Overexpression Of NF-κB-Inducing Kinase Research Articles

Non-canonical NF-κB contributes to endothelial pyroptosis and atherogenesis dependent on IRF-1

Cell inflammation and death are closely linked processes contributing to endothelial dysfunction, which plays a critical role in atherogenesis. Activation of the NLRP3 inflammasome causes pyroptosis, the Gasdermin D (GSDMD)-mediated inflammatory cell death. The non-canonical NF-κB pathway has been implicated in inflammation; however, its role in NLRP3 inflammasome-mediated endothelial dysfunction has not been investigated. This study investigated a role for the non-canonical NF-κB pathway in regulating endothelial pyroptosis as it relates to atherogenesis. Immunohistochemistry indicated inflammasome activation in the endothelial cells (EC) of human atherosclerotic arteries. Flow cytometry and Western blot analysis revealed that oxidized low-density lipoprotein (oxLDL) activated the NLRP3 inflammasome, concomitant with the activation of non-canonical NF-κB in primary human aortic EC. Interference of NF-κB inducing kinase (NIK), the key regulator of the non-canonical pathway, significantly attenuated oxLDL- or LPS/ATP-induced NLRP3 inflammasome activation, pyroptosis, IL-1β, and IL-18 secretion. In contrast, overexpression of NIK exacerbated these responses. Chromatin immunoprecipitation revealed that activation of the non-canonical NF-κB pathway upregulated the transcription factor IRF-1 through RelB/p52 binding to its promoter region at -782/-770. In addition to the known target CASP1, RNA sequencing further identified GSDMD as a target gene of IRF-1. IRF-1 but not RelB/p52 interacted with the GSDMD promoter at -526/-515 and the CASP1 promoter at -11/10 to promote the expression and CASP1-mediated activation of GSDMD. Consistent with the observations in cultured endothelium, endothelial-specific deficiency of NIK or IRF-1 attenuated atherosclerosis in high-fat diet-fed Apoe-null mice. These data demonstrate that the non-canonical NF-κB pathway contributes to NLRP3 inflammasome-mediated endothelial pyroptosis and the development of atherosclerosis through GSDMD activation in a manner dependent on IRF-1. Further investigation may facilitate the identification of specific therapeutic targets for atherosclerotic heart diseases.

NF-κB-inducing kinase (NIK) is activated in pancreatic β-cells but does not contribute to the development of diabetes

The transcription factor nuclear factor-κB (NF-κB) has a key role in the pathogenesis of diabetes and its complications. Although activation of the canonical NF-κB pathway in β-cells is generally deleterious, little is known about the role of the non-canonical NF-κB signalling and its main regulator, the NF-κB-inducing kinase (NIK), on pancreatic β-cell survival and function. Previous studies based on models of NIK overexpression in pancreatic islet cells showed that NIK induced either spontaneous β-cell death due to islet inflammation or glucose intolerance during diet-induced obesity (DIO) in mice. Therefore, NIK has been proposed as a potential target for diabetes therapy. However, no clear studies showed whether inhibition of NIK improves diabetes development. Here we show that genetic silencing of NIK in pancreatic β-cells neither modifies diabetes incidence nor inflammatory responses in a mouse model of immune-mediated diabetes. Moreover, NIK silencing in DIO mice did not influence body weight gain, nor glucose metabolism. In vitro studies corroborated the in vivo findings in terms of β-cell survival, function, and downstream gene regulation. Taken together, our data suggest that NIK activation is dispensable for the development of diabetes.

Hepatic NF-κB-Inducing Kinase and Inhibitor of NF-κB Kinase Subunit α Promote Liver Oxidative Stress, Ferroptosis, and Liver Injury.

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.

Targeting NF-κB-Inducing Kinase (NIK) in Immunity, Inflammation, and Cancer.

NF-κB-inducing kinase (NIK), the essential upstream kinase, which regulates activation of the noncanonical NF-κB pathway, has important roles in regulating immunity and inflammation. In addition, NIK is vital for maintaining cellular health through its control of fundamental cellular processes, including differentiation, growth, and cell survival. As such aberrant expression or regulation of NIK is associated with several disease states. For example, loss of NIK leads to severe immune defects, while the overexpression of NIK is observed in inflammatory diseases, metabolic disorders, and the development and progression of cancer. This review discusses recent studies investigating the therapeutic potential of NIK inhibitors in various diseases.

Activation of NF-κB-Inducing Kinase in Islet β Cells Causes β Cell Failure and Diabetes

Islet β cell death has been proved to contribute to diabetes. Studies suggest that the activation of nuclear factor κB (NF-κB)-inducing kinase (NIK) is involved in the β cell dysfunction encountered in obesity. However, the pathological significance of NIK activation in diabetes remains largely unknown. Here, we report that β cell-specific overexpression of NIK (β-NIK-OE) results in spontaneous diabetes in male mice at a young age (≥10weeks of age), which is likely due to insulin deficiency, β cell death, and insulitis. Importantly, inhibiting the kinase activation of NIK by the small molecule B022 prevents NIK- or H2O2-induced β cell death and also reduces streptozotocin (STZ)-induced β cell death while ameliorating hyperglycemia, suggesting that the kinase activity of NIK is essential in inducing islet inflammation, β cell death, and diabetes. In all, this study not only uncovers a role of NIK in β cell failure but also provides a potential therapeutic target for the treatment of diabetes.

1859-P: Hepatic NIK Augments Hepatocyte Ferroptosis and Hepatic Inflammation in Liver Disease

Nonalcoholic fatty liver disease (NAFLD) is a hepatic manifestation of obesity and diabetes. Hepatocyte injury and death pathologically mark a turning point from simple liver steatosis to nonalcoholic steatohepatitis (NASH). Chronic hepatocyte injury and hepatic inflammation drive liver fibrosis, leading to liver cirrhosis, end-stage liver failure, and/or hepatocellular carcinoma. We previously reported that NAFLD is associated with a marked elevation of NF-κB-inducing kinase (NIK) in the liver. NIK in turn augments the hepatic hyperglycemic response to counterregulatory hormone glucagon, contributing to hyperglycemia and diabetes progression. Here, we demonstrate that hepatic NIK also promotes hepatocyte injury and death, exacerbating liver disease. We found that hepatocyte-specific overexpression of NIK substantially increased hepatocyte death and liver inflammation in mice treated with acetaminophen (APAP), a hepatocyte-toxic drug. Conversely, hepatocyte-specific deletion of NIK significantly blunted APAP-induced liver injury and hepatitis. In line with these findings, in primary liver cell cultures, overexpression of NIK substantially enhanced APAP sensitivity to induce cell death. Notably, NIK overexpression dramatically increased liver reactive oxygen species (ROS) levels in APAP-treated mice, and ablation of hepatocyte NIK had the opposite effects. Treatment with antioxidant N-acetylcysteine abrogated the ability of NIK and APAP to induce hepatocyte death and liver inflammation. Given that ROS drives ferroptosis, we treated primary liver cells with ferroptosis inhibitors ferrostatin 1 and liproxtatin-1. Both ferrostatin 1 and liproxtatin-1 dose-dependently suppressed NIK/APAP-induced cell death. Collectively, our data suggest that aberrant activation of hepatic NIK augments hepatocyte ferroptosis, thereby promoting liver injury, liver inflammation, and liver disease progression. Disclosure X. Zhong: None. L. Rui: None.

Zebrafish NIK Mediates IFN Induction by Regulating Activation of IRF3 and NF-κB.

Type I IFN mediates the innate immune system to provide defense against viral infections. NF-κB-inducing kinase (NIK) potentiates the basal activation of endogenous STING, which facilitates the recruitment of TBK1 with the ectopically expressed IRF3 to induce IFN production. Moreover, NIK phosphorylates IKKα and confers its ability to phosphorylate p100 (also known as NF-κB2) in mammals. Our study demonstrated that NIK plays a critical role in IFN production in teleost fish. It was found that NIK interacts with IKKα in the cytoplasm and that IKKα phosphorylates the NIK at the residue Thr432, which is different from the mammals. Overexpression of NIK caused the activation of IRF3 and NF-κB, which in turn led to the production of IFN and IFN-stimulated genes (ISGs). Furthermore, the ectopic expression of NIK was observed to be associated with a reduced replication of the fish virus, whereas silencing of endogenous NIK had an opposite effect in vitro. Furthermore, NIK knockdown significantly reduced the expression of IFN and key ISGs in zebrafish larvae after spring viremia of carp virus infection. Additionally, the replication of spring viremia of carp virus was enhanced in NIK knockdown zebrafish larvae, leading to a lower survival rate. In summary, our findings revealed a previously undescribed function of NIK in activating IFN and ISGs as a host antiviral response. These findings may facilitate the establishment of antiviral therapy to combat fish viruses.

Discovery of a Potent and Selective NF-κB-Inducing Kinase (NIK) Inhibitor That Has Anti-inflammatory Effects in Vitro and in Vivo.

The overexpression of NIK plays a critical role in liver inflammatory diseases. Treatment of such diseases with small-molecule NIK inhibitors is a reasonable but underexplored approach. In this paper, we reported the discovery of a potent and selective NIK inhibitor 46 (XT2). 46 inhibited the NIK kinase with an IC50 value of 9.1 nM in vitro, and it also potently suppressed NIK activities in intact cells. In isogenic primary hepatocytes, treatment of 46 efficiently suppressed the expressions of NIK-induced genes. 46 was orally bioavailable in mice with moderate systemic exposure. In a NIK-associated mouse liver inflammation model, 46 suppressed CCl4-induced upregulation of ALT, a key biomarker of acute liver injury. 46 also decreased immune cell infiltration into the injured liver tissue. Overall, these studies provide examples that an NIK inhibitor is able to suppress toxin-induced liver inflammations, which indicates its therapeutic potentials for the treatment of liver inflammatory diseases.

Islet α-cell Inflammation Induced By NF-κB inducing kinase (NIK) Leads to Hypoglycemia, Pancreatitis, Growth Retardation, and Postnatal Death in Mice.

Islet α-cell dysfunction has been shown to contribute to type 2 diabetes; however, whether islet α-cell inflammation is involved in the occurrence of pancreatitis is largely unknown. The aims of this study were to investigate how NF-κB inducing kinase (NIK) regulates pancreatic α-cell function, both in vitro and in vivo, and to assess how islet α-cell inflammation induced by NIK affects the development of pancreatitis.Methods: We utilized adenovirus-mediated NIK overexpression, ELISA, qPCR, RNA-seq, and Western blot analyses to study the role of NIK in islet α cells in vitro. Islet α-cell-specific NIK overexpressing (α-NIK-OE) mice were generated, and pancreatic α/β-cell function and the occurrence of pancreatitis in these mice were assessed via ELISA, qPCR, and immunohistochemical analyses.Results: The LTβR/noncanonical NF-κB signaling pathway is present in islet α cells. Overexpression of NIK in αTC1-6 cells induces inflammation and cell death, contributing to a decrease in the expression and secretion of glucagon. Additionally, α-cell specific overexpression of NIK (α-NIK-OE) results in α-cell death, lower serum glucagon levels, and hypoglycemia in mice. Strikingly, α-NIK-OE mice also display a reduced β-cell mass, growth retardation, pancreatitis, and postnatal death.Conclusions: Islet α-cell specific overexpression of NIK results in islet α-cell dysfunction and causes islet β-cell death and pancreatitis, which are most likely due to paracrine secretion of cytokines and chemokines from islet α cells, thus leading to hypoglycemia, growth retardation, and postnatal death in mice.

Glucocorticoids increase skeletal muscle NF-κB inducing kinase (NIK): links to muscle atrophy.

Glucocorticoids (GC) are a frontline therapy for numerous acute and chronic diseases because of their demonstrated efficacy at reducing systemic inflammation. An unintended side effect of GC therapy is the stimulation of skeletal muscle atrophy. Pathophysiological mechanisms responsible for GC‐induced skeletal muscle atrophy have been extensively investigated, and the ability to treat patients with GC without unintended muscle atrophy has yet to be realized. We have reported that a single, standard‐of‐care dose of Methylprednisolone increases in vivo expression of NF‐κB‐inducing kinase (NIK), an important upstream regulatory kinase controlling NF‐κB activation, along with other key muscle catabolic regulators such as Atrogin‐1 and MuRF1 that induce skeletal muscle proteolysis. Here, we provide experimental evidence that overexpressing NIK by intramuscular injection of recombinant human NIK via adenoviral vector in mouse tibialis anterior muscle induces a 30% decrease in the average fiber cross‐sectional area that is associated with increases in mRNA expression of skeletal muscle atrophy biomarkers MuRF1, Atrogin‐1, myostatin and Gadd45. A single injection of GC induced NIK mRNA and protein within 2 h, with the increased NIK localized to nuclear and sarcolemmal locations within muscle fibers. Daily GC injections induced skeletal muscle fore limb weakness as early as 3 days with similar atrophy of muscle fibers as observed with NIK overexpression. NIK overexpression in primary human skeletal muscle myotubes increased skeletal muscle atrophy biomarkers, while NIK knockdown significantly attenuated GC‐induced increases in NIK and Atrogin‐1. These results suggest that NIK may be a novel, previously unrecognized mediator of GC‐induced skeletal muscle atrophy.

Carboxyl Terminus of HSC70-interacting Protein (CHIP) Down-regulates NF-κB-inducing Kinase (NIK) and Suppresses NIK-induced Liver Injury

Ser/Thr kinase NIK (NF-κB-inducing kinase) mediates the activation of the noncanonical NF-κB2 pathway, and it plays an important role in regulating immune cell development and liver homeostasis. NIK levels are extremely low in quiescent cells due to ubiquitin/proteasome-mediated degradation, and cytokines stimulate NIK activation through increasing NIK stability; however, regulation of NIK stability is not fully understood. Here we identified CHIP (carboxyl terminus of HSC70-interacting protein) as a new negative regulator of NIK. CHIP contains three N-terminal tetratricopeptide repeats (TPRs), a middle dimerization domain, and a C-terminal U-box. The U-box domain contains ubiquitin E3 ligase activity that promotes ubiquitination of CHIP-bound partners. We observed that CHIP bound to NIK via its TPR domain. In both HEK293 and primary hepatocytes, overexpression of CHIP markedly decreased NIK levels at least in part through increasing ubiquitination and degradation of NIK. Accordingly, CHIP suppressed NIK-induced activation of the noncanonical NF-κB2 pathway. CHIP also bound to TRAF3, and CHIP and TRAF3 acted coordinately to efficiently promote NIK degradation. The TPR but not the U-box domain was required for CHIP to promote NIK degradation. In mice, hepatocyte-specific overexpression of NIK resulted in liver inflammation and injury, leading to death, and liver-specific expression of CHIP reversed the detrimental effects of hepatic NIK. Our data suggest that CHIP/TRAF3/NIK interactions recruit NIK to E3 ligase complexes for ubiquitination and degradation, thus maintaining NIK at low levels. Defects in CHIP regulation of NIK may result in aberrant NIK activation in the liver, contributing to live injury, inflammation, and disease.

Mouse hepatocyte overexpression of NF-κB-inducing kinase (NIK) triggers fatal macrophage-dependent liver injury and fibrosis.

Damaged, necrotic, or apoptotic hepatocytes release damage-associated molecular patterns that initiate sterile inflammation, and liver inflammation drives liver injury and fibrosis. Here we identified hepatic nuclear factor kappa B (NF-κB)-inducing kinase (NIK), a Ser/Thr kinase, as a novel trigger of fatal liver inflammation. NIK is activated by a broad spectrum of stimuli. It was up-regulated in injured livers in both mice and humans. In primary mouse hepatocytes, NIK overexpression stimulated, independently of cell injury and death, release of numerous chemokines and cytokines that activated bone marrow-derived macrophages (BMDMs). BMDMs in turn secreted proapoptotic molecules that stimulated hepatocyte apoptosis. Hepatocyte-specific expression of the NIK transgene triggered massive liver inflammation, oxidative stress, hepatocyte apoptosis, and liver fibrosis, leading to weight loss, hypoglycemia, and death. Depletion of Kupffer cells/macrophages reversed NIK-induced liver destruction and death. the hepatocyte NIK-liver immune cell axis promotes liver inflammation, injury, and fibrosis, thus driving liver disease progression.

NF-κB–inducing kinase (NIK) promotes hyperglycemia and glucose intolerance in obesity by augmenting glucagon action

The canonical IKKβ/NF-κB1 pathway has been well documented to promote insulin resistance; however, the noncanonical NIK/NF-κB2 pathway is poorly understood in obesity. Additionally, the contribution of counterregulatory hormones, particularly glucagon, to hyperglycemia in obesity remains unclear. Here we show that NIK promotes glucagon responses in obesity. Hepatic NIK was abnormally-activated in mice with dietary or genetic obesity. Systemic deletion of NIK decreased glucagon responses and hepatic glucose production (HGP). Obesity is associated with increased glucagon responses, and liver-specific inhibition of NIK decreased glucagon responses and HGP and protected against hyperglycemia and glucose intolerance. Conversely, hepatocyte-specific overexpression of NIK increased glucagon responses and HGP. In isolated livers and primary hepatocytes, NIK also promoted glucagon action and glucose production, at least in part by increasing CREB stability. Therefore, overactivation of liver NIK in obesity promotes hyperglycemia and glucose intolerance by increasing the hyperglycemic response to glucagon and other factors that activate CREB.

Abstract 2889: NF-κB inducing kinase modulates melanoma tumorigenesis by regulating pro-survival factors through β-catenin

Abstract Nuclear factor-κB (NF-κB) contributes to the progression of many cancers due to its role in regulating the transcription of genes important for inflammation, proliferation, apoptosis, and survival. In melanoma, high NF-κB activity results from constitutive elevation of IKKβ activity, a major kinase of this pathway. Targeted deletion of IKKβ prevents Ras induced melanoma tumor formation in INK4a/ARF-/- mice, underscoring the importance of NF-κB in melanoma. We have demonstrated that the NF-κB inducing kinase (NIK) is elevated in many melanoma cell lines. To determine the role of NIK overexpression in melanoma, we knocked down NIK using shRNA in WM115 and Hs294T human melanoma cell lines. NIK knock-down melanoma cells exhibited increased apoptosis and decreased proliferation. Similarly, NIK depletion substantially decreased tumor burden of WM115 cells in a xenograft model. Analysis of gene expression microarray data suggests that NIK modulates expression of c-Myc, c-Met, CCND2 and CXCR4, genes which are crucial for melanoma progression. Consistent with the apoptotic phenotype of NIK knock-down melanoma cells, expression of survival factors, such as Bcl2, cIAP1 and survivin, were attenuated in NIK knock-down melanoma cells. Intriguingly, Bcl2 and survivin expression levels (mRNA and protein) were also diminished in NIK knock-out mouse embryonic fibroblasts (MEFs), suggesting that these pro-survival genes are under the regulation of NIK. However, NF-κB activity is not reduced in NIK knock down cells, possibly due to the compensatory activation of IKKβ. Thus, we explored the possibility that NIK affects expression of survival factors in a NF-κB independent manner. Our data suggest that NIK regulates subcellular localization of β-catenin and modulates expression of survival genes such as survivin. Our findings reveal a novel mechanism by which NIK modulates pro-survival genes and suggest that NIK may be a promising therapeutic target for melanoma malignancy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2889. doi:10.1158/1538-7445.AM2011-2889

NIK overexpression intrinsically inhibits regulatory T cell function (168.9)

Abstract Activation of the non-canonical, or alternative, NF-κB pathway depends on NF-κB-inducing kinase (NIK). We have shown that NIK is intrinsically essential for T cells to cause lethal graft-versus-host disease and to respond to OX40 costimulation. We went on to investigate the effects of NIK overexpression in T cells by crossing inducible NIK transgenic mice to CD4-Cre inducer mice and found that modest NIK overexpression limited to T cells (CD4-NIKtg) caused fatal autoimmunity by 4-weeks of age, resembling the phenotype described in FoxP3-/- and scurfy mice. We hypothesized that NIK overexpression in regulatory T cells (Treg) affects their suppressive phenotype. Here we show that NIKtg Treg cells obtained from healthy bone marrow chimeras fail to suppress WT conventional T cell (Tconv) proliferation in vitro. In addition, NIKtg Tconv obtained from these mice are resistant to suppression by WT Treg cells. Transient NIK overexpression in vitro also represses Treg cell function, and preliminary experiments suggest that NIK overexpression destabilizes the Treg phenotype. Lastly, we investigated the effect of more extreme NIK overexpression using a mutant NIK transgene that is not targeted for degradation (NIKΔTRAF3). Contrary to modest NIK upregulation, excessive NIK accumulation is lethal to T cells. Together, these results show that NIK plays a critical T-cell intrinsic role in both Tconv and Treg, and that its tight regulation is crucial for T cell regulation and survival.

Overexpression of NF-κB inducing kinase underlies constitutive NF-κB activation in lung cancer cells

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.

Overexpressed NF-κB–inducing kinase contributes to the tumorigenesis of adult T-cell leukemia and Hodgkin Reed-Sternberg cells

AbstractThe nuclear factor-κB (NF-κB) transcription factors play important roles in cancer development by preventing apoptosis and facilitating the tumor cell growth. However, the precise mechanisms by which NF-κB is constitutively activated in specific cancer cells remain largely unknown. In our current study, we now report that NF-κB–inducing kinase (NIK) is overexpressed at the pretranslational level in adult T-cell leukemia (ATL) and Hodgkin Reed-Sternberg cells (H-RS) that do not express viral regulatory proteins. The overexpression of NIK causes cell transformation in rat fibroblasts, which is abolished by a super-repressor form of IκBα. Notably, depletion of NIK in ATL cells by RNA interference reduces the DNA-binding activity of NF-κB and NF-κB–dependent transcriptional activity, and efficiently suppresses tumor growth in NOD/SCID/γcnull mice. These results indicate that the deregulated expression of NIK plays a critical role in constitutive NF-κB activation in ATL and H-RS cells, and suggest also that NIK is an attractive molecular target for cancer therapy.

Stabilization of Basally Translated NF-κB-inducing Kinase (NIK) Protein Functions as a Molecular Switch of Processing of NF-κB2 p100

The non-canonical pathway based on processing of NF-kappaB2 precursor protein p100 to generate p52 plays a critical role in controlling B cell function and lymphoid organogenesis. Activation of this unique pathway by extracellular stimuli requires NF-kappaB-inducing kinase (NIK) and de novo protein synthesis. However, how NIK is regulated is largely unknown. Here, we systematically analyzed NIK expression at different levels in the presence or absence of different NF-kappaB stimuli. We found that NIK mRNA is relatively abundant and undergoes constitutive protein synthesis in resting B cells. However, NIK protein is undetectable. Interestingly, protein expression of NIK is steadily induced by B cell-activating factor or CD40 ligand, two major physiological inducers of p100 processing, but not by mitogen phorbol 12-myristate 13-acetate/ionomycin or cytokine tumor necrosis factor alpha, two well known inducers of the canonical NF-kappaB signaling. Remarkably, both B cell-activating factor and CD40 ligand do not significantly induce expression of NIK at translational or transcriptional level but rather rescue the basally translated NIK protein from undergoing degradation. Furthermore, overexpressed or purified NIK protein triggers p100 processing in the presence of protein synthesis inhibitor. Taken together, these studies define one important mechanism of NIK regulation and the central role of NIK stabilization in the induction of p100 processing. These studies also provide the first evidence explaining why activation of the non-canonical NF-kappaB signaling is delayed and can be inhibited by protein synthesis inhibitor as well as why most classical NF-kappaB stimuli, including mitogens and tumor necrosis factor alpha, fail to induce p100 processing.

The heat-shock-induced suppression of the IκB/NF-κB cascade is due to inactivation of upstream regulators of IκBα through insolubilization

Heat shock (HS) was found to suppress the IκB/NF-κB cascade via the inhibition of IκB kinase (IKK) activity; however, the mechanism has not been clear. This study was undertaken to elucidate the detail of the mechanism involved. TNF-α-induced activation of IKK was suppressed by HS in human bronchial epithelial cells, and this was associated with the absence of IKK in the immunoprecipitates. It was not due to a degradation of IKK, but due to a conversion of IKK from a soluble to an insoluble form. IKK lost its activity rapidly upon exposure to HS in vitro. The time course of the insolubilization of IKK coincided with the decrease in IKK activity. However, inhibition of IKK insolubilization by the induction of thermotolerance did not reverse the HS-induced suppression of IKK activation and IκBα degradation. Upstream activators of IKK, such as NF-κB-inducing kinase (NIK) and IL-1R-associated kinase (IRAK) were also insolubilized by HS. The HS-induced insolubilization of NIK was not blocked by the induction of thermotolerance. Overexpression of NIK resumed TNF-α-induced activation of IKK in thermotolerant cells. These results indicate that the loss of activity of NIK, IRAK, and IKK through insolubilization is responsible for the HS-induced suppression of IκB/NF-κB pathway.

Tyrosine phosphorylation of I-kappa B kinase alpha/beta by protein kinase C-dependent c-Src activation is involved in TNF-alpha-induced cyclooxygenase-2 expression.

The signaling pathway involved in TNF-alpha-induced cyclooxygenase-2 (COX-2) expression was further studied in human NCI-H292 epithelial cells. A protein kinase C (PKC) inhibitor (staurosporine), tyrosine kinase inhibitors (genistein and herbimycin A), or a Src kinase inhibitor (PP2) attenuated TNF-alpha- or 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced COX-2 promoter activity. TNF-alpha- or TPA-induced I-kappaB kinase (IKK) activation was also blocked by these inhibitors, which reversed I-kappaBalpha degradation. Activation of c-Src and Lyn kinases, two Src family members, was inhibited by the PKC, tyrosine kinase, or Src kinase inhibitors. The dominant-negative c-Src (KM) mutant inhibited induction of COX-2 promoter activity by TNF-alpha or TPA. Overexpression of the constitutively active PKCalpha (PKCalpha A/E) or wild-type c-Src plasmids induced COX-2 promoter activity, and these effects were inhibited by the dominant-negative c-Src (KM), NF-kappaB-inducing kinase (NIK) (KA), or IKKbeta (KM) mutant. The dominant-negative PKCalpha (K/R) or c-Src (KM) mutant failed to block induction of COX-2 promoter activity caused by wild-type NIK overexpression. In coimmunoprecipitation experiments, IKKalpha/beta was found to be associated with c-Src and to be phosphorylated on its tyrosine residues after TNF-alpha or TPA treatment. Two tyrosine residues, Tyr(188) and Tyr(199), near the activation loop of IKKbeta, were identified to be crucial for NF-kappaB activation. Substitution of these residues with phenylalanines attenuated COX-2 promoter activity and c-Src-dependent phosphorylation of IKKbeta induced by TNF-alpha or TPA. These data suggest that, in addition to activating NIK, TNF-alpha also activates PKC-dependent c-Src. These two pathways cross-link between c-Src and NIK and converge at IKKalpha/beta, and go on to activate NF-kappaB, via serine phosphorylation and degradation of IkappaB-alpha, and, finally, to initiate COX-2 expression.