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NF-κB inducing kinase: A key regulator in the immune system and in cancer
Abstract
NF-κB inducing kinase (NIK) is a kinase that activates the canonical and non-canonical NF-κB pathways to control transcriptional expression of certain proteins such as cytokines, chemokines and NF-κB signaling molecules. Many advances have been made in understanding the molecular mechanisms by which the stability of NIK is regulated to affect downstream signaling. Genetic mouse models suggest that NIK plays an essential role in the regulation of the immune system as well as in the bone microenvironment. Increasing evidence links NIK to the tumorigenesis of hematological cancers, such as multiple myeloma, and solid tumors, such as pancreatic carcinoma and melanoma. Understanding the mechanism by which NIK is de-regulated will potentially provide therapeutic options for certain diseases such as autoimmunity and cancer.
... involved in immune, inflammatory, oxidative stress responses, cell proliferation, differentiation, growth, survival, apoptosis and other cellular processes (Collins et al. 2016). The mammalian proteins of the NF-κB family consist of p105 (precursor protein of p50), p100 (precursor of p52), RelA (p65), RelB and c-Rel (Thu and Richmond 2010). The NF-κB signalling pathway is stimulated by pathogenic factors, cytokines, ROS, and reactive nitrogen species. ...
... Upon activation by pathological stimuli, the IκB kinase phosphorylates the inhibitory kappa B (IκBs) protein of the cytoplasmic complex NF-κB/IκB to cause degradation of IκB and release of NF-κB protein heterodimers to translocate into the nucleus to activate gene transcription (Fig. 4). The IκB kinase is a primary target of reactive species, proinflammatory mediators such as IL-1β, TNFα and other pathogenic signals (Gloire and Legrand-Poels 2006;Thu and Richmond 2010;Collins et al. 2016;Jimi et al. 2019). Even so, a non-classical (non-canonical) or alternative pathway exists for the activation of NF-κB. ...
... The overall effect is associated with increase in the slow depolarising (pacemaker) potential (negative chronotropic effect) mainly due to reduced i f current, reduced Ca 2+ waves, and increased K + current (Foster et al. 2014;Manson et al. 2014;Welcome et al. 2015). Other abbreviations are similar to those in Fig. 1 member 5 (CD40) ligand and TNF-related weak inducer of apoptosis (Thu and Richmond 2010) resulting in cleavage of the p100 to produce p52 with subsequent dimerisation of the latter with RelB. The complex formed translocates into the nucleus to initiate gene transcription (Thu and Richmond 2010). ...
Heart diseases and related complications constitute a leading cause of death and socioeconomic threat worldwide. Despite intense efforts and research on the pathogenetic mechanisms of these diseases, the underlying cellular and molecular mechanisms are yet to be completely understood. Several lines of evidence indicate a critical role of inflammatory and oxidative stress responses in the development and progression of heart diseases. Nevertheless, the molecular machinery that drives cardiac inflammation and oxidative stress is not completely known. Recent data suggest an important role of cardiac bitter taste receptors (TAS2Rs) in the pathogenetic mechanism of heart diseases. Independent groups of researchers have demonstrated a central role of TAS2Rs in mediating inflammatory, oxidative stress responses, autophagy, impulse generation/propagation and contractile activities in the heart, suggesting that dysfunctional TAS2R signalling may predispose to cardiac inflammatory and oxidative stress disorders, characterised by contractile dysfunction and arrhythmia. Moreover, cardiac TAS2Rs act as gateway surveillance units that monitor and detect toxigenic or pathogenic molecules, including microbial components, and initiate responses that ultimately culminate in protection of the host against the aggression. Unfortunately, however, the molecular mechanisms that link TAS2R sensing of the cardiac milieu to inflammatory and oxidative stress responses are not clearly known. Therefore, we sought to review the possible role of TAS2R signalling in the pathophysiology of cardiac inflammation, oxidative stress, arrhythmia and contractile dysfunction in heart diseases. Potential therapeutic significance of targeting TAS2R or its downstream signalling molecules in cardiac inflammation, oxidative stress, arrhythmia and contractile dysfunction is also discussed.
... (11,12) Nuclear factor kappa B (NF-κB)-inducing kinase (NIK; also known as mitogen-activated protein kinase kinase kinase 14) is a serine/threonine kinase that mediates activation of the noncanonical NF-κB2 pathway. (13) NIK phosphorylates and activates inhibitor of kB (IkB) kinaseα (IKKα; also referred to as Chuk), and IKKα in turn activates transcription factor NF-κB2. (13)(14)(15) NIK is activated by a wide range of stimuli, including a subset of cytokines, numerous endogenous metabolites and exogenous substances, and various cellular stress-inducing agents. (13,15,16) Importantly, hepatic NIK is aberrantly activated in liver disease in mice and humans, including alcoholic liver injury, nonalcoholic fatty liver disease, hepatotoxin-induced liver injury, viral hepatitis, and autoimmune liver disease. ...
... (11,12) Nuclear factor kappa B (NF-κB)-inducing kinase (NIK; also known as mitogen-activated protein kinase kinase kinase 14) is a serine/threonine kinase that mediates activation of the noncanonical NF-κB2 pathway. (13) NIK phosphorylates and activates inhibitor of kB (IkB) kinaseα (IKKα; also referred to as Chuk), and IKKα in turn activates transcription factor NF-κB2. (13)(14)(15) NIK is activated by a wide range of stimuli, including a subset of cytokines, numerous endogenous metabolites and exogenous substances, and various cellular stress-inducing agents. (13,15,16) Importantly, hepatic NIK is aberrantly activated in liver disease in mice and humans, including alcoholic liver injury, nonalcoholic fatty liver disease, hepatotoxin-induced liver injury, viral hepatitis, and autoimmune liver disease. ...
... (13) NIK phosphorylates and activates inhibitor of kB (IkB) kinaseα (IKKα; also referred to as Chuk), and IKKα in turn activates transcription factor NF-κB2. (13)(14)(15) NIK is activated by a wide range of stimuli, including a subset of cytokines, numerous endogenous metabolites and exogenous substances, and various cellular stress-inducing agents. (13,15,16) Importantly, hepatic NIK is aberrantly activated in liver disease in mice and humans, including alcoholic liver injury, nonalcoholic fatty liver disease, hepatotoxin-induced liver injury, viral hepatitis, and autoimmune liver disease. (16)(17)(18)(19) We previously reported that a modest elevation of hepatic NIK in obesity augments hepatic glucose production, increasing the risk for type 2 diabetes. ...
Drug-induced hepatotoxicity limits development of new effective medications. Drugs and numerous endogenous/exogenous agents are metabolized/detoxified by hepatocytes, during which reactive oxygen species (ROS) are generated as a by-product. ROS has broad adverse effects on liver function and integrity, including damaging hepatocyte proteins, lipids, and DNA and promoting liver inflammation and fibrosis. ROS in concert with iron overload drives ferroptosis. Hepatic nuclear factor kappa B (NF-κB)-inducing kinase (NIK) is aberrantly activated in a broad spectrum of liver disease. NIK phosphorylates and activates inhibitor of NF-κB kinase subunit alpha (IKKα), and the hepatic NIK/IKKα cascade suppresses liver regeneration. However, the NIK/IKKα pathway has not been explored in drug-induced liver injury. Here, we identify hepatic NIK as a previously unrecognized mediator for acetaminophen (APAP)-induced acute liver failure. APAP treatment increased both NIK transcription and NIK protein stability in primary hepatocytes as well as in liver in mice. Hepatocyte-specific overexpression of NIK augmented APAP-induced liver oxidative stress in mice and increased hepatocyte death and mortality in a ROS-dependent manner. Conversely, hepatocyte-specific ablation of NIK or IKKα mitigated APAP-elicited hepatotoxicity and mortality. NIK increased lipid peroxidation and cell death in APAP-stimulated primary hepatocytes. Pretreatment with antioxidants or ferroptosis inhibitors blocked NIK/APAP-induced hepatocyte death. Conclusion: We unravel a previously unrecognized NIK/IKKα/ROS/ferroptosis axis engaged in liver disease progression.
Hepatic NIK is upregulated in response to hepatic toxicants. Ablation of hepatic NIK attenuates, whereas hepatocyte-specific overexpression of NIK aggravates, APAP-induced liver injury. NIK promotes hepatic oxidative stress and ferroptosis.
... In terms of non-canonical NF-κB disruption, BIRC3 mutations, similar to those found in CLL, affect NF-κB activation by stabilising NIK. Alternatively, mutations in TRAF3 lead to the stabilisation of NIK [121]. ...
... The absence of reports of toxicity from non-canonical NF-κB inhibitors is primarily due to the fact that so few of them have been published and expansive toxicological data has not yet been acquired. Developing inhibitors against the non-canonical NF-κB pathway has its challenges, particularly as IKKα plays a role in the canonical NF-κB pathway [161,162] and is also known to be involved in the phosphorylation of IKKβ in some settings [121]. Its roles independent of the NF-κB signalling pathway described earlier will also be impaired if it is inhibited as a phosphorylating entity. ...
... This may have advantages in a cancer setting as multiple roles of IKKα may be involved, but if discrimination between non-canonical NF-κB signalling and other IKKα outputs is the aim, inhibiting its kinase function may not be the answer, and targeting NIK may be the preferred route. NIK phosphorylates IKKβ to a much lesser extent than IKKα and has far fewer roles outside the non-canonical NF-κB pathway [121]. Furthermore, from a drug discovery perspective, a NIK inhibitor with no canonical NF-κB activity may be more tractable than a specific IKKα inhibitor due to the high level of homology between IKKα and IKKβ. ...
The cellular kinases inhibitory-κB kinase (IKK) α and Nuclear Factor-κB (NF-κB)-inducing kinase (NIK) are well recognised as key central regulators and drivers of the non-canonical NF-κB cascade and as such dictate the initiation and development of defined transcriptional responses associated with the liberation of p52-RelB and p52-p52 NF-κB dimer complexes. Whilst these kinases and downstream NF-κB complexes transduce pro-inflammatory and growth stimulating signals that contribute to major cellular processes, they also play a key role in the pathogenesis of a number of inflammatory-based conditions and diverse cancer types, which for the latter may be a result of background mutational status. IKKα and NIK, therefore, represent attractive targets for pharmacological intervention. Here, specifically in the cancer setting, we reflect on the potential pathophysiological role(s) of each of these kinases, their associated downstream signalling outcomes and the stimulatory and mutational mechanisms leading to their increased activation. We also consider the downstream coordination of transcriptional events and phenotypic outcomes illustrative of key cancer ‘Hallmarks’ that are now increasingly perceived to be due to the coordinated recruitment of both NF-κB-dependent as well as NF-κB–independent signalling. Furthermore, as these kinases regulate the transition from hormone-dependent to hormone-independent growth in defined tumour subsets, potential tumour reactivation and major cytokine and chemokine species that may have significant bearing upon tumour-stromal communication and tumour microenvironment it reiterates their potential to be drug targets. Therefore, with the emergence of small molecule kinase inhibitors targeting each of these kinases, we consider medicinal chemistry efforts to date and those evolving that may contribute to the development of viable pharmacological intervention strategies to target a variety of tumour types.
... [2][3][4] Among these, mutations in the genes encoding NF-κB-inducing kinase (NIK) or its negative regulators TRAF2, TRAF3, cIAP1, and cIAP2 lead to increased stability of NIK and subsequent aberrant activation of the non-canonical and canonical NF-κB pathways. [2][3][4][5][6][7] In addition to regulating NF-κB pathways, the NIK signaling pathway has been demonstrated to crosstalk with and activate other critical cancer-associated pathways including the MAPK-ERK 8,9 and JAK/STAT3. 10 Moreover, these pathways are highly interconnected at many levels, and have been demonstrated to be often persistently and simultaneously activated in many human cancers, including myeloma. ...
... Because NIK induction by pan-AKI was not associated with an increased activation of NF-κB pathways in 4 of 5 HMCL tested (except OPM-2), and yet NIK signaling has been demonstrated to crosstalk at different levels with other important prosurvival signaling pathways including MEK-ERK and STAT3 pathways, [8][9][10] we explored whether NIK induction by pan-AKI affected these pathways in MM cells. ...
Considering that Aurora kinase inhibitors are currently under clinical investigation in hematologic cancers, the identification of molecular events that limit the response to such agents is essential for enhancing clinical outcomes. Here, we discover a NF-κB-inducing kinase (NIK)-c-Abl-STAT3 signaling-centered feedback loop that restrains the efficacy of Aurora inhibitors in multiple myeloma. Mechanistically, we demonstrate that Aurora inhibition promotes NIK protein stabilization via downregulation of its negative regulator TRAF2. Accumulated NIK converts c-Abl tyrosine kinase from a nuclear proapoptotic into a cytoplasmic antiapoptotic effector by inducing its phosphorylation at Thr735, Tyr245 and Tyr412 residues, and, by entering into a trimeric complex formation with c-Abl and STAT3, increases both the transcriptional activity of STAT3 and expression of the antiapoptotic STAT3 target genes PIM1 and PIM2. This consequently promotes cell survival and limits the response to Aurora inhibition. The functional disruption of any of the components of the trimer NIK-c-Abl-STAT3 or the PIM survival kinases consistently enhances the responsiveness of myeloma cells to Aurora inhibitors. Importantly, concurrent inhibition of NIK or c-Abl disrupts Aurora inhibitor-induced feedback activation of STAT3 and sensitizes myeloma cells to Aurora inhibitors, implicating a combined inhibition of Aurora and NIK or c-Abl kinases as potential therapies for multiple myeloma. Accordingly, pharmacological inhibition of c-Abl together with Aurora resulted in substantial cell death and tumor regression in vivo The findings reveal an important functional interaction between NIK, Abl and Aurora kinases, and identify the NIK, c-Abl and PIM survival kinases as potential pharmacological targets for improving the efficacy of Aurora inhibitors in myeloma.
... NIK is the upstream kinase that regulates the activation of the non-canonical NF-κB signaling pathway and may suggest a role for non-canonical NF-κB signaling in immune cells after elraglusib treatment, which future studies could evaluate. It is known that the increased expression of NIK leads to an enhanced expression of chemokines and cytokines such as CCL3, TNF-α, and MCP-1, thus leading to the increased recruitment and proliferation of cytotoxic immune cells [37]. Moreover, the treatment of immune cells with elraglusib increased effector molecule secretion and increased expression of genes involved in cytotoxic granule exocytosis, cellular proliferation, and the modulation of NF-κB activity. ...
Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase that has been implicated in numerous oncogenic processes. GSK-3 inhibitor elraglusib (9-ING-41) has shown promising preclinical and clinical antitumor activity across multiple tumor types. Despite promising early-phase clinical trial results, there have been limited efforts to characterize the potential immunomodulatory properties of elraglusib. We report that elraglusib promotes immune cell-mediated tumor cell killing of microsatellite stable colorectal cancer (CRC) cells. Mechanistically, elraglusib sensitized CRC cells to immune-mediated cytotoxicity and enhanced immune cell effector function. Using western blots, we found that elraglusib decreased CRC cell expression of NF-κB p65 and several survival proteins. Using microarrays, we discovered that elraglusib upregulated the expression of proapoptotic and antiproliferative genes and downregulated the expression of cell proliferation, cell cycle progression, metastasis, TGFβ signaling, and anti-apoptotic genes in CRC cells. Elraglusib reduced CRC cell production of immunosuppressive molecules such as VEGF, GDF-15, and sPD-L1. Elraglusib increased immune cell IFN-γ secretion, which upregulated CRC cell gasdermin B expression to potentially enhance pyroptosis. Elraglusib enhanced immune effector function resulting in augmented granzyme B, IFN-γ, TNF-α, and TRAIL production. Using a syngeneic, immunocompetent murine model of microsatellite stable CRC, we evaluated elraglusib as a single agent or combined with immune checkpoint blockade (anti-PD-1/L1) and observed improved survival in the elraglusib and anti-PD-L1 group. Murine responders had increased tumor-infiltrating T cells, augmented granzyme B expression, and fewer regulatory T cells. Murine responders had reduced immunosuppressive (VEGF, VEGFR2) and elevated immunostimulatory (GM-CSF, IL-12p70) cytokine plasma concentrations. To determine the clinical significance, we then utilized elraglusib-treated patient plasma samples and found that reduced VEGF and BAFF and elevated IL-1 beta, CCL22, and CCL4 concentrations correlated with improved survival. Using paired tumor biopsies, we found that tumor-infiltrating immune cells had a reduced expression of inhibitory immune checkpoints (VISTA, PD-1, PD-L2) and an elevated expression of T-cell activation markers (CTLA-4, OX40L) after elraglusib treatment. These results address a significant gap in knowledge concerning the immunomodulatory mechanisms of GSK-3 inhibitor elraglusib, provide a rationale for the clinical evaluation of elraglusib in combination with immune checkpoint blockade, and are expected to have an impact on additional tumor types, besides CRC.
... 19,20 NIK is constitutively repressed by c-IAP1/2 proteins, which ubiquitinate NIK for proteasomal degradation via the adaptor proteins TNFR-associated factor 2 (TRAF2) and TRAF3. 16,21 By inducing the degradation of c-IAP1/2 proteins, IAP antagonists would release NIK to activate the non-canonical as well as the canonical NF-kB pathways, 16,[22][23][24] and both pathways could promote T cell survival, proliferation, and cytokine production such as TNF-a. 25 IAP antagonists have shown promising anti-tumor effects when combined with other immunostimulatory agents in preclinical settings, including in a GBM model, in which IAP antagonists synergized with oncolytic virus and immune checkpoint inhibitors to treat GBM in mice. ...
Antigen heterogeneity that results in tumor antigenic escape is one of the major obstacles to successful chimeric antigen receptor (CAR) T cell therapies in solid tumors including glioblastoma multiforme (GBM). To address this issue and improve the efficacy of CAR-T cell therapy for GBM, we developed an approach that combines CAR-T cells with inhibitor of apoptosis protein (IAP) antagonists, a new class of small molecules that mediate the degradation of IAPs, to treat GBM. Here, we demonstrated that the IAP antagonist birinapant could sensitize GBM cell lines and patient-derived primary GBM organoids to apoptosis induced by CAR-T cell-derived cytokines, such as tumor necrosis factor. Therefore, birinapant could enhance CAR-T cell-mediated bystander death of antigen-negative GBM cells, thus preventing tumor antigenic escape in antigen-heterogeneous tumor models in vitro and in vivo. In addition, birinapant could promote the activation of NF-κB signaling pathways in antigen-stimulated CAR-T cells, and with a birinapant-resistant tumor model, we showed that birinapant had no deleterious effect on CAR-T cell functions in vitro and in vivo. Overall, we demonstrated the potential of combining the IAP antagonist birinapant with CAR-T cells as a novel and feasible approach to overcoming tumor antigen heterogeneity and enhancing CAR-T cell therapy for GBM.
... Various research demonstrated the crucial role of the NIK and non-canonical NF-κB pathway in the maintenance of the hematopoietic stem cells (HSCs) population and regulation of B and T cells. Also, dysregulation of the alternative NF-κB pathway has been shown in some hematological malignancies such as acute myeloid leukemia (AML), multiple myeloma (MM), and Hodgkin-lymphoma (HL), and several solid tumors [92]. The mechanisms associated with the dysregulation of the NF-κB pathway in these tumors have recently been well-reviewed [93]. ...
The transcription factor nuclear factor-κB (NF-κB) is a critical regulator of the immune response, inflammation, cell growth, and survival. Canonical and non-canonical pathways, two NF-κB pathways, are activated through diverse stimulators and receptors. NF-κB activity is dysregulated in various inflammation-related diseases and cancers. It was found that the persistent NF-κB activity has a major role in proliferation, apoptosis inhibition, metastasis, and cell cycle disruption in cancer cells and also the survival of cancer stem cells (CSCs) within the tumors. Therefore, suppression of the NF-κB pathway could be a promising therapeutic target for cancer therapy. Different biological inhibitors (e.g., peptides, small molecules, antisense oligonucleotides (ASOs), and antibodies (Abs)) have been demonstrated to inhibit the NF-κB pathway. Low stability in the circulation system, weak availability, and poor cellular uptake of some inhibitors limit their therapeutic applications. To address these drawbacks nanocarrier systems are often formulated and applied in drug delivery as an effective therapeutic approach. Targeted nanosystems (i.e., small molecules, peptides, Abs and Aptamers (Aps) conjugated nanocarriers), as well as smart responsive nanocarriers, can improve the efficiency of therapeutics while reducing the off-target toxicity. This review describes the NF-κB signaling pathways and mechanisms of their over-activation in tumor initiation and progression. The NF-κB inhibitors and their clinical applications are also discussed. It also overviews different nanocarriers used as robust vehicles for the delivery of NF-κB inhibitors and anti-tumor agents to improve the bioavailability of drugs and selective targeting of cancer cells to repress NF-κB activity in tumor cells.
... The other gene, MAP3K14-AS1, also known as antisense RNA1 of MAP3K14, is a class of lncRNAs. MAP3K14 is a member of the mitogen-activated protein kinase family and plays a crucial role in the nonclassical NFκ B pathway, which can bind to TRAF2 and participate in mediating the response of TNF superfamily and IL-1 receptors to NFκ B signaling (45). MAP3K14-AS1 was rst reported as a circulating DNA marker for monitoring the treatment response of metastatic CRC (46). ...
Background
Blood-based methylation tests showed higher patient compliance and convenience than colonoscopy and stool DNA tests for colorectal cancer (CRC) detection. This study aimed to enhance the performance of plasma markers to detect CRCs using sense-antisense and dual-MGB probe (SADMP) technique.
Method
The current study consisted of 3 phases. The first phase was identifying eligible methylation markers. The second phase was assay development that incorporated the SADMP technique into methylation-specific PCR to enhance the target sensitivities in detecting plasma methylation signals. The third phase was assessing the test performance for CRC detection in training and validation cohorts.
Results
Hypermethylated NTMT1 and MAP3K14-AS1 were found in multiple CRC cohorts. The SADMP technique showed an approximately 2-fold increase in detecting methylation signals than single-strand and single-MGB probe techniques. The established MethyDT test obtained an average sensitivity of 84.47% for CRC detection, higher than any single target alone without a significant attenuation of specificity (average specificities of 91.81% for NTMT1 and 96.93% for MAP3K14-AS1 vs. 89.76% for MethyDT). For early (I-II) and late- (III-IV) stage CRC, the test sensitivities were 82.61% and 88.64%, respectively, in validation cohort. Meanwhile, the test sensitivity was independent of patient age and gender.
Conclusion
The SADMP technique enhanced the detection of methylation signals, and the MethyDT test showed potential utility as a promising noninvasive tool for CRC detection.
... Additionally, NIK also activates the MAPK pathway and suppresses the JAK2/STAT3 pathway 20,21 . Functionally, lymphoid NIK critically regulates immune system development and immune response 10,22 . Global NIK knockout impairs lymph node and thymus developments 23 . ...
Excessive cholangiocyte expansion (ductular reaction) promotes liver disease progression, but the underlying mechanism is poorly understood. Here we identify biliary NF-κB-inducing kinase (NIK) as a pivotal regulator of ductular reaction. NIK is known to activate the noncanonical IKKα/NF-κB2 pathway and regulate lymphoid tissue development. We find that cholangiocyte NIK is upregulated in mice with cholestasis induced by bile duct ligation (BDL), 5-diethoxycarbonyl-1,4-dihydrocollidine (DDC), or α-naphtyl-isothiocyanate (ANIT). DDC, ANIT, or BDL induces ductular reaction, liver injury, inflammation, and fibrosis in mice. Cholangiocyte-specific deletion of NIK, but not IKKα, blunts these pathological alterations. NIK inhibitor treatment similarly ameliorates DDC-induced ductular reaction, liver injury, and fibrosis. Biliary NIK directly increases cholangiocyte proliferation while suppressing cholangiocyte death, and it also promotes secretion of cholangiokines from cholangiocytes. Cholangiokines stimulate liver macrophages and hepatic stellate cells, augmenting liver inflammation and fibrosis. These results unveil a NIK/ductular reaction axis and a NIK/cholangiokine axis that promote liver disease progression.
... MAP3K14 is a central activator of the non-canonical NF-κB pathway. It can promote tumorigenesis by regulating cell proliferation and survival [52,53]. NFKB2 is a member of the NF-κB family transcription factor complexes, which can promote the proliferation of tumor cells and inhibit apoptosis [54,55]. ...
CDK12 is a cyclin-dependent kinase that plays critical roles in DNA replication, transcription, mRNA splicing, and DNA damage repair. CDK12 genomic changes, including mutation, amplification, deletion, and fusion, lead to various cancers, such as colorectal cancer, gastric cancer, and ovarian cancer. An increasing number of CDK12 inhibitors have been reported since CDK12 was identified as a biomarker and cancer therapeutic target. A major challenge lies in that CDK12 and CDK13 share highly similar sequences, which leads to great difficulties in the development of highly selective CDK12 inhibitors. In recent years, great efforts were made in developing selective CDK12 blockers. Techniques including PROTAC and molecular glue degraders were also applied to facilitate their development. Also, the drug combination strategy of CDK12 small molecule inhibitors were studied. This review discusses the latest studies on CDK12 inhibitors and analyzes their structure-activity relationships, shedding light on their further development.
... 27 Inappropriate activation of NF-kB leads to tumor proliferation, invasion, and metastasis. 28,29 Early studies showed that Cdc42 regulates specifically in the NF-kB-dependent transcription. 30,31 Therefore, in view of the special role of CIP4 and Cdc42 in invadopodia formation, along with the unknown downstream signaling pathway, whether NF-kB is in response to the activated Cdc42 interacting with CIP4 to promote CRC metastasis attracted our interest. ...
Cdc42-interacting protein 4 (CIP4), a member of the F-BAR family which plays an important role in regulating cell membrane and actin, has been reported to interact with Cdc42 and closely associated with tumor invadopodia formation. In this study, we found that CIP4 expression was significantly higher in human CRC tissues and correlated with the CRC infiltrating depth and metastasis as well as the lower survival rate in patients. In cultured CRC cells, knockdown of CIP4 inhibited cell migration and invasion ability in vitro and the tumor metastasis in vivo, while overexpression of CIP4 promoted invadopodia formation and matrix degradation ability. We then identified GTP-Cdc42 as a directly interactive protein of CIP4, which was upregulated and recruited by CIP4. Furthermore, activated NF-κB signaling pathway was found in CIP4 overexpression CRC cells contributing to invadopodia formation while inhibition of either CIP4 or Cdc42 led to suppression of NF-κB pathway resulted in decrease quantity of invadopodia. Our findings suggested that CIP4 targets to recruit GTP-Cdc42 and directly combines with it to accelerate invadopodia formation and function by activating NF-κB signaling pathway, thus promoting CRC infiltration and metastasis.
... The nuclear translocation of RelB-p52 heterodimer then initiates the expression of target genes (4, 5) ( Figure 1). NIK is a key molecule for non-canonical NF-kB pathway activation, and this process requires both NIK expression and kinase activity (6,7). Expression of human NIK-dominant inactivated mutant (NIKKA, KK429/430AA) results in the blockage of the noncanonical NF-kB pathway (8). ...
The non-canonical nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway is an important component of NF-κB transcription complex. Activation of this pathway mediates the development and function of host immune system involved in inflammation and viral infection. During hepatitis B virus (HBV) infection, there is a complex interaction between infected hepatocytes and the immune cells, which can hinder antiviral immune responses and is associated with pathological changes in liver tissue. Consistently, the host immune system is closely related to the severity of liver damage and the level of viral replication. Previous studies indicated that the non-canonical NF-κB signaling pathway was affected by HBV and might play an important regulatory role in the antiviral immunity. Therefore, systematically elucidating the interplay between HBV and non-canonical NF-κB signaling will contribute the discovery of more potential therapeutic targets and novel drugs to treat HBV infection.
... Transgenic mice bearing a NIK allele lacking the TRAF3 binding motif (NT3) downstream of a loxP/Neo-STOP/loxP cassette knocked into the ROSA26 locus permit targeted expression of a constitutively active form of NIK with any Cre-recombinase driver [21]. Given the critical function of NIK in multiple myeloma [15], it was quite surprising that NT3 mice crossed to CD19-Cre (pre B-cell) presented only with B-cell hyperplasia and not lymphoma or myeloma [21]. However, other studies using CD19-Cre show that insults to either Bcl6 [22] or Notch [23], in conjunction with the NT3 transgene, did lead to oncogenic transformation. ...
Aberrant NF-κB signaling fuels tumor growth in multiple human cancer types including both hematologic and solid malignancies. Chronic elevated alternative NF-κB signaling can be modeled in transgenic mice upon activation of a conditional NF-κB-inducing kinase (NIK) allele lacking the regulatory TRAF3 binding domain (NT3). Here, we report that expression of NT3 in the mesenchymal lineage with Osterix (Osx/Sp7)-Cre or Fibroblast-Specific Protein 1 (FSP1)-Cre caused subcutaneous, soft tissue tumors. These tumors displayed significantly shorter latency and a greater multiple incidence rate in Fsp1-Cre;NT3 compared to Osx-Cre;NT3 mice, regardless of sex. Histological assessment revealed poorly differentiated solid tumors with some spindled patterns, as well as robust RelB immunostaining, confirming activation of alternative NF-κB. Even though NT3 expression also occurs in the osteolineage in Osx-Cre;NT3 mice, we observed no bony lesions. The staining profiles and pattern of Cre expression in the two lines pointed to a mesenchymal tumor origin. Immunohistochemistry revealed that these tumors stain strongly for alpha-smooth muscle actin (αSMA), although vimentin staining was uniform only in Osx-Cre;NT3 tumors. Negative CD45 and S100 immunostains precluded hematopoietic and melanocytic origins, respectively, while positive staining for cytokeratin 19 (CK19), typically associated with epithelia, was found in subpopulations of both tumors. Principal component, differential expression, and gene ontology analyses revealed that NT3 tumors are distinct from normal mesenchymal tissues and are enriched for NF-κB related biological processes. We conclude that constitutive activation of the alternative NF-κB pathway in the mesenchymal lineage drives spontaneous sarcoma and provides a novel mouse model for NF-κB related sarcomas.
... NF-kB is a critical cell signaling pathway involving in many cellular activities and can result in cancer if not appropriately regulated (27). Inappropriate activation of NF-kB leads to tumor proliferation, invasion and metastasis (28,29). Early studies showed that Cdc42 regulates speci cally in the NF-kB-dependent transcription (30,31). ...
Background
CIP4 (Cdc42-interacting protein 4), a member of the F-BAR family which plays an important role in regulating cell membrane and actin, has been reported to interact with Cdc42 and closely associated with tumor invadopodia formation. However, the specific mechanism of the interaction between CIP4 and Cdc42 as well as the downstream signaling pathway in response in colorectal cancer (CRC) remains unknown, which is worth exploring for its impact on tumor infiltration and metastasis.
Methods
Immunohistochemistry and western blot analyses were performed to detect the expression of CIP4 and Cdc42. Their relationship with CRC clinicopathological characteristics was further analyzed. Wound-healing, transwell migration and invasion assays tested the effect of CIP4 on cells migration and invasion ability in vitro, and the orthotopic xenograft colorectal cancer mouse mode evaluated the tumor metastasis in vivo. The invadopodia formation and function were assessed by immunofluorescence, scanning electron microscopy (SEM) and matrix degradation assay. The interaction between CIP4 and Cdc42 was confirmed by co-immunoprecipitation (co-IP) and GST-Pull down assays. Immunofluorescence was used to observed the colocalization of CIP4, GTP-Cdc42 and invadopodia. The related downstream signaling pathway was investigated by western blot and immunofluorescence.
Results
CIP4 expression was significantly higher in human colorectal cancer tissues and correlated with the CRC infiltrating depth and metastasis as well as the lower survival rate in patients. In cultured CRC cells, knockdown of CIP4 inhibited cell migration and invasion ability in vitro and the tumor metastasis in vivo, while overexpression of CIP4 confirmed the opposite situation by promoting invadopodia formation and matrix degradation ability. In addition, we identified GTP-Cdc42 as a directly interactive protein of CIP4, which was upregulated and recruited by CIP4 to participate in this process. Furthermore, activated NF-κB signaling pathway was found in CIP4 overexpression CRC cells contributing to invadopodia formation while inhibition of either CIP4 or Cdc42 led to suppression of NF-κB pathway resulted in decrease quantity of invadopodia.
Conclusion
Our findings suggested that CIP4 targets to recruit GTP-Cdc42 and directly combines with it to accelerate invadopodia formation and function by activating NF-κB signaling pathway, thus promoting CRC infiltration and metastasis.
... NF-κB transcription factor is a kind of structurally related transcription factor in eukaryotes, which regulates the expression of more than 150 genes in cellular processes (Thu and Richmond, 2010). When the cell is stimulated, the IκB kinase (IKK) complex is activated, and IκB is phosphorylated and dissociated from NF-κB under the catalysis of IKK, thereby converting NF-κB into an activated form, and the activated NF-κB is transported (Maijer et al., 2015). ...
Vaccination is still the most successful strategy to prevent and control the spread of infectious diseases by generating an adequate protective immune response. However, vaccines composed of antigens alone can only stimulate weak immunogenicity to prevent infection in many cases. Adjuvant can enhance the immunogenicity of the antigens. Therefore, adjuvant is urgently needed to strengthen the immune response of the vaccines. An ideal adjuvant should be safe, cheap, biodegradable and biologically inert. In addition to having a long shelf life, it can also promote cellular and humoral immune responses. Traditional Chinese medicine (TCM) has many different ingredients, such as glycosides, polysaccharides, acids, terpenes, polyphenols, flavonoids, alkaloids, and so on. TCM polysaccharides are one of the main types of biologically active substances. They have a large range of pharmacological activities, especially immunomodulatory. TCM polysaccharides can regulate the immune system of animals by binding to multiple receptors on the surface of immune cells and activating different signal pathways. This review focuses on a comprehensive summary of the most recent developments in vaccine adjuvant effects of polysaccharides from many important TCM, such as Artemisia rupestris L., Cistanche deserticola, Pinus massoniana, Chuanminshen violaceum, Astragalus, Ganoderma lucidum, Codonopsis pilosula, Lycium barbarum, Angelica, Epimedium, and Achyranthes bidentata. Moreover, this review also introduces their immunomodulatory effects and the molecular mechanisms of action on animal bodies, which showed that TCM polysaccharides can activate macrophages, the signal pathway of T/B lymphocytes, regulate the signal pathway of natural killer cells, activate the complement system, and so on.
... Previous reviews have shown that NF-KB signaling pathway is associated with cell proliferation, invasion, anti-apoptosis, inflammation, angiogenesis, and chemotherapy resistance in PC [27]. Moreover, NF-KB signaling pathway is also significantly related to chemotherapy sensitivity and survival of PC, and can serve as a drug target of PC targeted therapy [28][29][30][31][32]. A systematic review report summed up a lot of evidence that NK cells play a critical role in PDAC and their potential therapeutic impact [33]. ...
Objective: The principal objective of this project was to investigate the prognostic value of UXT antisense RNA 1 (UXT-AS1) in pancreatic ductal adenocarcinoma (PDAC), as well as its biological function mechanisms and the screening of targeted drugs using The Cancer Genome Atlas (TCGA) PDAC genome-wide RNA sequencing (RNA-seq) dataset.
Methods: We used TCGA 112 early stage PDAC patients to screen the prognostic value of UXT-AS1. Biological functions and mechanisms of UXT-AS1 were investigated by co-expression analysis, differentially expressed genes (DEGs) and gene set enrichment analysis, while targeted drug screening was investigated by connectivity Map (CMap).
Results: By analyzing the dataset from TCGA cohort, we found that UXT-AS1 was significantly up-regulated in pancreatic cancer tissues. Multivariate survival analysis demonstrated that PDAC patients with high UXT-AS1 expression had an unfavourable prognosis (adjusted P=0.033, HR=1.830, 95%CI=1.051-3.188). Genome-wide co-expression analysis and gene set enrichment analysis suggested that UXT-AS1 may act as a pivotal part in PDAC by participating in nuclear factor kappa beta, regulation of tumor necrosis factor, cell adhesion, T cell receptor signaling pathway, and numerous immune-related biological processes and signaling pathways. Functional enrichment analysis of DEGs between high- and low-UXT-AS1 expression groups suggested that these DEGs were significant enriched in B cell receptor complex, response to drug chemical carcinogenesis and drug metabolism-cytochrome P450. CMap analysis revealed that quipazine and terazosin may be targeted drugs for UXT-AS1 in PDAC.
Conclusion: Our current study has identified UXT-AS1 as a novel biomarker for the prognosis of early stage PDAC. We also clarified its biological functional mechanisms and identified two targeted drugs of UXT-AS1 in PDAC.
... Although NIK is well established as a positive regulator of the inhibitor of kB kinase (IKK) complex, which is required for activation of NF-κB signaling, IKK-independent functions for NIK have also been described 20,27 . Thus, we sought to determine whether NIK-dependent regulation of mitochondrial metabolism is mediated by IKK and downstream NF-κB signaling. ...
Cancers, including glioblastoma multiforme (GBM), undergo coordinated reprogramming of metabolic pathways that control glycolysis and oxidative phosphorylation (OXPHOS) to promote tumor growth in diverse tumor microenvironments. Adaptation to limited nutrient availability in the microenvironment is associated with remodeling of mitochondrial morphology and bioenergetic capacity. We recently demonstrated that NF-κB-inducing kinase (NIK) regulates mitochondrial morphology to promote GBM cell invasion. Here, we show that NIK is recruited to the outer membrane of dividing mitochondria with the master fission regulator, Dynamin-related protein1 (DRP1). Moreover, glucose deprivation-mediated metabolic shift to OXPHOS increases fission and mitochondrial localization of both NIK and DRP1. NIK deficiency results in decreased mitochondrial respiration, ATP production, and spare respiratory capacity (SRC), a critical measure of mitochondrial fitness. Although IκB kinase α and β (IKKα/β) and NIK are required for OXPHOS in high glucose media, only NIK is required to increase SRC under glucose deprivation. Consistent with an IKK-independent role for NIK in regulating metabolism, we show that NIK phosphorylates DRP1-S616 in vitro and in vivo. Notably, a constitutively active DRP1-S616E mutant rescues oxidative metabolism, invasiveness, and tumorigenic potential in NIK −/− cells without inducing IKK. Thus, we establish that NIK is critical for bioenergetic stress responses to promote GBM cell pathogenesis independently of IKK. Our data suggest that targeting NIK may be used to exploit metabolic vulnerabilities and improve therapeutic strategies for GBM.
... IKKa in turns phosphorylates the RelB (NFkB2) precursor p100, resulting in the cleavage and maturation of RelB (85). Thus, NIK is responsible for activation of the atypical NFkB pathway, which induces the expression of target genes, such as Ccl2 (C-C motif chemokine ligand 2), to promote macrophage infiltration and inflammation (86)(87)(88). Interestingly, TBK1 directly phosphorylates NIK, leading to its degradation (62,63). ...
Adipocytes and adipose tissue play critical roles in the regulation of metabolic homeostasis. In obesity and obesity-associated metabolic diseases, immune cells infiltrate into adipose tissues. Interaction between adipocytes and immune cells re-shapes both metabolic and immune properties of adipose tissue and dramatically changes metabolic set points. Both the expression and activity of the non-canonical IKK family member TBK1 are induced in adipose tissues during diet-induced obesity. TBK1 plays important roles in the regulation of both metabolism and inflammation in adipose tissue and thus affects glucose and energy metabolism. Here we review the regulation and functions of TBK1 and the molecular mechanisms by which TBK1 regulates both metabolism and inflammation in adipose tissue. Finally, we discuss the potential of a TBK1/IKKε inhibitor as a new therapy for metabolic diseases.
... This pathway is stimulated -amongst others -by a subset of TNF-R family members like CD40 and the B-cell activating receptor (BAFFR) and mainly relies on an interplay between TRAF3, the NF-κB-inducing kinase (NIK), and a homodimeric IKKα complex [55]. Noncanonical NF-κB activation typically results in RelB:p52 heterodimers translocating to the nucleus to regulate a specific set of target genes [55,63,64]. While canonical NF-κB is usually attributed to inflammation and noncanonical NF-κB to development, both signaling pathways a crucial for maintaining cellular processes and alterations have been linked to a variety of diseases, including immunodeficiency and cancer [65]. ...
Das angeborene Immunsystem detektiert Pathogen-assoziierte molekulare Muster und initiiert schnelle Zytokin-basierte Abwehrmechanismen. So wird im Zytosol virale DNA durch den Sensor cGAS erkannt, virale RNA durch RIG-I. Diese Rezeptoren vermitteln Signale an die Adaptorprotein STING bzw. MAVS. Die Adaptoren sind in unterschiedlichem Mass von TRAF-Proteinen abhängig, um Signale an nachgeordnete Kinasen weiterzuleiten. Diese Enzyme aktivieren insbesondere IRF3 und NF- κB. Die aktivierten Transkriptionsfaktoren induzieren gemeinsam eine für die angeborene Immunantwort typische IFN-β-Produktion. Gamma-Herpesviren wie das humane Epstein-Barr-Virus (EBV) besitzen ein onkogenes Potential, das durch ihre sogenannten Onkoproteine vermittelt wird. Das primäre EBV-Onkoprotein LMP1 und auch das nahe verwandte Tio-Protein interagieren mit TRAFs um NF-κB zu aktiveren. Die gemeinsam genutzten Kofaktoren führten zur Hypothese, dass Tio und LMP1 mit den cGAS-STING- und RIG-I-MAVS-Signalübertragungswegen interferieren. Daher wurden in dieser Studie HEK293-Zellen (Wildtyp und TRAF6-knockout) verwendet für NF-κB-, IRF3- und IFN-β-Promotor-Reporterassays, IFN-β RT-qPCRs, Immunoblot- und Immunopräzipitationsanalysen. Die intrazelluläre Lokalikation wurde mittels Immunofluoreszenz-Färbung von HeLa-Zellen überprüft. Tio verstärkte die STING-vermittelte IFN-β-Aktivierung deutlich, speziell durch gesteigerte NF-κB Aktivität. Alle Effekte von STING und Tio auf IFN-β und die NF-κB-Aktivität waren TRAF6- abhängig, während die IRF3-Induktion durch STING komplett TRAF6-unabhängig und nicht von Tio beeinflusst war. Dagegen reprimierte Tio im Zusammenhang mit MAVS die IFN-β-Induktion über eine Verminderung der IRF3-Aktivität. Diese Reduktion war wieder TRAF6-abhängig. Begleitend zeigte sich, dass Tio TRAF6 aus dem mitochondrialen MAVS-Signalkomplex herauslöste und die TBK1- induzierte IRF3-Aktivierung unterbrochen wurde. Die MAVS-modulierende Wirkung basierte auf Tio’s speziellem TRAF6-Bindemotiv, welches Tio vermutlich zum dominanten TRAF6-Interaktor macht. LMP1 verstärkte ebenfalls die NF-κB- und IFN-β-Aktitivierung durch STING, beschränkte jedoch die gleichzeitige IRF3-Induktion. Das EBV-Onkoprotein reprimierte die MAVS-vermittelte IRF3- Aktivierung und IFN-β-Induktion wesentlich effizienter als Tio. Dementsprechend führte auch LMP1 zu einer Entfernung von TRAF6 aus dem mitochondrialen Signalkomplex, was für einen konservierten gamma-herpesviralen Mechanismus spricht. Die verstärkte Repression durch LMP1 ließe sich dadurch erklären, dass LMP1 zusätzlich über einen TRAF6-unabhängigen Weg die IRF3-Aktivierung und über einen TRAF6-abhängigen Weg das bereits aktivierte IRF3 hemmte. Außerdem wurde LMP1 im Komplex mit IRF3 nachgewiesen, was auf weitere Mechanismen zur Modulation von IRF3 hindeutet. Insgesamt wurde in dieser Studie die bisher unbekannte Funktion herpesviraler Onkoproteine als Immunregulatoren beschrieben, welche die Immunantwort des Wirtes formen und eine für die virale Persistenz vorteilhafte Mikroumgebung schaffen könnten. Im Hinblick auf LMP1 könnte sich daraus ein Beitrag zum besseren Verständnis der EBV-Latenz ergeben.
... Both kinases phosphorylate p100, resulting in the partial proteolysis of p100. The resulting protein p52 heterodimerizes with RelB to mediate gene expression in the non-canonical arm of NF-κB [6]. ...
Purpose:
To investigate the role of an autophagy/lysosome pathway in NF-κB pathway blocked pancreatic cancer Panc-1 cells.
Methods:
The inhibitory effects of SN50 on pancreatic cancer cell line Panc-1 were detected by MTT assay. After SN50 treatment, autophagy activation was observed by MDC staining and transmission electron microscope (TEM). The expression of light chain 3 (LC3) was detected by immunofluorescence staining. Western blotting analyses were used to detect the expression of apoptosis-related protein p53 and autophagy-related proteins LC3, p62, and Beclin1.
Results:
Panc-1 cell activity was inhibited after SN50 treatment. The inhibition ratios of Panc-1 cells were (25.76±5.53)%, (34.35±4.49)% and (45.22±1.76)% after treatment of SN50 for 6 h, 12 h, and 24 h, and all changes were significant (P<0.05). Western blotting analysis showed that expressions of apoptotic protein p53, autophagic protein LC3, and Beclin 1 were increased, but the expression of p62 was down-regulated in Panc-1 cells. After SN50 treatment, immunofluorescence showed staining of microtubule-related protein 1 LC3, and MDC fluorescence staining showed increased autophagy bubbles labeled with MDC. Transmission electron microscope (TEM) was used to observe ultrastructure of Panc-1 cells that underwent autophagy after SN50 treatment.
Conclusion:
The activation of NF-κB was blocked by the inhibitor of p65 nuclear translocation, which activated autophagy and induced autophagic cell death in pancreatic cancer Panc-1 cell line.
... [18][19][20][21] Analyses of a spontaneous mutant mouse line, alymphoplasia, (aly/aly), which possesses a loss of function point mutation in the NIK gene, 22,23 have uncovered the essential roles of NIK in multiple levels of T-cell response. 16,24 These mutant mice have disorganized thymi, with very few mTECs, and display autoimmune symptoms with infiltration of lymphoid cells into several organs, especially exocrine systems such as the liver, pancreas, lung, salivary gland or lacrimal gland. 13,[25][26][27] As Sekai et al. 27 have clearly demonstrated, autoimmunity in aly/aly stems from lack of mTEC progenitors, leading to impaired deletion of some auto-reactive T-cell clones, although negative selection of T-cells specific for ubiquitous antigens may still occur. ...
NF‐κB‐inducing kinase (NIK) is known to be a critical regulator of multiple aspects of the immune response. Although the role of NIK in the development of medullary thymic epithelial cells (mTECs) has been well documented, the impact of NIK on the differentiation and function of cortical thymic epithelial cells (cTECs) remains ambiguous. To investigate the possible involvement of NIK in cTEC differentiation, we have compared the gene expression and function of cTECs from an NIK‐mutant mouse, alymphoplasia (aly/aly) with those of cTECs from wild type (WT) mice. Flow cytometric analyses revealed that expression levels of MHC class II, but not MHC class I or other TEC markers, were higher in aly/aly cells than in WT cells. Notably, the proportion of MHC class IIhi+ cTECs was elevated in aly/aly mice. We also demonstrated that expression of Ccl5 mRNA in the MHC class IIhi+ subset of aly/aly cTECs was decreased compared with that in WT cells, implying an abnormal pattern of gene expression in aly/aly cTECs. Analyses of bone marrow chimera using aly/aly or aly/+ mice as hosts suggested that Vβ usage and CD5 expression on WT T cells were altered when they matured in aly/aly thymi. These results collectively indicate that NIK may be involved in controlling the function of cTEC in selecting a proper T cell repertoire.
... More specifically, both TNF receptor-associated factor 1 (Traf1) and Traf2 were upregulated by TNF-α treatment. Furthermore, we found that ILC2s upregulated NIK (encoded by Map3k14), a crucial member of the NF-κB pathway and regulator of the immune system (Thu and Richmond, 2010). Notably, the non-canonical NF-κB genes Nfkb2 and Relb were statistically upregulated by TNF-α, whereas the canonical NF-κB genes Nfkb1 and Rela were either downregulated or unchanged ( Figure 5D). ...
Group 2 innate lymphoid cells (ILC2s) can initiate pathologic inflammation in allergic asthma by secreting copious amounts of type 2 cytokines, promoting lung eosinophilia and airway hyperreactivity (AHR), a cardinal feature of asthma. We discovered that the TNF/TNFR2 axis is a central immune checkpoint in murine and human ILC2s. ILC2s selectively express TNFR2, and blocking the TNF/TNFR2 axis inhibits survival and cytokine production and reduces ILC2-dependent AHR. The mechanism of action of TNFR2 in ILC2s is through the non-canonical NF-κB pathway as an NF-κB-inducing kinase (NIK) inhibitor blocks the costimulatory effect of TNF-α. Similarly, human ILC2s selectively express TNFR2, and using hILC2s, we show that TNFR2 engagement promotes AHR through a NIK-dependent pathway in alymphoid murine recipients. These findings highlight the role of the TNF/TNFR2 axis in pulmonary ILC2s, suggesting that targeting TNFR2 or relevant signaling is a different strategy for treating patients with ILC2-dependent asthma.
... This is triggered by activation of IKKα by NF-κB-inducing kinase (NIK). IKK activation in turn leads to p100 phosphorylation, ubiquitination, and processing, which yields active p52 protein able to form heterodimers with RelB; resulting p52/RelB non-canonical NF-κB complexes translocate into the nucleus and activates target genes [71]. ...
The risk of developing adult T-cell leukemia/lymphoma (ATLL) in individuals infected with human T-cell lymphotropic virus 1 (HTLV-1) is about 3–5%. The mechanisms by which the virus triggers this aggressive cancer are still an area of intensive investigation. The viral protein Tax-1, together with additional regulatory proteins, in particular HTLV-1 basic leucine zipper factor (HBZ), are recognized as relevant viral factors required for both viral replication and transformation of infected cells. Tax-1 deregulates several cellular pathways affecting the cell cycle, survival, and proliferation. The effects of Tax-1 on the NF-κB pathway have been thoroughly studied. Recent studies also revealed the impact of Tax-1 and HBZ on microRNA expression. In this review, we summarize the recent progress in understanding the contribution of HTLV-1 Tax- and HBZ-mediated deregulation of NF-κB and the microRNA regulatory network to HTLV-1 pathogenesis.
... The various stimuli like stress, cytokines, and ROS can activate NF-κB through two major signaling pathways, the canonical and non-canonical (or alternative) pathways, both being important for regulating immune and inflammatory responses [80]. A NF-κB-inducing kinase (NIK) is a kinase that activates the canonical and non-canonical NF-κB pathways [81]. NIK phosphorylates and activates IκB kinase complex (IKKα) homodimers [82]. ...
With the progress of nanotechnology, the adverse effects of nanoscale materials are receiving much attention. Inhibition of toll-like receptor 4 (TLR-4)/nuclear factor kappa B (NF-κB) signaling is a hallmark for downregulating the expression of many inflammatory genes implicated in oxidative stress. Therefore, the present study aimed to demonstrate the influence of grape seed proanthocyanidin extract (GSE) on the hepatic TLR-4/ NF-κB signaling pathway in TiO2-NP-induced liver damage in rats. Forty male Albino rats were divided into 4 groups (n = 10): G1 was used as a control, G2 received TiO2-NPs (500 mg/kg/day orally) from the 17th to 30th day (acute toxicity), G3 received GSE (75 mg/kg/day orally) for 30 days, and G4 pre- and co-treated with GSE (for 30 days) and TiO2-NPs (from the 17th to 30th day), with the aforementioned doses. TiO2-NPs induced severe hepatic injury that was indicated by biochemical alterations in serum liver markers (acetylcholinesterase, ALT, ALP, total proteins, albumin, and direct bilirubin), oxidative stress indicators (MDA, GSH, and catalase), and histopathological alterations as well. Moreover, TiO2-NPs triggered an inflammatory response via the upregulation of TLR-4, NF-κB, NIK, and TNF-α mRNA expressions. Pre- and co-treatments with GSE alleviated the detrimental effects of TiO2-NPs which were enforced by the histopathological improvements. These results indicated that GSE effectively protected against TiO2-NP-induced hepatotoxicity via the inhibition of TLR-4/NF-κB signaling and hence suppressed the production of pro inflammatory cytokines such as TNF-α and improved the antioxidant status of the rats.
... NIK can activate both canonical and non-canonical pathways by inducing phosphorylation and degradation of inhibitor of κB (IκB). The canonical pathway is mediated by the transcriptional activity of the p50:p65 dimer, whereas the noncanonical pathway is transcriptionally controlled by the p52:RelB dimer (183,184). Physiologically, NIK plays an important role in the maintenance of the embryonic pluripotent stem cell state and mammary gland development. This may suggest a potential role for NIK in maintenance of BCSCs (185)(186)(187)(188). NIK-IKKα was shown to regulate ErbB2-induced mammary tumorigenesis in a preclinical model through the nuclear export of p27/kip1 which supports the proliferation and expansion of BCSCs (189). ...
Breast cancer stem cells (BCSCs) play a vital role in tumor progression and metastasis. They are heterogeneous and inherently radio- and chemoresistant. They have the ability to self-renew and differentiate into non-BCSCs. These determinants of BCSCs including the plasticity between the mesenchymal and epithelial phenotypes often leads to minimal residual disease (MRD), tumor relapse, and therapy failure. By studying the resistance mechanisms in BCSCs, a combinatorial therapy can be formulated to co-target BCSCs and bulk tumor cells. This review addresses breast cancer stemness and molecular underpinnings of how the cancer stemness can lead to pharmacological resistance. This might occur through rewiring of signaling pathways and modulated expression of various targets that support survival and self-renewal, clonogenicity, and multi-lineage differentiation into heterogeneous bulk tumor cells following chemotherapy. We explore emerging novel and alternative molecular targets against BC stemness and chemoresistance involving survival, drug efflux, metabolism, proliferation, cell migration, invasion, and metastasis. Strategic targeting of such vulnerabilities in BCSCs may overcome the chemoresistance and increase the longevity of the metastatic breast cancer patients.
... TRAF3 loss-of-function mutations are commonly observed in B-cell lymphoma [69]. TRAF3 is constitutively associated with NIK, which is also an important regulator of the noncanonical/NF-κB2 pathway induced by IL-1 or TNF superfamily members [70,71], in a dynamic manner, and the TRAF3-NIK interaction leads to NIK degradation by the proteasome [72]. Stimulation with both anti-CD40 and BAFF caused persistent degradation of TRAF3 and increased stability, reversed the blockage of NIK and resulted in activation of the downstream NF-κB signaling molecules [73]. ...
Full activation of T lymphocytes requires signals from both T cell receptors and costimulatory molecules. In addition to CD28, several T cell molecules could deliver costimulatory signals, including CD154, which primarily interacts with CD40 on B-cells. CD40 is a critical molecule regulating several B-cell functions, such as antibody production, germinal center formation and cellular proliferation. Upregulated expression of CD40 and CD154 occurs in immune effector cells and non-immune cells in different autoimmune diseases. In addition, therapeutic benefits have been observed by blocking the CD40-CD154 interaction in animals with collagen-induced arthritis. Given the therapeutic success of the biologics abatacept, which blocks CD28 costimulation, and rituximab, which deletes B cells in the treatment of autoimmune arthritis, the inhibition of the CD40-CD154 axis has two advantages, namely, attenuating CD154-mediated T cell costimulation and suppressing CD40-mediated B-cell stimulation. Furthermore, blockade of the CD40-CD154 interaction drives the conversion of CD4+ T cells to regulatory T cells that mediate immunosuppression. Currently, several biological products targeting the CD40-CD154 axis have been developed and are undergoing early phase clinical trials with encouraging success in several autoimmune disorders, including autoimmune arthritis. This review addresses the roles of the CD40-CD154 axis in the pathogenesis of autoimmune arthritis and its potential as a therapeutic target.
... Functional enrichment of the candidate region at LG19 using the turbot transcriptome as a reference revealed 32 genes with functions related to the immune or defence system. These functions were associated with (i) tissue regeneration; (ii) response to wounding; (iii) leukotriene production involved in inflammatory response (Funk, 2001); (iv) activation of NF-kappaB-inducing kinase activity involved in the regulation of the immune system (Thu and Richmond, 2010); (v) toll-like receptor 21 signalling pathway involved in the innate immune response (Akira and Takeda, 2004); (vi) activation of MAPK activity involved in innate immune response (Vidal et al., 2001); and (vii) cellular defence response. Seven of these genes had been previously suggested to be involved in the response to infectious diseases in turbot (Supplementary Table 1). ...
Selective breeding for improving host responses to infectious pathogens is a promising option for disease control. In fact, disease resilience, the ability of a host to survive or cope with infectious challenge, has become a highly desirable breeding goal. However, resilience is a complex trait composed of two different host defence mechanisms, namely resistance (the ability of a host to avoid becoming infected or diseased) and endurance (the ability of an infected host to survive the infection). While both could be targeted for genetic improvement, it is currently unknown how they contribute to survival, as reliable estimates of genetic parameters for both traits obtained simultaneously are scarce. A difficulty lies in obtaining endurance phenotypes for genetic analyses. In this study, we present the results from an innovative challenge test carried out in turbot whose design allowed disentangling the genetic basis of resistance and endurance to Philasterides dicentrarchi, a parasite causing scuticociliatosis that leads to substantial economic losses in the aquaculture industry. A noticeable characteristic of the parasite is that it causes visual signs that can be used for disentangling resistance and endurance. Our results showed the existence of genetic variation for both traits (heritability = 0.26 and 0.12 for resistance and endurance, respectively) and for the composite trait resilience (heritability = 0.15). The genetic correlation between resistance and resilience was very high (0.90) indicating that both are at a large extent the same trait, but no significant genetic correlation was found between resistance and endurance. A total of 18,125 SNPs obtained from 2b-RAD sequencing enabled genome-wide association analyses for detecting QTLs controlling the three traits. A candidate QTL region on linkage group 19 that explains 33% of the additive genetic variance was identified for resilience. The region contains relevant genes related to immune response and defence mechanisms. Although no significant associations were found for resistance, the pattern of association was the same as for resilience. For endurance, one significant association was found on linkage group 2. The accuracy of genomic breeding values was also explored for resilience, showing that it increased by 12% when compared with the accuracy of pedigree-based breeding values. To our knowledge, this is the first study in turbot disentangling the genetic basis of resistance and endurance to scuticociliatosis.
... After cytokine induction, NIK undergoes conformational changes and regulates NF-κB signal transduction through constitutive kinase activity [60]. However, the hyperactive NIK leads to autoimmune disease and cancer [61]. ...
STK16 (Ser/Thr kinase 16, also known as Krct/PKL12/MPSK1/TSF-1) is a myristoylated and palmitoylated Ser/Thr protein kinase that is ubiquitously expressed and conserved among all eukaryotes. STK16 is distantly related to the other kinases and belongs to the NAK kinase family that has an atypical activation loop architecture. As a membrane-associated protein that is primarily localized to the Golgi, STK16 has been shown to participate in the TGF-β signaling pathway, TGN protein secretion and sorting, as well as cell cycle and Golgi assembly regulation. This review aims to provide a comprehensive summary of the progress made in recent research about STK16, ranging from its distribution, molecular characterization, post-translational modification (fatty acylation and phosphorylation), interactors (GlcNAcK/DRG1/MAL2/Actin/WDR1), and related functions. As a relatively underexplored kinase, more studies are encouraged to unravel its regulation mechanisms and cellular functions.
... Together these results suggest that mutations of MAP3K14 including G33G and G53G disrupt the regulatory motifs and possibly some unknown DNA elements that are necessary for negative regulation of NIK, which enhance the stability of the protein leading to constitutive activation of NF-kB signaling. Constitutive activation of NF-kB signaling is one of the key mechanisms involved in the development of most types of lymphoma such as MM, MZL, CLL, ABC-DLBCL, and MCL(Thu and Richmond 2010). ...
Mantle cell lymphoma (MCL) is a rare, aggressive and incurable type of cancer with a high incidence of systemic dissemination and poor survival outcomes. Understanding the genetics and biology of MCL is necessary for development of suitable biomarkers and therapies. A limited number of sequencing-based studies were performed on MCL recently, which improved the knowledge on causal mutations and molecular mechanisms of MCL. However, because of the small cohort sizes of these studies and use of outdated computational tools and databases have limited the findings of these studies. The objective of our study was to identify the genetic alterations and molecular pathways that are associated with the development, progression, and dissemination of MCL by using leading-edge bioinformatics tools, up-to-date molecular biology databases, and a large sample size. Presented here is a meta-analysis of whole exome sequencing data from tumor biopsies of 67 MCL patients, which resulted in the identification of several novel candidate driver genes that were targets of recurrent mutations in MCL such as SP140, S1PR1, PTPRD, HNRNPH1, LRP1B, FAT1, MAP3K14, and DST. Our analysis revealed four mutation hotspots in the first exon of MAP3K14 in MCL, five other types of B-cell lymphoma and leukemia, and canine lymphoma. Our research showed that the recurrent mutations of MAP3K14 that have activating effect on the protein, may be associated with a risk of relapse. This finding suggests that the recurrent mutations of MAP3K14 may have application for prediction of disease outcome, response to therapy, and the transformation of the leukemic variant of MCL to the more aggressive subtype of the disease.
Finally, we detected a significant accumulation of mutations in signaling pathways with roles in mechanisms of tumor metastasis, such as Rho GTPase mediated signaling, focal adhesion, G-protein coupled receptor signaling, cAMP-PKA signaling, ERK-MAPK signaling, ROBO-Slit signaling, and JAK-STAT signaling. These findings offer new insights into the understanding of the driver genes and molecular mechanisms underlying the aggressive clinical course of MCL and may have implications for the development of therapies.
... NF-κB is able to be activated by a variety of factors, including pro-inflammatory cytokines, growth factors and cell stress; it may also be activated by chemotherapeutic drugs, including daunorubicin, doxorubicin and cisplatin and other chemotherapy drugs (19). NF-κB is also a key regulator of apoptosis, and may induce the expression of anti-apoptotic factors, including survivin and Bcl-2 (20). NF-κB activation is strictly regulated by inhibitor of NF-κB, therefore conventional chemotherapy drugs are often accompanied by NF-κB inhibitors, to inhibit the NF-κB signaling pathway, decrease local recurrence and improve patient survival rate (21). ...
Melanoma is highly malignant, particularly prone to metastasizing to the skin. The incidence of melanoma varies markedly between countries, and is relatively low in China. The aim of the present study was to investigate the antitumorigenic effect of damnacanthal on melanoma cells, and its molecular mechanism. MUM-2B cells were treated with 0-20 µM damnacanthal for 12, 24 and 48 h. In vitro, it was demonstrated that damnacanthal inhibited proliferation and promoted apoptosis of melanoma cells in a dose- and time-dependent manner. Damnacanthal treatment increased caspase-3/8 and 9 activity, and promoted B-cell lymphoma 2-associated X protein, tumor protein p53 (p53) and p21 protein expression levels in melanoma cells. Damnacanthal treatment also resulted in downregulated nuclear factor-κB (NF-κB), cyclin D and cyclin E protein expression in melanoma cells. In conclusion, the results of the present study demonstrated that the antitumorigenic activity of damnacanthal on melanoma cells is executed via the p53/p21 and NF-κB/cyclin/ caspase-3 signaling pathways.
... The regulation of the canonical NF-κB pathway has a great influence in multiple immunological pathways [45][46][47]. NIK as the key enzyme of the non-canonical NF-κB pathway has been shown to have a stabilizing and amplifying effect also on the canonical NF-κB pathway [48]. The low levels of NIK in most cell types during steady state and the survival of mice and humans with mutations in NIK, make NIK an attractive potential target in the treatment of autoimmune diseases. ...
NF-κB inducing kinase (NIK) is the key protein of the non-canonical NF-κB pathway and is important for the development of lymph nodes and other secondary immune organs. We elucidated the specific role of NIK in T cells using T-cell specific NIK-deficient (NIKΔT) mice. Despite showing normal development of lymphoid organs, NIKΔT mice were resistant to induction of CNS autoimmunity. T cells from NIKΔT mice were deficient in late priming, failed to up-regulate T-bet and to transmigrate into the CNS. Proteomic analysis of activated NIK-/- T cells showed de-regulated expression of proteins involved in the formation of the immunological synapse: in particular, proteins involved in cytoskeleton dynamics. In line with this we found that NIK-deficient T cells were hampered in phosphorylation of Zap70, LAT, AKT, ERK1/2 and PLCγ upon TCR engagement. Hence, our data disclose a hitherto unknown function of NIK in T-cell priming and differentiation.
... Another report concludes that autophagy regulates odontoblast differentiation by suppressing NF-κB p65 activation in inflammatory environments [55]. In some other reports, autophagy inhibits the activation of NF-κB p65 by degradation of NF-κB signaling components, such as IκB kinases that are necessary for triggering the translocation of NF-κB and its phosphorylation [56][57][58]. ...
Preadipocyte migration is a fundamental and important process for the development of tissue organization, especially in the development of primitive adipose tissue and adipocyte tissue wound healing. However, excessive migration may result in abnormal development and fibrosis-related diseases such as hypertrophic scar. We previously reported that type I collagen (collagen I) enhanced migration of 3T3-L1 preadipocytes via phosphorylation and/or acetylation of NF-κB p65, and the enhanced cell migration is repressed by silibinin treatment through sirt1. It is known that sirt1 has an ability to deacetylate acetylated NF-κB p65, but little is known about the effect of sirt1 on phosphorylated NF-κB p65. This study aims to examine the potential effect of sirt1 on the regulation of phosphorylated NF-κB p65 and the underlying mechanism. Autophagy is involved in many physiological and pathological processes, including regulation of cell migration as well as in cellular homeostasis. The present study demonstrates that silibinin induces autophagy in a dose-dependent manner in 3T3-L1 cells. Autophagy is under the regulation of sirt1/AMPK pathway, and inhibits collagen I-enhanced migration of 3T3-L1 cells through negative regulation of NF-κB p65 phosphorylation but not acetylation. The expression of peroxisome proliferator-activated receptor α (PPARα) is up-regulated with silibinin accompanying up-regulation of autophagy through activating sirt1 in 3T3-L1 cells. Taken together, these findings indicate that silibinin-induced autophagy is mediated by up-regulation of PPARα-sirt1-AMPK, contributing to repression of type I collagen-enhanced migration in murine 3T3-L1 preadipocytes through down-regulation of phosphorylated NF-κB p65.
The noncanonical NFκB signaling pathway mediates the biological functions of diverse cell survival, growth, maturation, and differentiation factors that are important for the development and maintenance of hematopoietic cells and immune organs. Its dysregulation is associated with a number of immune pathologies and malignancies. Originally described as the signaling pathway that controls the NFκB family member RelB, we now know that noncanonical signaling also controls NFκB RelA and cRel. Here, we aim to clarify our understanding of the molecular network that mediates noncanonical NFκB signaling and review the human diseases that result from a deficient or hyper‐active noncanonical NFκB pathway. It turns out that dysregulation of RelA and cRel, not RelB, is often implicated in mediating the resulting pathology.
This article is categorized under: Immune System Diseases > Molecular and Cellular Physiology
Cancer > Molecular and Cellular Physiology
Immune System Diseases > Stem Cells and Development
Cis-regulatory elements (CREs) have an important role in human adaptation to the living environment. However, the lag in population genomic cohort studies and epigenomic studies, hinders the research in the adaptive analysis of CREs in human populations. In this study, we collected 4,013 unrelated individuals and performed a comprehensive analysis of adaptive selection of genome-wide CREs in the Han Chinese. In total, 12.34% of genomic regions are under the influence of adaptive selection, where 1.00% of enhancers and 2.06% of promoters are under positive selection, and 0.06% of enhancers and 0.02% of promoters are under balancing selection. Gene ontology enrichment analysis of these CREs under adaptive selection reveals that many positive selections in the Han Chinese occur in pathways involved in cell-cell adhesion processes, and many balancing selections are related to immune processes. Two classes of adaptive CREs related to cell adhesion were in-depth analysed, one is the adaptive enhancers derived from neanderthal introgression, leads to lower hyaluronidase level in skin, and brings better performance on UV-radiation resistance to the Han Chinese. Another one is the CREs regulating wound healing, and the results suggest the positive selection inhibits coagulation and promotes angiogenesis and wound healing in the Han Chinese. Finally, we found that many pathogenic alleles, such as risky alleles of type 2 diabetes or schizophrenia, remain in the population due to the hitchhiking effect of positive selections. Our findings will help deepen our understanding of the adaptive evolution of genome regulation in the Han Chinese.
IκB kinases (IKKs), specifically IKKα and IKKβ, have long been recognized for their pivotal role in the NF-κB pathway, orchestrating immune and inflammatory responses. However, recent years have unveiled their dual role in cancer, where they can act as both promoters and suppressors of tumorigenesis. In addition, the interplay with pathways such as the MAPK and PI3K pathways underscores the complexity of IKK regulation and its multifaceted role in both inflammation and cancer. By exploring the molecular underpinnings of these processes, we can better comprehend the complex interplay between IKKs, tumor development, immune responses, and the development of more effective therapeutics. Ultimately, this review explores the dual role of IκB kinases in cancer, focusing on the impact of phosphorylation events and crosstalk with other signaling pathways, shedding light on their intricate regulation and multifaceted functions in both inflammation and cancer.
The pathophysiology of intracranial aneurysms (IA) has been proven to be closely linked to hemodynamic stress and inflammatory pathways, most notably the NF-kB pathway. Therefore, it is a potential target for therapeutic intervention. In the present review, we investigated alterations in the vascular smooth muscle cells (VSMCs), extracellular matrix, and endothelial cells by the mediators implicated in the NF-kB pathway that lead to the formation, growth, and rupture of IAs. We also present an overview of the NF-kB pathway, focusing on stimuli and transcriptional targets specific to IAs, as well as a summary of the current strategies for inhibiting NF-kB activation in IAs. Our report adds to previously reported data and future research directions for treating IAs using compounds that can suppress inflammation in the vascular wall.
Background:
Early brain injury including neuronal apoptosis is a main contributor to neurological deterioration after subarachnoid hemorrhage (SAH). This study was aimed to investigate whether EGFR (epidermal growth factor receptor)/NFκB (nuclear factor-kappa B) inducing kinase (NIK)/NFκB (p65 and p50) pathway is involved in the neuronal apoptosis after SAH in mice.
Methods:
C57BL/6 adult male mice underwent endovascular perforation SAH modeling or sham-operation (n=286), and 86 mild SAH mice were excluded. In experiment 1, vehicle or an EGFR inhibitor (632.0 ng AG1478) was administered intraventricularly at 30 minutes postmodeling. At 24 or 72 hours, after neurological score was tested, brain water content, double immunolabeling with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and a neuronal marker antimicrotubule-associated protein-2 antibody, Western blotting using whole tissue lysate or nuclear protein extraction of the left cortex, and immunohistochemistry for cleaved caspase-3, phosphorylated (p-) EGFR, NIK, p-NFκB p65, and NFκB p105/50 were evaluated. In experiment 2, after sham or SAH modeling, AG1478+vehicle or AG1478+4.0 ng EGF was administered intraventricularly. The brain was used for TUNEL staining and immunohistochemistry after 24-hour observation.
Results:
SAH group showed deteriorated neurological score (P<0.01, Mann-Whitney U test), more TUNEL- and cleaved caspase-3-positive neurons (P<0.01, ANOVA), and higher brain water content (P<0.01, Mann-Whitney U test), and these observations were improved in SAH-AG1478 group. Western blotting showed that expression levels of p-EGFR, p-p65, p50, and nuclear-NIK were increased after SAH (P<0.05, ANOVA), and decreased by AG1478 administration. Immunohistochemistry revealed these molecules localized in degenerating neurons. EGF administration resulted in neurological deterioration, increased TUNEL-positive neurons, and activation of EGFR, NIK, and NFκB.
Conclusions:
Activated EGFR, nuclear-NIK, and NFκB expressions were observed in cortical degenerating neurons after SAH, and were decreased by administration of AG1478, associated with suppression of TUNEL- and cleaved caspase-3-positive neurons. EGFR/NIK/NFκB pathway is suggested to be involved in neuronal apoptosis after SAH in mice.
Inhibition of GSK-3 using small-molecule elraglusib has shown promising preclinical antitumor activity. Using in vitro systems, we found that elraglusib promotes immune cell-mediated tumor cell killing, enhances tumor cell pyroptosis, decreases tumor cell NF-κB-regulated survival protein expression, and increases immune cell effector molecule secretion. Using in vivo systems, we observed synergy between elraglusib and anti-PD-L1 in an immunocompetent murine model of colorectal cancer. Murine responders had more tumor-infiltrating T-cells, fewer tumor-infiltrating Tregs, lower tumorigenic circulating cytokine concentrations, and higher immunostimulatory circulating cytokine concentrations. To determine the clinical significance, we utilized human plasma samples from patients treated with elraglusib and correlated cytokine profiles with survival. Using paired tumor biopsies, we found that CD45+ tumor-infiltrating immune cells had lower expression of inhibitory immune checkpoints and higher expression of T-cell activation markers in post-elraglusib patient biopsies. These results introduce several immunomodulatory mechanisms of GSK-3 inhibition using elraglusib, providing a rationale for the clinical evaluation of elraglusib in combination with immunotherapy.
Statement of significance
Pharmacologic inhibition of GSK-3 using elraglusib sensitizes tumor cells, activates immune cells for increased anti-tumor immunity, and synergizes with anti-PD-L1 immune checkpoint blockade. These results introduce novel biomarkers for correlations with response to therapy which could provide significant clinical utility and suggest that elraglusib, and other GSK-3 inhibitors, should be evaluated in combination with immune checkpoint blockade.
Baicalin is a main active ingredient of the dried root of Scutellaria and has been extensively employed in Traditional Chinese Medicine for the treatment of asthma, fever, and psoriasis. Based on the reports of antioxidant, anti-inflammatory, anti-infection, and anti-tumor activities of baicalin, we have explored its radioprotective efficacy using in vitro and in vivo experimental model systems. In the present study, we have investigated the radioprotective, immunomodulatory, and anti-inflammatory properties of baicalin using wet lab and in silico approaches. It was observed that pre-treatment of murine splenic lymphocytes with baicalin protected cells against radiation-induced cell death possibly by decreasing the cellular reactive oxygen species levels. Prophylactic oral administration of baicalin offered significant increase in endogenous spleen colony counts and an enhancement in the survival of mice. We have also observed that baicalin suppressed mitogen-induced splenic lymphocyte proliferation and IL-2 production. It also inhibited the production of nitric oxide in RAW 264.7 cells in response to elicitation of lipopolysaccharide. Further, in silico study was performed to evaluate the possible mechanism of radioprotection and immunomodulation by selecting different pro-inflammatory mediators such as COX2, Lck, NIK, and IKK-β which have a significant role in radioprotection, lymphocyte activation, and inflammation. Our molecular docking and molecular dynamics study show that baicalin has a significant predicted binding affinity with COX2, Lck, NIK, and IKK-β. These in silico results can explain the experimentally observed radioprotective, immunosuppressive, and anti-inflammatory properties of baicalin. Thus, radioprotection offered by baicalin may be because of its antioxidant, anti-inflammatory, and immunomodulatory properties.
Das duktale Adenokarzinom des Pankreas stellt weiterhin trotz aller medizinischen Entwicklungen eine Herausforderung in der Diagnostik und Therapie bei einer nahezu identischen Inzidenz und Mortalität dar. Auch die Genese der Erkrankung ist bis zum heutigen Tag nicht geklärt. Als eine mögliche Ursache wird das inflammatorische Mikromilieu diskutiert, bzgl. dessen Entstehung und Aufrechterhaltung ebenfalls noch Unklarheiten bestehen. Als möglicher Trigger hierfür kommen die endosomalen Toll like-Rezeptoren 7 und 8 in Frage, die sowohl in ihrer Immunfunktion virale RNA-Bestandteile, so genannte pathogen-associated molecular patterns, als auch damage-associated molecular patterns, d.h. RNA-Fragmente von geschädigten oder sterbenden Zellen erkennen können. Durch ihre Stimulation kommt es zu einer Immunantwort. Im Rahmen dieser Arbeit wurde die Auswirkung der Stimulation von TLR 7 und TLR 8 exprimierenden PANC-1-Zellen bzgl. des Wachstumsverhaltens und der Chemosensibilität auf 5-FU untersucht. Es konnte gezeigt werden, dass mit einem spezifischen TLR7 und TLR8-Agonisten (R848) das Wachstum signifikant im Vergleich zu unbehandelten Zellen gesteigert werden konnte. Das dieser Effekt abhängig von der Expression von TLR7 und TLR8 war, konnte dadurch bewiesen werden, dass PANC-1 Zellen ohne die Expression von TLR7 oder TLR8 sowie mittels siRNA-Knockdown für TLR7 oder TLR8 behandelte TLR7- oder TLR8-exprimierende PANC1-Zellen kein gesteigertes Wachstum zeigten. Die Chemosensibilität auf 5-FU in einer LD50-Dosierung war bei den stimulierten Zellen im Vergleich zu den unstimulierten Zellen signifikant reduziert. Auf molekularer Ebene war ein Trend hinsichtlich eines Anstiegs Apoptose-inhibierender, Wachstums-fördernder und Inflammation-aufrechterhaltender Faktoren (IL-6, NF-kB, COX-2) zu erkennen. Zusammenfassend konnte gezeigt werden, dass die Stimulation von Toll like-Rezeptoren 7 und 8 exprimierenden Pankreaskarzinomzellen mit einem weiteren Tumorwachstum sowie einer reduzierten Chemosensibilität sowie daraus resultierenden schlechten Therapieansprechen vergesellschaftet sein können.
Autoimmune and inflammatory diseases place a huge burden on the healthcare system. Small molecule (SM) therapeutics provide much needed complementary treatment options for these diseases. This digest series highlights the latest progress in the discovery and development of safe and efficacious SMs to treat autoimmune and inflammatory diseases with each part representing a class of SMs, namely: 1) protein kinases; 2) nucleic acid-sensing pathways; and 3) soluble ligands and receptors on cell surfaces. In this first part of the series, the focus is on kinase inhibitors that emerged between 2018 and 2020, and which exhibit increased target and tissue selectivity with the aim of increasing their therapeutic index.
Two different signaling pathways lead to the activation of the transcription factor NF-κB, initiating distinct biological responses: The canonical NF-κB pathway activation has been implicated in host immunity and inflammatory responses, whereas the noncanonical pathway activation has been involved in lymphoid organ development and B-cell maturation, as well as in the development of chronic inflammatory diseases and some hematologic cancers. The NF-κB-inducing kinase (NIK) is a cytoplasmic Ser/Thr kinase and is a key regulator of the noncanonical pathway. NIK activation results in the processing of the p100 subunit to p52, leading to the formation of the RelB/p52 complex and noncanonical pathway activation. Because of its role in the development of lymphoid malignancies, this kinase has always been considered as an attractive target for the treatment of certain types of cancers and immune diseases. We at Takeda have pursued a drug discovery program to identify small-molecule inhibitors against NIK. This report provides an overview of the data generated from our screening campaign using a small fragment library. Most importantly, we also provide a kinetic analysis of published compounds and chemical series developed at Takeda that are associated with a slow tight-binding mechanism and excellent cellular potency.
Disruption of tissue function activates cellular stress which triggers a number of mechanisms that protect the tissue from further damage. These mechanisms involve a number of homeostatic modules, which are regulated at the level of gene expression by the transactivator NF-κB. This transcription factor shifts between activation and repression of discrete, cell-dependent gene expression clusters. Some of its target genes provide feedback to NF-κB itself, thereby strengthening the inflammatory response of the tissue and later terminating inflammation to facilitate restoration of tissue homeostasis. Disruption of key feedback modules for NF-κB in certain cell types facilitates the survival of clones with genomic aberrations, and protects them from being recognized and eliminated by the immune system, to enable thereby carcinogenesis.
Osteoporosis is a pathological loss of bone mass due to an imbalance in bone remodeling where osteoclast-mediated bone resorption exceeds osteoblast-mediated bone formation resulting in skeletal fragility and fractures. Anti-resorptive agents such as bisphosphonates and SERMs, and anabolic drugs that stimulate bone formation, including PTH analogues and sclerostin inhibitors, are current treatments for osteoporosis. Despite their efficacy, severe side effects and loss of potency may limit the long term usage of a single drug. Sequential and combinational use of current drugs, such as switching from an anabolic to an anti-resorptive agent, may provide an alternative approach. Moreover, there are novel drugs being developed against emerging new targets such as Cathepsin K and 17β-HSD2 that may have less side effects. This review will summarize the molecular mechanisms of osteoporosis, current drugs for osteoporosis treatment, and new drug development strategies.
A set of NF-κB-inducing kinase (NIK) inhibitors was used to develop a molecular docking-based QSAR model by using nonlinear regression method. The accuracy of the QSAR model was remarkably improved by integrating the docking scores and key interaction profiles. Two indole-aminopyrimidine derivatives 32a and 32b predicted as NIK inhibitors were synthesized and biologically evaluated. The significant correlationship between experimental data and MD-SVR model-predicted results were observed. The binding mode of 32a and 32b with NIK were further investigated by dynamic simulations. Compound 32b was proposed as a promising lead for the findings of highly potent inhibitors.
NF-κB-inducing kinase (NIK) is a protein kinase central to the non-canonical NF-κB pathway downstream from multiple TNF receptor family members, including BAFF, which have been associated with B cell survival and maturation, dendritic cell activation, secondary lymphoid organ development, and bone metabolism. We report herein the discovery of a lead chemical series of NIK-inhibitors that was identified through a scaffold-hopping strategy using structure-based design. Electronic and steric properties of lead compounds were modified to address glutathione conjugation and amide hydrolysis. These highly potent compounds exhibited selective inhibition of LTβR-dependent p52 translocation and transcription of NF-κB2 related genes. Compound 4f is shown to have a favorable pharmacokinetic profile across species and to inhibit BAFF-induced B cell survival in vitro and reduce splenic marginal zone B cells in vivo.
Small molecule inhibitors targeting autoimmune and inflammatory processes have been an area of intense focus within academia and industry. Much of this work has been aimed at key kinases operating as central nodes in inflammatory signaling pathways. While this focus has led to over thirty FDA-approved small molecule kinase inhibitors, only one is currently approved for autoimmune and inflammatory diseases. Despite this lack of success, there remains tremendous reason for excitement. Our growing understanding of the biology involved in the inflammatory response, the factors that lead to safer small molecule kinase inhibitors and the availability of selective tool molecules for interrogating specific nodes and pathways are all pushing the field forward. This article focuses towards recent developments requiring novel approaches to create safe and effective small molecule kinase inhibitors and where further work is needed to realize the promise of small molecule kinase inhibitors for patient benefit.
There is considerable evidence that osteoclasts are involved in the pathogenesis of focal bone erosion in rheumatoid arthritis. Tumor necrosis factor-related activation-induced cytokine, also known as receptor activator of nuclear factor-kappaB ligand (TRANCE/RANKL) is an essential factor for osteoclast differentiation. In addition to its role in osteoclast differentiation and activation, TRANCE/RANKL also functions to augment T-cell dendritic cell cooperative interactions. To further evaluate the role of osteoclasts in focal bone erosion in arthritis, we generated inflammatory arthritis in the TRANCE/RANKL knockout mouse using a serum transfer model that bypasses the requirement for T-cell activation. These animals exhibit an osteopetrotic phenotype characterized by the absence of osteoclasts. Inflammation, measured by clinical signs of arthritis and histopathological scoring, was comparable in wild-type and TRANCE/RANKL knockout mice. Microcomputed tomography and histopathological analysis demonstrated that the degree of bone erosion in TRANCE/RANKL knockout mice was dramatically reduced compared to that seen in control littermate mice. In contrast, cartilage erosion was present in both control littermate and TRANCE/RANKL knockout mice. These results confirm the central role of osteoclasts in the pathogenesis of bone erosion in arthritis and demonstrate distinct mechanisms of cartilage destruction and bone erosion in this animal model of arthritis.
The alymphoplasia (aly) mutation of mouse is autosomal recessive and characterized by the systemic absence of lymph nodes (LN) and Peyer's patches (PP) and disorganized splenic and thymic structures with immunodeficiency. Although recent reports have shown that the interaction between lymphotoxin (LT) and the LT β-receptor (Ltβr, encoded by Ltbr ) provides a critical signal for LN genesis in mice, the aly locus on chromosome 11 (ref. 11) is distinct from those for LT and its receptor. We found that the aly allele carries a point mutation causing an amino acid substitution in the carboxy-terminal interaction domain of Nf-κb-inducing kinase (Nik, encoded by the gene Nik). Transgenic complementation with wild-type Nik restored the normal structures of LN, PP, spleen and thymus, and the normal immune response in aly/aly mice. In addition, the aly mutation in a kinase domain-truncated Nik abolished its dominant-negative effect on Nf-κb activation induced by an excess of Ltβr. Our observations agree with previous reports that Ltβr-deficient mice showed defects in LN genesis and that Nik is a common mediator of Nf-κb activation by the tumour necrosis factor (TNF) receptor family. Nik is able to interact with members of the TRAF family (Traf1, 2, 3, 5 and 6; ref. 13), suggesting it acts downstream of TRAF-associating receptor signalling pathways, including Tnfr ( ref. 12), Cd40 (Refs 14, 15), Cd30 (Refs 16, 17) and Ltβr (refs 18, 19, 20, 21). The phenotypes of aly/aly mice are more severe than those of Ltbr–/– mice, however, indicating involvement of Nik in signal transduction mediated by other receptors.
Previous evidence suggested that NF-κB-inducing kinase (NIK) might regulate IL-2 synthesis. However, the molecular mechanism is not understood. In this study, we show that NIK is involved in CD3 plus CD28 activation of IL-2 transcription. Splenic T cells from aly/aly mice (that have a defective NIK protein) have a severe impairment in IL-2 and GM-CSF but not TNF secretion in response to CD3/CD28. This effect takes place at the transcriptional level as overexpression of alyNIK inhibits IL-2 promoter transcription. NIK activates the CD28 responsive element (CD28RE) of the IL-2 promoter and strongly synergizes with c-Rel in this activity. We found that NIK interacts with the N-terminal domain of c-Rel, mapping this interaction to aa 771–947 of NIK. Moreover, NIK phosphorylates the c-Rel C-terminal transactivation domain (TAD) and induces Gal4-c-Rel-transactivating activity. Anti-CD28 activated Gal4-c-Rel transactivation activity, and this effect was inhibited by a NIK-defective mutant. Deletion studies mapped the region of c-Rel responsive to NIK in aa 456–540. Mutation of several serines, including Ser471, in the TAD of c-Rel abrogated the NIK-enhancing activity of its transactivating activity. Interestingly, a Jurkat mutant cell line that expresses one of the mutations of c-Rel (Ser471Asn) has a severe defect in IL-2 and CD28RE-dependent transcription in response to CD3/CD28 or to NIK. Our results support that NIK may be controlling CD28RE-dependent transcription and T cell activation by modulating c-Rel phosphorylation of the TAD. This leads to more efficient transactivation of genes which are dependent on CD28RE sites where c-Rel binds such as the IL-2 promoter.
Nontypeable Hemophilus influenzae (NTHi) is an important human pathogen in both children and adults. In children, it causes otitis media, the most common childhood
infection and the leading cause of conductive hearing loss in the United States. In adults, it causes lower respiratory tract
infections in the setting of chronic obstructive pulmonary disease, the fourth leading cause of death in the United States.
The molecular mechanisms underlying the pathogenesis of NTHi-induced infections remain undefined, but they may involve activation
of NF-κB, a transcriptional activator of multiple host defense genes involved in immune and inflammatory responses. Here,
we show that NTHi strongly activates NF-κB in human epithelial cells via two distinct signaling pathways, NF-κB translocation-dependent
and -independent pathways. The NF-κB translocation-dependent pathway involves activation of NF-κB inducing kinase (NIK)–IKKα/β
complex leading to IκBα phosphorylation and degradation, whereas the NF-κB translocation-independent pathway involves activation
of MKK3/6–p38 mitogen-activated protein (MAP) kinase pathway. Bifurcation of NTHi-induced NIK–IKKα/β-IκBα and MKK3/6–p38 MAP
kinase pathways may occur at transforming growth factor-β activated kinase 1 (TAK1). Furthermore, we show that toll-like receptor
2 (TLR2) is required for NTHi-induced NF-κB activation. In addition, several key inflammatory mediators including IL-1β, IL-8,
and tumor necrosis factor-α are up-regulated by NTHi. Finally, P6, a 16-kDa lipoprotein highly conserved in the outer membrane
of all NTHi and H. influenzae type b strains, appears to also activate NF-κB via similar signaling pathways. Taken together, our results demonstrate that
NTHi activates NF-κB via TLR2–TAK1-dependent NIK–IKKα/β-IκBα and MKK3/6–p38 MAP kinase signaling pathways. These studies may
bring new insights into molecular pathogenesis of NTHi-induced infections and open up new therapeutic targets for these diseases.
NF-κB is activated by various stimuli including inflammatory cytokines and stresses. A key step in the activation of NF-κB
is the phosphorylation of its inhibitors, IκBs, by an IκB kinase (IKK) complex. Recently, two closely related kinases, designated
IKKα and IKKβ, have been identified to be the components of the IKK complex that phosphorylate critical serine residues of
IκBs for degradation. A previously identified NF-κB-inducing kinase (NIK), which mediates NF-κB activation by TNFα and IL-1,
has been demonstrated to activate IKKα. Previous studies showed that mitogen-activated protein kinase/ERK kinase kinase-1
(MEKK1), which constitutes the c-Jun N-terminal kinase/stress-activated protein kinase pathway, also activates NF-κB by an
undefined mechanism. Here, we show that overexpression of MEKK1 preferentially stimulates the kinase activity of IKKβ, which
resulted in phosphorylation of IκBs. Moreover, a catalytically inactive mutant of IKKβ blocked the MEKK1-induced NF-κB activation.
By contrast, overexpression of NIK stimulates kinase activities of both IKKα and IKKβ comparably, suggesting a qualitative
difference between NIK- and MEKK1-mediated NF-κB activation pathways. Collectively, these results indicate that NIK and MEKK1
independently activate the IKK complex and that the kinase activities of IKKα and IKKβ are differentially regulated by two
upstream kinases, NIK and MEKK1, which are responsive to distinct stimuli.
The multiplicity of Notch receptors raises the question of the contribution of specific isoforms to T-cell development. Notch3 is expressed in CD4-8- thymocytes and is down-regulated across the CD4-8- to CD4+8+ transition, controlled by pre-T-cell receptor signaling. To determine the effects of Notch3 on thymocyte development, transgenic mice were generated, expressing lck promoter-driven intracellular Notch3. Thymuses of young transgenics showed an increased number of thymocytes, particularly late CD4-8- cells, a failure to down-regulate CD25 in post-CD4-8- subsets and sustained activity of NF-B. Subsequently, aggressive multicentric T-cell lymphomas developed with high penetrance. Tumors sustained characteristics of immature thymocytes, including expression of CD25, pT and activated NF-B via IKK-dependent degradation of IB and enhancement of NF-B-dependent anti-apoptotic and proliferative pathways. Together, these data identify activated Notch3 as a link between signals leading to NF-B activation and T-cell tumorigenesis. The phenotypes of pre-malignant thymocytes and of lymphomas indicate a novel and particular role for Notch3 in co-ordinating growth and differentiation of thymocytes, across the pre-T/T cell transition, consistent with the normal expression pattern of Notch3.
Cellular inhibitor of apoptosis (cIAP) proteins, cIAP1 and cIAP2, are important regulators of TNF superfamily signaling and
are amplified in a number of tumor types. They are targeted by IAP antagonist compounds that are undergoing clinical trials.
IAP antagonist compounds trigger cIAP auto-ubiquitylation and degradation. The TNFSF member TWEAK induces lysosomal degradation
of TRAF2 and cIAPs, leading to elevated NIK levels and activation of non-canonical NF-κB. To investigate the role of the ubiquitin
ligase RING domain of cIAP1 in these pathways, we used cIAP deleted cells reconstituted with cIAP1 point mutants designed
to interfere with the ability of the RING to dimerize or to interact with E2 enzymes. We show that RING dimerization and E2
binding are required for IAP antagonists to induce cIAP1 degradation and to protect cells from TNF induced cell death. The
RING functions of cIAP1 are required for full TNF induced activation of NF-κB, however delayed activation of NF-κB still occurs
in cIAP1 and 2 double knock-out cells. The RING functions of cIAP1 are also required to prevent constitutive activation of
non-canonical NF-κB by targeting NIK for proteasomal degradation. However, in cIAP DKO cells TWEAK was still able to increase
NIK levels demonstrating that NIK can be regulated by cIAP independent pathways. Finally we show that, unlike IAP antagonists,
TWEAK was able to induce degradation of cIAP1 RING mutants. These results emphasize the critical importance of the RING of
cIAP1 in many signaling scenarios, but also demonstrate that in some pathways RING functions are not required
CD27 is a member of the tumor necrosis factor (TNF) receptor superfamily and is expressed on T, B, and NK cells. The signal
via CD27 plays pivotal roles in T-T and T-B cell interactions. Here we demonstrate that overexpression of CD27 activates NF-κB
and stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK). Deletion analysis of the cytoplasmic domain of CD27
revealed that the C-terminal PIQEDYR motif was indispensable for both NF-κB and SAPK/JNK activation and was also required
for the interaction with TNF receptor-associated factor (TRAF) 2 and TRAF5, both of which have been implicated in NF-κB activation
by members of the TNF-R superfamily. Co-transfection of a dominant negative TRAF2 or TRAF5 blocked NF-κB and SAPK/JNK activation
induced by CD27. Recently, a TRAF2-interacting kinase has been identified, termed NF-κB-inducing kinase (NIK). A kinase-inactive
mutant NIK blocked CD27-, TRAF2-, and TRAF5-mediated NF-κB and SAPK/JNK activation. These results indicate that TRAF2 and
TRAF5 are involved in NF-κB and SAPK/JNK activation by CD27, and NIK is a common downstream kinase of TRAF2 and TRAF5 for
NF-κB and SAPK/JNK activation.
Previous evidence suggested that NF-kappaB-inducing kinase (NIK) might regulate IL-2 synthesis. However, the molecular mechanism is not understood. In this study, we show that NIK is involved in CD3 plus CD28 activation of IL-2 transcription. Splenic T cells from aly/aly mice (that have a defective NIK protein) have a severe impairment in IL-2 and GM-CSF but not TNF secretion in response to CD3/CD28. This effect takes place at the transcriptional level as overexpression of alyNIK inhibits IL-2 promoter transcription. NIK activates the CD28 responsive element (CD28RE) of the IL-2 promoter and strongly synergizes with c-Rel in this activity. We found that NIK interacts with the N-terminal domain of c-Rel, mapping this interaction to aa 771-947 of NIK. Moreover, NIK phosphorylates the c-Rel C-terminal transactivation domain (TAD) and induces Gal4-c-Rel-transactivating activity. Anti-CD28 activated Gal4-c-Rel transactivation activity, and this effect was inhibited by a NIK-defective mutant. Deletion studies mapped the region of c-Rel responsive to NIK in aa 456-540. Mutation of several serines, including Ser471, in the TAD of c-Rel abrogated the NIK-enhancing activity of its transactivating activity. Interestingly, a Jurkat mutant cell line that expresses one of the mutations of c-Rel (Ser471Asn) has a severe defect in IL-2 and CD28RE-dependent transcription in response to CD3/CD28 or to NIK. Our results support that NIK may be controlling CD28RE-dependent transcription and T cell activation by modulating c-Rel phosphorylation of the TAD. This leads to more efficient transactivation of genes which are dependent on CD28RE sites where c-Rel binds such as the IL-2 promoter.
IL-17-secreting CD4(+) T cells are critically involved in inflammatory immune responses. Development of these cells is promoted in vivo and in vitro by IL-23 or TGFbeta1 plus IL-6. Despite growing interest in this inflammatory Th subset, little is known about the transcription factors that are required for their development. We demonstrate that Stat3 is required for programming the TGFbeta1 plus IL-6 and IL-23-stimulated IL-17-secreting phenotype, as well as for RORgammat expression in TGFbeta1 plus IL-6-primed cells. Moreover, retroviral transduction of a constitutively active Stat3 into differentiating T cell cultures enhances IL-17 production from these cells. We further show that Stat4 is partially required for the development of IL-23-, but not TGFbeta1 plus IL-6-primed IL-17-secreting cells, and is absolutely required for IL-17 production in response to IL-23 plus IL-18. The requirements for Stat3 and Stat4 in the development of these IL-17-secreting subsets reveal additional mechanisms in Th cell fate decisions during the generation of proinflammatory cell types.
Activation of the transcription factor NF-kappaB by inflammatory cytokines involves the successive action of NF-kappaB-inducing kinase (NIK) and two IkappaB kinases, IKK-alpha and IKK-beta. Here we show that NIK preferentially phosphorylates IKK-alpha over IKK-beta, leading to the activation of IKK-alpha kinase activity. This phosphorylation of IKK-alpha occurs specifically on Ser-176 in the activation loop between kinase subdomains VII and VIII. A mutant form of IKK-alpha containing alanine at residue 176 cannot be phosphorylated or activated by NIK and acts as a dominant negative inhibitor of interleukin 1- and tumor necrosis factor-induced NF-kappaB activation. Conversely, a mutant form of IKK-alpha containing glutamic acid at residue 176 is constitutively active. Thus, the phosphorylation of IKK-alpha on Ser-176 by NIK may be required for cytokine-mediated NF-kappaB activation.
Cellular inhibitor of apoptosis (cIAP) proteins, cIAP1 and cIAP2, are important regulators of tumor necrosis factor (TNF) superfamily (SF) signaling and are amplified in a number of tumor types. They are targeted by IAP antagonist compounds that are undergoing clinical trials. IAP antagonist compounds trigger cIAP autoubiquitylation and degradation. The TNFSF member TWEAK induces lysosomal degradation of TRAF2 and cIAPs, leading to elevated NIK levels and activation of non-canonical NF-kappaB. To investigate the role of the ubiquitin ligase RING domain of cIAP1 in these pathways, we used cIAP-deleted cells reconstituted with cIAP1 point mutants designed to interfere with the ability of the RING to dimerize or to interact with E2 enzymes. We show that RING dimerization and E2 binding are required for IAP antagonists to induce cIAP1 degradation and protect cells from TNF-induced cell death. The RING functions of cIAP1 are required for full TNF-induced activation of NF-kappaB, however, delayed activation of NF-kappaB still occurs in cIAP1 and -2 double knock-out cells. The RING functions of cIAP1 are also required to prevent constitutive activation of non-canonical NF-kappaB by targeting NIK for proteasomal degradation. However, in cIAP double knock-out cells TWEAK was still able to increase NIK levels demonstrating that NIK can be regulated by cIAP-independent pathways. Finally we show that, unlike IAP antagonists, TWEAK was able to induce degradation of cIAP1 RING mutants. These results emphasize the critical importance of the RING of cIAP1 in many signaling scenarios, but also demonstrate that in some pathways RING functions are not required.
NF-kappaB transcription factors are key regulators of cellular proliferation and frequently contribute to oncogenesis. The herpesviral oncoprotein Tio, which promotes growth transformation of human T cells in a recombinant herpesvirus saimiri background, potently induces canonical NF-kappaB signaling through membrane recruitment of the ubiquitin ligase tumor necrosis factor receptor-associated factor 6 (TRAF6). Here, we show that, in addition to Tio-TRAF6 interaction, the Tio-induced canonical NF-kappaB signal requires the presence of the regulatory subunit of the inhibitor of kappaB kinase (IKK) complex, NF-kappaB essential modulator (NEMO), and the activity of its key kinase, IKKbeta, to up-regulate expression of endogenous cellular inhibitor of apoptosis 2 (cIAP2) and interleukin 8 (IL-8) proteins. Dependent on TRAF6 and NEMO, Tio enhances the expression of the noncanonical NF-kappaB proteins, p100 and RelB. Independent of TRAF6 and NEMO, Tio mediates stabilization of the noncanonical kinase, NF-kappaB-inducing kinase (NIK). Concomitantly, Tio induces efficient processing of the p100 precursor molecule to its active form, p52, as well as DNA binding of nuclear p52 and RelB. In human T cells transformed by infection with a Tio-recombinant virus, sustained expression of p100, RelB, and cIAP2 depends on IKKbeta activity, yet processing to p52 remains largely unaffected by IKKbeta inhibition. However, long term inhibition of IKKbeta disrupts the continuous growth of the transformed cells and induces cell death. Hence, the Tio oncoprotein triggers noncanonical NF-kappaB signaling through NEMO-dependent up-regulation of p100 precursor and RelB, as well as through NEMO-independent generation of p52 effector.
The lymphotoxin-beta receptor (LTbetaR) activates the NF-kappaB2 transcription factors, p100 and RelB, by regulating the NF-kappaB-inducing kinase (NIK). Constitutive proteosomal degradation of NIK limits NF-kappaB activation in unstimulated cells by the ubiquitin:NIK E3 ligase comprised of subunits TNFR-associated factors (TRAF)3, TRAF2, and cellular inhibitor of apoptosis (cIAP). However, the mechanism releasing NIK from constitutive degradation remains unclear. We found that insertion of a charge-repulsion mutation in the receptor-binding crevice of TRAF3 ablated binding of both LTbetaR and NIK suggesting a common recognition site. A homologous mutation in TRAF2 inhibited cIAP interaction and blocked NIK degradation. Furthermore, the recruitment of TRAF3 and TRAF2 to the ligated LTbetaR competitively displaced NIK from TRAF3. Ligated LTbetaR complexed with TRAF3 and TRAF2 redirected the specificity of the ubiquitin ligase reaction to polyubiquitinate TRAF3 and TRAF2, leading to their proteosomal degradation. Stimulus-dependent degradation of TRAF3 required the RING domain of TRAF2, but not of TRAF3, implicating TRAF2 as a key E3 ligase in TRAF turnover. The combined action of competitive displacement of NIK and TRAF degradation halted NIK turnover, and promoted its association with IKKalpha and signal transmission. These results indicate the LTbetaR modifies the ubiquitin:NIK E3 ligase, and also acts as an allosteric regulator of the ubiquitin:TRAF E3 ligase.
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.
B-cell CLL/lymphoma 10 (BCL10), the caspase recruitment domain (CARD)-containing protein involved in the etiology of the mucosa-associated lymphoid tissue (MALT) lymphomas, has been implicated in inflammatory processes in epithelial cells, as well as in immune cells. Experiments in this report indicate that BCL10 is required for activation of nuclear factor (NF)-kappaB by both canonical and noncanonical pathways, following stimulation by the sulfated polysaccharide carrageenan (CGN). In wild type and IkappaB-kinase (IKK)alpha(-/-) mouse embryonic fibroblasts, increases in phospho-IkappaBalpha, nuclear NF-kappaB p65 (RelA) and p50, and KC, the mouse analog of human interleukin-8, were markedly reduced by silencing BCL10 or by exposure to the free radical scavenger Tempol. In IKKbeta(-/-) cells, BCL10 silencing, but not Tempol, reduced the CGN-induced increases in KC, phospho-NF-kappaB-inducing kinase (NIK), cytoplasmic NF-kappaB p100, and nuclear NF-kappaB p52 and RelB, suggesting a BCL10 requirement for activation of the noncanonical pathway. In NCM460 cells, derived from normal, human colonic epithelium, the CGN-induced increases in NF-kappaB family members, p65, p50, p52, and RelB, were inhibited by BCL10 silencing. Although enzyme-linked immunosorbent assay and confocal images demonstrated no change in total NIK following CGN, increases in phospho-NIK in the wild type, IKKbeta(-/-) and IKKalpha(-/-) cells were inhibited by silencing BCL10. These findings indicate an upstream signaling role for BCL10, in addition to its effects on IKKgamma, the regulatory component of the IKK signalosome, and a requirement for BCL10 in both canonical and noncanonical pathways of NF-kappaB activation. Also, the commonly used food additive carrageenan can be added to the short list of known activators of both pathways.
Bone undergoes a continuous cycle of renewal, and osteoclasts--the cells responsible for bone resorption--play a pivotal role in bone homeostasis. This resorption is largely mediated by inflammatory cytokines such as TNF-alpha. In this issue of the JCI, Yao et al. demonstrate that the NF-kappaB precursor protein NF-kappaB2 (p100) acts as a negative regulator of osteoclastogenesis (see the related article beginning on page 3024). TNF-alpha induced a sustained accumulation of p100 in osteoclast precursors, and TNF-alpha-induced osteoclast formation was markedly increased in Nfkb2-/- mice. They also found that TNF receptor-associated factor 3 (TRAF3) is involved in the posttranslational regulation of p100 expression. These results suggest that blockade of the processing of p100 is a novel strategy to treat TNF-alpha-related bone diseases such as RA.
TNF and RANKL mediate bone destruction in common bone diseases, including osteoarthritis and RA. They activate NF-kappaB canonical signaling directly in osteoclast precursors (OCPs) to induce osteoclast formation in vitro. However, unlike RANKL, TNF does not activate the alternative NF-kappaB pathway efficiently to process the IkappaB protein NF-kappaB p100 to NF-kappaB p52, nor does it appear to induce osteoclast formation in vivo in the absence of RANKL. Here, we show that TNF limits RANKL- and TNF-induced osteoclast formation in vitro and in vivo by increasing NF-kappaB p100 protein accumulation in OCPs. In contrast, TNF induced robust osteoclast formation in vivo in mice lacking RANKL or RANK when the mice also lacked NF-kappaB p100, and TNF-Tg mice lacking NF-kappaB p100 had more severe joint erosion and inflammation than did TNF-Tg littermates. TNF, but not RANKL, increased OCP expression of TNF receptor-associated factor 3 (TRAF3), an adapter protein that regulates NF-kappaB p100 levels in B cells. TRAF3 siRNA prevented TNF-induced NF-kappaB p100 accumulation and inhibition of osteoclastogenesis. These findings suggest that upregulation of TRAF3 or NF-kappaB p100 expression or inhibition of NF-kappaB p100 degradation in OCPs could limit bone destruction and inflammation-induced bone loss in common bone diseases.
Pancreatic cancer has one of the poorest prognoses among human neoplasms. Constitutive activation of NF-kappaB is frequently observed in pancreatic cancer cells and is involved in their malignancy. However, little is known about the molecular mechanism of this constitutive NF-kappaB activation. Here, we show that the alternative pathway is constitutively activated and NF-kappaB-inducing kinase (NIK), a mediator of the alternative pathway, is significantly expressed in pancreatic cancer cells. siRNA-mediated silencing of NIK expression followed by subcellular fractionation revealed that NIK is constitutively involved in the processing of p100 and nuclear transport of p52 and RelB in pancreatic cancer cells. In addition, NIK silencing significantly suppressed proliferation of pancreatic cancer cells. These results clearly indicate that NIK is involved in the constitutive activation of the alternative pathway and controls cell proliferation in pancreatic cancer cells. Therefore, NIK might be a novel target for the treatment of pancreatic cancer.
Th17 cells play an important role in mediating autoimmune diseases, but the molecular mechanism underlying Th17 differentiation is incompletely understood. We show here that NF-kappaB-inducing kinase (NIK), which is known to regulate B-cell maturation and lymphoid organogenesis, is important for the induction of Th17 cells. NIK-deficient naive CD4 T cells are attenuated in the differentiation to Th17 cells, although they are competent in committing to the other effector lineages. Consistently, NIK knockout mice are resistant to experimental autoimmune encephalomyelitis, a disease model that involves the function of Th17 cells. This phenotype was also detected in Rag2 knockout mice reconstituted with NIK-deficient T cells, confirming a T-cell intrinsic defect. We further show that NIK mediates synergistic activation of STAT3 by T-cell receptor and IL-6 receptor signals. NIK deficiency attenuates activation of STAT3 and induction of STAT3 target genes involved in Th17-commitment program. These findings establish NIK as an important signaling factor that regulates Th17 differentiation and experimental autoimmune encephalitis induction.
Tumor necrosis factor (TNF) receptor-associated factor 3 (TRAF3) regulates both innate and adaptive immunity by modulating signaling by Toll-like receptors (TLR) and TNF receptors. TRAF3 was recently identified as a tumor suppressor in human multiple myeloma, suggesting a prominent role in plasma cell homeostasis. We have generated transgenic mice expressing human TRAF3 in lymphocytes. These mice are normal at birth, but they develop over time plasmacytosis and hypergammaglobulinemia, as well as systemic inflammation and tertiary lymphoid organ formation. The analysis of the humoral responses of the TRAF3 mice demonstrated increased responses to T-dependent and T-independent antigens with increased production of antigen-specific immunoglobulin Gs (IgGs) compared with wild-type mice. Furthermore, TLR-mediated IgG production is also increased in TRAF3 B cells. In addition, TRAF3 mice develop autoimmunity and are predisposed to cancer, particularly squamous cell carcinomas of the tongue ( approximately 50% incidence) and salivary gland tumors. In summary, TRAF3 renders B cells hyperreactive to antigens and TLR agonists, promoting autoimmunity, inflammation, and cancer, hereby providing a new model for studying de novo carcinogenesis promoted by B cell-initiated chronic inflammation.
Recent studies suggest that nuclear factor kappaB-inducing kinase (NIK) is suppressed through constitutive proteasome-mediated degradation regulated by TRAF2, TRAF3 and cIAP1 or cIAP2. Here we demonstrated that the degradation of NIK occurs upon assembly of a regulatory complex through TRAF3 recruitment of NIK and TRAF2 recruitment of cIAP1 and cIAP2. In contrast to TRAF2 and TRAF3, cIAP1 and cIAP2 seem to play redundant roles in the degradation of NIK, as inhibition of both cIAPs was required for noncanonical NF-kappaB activation and increased survival and proliferation of primary B lymphocytes. Furthermore, the lethality of TRAF3 deficiency in mice could be rescued by a single NIK gene, highlighting the importance of tightly regulated NIK.
The adaptor and signaling proteins TRAF2, TRAF3, cIAP1 and cIAP2 may inhibit alternative nuclear factor-kappaB (NF-kappaB) signaling in resting cells by targeting NF-kappaB-inducing kinase (NIK) for ubiquitin-dependent degradation, thus preventing processing of the NF-kappaB2 precursor protein p100 to release p52. However, the respective functions of TRAF2 and TRAF3 in NIK degradation and activation of alternative NF-kappaB signaling have remained elusive. We now show that CD40 or BAFF receptor activation result in TRAF3 degradation in a cIAP1-cIAP2- and TRAF2-dependent way owing to enhanced cIAP1, cIAP2 TRAF3-directed ubiquitin ligase activity. Receptor-induced activation of cIAP1 and cIAP2 correlated with their K63-linked ubiquitination by TRAF2. Degradation of TRAF3 prevented association of NIK with the cIAP1-cIAP2-TRAF2 ubiquitin ligase complex, which resulted in NIK stabilization and NF-kappaB2-p100 processing. Constitutive activation of this pathway causes perinatal lethality and lymphoid defects.
BAFF-R-dependent activation of the alternative NF-κB pathway plays an essential role in mature B cell survival. Mutations leading to overexpression of NIK and deletion of the TRAF3 gene are implicated in human multiple myeloma. We show that overexpression of NIK in mouse B lymphocytes amplifies alternative NF-κB activation and peripheral B cell numbers in a BAFF-R-dependent manner, whereas uncoupling NIK from TRAF3-mediated control causes maximal p100 processing and dramatic hyperplasia of BAFF-R-independent B cells. NIK controls alternative NF-κB signaling by increasing the protein levels of its negative regulator TRAF3 in a dose-dependent fashion. This mechanism keeps NIK protein levels below detection even when they cause B cell hyperplasia, so that contributions of NIK to B cell pathologies can easily be overlooked.
• IκB kinase
• NF-κB
• Hyperplasia
• p100 processing
• knockin
The alymphoplasia (aly) mutation of mice causes the systemic absence of lymph nodes, Peyer's patches and well-defined lymphoid follicles in the
spleen. We found that antibody responses are elicited, albelt weakly, to either T cell-dependent or T cell-Independent antigen
by aly/aly mutants. However, isotype switching was defective. The T cell-dependent immune response was not elicited in splenectomized
aly/aly mice. Neither hypermutation nor germinal center formation was observed in aly/aly mice. These results suggest that T–B collaboration requires either lymph nodes or spleen, and that hypermutation and affinity
maturation depend on germinal center formation.
Several members of the tumour-necrosis/nerve-growth factor (TNF/NGF) receptor family activate the transcription factor NF-kappaB through a common adaptor protein, Traf2 (refs 1-5), whereas the interleukin 1 type-I receptor activates NF-kappaB independently of Traf2 (ref. 4). We have now cloned a new protein kinase, NIK, which binds to Traf2 and stimulates NF-kappaB activity. This kinase shares sequence similarity with several MAPKK kinases. Expression in cells of kinase-deficient NIK mutants fails to stimulate NF-kappaB and blocks its induction by TNF, by either of the two TNF receptors or by the receptor CD95 (Fas/Apo-1), and by TRADD, RIP and MORT1/FADD, which are adaptor proteins that bind to these receptors. It also blocked NF-kappaB induction by interleukin-1. Our findings indicate that NIK participates in an NF-kappaB-inducing signalling cascade common to receptors of the TNF/NGF family and to the interleukin-1 type-I receptor.
The role of the spleen and of other organized secondary lymphoid organs for the induction of protective antiviral immune responses was evaluated in orphan homeobox gene 11 knockout mice (Hox11(-/-)) lacking the spleen, and in homozygous alymphoplastic mutant mice (aly/aly) possessing a structurally altered spleen but lacking lymph nodes and Peyer's patches. Absence of the spleen had no major effects on the immune response, other than delaying the antibody response by 1-2 d. In aly/aly mice, the thymus-independent IgM response against vesicular stomatitis virus (VSV) was delayed and reduced, whereas the T-dependent switch to the protective IgG was absent. Therefore, aly/aly mice were highly susceptible to VSV infection. Since aly/aly spleen cells yielded neutralizing IgM and IgG after adoptive transfer into recipients with normally structured secondary lymphoid organs, these data suggest that the structural defect was mainly responsible for inefficient T-B cooperation. Although aly/aly mice generated detectable, but reduced, CTL responses after infection with vaccinia virus (VV) and lymphocytic choriomeningitis virus (LCMV), the elimination of these viruses was either delayed (VV) or virtually impossible (LCMV); irrespective of the dose or the route of infection, aly/aly mice developed life-long LCMV persistence. These results document the critical role of organized secondary lymphoid organs in the induction of naive T and B cells. These structures also provide the basis for cooperative interactions between antigen-presenting cells, T cells, and B cells, which are a prerequisite for recovery from primary virus infections via skin or via blood.
TNF-induced activation of the transcription factor NF-kappaB and the c-jun N-terminal kinase (JNK/SAPK) requires TNF receptor-associated factor 2 (TRAF2). The NF-kappaB-inducing kinase (NIK) associates with TRAF2 and mediates TNF activation of NF-kappaB. Herein we show that NIK interacts with additional members of the TRAF family and that this interaction requires the conserved "WKI" motif within the TRAF domain. We also investigated the role of NIK in JNK activation by TNF. Whereas overexpression of NIK potently induced NF-kappaB activation, it failed to stimulate JNK activation. A kinase-inactive mutant of NIK was a dominant negative inhibitor of NF-kappaB activation but did not suppress TNF- or TRAF2-induced JNK activation. Thus, TRAF2 is the bifurcation point of two kinase cascades leading to activation of NF-kappaB and JNK, respectively.
NF-kappaB corresponds to an inducible eukaryotic transcription factor complex that is negatively regulated in resting cells by its physical assembly with a family of cytoplasmic ankyrin-rich inhibitors termed IkappaB. Stimulation of cells with various proinflammatory cytokines, including tumor necrosis factor alpha (TNF-alpha), induces nuclear NF-kappaB expression. TNF-alpha signaling involves the recruitment of at least three proteins (TRADD, RIP, and TRAF2) to the type 1 TNF-alpha receptor tail, leading to the sequential activation of the downstream NF-kappaB-inducing kinase (NIK) and IkappaB-specific kinases (IKKalpha and IKKbeta). When activated, IKKalpha and IKKbeta directly phosphorylate the two N-terminal regulatory serines within IkappaB alpha, triggering ubiquitination and rapid degradation of this inhibitor in the 26S proteasome. This process liberates the NF-kappaB complex, allowing it to translocate to the nucleus. In studies of NIK, we found that Thr-559 located within the activation loop of its kinase domain regulates NIK action. Alanine substitution of Thr-559 but not other serine or threonine residues within the activation loop abolishes its activity and its ability to phosphorylate and activate IKKalpha. Such a NIK-T559A mutant also dominantly interferes with TNF-alpha induction of NF-kappaB. We also found that ectopically expressed NIK both spontaneously forms oligomers and displays a high level of constitutive activity. Analysis of a series of NIK deletion mutants indicates that multiple subregions of the kinase participate in the formation of these NIK-NIK oligomers. NIK also physically assembles with downstream IKKalpha; however, this interaction is mediated through a discrete C-terminal domain within NIK located between amino acids 735 and 947. When expressed alone, this C-terminal NIK fragment functions as a potent inhibitor of TNF-alpha-mediated induction of NF-kappaB and alone is sufficient to disrupt the physical association of NIK and IKKalpha. Together, these findings provide new insights into the molecular basis for TNF-alpha signaling, suggesting an important role for heterotypic and possibly homotypic interactions of NIK in this response.
IκB kinases (IKKα and IKKβ) are key components of the IKK complex that mediates activation of the transcription factor NF-κB
in response to extracellular stimuli such as inflammatory cytokines, viral and bacterial infection, and UV irradiation. Although
NF-κB-inducing kinase (NIK) interacts with and activates the IKKs, the upstream kinases for the IKKs still remain obscure.
We identified mitogen-activated protein kinase kinase kinase 1 (MEKK1) as an immediate upstream kinase of the IKK complex.
MEKK1 is activated by tumor necrosis factor alpha (TNF-α) and interleukin-1 and can potentiate the stimulatory effect of TNF-α
on IKK and NF-κB activation. The dominant negative mutant of MEKK1, on the other hand, partially blocks activation of IKK
by TNF-α. MEKK1 interacts with and stimulates the activities of both IKKα and IKKβ in transfected HeLa and COS-1 cells and
directly phosphorylates the IKKs in vitro. Furthermore, MEKK1 appears to act in parallel to NIK, leading to synergistic activation
of the IKK complex. The formation of the MEKK1-IKK complex versus the NIK-IKK complex may provide a molecular basis for regulation
of the IKK complex by various extracellular signals.
Both lymphotoxin-alpha (LTalpha)-deficient mice and alymphoplasia (aly) mice, a natural mutant strain, manifest a quite similar phenotype: lack of lymph nodes (LN) and Peyer's patches (PP), with disturbed spleen architecture. The mechanisms underlying the defective lymphoid organogenesis in these mice were investigated by generating aggregation chimeras; ex vivo fused morulae were implanted into pseudo-pregnant host females and allowed to develop to term. Chimeric mice between LTalpha-deficient mice and wild-type mice restored LN and PP almost completely, suggesting that LTalpha expressed by circulating bone marrow-derived cells is essential for lymphoid organogenesis as well as for organization of spleen architecture. By contrast, chimeric mice between aly mice and wild-type mice showed only limited restoration of LN and PP. This suggests that the putative aly gene product does not act as a circulating ligand for lymphoid organogenesis, like LTalpha. Rather, abnormal development of lymphoid organs in aly mice seems most likely due to the defective development of the incipient stromal cells of the LN and PP. Supporting this hypothesis, up-regulation of VCAM-1 on aly mouse embryonic fibroblasts by signals through LTbetaR, which is exclusively expressed by nonlymphoid cells, was disturbed. These studies demonstrate that LTalpha and the putative aly gene product together control lymphoid organogenesis with a close mechanistic relationship in their biochemical pathways through governing the distinct cellular compartments, the former acting as a circulating ligand and the latter as a LTbetaR-signaling molecule expressed by the stroma of the lymphoid organs.
The proto-oncogene Cot/Tpl-2 encodes a MAP3K-related serine-threonine kinase. Expression of wild type Cot activates the IκB kinases (IKK) leading to induction of NF-κB. Conversely, expression of kinase-deficient Cot inhibits CD3/CD28 but not TNFα induction of NF-κB. These findings suggest the selective involvement of Cot/Tpl-2 or a closely related kinase in the CD3/CD28 costimulatory pathway leading to induced nuclear expression of NF-κB. In contrast, a kinase-deficient mutant of the NF-κB-inducing kinase (NIK) inhibits both CD3/CD28 and TNFα signaling, indicating that these pathways converge at or prior to the action of NIK. Consistent with such a sequential function of these two kinases, Cot physically assembles with and phosphorylates NIK in vivo.
Processing of the nfκb2 gene product p100 to generate p52 is an important step in NF-κB regulation. We show that this step is negatively regulated by a processing-inhibitory domain (PID) within p100 and positively regulated by the NF-κB-inducing kinase (NIK). While the PID suppresses the constitutive processing of p100, NIK induces p100 processing by stimulating site-specific phosphorylation and ubiquitination of this precursor protein. Further, a natural mutation of the gene encoding NIK in alymphoplasia (aly) mice cripples the function of NIK in p100 processing, causing a severe defect in p52 production. These data suggest that NIK is a specific kinase regulating p100 processing and explain why the aly and nfκb2 knockout mice exhibit similar immune deficiencies.
Pluripotent mesenchymal stem cells in bone marrow differentiate into adipocytes, osteoblasts and other cells. Balanced cytodifferentiation of stem cells is essential for the formation and maintenance of bone marrow; however, the mechanisms that control this balance remain largely unknown. Whereas cytokines such as interleukin-1 (IL-1) and tumour-necrosis factor-α (TNF-α) inhibit adipogenesis, the ligand-induced transcription factor peroxisome proliferator-activated receptor-γ (PPAR-γ), is a key inducer of adipogenesis. Therefore, regulatory coupling between cytokine- and PPAR-γ-mediated signals might occur during adipogenesis. Here we show that the ligand-induced transactivation function of PPAR-γ is suppressed by IL-1 and TNF-α, and that this suppression is mediated through NF-κB activated by the TAK1/TAB1/NF-κB-inducing kinase (NIK) cascade, a downstream cascade associated with IL-1 and TNF-α signalling. Unlike suppression of the PPAR-γ transactivation function by mitogen-activated protein kinase-induced growth factor signalling through phosphorylation of the A/B domain, NF-κB blocks PPAR-γ binding to DNA by forming a complex with PPAR-γ and its AF-1-specific co-activator PGC-2. Our results suggest that expression of IL-1 and TNF-α in bone marrow may alter the fate of pluripotent mesenchymal stem cells, directing cellular differentiation towards osteoblasts rather than adipocytes by suppressing PPAR-γ function through NF-κB activated by the TAK1/TAB1/NIK cascade.
We have found a new spontaneous autosomal recessive mutation in mice that causes a systemic absence of lymph nodes and Peyer's patches. The name “alymphoplasia”, with the gene symbol “uly”, is proposed for this mutant. The spleen of alylay mice is devoid of well-defined lymphoid follicles, and the thymus does not show a clear cortical-medullary distinction. The mutant homozygotes are deficient in both humoral and cell-mediated immune functions, and are highly susceptible to infections. They have a reduced level of IgM and severely depressed levels of IgG and IgA in their sera, and do not reject allogeneic skin grafts. However, they have mature T and B cells as determined from their cell surface antigens. The results of bone marrow transplantation experiments suggest a mesenchymal disorder as a possible cause of the lack of lymph nodes and of immunodeficiency in the aly mouse. The aly mutant mouse may be a useful animal model of primary immunodeficiency, as are the nu (nude) and scid (severe combined immunodeficiency) mice.
NF-κB-inducing kinase (NIK)-mediated IKKα phosphorylation activates the alternative NF-κB pathway, which is characterized by nuclear translocation of p52:RelB heterodimers. This alternative pathway is initiated by a select few receptors, including LT-βR, BAFF-R, and CD40. Although NIK, IKKα, and p52 are all critical regulators of LT-βR signaling in stromal cells during humoral immune responses, lymphocytes require NIK, but not p52, for optimal Ig production. This disparity suggests that NIK possesses critical cell-type-specific functions that do not depend on NF-κB. Here we use mice bearing targeted mutations of the IKKα activation loop Ser176/180 (IKKαAA) to address the B cell-intrinsic functions of NIK–IKKα signaling in vivo. We find that IKKαAA B cells mount normal primary antibody responses but do not enter germinal centers. This defect likely derives from ineffective early T–B cell collaboration and leads to impaired generation of humoral memory and relatively short-lived, low-affinity antibody production. Our findings contrast with those obtained by using p52−/− B cells, which mount normal Ig responses, and alymphoplasia (NIK mutant) B cells, which produce very little primary Ig. Thus, the NIK–IKKα–p52 axis is not as linear and exclusive as previous studies suggest, and IKKα possesses critical NF-κB-independent functions in B cells.
• germinal center
To examine the role of the TNF-R superfamily signaling protein TRAF2 in mature B cell development and NF-kappaB activation, conditionally TRAF2-deficient mice were produced. B cells lacking TRAF2 expression in these mice possessed a selective survival advantage, accumulated in the lymph nodes and splenic marginal zone, were larger in size, and expressed increased levels of CD21/35. These TRAF2-deficient B cells could not proliferate or activate the canonical NF-kappaB pathway in response to CD40 ligation. By contrast, noncanonical NF-kappaB activation was constitutively hyperactive, with TRAF2-deficient B cells exhibiting close to maximal processing of NF-kappaB2 from p100 to p52 and high levels of constitutive p52 and RelB DNA binding activity. These findings establish TRAF2 as a multifunctional regulator of NF-kappaB activation that mediates activation of the canonical pathway but acts as a negative regulator of the noncanonical pathway. This dual functionality explains the contrasting roles of TRAF2 in B cell maturation and activation.
The present study investigates roles for NF-κB inducing kinase (NIK) in constitutive NF-κB activation in lung cancer cells. A wealth of evidence showed that NF-κB is often constitutively activated in human cancer cells, including non-small cell lung cancer tissue specimens and cell lines, which may lead to deregulated apoptosis and enhanced resistance of tumor cells to chemotherapy. However, the mechanisms of NF-κB activation in lung cancer cells remain largely unknown. We report here that NF-κB inducing kinase (NIK) is aberrantly expressed at the pre-translational level in non-small cell lung cancer (NSCLC) cell lines. Depletion of NIK by RNA interference remarkably diminished nuclear NF-κB DNA binding activity and reporter gene expression. NIK depletion induced apoptosis in A549 cells, reduced the matrix metalloproteinase 9 (MMP-9) and survivin mRNA expression and affected efficiency of anchorage-independent H1299 cell growth, suggesting a role for NIK in the manifestation of oncogenic phenotype. These results indicate that NIK plays a key role in constitutive NF-κB activation in NSCLC cells and implicate NIK as a molecular target for lung cancer therapy.
Mutations involving the nuclear factor-kappaB (NF-kappaB) pathway are present in at least 17% of multiple myeloma (MM) tumors and 40% of MM cell lines (MMCLs). These mutations, which are apparent progression events, enable MM tumors to become less dependent on bone marrow signals that activate NF-kappaB. Studies on a panel of 51 MMCLs provide some clarification of the mechanisms through which these mutations act and the significance of classical versus alternative activation of NF-kappaB. First, only one mutation (NFKB2) selectively activates the alternative pathway, whereas several mutations (CYLD, NFKB1, and TACI) selectively activate the classical pathway. However, most mutations affecting NF-kappaB-inducing kinase (NIK) levels (NIK, TRAF2, TRAF3, cIAP1&2, and CD40) activate the alternative but often both pathways. Second, we confirm the critical role of TRAF2 in regulating NIK degradation, whereas TRAF3 enhances but is not essential for cIAP1/2-mediated proteasomal degradation of NIK in MM. Third, using transfection to selectively activate the classical or alternative NF-kappaB pathways, we show virtually identical changes in gene expression in one MMCL, whereas the changes are similar albeit nonidentical in a second MMCL. Our results suggest that MM tumors can achieve increased autonomy from the bone marrow microenvironment by mutations that activate either NF-kappaB pathway.
Constitutive nuclear factor (NF)-kappaB activation is thought to be involved in survival, invasion, and metastasis in various types of cancers. However, neither the subtypes of breast cancer cells with constitutive NF-kappaB activation nor the molecular mechanisms leading to its constitutive activation have been clearly defined. Here, we quantitatively analyzed basal NF-kappaB activity in 35 human breast cancer cell lines and found that most of the cell lines with high constitutive NF-kappaB activation were categorized in the estrogen receptor negative, progesterone receptor negative, ERBB2 negative basal-like subtype, which is the most malignant form of breast cancer. Inhibition of constitutive NF-kappaB activation by expression of IkappaBalpha super-repressor reduced proliferation of the basal-like subtype cell lines. Expression levels of mRNA encoding NF-kappaB-inducing kinase (NIK) were elevated in several breast cancer cell lines, and RNA interference-mediated knockdown of NIK reduced NF-kappaB activation in a subset of the basal-like subtype cell lines with upregulated NIK expression. Taken together, these results suggest that constitutive NF-kappaB activation, partially dependent on NIK, is preferentially involved in proliferation of basal-like subtype breast cancer cells and may be a useful therapeutic target for this subtype of cancer.
Medullary thymic epithelial cells (mTECs) establish T cell self-tolerance through the expression of autoimmune regulator (Aire) and peripheral tissue-specific self-antigens. However, signals underlying mTEC development remain largely unclear. Here, we demonstrate crucial regulation of mTEC development by receptor activator of NF-kappaB (RANK) and CD40 signals. Whereas only RANK signaling was essential for mTEC development during embryogenesis, in postnatal mice, cooperation between CD40 and RANK signals was required for mTEC development to successfully establish the medullary microenvironment. Ligation of RANK or CD40 on fetal thymic stroma in vitro induced mTEC development in a tumor necrosis factor-associated factor 6 (TRAF6)-, NF-kappaB inducing kinase (NIK)-, and IkappaB kinase beta (IKKbeta)-dependent manner. These results show that developmental-stage-dependent cooperation between RANK and CD40 promotes mTEC development, thereby establishing self-tolerance.
Activation of the transcription factor nuclear factor kappa B (NF-kappaB) by inflammatory cytokines requires the successive action of NF-kappaB-inducing kinase (NIK) and IkappaB kinase-alpha (IKK-alpha). A widely expressed protein kinase was identified that is 52 percent identical to IKK-alpha. IkappaB kinase-beta (IKK-beta) activated NF-kappaB when overexpressed and phosphorylated serine residues 32 and 36 of IkappaB-alpha and serines 19 and 23 of IkappaB-beta. The activity of IKK-beta was stimulated by tumor necrosis factor and interleukin-1 treatment. IKK-alpha and IKK-beta formed heterodimers that interacted with NIK. Overexpression of a catalytically inactive form of IKK-beta blocked cytokine-induced NF-kappaB activation. Thus, an active IkappaB kinase complex may require three distinct protein kinases.
Several mitogen-activated protein kinase kinase kinases (MAPKKKs), including NF-kappa B-inducing kinase (NIK), play critical roles in NF-kappa B activation. We isolated cDNA for human TGF-beta activated kinase 1 (TAK1), a member of the MAPKKK family, and evaluated its ability to stimulate NF-kappa B activation. Overexpression of TAK1 together with its activator protein, TAK1 binding protein 1 (TAB1), induced the nuclear translocation of NF-kappa B p50/p65 heterodimer accompanied by the degradation of I kappa B alpha and I kappa B beta, and the expression of kappa B-dependent reporter gene. A dominant negative mutant of NIK did not inhibit TAK1-induced NF-kappa B activation. These results suggest that TAK1 induces NF-kappa B activation through a novel NIK-independent signaling pathway.
Tumor necrosis factor α (TNF-α) binding to the TNF receptor (TNFR) potentially initiates apoptosis and activates the transcription
factor nuclear factor kappa B (NF-κB), which suppresses apoptosis by an unknown mechanism. The activation of NF-κB was found
to block the activation of caspase-8. TRAF1 (TNFR-associated factor 1), TRAF2, and the inhibitor-of-apoptosis (IAP) proteins
c-IAP1 and c-IAP2 were identified as gene targets of NF-κB transcriptional activity. In cells in which NF-κB was inactive,
all of these proteins were required to fully suppress TNF-induced apoptosis, whereas c-IAP1 and c-IAP2 were sufficient to
suppress etoposide-induced apoptosis. Thus, NF-κB activates a group of gene products that function cooperatively at the earliest
checkpoint to suppress TNF-α–mediated apoptosis and that function more distally to suppress genotoxic agent–mediated apoptosis.
The transcription factor NF-kappaB coordinates the activation of numerous genes in response to pathogens and pro-inflammatory cytokines, and is, therefore, vital in the development of acute and chronic inflammatory diseases. NF-kappaB is activated by phsophorylation of its inhibitory subunit, IkappaB-alpha, on serine residues 32 and 36 by cytokine-activated IKB kinases (IKKs); this phosphorylation precedes rapid degradation of IkappaB. IKK-alpha and IKK-beta isozymes are found in large complexes of relative molecular mass 700,000-900,000 (M(r) 70K-90K), but little is known about other components that organize and regulate these complexes. IKK-alpha was independently discovered as a NF-kappaB-inducing kinase (NIK)-associated protein in a yeast two-hybrid screen, and IKK-beta was also identified by homology screening. It is, however, unknown whether NIK is part of the IKK complex. Here we isolate large, interleukin-1-inducible IKK complexes that contain NIK, IKK-alpha, IKK-beta, IkappaB-alpha, NF-kappaB/RelA and a protein of M(r) 150K. This latter component is a new protein, termed IKK-complex-associated protein (IKAP), which can bind NIK and IKKs and assemble them into an active kinase complex. We show that IKAP is a scaffold protein and a regulator for three different kinases involved in pro-inflammatory cytokine signalling.
FIP-3 (14.7K interacting protein) was discovered during a search for cell proteins that could interact with an adenovirus protein (Ad E3-14.7K) that had been shown to prevent tumor necrosis factor (TNF)-alpha-induced cytolysis. FIP-3, which contains leucine zippers and a zinc finger domain, inhibits both basal and induced transcriptional activity of NF-kappaB and causes a late-appearing apoptosis with unique morphologic manifestations. Ad E3-14.7K can partially reverse apoptotic death induced by FIP-3. FIP-3 also was shown to bind to other cell proteins, RIP and NIK, which previously had been described as essential components of TNF-alpha-induced NF-kappaB activation. In addition, FIP-3 inhibited activation of NF-kappaB induced by TNF-alpha, the TNFR-1 receptor, RIP, NIK, and IKKbeta, as well as basal levels of endogenous NF-kappaB in 293 cells. Because the activation of NF-kappaB has been shown to inhibit apoptosis, FIP-3 appears both to activate a cell-death pathway and to inhibit an NF-kappaB-dependent survival mechanism.
The proto-oncogene Cot/Tpl-2 encodes a MAP3K-related serine-threonine kinase. Expression of wild type Cot activates the IkappaB kinases (IKK) leading to induction of NF-kappaB. Conversely, expression of kinase-deficient Cot inhibits CD3/CD28 but not TNF alpha induction of NF-kappaB. These findings suggest the selective involvement of Cot/Tpl-2 or a closely related kinase in the CD3/CD28 costimulatory pathway leading to induced nuclear expression of NF-kappaB. In contrast, a kinase-deficient mutant of the NF-kappaB-inducing kinase (NIK) inhibits both CD3/CD28 and TNF alpha signaling, indicating that these pathways converge at or prior to the action of NIK. Consistent with such a sequential function of these two kinases, Cot physically assembles with and phosphorylates NIK in vivo.
Interleukin-1 (IL-1) is a proinflammatory cytokine that has several effects in the inflammation process. When it binds to its cell-surface receptor, IL-1 initiates a signalling cascade that leads to activation of the transcription factor NF-kappaB and is relayed through the protein TRAF6 and a succession of kinase enzymes, including NF-kappaB-inducing kinase (NIK) and I kappaB kinases (IKKs). However, the molecular mechanism by which NIK is activated is not understood. Here we show that the MAPKK kinase TAK1 acts upstream of NIK in the IL-1-activated signalling pathway and that TAK1 associates with TRAF6 during IL-1 signalling. Stimulation of TAK1 causes activation of NF-kappaB, which is blocked by dominant-negative mutants of NIK, and an inactive TAK1 mutant prevents activation of NF-kappaB that is mediated by IL-1 but not by NIK. Activated TAK1 phosphorylates NIK, which stimulates IKK-alpha activity. Our results indicate that TAK1 links TRAF6 to the NIK-IKK cascade in the IL-1 signalling pathway.
IκB [inhibitor of nuclear factor κB (NF-κB)] kinase (IKK) phosphorylates IκB inhibitory proteins, causing their degradation
and activation of transcription factor NF-κB, a master activator of inflammatory responses. IKK is composed of three subunits—IKKα
and IKKβ, which are highly similar protein kinases, and IKKγ, a regulatory subunit. In mammalian cells, phosphorylation of
two sites at the activation loop of IKKβ was essential for activation of IKK by tumor necrosis factor and interleukin-1. Elimination
of equivalent sites in IKKα, however, did not interfere with IKK activation. Thus, IKKβ, not IKKα, is the target for proinflammatory
stimuli. Once activated, IKKβ autophosphorylated at a carboxyl-terminal serine cluster. Such phosphorylation decreased IKK
activity and may prevent prolonged activation of the inflammatory response.