Fig 2: Constitutive activation of NIK induces ectopic colonic M-cells and predisposes to colitis. a Schematic representation of the mice with intestinal epithelial-specific overexpression of NIK (NikVilRosa?T3) generated by crossing mice with cDNA harboring mutation in TRAF3 binding site of NIK driven by loxP-Rosa26 promoter-loxP with mice expressing Cre recombinase driven by Villin promoter. b Western blot analysis of NIK and p52 in the small intestine (SI) and colon (Col) of 6-week-old NikVilRosa?T3 mice. c, d qPCR analysis of M-cell markers (c) and GP2 staining (d) in the colon of NikVilRosa?T3 mice. Images were taken at ×10 magnification. e ELISA for IL17 in the colon and serum of 6-week-old untreated NikVilRosa?T3 mice. f–h Body weight (f), colon length (g), and H&E analysis (h) in 7-week-old NikVilRosa?T3 mice that were treated with DSS for 6 days. H&E images were taken at ×10 magnification. Results are expressed as mean ± SEM. Significance was determined using t test. *P < 0.05; **P < 0.01; ***P < 0.001. i Intesinal epithelial NIK-RANK signaling is critical to maintain M-cells and local and systemic IL17 and IgA response. Intestinal epithelial non-canonical NF?B signaling is essential to maintain the antigen sampling M-cells in the Peyer’s patch. Loss of M-cells in the Peyer’s patches is associated with a decrease in IL17 expression in T-cells. Loss of epithelial NIK signaling diminishes both local and systemic IL17 and IgA response resulting in increased susceptibility to colitis and polymicrobial sepsis. Conversely, constitutive activation of epithelial NIK also exacerbates colitis, which is associated with induction in ectopic colonic M-cells and IL-17 levels. Thus, intestinal NIK signaling plays a rheostatic role in maintaining intestinal homeostasis by regulating the immune and inflammatory signatures

Fig 3: CnAα interacts with NIK through its phosphatase domain.A. Co-immunoprecipitation of CnAα and its mutants (ΔN and ΔCA) with NIK. CnAα and its mutants expressed in cells are indicated at the top of panels. Control indicates the Myc-tagged expression vector. The upper left panel (Co-IP) shows western blotting of immunoprecipitates using the anti-Flag antibody to detect co-immunoprecipitation of Myc-tagged CnAα and its mutants. The lower left panel shows western blotting of immunoprecipitates using the anti-Flag antibody to detect Flag-tagged NIK. The upper right panel shows western blotting of total cell lysates using the anti-Myc antibody. The lower right panels show western blotting of immunoprecipitates using the anti-Flag antibody to detect Flag-tagged NIK. Arrows indicate bands of IgG chains used for immunoprecipitation. Results of one representative experiment of three are shown. Blots are cropped for clarity. Full-length blots of key data are presented in Supplementary Figure 2. B. Schematics of CnAα and its deletion mutants used in this study. “Phosphatase” indicates the phosphatase domain containing the catalytic domain and regulatory subunit-binding domain. “CaM” indicates a potential calmodulin-binding domain. “AI” indicates the auto-inhibitory domain. The Flag tag (abbreviated in this figure) was connected to the N-terminus of the wild-type protein and mutants. The binding ability of each protein for NIK, as determined in Fig. 2A, is indicated at the right of each structure. “+” indicates positive for binding, and “−” indicates negative for binding.

Fig 4: Overexpressing human NIK in mouse tibialis anterior induces muscle atrophy. (A) NIK mRNA levels presented as cycle times three weeks after injection of AAV-NIK or AAV-GFP. (B) Representative immunohistochemical images denoting dystrophin (red), GFP (green) and DAPI (blue) in cross-sections of the tibialis anterior following administration of AAV-NIK or AAV-GFP. Scale bar = 50 µm. (C) Percentage of fibers-expressing GFP presented as mean ± SD. ND, none detected. (D) Representative immunohistochemical images denoting dystrophin (green), NIK (red) and DAPI (blue) in cross-sections of the tibialis anterior following administration of AAV-NIK or AAV-GFP. Scale bar = 50 µm. (E) Percentage of fibers-expressing NIK perinuclear staining presented as mean ± SD. (F) Average myofiber cross-sectional area (CSA) in µm2 of NIK- and NIK+ fibers following administration of AAV-NIK. Data are presented as mean ± SD. ***Significant effect of AAV-NIK administration, P < 0.001. N = 8 mice/group.

Fig 5: (A) Principal component analysis of gene expression profiles for high NIK-expressing MCF7 cells, low NIK-expressing MCF7 cells and MCF7 control cells. (B) Clustering diagram of samples and differentially expressed genes in MCF7 cells overexpressing NIK and NIK-deficient MCF7 cells. Log-Fold Changes of differentially expressed genes are depicted in a heat map, scaling from low (green) to high (red). Experiments were performed in three independent transiently-transfected MCF7 cell lines (C). Graph showing the fold change of stem cells-associated genes in high NIK-expressing cells and (D) low NIK-expressing cells. (E) RT-qPCR analysis of EGR1, TCN1, DUSP6, GDF15, CSPG4 and MAP2K6 in MCF7 overexpressing-NIK cells, MCF7 control cells, NIK-deficient MCF7 cells (shNIK1 and shNIK2) and MCF7 control cells (shLuc). All RT-qPCR were normalized to TBP. (n = 3, error bars are +/- s.d, p < 0.05). (G) IPA network of top gene networks from NIK-overexpressing cells. (H) IPA network of top gene networks from NIK-depleted cells. Note that ERK1/2 is a central node in both networks. Red color indicates induced genes and green color represents suppressed genes.