Nucleoredoxin negatively regulates Toll-like receptor 4 signaling via recruitment of flightless-I to myeloid differentiation primary response gene (88)

Abstract

We previously characterized nucleoredoxin (NRX) as a negative regulator of the Wnt signaling pathway through Dishevelled (Dvl). We perform a comprehensive search for other NRX-interacting proteins and identify Flightless-I (Fli-I) as a novel NRX-binding partner. Fli-I binds to NRX and other related proteins, such as Rod-derived cone viability factor (RdCVF), whereas Dvl binds only to NRX. Endogenous NRX and Fli-I in vivo interactions are confirmed. Both NRX and RdCVF link Fli-I with myeloid differentiation primary response gene (88) (MyD88), an important adaptor protein for innate immune response. NRX and RdCVF also potentiate the negative effect of Fli-I upon lipopolysaccharide-induced activation of NF-kappaB through the Toll-like receptor 4/MyD88 pathway. Embryonic fibroblasts derived from NRX gene-targeted mice show aberrant NF-kappaB activation upon lipopolysaccharide stimulation. These results suggest that the NRX subfamily of proteins forms a link between MyD88 and Fli-I to mediate negative regulation of the Toll-like receptor 4/MyD88 pathway.

FIGURE 1.

NRX co-precipitated proteins. A, schematics of TRX, NRX, RdCVF, C9orf121, and TryX. Amino acid residue numbers in each protein are also shown. B, lysate of NIH3T3 cells stably expressing FLAG-NRX or GFP immunoprecipitated with anti-FLAG antibody. The precipitated proteins were separated by SDS-PAGE and visualized by silver staining. The proteins corresponding to the bands (indicated by arrows) were subjected to MALDI-MS/MS analyses. C, results of the MALDI-MS/MS analyses. The names of the identified proteins, their calculated mass, the number of the peptides identified, and score are shown.

FIGURE 2.

Interaction of Fli-I with NRX and its family member proteins. A, recombinant Fli-I proteins were incubated with GST- or GST-NRX-immobilized beads. After washing, the bound proteins were subjected to SDS-PAGE, followed by immunoblotting (IB) with anti-Fli-I antibody (upper panel). The purity of recombinant Fli-I, GST, and GST-NRX proteins, as determined by Coomassie staining, is also shown (lower panels). B, schematic representation of NRX mutants. C, Myc-Fli-I transfected into COS7 cells with FLAG-tagged WT or mutant forms of NRX. Cell lysates were immunoprecipitated (IP) with anti-FLAG antibody followed by immunoblotting. The relative amounts of co-precipitated Myc-Fli-I (right) were determined by densitometry (mean ± S.E. (error bars) of co-precipitated versus input from three independent experiments). *, p < 0.05 (Student's t test); n.s., not significant. D, MEF lysates subjected to immunoprecipitation with anti-NRX antibody. The precipitates were separated by SDS-PAGE followed by immunoblotting with anti-Fli-I antibody. In this experiment, anti-NRX antibodies were covalently attached to the beads to avoid nonspecific signals. E, lysates of COS7 cells transfected with Myc-Fli-I and FLAG-tagged TRX, NRX, RdCVF, and C9orf121 subjected to immunoprecipitation with anti-FLAG antibody, followed by immunoblotting with the indicated antibodies.

FIGURE 3.

NRX links Fli-I to MyD88. A and B, COS7 cells were transfected with FLAG-Fli-I, Myc-MyD88, and Myc-NRX (A) or Myc-RdCVF (B). The cell lysates were subjected to anti-FLAG immunoprecipitation (IP). IB, immunoblotting. C, Myc-MyD88 and FLAG-tagged WT or mutant forms of NRX were transfected into COS7 cells, and their lysates were immunoprecipitated with anti-FLAG antibody followed by immunoblotting. D, lysates from MEFs were subjected to immunoprecipitation with anti-NRX antibody. The precipitates were separated by SDS-PAGE followed by anti-MyD88 immunoblotting. In this experiment, anti-NRX antibodies were covalently attached to the beads to avoid nonspecific signals. E, purified MBP-MyD88 proteins were immobilized onto beads and then incubated with purified GST-Fli-I and/or His-NRX proteins. The bound proteins were detected by immunoblotting with the indicated antibodies. The purity of recombinant GST-Fli-I, His-NRX, MBP, and MBP-MyD88, determined by Coomassie staining, is shown.

FIGURE 4.

NRX synergistically suppresses the TLR4/MyD88 pathway with Fli-I. A–C, MEFs were transfected with the NF-κB reporter plasmid and the indicated expression constructs. The cells were stimulated with LPS (0.1 μg/ml, 6 h) before the reporter assays were performed. *, p < 0.05; **, p < 0.01; ***, p < 0.001 (Student's t test). Error bars, S.E. D, WT (+/+) or NRX-deficient (−/−) MEFs were transfected with the NF-κB reporter plasmid. *, p < 0.05 (Student's t test). E, WT or NRX^−/− MEFs were treated with 0.1 μg/ml LPS for the indicated time periods, and their lysates were subjected to immunoblotting (IB) with anti-IκBα and anti-NRX antibodies. F, WT or NRX^−/− MEFs were treated with LPS (1 μg/ml, 10 min), and their lysates were immunoprecipitated with anti-MyD88 antibody followed by immunoblotting with anti-Fli-I antibody. In this experiment, anti-MyD88 antibodies were covalently attached to the beads to avoid nonspecific signals.

FIGURE 5.

Schematic illustration of the role of NRX, Fli-I, and MyD88 on TLR4 signaling. Left, in resting state (−LPS), IκBα associates with NF-κB and retains it in the cytosol. Center, LPS binding to TLR4 induces the recruitment of MyD88 to TLR4, which leads to the degradation of IκBα. NF-κB free from IκBα then moves into the nucleus and activates the transcription of various target genes. In WT cells, Fli-I forms a complex with MyD88 through NRX and avoids the unnecessary activation. Right, in NRX-deficient (NRX^−/−) cells, Fli-I cannot sequester MyD88 from TLR4, and thus the hyperactivation of TLR4/MyD88 signaling occurs upon LPS stimulation.