Deregulated NF-κB Activation Research Articles

Dexamethasone Facilitates NF-κB Signal Pathway in TNF-α Stimulated Rotator Cuff Tenocytes.

Corticosteroids are commonly used for pain control in rotator cuff tear. Deregulated NF-κB activation is a hallmark of chronic inflammatory diseases and has been responsible for the pathogenesis of rotator cuff tear. The Dexamethasone(DEXA) is a synthetic corticosteroid. The purpose of this study was to examine the exact effect of dexamethasone on NF-κB signaling in rotator cuff tear. We measured NF-κB expression in four groups: control, TNF-α-treated, DEXA-treated, and combined treatment with TNF-α and DEXA. Tenocytes were isolated from patients with rotator cuff tears and pre-incubated with TNF-α (10 ng/ml), DEXA (1 µM), or both of them for 10 min, 1 h, and 2 h. Expression of p65, p50, and p52 in the nuclei and cytosol was analyzed by western blotting and immunofluorescence imaging using confocal microscopy. We also evaluated nucleus/cytosol (N/C) ratios of p65, p50, and p52. In our study, the combined treatment with DEXA and TNF-α showed increased N/C ratios of p65, p50, and p52 compared with those in the TNF-α group at all time points. Additionally, in the DEXA group, N/C ratios of p65, p50, and p52 gradually increased from 10 min to 2 h. In conclusion, DEXA promoted the nuclear localization of p65, p50, and p52, but was not effective in inhibiting the inflammatory response of TNF-α-stimulated rotator cuff tear.

Constitutive Activation of NF-κB Pathway in Hematopoietic Stem Cells Causes Loss of Quiescence and Deregulated Transcription Factor Networks.

Identifying physiological roles of specific signaling pathways that regulate hematopoietic stem cell (HSC) functions may lead to new treatment strategies and therapeutic interventions for hematologic disorders. Here, we provide genetic evidence that constitutive activation of NF-κB in HSCs results in reduced pool size, repopulation capacities, and quiescence of HSCs. Global transcriptional profiling and bioinformatics studies identified loss of ‘stemness’ and ‘quiescence’ signatures in HSCs with deregulated NF-κB activation. In particular, gene set enrichment analysis identified upregulation of cyclin dependent kinase- Ccnd1 and down regulation of cyclin dependent kinase inhibitor p57kip2. Interestingly, constitutive activation of NF-κB is sufficient to alter the regulatory circuits of transcription factors (TFs) that are critical to HSC self-renewal and functions. Molecular studies identified Junb, as one of the direct targets of NF-κB in hematopoietic cells. In essence, these studies demonstrate that aberrant activation of NF-κB signals impairs HSC quiescence and functions and alters the ‘TF networks’ in HSCs.

NF-κB Activation in Lymphoid Malignancies: Genetics, Signaling, and Targeted Therapy.

The NF-κB transcription factor family plays a crucial role in lymphocyte proliferation and survival. Consequently, aberrant NF-κB activation has been described in a variety of lymphoid malignancies, including diffuse large B-cell lymphoma, Hodgkin lymphoma, and adult T-cell leukemia. Several factors, such as persistent infections (e.g., with Helicobacter pylori), the pro-inflammatory microenvironment of the cancer, self-reactive immune receptors as well as genetic lesions altering the function of key signaling effectors, contribute to constitutive NF-κB activity in these malignancies. In this review, we will discuss the molecular consequences of recurrent genetic lesions affecting key regulators of NF-κB signaling. We will particularly focus on the oncogenic mechanisms by which these alterations drive deregulated NF-κB activity and thus promote the growth and survival of the malignant cells. As the concept of a targeted therapy based on the mutational status of the malignancy has been supported by several recent preclinical and clinical studies, further insight in the function of NF-κB modulators and in the molecular mechanisms governing aberrant NF-κB activation observed in lymphoid malignancies might lead to the development of additional treatment strategies and thus improve lymphoma therapy.

API2-MALT1 oncoprotein promotes lymphomagenesis via unique program of substrate ubiquitination and proteolysis.

Lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma) is the most common extranodal B cell tumor and accounts for 8% of non-Hodgkin's lymphomas. Gastric MALT lymphoma is the best-studied example and is a prototypical neoplasm that occurs in the setting of chronic inflammation brought on by persistent infection or autoimmune disease. Cytogenetic abnormalities are commonly acquired during the course of disease and the most common is chromosomal translocation t(11;18)(q21;q21), which creates the API2-MALT1 fusion oncoprotein. t(11;18)-positive lymphomas can be clinically aggressive and have a higher rate of dissemination than t(11;18)-negative tumors. Many cancers, including MALT lymphomas, characteristically exhibit deregulated over-activation of cellular survival pathways, such as the nuclear factor-κB (NF-κB) pathway. Molecular characterization of API2-MALT1 has revealed it to be a potent activator of NF-κB, which is required for API2-MALT1-induced cellular transformation, however the mechanisms by which API2-MALT1 exerts these effects are only recently becoming apparent. The API2 moiety of the fusion binds tumor necrosis factor (TNF) receptor associated factor (TRAF) 2 and receptor interacting protein 1 (RIP1), two proteins essential for TNF receptor-induced NF-κB activation. By effectively mimicking ligand-bound TNF receptor, API2-MALT1 promotes TRAF2-dependent ubiquitination of RIP1, which then acts as a scaffold for nucleating and activating the canonical NF-κB machinery. Activation occurs, in part, through MALT1 moiety-dependent recruitment of TRAF6, which can directly modify NF-κB essential modulator, the principal downstream regulator of NF-κB. While the intrinsic MALT1 protease catalytic activity is dispensable for this canonical NF-κB signaling, it is critical for non-canonical NF-κB activation. In this regard, API2-MALT1 recognizes NF-κB inducing kinase (NIK), the essential upstream regulator of non-canonical NF-κB, and cleaves it to generate a stable, constitutively active fragment. Thus, API2-MALT1 harnesses multiple unique pathways to achieve deregulated NF-κB activation. Emerging data from our group and others have also detailed additional gain-of-function activities of API2-MALT1 that extend beyond NF-κB activation. Specifically, API2-MALT1 recruits and subverts multiple other signaling factors, including LIM domain and actin-binding protein 1 (LIMA1) and Smac/DIABLO. Like NIK, LIMA1 represents a unique substrate for API2-MALT1 protease activity, but unlike NIK, its cleavage sets in motion a major NF-κB-independent pathway for promoting oncogenesis. In this review, we highlight the most recent results characterizing these unique and diverse gain-of-function activities of API2-MALT1 and how they contribute to lymphomagenesis.

NF-κB in inflammation and renal diseases.

Nuclear factor κB (NF-κB) is a family of inducible transcription factors that plays a vital role in different aspects of immune responses. NF-κB is normally sequestered in the cytoplasm as inactive complexes via physical association with inhibitory proteins termed IκBs. In response to immune and stress stimuli, NF-κB members become activated via two major signaling pathways, the canonical and noncanonical pathways, and move to the nucleus to exert transcriptional functions. NF-κB is vital for normal immune responses against infections, but deregulated NF-κB activation is a major cause of inflammatory diseases. Accumulated studies suggest the involvement of NF-κB in the pathogenesis of renal inflammation caused by infection, injury, or autoimmune factors. In this review, we discuss the current understanding regarding the activation and function of NF-κB in different types of kidney diseases.

Recurrent alterations ofTNFAIP3(A20) in T-cell large granular lymphocytic leukemia

The pathogenesis of T-cell large granular lymphocytic leukemia (T-LGL) is poorly understood, as STAT3 mutations are the only known frequent genetic lesions. Here, we identified non-synonymous alterations in the TNFAIP3 tumor suppressor gene in 3 of 39 T-LGL. In two cases these were somatic mutations, in one case the somatic origin was likely. A further case harbored a SNP that is a known risk allele for autoimmune diseases and B cell lymphomas. Thus, TNFAIP3 mutations represent recurrent genetic lesions in T-LGL that affect about 8% of cases, likely contributing to deregulated NF-κB activity in this leukemia.

NFκB regulates expression of Polo-like kinase 4

Activation of the NFκB signaling pathway allows the cell to respond to infection and stress and can affect many cellular processes. As a consequence, NFκB activity must be integrated with a wide variety of parallel signaling pathways. One mechanism through which NFκB can exert widespread effects is through controlling the expression of key regulatory kinases. Here we report that NFκB regulates the expression of genes required for centrosome duplication, and that Polo-like kinase 4 (PLK4) is a direct NFκB target gene. RNA interference, chromatin immunoprecipitation, and analysis of the PLK4 promoter in a luciferase reporter assay revealed that all NFκB subunits participate in its regulation. Moreover, we demonstrate that NFκB regulation of PLK4 expression is seen in multiple cell types. Significantly long-term deletion of the NFκB2 (p100/p52) subunit leads to defects in centrosome structure. This data reveals a new component of cell cycle regulation by NFκB and suggests a mechanism through which deregulated NFκB activity in cancer can lead to increased genomic instability and uncontrolled proliferation.

Oncogenic herpesviruses sending mixed signals.

Human herpesvirus infections are generally asymptomatic in immune-competent hosts; however, immune dysfunction can unveil inherent oncogenic properties associated with Epstein-Barr virus (EBV) and Kaposi’s sarcoma herpesvirus (KSHV) infection. Although these viruses were first discovered in human tumor specimens in 1964 and 1994, respectively, it was discovered later on that large proportions of the human population are persistently infected with EBV and to a lesser extent KSHV. To colonize immune-competent hosts, EBV and KSHV drive proliferation of newly infected cells by latent transcription programs. EBV infection is principally limited to B cells, and few infected epithelial cells in the oral mucosa that produce infectious virus for host–host transmission via the saliva. In contrast, KSHV has a broader spectrum of target cells while transmission and life cycle are less well understood. However, these viruses share a host-colonization and persistence strategy that involves activation of NF-κB, a critical survival factor for developing human B cells. Deregulated NF-κB activation can cause uncontrolled B-cell proliferation and secretion of proinflammatory factors that support cancer development. Apart from protumorigenic soluble factors, many tumor cells release endosome-derived vesicles, called exosomes, which mediate intercellular communication. In PNAS, Meckes et al. describe that EBV- and KSHV-infected lymphoma cells secrete exosomes with distinct repertoires of cellular proteins (1). In light of recent studies demonstrating that tumor cell-derived exosomes exert protumorigenic signaling properties (2), the data presented by Meckes et al. suggest that virus and tumor cells share common strategies in shaping a permissive microenvironment by modulating the cargo of secreted exosomes (Fig. 1).

NIK Controls Classical and Alternative NF-κB Activation and Is Necessary for the Survival of Human T-cell Lymphoma Cells

Peripheral T-cell lymphomas (PTCL) are a heterogeneous entity of neoplasms with poor prognosis, a lack of effective therapies, and a largely unknown molecular pathology. Deregulated NF-κB activity has been associated with several lymphoproliferative diseases, but its importance in T-cell lymphomagenesis is poorly understood. We investigated the function of the NF-κB-inducing kinase (NIK), in this pathway and its role as a potential molecular target in T-cell lymphomas. We used immunohistochemistry to analyze the expression of different NF-κB members in primary human PTCL samples and to study its clinical impact. With the aim of inhibiting the pathway, we used genetic silencing of NIK in several T-cell lymphoma cell lines and observed its effect on downstream targets and cell viability. We showed that the NF-κB pathway was activated in a subset of PTCLs associated with poor overall survival. NIK was overexpressed in a number of PTCL cell lines and primary samples, and a pivotal role for NIK in the survival of these tumor cells was unveiled. NIK depletion led to a dramatic induction of apoptosis in NIK-overexpressing cell lines and also showed a more pronounced effect on cell survival than inhibitor of kappa B kinase (IKK) knockdown. NIK silencing induced a blockage of both classical and alternative NF-κB activation and reduced expression of several prosurvival and antiapoptotic factors. The results of the present study indicate that NIK could be a promising therapeutic target in these aggressive malignancies.

Genetic lesions of the TRAF3 and MAP3K14 genes in classical Hodgkin lymphoma

Hodgkin and Reed/Sternberg (HRS) cells in classical Hodgkin lymphoma (cHL) show constitutive activation of nuclear factor (NF)-κB. Several genetic lesions contribute to this deregulated NF-κB activity. Here, we analysed two further NF-κB regulators for genetic lesions, the inhibitory factor TRAF3 and the key signalling component of the alternative NF-κB pathway, MAP3K14 (NIK). Single nucleotide polymorphism (SNP) array analysis of cHL cell lines revealed a uniparental disomy of the long arm of chromosome 14 associated with a biallelic deletion of TRAF3 located on this chromosome in cell line U-HO1. Cloning of the deletion breakpoint showed a 123371 bp deletion. No inactivating mutations of TRAF3 were found in six other cHL cell lines or in microdissected HRS cells from seven cHL. However, in primary cHL samples interphase cytogenetic analyses revealed signal patterns indicating monoallelic deletion of TRAF3 in 3/20 other cases. SNP array analysis revealed a gain of copy number for MAP3K14 in three cHL cell lines. Gains of MAP3K14 were detected in 5/16 cases of primary cHL. In conclusion, in rare instances, HRS cells harbour inactivating mutations of the TRAF3 gene and recurrently show gains of MAP3K14, indicating that more components of NF-κB signalling show genetic lesions in HRS cells than previously known.

A special issue on NF-κB signaling and function

A special issue on NF-κB signaling and function

NF-κB as a target for oncogenic viruses.

NF-κB is a pivotal transcription factor that controls cell survival and proliferation in diverse physiological processes. The activity of NF-κB is tightly controlled through its cytoplasmic sequestration by specific inhibitors, IκBs. Various cellular stimuli induce the activation of an IκB kinase, which phosphorylates IκBs and triggers their proteasomal degradation, causing nuclear translocation of activated NF-κB. Under normal conditions, the activation of NF-κB occurs transiently, thus ensuring rapid but temporary induction of target genes. Deregulated NF-κB activation contributes to the development of various diseases, including cancers and immunological disorders. Accumulated studies demonstrate that the NF-κB signaling pathway is a target of several human oncogenic viruses, including the human T cell leukemia virus type 1, the Kaposi sarcoma-associated herpesvirus, and the Epstein-Bar virus. These viruses encode specific oncoproteins that target different signaling components of the NF-κB pathway, leading to persistent activation of NF-κB. This chapter will discuss the molecular mechanisms by which NF-κB is activated by the viral oncoproteins.

NIK and the Alternative NF-κB Pathway in Human Lymphomas.

Abstract 3943Poster Board III-879The nuclear factor κB (NF-κB) family of transcription factors is required for the development of T and B lymphocytes and the regulation of the innate and adaptive immune response. Deregulated NF-κB activity has been associated with a number of malignancies and several types of lymphomas depend on NF-κB activity for cell proliferation and survival. A number of studies have been published in the last few years demonstrating the importance of the alternative NF-κB pathway in lymphomas. The NF-κB-Inducing Kinase (NIK or MAP3K14) is a serine/threonine kinase that is essential for the activation of the alternative NF-κB pathway. NIK induces the phosphorylation of the NF-κB member p100, which is followed by the processing of p100 to p52 and its subsequent nuclear translocation. Gene expression data from 106 lymphoma samples with different diagnosis (diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), mucosa-associated lymphoid tissue (MALT), nodal marginal zone lymphoma (NMZL) and chronic lymphocytic leukemia (CLL)) were previously generated using Agilent oligonucleotide microarrays. Data analysis indicated variability in NIK expression among the different lymphoma samples, showing a higher expression in CLL and FL samples. NIK expression was closely associated with the expression of other members of the alternative NF-κB pathway, such as NFKB2, RELB and CD40. To investigate the correlation between NIK gene expression and functional pathways, we performed a gene set enrichment analysis (GSEA). We found that the expression of NIK was positively and significantly correlated with several biologically important pathways in both lymphomagenesis and normal leukocyte development and function, such as B- and T-cell receptor pathways, CD40 signaling pathway, and the classical and alternative NF-κB pathways. Twenty seven lymphoma-derived cell lines were examined by Western blot for expression of NIK and p100/p52. The pathway was found to be frequently activated in these cell lines since high levels of p52 were detected in the majority of MCL (5/9 cell lines), Hodgkin lymphoma (3/3), CLL (1/1), DLBCL (7/9) and T-cell lymphoma (5/5) cell lines. Two-thirds of these p52-positive cell lines also expressed NIK and three of them expressed a truncated form of p100. The expression of NIK and p52 was also associated with Epstein-Barr virus (EBV) infection, given that four out of five EBV-positive cell lines showed elevated levels of these proteins. p100/p52 expression was immunohistochemically analyzed using tissue microarrays including paraffin-embedded tissues from totally 356 DLBCL patients. Examination of these samples indicated that the alternative pathway is activated in a subset of these tumors, with nuclear p52 expressed in 17% of the cases. A significant positive correlation between EBV-infection and p52 expression was established and the majority of the p52-positive cases also expressed nuclear p50, suggesting that both the alternative and classical NF-κB pathways are frequently activated in the same tumors. Alternative and classical NF-κB activation was more frequently present in the non-GC DLBCL type, but also observed in a significant proportion of the GC-DLBCL cases. Furthermore, array based comparative genomic hybridization (aCGH) revealed mono-allelic loss of TRAF3, a negative regulator of NIK, in 5 out of 22 DLBCL cases. Taken together, our results show that the alternative NF-κB pathway is activated in a subset of human lymphoma samples and cell lines. This highlights the relevance of NIK as a potential therapeutic target in lymphoid malignancies. Disclosures:No relevant conflicts of interest to declare.

Lucifer's Labyrinth—Ten Years of Path Finding in Cell Death

Cell death, as a foil to cell expansion, has become one of the most intensively studied research areas of modern biology. Almost every aspect of life is intimately enmeshed with the proper regulation of cell death, which either contributes to or forms the basis of most human disease pathogenesis. Now, with our ever-expanding knowledge of the various death pathways, comes the hope that we might harness cell death--by either inhibition or promotion--to translate the concepts into cure.

Deregulated NF-κB activity in haematological malignancies

The NF-κB family of transcription factors plays key roles in the control of cell proliferation and apoptosis. Constitutive NF-κB activation is a common feature for most haematological malignancies and is therefore believed to be a crucial event for enhanced proliferation and survival of these malignant cells. In this review, we will describe the molecular mechanisms underlying NF-κB deregulation in haematological malignancies and will highlight what is still unclear in this field, 20 years after the discovery of this transcription factor.