NF-κB Dimers Research Articles

Hidden Native State Ensembles of NFκB Dimers Provide Insights into their Different DNA-Binding Affinities

Hidden Native State Ensembles of NFκB Dimers Provide Insights into their Different DNA-Binding Affinities

Resolving the NFκB Heterodimer Binding Paradox: Strain and Frustration Guide the Binding of Dimeric Transcription Factors.

Many eukaryotic transcription factors function after forming oligomers. The choice of protein partners is a nonrandom event that has distinct functional consequences for gene regulation. In the present work we examine three dimers of transcription factors in the NFκB family: p50p50, p50p65, and p65p65. The NFκB dimers bind to a myriad of genomic sites and switch the targeted genes on or off with precision. The p65p50 heterodimer of NFκB is the strongest DNA binder, and its unbinding is controlled kinetically by molecular stripping from the DNA induced by IκB. In contrast, the homodimeric forms of NFκB, p50p50 and p65p65, bind DNA with significantly less affinity, which places the DNA residence of the homodimers under thermodynamic rather than kinetic control. It seems paradoxical that the heterodimer should bind more strongly than either of the symmetric homodimers since DNA is a nearly symmetric target. Using a variety of energy landscape analysis tools, here we uncover the features in the molecular architecture of NFκB dimers that are responsible for these drastically different binding free energies. We show that frustration in the heterodimer interface gives the heterodimer greater conformational plasticity, allowing the heterodimer to better accommodate the DNA. We also show how the elastic energy and mechanical strain in NFκB dimers can be found by extracting the principal components of the fluctuations in Cartesian coordinates as well as fluctuations in the space of physical contacts, which are sampled via simulations with a predictive energy landscape Hamiltonian. These energetic contributions determine the specific detailed mechanisms of binding and stripping for both homo- and heterodimers.

DRES-17. BCL3 IS A 19q PASSENGER THAT PREDICTS RESPONSE TO ALKYLATING CHEMOTHERAPY IN GBM

Despite the universal use of temozolomide (TMZ) in GBM, many patients experience minimal benefit. Moreover, TMZ can be deleterious due to its mutagenic nature. These findings underline the importance of predicting which patients will benefit from TMZ. DATA: Bcl-3 was identified as being involved in cytotoxicity by TMZ due to its unique interaction with NF-κB residues modified following treatment. Up- and down-regulation of Bcl-3 in patient-derived glioma stem-like cells (GSCs) demonstrates that Bcl-3 blocks cytotoxicity by TMZ in vitro and in an intracranial PDX. Analysis of multiple public GBM datasets demonstrates that BCL3 mRNA expression is a marker of outcome, a finding confirmed on multivariate analysis incorporating MGMT promoter methylation, age and IDH mutation among other variables. Most notably, separation of patients in TCGA by treatment modality demonstrates that BCL3 is only informative in patients who received alkylating chemotherapy not untreated patients or those receiving ionizing radiation (IR) alone, a finding significant on multivariate analysis and validated in REMBRANDT. To examine Bcl-3 protein, TMAs were constructed incorporating 86 GBMs. Bcl-3 immunohistochemistry graded on a 4-tier system and dichotomized significantly separates patients into survival groups (P<0.0001; HR, 5.418; 95%CI, 2.747–9.647). In GBM, Bcl-3 mRNA and protein level is regulated by gene copy number determined in public datasets and in TMAs by FISH. CNAs of BCL3 occur due to changes directed to a broad region of 19q13, an area deleted in 20–40% of all types of glioma. Mechanistically, Bcl-3 promotes resistance by inducing mesenchymal differentiation via promoter-specific NF-κB dimer exchange, shown in GSCs and confirmed in TCGA GBM. BCL3 is an independent predictor of response to TMZ in GBM whose expression is regulated by CNA targeted to 19q13 that inadvertently affect BCL3. Bcl-3 expression level represents a cell-intrinsic modulator of NF-κB activity that promotes mesenchymal differentiation and resistance to therapy.

DNA-binding affinity and transcriptional activity of the RelA homodimer of nuclear factor κB are not correlated

The nuclear factor κB (NF-κB) transcription factor family regulates genes involved in cell proliferation and inflammation. The promoters of these genes often contain NF-κB-binding sites (κB sites) arranged in tandem. How NF-κB activates transcription through these multiple sites is incompletely understood. We report here an X-ray crystal structure of homodimers comprising the RelA DNA-binding domain containing the Rel homology region (RHR) in NF-κB bound to an E-selectin promoter fragment with tandem κB sites. This structure revealed that two dimers bind asymmetrically to the symmetrically arranged κB sites at which multiple cognate contacts between one dimer to the corresponding DNA are broken. Because simultaneous RelA-RHR dimer binding to tandem sites in solution was anti-cooperative, we inferred that asymmetric RelA-RHR binding with fewer contacts likely indicates a dissociative binding mode. We found that both κB sites are essential for reporter gene activation by full-length RelA homodimer, suggesting that dimers facilitate DNA binding to each other even though their stable co-occupation is not promoted. Promoter variants with altered spacing and orientation of tandem κB sites displayed unexpected reporter activities that were not explained by the solution-binding pattern of RelA-RHR. Remarkably, full-length RelA bound all DNAs with a weaker affinity and specificity. Moreover, the transactivation domain played a negative role in DNA binding. These observations suggest that other nuclear factors influence full-length RelA binding to DNA by neutralizing the transactivation domain negative effect. We propose that DNA binding by NF-κB dimers is highly complex and modulated by facilitated association-dissociation processes.

Regulation of NF-κB by the p105-ABIN2-TPL2 complex and RelAp43 during rabies virus infection.

At the crossroad between the NF-κB and the MAPK pathways, the ternary complex composed of p105, ABIN2 and TPL2 is essential for the host cell response to pathogens. The matrix protein (M) of field isolates of rabies virus was previously shown to disturb the signaling induced by RelAp43, a NF-κB protein close to RelA/p65. Here, we investigated how the M protein disturbs the NF-κB pathway in a RelAp43-dependant manner and the potential involvement of the ternary complex in this mechanism. Using a tandem affinity purification coupled with mass spectrometry approach, we show that RelAp43 interacts with the p105-ABIN2-TPL2 complex and we observe a strong perturbation of this complex in presence of M protein. M protein interaction with RelAp43 is associated with a wide disturbance of NF-κB signaling, involving a modulation of IκBα-, IκBβ-, and IκBε-RelAp43 interaction and a favored interaction of RelAp43 with the non-canonical pathway (RelB and p100/p52). Monitoring the interactions between host and viral proteins using protein-fragment complementation assay and bioluminescent resonance energy transfer, we further show that RelAp43 is associated to the p105-ABIN2-TPL2 complex as RelAp43-p105 interaction stabilizes the formation of a complex with ABIN2 and TPL2. Interestingly, the M protein interacts not only with RelAp43 but also with TPL2 and ABIN2. Upon interaction with this complex, M protein promotes the release of ABIN2, which ultimately favors the production of RelAp43-p50 NF-κB dimers. The use of recombinant rabies viruses further indicates that this mechanism leads to the control of IFNβ, TNF and CXCL2 expression during the infection and a high pathogenicity profile in rabies virus infected mice. All together, our results demonstrate the important role of RelAp43 and M protein in the regulation of NF-κB signaling.

LB982 Novel mechanism of self inhibition by NF-κB c-Rel

Dimers of the NF-κB/Rel transcription factor family regulate a wide range of cellular processes ranging from cytokine production and inflammation to cell survival and proliferation. Each of the fifteen possible NF-κB dimers exhibit slight differences in κB-site specificity, and thus have different target gene expression profiles. Although NF-κB dimer specificity has been somewhat well studied, the mechanisms regulating dimer specific NF-κB transactivation after its translocation into the nucleus is as of now fairly opaque. We have identified a novel mechanism by which NF-κB c-Rel can cross-regulate another NF-κB family member, p65 (RelA). In both mouse embryonic fibroblasts as well as keratinocytes, the absence of c-Rel greatly increases TNF-α-induced p65-targeted gene expression. This is accompanied by both an increase in p65 nuclear translocation and activation. Cross-inhibition of inflammatory gene expression by subunits that are classically thought to be transactivators have interesting implications on many pathologies, including those affecting the skin. Our findings demonstrate a novel role of c-Rel in the suppression of the pro-inflammatory NF-κB profile, and have begun to elucidate a mechanism by which this inhibition occurs, and in the future may provide new therapeutic targets for interfering or encouraging dimer specific interactions of NF-κB.

A green tea polyphenol epigallocatechin-3-gallate enhances neuroregeneration after spinal cord injury by altering levels of inflammatory cytokines

Spinal cord injury (SCI) is a debilitating condition which is characterized by an extended secondary injury due to the presence of inflammatory local milieu. Epigallocatechin gallate (EGCG) appears to possess strong neuroprotective properties. Here, we evaluated the beneficial effect of EGCG on recovery from SCI. Male Wistar rats were given either EGCG or saline directly to the injured spinal cord and thereafter a daily IP injection. Behavior recovery was monitored by BBB, plantar, rotarod and flat-beam tests. The levels of inflammatory cytokines were determined on days 1, 3, 7, 10 and 14 after SCI. Additionally, NF-κB pathway activity was evaluated. The results demonstrated that EGCG-treated rats displayed a superior behavioral performance in a flat beam test, higher axonal sprouting and positive remodelation of glial scar. Cytokine analysis revealed a reduction in IL-6, IL2, MIP1α and RANTES levels on days 1 and 3, and an upregulation of IL-4, IL-12p70 and TNFα 1 day following SCI in EGCG-treated rats. Treatment with EGCG was effective in decreasing the nuclear translocation of subunit p65 (RelA) of the NF-κB dimer, and therefore canonical NF-κB pathway attenuation. A significant increase in the gene expression of growth factors (FGF2 and VEGF), was noted in the spinal cord of EGCG-treated rats. Further, EGCG influenced expression of M1 and M2 macrophage markers. Our results have demonstrated a therapeutic value of EGCG in SCI, as observed by better behavioral performance measured by flat beam test, modulation of inflammatory cytokines and induction of higher axonal sprouting.

Hematopoietic stem cell transplantation for RelB deficiency

Hematopoietic stem cell transplantation for RelB deficiency

Role of Bcl-3 in T cells upon challenge with lymphocytic choriomeningitis virus

Abstract Bcl-3 is an atypical member of the IkB family. Classical IkBs inhibit trans-activating NF-kB dimers such as p50/RelA largely via cytoplasmic retention; they are degraded in response to many signals, which allows nuclear entry of NF-kB. By contrast, Bcl-3 is not degraded and acts in the nucleus to modulate transcription of NF-kB target genes by associating with p50/NF-kB1 and p52/NF-kB2 homodimers. The latter complexes do not trans-activate by themselves, and association with Bcl-3 may promote or inhibit transcription in a highly gene- and context-dependent manner. Bcl-3 has profound biologic roles. It is associated with recurrent translocations in some B-cell tumors and Bcl-3−/− mice exhibit notably impaired adaptive and innate immune responses. Previously, we have shown that Bcl-3 regulates plasticity and pathogenicity of autoimmune CD4 T cells in the T cell transfer-induced colitis model as well as in EAE, a model for multiple sclerosis. However, the detailed molecular controls of Bcl-3 in T cell differentiation and its antigenic responses are not well understood. To evaluate the possible role of Bcl-3 in antigen-specific T cell responses, mice lacking Bcl-3 specifically in T cells were challenged with acute lymphocytic choriomeningitis virus (LCMV) infection. Preliminary data revealed that Bcl-3−/− CD8 T cells had comparable primary antiviral responses at day 8 post infection. However, loss of Bcl-3 in T cells significantly reduced the proportion of LCMV-specific CD8 T cells in spleens 6 weeks post infection. This suggests a crucial role of Bcl-3 in long-term maintenance of antigen-specific CD8 T cells. We will present initial investigations into mechanisms by which Bcl-3 may help to maintain antiviral T cell responses over time.

The role of the SWI/SNF chromatin remodeling complex in maintaining the stemness of glioma initiating cells

Glioma initiating cells (GICs) are thought to contribute to therapeutic resistance and tumor recurrence in glioblastoma, a lethal primary brain tumor in adults. Although the stem-like properties of GICs, such as self-renewal and tumorigenicity, are epigenetically regulated, the role of a major chromatin remodeling complex in human, the SWI/SNF complex, remains unknown in these cells. We here demonstrate that the SWI/SNF core complex, that is associated with a unique corepressor complex through the d4-family proteins, DPF1 or DPF3a, plays essential roles in stemness maintenance in GICs. The serum-induced differentiation of GICs downregulated the endogenous expression of DPF1 and DPF3a, and the shRNA-mediated knockdown of each gene reduced both sphere-forming ability and tumor-forming activity in a mouse xenograft model. Rescue experiments revealed that DPF1 has dominant effects over DPF3a. Notably, whereas we have previously reported that d4-family members can function as adaptor proteins between the SWI/SNF complex and NF-κB dimers, this does not significantly contribute to maintaining the stemness properties of GICs. Instead, these proteins were found to link a corepressor complex containing the nuclear receptor, TLX, and LSD1/RCOR2 with the SWI/SNF core complex. Collectively, our results indicate that DPF1 and DPF3a are potential therapeutic targets for glioblastoma.

Elevated levels of Bcl-3 inhibits Treg development and function resulting in spontaneous colitis

Bcl-3 is an atypical NF-κB family member that regulates NF-κB-dependent gene expression in effector T cells, but a cell-intrinsic function in regulatory T (Treg) cells and colitis is not clear. Here we show that Bcl-3 expression levels in colonic T cells correlate with disease manifestation in patients with inflammatory bowel disease. Mice with T-cell-specific overexpression of Bcl-3 develop severe colitis that can be attributed to defective Treg cell development and function, leading to the infiltration of immune cells such as pro-inflammatory γδT cells, but not αβ T cells. In Treg cells, Bcl-3 associates directly with NF-κB p50 to inhibit DNA binding of p50/p50 and p50/p65 NF-κB dimers, thereby regulating NF-κB-mediated gene expression. This study thus reveals intrinsic functions of Bcl-3 in Treg cells, identifies Bcl-3 as a potential prognostic marker for colitis and illustrates the mechanism by which Bcl-3 regulates NF-κB activity in Tregs to prevent colitis.

NFκB and IκBα Engage in an ‘I Fold You, You Fold Me’ Interaction Excluding DNA

Binding of IκBα to NFκB robustly causes cessation of NFκB signalling. This phenomenon is facilitated by the sequestering of p50/p65 NFκB dimers in the cytosol via IκBα's masking of the p65 NLS. Interestingly, IκBα is one of the genes upregulated by NFκB binding to its promoter upon translocation into the nucleus; IκBα then facilitates dissociation of NFκB from DNA, creating a negative feedback loop to turn off the NFκB response. NFκB dimers consist of two domains, a dimerization domain and an N-terminal domain (NTD), which are responsible for dimerization/IκBα binding and DNA binding respectively.

Signal Integration by the IκB Protein Pickle Shapes Drosophila Innate Host Defense.

SummaryPattern recognition receptors are activated following infection and trigger transcriptional programs important for host defense. Tight regulation of NF-κB activation is critical to avoid detrimental and misbalanced responses. We describe Pickle, a Drosophila nuclear IκB that integrates signaling inputs from both the Imd and Toll pathways by skewing the transcriptional output of the NF-κB dimer repertoire. Pickle interacts with the NF-κB protein Relish and the histone deacetylase dHDAC1, selectively repressing Relish homodimers while leaving other NF-κB dimer combinations unscathed. Pickle’s ability to selectively inhibit Relish homodimer activity contributes to proper host immunity and organismal health. Although loss of pickle results in hyper-induction of Relish target genes and improved host resistance to pathogenic bacteria in the short term, chronic inactivation of pickle causes loss of immune tolerance and shortened lifespan. Pickle therefore allows balanced immune responses that protect from pathogenic microbes while permitting the establishment of beneficial commensal host-microbe relationships.

Abstract 1289: The synthetic peptide CIGB-300 inhibits nuclear factor κB (NF-κB) affecting the survival and chemoresistance of human lung cancer cells

Abstract Lung cancer is the leading cause of cancer deaths worldwide and despite significant progress, current therapies are limited in efficacy. The CK2 Ser/Thr kinase has been historically linked with cancer. It is involved in cell proliferation, survival and apoptosis by modulating diverse signaling pathways, including Wnt and NF-κB among the most relevant. CIGB-300 is an antitumor peptide with a novel mechanism of action, capable of binding to CK2 substrates thus preventing the enzyme activity. Previously, we have determined that CIGB-300 induces apoptosis through caspase-3 activation in different lung cancer cell lines. Moreover, CIGB-300 strongly inhibited RelA/NF-κB (p65) nuclear translocation, even in the presence of a phorbol-ester activating stimulus. NF-κB activation is known to reduce chemotherapy efficiency in different malignancies, including lung cancer. Based on this evidence, we hypothesize that supplementing cisplatin with CIGB-300 would improve the treatment efficiency. Indeed, we observed by Western blot that nuclear p65 levels were highly increased after treating human NCI-H125 cells with cisplatin. Moreover, when cells were treated with cisplatin plus CIGB-300, NF-κB activation was completely abolished. Therefore, the CIGB-300 effect on NF-κB signaling pathway prevails over cisplatin. These promising results on NF-κB inhibition led us to evaluate the combined treatment in chemoresistant setting. For this purpose we developed a cisplatin resistant A549 lung cancer cell line (A549-Rcisp) by the chronic administration of cisplatin during six months. A549-Rcisp viability was 40% higher than parental cells, confirming the cisplatin-acquired resistance. Remarkably, cisplatin resistant cells showed a significant increase in CIGB-300 sensitivity as compared to the parental cell line (p&amp;lt;0.01; Student's t-test). More on, only A549-Rcisp showed an increased p65 nuclear level after cisplatin treatment, suggesting that both cisplatin resistance and CIGB-300 sensitization might be linked to the NF-kB transcription factor. Given that NF-κB dimer stability is regulated by the proteasome-selective proteolysis of its inhibitory proteins, we studied the effect of CIGB-300 on this process. Surprisingly, we observed a significant increase on protease activities associated with the proteasome after 30 minutes of CIGB-300 treatment. Thus, proteasome complex is a newly identified target of CIGB-300 that could be relevant for its mechanism of action and deserves further exploration in order to determine the association with the observed perturbation of different signaling pathways. Citation Format: Stéfano M. Cirigliano, María Inés Díaz Bessone, Carolina Flumian, Damián E. Berardi, Silvio Perea, Elisa Bal De Kier Joffé, Hernán Farina, Laura Todaro, Alejandro Urtreger. The synthetic peptide CIGB-300 inhibits nuclear factor κB (NF-κB) affecting the survival and chemoresistance of human lung cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1289.

Modulation of Macrophage Inflammatory Nuclear Factor κB (NF-κB) Signaling by Intracellular Cryptococcus neoformans

Cryptococcus neoformans (Cn) is a common facultative intracellular pathogen that can cause life-threatening fungal meningitis in immunocompromised individuals. Shortly after infection, Cn is detectable as both extra- and intracellular yeast particles, with Cn being capable of establishing long-lasting latent infections within host macrophages. Although recent studies have shown that shed capsular polysaccharides and intact extracellular Cn can compromise macrophage function through modulation of NF-κB signaling, it is currently unclear whether intracellular Cn also affects NF-κB signaling. Utilizing live cell imaging and computational modeling, we find that extra- and intracellular Cn support distinct modes of NF-κB signaling in cultured murine macrophages. Specifically, in RAW 264.7 murine macrophages treated with extracellular glucuronoxylomannan (GXM), the major Cn capsular polysaccharide, LPS-induced nuclear translocation of p65 is inhibited, whereas in cells with intracellular Cn, LPS-induced nuclear translocation of p65 is both amplified and sustained. Mathematical simulations and quantification of nascent protein expression indicate that this is a possible consequence of Cn-induced "translational interference," impeding IκBα resynthesis. We also show that long term Cn infection induces stable nuclear localization of p65 and IκBα proteins in the absence of additional pro-inflammatory stimuli. In this case, nuclear localization of p65 is not accompanied by TNFα or inducible NOS (iNOS) expression. These results demonstrate that capsular polysaccharides and intact intracellular yeast manipulate NF-κB via multiple distinct mechanisms and provide new insights into how Cn might modulate cellular signaling at different stages of an infection.

Regulation of the Adaptive Immune Response by the IκB Family Protein Bcl-3.

Bcl-3 is a member of the IκB family of proteins and an important regulator of Nuclear Factor (NF)-κB activity. The ability of Bcl-3 to bind and regulate specific NF-κB dimers has been studied in great depth, but its physiological roles in vivo are still not fully understood. It is, however, becoming clear that Bcl-3 is essential for the proper development, survival and activity of adaptive immune cells. Bcl-3 dysregulation can be observed in a number of autoimmune pathologies, and Bcl3-deficient animals are more susceptible to bacterial and parasitic infection. This review will describe our current understanding of the roles played by Bcl-3 in the development and regulation of the adaptive immune response, including lymphoid organogenesis, immune tolerance, lymphocyte function and dendritic cell biology.

A roadmap of constitutive NF-κB activity in Hodgkin lymphoma: Dominant roles of p50 and p52 revealed by genome-wide analyses.

BackgroundNF-κB is widely involved in lymphoid malignancies; however, the functional roles and specific transcriptomes of NF-κB dimers with distinct subunit compositions have been unclear.MethodsUsing combined ChIP-sequencing and microarray analyses, we determined the cistromes and target gene signatures of canonical and non-canonical NF-κB species in Hodgkin lymphoma (HL) cells.ResultsWe found that the various NF-κB subunits are recruited to regions with redundant κB motifs in a large number of genes. Yet canonical and non-canonical NF-κB dimers up- and downregulate gene sets that are both distinct and overlapping, and are associated with diverse biological functions. p50 and p52 are formed through NIK-dependent p105 and p100 precursor processing in HL cells and are the predominant DNA binding subunits. Logistic regression analyses of combinations of the p50, p52, RelA, and RelB subunits in binding regions that have been assigned to genes they regulate reveal a cross-contribution of p52 and p50 to canonical and non-canonical transcriptomes. These analyses also indicate that the subunit occupancy pattern of NF-κB binding regions and their distance from the genes they regulate are determinants of gene activation versus repression. The pathway-specific signatures of activated and repressed genes distinguish HL from other NF-κB-associated lymphoid malignancies and inversely correlate with gene expression patterns in normal germinal center B cells, which are presumed to be the precursors of HL cells.ConclusionsWe provide insights that are relevant for lymphomas with constitutive NF-κB activation and generally for the decoding of the mechanisms of differential gene regulation through canonical and non-canonical NF-κB signaling.Electronic supplementary materialThe online version of this article (doi:10.1186/s13073-016-0280-5) contains supplementary material, which is available to authorized users.

Signaling via the NFκB system.

The nuclear factor kappa B (NFκB) family of transcription factors is a key regulator of immune development, immune responses, inflammation, and cancer. The NFκB signaling system (defined by the interactions between NFκB dimers, IκB regulators, and IKK complexes) is responsive to a number of stimuli, and upon ligand-receptor engagement, distinct cellular outcomes, appropriate to the specific signal received, are set into motion. After almost three decades of study, many signaling mechanisms are well understood, rendering them amenable to mathematical modeling, which can reveal deeper insights about the regulatory design principles. While other reviews have focused on upstream, receptor proximal signaling (Hayden MS, Ghosh S. Signaling to NF-κB. Genes Dev 2004, 18:2195-2224; Verstrepen L, Bekaert T, Chau TL, Tavernier J, Chariot A, Beyaert R. TLR-4, IL-1R and TNF-R signaling to NF-κB: variations on a common theme. Cell Mol Life Sci 2008, 65:2964-2978), and advances through computational modeling (Basak S, Behar M, Hoffmann A. Lessons from mathematically modeling the NF-κB pathway. Immunol Rev 2012, 246:221-238; Williams R, Timmis J, Qwarnstrom E. Computational models of the NF-KB signalling pathway. Computation 2014, 2:131), in this review we aim to summarize the current understanding of the NFκB signaling system itself, the molecular mechanisms, and systems properties that are key to its diverse biological functions, and we discuss remaining questions in the field. WIREs Syst Biol Med 2016, 8:227-241. doi: 10.1002/wsbm.1331 For further resources related to this article, please visit the WIREs website.

The RelA/cRel nuclear factor-κB (NF-κB) dimer, crucial for inflammation resolution, mediates the transcription of the key enzyme in melatonin synthesis in RAW 264.7 macrophages.

Lipopolysaccharide (LPS) modulates the transcription of the gene that codifies the enzyme arylalkylamine-N-acetyltransferase (AA-NAT) through nuclear translocation of the transcription factor nuclear factor-κ-light-chain-enhancer of activated B cells (NF-κB). AA-NAT converts serotonin to N-acetylserotonin, the ultimate precursor of melatonin. Activation of kappa B elements (aa-nat-κB), localized in the promoter (nat-κB1 and nat-κB2), leads to Aa-nat transcription in RAW 264.7 macrophages. Competitive electrophoretic mobility shift assay (EMSA) with oligonucleotide probes corresponding to each of the two elements, as well as a NF-κB consensus corresponding probe, revealed different specificities for each κB element. In addition, activator protein-1 (AP-1) as well as signal transducers and activator of transcription-1 and 3 (STAT-1; STAT-3) competed with NF-κB for binding to nat-κB1, while only STAT-3 competed with NF-κB for binding to nat-κB2. According to co-immunoprecipitation (ChiP) assays, these two sites are able to distinguish NF-κB subunits. The sequence nat-κB1 bound dimers containing p52, RelA, and cRel, while nat-κB2 bound preferentially p50, p52, and RelA, and did not bind cRel. The expression of RelA and cRel is essential for the induction of Aa-nat expression and melatonin synthesis. Considering that the expression of cRel is induced by the earlier expressed p50/RelA, the differential effects of NF-κB dimers may be intimately associated with the temporal regulation of inflammatory responses, with the resolution phase being associated with paracrine and autocrine melatonin effects. Such data suggest that the proven effects of exogenous melatonin in the resolution phase of inflammation are paralleled by the effects of locally synthesized melatonin in immune cells.

Dioxonaphthoimidazoliums AB1 and YM155 disrupt phosphorylation of p50 in the NF-κB pathway.

The NF-κB pathway is overexpressed in non-small cell lung cancers (NSCLC) and contributes to the poor prognosis and high mortality characterizing this malignancy. Silencing the p50 and p65 NF-κB subunits in the NSCLC H1299 cell line led to profound loss in cell viability and downregulated anti-apoptotic proteins survivin and Mcl1. We also showed that a survivin suppressant, the dioxonaphthoimidazolium YM155, and its structural analog AB1 arrested the growth of H1299 cells at nanomolar concentrations. Both compounds were apoptogenic and suppressed survivin and other anti-apoptotic proteins (Mcl1, Bcl-2, Bcl-xl) in a dose- and/or time-dependent manner. YM155 and AB1 did not affect the expression of key proteins (IκBα, p65, p50) involved in NF-κB signaling. Stable IκBα levels suggest that the NF-κB/IκB complex and proteins upstream of IκBα, were not targeted. Neither did the compounds intercept the nuclear translocation of the p50 and p65 subunits. On the other hand, YM155 and AB1 suppressed the phosphorylation of the p50 subunit at Ser337 which is critical in promoting the binding of NF-κB dimers to DNA. Both compounds duly impeded the binding of NF-κB dimers to DNA and attenuated transcriptional activity of luciferase-transfected HEK293 cells controlled by NF-κB response elements. We propose that the “silencing” the NF-κB pathway effected by these compounds contributed to their potent apoptogenic effects on H1299. Notwithstanding, the mechanism(s) involved in their ability to abolish phosphorylation of p50 remains to be elucidated. Taken together, these results disclose a novel facet of functionalized dioxonaphthoimidazoliums that could account for their potent cell killing property.