A conserved core region of the scaffold NEMO is essential for signal-induced conformational change and liquid-liquid phase separation.

Scaffold proteins help mediate interactions between protein partners, often to optimize intracellular signaling. Herein, we use comparative, biochemical, biophysical, molecular, and cellular approaches to investigate how the scaffold protein NEMO contributes to signaling in the NF-κB pathway. Comparison of NEMO and the related protein optineurin from a variety of evolutionarily distant organisms revealed that a central region of NEMO, called the Intervening Domain (IVD), is conserved between NEMO and optineurin. Previous studies have shown that this central core region of the IVD is required for cytokine-induced activation of IκB kinase (IKK). We show that the analogous region of optineurin can functionally replace the core region of the NEMO IVD. We also show that an intact IVD is required for the formation of disulfide-bonded dimers of NEMO. Moreover, inactivating mutations in this core region abrogate the ability of NEMO to form ubiquitin-induced liquid-liquid phase separation droplets in vitro and signal-induced puncta in vivo. Thermal and chemical denaturation studies of truncated NEMO variants indicate that the IVD, while not intrinsically destabilizing, can reduce the stability of surrounding regions of NEMO due to the conflicting structural demands imparted on this region by flanking upstream and downstream domains. This conformational strain in the IVD mediates allosteric communication between the N- and C-terminal regions of NEMO. Overall, these results support a model in which the IVD of NEMO participates in signal-induced activation of the IKK/NF-κB pathway by acting as a mediator of conformational changes in NEMO.

Starvation differentially affects gene expression, immunity and pathogen susceptibility across symbiotic states in a model cnidarian.

Mutualistic symbioses between cnidarians and photosynthetic algae are modulated by complex interactions between host immunity and environmental conditions. Here, we investigate how symbiosis interacts with food limitation to influence gene expression and stress response programming in the sea anemone Exaiptasia pallida (Aiptasia). Transcriptomic responses to starvation were similar between symbiotic and aposymbiotic Aiptasia; however, aposymbiotic anemone responses were stronger. Starved Aiptasia of both symbiotic states exhibited increased protein levels of immune-related transcription factor NF-κB, its associated gene pathways, and putative target genes. However, this starvation-induced increase in NF-κB correlated with increased immunity only in symbiotic anemones. Furthermore, starvation had opposite effects on Aiptasia susceptibility to pathogen and oxidative stress challenges, suggesting distinct energetic priorities under food scarce conditions. Finally, when we compared starvation responses in Aiptasia to those of a facultative coral and non-symbiotic anemone, 'defence' responses were similarly regulated in Aiptasia and the facultative coral, but not in the non-symbiotic anemone. This pattern suggests that capacity for symbiosis influences immune responses in cnidarians. In summary, expression of certain immune pathways-including NF-κB-does not necessarily predict susceptibility to pathogens, highlighting the complexities of cnidarian immunity and the influence of symbiosis under varying energetic demands.

An innate ability: How do basal invertebrates manage their chronic exposure to microbes?

Homologs of mammalian innate immune sensing and downstream pathway proteins have been discovered in a variety of basal invertebrates, including cnidarians and sponges, as well as some single-celled protists. Although the structures of these proteins vary among the basal organisms, many of the activities found in their mammalian counterparts are conserved. This is especially true for the Toll-like receptor (TLR) and cGAS-STING pathways that lead to downstream activation of transcription factor NF-κB. In this short perspective, we describe the evidence that TLR and cGAS-STING signaling to NF-κB is also involved in immunity in basal animals, as well as in the maintenance of microbial symbionts. Different from terrestrial animals, immunity in many marine invertebrates might have a constitutively active state (to protect against continual exposure to resident or waterborne microbes), as well as a hyperactive state that can be induced by pathogens at both transcriptional and posttranscriptional levels. Research on basal immunity may be important for (1) understanding different approaches that organisms take to sensing and protecting against microbes, as well as in maintaining microbial symbionts; (2) the identification of novel antimicrobial effector genes and processes; and (3) the molecular pathways that are being altered in basal marine invertebrates in the face of the effects of a changing environment.

Scaffold proteins as dynamic integrators of biological processes.

Scaffold proteins act as molecular hubs for the docking of multiple proteins to organize efficient functional units for signaling cascades. Over 300 human proteins have been characterized as scaffolds, acting in a variety of signaling pathways. While the term scaffold implies a static, supportive platform, it is now clear that scaffolds are not simply inert docking stations but can undergo conformational changes that affect their dependent signaling pathways. In this review, we catalog scaffold proteins that have been shown to undergo actionable conformational changes, with a focus on the role that conformational change plays in the activity of the classic yeast scaffold STE5, as well as three human scaffold proteins (KSR, NEMO, SHANK3) that are integral to well-known signaling pathways (RAS, NF-κB, postsynaptic density). We also discuss scaffold protein conformational changes vis-à-vis liquid-liquid phase separation. Changes in scaffold structure have also been implicated in human disease, and we discuss how aberrant conformational changes may be involved in disease-related dysregulation of scaffold and signaling functions. Finally, we discuss how understanding these conformational dynamics will provide insight into the flexibility of signaling cascades and may enhance our ability to treat scaffold-associated diseases.

Sea anemone model has a single Toll-like receptor that can function in pathogen detection, NF-κB signal transduction, and development.

In organisms from insects to vertebrates, Toll-like receptors (TLRs) are primary pathogen detectors that activate downstream pathways, specifically those that direct expression of innate immune effector genes. TLRs also have roles in development in many species. The sea anemone Nematostella vectensis is a useful cnidarian model to study the origins of TLR signaling because its genome encodes a single TLR and homologs of many downstream signaling components, including the NF-κB pathway. We have characterized the single N. vectensis TLR (Nv-TLR) and demonstrated that it can activate canonical NF-κB signaling in human cells. Furthermore, we show that the intracellular Toll/IL-1 receptor (TIR) domain of Nv-TLR can interact with the human TLR adapter proteins MAL and MYD88. We demonstrate that the coral pathogen Vibrio coralliilyticus causes a rapidly lethal disease in N. vectensis and that heat-inactivated V. coralliilyticus and bacterial flagellin can activate a reconstituted Nv-TLR-to-NF-κB pathway in human cells. By immunostaining of anemones, we show that Nv-TLR is expressed in a subset of cnidocytes and that many of these Nv-TLR-expressing cells also express Nv-NF-κB. Additionally, the nematosome, which is a Nematostella-specific multicellular structure, expresses Nv-TLR and many innate immune pathway homologs and can engulf V. coralliilyticus Morpholino knockdown indicates that Nv-TLR also has an essential role during early embryonic development. Our characterization of this primitive TLR and identification of a bacterial pathogen for N. vectensis reveal ancient TLR functions and provide a model for studying the molecular basis of cnidarian disease and immunity.

A Central Region of NF-κB Essential Modulator Is Required for IKKß-Induced Conformational Change and for Signal Propagation.

NF-κB essential modulator (NEMO) regulates NF-κB signaling by acting as a scaffold for the kinase IKKß to direct its activity toward the NF-κB inhibitor, IκBα. Here, we show that a highly conserved central region of NEMO termed the intervening domain (IVD, amino acids 112-195) plays a key role in NEMO function. We determined a structural model of full-length NEMO by small-angle X-ray scattering and show that full-length, wild-type NEMO becomes more compact upon binding of a peptide comprising the NEMO binding domain of IKKß (amino acids 701-745). Mutation of conserved IVD residues (9SG-NEMO) disrupts this conformational change in NEMO and abolishes the ability of NEMO to propagate NF-κB signaling in cells, although the affinity of 9SG-NEMO for IKKß compared to that of the wild type is unchanged. On the basis of these results, we propose a model in which the IVD is required for a conformational change in NEMO that is necessary for its ability to direct phosphorylation of IκBα by IKKß. Our findings suggest a molecular explanation for certain disease-associated mutations within the IVD and provide insight into the role of conformational change in signaling scaffold proteins.

Evolutionary Origins of Toll-like Receptor Signaling.

Toll-like receptors (TLRs) are transmembrane pattern recognition receptors that are best known for their roles in innate immunity for the detection of and defense against microbial pathogens. However, TLRs also have roles in many nonimmune processes, most notably development. TLRs direct both immune and developmental programs by activation of downstream signaling pathways, often by activation of the NF-κB pathway. There are two primary TLR subtypes: 1) TLRs with multiple cysteine clusters in their ectodomain (mccTLRs) and 2) TLRs with a single cysteine cluster in their ectodomain (sccTLRs). For some time, it has been known that TLRs and the biological processes that they control are conserved in organisms from insects to mammals. However, genome and transcriptome sequencing has revealed that many basal metazoans also have TLRs and downstream NF-κB signaling components. In this review, we discuss what is known about the structure, biological function, and downstream signaling pathways of TLRs found in phyla from Porifera through Annelida. From these analyses, we hypothesize that mccTLRs emerged in the phylum Cnidaria, that sccTLRs evolved in the phylum Mollusca, and that TLRs have dual immune and developmental biological functions in organisms as ancient as cnidarians.

NF-κB: where did it come from and why?

The vast majority of research on nuclear factor κB (NF-κB) signaling in the past 25 years has focused on its roles in normal and disease-related processes in vertebrates, especially mice and humans. Recent genome and transcriptome sequencing efforts have shown that homologs of NF-κB transcription factors, inhibitor of NF-κB (IκB) proteins, and IκB kinases are present in a variety of invertebrates, including several in phyla simpler than Arthropoda, the phylum containing insects such Drosophila. Moreover, many invertebrates also contain genes encoding homologs of upstream signaling proteins in the Toll-like receptor signaling pathway, which is well-known for its downstream activation of NF-κB for innate immunity. This review describes what we now know or can infer and speculate about the evolution of the core elements of NF-κB signaling as well as the biological processes controlled by NF-κB in invertebrates. Further research on NF-κB in invertebrates is likely to uncover information about the evolutionary origins of this key human signaling pathway and may have relevance to our management of the responses of ecologically and economically important organisms to environmental and adaptive pressures.

Multiple myeloma: lusting for NF-kappaB.

Multiple myeloma (MM) is a late-stage B cell malignancy that has received much attention recently because of its therapeutic susceptibility to proteasome inhibitors. Two papers in this issue of Cancer Cell show that primary MM samples and MM cell lines frequently have mutations in genes encoding regulators and effectors of NF-kappaB signaling, and that these mutations lead to chronic NF-kappaB target gene expression, which is required for the viability of these MM tumor cells. These results reveal the molecular basis for constitutive NF-kappaB activity in many MMs and further validate the NF-kappaB signaling pathway as an appropriate target for MM therapy.

Inhibition of Oncogenic Transcription Factor REL by the Natural Product Derivative Calafianin Monomer 101 Induces Proliferation Arrest and Apoptosis in Human B-Lymphoma Cell Lines.

Increased activity of transcription factor NF-κB has been implicated in many B-cell lymphomas. We investigated effects of synthetic compound calafianin monomer (CM101) on biochemical and biological properties of NF-κB. In human 293 cells, CM101 selectively inhibited DNA binding by overexpressed NF-κB subunits REL (human c-Rel) and p65 as compared to NF-κB p50, and inhibition of REL and p65 DNA binding by CM101 required a conserved cysteine residue. CM101 also inhibited DNA binding by REL in human B-lymphoma cell lines, and the sensitivity of several B-lymphoma cell lines to CM101-induced proliferation arrest and apoptosis correlated with levels of cellular and nuclear REL. CM101 treatment induced both phosphorylation and decreased expression of anti-apoptotic protein Bcl-XL, a REL target gene product, in sensitive B-lymphoma cell lines. Ectopic expression of Bcl-XL protected SUDHL-2 B-lymphoma cells against CM101-induced apoptosis, and overexpression of a transforming mutant of REL decreased the sensitivity of BJAB B-lymphoma cells to CM101-induced apoptosis. Lipopolysaccharide-induced activation of NF-κB signaling upstream components occurred in RAW264.7 macrophages at CM101 concentrations that blocked NF-κB DNA binding. Direct inhibitors of REL may be useful for treating B-cell lymphomas in which REL is active, and may inhibit B-lymphoma cell growth at doses that do not affect some immune-related responses in normal cells.

Disulfide-mediated stabilization of the IκB kinase binding domain of NF-κB essential modulator (NEMO).

Human NEMO (NF-κB essential modulator) is a 419 residue scaffolding protein that, together with catalytic subunits IKKα and IKKß, forms the IκB kinase (IKK) complex, a key regulator of NF-κB pathway signaling. NEMO is an elongated homodimer comprising mostly α-helix. It has been shown that a NEMO fragment spanning residues 44-111, which contains the IKKα/ß binding site, is structurally disordered in the absence of bound IKKß. Herein we show that enforcing dimerization of NEMO1-120 or NEMO44-111 constructs through introduction of one or two interchain disulfide bonds, through oxidation of the native Cys54 residue and/or at position 107 through a Leu107Cys mutation, induces a stable α-helical coiled-coil structure that is preorganized to bind IKKß with high affinity. Chemical and thermal denaturation studies showed that, in the context of a covalent dimer, the ordered structure was stabilized relative to the denatured state by up to 3 kcal/mol. A full-length NEMO-L107C protein formed covalent dimers upon treatment of mammalian cells with H2O2. Furthermore, NEMO-L107C bound endogenous IKKß in A293T cells, reconstituted TNF-induced NF-κB signaling in NEMO-deficient cells, and interacted with TRAF6. Our results indicate that the IKKß binding domain of NEMO possesses an ordered structure in the unbound state, provided that it is constrained within a dimer as is the case in the constitutively dimeric full-length NEMO protein. The stability of the NEMO coiled coil is maintained by strong interhelix interactions in the region centered on residue 54. The disulfide-linked constructs we describe herein may be useful for crystallization of NEMO's IKKß binding domain in the absence of bound IKKß, thereby facilitating the structural characterization of small-molecule inhibitors.

NF-κB is required for cnidocyte development in the sea anemone Nematostella vectensis.

The sea anemone Nematostella vectensis (Nv) is a leading model organism for the phylum Cnidaria, which includes anemones, corals, jellyfishes and hydras. A defining trait across this phylum is the cnidocyte, an ectodermal cell type with a variety of functions including defense, prey capture and environmental sensing. Herein, we show that the Nv-NF-κB transcription factor and its inhibitor Nv-IκB are expressed in a subset of cnidocytes in the body column of juvenile and adult anemones. The size and distribution of the Nv-NF-κB-positive cnidocytes suggest that they are in a subtype known as basitrichous haplonema cnidocytes. Nv-NF-κB is primarily cytoplasmic in cnidocytes in juvenile and adult animals, but is nuclear when first detected in the 30-h post-fertilization embryo. Morpholino-mediated knockdown of Nv-NF-κB expression results in greatly reduced cnidocyte formation in the 5 day-old animal. Taken together, these results indicate that NF-κB plays a key role in the development of the phylum-specific cnidocyte cell type in Nematostella, likely by nuclear Nv-NF-κB-dependent activation of genes required for cnidocyte development.

Histone acetyltransferase-deficient p300 mutants in diffuse large B cell lymphoma have altered transcriptional regulatory activities and are required for optimal cell growth.

BACKGROUND: Recent genome-wide studies have shown that approximately 30% of diffuse large B-cell lymphoma (DLBCL) cases harbor mutations in the histone acetyltransferase (HAT) coactivators p300 or CBP. The majority of these mutations reduce or eliminate the catalytic HAT activity. We previously demonstrated that the human DLBCL cell line RC-K8 expresses a C-terminally truncated, HAT-defective p300 protein (p300ΔC-1087), whose expression is essential for cell proliferation. METHODS: Using results from large-scale DLBCL studies, we have identified and characterized a second C-terminally truncated, HAT-defective p300 mutant, p300ΔC-820, expressed in the SUDHL2 DLBCL cell line. Properties of p300ΔC-820 were characterized in the SUDHL2 DLBCL cell line by Western blotting, co-immunoprecipitation, and shRNA gene knockdown, as well by using cDNA expression vectors for p300ΔC-820 in pull-down assays, transcriptional reporter assays, and immunofluorescence experiments. A mass spectrometry-based method was used to compare the histone acetylation profile of DLBCL cell lines expressing various levels of wild-type p300. RESULTS: We show that the SUDHL2 cell line expresses a C-terminally truncated, HAT-defective form of p300 (p300ΔC-820), but no wild-type p300. The p300ΔC-820 protein has a wild-type ability to localize to subnuclear "speckles," but has a reduced ability to enhance transactivation by transcription factor REL. Knockdown of p300ΔC-820 in SUDHL2 cells reduced their proliferation and soft agar colony-forming ability. In RC-K8 cells, knockdown of p300ΔC-1087 resulted in increased expression of mRNA and protein for REL target genes A20 and IκBα, two genes that have been shown to limit the growth of RC-K8 cells when overexpressed. Among a panel of B-lymphoma cell lines, low-level expression of full-length p300 protein, which is characteristic of the SUDHL2 and RC-K8 cells, was associated with decreased acetylation of histone H3 at lysines 14 and 18. CONCLUSIONS: The high prevalence of p300 mutations in DLBCL suggests that HAT-deficient p300 activity defines a subtype of DLBCL, which we have investigated using human DLBCL cell lines RC-K8 and SUDHL2. Our results suggest that truncated p300 proteins contribute to DLBCL cell growth by affecting the expression of specific genes, perhaps through a mechanism that involves alterations in global histone acetylation.

Mutation of nonessential cysteines shows that the NF-κB essential modulator forms a constitutive noncovalent dimer that binds IκB kinase-ß with high affinity.

NEMO (NF-κB essential modulator) associates with catalytic subunits IKKα and IKKß to form the IκB kinase (IKK) complex and is a key regulator of NF-κB pathway signaling. Biochemical and structural characterization of NEMO has been challenging, however, leading to conflicting data about basic biochemical properties such as the oligomeric state of active NEMO and its binding affinity for IKKß. We show that up to seven of NEMO's 11 cysteine residues can be mutated to generate recombinant full-length NEMO that is highly soluble and active. Using a fluorescence anisotropy binding assay, we show that full-length NEMO binds a 44-mer peptide encompassing residues 701-745 of IKKß with a K(D) of 2.2 ± 0.8 nM. The IKKß binding affinities of mutants with five and seven Cys-to-Ala substitutions are indistinguishable from that of wild-type NEMO. Moreover, when expressed in NEMO -/- fibroblasts, the five-Ala and seven-Ala NEMO mutants can interact with cellular IKKß and restore NF-κB signaling to provide protection against tumor necrosis factor α-induced cell death. Treatment of the NEMO-reconstituted cells with H2O2 led to the formation of covalent dimers for wild-type NEMO and the five-Ala mutant, but not for the seven-Ala mutant, confirming that Cys54 and/or Cys347 can mediate interchain disulfide bonding. However, the IKKß binding affinity of NEMO is unaffected by the presence or absence of interchain disulfide bonding at Cys54, which lies within the IKKß binding domain of NEMO, or at Cys347, indicating that NEMO exists as a noncovalent dimer independent of the redox state of its cysteines. This conclusion was corroborated by the observation that the secondary structure content of NEMO and its thermal stability were independent of the presence or absence of interchain disulfide bonds.

Identification of an NF-κB p50/p65-responsive site in the human MIR155HG promoter.

BACKGROUND: MicroRNA-155 (miR-155) is the diced product of the MIR155HG gene. miR-155 regulates the expression of many immune-specific transcripts, is overexpressed in many human lymphomas, and has oncogenic activity in mouse transgenic models. MIR155HG has been proposed to be a target gene for transcription factor NF-κB largely due to the positive correlation between high nuclear NF-κB activity and increased miR-155 expression following treatment with NF-κB inducers or in subsets of hematopoietic cancers. Nevertheless, direct regulation of the human MIR155HG promoter by NF-κB has not been convincingly demonstrated previously. RESULTS: This report shows that induction of NF-κB activity rapidly leads to increased levels of both primary MIR155HG mRNA and mature miR-155 transcripts. We have mapped an NF-κB-responsive element to a position approximately 178 nt upstream of the MIR155HG transcription start site. The -178 site is specifically bound by the NF-κB p50/p65 heterodimer and is required for p65-induced reporter gene activation. Moreover, the levels of miR-155 in nine human B-lymphoma cell lines generally correlate with increased nuclear NF-κB proteins. CONCLUSION: Overall, the identification of an NF-κB-responsive site in the MIR155HG proximal promoter suggests that MIR155HG is a direct NF-κB target gene in vivo. Understanding NF-κB-mediated regulation of miR-155 could lead to improved immune cell-related diagnostic tools and targeted therapies.

A report from the second Nematostella vectensis research conference.

This report summarizes information discussed at the second Nematostella vectensis research conference, which took place on August 27, 2012 in Boston, MA, USA. The startlet sea anemone Nematostella is emerging as one of leading model organisms among cnidarians, in part because of the extensive genome and transcriptome resources that are becoming available for Nematostella, which were the focus of several presentations. In addition, research was presented on the use of Nematostella in developmental, regeneration, signal transduction, host-symbiont, and gene-environment interaction studies.

Starvation decreases immunity and immune regulatory factor NF-κB in the starlet sea anemone Nematostella vectensis.

Lack of proper nutrition has important consequences for the physiology of all organisms, and nutritional status can affect immunity, based on many studies in terrestrial animals. Here we show a positive correlation between nutrition and immunity in the sea anemone Nematostella vectensis. Gene expression profiling of adult anemones shows downregulation of genes involved in nutrient metabolism, cellular respiration, and immunity in starved animals. Starved adult anemones also have reduced protein levels and activity of immunity transcription factor NF-κB. Starved juvenile anemones have increased sensitivity to bacterial infection and also have lower NF-κB protein levels, as compared to fed controls. Weighted Gene Correlation Network Analysis (WGCNA) is used to identify significantly correlated gene networks that were downregulated with starvation. These experiments demonstrate a correlation between nutrition and immunity in an early diverged marine metazoan, and the results have implications for the survival of marine organisms as they encounter changing environments.

A Conserved Core Region of the Scaffold NEMO is Essential for Signal-induced Conformational Change and Liquid-liquid Phase Separation.

Scaffold proteins help mediate interactions between protein partners, often to optimize intracellular signaling. Herein, we use comparative, biochemical, biophysical, molecular, and cellular approaches to investigate how the scaffold protein NEMO contributes to signaling in the NF-κB pathway. Comparison of NEMO and the related protein optineurin from a variety of evolutionarily distant organisms revealed that a central region of NEMO, called the Intervening Domain (IVD), is conserved between NEMO and optineurin. Previous studies have shown that this central core region of the IVD is required for cytokine-induced activation of IκB kinase (IKK). We show that the analogous region of optineurin can functionally replace the core region of the NEMO IVD. We also show that an intact IVD is required for the formation of disulfide-bonded dimers of NEMO. Moreover, inactivating mutations in this core region abrogate the ability of NEMO to form ubiquitin-induced liquid-liquid phase separation droplets in vitro and signal-induced puncta in vivo. Thermal and chemical denaturation studies of truncated NEMO variants indicate that the IVD, while not intrinsically destabilizing, can reduce the stability of surrounding regions of NEMO, due to the conflicting structural demands imparted on this region by flanking upstream and downstream domains. This conformational strain in the IVD mediates allosteric communication between N- and C-terminal regions of NEMO. Overall, these results support a model in which the IVD of NEMO participates in signal-induced activation of the IKK/NF-κB pathway by acting as a mediator of conformational changes in NEMO.

NF-kappaB down-regulates expression of the B-lymphoma marker CD10 through a miR-155/PU.1 pathway.

Cell-surface protein CD10 is a prognostic marker for diffuse large B-cell lymphoma (DLBCL), where high expression of CD10 is found in the germinal center B-cell (GCB) subtype and CD10 expression is low or absent in the activated B-cell (ABC) subtype. As compared with the GCB subtype, patients with ABC DLBCL have a poorer prognosis after standard treatment, and ABC tumor cells have higher NF-κB activity. Herein, we show that increased expression of the NF-κB target micro-RNA miR-155 is correlated with reduced expression of transcription factor PU.1 and CD10 in several B-lymphoma cell lines. Moreover, electromobility shift assays and luciferase reporter assays indicate that PU.1 can directly activate expression from the CD10 promoter. Expression of a DLBCL-derived mutant of the adaptor CARD11 (a constitutive activator of NF-κB) in the GCB-like human BJAB cell line or v-Rel in the chicken DT40 B-lymphoma cell line causes reduced expression of PU.1. The CARD11 mutant also causes a decrease in CD10 levels in BJAB cells. Similarly, overexpression of miR-155, which is known to down-regulate PU.1, leads to reduced expression of CD10 in BJAB cells. Finally, we show that CD10 expression is reduced in BJAB cells after treatment with the NF-κB inducer lipopolysaccharide (LPS). Additionally, miR-155 is induced by LPS treatment or expression of the CARD11 mutant in BJAB cells. These results point to an NF-κB-dependent mechanism for down-regulation of CD10 in B-cell lymphoma: namely, that increased NF-κB activity leads to increased miR-155, which results in decreased PU.1, and consequently reduced CD10 mRNA and protein.