NLRP12 Suppresses Colon Inflammation and Tumorigenesis through the Negative Regulation of Noncanonical NF-κB Signaling

RIPK1 is involved in signaling from TNF and TLR family receptors. After receptor ligation, RIPK1 not only modulates activation of both canonical and NIK-dependent NF-κB, but also regulates caspase-8 activation and cell death. Although overexpression of RIPK1 can cause caspase-8-dependent cell death, when RIPK1(-/-) cells are exposed to TNF and low doses of cycloheximide, they die more readily than wild-type cells, indicating RIPK1 has pro-survival as well as pro-apoptotic activities. To determine how RIPK1 promotes cell survival, we compared wild-type and RIPK1(-/-) cells treated with TNF. Although TRAF2 levels remained constant in TNF-treated wild-type cells, TNF stimulation of RIPK1(-/-) cells caused TRAF2 and cIAP1 to be rapidly degraded by the proteasome, which led to an increase in NIK levels. This resulted in processing of p100 NF-κB2 to p52, a decrease in levels of cFLIP(L), and activation of caspase-8, culminating in cell death. Therefore, the pro-survival effect of RIPK1 is mediated by stabilization of TRAF2 and cIAP1.

NF-κB-inducing kinase (NIK) is a central component in the non-canonical NF-κB signaling pathway. Excessive NIK activity is implicated in various disorders, such as autoimmune conditions and cancers. Here, we report the first crystal structure of truncated human NIK in complex with adenosine 5'-O-(thiotriphosphate) at a resolution of 2.5 Å. This truncated protein is a catalytically active construct, including an N-terminal extension of 60 residues prior to the kinase domain, the kinase domain, and 20 residues afterward. The structure reveals that the NIK kinase domain assumes an active conformation in the absence of any phosphorylation. Analysis of the structure uncovers a unique role for the N-terminal extension sequence, which stabilizes helix αC in the active orientation and keeps the kinase domain in the catalytically competent conformation. Our findings shed light on the long-standing debate over whether NIK is a constitutively active kinase. They also provide a molecular basis for the recent observation of gain-of-function activity for an N-terminal deletion mutant (ΔN324) of NIK, leading to constitutive non-canonical NF-κB signaling with enhanced B-cell adhesion and apoptosis resistance.

Hemochromatosis, an inherited iron-overload disease, causes tissue damage due to deposition of iron in toxic levels in most tissues including the liver, heart, kidney, pancreas and colon. Free iron, both in the inorganic form (i.e, non-transferrin-bound and non-ferritin-bound) and organic form (i.e. heme), facilitates Fenton reaction to generate hydroxyl radicals and to induce cellular oxidative damage that is observed in carcinogenesis. The primary site of iron deposition in hemochromatosis patients is liver, and therefore, liver cancer formation in these patients has been studied extensively. However, little is known about the iron-elicited colonic pathologies, mainly colonic inflammation and colon cancer. Here we postulate that iron overload as observed in hemochromatosis disrupts healthy colonic homeostasis and exacerbates the development of colon inflammation and colon cancer. To test our hypothesis, we examined the progression and severity of colitis and colon cancer in Hfe-/- mouse and the wild type control. Hfe-/- mouse is a model for classical hemochromatosis. Besides serum and liver, high concentrations of iron and heme deposits are found in the colon of Hfe-/- mouse. Experimental colitis was induced by administration of Dextran Sodium Sulfate (DSS) in drinking water. Colitis-associated colon cancer was initiated by the intraperitoneal injection of carcinogen, azoxymethane (AOM) and the carcinogenesis was driven by DSS administration. Colonic inflammation was more severe in the Hfe-/- mouse than in control. In addition, hemochromatosis mouse developed more and larger colonic polyps than the control group. We observed that this high susceptibility to colitis and colon cancer in Hfe-/- but not wild type mouse, lies in the differences in strains regarding the bacterial composition and colonic defense machinery. 16S ribosomal RNA sequencing of fecal bacteria revealed that the microbiota composition of Hfe-/- mouse altered to favor the pathogenic bacteria that belong to phyla Proteobacteria and TM7. In addition, the Hfe-/- proinflammatory bacteria adhered to colon and thus increased the colonic bacterial load. This phenomenon, addressed as bacterial dysbiosis, is considered a hallmark of colon inflammation and colon cancer. Furthermore, we observed that the colonic epithelial cells of Hfe-/- mouse had a lower expression of antimicrobial peptides and thus a defective first line defense against the pathogens. Finally, hemochromatosis mouse colon under inflammation released higher concentration of the pro-inflammatory cytokines that contributed to the creation of the hostile colonic environment. In summary, iron overload as seen in hemochromatosis, impairs the colonic defense machinery and it causes bacterial dysbiosis, thus providing the ideal environment for the colitis and colon cancer development. Citation Format: Bojana Ristic, Sathish Sivaprakasam, Rao Kottapalli, Abdul Hamood, Vadivel Ganapathy. Bacterial dysbiosis in the mouse model of hemochromatosis: Increased risk of colitis and colitis-associated colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1479.

Tumor necrosis factor (TNF) receptor-associated factors (TRAFs) are critical signaling adaptors downstream of many receptors in the TNF receptor and interleukin-1 receptor/Toll-like receptor superfamilies. Whereas TRAF2, 5, and 6 are activators of the canonical NF-kappaB signaling pathway, TRAF3 is an inhibitor of the noncanonical NF-kappaB pathway. The contribution of the different domains in TRAFs to their respective functions remains unclear. To elucidate the structural and functional specificities of TRAF3, we reconstituted TRAF3-deficient cells with a series of TRAF3 mutants and assessed their abilities to restore TRAF3-mediated inhibition of the noncanonical NF-kappaB pathway as measured by NF-kappaB-inducing kinase (NIK) protein levels and processing of p100 to p52. We found that a structurally intact RING finger domain of TRAF3 is required for inhibition of the noncanonical NF-kappaB pathway. In addition, the three N-terminal domains, but not the C-terminal TRAF domain, of the highly homologous TRAF5 can functionally replace the corresponding domains of TRAF3 in suppression of the noncanonical NF-kappaB pathway. This functional specificity correlates with the specific binding of TRAF3, but not TRAF5, to the previously reported TRAF3 binding motif in NIK. Our studies suggest that both the RING finger domain activity and the specific binding of the TRAF domain to NIK are two critical components of TRAF3 suppression of NIK protein levels and the processing of p100 to p52.

The current paradigm of noncanonical NF-κB signaling suggests that the loss of TRAF2, TRAF3 or cIAP1 and cIAP2 leads to stabilization of NF-κB-inducing kinase (NIK) to activate the noncanonical pathway. Although a crucial role of RIP1 in the TNFα-induced canonical NF-κB pathway has been well established, its involvement in noncanonical activation of NF-κB through the TNFR1 receptor, is unknown. Here we show that TNFα is capable of activating the noncanonical NF-κB pathway, but that activation of this pathway is negatively regulated by RIP1. In the absence of RIP1, TNFR1 stimulation leads to activation of the noncanonical NF-κB pathway through TRAF2 degradation, leading to NIK stabilization, IKKα phosphorylation and the processing of p100 to generate p52. Thus although RIP1(-/-) mouse embryonic fibroblasts are sensitive at early time points to cell death induced by TNFα, probably as a result of lack of canonical NF-κB activation, the late activation of the noncanonical NF-κB pathway protects the remaining cells from further cell death. The TNFR1-dependent noncanonical NF-κB activation in RIP1(-/-) cells suggests that there is functional interplay between the two NF-κB pathways during TNFR1 signaling, which might regulate the number and kinds of NF-κB transcription factors and thus finely control NF-κB-dependent gene transcription.

SMAC antagonization of cIAP1/2 in TH 17cells upregulates cell adhesion and cytoskeleton genes through the NIK-RelB and p52 axis. SMAC also increases the homotypic interactions of TH 17cells through a non-canonical NF-κB- and integrin-mediated mechanism resulting in increased ability of TH 17cells to withstand shear stress.

Components of lymphotoxin beta receptor (LTBR)-associated signaling complexes, including TRAF2, TRAF3, NIK, IKK1, and IKK2 have been shown to participate in the coupling of LTBR to NFkappaB. Here, we report that TRAF3 functions as a negative regulator of LTBR signaling via both canonical and non-canonical NFkappaB pathways by two distinct mechanisms. Analysis of NFkappaB signaling in cell lines with functionally intact NFkappaB pathway but lacking LTBR-mediated induction of NFkappaB target genes revealed an inverse association of cellular TRAF3 levels with LTBR-specific defect in canonical NFkappaB activation. Increased expression of TRAF3 correlated with its increased recruitment to LTBR-induced signaling complexes, decreased recruitment of TRAF2, and attenuated phosphorylation of IkappaB alpha and RelA. In contrast, activation of NFkappaB by TNF did not depend on TRAF3 levels. siRNA-mediated depletion of TRAF3 promoted recruitment of TRAF2 and IKK1 to activated LTBR, enabling LTBR-inducible canonical NFkappaB signaling and NFkappaB target gene expression. TRAF3 knock-down also increased mRNA and protein expression of several non-canonical NFkappaB components, including NFkappaB2/p100, RelB, and NIK, accompanied by processing of NFkappaB2/p100 into p52. These effects of TRAF3 depletion did not require LTBR signaling and were consistent with autonomous activation of the non-canonical NFkappaB pathway. Our data illustrate the function of TRAF3 as a dual-mode repressor of LTBR signaling that controls activation of canonical NFkappaB, and de-repression of the intrinsic activity of non-canonical NFkappaB. Modulation of cellular TRAF3 levels may thus contribute to regulation of NFkappaB-dependent gene expression by LTBR by affecting the balance of LTBR-dependent activation of canonical and non-canonical NFkappaB pathways.

NF-κB pathway consists of canonical and non-canonical pathways. The canonical NF-κB is activated by various stimuli, transducing a quick but transient transcriptional activity, to regulate the expression of various proinflammatory genes and also serve as the critical mediator for inflammatory response. Meanwhile, the activation of the non-canonical NF-κB pathway occurs through a handful of TNF receptor superfamily members. Since the activation of this pathway involves protein synthesis, the kinetics of non-canonical NF-κB activation is slow but persistent, in concordance with its biological functions in the development of immune cell and lymphoid organ, immune homeostasis and immune response. The activation of the canonical and non-canonical NF-κB pathway is tightly controlled, highlighting the vital roles of ubiquitination in these pathways. Emerging studies indicate that dysregulated NF-κB activity causes inflammation-related diseases as well as cancers, and NF-κB has been long proposed as the potential target for therapy of diseases. This review attempts to summarize our current knowledge and updates on the mechanisms of NF-κB pathway regulation and the potential therapeutic application of inhibition of NF-κB signaling in cancer and inflammatory diseases.

Peripheral artery disease (PAD) is atherosclerotic occlusion of vessel outside the heart and most commonly affects the lower extremities. Diabetes (DM) accelerates the course and severity of PAD. Studies have shown that vascular endothelial cell NF-κB activity is required for post ischemic adaptation in experimental PAD. To better understand how DM contributes to PAD severity, we investigated the role of DM hyperglycemia in the activation of NF-κB under ischemic conditions. Induced ischemia in human vascular endothelial cell (HUVEC) cultures increased components of both canonical and non-canonical NF-κB pathways in the nucleus (p65 1.0 ± 0.1 vs 1.5 ± 0.2, p< 0.05, RelB 1.0 ± 0.1 vs 1.5 ± 0.2, p<0.01). Similarly, HUVEC acutely exposed to high glucose (HG, 25 mM) activated both canonical (IκB-α degradation, normal vs. HG 1.25 ± 0.02 vs 0.9 ± 0.0, p<0.05) and non-canonical NF-κB (p100 degradation, normal vs HG 0.021±0.001 vs 0.016±0.000, p<0.05) pathways. Prolonged exposure (3 days) of HUVEC to high glucose before ischemia resulted in impaired NF-κB activation as evident from decreased IκB phosphorylation (pIκB/IκB in normal glucose and ischemia 1.56 ± 0.22 vs 1.12 ± 0.35, p<0.01). To understand the signaling pathways underlying the ischemic activation of the NF-κB pathway, we used an array of antibodies to phosphoproteins involved in the inflammatory pathway. Compared to the lysates from cells grown in normal glucose, the lysates from cells grown in prolonged high glucose had dramatically increased phosphorylation of PKC-β2 (PKC-β2pSer661, 8-fold increase). To test whether this increase in PKC-β2pSer66 impairs NF-κB activation by ischemia, we treated HUVECS with prolonged high glucose exposure and ruboxystaurin (Rbx) (20 nM), an inhibitor of PKC-β2 phosphorylation, prior to ischemic exposure. Immunoblotting results confirmed that inhibition of PKC-β2 phosphorylation enhanced the ischemia induced NF-κB activation in HUVEC in this condition. We then tested the effect of Rbx on PKC-β2 phosphorylation and NF-κB activation in vivo in Akita mice, a model for type 1 diabetes. Consistent with our in vitro findings, in experimental PAD, NF-κB activity in the ischemic hind limb of Akita mice was significantly lower than those of the wild type (WT) mice as measured by IκB-α degradation (WT ischemic vs Akita ischemic; 0.04 ± 0.03 vs 0.10 ± 0.04 p<0.05). However, treatment of Akita mice with Rbx increased NF-κB activation in the ischemic hind limb (Akita ischemic 0.10 ± 0.04 vs ischemic+ Rbx 0.05 ± 0.02, p<0.05). Moreover, compared to the WT mice, the untreated Akita mice showed an impaired perfusion in the ischemic limbs (% perfusion recovery, WT vs Akita; 80.1 ± 10.3 vs 55.7 ± 10.1, p<0.05, n=5-8) that was improved in Rbx treated Akita mice (96.3 ± 2.3, p<0.01). Thus, hyperglycemic conditions increase PKC-β2pSer66 in endothelial cells attenuating salutary NF-κB activation contributing to poor PAD outcomes in DM.

Ligation of the lymphotoxin-β receptor (LTβR) by LIGHT (lymphotoxin-related inducible ligand that competes for glycoprotein D binding to herpes virus entry mediator on T cells (TNFSF14)) activates the noncanonical (NC) NF-κB (nuclear factor-κB) pathway and up-regulates CXCL12 gene expression by human umbilical vein endothelial cells (HUVEC). In contrast, TNF only activates classical NF-κB signaling and does not up-regulate CXCL12. To determine whether cross-talk between the classical and NC pathways affects CXCL12 expression, we investigated the effects of TNF on LIGHT signaling in HUVEC. We show here that TNF inhibits both basal and LIGHT-induced CXCL12 expression. Negative regulation by TNF requires the classical NF-κB pathway as inhibition of basal and induced CXCL12 was reversed in HUVEC-expressing dominant negative IκB (inhibitor of NF-κB) kinase (IKK)β (IKKβ(K44M)). TNF did not inhibit the NC NF-κB pathway activation as LIGHT-induced p100 processing to p52 was intact; however, TNF either alone or together with LIGHT up-regulated p100 and RelB expression and induced the nuclear localization of p100-RelB complexes. Enhanced p100 and RelB expression was inhibited by IKKβ(K44M), which led us to question whether the IκB function of elevated p100 mediates the inhibition of CXCL12 expression by TNF. We retrovirally transduced HUVEC to express p100 at a level similar to that up-regulated by TNF; however, basal and LIGHT-induced CXCL12 expression was normal in the transduced cells. In contrast, ectopic RelB expression recapitulated the effects of TNF on NC signaling and inhibited basal and LIGHT-induced CXCL12 expression by HUVEC. Our findings therefore demonstrate that TNF-induced classical NF-κB signaling up-regulates RelB expression that inhibits both basal and NC NF-κB-dependent CXCL12 expression.

IAP and HDAC inhibitors interact synergistically in myeloma cells through noncanonical NF-κB– and caspase-8–dependent mechanisms

TNF-like weak inducer of apoptosis (TWEAK) receptor Fn14 is expressed by podocytes and Fn14 deficiency protects from experimental proteinuric kidney disease. However, the downstream effectors of TWEAK/Fn14 in podocytes are poorly characterized. We have explored TWEAK activation of non-canonical NFκB signaling in cultured podocytes. In cultured podocytes, TWEAK increased the expression of the chemokines CCL21, CCL19 and RANTES in a time-dependent manner. The inhibitor of canonical NFκB activation parthenolide inhibited the CCL19 and the early RANTES responses, but not the CCL21 or late RANTES responses. In this regard, TWEAK induced non-canonical NFκB activation in podocytes, characterized by NFκB2/p100 processing to NFκB2/p52 and nuclear migration of RelB/p52. Silencing by a specific siRNA of NIK, the upstream kinase of the non-canonical NFκB pathway, prevented CCL21 upregulation but did not modulate CCL19 or RANTES expression in response to TWEAK, thus establishing CCL21 as a non-canonical NFκB target in podocytes. Increased kidney Fn14 and CCL21 expression was also observed in rat proteinuric kidney disease induced by puromycin, and was localized to podocytes. In conclusion, TWEAK activates the non-canonical NFκB pathway in podocytes, leading to upregulation of CCL21 expression. The non-canonical NFκB pathway should be explored as a potential therapeutic target in proteinuric kidney disease.

The signaling cascades triggered by OX40 in the control of diverse responses from initial T cell activation to the generation of memory and regulatory T cells remain incompletely defined. OX40 preferentially activates and sustains the non-canonical NF-κB pathway, but the underlying mechanisms remain unknown. Here, we found that in primary CD4+ T cells activated under OX40 costimulation, TRAF6 and cIAP2 are two essential molecules and both cooperatively activate the non-canonical NF-κB pathway. Mechanistically, OX40 directly recruited TRAF6 and TRAF2/TRAF3/cIAP2/NIK complexes in which TRAF6 acted as the E3 ligase to mediate Lys63-linked polyubiquitination of cIAP2. Consequently, cIAP2 used its E3 ligase activity to promote TRAF3 Lys48-linked polyubiquitination and degradation. The UBA domain of cIAP2 inhibited the E3 ligase activity of cIAP2 targeting to TRAF3 and this autoinhibition could be released by the C-terminal RING domain polyubiquitination mediated by TRAF6. The lysines 585 and 594 at cIAP2 C-terminus were two main ubiquitin acceptor sites for TRAF6-mediated polyubiquitination, as combined mutation of such sites abolished Lys48-linked TRAF3 polyubiquitination. Thus, in TRAF6 knockout T cells, OX40 failed to induce sufficient non-canonical NF-κB activation and the downstream IL-9 production due to insufficient TRAF3 degradation. Our study identifies a previously unknown mechanism of OX40 controlled non-canonical NF-κB activation in T cells.

To determine effects of (1) parenteral nutrition (PN), (2) exogenous Lymphotoxin β receptor (LTβR) stimulation in PN animals, and (3) exogenous LTβR blockade in chow animals on NF-κB activation pathways and products: MAdCAM-1, chemokine (C-C motif) Ligand (CCL) 19, CCL20, CCL25, interleukin (IL)-4, and IL-10. LT stimulates LTβR in Peyer's patches (PP) to activate NF-κB via the noncanonical pathway. The p100/RelB precursor yields p52/RelB producing MAdCAM-1, cytokines, and chemokines important in cell trafficking. TNFα, IL-1β, and bacterial products stimulate the inflammatory canonical NF-κB pathway producing p65/p50 and c-Rel/p50. PN decreases LTβR, MAdCAM-1, and chemokines in PP and lowers small intestinal IgA compared with chow. Canonical (p50 and p65) and noncanonical (p52 and Rel B) NF-κB proteins in PP were analyzed by TransAM NF-κB kit after 5 days of chow or PN, 2 days of LTβR stimulation or 3 days of LTβR blockade. MAdCAM-1, chemokines, and cytokines in PP were measured by ELISA after LTβR stimulation or blockade. PN significantly reduced all NF-κB proteins in PP compared with chow. Exogenous LTβR stimulation during PN increased p50, p52, Rel B, MAdCAM-1, IL-4, and IL-10 in PP, but not p65, CCL19, CCL20, or CCL25 compared with PN. LTβR blockade reduced noncanonical products (p52 and Rel B), MAdCAM-1, CCL19, CCL20, CCL25, IL-4, and IL-10 but had no effect on the inflammatory pathway (p50 and p65) compared with chow. Lack of enteral stimulation during PN decreases both canonical and noncanonical NF-κB pathways in PP. LTβR stimulation during PN feeding completely restores PP noncanonical NF-κB activity, MAdCAM-1, IL-4, IL-10, and partly the canonical pathway. LTβR blockade decreases the noncanonical NF-κB activity, MAdCAM-1, chemokines, and cytokines without effect on the canonical NF-κB activity in PP.

Hemostatic Factors Support Colitis and Colitis-Associated Colon Cancer