NF-κB-inducing Kinase Research Articles

Hodgkin lymphoma requires stabilized NIK and constitutive RelB expression for survival

AbstractWe have analyzed the role of the REL family members in Hodgkin lymphoma (HL). shRNA targeting of each REL member showed that HL was uniquely dependent on relB, in contrast to several other B-cell lymphomas. In addition, relA and c-rel shRNA expression also decreased HL cell viability. In exploring relB activation further, we found stable NF-κB inducing kinase (NIK) protein in several HL cell lines and that NIK shRNA also affected HL cell line viability. More importantly, 49 of 50 HL patient biopsies showed stable NIK protein, indicating that NIK and the noncanonical pathway are very prevalent in HL. Lastly, we have used a NIK inhibitor that reduced HL but not other B-cell lymphoma cell viability. These data show that HL is uniquely dependent on relB and that the noncanonical pathway can be a therapeutic target for HL. Furthermore, these results show that multiple REL family members participate in the maintenance of a HL phenotype.

Understanding NIK Regulation from Its Structure

In this issue of Structure, de Leon-Boenig and colleagues bring to light several key characteristics of NF-κB inducing kinase (NIK), which is at the center of many pathological disorders, using an impressive body of structural and functional studies. Structures of NIK described here provide deep insights into the mechanism of NIK activity regulation and may facilitate the design of new generation inhibitors for therapeutic use.

The Crystal Structure of the Catalytic Domain of the NF-κB Inducing Kinase Reveals a Narrow but Flexible Active Site

The NF-κB inducing kinase (NIK) regulates the non-canonical NF-κB pathway downstream of important clinical targets including BAFF, RANKL, and LTβ. Despite numerous genetic studies associating dysregulation of this pathway with autoimmune diseases and hematological cancers, detailed molecular characterization of this central signaling node has been lacking. We undertook a systematic cloning and expression effort to generate soluble, well-behaved proteins encompassing the kinase domains of human and murine NIK. Structures of the apo NIK kinase domain from both species reveal an active-like conformation in the absence of phosphorylation. ATP consumption and peptide phosphorylation assays confirm that phosphorylation of NIK does not increase enzymatic activity. Structures of murine NIK bound to inhibitors possessing two different chemotypes reveal conformational flexibility in the gatekeeper residue controlling access to a hydrophobic pocket. Finally, a single amino acid difference affects the ability of some inhibitors to bind murine and human NIK with the same affinity.

DMA, a Bisbenzimidazole, Offers Radioprotection by Promoting NFκB Transactivation through NIK/IKK in Human Glioma Cells

BackgroundIonizing radiation (IR) exposure often occurs for human beings through occupational, medical, environmental, accidental and/or other sources. Thus, the role of radioprotector is essential to overcome the complex series of overlapping responses to radiation induced DNA damage.Methods and ResultsTreatment of human glioma U87 cells with DMA (5- {4-methylpiperazin-1-yl}-2-[2′-(3, 4-dimethoxyphenyl)-5′-benzimidazolyl] in the presence or absence of radiation uncovered differential regulation of an array of genes and proteins using microarray and 2D PAGE techniques. Pathway construction followed by relative quantitation of gene expression of the identified proteins and their interacting partners led to the identification of MAP3K14 (NFκB inducing kinase, NIK) as the candidate gene affected in response to DMA. Subsequently, over expression and knock down of NIK suggested that DMA affects NFκB inducing kinase mediated phosphorylation of IKKα and IKKβ both alone and in the presence of ionizing radiation (IR). The TNF-α induced NFκB dependent luciferase reporter assay demonstrated 1.65, 2.26 and 3.62 fold increase in NFκB activation at 10, 25 and 50 µM DMA concentrations respectively, compared to control cells. This activation was further increased by 5.8 fold in drug + radiation (50 µM +8.5 Gy) treated cells in comparison to control. We observed 51% radioprotection in control siRNA transfected cells that attenuated to 15% in siRNA NIK treated U87 cells, irradiated in presence of DMA at 24 h.ConclusionsOur studies show that NIK/IKK mediated NFκB activation is more intensified in cells over expressing NIK and treated with DMA, alone or in combination with ionizing radiation, indicating that DMA promotes NIK mediated NFκB signaling. This subsequently leads to the radioprotective effect exhibited by DMA.

The nuclear factor- B pathway is involved in matrix metalloproteinase-9 expression in RU486-induced endometrium breakdown in mice

Progesterone-withdrawal (WP)-induced endometrial breakdown occurs in both physiological and pathological processes such as menstruation and abortion. However, the underlying mechanisms are not clearly understood. As the nuclear factor-κB (NF-κB) pathway has been proposed to play a role in endometrial breakdown, we tested this hypothesis using RU486-induced mouse menstruation-like model. The activation of NF-κB was evaluated by immunohistochemistry, western blot and immunofluorescence. The expression of matrix metalloproteinase-9 (MMP9) was analyzed by real-time PCR and its proteins by gelatin zymography and western blot. Chromatin immunoprecipitation was used to investigate the direct binding of NF-κB to MMP9 gene promoter. Inhibitors of NF-κB were used to block its signal in vivo and in vitro to analyze the function of NF-κB in the tissue breakdown process. Administration of RU486 resulted in increased phospho-IκB levels and nuclear translocation of p65 in decidual stromal cells, accompanied by the up-regulation of NF-κB inducing kinase and IκB kinase β mRNA. The NF-κB inhibitor, 'pyrrolidine dithiocarbamate' partially suppressed the RU486-induced endometrial breakdown, thus verifying the role of this pathway in vivo. MMP9 was up- and down-regulated following the NF-κB activation and inhibition, respectively. RU486 stimulated recruitment of NF-κB p65 to the MMP9 promoter and further increased its expression. Effects of NF-κB activation and inactivation on MMP9 expression were further explored in human stromal cells in vitro. A similar MMP9 expression pattern was observed in cultured human, as well as mouse, decidual stromal cells following RU486 treatment. The activation of the NF-κB pathway induces downstream target genes, including MMP9 from stromal cells to facilitate tissue breakdown in mouse uterus, highlighting the likelihood that this regulatory pattern exists in the human endometrium.

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.

NIK Prevents the Development of Hypereosinophilic Syndrome-like Disease in Mice Independent of IKKα Activation

Immune cell-mediated tissue injury is a common feature of different inflammatory diseases, yet the pathogenetic mechanisms and cell types involved vary significantly. Hypereosinophilic syndrome (HES) represents a group of inflammatory diseases that is characterized by increased numbers of pathogenic eosinophilic granulocytes in the peripheral blood and diverse organs. On the basis of clinical and laboratory findings, various forms of HES have been defined, yet the molecular mechanism and potential signaling pathways that drive eosinophil expansion remain largely unknown. In this study, we show that mice deficient of the serine/threonine-specific protein kinase NF-κB-inducing kinase (NIK) develop a HES-like disease, reflected by progressive blood and tissue eosinophilia, tissue injury, and premature death at around 25-30 wk of age. Similar to the lymphocytic form of HES, CD4(+) T cells from NIK-deficient mice express increased levels of Th2-associated cytokines, and eosinophilia and survival of NIK-deficient mice could be prevented completely by genetic ablation of CD4(+) T cells. Experiments based on bone marrow chimeric mice, however, demonstrated that inflammation in NIK-deficient mice depended on radiation-resistant tissues, implicating that NIK-deficient immune cells mediate inflammation in a nonautonomous manner. Surprisingly, disease development was independent of NIK's known function as an IκB kinase α (IKKα) kinase, because mice carrying a mutation in the activation loop of IKKα, which is phosphorylated by NIK, did not develop inflammatory disease. Our data show that NIK activity in nonhematopoietic cells controls Th2 cell development and prevents eosinophil-driven inflammatory disease, most likely using a signaling pathway that operates independent of the known NIK substrate IKKα.

Delphinidin blocks IL-1β-induced activation of NF-κB by modulating the IKKβ gene expression and the activation of NIK in human chondrocytes

Purpose: Osteoarthritis (OA) is a highly prevalent disease that affects the quality of life of its victims. Interleukin-1 β (IL-1β) plays a major role in the pathogenesis of OA and is present in the arthritic joints at high levels. IL-1β has been shown to cause increased production of catabolic enzymes such as MMPs, and inflammatory mediators such as prostaglandins and nitric oxide through the activation of several signaling pathways including NF-κB. Novel approaches using plant derived polyphenols to inhibit signal transduction pathways have shown promise and could overcome some of the toxicity issues related to current therapies. One key signaling mechanism implicated in OA that could be a target of orally bioavailable inhibitors is the NF-κB pathway. Delphinidin (2-(3,4,5-trihydroxyphenyl)chromenylium-3,5,7-triol) is an anthocyanidin that is widely distributed in pigmented fruits and flowers and has been shown to possess anti-inflammatory activity and block the activation of NF-κB in cancer cells. No such study with human chondrocytes has been reported. In the present study using primary human chondrocytes we determined (a) the chondroprotective effect of Delphinidin; and (b) mechanism of NF-κB inhibition in IL-1β-stimulated human chondrocytes. Methods: Chondrocytes were derived by enzymatic digestion of human cartilage (OA chondrocytes) from patients undergoing total knee arthroplasty. OA chondrocytes were pretreated with Delphinidin for different time points and were then stimulated with IL-1β (10ng/ml) in vitro and total RNA or cell lysate was prepared following our standard protocols. To determine the gene expression, single stranded cDNA was synthesized and the expression of target mRNAs (COX-2, MMP-13, iNOS) was quantified using TaqMan Assays. HeLa cells were transfected with promoter reporter vectors and negative control vectors using the Fugene-6 reagent and the effect on promoter activity was verified by Luciferase assay. Activation of NF-κB p65 was determined by specific ELISA-based DNA binding assay. Total protein levels were determined by detergent compatible Biorad Protein Assay. Nitric oxide production was determined by Griess reaction. Protein expression and the phosphorylated forms of different kinases were determined by Western immunoblotting. Data were analyzed using Origin 6.1 software package and p<0.05 was considered significant. Results: Pretreatment of chondrocytes with Delphinidin (50 μg/ml) for 2 hrs significantly inhibited the IL-1β induced expression of COX-2, iNOS and MMP-13 and the production of nitric oxide. Delphinidin also inhibited the phosphorylation of IKKβ and was also a potent suppressor of IKKβ gene and protein expression in OA chondrocytes. IKK promoter had a strong activity in HeLa cells and this activity and production of the Luciferase reporter enzyme was blocked by pre-treatment with Delphinidin. Functional pathway analyses showed that IL-1β-induced phosphorylation of NF-κB-inducing Kinase (NIK), which interacts with and activates IKKα and IKKβ which then phosphorylate IκB, was inhibited by pretreatment of OA chondrocytes with Delphinidin. In agreement with these findings, pre-treatment with Delphinidin inhibited the phosphorylation and degradation of IκB, as determined by Western immunoblotting, and the activation and the DNA binding activity of NF-κB p65 determined by a highly specific ELISA based assay. Conclusions: Taken together, the data presented here identifies a novel mechanism of NF-κB inhibition by Delphinidin by inhibiting NIK, a kinase upstream of IKK complex in the NF-κB activation pathway. Given the important role played by IL-1β and NF-κB in OA, these results may provide important clues to develop useful pharmacological inhibitors of NF-κB for the prevention and/or treatment of OA.

The noncanonical NF-κB pathway.

The noncanonical nuclear factor-κB (NF-κB) signaling pathway mediates activation of the p52/RelB NF-κB complex and, thereby, regulates specific immunological processes. This NF-κB pathway relies on the inducible processing of NF-κB2 precursor protein, p100, as opposed to the degradation of IκBα in the canonical NF-κB pathway. A central signaling component of the noncanonical NF-κB pathway is NF-κB-inducing kinase (NIK), which functions together with a downstream kinase, IKKα (inhibitor of NF-κB kinase α), to induce phosphorylation-dependent ubiquitination and processing of p100. Under normal conditions, NIK is targeted for continuous degradation by a tumor necrosis factor (TNF) receptor-associated factor-3 (TRAF3)-dependent E3 ubiquitin ligase. In response to signals mediated by a subset of TNF receptor superfamily members, NIK becomes stabilized as a result of TRAF3 degradation, leading to the activation of noncanonical NF-κB. This review discusses both the historical perspectives and the recent progress in the regulation and biological function of the noncanonical NF-κB pathway.

A Noncanonical Flt3ITD/NF-κB Signaling Pathway Represses DAPK1 in Acute Myeloid Leukemia

Death-associated protein kinase 1 (DAPK1), a tumor suppressor, is a rate-limiting effector in an endoplasmic reticulum (ER) stress-dependent apoptotic pathway. Its expression is epigenetically suppressed in several tumors. A mechanistic basis for epigenetic/transcriptional repression of DAPK1 was investigated in certain forms of acute myeloid leukemia (AML) with poor prognosis, which lacked ER stress-induced apoptosis. Heterogeneous primary AMLs were screened to identify a subgroup with Flt3ITD in which repression of DAPK1, among NF-κB-and c-Jun-responsive genes, was studied. RNA interference knockdown studies were carried out in an Flt3ITD(+) cell line, MV-4-11, to establish genetic epistasis in the pathway Flt3ITD-TAK1-DAPK1 repression, and chromatin immunoprecipitations were carried out to identify proximate effector proteins, including TAK1-activated p52NF-κB, at the DAPK1 locus. AMLs characterized by normal karyotype with Flt3ITD were found to have 10- to 100-fold lower DAPK1 transcripts normalized to the expression of c-Jun, a transcriptional activator of DAPK1, as compared with a heterogeneous cytogenetic category. In addition, Meis1, a c-Jun-responsive adverse AML prognostic gene signature was measured as control. These Flt3ITD(+) AMLs overexpress relB, a transcriptional repressor, which forms active heterodimers with p52NF-κB. Chromatin immunoprecipitation assays identified p52NF-κB binding to the DAPK1 promoter together with histone deacetylase 2 (HDAC2) and HDAC6 in the Flt3ITD(+) human AML cell line MV-4-11. Knockdown of p52NF-κB or its upstream regulator, NF-κB-inducing kinase (NIK), de-repressed DAPK1. DAPK1-repressed primary Flt3ITD(+) AMLs had selective nuclear activation of p52NF-κB. Flt3ITD promotes a noncanonical pathway via TAK1 and p52NF-κB to suppress DAPK1 in association with HDACs, which explains DAPK1 repression in Flt3ITD(+) AML.

A novel tumor suppressor miRNA miR-520e contributes to suppression of hepatoma

Hepatocellular carcinoma (HCC), one of the most common cancers worldwide, represents the third cause of cancer-related death1. Curative options for HCC are limited and exclusively available for patients carrying an early stage HCC. In advanced stages, traditional chemotherapy proved to be only marginally effective or even toxic. Thus, the identification of new treatment options is needed. New targets for non-conventional treatment will necessarily take advantage of progresses on the molecular pathogenesis of HCC2. MicroRNAs (miRNAs) are short non-coding RNAs 19–25 nucleotides in length, which negatively regulate gene expression by pairing with complementary sequences within the 3′-untranslated regions (UTRs) of targeted transcripts at the post-transcriptional and/or translational level3, 4. miRNAs expression is frequently deregulated in many types of human cancers, and plays critical roles in tumorigenesis, which functions either as tumor suppressors or as oncogenes. In the recent years, several studies have revealed that the expression of miRNAs is deregulated in human HCC in comparison with matched non-neoplastic tissue5. More and more miRNAs involved in hepatocarcinogenesis were identified in HCC tissues. Xu et al indicated that miR-195 may suppress tumorigenicity and regulate G1/S transition of HCC cells by repressing Rb-E2F signaling involving targeting multiple molecules, such as cyclin D1, CDK6, and E2F36. Liang et al reported that miR-125b was able to suppress tumor growth in hepatic carcinogenesis through the suppression of oncogene LIN28B expression7. miR-637 acts as a tumor suppressor, through the suppressive effect on HCC mediated by disruption of Stat3 activation8. miR-99a acts as a prospective prognosis predictor of HCC and potential tumor suppressor for HCC therapeutics, which induces cell cycle arrest by directly targeting IGF-1R and mTOR9. Thus, those miRNAs may function in a network in hepatocarcinogenesis. In a recent issue of Oncogene, Zhang's group identified a novel tumor suppressor miRNA miR-520e in hepatoma cells. Zhang and colleagues demonstrated that the miR-520e was dramatically decreased in the examined hepatoma cells and clinical hepatocellular carcinoma (HCC) tissues10. In cancer, aberrant DNA hypermethylation of tumor suppressor genes, global genomic DNA hypomethylation and disruption of the histone modification patterns are the main epigenetic alterations, which have consequently been widely studied. Some miRNAs are downregulated in cancer and act as bona fide tumor suppressor genes, and this knowledge led to the proposal of the hypothesis that miRNAs could be silenced by epigenetic mechanisms11. The miR-520e gene is located in 19q13.42 and is reported to be in chromosome 19 miRNA cluster (C19MC), the largest human miRNA gene cluster discovered so far. Their finding presented important information for the identification of potential mechanism of downregulation of miR-520e. The silencing of miR-520e was attributed to the methylation status of CpG islands, which are localized in the region of miR-520e promoter. Next, the paper used MTT assay, EdU assay, colony formation assay and analysis of tumorigenicity in nude mice to examine the potential role of miR-520e in tumorigenesis. They found that miR-520e suppresses growth of hepatoma cells in vitro and in vivo. Then, they revealed that NF-κB-inducing kinase (NIK) was a direct target gene of miR-520e. The NIK is a member of mitogen-activated protein kinase kinase kinase (MAP3K) family. NIK is usually constitutively and rapidly degraded by its TRAF3-mediated recruitment to TRAF2, which recruits cIAPs 1 and 2. NIK ubiquitination by these cIAPs promotes its proteosomal degradation. Stabilized NIK phosphorylates IKKα homodimers, which in turn phosphorylate p100 on its C-terminus, resulting in p100 ubiquitination and partial proteolytic processing by the proteosome to p52. The activated p52 then activates transcription when it associates with its binding partner (primarily RelB). Usually, NIK has a vital role in the alternative NF-κB activation pathway12, 13. However, in this study they observed that NIK was able to activate NF-κB/p65 via ERK1/2 signaling. Taken together, the finding suggests that miR-520e suppresses hepatoma cell growth through NIK/p-ERK1/2/NF-κB signaling pathway. Finally, they observed that the growth of tumor could be efficiently inhibited by direct injection with miR-520e in mice, suggesting that miR-520e might be a novel candidate for HCC therapy. As the important roles of miRNAs in cancer are gradually revealed, their potential applications as useful and effective targets have generated great interest in cancer gene therapy strategies, as well as diagnosis, classification, prognosis and risk factor evaluations. In cell culture and xenograft mice models, the miRNA could cause tumor regression. One of the major problems of miRNA therapy is the delivery to only a restricted number of cells, which will certainly limit the therapeutic efficacy and provide only partial knock-down14. Thus, additional work needs to be carried out to improve the delivery of miRNA including novel chemical modifications of miRNA targeting molecules, development of novel devices for sustained delivery of therapeutics and engineering of novel viral vectors. The current large number of intense research and development activities in the area of miRNA therapy will further strengthen the possibilities of affecting modern medicine.

TNF receptor family signaling in the development and functions of medullary thymic epithelial cells

Thymic epithelial cells (TECs) provide the microenvironment required for the development of T cells in the thymus. A unique property of medullary thymic epithelial cells (mTECs) is their expression of a wide range of tissue-restricted self-antigens, critically regulated by the nuclear protein AIRE, which contributes to the selection of the self-tolerant T cell repertoire, thereby suppressing the onset of autoimmune diseases. The TNF receptor family (TNFRF) protein receptor activator of NF-κB (RANK), CD40 and lymphotoxin β receptor (LtβR) regulate the development and functions of mTECs. The engagement of these receptors with their specific ligands results in the activation of the NF-κB family of transcription factors. Two NF-κB activation pathways, the classical and non-classical pathways, promote the development of mature mTECs induced by these receptors. Consistently, TNF receptor-associated factor (TRAF6), the signal transducer of the classical pathway, and NF-κB inducing kinase (NIK), the signal transducer of the non-classical pathway, are essential for the development of mature mTECs. This review summarizes the current understanding of how the signaling by the TNF receptor family controls the development and functions of mTEC.

Polycomb-Mediated Loss of miR-31 Activates NIK-Dependent NF-κB Pathway in Adult T Cell Leukemia and Other Cancers

Constitutive NF-κB activation has causative roles in adult T cell leukemia (ATL) caused by HTLV-1 and other cancers. Here, we report a pathway involving Polycomb-mediated miRNA silencing and NF-κB activation. We determine the miRNA signatures and reveal miR-31 loss in primary ATL cells. MiR-31 negatively regulates the noncanonical NF-κB pathway by targeting NF-κB inducing kinase (NIK). Loss of miR-31 therefore triggers oncogenic signaling. In ATL cells, miR-31 level is epigenetically regulated, and aberrant upregulation of Polycomb proteins contribute to miR-31 downregulation in an epigenetic fashion, leading to activation of NF-κB and apoptosis resistance. Furthermore, this emerging circuit operates in other cancers and receptor-initiated NF-κB cascade. Our findings provide a perspective involving the epigenetic program, inflammatory responses, and oncogenic signaling.

A new alternative NF-ΚB Pathway mediated the neuroprotection of GDNF on 6-OHDA-induced DA neurons neurotoxicity

Glial cell line-derived neurotrophic factor (GDNF) is a potent protective factor for dopaminergic (DA) neurons, but the signaling mechanisms underlying the effect of GDNF on these neurons remain obscure. Here, both our in vivo and in vitro studies demonstrate that the majority of DA neurons express the NF-κB-inducing kinase (NIK), which is the essential kinase for mediating activation of the new alternative NF-κB signaling pathway. Additionally, we also show that GDNF induced the time/dose-dependent phosphorylation of IκB kinase α (IKKα) and p100, facilitated the processing of p100 to p52 and accelerated the translocation of NF-κB dimmers into the nuclei of DA neurons. We furtherly found that the dimmer which translocate into the nucleus was RelA/p52 not RelB/p52. Meanwhile, the attenuation of 6-OHDA-induced DA neuronal apoptosis due to GDNF was reversed subsequent to the inhibition of p100 expression by RNAi while the neuroprotective effect of GDNF on injured DA neurons was strengthened by the overexpression of p100. Our data, therefore, indicate that a new alternative NF-κB signaling pathway, which was not the classic pathway but different from the non-canonical pathway, exists in DA neurons and mediates the neuroprotective effect of GDNF on these neurons.

Effects of polysaccharides from Morchella conica on nitric oxide production in lipopolysaccharide-treated macrophages

Morchella conica is a species of rare edible mushroom whose multiple medicinal functions have been proven. However, reports barely mention the mechanisms of these functions. In this study, the effects of two polysaccharides from M. conica (PMCs) on nitric oxide (NO) production in lipopolysaccharide (LPS)-treated macrophages were investigated. The results showed that 50-200 μg/ml of the extracellular polysaccharide (EPMC) and 25-200 μg/ml of the intracellular polysaccharide (IPMC) significantly inhibited NO production. Accordingly, the signal mechanisms were also explored. It was found that 100 μg/ml of EPMC and 25 μg/ml of IPMC could efficiently down-regulate the inducible nitric oxide synthase (iNOS) expression and nuclear factor-κB (NF-κB) DNA-binding activity and up-regulate heme oxygenase 1 (HO-1) expression. Moreover, by using a HO-1 inhibitor NaPP to treat the cells, the PMC-inhibited NO production and iNOS expression, rather than NF-κB activation, were released partially, indicating that HO-1 probably medicates the inhibition of PMCs on iNOS and NO. Besides, EPMC also significantly suppressed the phosphorylation of p38 mitogen-activated protein kinase (p38), c-jun N-terminal kinase, mitogen-activated protein kinase kinase 4, and expression of NF-κB inducing kinase, while IPMC seemed to show no regular effect on p38. In conclusion, PMCs inhibited NO production in LPS-induced macrophages through regulating a series of signal pathways, suggesting that PMCs play a potential role on immunomodulation and treating related diseases.

NF-κB–inducing kinase plays an essential T cell–intrinsic role in graft-versus-host disease and lethal autoimmunity in mice

NF-κB–inducing kinase (NIK) is an essential upstream kinase in noncanonical NF-κB signaling. NIK-dependent NF-κB activation downstream of several TNF receptor family members mediates lymphoid organ development and B cell homeostasis. Peripheral T cell populations are normal in the absence of NIK, but the role of NIK during in vivo T cell responses to antigen has been obscured by other developmental defects in NIK-deficient mice. Here, we have identified a T cell–intrinsic requirement for NIK in graft-versus-host disease (GVHD), wherein NIK-deficient mouse T cells transferred into MHC class II mismatched recipients failed to cause GVHD. Although NIK was not necessary for antigen receptor signaling, it was absolutely required for costimulation through the TNF receptor family member OX40 (also known as CD134). When we conditionally overexpressed NIK in T cells, mice suffered rapid and fatal autoimmunity characterized by hyperactive effector T cells and poorly suppressive Foxp3(+) Tregs. Together, these data illuminate a critical T cell–intrinsic role for NIK during immune responses and suggest that its tight regulation is critical for avoiding autoimmunity.

Hematopietic Stem Cells with Defects in the NF-κB Alternative Pathway Show a Deficient Repopulation Capacity

Abstract 1269Hematopoietic stem cells (HSCs) maintain the production of all blood cells through the lifespan of an organism, and regenerate the whole hematopoietic system after stressful episodes such as high dose chemotherapy or upon transplantation. The functions of HSCs in these 2 situations, steady-state and under stress, are controlled by a variety of molecules, which may provide different contribution to each process. We investigated whether the NF-kB alternative pathway might have a role in HSCs functions, using mice deficient for two components of this pathway: NF-kB-inducing kinase (NIK) or p52. The activation of NIK is generally known as the alternative (or non-canonical) NF-kB pathway, and drives the post-translational processing of p100 to mature p52, which results in the translocation to the nucleus of p52-containing complexes such as p52/RelB. Apart from the already reported defects in B-cell maturation, both NIK- and p52-deficient mice did not present major disturbances in blood cells numbers. The absolute numbers of marrow cells were not different among the knocked-out and the wild-type mice. We first studied the compartment of marrow cells known to be enriched for HSCs, either lineage-depleted Sca1-positive ckit-positive cells (LSK), or CD150 positive CD48 negative cells. The proportions of marrow cells with the immunophenotype of HSCs in either NIK-deficient or p52-deficient mice were similar to those in control mice. The amount of clonogeneic progenitor cells in the marrow was assessed in standard CFU-GM cultures, and gave no differences in output in any of the mice studied. We set up in vitro liquid cultures with murine stem cell factor and human interleukin-11, and determined the cellular production weekly. Cultures started with NIK-deficient marrow cells produced significantly less numbers of cells and CFU-GM, compared with those started with wild type marrow. This deficit in hematopoietic capacity was further confirmed in a more stringent assay of HSC function, the in vivo competitive repopulation assay. Equal numbers of lineage-depleted (Lin-) CD45.2 marrow cells of either NIK-deficient, p52-deficient or wild-type mice, were mixed with Lin- CD45.1 marrow cells of syngeneic mice, and transplanted into lethally irradiated CD45.1 recipients. Four months after transplant, the chimeric status and the hematopoietic lineage repopulation of CD45.2 cells was assessed in peripheral blood (PB). NIK- or p52-deficient HSCs repopulated the B-, T- and myeloid-lineages but at significantly lower levels when compared to wild type HSCs. Total donor CD45.2 cells and total CD45.2 LSK cells were also significantly lower in the marrows of mice transplanted with NIK- or p52-deficient HSCs versus those of controls. We used the marrows of the repopulated mice for secondary transplants, and confirmed the defect in the repopulating capacity of NIK- and p52-deficient HSCs. Our results suggest that the NF-kB alternative pathway plays a role in the function of HSCs, and this role may be important under stress conditions. Disclosures:No relevant conflicts of interest to declare.

The NF-κB-Inducing Kinase (NIK) Is Involved in Both Classical and Alternative NF-κB Activation and Is Necessary for the Survival of T Cell Lymphoma Cells,

Abstract 3665Nuclear factor κB (NF-κB) signaling has a critical role in the development and progression of several types of cancers. T cell lymphomas have been associated with NF-κB activity and its inhibition has been suggested as a possible therapeutic strategy to be further evaluated. However, the causes of NF-κB activation and the importance of this signaling pathway in T cell lymphomagenesis are still poorly understood. The NF-κB-inducing kinase, NIK, is a serine/threonine kinase that is crucial for the activation of the alternative NF-κB pathway but can also be involved in classical pathway activation. We evaluated the expression of nuclear p50 (classical NF-κB activation) and p52 (alternative NF-κB activation) in 30 primary T cell lymphoma samples and observed that 80.0% and 80.6% of cases showed nuclear staining of p50 and p52, respectively. Subsequently, using T-cell lymphoma cell lines, we investigated whether this classical and alternative NF-κB activation could be dependent on NIK expression. A strong overexpression of NIK mRNA in T cell lymphoma cell lines, compared to purified healthy donor T cells, was observed. This overexpression was associated with both classical (nuclear p50 and p65) and alternative (nuclear p52 and RelB) NF-κB activation. Knockdown of NIK in T cell lymphoma cells resulted in decreased processing of both p100 and p105, as well as a decrease in DNA binding activity of both classical and alternative NF-κB members. Furthermore, NIK knockdown lead to a dramatic increase in cell death in NIK overexpressing T cell lymphoma cell lines. These results suggest that NIK is involved in both classical and alternative NF-κB activation and is a putative therapeutic target in T cell lymphomas. Disclosures:No relevant conflicts of interest to declare.

A Constitutivly Active NFkB Non Canonical Pathway Is Maintained by RelB Repression of TRAF2 to Prevent Autophagy-Mediated Cell Death in Hodgkin Lymphomas

Abstract 428NFkB is a pleitropic transcription factor (TF) known to play major roles in diverse cell processes such as proliferation, apoptosis and inflammation. The REL or NFkB family is comprised of hetero or homo dimeric combinations of five members: RELA (p65), RELB, c-REL, NFKB1 (p50) and NFkB2 (p52).Despite considerable evidence supporting the role of the REL members in the immune system and lymphomagenesis, it has never been accurately established whether specific NFkB dimers control a particular set of target genes that account for the biological functions known to be mediated by these TFs. Furthermore, it is not clear whether NFkB controls different targets in distinct subsets of germinal center (GC) B cell derived lymphomas.We performed loss of function experiments delivering specific shRNAs to knock down each REL member in lymphoma cell lines modeling sequential stages of the GC B cell maturation: Mantle Cell Lymphoma (MCL), Diffuse Large B Cell Lymphoma [DLBCL including ABC-Like (Activated B Cell), GCB-Like (Germinal Center B Cell) and PMBL (Primary Mediastinal B cell Lymphoma)], Burkitt Lymphoma (BL) and Hodgkin Lymphoma (HL). All the NHL subsets showed a dependency on RelA and c-Rel to survive but were largely unaffected by RelB depletion. Only HL cells were dramatically sensitive to RelB knock-down.ChIP-seq analysis uncovered an extensive NFkB network in BJAB (GCB), HBL-1 (ABC), U-H01 (HL) and human primary centroblasts. This data shaped two network architectures: a RelB hierarchical network in HL and a c-rel hierarchy in NHL. Out of 4,581 genes that had relB binding (+/− 2kB TSS) in HL cells, more than 70% were only relB targets in HL as compared to NHL cells.Microarray analysis of these cell lines following RelA, RelB or c-Rel knockdown was merged with our ChIP-seq data. The overlap of RelB peaks and RelB shRNA downregulated targets showed that RelB directly controlled cell cycle regulators and apoptosis inhibitors including CCND3, CDK6, BCL2 and BCLxL. Interestingly, RelB maintained HL cell viability through BCL2 induction since expression of a BCL2 cDNA rescued HL cell lines from the shRNA RelB lethality.Strikingly, the RelB induced BCL2 and BCLxL protein down regulation in HL cells does not kill these cells by predicted apoptotic mechanisms. Morphological and molecular evidence indicated that RelB inhibition lead to cell death by autophagy. Electron microscopy revealed intact nucleus with lack of condensed chromatin and citosolic vacuoles. After RelB inhibition, the well known autophagy marker L3C, is completely degraded meanwhile ATG7 protein expression, a key player in the autophagosomes development, is enhanced. RelB suppressed BCL2/BCL-xL dependent autophagic cell death by maintaining high levels of these two pro-survival molecules interacting with the autophagy executing protein Beclin-1. The interaction is disrupted by the RelB shRNA.We found that RelB is engaged in a positive regulatory loop that keeps the non canonical arm of the NFkB pathway on.Activation of the alternative pathway centers on the modulation of NIK (NFkB inducing kinase). In physiological conditions, NIK undergoes constitutive ubiquitin-dependent degradation through the TRAF-2/TRAF-3/c-IAP-1/c-IAP-2 complex, which keeps its abundance below the threshold required for its function. We found that HL cell lines lack TRAF-2 or TRAF-3 protein expression which leads to stabilization and accumulation of NIK. RelB knock down is followed by up-regulation and/or re-expression of TRAF members and depletion of NIK protein. Remarkably, lymph nodes of HL patient biopsies showed NIK protein citosolic expression by IHC in the Reed-Sternberg Cells, indicating stabilization and accumulation of NIK that accounts for constitutive NFkB-2/p100b processing to release active p52-RelB dimers.In summary, we found that HL is the only GC B cell derived malignancy that relies on RelB for viability, and thus differentiates Hodgkin Lymphomas from non-Hodgkin lymphomas. These data argue that each REL member has specific and unique functions and targets, as we predicted from the lack of similarities in their transactivation domains. Of clinical relevance, HL patient samples reproduced the alternative NFkB pathway molecular alterations that we initially observed in HL cell lines.Furthermore, the unique Hodgkin Lymphoma RelB dependency makes the non canonical NF-kB signaling pathway an attractive therapeutic target. Disclosures:No relevant conflicts of interest to declare.

Characterization and functional analysis of two PKR genes in fugu ( Takifugu rubripes)

PKR (protein kinase R) is a serine–threonine kinase that inhibits protein synthesis by the phosphorylation of the eukaryotic translation initiation factor 2-alpha (eIF2α), and activates NFκB by inducing NFκB-inducing kinase and IκB (inhibitor of NFκB) kinase. This can lead to antiviral and anti-proliferative effects. In this study, the complete sequence and organization of two fugu PKR genes (fPKRs) were determined by in silico analysis and conventional PCR. The full-length fPKR1 and fPKR2 genes were 3832 bp and 4325 bp, which encoded 523 and 492 amino acids, respectively. Both encoded two dsRNA binding domains and a Serine/Threonine protein kinase domain, and showed very high similarity to green spotted puffer PKRs. Gene expression of the two fPKRs was measured by quantitative real-time PCR on tissue samples from healthy fish and peripheral blood leukocytes stimulated with polyinosinic:polycytidylic acid (PolyI:C) or lipopolysaccharides (LPS). The fPKRs were highly expressed in the skin and fPKR2 was significantly induced in PBLs by PolyI:C but not by LPS. The fPKRs inhibited translation of a luciferase reporter gene in a dose-dependent manner and induced transcriptional activity of a mammalian NFκB luciferase reporter. These results demonstrate that two PKRs in a single species can both be independently, but not equally, functional and support the hypothesis that fish PKRs have roles in the innate immune response similar to those of mammalian PKRs.