Non-canonical NF-κB signaling pathway

The non-canonical NF-κB pathway is an important arm of NF-κB signaling that predominantly targets activation of the p52/RelB NF-κB complex. This pathway depends on the inducible processing of p100, a molecule functioning as both the precursor of p52 and a RelB-specific inhibitor. A central signaling component of the non-canonical pathway is NF-κB-inducing kinase (NIK), which integrates signals from a subset of TNF receptor family members and activates a downstream kinase, IκB kinase-α (IKKα), for triggering p100 phosphorylation and processing. A unique mechanism of NIK regulation is through its fate control: the basal level of NIK is kept low by a TRAF-cIAP destruction complex and signal-induced non-canonical NF-κB signaling involves NIK stabilization. Tight control of the fate of NIK is important, since deregulated NIK accumulation is associated with lymphoid malignancies.     

NFκB signaling regulates embryonic and adult neurogenesis

Both embryonic and adult neurogenesis involves the self-renewal/proliferation,survival,migration and lineage differentiation of neural stem/progenitor cells.Such dynamic process is tightly regulated by...     

RIOK3 interacts with caspase-10 and negatively regulates the NF-κB signaling pathway

RIOK3 was initially characterized as a homolog of Aspergillus nidulans sudD and showed down-regulation at the invasive front of malignant melanomas, but the molecular mechanism remains elusive. Here, we report that overexpression of RIOK3 inhibits TNFα-induced NF-κB activation, but down-regulation of endogenous RIOK3 expression by siRNA potentiates it. A yeast two-hybrid experiment revealed that RIOK3 interacted with caspase-10, and further, a GST pull-down assay and endogenous coimmunoprecipitation validated the interaction. We subsequently showed that the interaction was mediated by the RIO domain of RIOK3 and each death effector domain of caspase-10. Interestingly, our data demonstrated that RIOK3 suppressed caspase-10-mediated NF-κB activation by competing RIP1 and NIK to bind to caspase-10. Importantly, the kinase activity of RIOK3 was confirmed to be relevant to NF-κB signaling. Taken together, our findings strongly suggest that RIOK3 negatively regulates NF-κB signaling pathway activated by TNFα dependent on its kinase activity and NF-κB signaling pathway activated by caspase-10 independent of its kinase activity.     

Herbal melanin activates TLR4/NF-κB signaling pathway

Expression of many pro-inflammatory cytokines is controlled by the NF-κB signaling pathway. NF-κB is induced by LPS through activation of TLR4. Melanins extracted from fungal, plant and human sources modulate cytokine production and activate NF-κB pathway. We showed that a herbal melanin (HM) from Nigella sativa L. modulates cytokine production and suggested it as a ligand for TLR4. In this study we investigated the possibility that the HM-induced cytokine production is via an NF-κB signaling pathway. We found that HM induced the degradation of IκBα, a key step in the activation of NF-κB. Moreover, addition of IκB kinase (IKK) specific inhibitors effectively inhibited the observed HM-induced production of IL-8 and IL-6 by TLR4-transfected HEK293 cells and THP-1 cells. Our results have also shown that HM induced cleavage of caspase 8, and that this cleavage was partially abrogated by IKK inhibitors. We suggest that HM can modulate the inflammatory response by inducing IL-8 and IL-6 production via TLR4-dependent activation of the NF-κB signaling pathway.     

TAK1 ubiquitination regulates doxorubicin-induced NF-κB activation

Chemotherapeutic agents- and radiation therapy-induced NF-κB activation in cancer cells contributes to aggressive tumor growth and resistance to chemotherapy and ionizing radiation during cancer treatment. TAK1 has been shown to be required for genotoxic stress-induced NF-κB activation. However, whether TAK1 ubiquitination is involved in genotoxic stress-induced NF-κB activation remains unknown. Herein, we demonstrate that TAK1 ubiquitination plays an important role in the positive and negative regulation of doxorubicin (Dox)-induced NF-κB activation. We found that TAK1 was required for Dox-induced NF-κB activation. At the early stage of Dox treatment, Dox induced Lys63-linked TAK1 polyubiquitination at lysine 158 residue. USP4 inhibited Dox-induced TAK1 Lys63-linked polyubiquitination and knockdown of USP4 enhanced Dox-induced NF-κB activation. At the late stage of Dox treatment, Dox induced Lys48-linked TAK1 polyubiquitination to promote TAK1 degradation. ITCH inhibited Dox-induced NF-κB activation by promoting Lys48-linked TAK1 polyubiquitination and its subsequent degradation. Our study indicates that TAK1 ubiquitination plays critical roles in the regulation of Dox-induced NF-κB activation. Thus, intervention of TAK1 kinase activity or TAK1 Lys63-linked polyubiquitination pathways might greatly enhance the therapeutic efficacy of Dox.     

miR-486 sustains NF-κB activity by disrupting multiple NF-κB-negative feedback loops

Deubiquitinases, such as CYLD, A20 and Cezanne, have emerged as important negative regulators that balance the strength and the duration of NF-κB signaling through feedback mechanisms. However, how these serial feedback loops are simultaneously disrupted in cancers, which commonly exhibit constitutively activated NF-κB, remains puzzling. Herein, we report that miR-486 directly suppresses NF-κB-negative regulators, CYLD and Cezanne, as well as multiple A20 activity regulators, including ITCH, TNIP-1, TNIP-2 and TNIP-3, resulting in promotion of ubiquitin conjugations in NF-κB signaling and sustained NF-κB activity. Furthermore, we demonstrate that upregulation of miR-486 promotes glioma aggressiveness both in vitro and in vivo through activation of NF-κB signaling pathway. Importantly, miR-486 levels in primary gliomas significantly correlate with NF-κB activation status. These findings uncover a novel mechanism for constitutive NF-κB activation in gliomas and support a functionally and clinically relevant epigenetic mechanism in cancer progression.     

Deubiquitinating enzyme A20 negatively regulates NF-κB signaling in skeletal muscle in mdx mice

Duchenne muscular dystrophy (DMD) is caused by the lack of a functional dystrophin protein that results in muscle fiber membrane disruption and, ultimately, degeneration. Regeneration of muscle fibers fails progressively, and muscle tissue is replaced with connective tissue. As a result, DMD causes progressive limb muscle weakness and cardiac and respiratory failure. The absence of dystrophin from muscle fibers triggers the chronic activation of the nuclear factor of kappa B (NF-κB). Chronic activation of NF-κB in muscle leads to infiltration of macrophages, up-regulation of the ubiquitin-proteosome system, and down-regulation of the helix-loop-helix muscle regulatory factor, MyoD. These processes, triggered by NF-κB activation, promote muscle degeneration and failure of muscle regeneration. A20 (TNFAIP3) is a critical negative regulator of NF-κB. In this study, we characterize the role of A20 in regulating NF-κB activation in skeletal muscle, identifying a novel role in muscle regeneration. A20 is highly expressed in regenerating muscle fibers, and knockdown of A20 impairs muscle differentiation in vitro, which suggests that A20 expression is critically important for regeneration of dystrophic muscle tissue. Furthermore, down-regulation of the classic pathway of NF-κB activation is associated with up-regulation of the alternate pathway in regenerating muscle fibers, suggesting a mechanism by which A20 promotes muscle regeneration. These results demonstrate the important role of A20 in muscle fiber repair and suggest the potential of A20 as a therapeutic target to ameliorate the pathology and clinical symptoms of DMD.     

Targeting NF-κB/COX-2 signaling by soyasaponin I alleviates diclofenac-induced gastric ulceration in male albino rats

Gastric ulceration is a prevalent worldwide clinical presentation due to altered gastric defense mechanisms. Nonsteroidal anti-inflammatory drugs are one of the common causes of gastric ulcers mediated by the release of inflammatory mediators. The study aimed to investigate the potential protective effect of soyasaponin I (soya) against diclofenac (DIC)-induced gastric ulcer in rats and to highlight the underlying mechanisms. The experiment was conducted on 40 male Wistar albino rats, equally distributed into five groups: control, DIC-induced ulcer (9 mg/kg/d, orally, twice daily for 3 days), ulcer/soya-, ulcer/ranitidine-, and ulcer/soya/selective nuclear factor kappa B inhibitor (JSH-23)-treated groups. The doses of soya, ranitidine, and JSH were 20, 25, and 5 mg/kg/d, respectively, given orally. Gastric specimens were prepared for gene and histological study and for biochemical analysis of gastric prostaglandin E2 (PGE2), oxidative markers, and inflammatory cytokines. The gastric samples were formalin-fixed, paraffin-embedded, and subjected to hematoxylin and eosin (H&E), PAS staining, and immunohistochemical assay for identification of nuclear factor kappa B (NF-κB), cyclooxygenase-2 (COX-2), and proliferation marker (Ki67) expressions. The findings revealed decreased gastric PGE2 and altered inflammatory and oxidative markers in the ulcer model group. The H&E staining showed mucosal injury characterized by mucosal surface defects and inflammatory cell infiltrations. The polymerase chain reaction (PCR) and immunohistochemistry demonstrated an upregulation of NF-κB and COX-2 expression at gene/protein levels; meanwhile, Ki67 downregulation. The soya-treated group showed maintained biochemical, histological, and PCR findings comparable to the ranitidine-treated group. The JSH-23-treated group still showed partial gastric protection with biochemical and immunohistochemical changes. Soyasaponin I ameliorated DIC-induced gastric ulcers by targeting the COX-2 activity through modulation of NF-κB signaling.     

P2X7 receptor positively regulates MyD88-dependent NF-κB activation

Recent studies have demonstrated that P2X7 plays a critical role in the immune system. Here, our results showed that P2X7 activated a NF-κB - but not an IFN-β-dependent luciferase reporter gene in HEK293T cells. P2X7 was involved in the LPS- and ATP-induced NF-κB activation but did not significantly impact the response to Zymosan in RAW264.7 cells. The activation of NF-κB and IFN-β induced by myeloid differentiation primary-response protein 88 (MyD88) was enhanced by P2X7 co-expression. The siRNA silencing MyD88 almost abolished the NF-κB activation induced by P2X7, and co-immunoprecipitation showed that P2X7 interacted with MyD88. The amino acids in the C-terminus, especially the LPS-binding region of P2X7, were critical for the cellular localization and immune function of P2X7. P2X7ΔC (190 amino acids deleted in the C-terminus) and P2X7 G586A variants localized throughout the cytoplasma with a little aggregation, which differs from the cell membrane localization of wild type P2X7. Both of them could not localize to Golgi or endoplasmic reticulum. P2X7ΔC and P2X7 G586A had impaired proteolytic cleavage of caspase-1 into the functional p20 subunit, which can activate pro-inflammatory cytokines such as IL-1β. P2X7 G586A also showed a slight interaction with MyD88 in our co-immunoprecipitation experiment. This interaction might result in the attenuated activation of NF-κB and IFN-β induced by MyD88.     

Molecular evolution of the NF-κB signaling system

The mechanisms of innate immunity in vertebrates show certain overall resemblances to immune mechanisms of insects. Two hypotheses have been proposed to explain these resemblances. (1) According to the evolutionary continuity hypothesis, innate immune mechanisms evolved in the common ancestor of vertebrates and insects and have been conserved since that time. (2) In the independent-evolution hypothesis, the mechanisms of innate immunity in vertebrates evolved independently from invertebrate immune mechanisms. Phylogenetic analysis of five gene families (Pelle, Rel, IkappaB, Toll, and TRAF) whose members are involved in NF-kappaB signaling in vertebrates and insects were used to decide between these hypotheses. The phylogenies of the Rel and TRAF families strongly supported independent evolution of immune functions in vertebrates and invertebrates, and, except for a possible case in the Pelle family, orthologous molecules having immune functions in both vertebrates and invertebrates were not found. The results suggest that NF-kappaB represents an ancient, generalized signaling system that has been co-opted for immune system roles independently in vertebrate and insect lineages.     

Expanding role of ubiquitination in NF-κB signaling

Best known for its role in targeting protein degradation by the proteasome, ubiquitin modification has also emerged as an important mechanism that regulates cell signaling through proteasome-independent mechanisms. The role of ubiquitin as a versatile signaling tag is characteristically illustrated in the NF-κB pathways, which regulate a variety of physiological and pathological processes in response to diverse stimuli. Here, we review the role of ubiquitination in different steps of the NF-κB signaling cascades, focusing on recent advances in understanding the mechanisms of protein kinase activation by polyubiquitin chains in different pathways that converge on NF-κB.     

Deubiquitinases in the regulation of NF-κB signaling

Nuclear factor-kappa B (NF-κB) is a critical regulator of multiple biological functions including innate and adaptive immunity and cell survival. Activation of NF-κB is tightly regulated to preclude chronic signaling that may lead to persistent inflammation and cancer. Ubiquitination of key signaling molecules by E3 ubiquitin ligases has emerged as an important regulatory mechanism for NF-κB signaling. Deubiquitinases (DUBs) counteract E3 ligases and therefore play a prominent role in the downregulation of NF-κB signaling and homeostasis. Understanding the mechanisms of NF-κB downregulation by specific DUBs such as A20 and CYLD may provide therapeutic opportunities for the treatment of chronic inflammatory diseases and cancer.