were calculated in immunoprecipitates. Images are representative of three independent experiments. n = 3 over three independent experiments. (F) Immunoblot analysis and quantification of endogenous IKKβ after immunoprecipitation with RMP from whole cell lysates (input) from RAW264.7 cells after treatment with LPS. Ratios of IKKβ/RMP were calculated. Images are representative of three independent experiments. n = 3 over three independent experiments. (G) NF-κB luciferase reporter analysis of HEK-293T cells co-transfected with TLR4, His-mouse RMP WT, His-mouse RMP-S439A, or His-mouse RMP S439E after LPS stimulation. n = 3 over three independent experiments. (H) GST, GST-mouse RMP WT, or GST-mouse RMP S439A proteins were incubated with active His-IKKβ in a kinase assay, and then the luminescence of ADP was detected. n = 4 over three independent experiments. (I) The phosphorylation state of mouse RMP WT and RMP S439A in the kinase assay were detected by Phos-tag SDS-PAGE electrophoresis. Red arrows indicate the phosphorylated bands of RMP. Images are representative of three independent experiments. n = 3 over three independent experiments. Data in (**D-F**) are analyzed by unpaired two-tailed t-test. Data in (G, H) are analyzed by a one-way ANOVA test. *P < 0.05; **P < 0.01; ***P < 0.001

We further explored the association between RMP and IKKβ under LPS stimulation, since LPS could induce the phosphorylation of RMP and IKKβ. LPS stimulation attenuated the interaction between IKKβ and RMP in RAW264.7 cells (Fig. 6E, F), and similar results were also observed in HEK-293T cells co-transfected with RMP, IKKβ, and TLR4 plasmids (Fig. S5D, E), suggesting that RMP separated from the IKK complex after LPS stimulation. These results were verified by immunofluorescence staining assays (Fig. S5F). The association between RMP and IKKβ partially recovered after LPS stimulation (Fig. S5F, G), suggesting a dynamic change in the RMP phosphorylation state may affect their association.

Human RMP protein can be phosphorylated at Ser³⁷², Ser⁴⁴², and Thr³⁷³ sites by mTOR and S6K1 [20, 30]. Since the amino acid sequences of mouse RMP and human RMP are partially different, the Ser³⁷² and Ser⁴⁴² sites of human RMP correspond to the Ser³⁶⁹ and Ser⁴³⁹ sites of mouse RMP, whereas the Thr³⁷³ site of human RMP has no counterpart on the mouse RMP protein. To confirm which site of RMP is indispensable for regulating NF-κB signaling, mouse RMP S369A, S439A, and human RMP S372A plasmids were constructed. Their binding properties with IKKβ and function in regulating NF-κB signaling were tested. Increased binding to IKKβ was only observed in the immunoprecipitates of mouse RMP S439A (Fig. S6A). Moreover, there was no significant difference in the effect of mouse RMP WT and S369A mutants on LPS-induced IKKβ/NF-κB signaling, whereas mouse RMP S439A mutant reversed the effects of mouse RMP WT (Fig. S6B). Furthermore, inhibition of mTOR signaling also did not affect the binding of RMP to IKKβ (Fig. S6C). Next, we explored whether phosphorylation of RMP altered the downstream signaling of IKK. NF-κB-luc reporter assays revealed that RMP-WT decreased NF-κB signaling which was counteracted by the mouse RMP-S439A mutant (Fig. 6G). These results suggested that RMP inhibited NF-κB signaling through its phosphorylation at the Ser⁴³⁹ site.

To determine whether IKKβ can directly phosphorylate the RMP protein, we performed in vitro ADP-Glo kinase assays in which control GST, GST-RMP-WT, and GST-RMP-S439A proteins were incubated with active IKKβ in an ADP-Glo kinase assay. The luminescence signal was positively correlated with the amount of ADP converted from ATP in the kinase reaction. We found that the RMP-WT protein increased the luminescence signal, whereas the S439A mutant did not (Fig. 6H). Moreover, the products of kinase assays were analyzed by Phospho-tag SDS-PAGE electrophoresis, in which the phosphorylated proteins were trapped by metal ions, separating them from their non-phosphorylated homologs. Immunoblotting showed that the RMP-WT protein could be phosphorylated by IKKβ kinase assay, whereas the RMP-S439A mutant was less responsive to this treatment (Fig. 6I). In addition, we observed that RMP did not affect the interaction of IKK subunits in co-immunoprecipitation assays, but inhibited the IKK activity in immunoprecipitation kinase assays, indicating RMP affected the activation of IKK complex rather than the formation (Fig. S6D, E). Taken together, IKKβ acted as an RMP kinase that phosphorylated RMP at the Ser⁴³⁹ site, and this phosphorylation was a negative feedback mechanism of IKKβ signaling.

The C-terminal region of RMP binds to the kinase domain of IKKβ

To identify the structural domain of the protein responsible for the interaction between RMP and IKKβ, truncations of mouse RMP were constructed according to the functional domains (Fig. 7A). We co-transfected IKKβ with various RMP truncations into HEK-293T cells and then performed co-immunoprecipitation experiments. The results showed that RMP-FL (1-531 aa), RMP-D3 (287–531 aa), and RMP-D5 (162–531 aa) interacted with IKKβ, whereas the N-terminal regions RMP-D1 (1-161 aa), RMP-D2 (162–286 aa), and RMP-D4 (1-286 aa) failed (Fig. 7B, C), suggesting that the C-terminal region of RMP is required for binding to IKKβ. The C-terminal truncations RMP-D3 (287–531 aa) and RMP-D5 (162–531 aa) both contain a Ser⁴³⁹ site targeted by active IKKβ, raising the possibility that RMP Ser⁴³⁹ site is vital for binding to IKKβ.

Fig. 7 — The C-terminal region of RMP binds to the kinase domain of IKKβ. (A) Schematic map of full-length and truncated mouse RMP. All truncated mutants were fused with His-tag. (B) Immunoblot analysis after immunoprecipitation with His-tag antibody of whole cell lysates (input) from HEK-293T cells. Cells were co-transfected with His-tagged mouse RMP truncated mutants and HA-tagged IKKβ. Images are representative of three independent experiments. (C) Immunoblot analysis after immunoprecipitation with HA-tag antibody of whole cell lysates (input) from HEK-293T cells. Cells were co-transfected with His-tagged mouse RMP truncated mutants and HA-tagged IKKβ. Images are representative of three independent experiments. Red asterisks indicate the immunoprecipitated bands. (D) Schematic map of full-length and truncated mouse IKKβ. All truncated mutants were fused with the HA-tag. (E) Immunoblot analysis after immunoprecipitation with HA-tag antibody of whole cell lysates (input) from HEK-293T cells. Cells were co-transfected with HA-tagged IKKβ truncated mutants and His-tagged mouse RMP. Images are representative of three independent experiments. (F) Immunoblot analysis after immunoprecipitation with His-tag antibody of whole cell lysates (input) from HEK-293T cells. Cells were co-transfected with HA-tagged IKKβ truncated mutants and His-tagged mouse RMP. Images are representative of three independent experiments. (G) Immunoblot analysis after immunoprecipitation with HA-tag antibody of whole cell lysates (input) from HEK-293T cells. Cells were co-transfected with HA-tagged IKKβ-D1 and His-tagged mouse RMP-D3. Images are representative of three independent experiments. (H) Immunoblot analysis after immunoprecipitation with His-tag antibody of whole cell lysates (input) from HEK-293T cells. Cells were co-transfected with HA-tagged IKKβ-D1 and His-tagged mouse RMP-D3. Images are representative of three independent experiments

To further explore the mechanisms by which the RMP structural domain containing the Ser⁴³⁹ site binds to IKKβ, and based on the assumption that the kinase structural domain of IKKβ may be responsible for this interaction, we constructed several IKKβ truncations containing a kinase domain (KD), leucine-zipper (LZ) domain, or helix-loop-helix (HLH) domain (Fig. 7D). RMP interacted with the kinase domain of IKKβ (IKKβ-KD) but not with the IKKβ-HLH or IKKβ-LZ domain (Fig. 7E, F). We co-transfected RMP-D3 (287–531 aa) and the IKKβ-KD domain into HEK-293T cells and confirmed the interaction between the two regions (Fig. 7G, H). Together, these data verified that the C-terminal region of RMP binds to the kinase domain of IKKβ.

Inhibition of LPS-induced activation of IKKβ requires the RMP/PP2A complex

The above data revealed that RMP acts as a downstream substrate of IKK kinase and attenuates LPS-induced inflammation. Next, we sought to investigate the mechanism by which RMP inhibits IKKβ phosphorylation. It has been reported that IKKβ can form signaling complexes with PP1γ and/or PP2A in response to TNFR/TLR activation, and these protein serine/threonine phosphatases negatively regulate NF-κB signaling through the dephosphorylation of IKKβ [31, 32]. Similarly, RMP also interacts with PP1γ and/or PP2A and is involved in their functional regulation [20, 24]. We speculated that RMP may act as a mediator of IKKβ inactivation by recruiting members of protein phosphatase. Indeed, we identified that PP2Ab, but not PP2Aa, PP2Ac, or PP1γ, could be precipitated by RMP and IKKβ antibodies in RAW264.7 cells (Fig. 8A), indicating that RMP, IKKβ, and PP2Ab form a complex and that the PP2A holoenzyme may be involved in RMP-mediated inactivation of IKKβ. Then we discovered that RMP overexpression strengthened the interaction between IKKβ and PP2Ab (Fig. 8B-E), indicating that RMP promoted the formation of the IKK-PP2A complex. To verify that the PP2A holoenzyme dephosphorylates IKKβ and affects NF-κB signaling, we treated THP-1 cells with a PP2A inhibitor or overexpressed PP2Ab in THP-1 cells. Immunoprecipitation experiments showed that under LPS treatment, PP2A inhibitor increased IKKβ phosphorylation and decreased the binding of IKKβ to RMP (Fig. S7A, B). Together, these data suggested that PP2A inhibited the phosphorylation of IKK and promoted the interaction between IKKβ and RMP.

Fig. 8 — Inhibition of LPS-induced activation of IKKβ requires the RMP/PP2A signaling complex. (A) Immunoblot analysis of endogenous PP2Aa, PP2Ab, PP2Ac, and PP1γ after immunoprecipitation with RMP or IKKβ antibodies from whole cell lysates (input) from RAW264.7 cells. Images are representative of three independent experiments. (B) Immunoblot analysis of the PP2Ab after immunoprecipitation with HA-tag antibody. HEK-293T cells were co-transfected HA-tagged IKKβ and Flag-tagged PP2Ab with or without His-tagged mouse RMP. Images are representative of three independent experiments. (C) Quantitative analysis of relative PP2Ab enrichment by the HA-tag (IKKβ) antibody. Ratios of PP2Ab/IKKβ were calculated. n = 3 over three independent experiments. (D) Immunoblot analysis of the IKKβ after immunoprecipitation with Flag-tag antibody. HEK-293T cells were co-transfected HA-tagged IKKβ and Flag-tagged PP2Ab with or without His-tagged mouse RMP. Images are representative of three independent experiments. (E) Quantitative analysis of relative IKKβ enrichment by the Flag-tag (PP2Ab) antibody. Ratios of IKKβ/PP2Ab were calculated. n = 3 over three independent experiments. (F) Left: NF-κB-responsive luciferase reporter analysis of THP-1 cells overexpressing human RMP WT treated with PMA and LPS in the presence or absence of PP2A inhibitor; Right: NF-κB-responsive luciferase reporter analysis of THP-1 cells expressing the RMP shRNA (shRMP) and PP2Ab plasmid and then treated with PMA and LPS. n = 3 over three independent experiments. (G) Immunoblot analysis and quantification of phosphorylated (p-) and total IKK, P65 in the lysates of THP-1 cells overexpressing human RMP. Cells were treated with PMA and LPS with or without PP2A inhibitor (LB100). Images are representative of three independent experiments. n = 3 over three independent experiments. (H) Immunoblot analysis and quantification of phosphorylated (p-) and total IKKβ, p-65 in the lysates of THP-1 cells expressing human RMP shRNA (shRMP) and PP2Ab plasmid. Cells were then treated with PMA and LPS. Images are representative of three independent experiments. n = 3 over three independent experiments. (I) KDs between IKKβ and GST-RMP WT or GST-RMP S439A were detected by SPR. n = 3 over three independent experiments. (J) KDs between PP2Ab and GST-RMP WT, GST-RMP S439A, or His-IKKβ were detected by SPR. n = 3 over three independent experiments. (K) The model of RMP-IKK-PP2Ab complex in the regulation of TLR4-NF-κB signaling pathway. WCL, Whole cell lysates. KD, dissociation constant; Kon, association rate constant; Kdis, dissociation rate constant. KD = Kdis/Kon. Data in (E, G, H) are presented as mean ± SEM and analyzed by unpaired two-tailed t-test. Data in (F) are presented as mean ± SEM and analyzed by one-way ANOVA test. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant

To further probe whether PP2A was responsible for the RMP-mediated inhibition of IKKβ and NF-κB signaling, NF-κB-luc reporter gene assays and immunoblotting were performed in THP-1 cells. Consequently, the PP2A inhibitor restored the inhibitory effect of RMP overexpression on IKK/NF-κB signaling under LPS treatment (Fig. 8F, G). Similarly, under LPS treatment, PP2Ab overexpression could limit the excessive activation of IKK/NF-κB signaling (Fig. 8F, H). In conclusion, these results indicated a potential mechanism for RMP-induced IKK inactivation and suggested that RMP recruits the protein phosphatase PP2A to form the IKK-RMP-PP2A complex, which maintains IKK in its default inactive state in macrophages.

To directly confirm the association between IKKβ, RMP, and PP2Ab proteins, surface plasmon resonance (SPR) was employed to detect the binding characteristics and affinity constants between IKKβ, PP2Ab, and RMP WT or RMP S439A. RMP could bind to IKKβ in vitro and RMP mutation at the Ser⁴³⁹ site enhanced their binding (Fig. 8I and Fig. S7C, D), consistent with results of co-immunoprecipitation and GST-pulldown assay. Moreover, PP2Ab, RMP, and IKKβ potentially bound to each other in vitro to form a complex (Fig. 8J and Fig. S7E-G).

Collectively, we proposed a model that IKKβ, RMP, and PP2Ab could form a complex in macrophages based on our in vivo and in vitro data. In the resting state, RMP would be associated with IKKβ and recruit PP2A to maintain IKKβ in an unphosphorylated inactive state. Treatment with LPS led to RMP phosphorylation at the Ser⁴³⁹ site by IKKβ and the release of both RMP and PP2A from the complex, permitting phosphorylated IKK complex to activate IκB, leading to the degradation of IκB and the nuclear translocation of NF-κB (Fig. 8K).

Discussion

TLRs are known to elicit conserved inflammatory pathways, which culminate in the activation of diverse signaling pathways, such as IKK/NF-κB, MAPK (JNK, ERK, and P38), PI3K-AKT and other pathways [9]. However, aberrant activation of TLRs disturbs immune homeostasis, which leads to the development of autoimmune and inflammatory diseases [33–35]. Multiple mechanisms have been reported to prevent inappropriate activation of TLRs, including kinase activity alteration, signal protein degradation, epigenetic modification, and transcriptional regulation [3, 9, 36–38]. Although overwhelming evidence shows that IKKβ is essential for the activation of NF-κB [12, 39, 40], relatively little is known about its negative regulation. In the present study, our data revealed a homeostatic mechanism in which RMP attenuates TLR-mediated NF-κB signaling through an inhibitory interaction with IKKβ.

The RMP gene was first isolated and cloned from a human HepG2 cDNA and was found to counteract the transactivation of the hepatitis B virus X protein [16]. The primary structure of RMP contains all features of an α class PFD, an RPB5-binding region, and a long acidic C-terminal domain [19, 21]. RMP was found to be a part of the R2TP/PDFL complex and functioned as a chaperone that coordinated the interaction of proteins involved in transcription such as TFIIF, the Paf-1 complex, and the RNA polymerase II [16, 41, 42]. It has also been suggested that RMP could be phosphorylated by S6K1 at Ser³⁷¹ to limit the mTOR signaling [20]. Thus, evidence suggests that RMP can also act as a substrate for several important kinases, and not simply as a chaperone. RMP was observed to associate with P65 and RPB5, which assemble in the pre-transcriptional initiation complex of IL-6 [23], indicating that RMP is involved in NF-κB signaling. However, since these studies were performed in parenchymal cells, the regulatory role of RMP in mesenchymal cells, particularly in immunocyte activation, remains to be fully elucidated.

We found that RMP attenuated IKK phosphorylation and then NF-κB activation by selectively interacting with IKKβ. The IKK complex is composed of the kinase subunits IKKα and IKKβ as well as the regulatory subunit IKKγ. Also, our exogenous and endogenous co-immunoprecipitation results revealed that RMP was also slightly bound to IKKα/γ, so we speculated that RMP may indirectly interact with these two subunits by IKKβ. Few studies have discovered proteins that directly downregulate IKK activity. In our study, we found that RMP preferentially bound to the KD domain of IKKβ and inhibited its phosphorylation and activity.

CUE domain-containing 2 (CUEDC2) interacts with IKKα/β but not IKKγ and recruits PP1 to deactivate the IKK complex by dephosphorylating IKKα/β [43]. Given that RMP was shown to interact with the protein phosphatases PP1 and PP2A [20, 24], which were reported to associate with IKKβ and deactivate the IKK complex [31, 32], we then explored whether RMP elicited a similar function as CUEDC2 on IKK kinases. We revealed that PP2Ab (rather than PP2Aa, PP2Ac, or PP1) interacted with RMP in macrophages. As a heterotrimer, PP2A is composed of three subunits: a structural A subunit (PP2Aa), a regulatory B-type subunit (PP2Ab), and a catalytic C subunit (PP2Ac). To further prove that PP2A was responsible for the RMP-mediated dephosphorylation of IKK, a PP2A inhibitor was used to treat RMP-overexpressing or RMP-knockdown macrophages, and results confirmed that the inhibitory role of RMP on the phosphorylation of IKKβ was dependent on PP2A enzymatic activation.

The interaction between RMP and IKKβ appeared to be dynamic since LPS treatment decreased the interaction between RMP and IKKβ. We observed altered phosphorylation of RMP protein at the Ser⁴³⁹ site induced by LPS stimulation. Treatment with IKK inhibitors and in vitro IKK kinase assays confirmed that IKKβ was a functional kinase targeting the RMP. The S439 site is located at the C-terminal domain of RMP, which is responsible for interaction with the KD domain of IKKβ. When stimulated by LPS (an agonist of TLR4), Pam3csk4 (an agonist of TLR1/2), or Resiquimod (an agonist of TLR7/8), TLRs are activated, which in turn initiates a downstream kinase signaling cascade that includes IKKβ. Activated IKKβ begins to phosphorylate the RMP protein, and this phosphorylation may lead to conformational changes in RMP that reduce its ability to bind to IKKβ. To confirm this hypothesis, we mutated the Ser⁴³⁹ site in RMP and showed that the mutant bound more tightly to IKKβ than the wild-type RMP, regardless of treatment with LPS. The RMP S439A mutant also inhibited LPS-induced IKK activation, indicating that the Ser⁴³⁹ site is a checkpoint for RMP-mediated inflammatory signaling. Consequently, a working model was proposed to illustrate how RMP modulated NF-κB signaling. In resting macrophage cells, RMP binds to the kinase domain of IKKβ and recruits PP2A to prevent the phosphorylation of IKKβ. After LPS stimulation, however, RMP was phosphorylated by IKKβ at the Ser⁴³⁹ site and dissociated from IKKβ, allowing downstream NF-κB signaling activation. Therefore, RMP serves as a checkpoint in TLR signaling and is a key regulator of innate immunity.

In addition, RMP promotes IL-6 transcription in hepatocellular carcinoma (HCC) cell lines through the NF-κB pathway [31, 32]. We discovered that RMP negatively regulated the TLR4-NF-κB pathway in macrophages, which led to alterations in IL-6 transcription. To our knowledge, HCC cells lack TLR4 receptors and cannot be activated by the corresponding ligands. Therefore, we speculated that RMP plays different roles in diverse cell types through particular signaling pathways. Conclusion.

Collectively, our findings revealed the role of RMP in the negative regulation of NF-κB signaling and elucidated the molecular mechanism by which RMP dynamically interacts with the IKK complex in a phosphorylation-dependent manner. Thus, RMP may represent a useful therapeutic target for microbial infections and inflammation-associated diseases.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material ^{(3.9MB, docx)}

Acknowledgements

We thank statistician Cheng Wu (Department of Health Statistics, Naval Medical University) for the support of the statistical analyses in this study.

Abbreviations

RMP: RNA polymerase II subunit 5 (RPB5) mediating protein
NF-κB: Nuclear factor κB
IKK: IκB kinase
LPS: Lipopolysaccharide
CLP: Cecal ligation and perforation
HE: Hematoxylin-eosin
WB: Western-blot
RT-PCR: Reverse transcription-polymerase chain reaction
Co-IP: Co‑immunoprecipitation
GST: Pulldown Glutathione S transferase pulldown
SPR: Surface plasmon resonance
PP2A: Phosphatase 2 A
TLR4: Toll-like receptor 4
PAMPs: Pathogen-associated molecular patterns
MYD88: Myeloid differential protein-88
TRIF: TIR-domain-containing adapter-inducing interferon-β
MAPKs: Mitogen-activated protein kinases
IRF3: Interferon regulatory factor 3
TNFα: Tumor necrosis factor α
IL: Interleukin
URL: Unconventional prefoldin RBP5 interactor
PAQosome: Particle for Arrangement of Quaternary Structure
mTOR: Mammalian target of rapamycin
S6K1: Ribosomal protein S6 Kinase 1
IGF1: Insulin-like growth factor 1
Luc: Luciferases
pMφ: Peritoneal macrophages
KO: Knockout
WT: Wild type
λPPase: Lambda phosphatase
KD: Kinase domain
LZ: Leucine-zipper
HLH: Helix-loop-helix
CUEDC2: CUE domain-containing 2
HCC: Hepatocellular carcinoma

Author contributions

SJ Pang, TY Jiang, XW Cui, and NG Wang performed the experiments. N Yang and NG Wang provided materials. YF Pan and H Wang intellectually contributed throughout the project. TY Jiang, SJ Pang, and LW Dong analyzed data and wrote the manuscript. TY Jiang, N Yang and LW Dong were responsible for research supervision, coordination, and strategy.

Funding

This work was supported by grants from the National Natural Science Foundation of China (32270814, 82203586, 82403719, 92359301 and 91859205), the Shanghai Rising-Star Program (24QA2711800), the National Key R&D Program of China (2022YFC2503704), Project of Shanghai Research Center (2023ZZ02005), and the special clinic project of Shanghai (20234Y0264).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

All animal operating procedures complied with the ethical standards of the Institutional Animal Care and Use Committee of the Naval Medical University. Animal experiments were approved by the Ethics Board of the Eastern Hepatobiliary Surgery Hospital.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Shu-jie Pang, Tian-yi Jiang, Nai-guo Wang and Xiao-wen Cui contributed equally to this work.

Contributor Information

Tian-yi Jiang, Email: jiangtianyi@smmu.edu.cn.

Ning Yang, Email: lancet00@163.com.

Li-wei Dong, Email: dlw@smmu.edu.cn.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material ^{(3.9MB, docx)}

Data Availability Statement

No datasets were generated or analysed during the current study.

[CR1] 1.Medzhitov R. Recognition of microorganisms and activation of the immune response. Nature. 2007;449:819–26. [DOI] [PubMed] [Google Scholar]

[CR2] 2.Poltorak A, He X, Smirnova I, Liu MY, Van Huffel C, Du X, et al. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science. 1998;282:2085–8. [DOI] [PubMed] [Google Scholar]

[CR3] 3.Kawai T, Akira S. Toll-like receptor and RIG-I-like receptor signaling. Ann N Y Acad Sci. 2008;1143:1–20. [DOI] [PubMed] [Google Scholar]

[CR4] 4.Karin M, Lawrence T, Nizet V. Innate immunity gone awry: linking microbial infections to chronic inflammation and cancer. Cell. 2006;124:823–35. [DOI] [PubMed] [Google Scholar]

[CR5] 5.Liew FY, Xu D, Brint EK, O’Neill LA. Negative regulation of toll-like receptor-mediated immune responses. Nat Rev Immunol. 2005;5:446–58. [DOI] [PubMed] [Google Scholar]

[CR6] 6.Rudd KE, Johnson SC, Agesa KM, Shackelford KA, Tsoi D, Kievlan DR, et al. Global, regional, and National sepsis incidence and mortality, 1990–2017: analysis for the global burden of disease study. Lancet. 2020;395:200–11. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR7] 7.Singer M, Deutschman CS, Seymour CW, Shankar-Hari M, Annane D, Bauer M, et al. The third international consensus definitions for Sepsis and septic shock (Sepsis-3). JAMA. 2016;315:801–10. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR8] 8.van der Poll T, van de Veerdonk FL, Scicluna BP, Netea MG. The immunopathology of sepsis and potential therapeutic targets. Nat Rev Immunol. 2017;17:407–20. [DOI] [PubMed] [Google Scholar]

[CR9] 9.Takeda K, Kaisho T, Akira S. Toll-like receptors. Annu Rev Immunol. 2003;21:335–76. [DOI] [PubMed] [Google Scholar]

[CR10] 10.Chen G, Goeddel DV. TNF-R1 signaling: a beautiful pathway. Science. 2002;296:1634–5. [DOI] [PubMed] [Google Scholar]

[CR11] 11.Karin M, Ben-Neriah Y. Phosphorylation Meets ubiquitination: the control of NF-[kappa]B activity. Annu Rev Immunol. 2000;18:621–63. [DOI] [PubMed] [Google Scholar]

[CR12] 12.Mercurio F, Zhu H, Murray BW, Shevchenko A, Bennett BL, Li J, et al. IKK-1 and IKK-2: cytokine-activated IkappaB kinases essential for NF-kappaB activation. Science. 1997;278:860–6. [DOI] [PubMed] [Google Scholar]

[CR13] 13.Ashall L, Horton CA, Nelson DE, Paszek P, Harper CV, Sillitoe K, et al. Pulsatile stimulation determines timing and specificity of NF-kappaB-dependent transcription. Science. 2009;324:242–6. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR14] 14.Shinohara H, Behar M, Inoue K, Hiroshima M, Yasuda T, Nagashima T, et al. Positive feedback within a kinase signaling complex functions as a switch mechanism for NF-kappaB activation. Science. 2014;344:760–4. [DOI] [PubMed] [Google Scholar]

[CR15] 15.Hoffmann A, Levchenko A, Scott ML, Baltimore D. The IkappaB-NF-kappaB signaling module: Temporal control and selective gene activation. Science. 2002;298:1241–5. [DOI] [PubMed] [Google Scholar]

[CR16] 16.Dorjsuren D, Lin Y, Wei W, Yamashita T, Nomura T, Hayashi N, et al. RMP, a novel RNA polymerase II subunit 5-interacting protein, counteracts transactivation by hepatitis B virus X protein. Mol Cell Biol. 1998;18:7546–55. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR17] 17.Mita P, Savas JN, Ha S, Djouder N, Yates JR 3rd, Logan SK. Analysis of URI nuclear interaction with RPB5 and components of the R2TP/prefoldin-like complex. PLoS ONE. 2013;8:e63879. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR18] 18.Gauthier MS, Cloutier P, Coulombe B. Role of the paqosome in regulating arrangement of protein quaternary structure in health and disease. Adv Exp Med Biol. 2018;1106:25–36. [DOI] [PubMed] [Google Scholar]

[CR19] 19.Boulon S, Pradet-Balade B, Verheggen C, Molle D, Boireau S, Georgieva M, et al. HSP90 and its R2TP/Prefoldin-like cochaperone are involved in the cytoplasmic assembly of RNA polymerase II. Mol Cell. 2010;39:912–24. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR20] 20.Djouder N, Metzler SC, Schmidt A, Wirbelauer C, Gstaiger M, Aebersold R, et al. S6K1-mediated disassembly of mitochondrial URI/PP1gamma complexes activates a negative feedback program that counters S6K1 survival signaling. Mol Cell. 2007;28:28–40. [DOI] [PubMed] [Google Scholar]

[CR21] 21.Gstaiger M, Luke B, Hess D, Oakeley EJ, Wirbelauer C, Blondel M, et al. Control of nutrient-sensitive transcription programs by the unconventional prefoldin URI. Science. 2003;302:1208–12. [DOI] [PubMed] [Google Scholar]

[CR22] 22.Tummala KS, Gomes AL, Yilmaz M, Grana O, Bakiri L, Ruppen I, et al. Inhibition of de Novo NAD(+) synthesis by oncogenic URI causes liver tumorigenesis through DNA damage. Cancer Cell. 2014;26:826–39. [DOI] [PubMed] [Google Scholar]

[CR23] 23.Zhang J, Pan YF, Ding ZW, Yang GZ, Tan YX, Yang C, et al. RMP promotes venous metastases of hepatocellular carcinoma through promoting IL-6 transcription. Oncogene. 2015;34:1575–83. [DOI] [PubMed] [Google Scholar]

[CR24] 24.Mita P, Savas JN, Briggs EM, Ha S, Gnanakkan V, Yates JR 3, et al. URI regulates KAP1 phosphorylation and transcriptional repression via PP2A phosphatase in prostate Cancer cells. J Biol Chem. 2016;291:25516–28. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR25] 25.Theurillat JP, Metzler SC, Henzi N, Djouder N, Helbling M, Zimmermann AK, et al. URI is an oncogene amplified in ovarian cancer cells and is required for their survival. Cancer Cell. 2011;19:317–32. [DOI] [PubMed] [Google Scholar]

[CR26] 26.Xue YL, Guan ZQ, Meng XQ. Overexpression of PTEN enhanced gemcitabine-induced apoptosis by promoting autophagy in osteosarcoma cell line MG-63. Int J Clin Exp Med. 2016;9:23258–66. [Google Scholar]

[CR27] 27.Kinoshita E, Kinoshita-Kikuta E, Takiyama K, Koike T. Phosphate-binding tag, a new tool to visualize phosphorylated proteins. Mol Cell Proteom. 2006;5:749–57. [DOI] [PubMed] [Google Scholar]

[CR28] 28.Nedeva C, Menassa J, Duan M, Liu C, Doerflinger M, Kueh AJ, et al. TREML4 receptor regulates inflammation and innate immune cell death during polymicrobial sepsis. Nat Immunol. 2020;21:1585–96. [DOI] [PubMed] [Google Scholar]

[CR29] 29.Zandi E, Rothwarf DM, Delhase M, Hayakawa M, Karin M. The IkappaB kinase complex (IKK) contains two kinase subunits, IKKalpha and ikkbeta, necessary for IkappaB phosphorylation and NF-kappaB activation. Cell. 1997;91:243–52. [DOI] [PubMed] [Google Scholar]

[CR30] 30.Zhou H, Di Palma S, Preisinger C, Peng M, Polat AN, Heck AJ, et al. Toward a comprehensive characterization of a human cancer cell phosphoproteome. J Proteome Res. 2013;12:260–71. [DOI] [PubMed] [Google Scholar]

[CR31] 31.Li Q, Zheng Z, Liu Y, Zhang Z, Liu Q, Meng J, et al. 2 C proteins of enteroviruses suppress IKKbeta phosphorylation by recruiting protein phosphatase 1. J Virol. 2016;90:5141–51. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR32] 32.Witt J, Barisic S, Schumann E, Allgower F, Sawodny O, Sauter T, et al. Mechanism of PP2A-mediated IKK beta dephosphorylation: a systems biological approach. BMC Syst Biol. 2009;3:71. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR33] 33.Cook DN, Pisetsky DS, Schwartz DA. Toll-like receptors in the pathogenesis of human disease. Nat Immunol. 2004;5:975–9. [DOI] [PubMed] [Google Scholar]

[CR34] 34.Perkins DJ, Vogel SN. Inflammation: Species-specific TLR signalling -- insight into human disease. Nat Rev Rheumatol. 2016;12:198–200. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR35] 35.Li J, Wang X, Zhang F, Yin H. Toll-like receptors as therapeutic targets for autoimmune connective tissue diseases. Pharmacol Ther. 2013;138:441–51. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR36] 36.Du M, Yuan L, Tan X, Huang D, Wang X, Zheng Z, et al. The LPS-inducible LncRNA Mirt2 is a negative regulator of inflammation. Nat Commun. 2017;8:2049. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR37] 37.Lin H, Jiang M, Liu L, Yang Z, Ma Z, Liu S, et al. The long noncoding RNA Lnczc3h7a promotes a TRIM25-mediated RIG-I antiviral innate immune response. Nat Immunol. 2019;20:812–23. [DOI] [PubMed] [Google Scholar]

[CR38] 38.Zhao X, Huo R, Yan X, Xu T. IRF3 negatively regulates Toll-Like Receptor-Mediated NF-kappaB signaling by targeting TRIF for degradation in teleost fish. Front Immunol. 2018;9:867. [DOI] [PMC free article] [PubMed] [Google Scholar]

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PERMALINK

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

Shu-jie Pang

Tian-yi Jiang

Nai-guo Wang

Xiao-wen Cui

Hui Wang

Yu-fei Pan

Ning Yang

Li-wei Dong

Abstract

Background

Methods

Results

Conclusions

Supplementary Information

Background

Methods

Cell culture and treatment

Isolation of pMφs and spleen cells

Animals

Plasmids, DNA transfection, and lentivirus infection

Antibodies and reagents

Immunoblot analysis and co-immunoprecipitation

Luciferase reporter gene assay

RNA extraction and RT-PCR

Cytokine measurement

Mass spectrometry of phosphoproteins

Phos-tag SDS-PAGE

LPS-induced lethal septic shock

Establishment of CLP-induced sepsis and bacterial load measurement

Flow cytometry

Histology and injury evaluation of lung

GST pull-down assay

ADP-Glo kinase assay

Immunofluorescence staining

Surface plasmon resonance analysis

Statistical analysis

Results

RMP is a negative regulator of LPS-induced NF-κB signaling activation

Fig. 1.

Genetic knockout of Rmp enhances LPS-induced NF-κB signaling in macrophages

Fig. 2.

Myeloid cell-specific deletion of Rmp promotes susceptibility to lethal LPS shock in mice

Fig. 3.

Myeloid cell-specific deletion of Rmp augments mortality in polymicrobial sepsis

Fig. 4.

Inflammatory stimulation induces RMP phosphorylation in macrophages

Fig. 5.

IKKβ physically associates with and phosphorylates RMP

Fig. 6.

The C-terminal region of RMP binds to the kinase domain of IKKβ

Fig. 7.

Inhibition of LPS-induced activation of IKKβ requires the RMP/PP2A complex

Fig. 8.

Discussion

Electronic supplementary material

Acknowledgements

Abbreviations

Author contributions

Funding

Data availability

Declarations

Ethics approval and consent to participate

Consent for publication

Competing interests

Footnotes

Contributor Information

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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