RelA Subunit Research Articles

Monoubiquitination of nuclear RelA negatively regulates NF-κB activity independent of proteasomal degradation

Termination and resolution of inflammation are tightly linked to the inactivation of one of its strongest inducers, NF-κB. While canonical post-stimulus inactivation is achieved by upregulation of inhibitory molecules that relocate NF-κB complexes to the cytoplasm, termination of the NF-κB response can also be accomplished directly in the nucleus by posttranslational modifications, e.g., ubiquitination of the RelA subunit. Here we reveal a functional role for RelA monoubiquitination in regulating NF-κB activity. By employing serine-to-alanine mutants, we found that hypo-phosphorylated nuclear RelA is monoubiquitinated on multiple lysine residues. Ubiquitination was reversed by IκBα expression and was reduced when nuclear translocation was inhibited. RelA monoubiquitination decreased NF-κB transcriptional activity despite prolonged nuclear presence and independently of RelA degradation, possibly through decreased CREB-binding protein (CBP) co-activator binding. Polyubiquitin-triggered proteasomal degradation has been proposed as a model for RelA inactivation. However, here we show that proteasomal inhibition, similar to RelA hypo-phosphorylation, resulted in nuclear translocation and monoubiquitination of RelA. These findings indicate a degradation-independent mechanism for regulating the activity of nuclear RelA by ubiquitination.

Free radicals enzymatically triggered by Clonorchis sinensis excretory–secretory products cause NF-κB-mediated inflammation in human cholangiocarcinoma cells

Chronic clonorchiasis, caused by direct and continuous contact with Clonorchis sinensis worms and their excretory–secretory products, is associated with hepatobiliary damage, inflammation, periductal fibrosis and even development of cholangiocarcinoma. Our previous report revealed that intracellular reactive oxygen species were generated in C. sinensis excretory–secretory product-treated human cholangiocarcinoma cells; however, their endogenous sources and pathophysiological roles in host cells were not determined. In the present study, we found that treatment of human cholangiocarcinoma cells with excretory–secretory products triggered increases in free radicals via a time-dependent activation of NADPH oxidase, xanthine oxidase and inducible nitric oxide synthase. This increase in free radicals substantially promoted the degradation of cytosolic IκB-α, nuclear translocation of nuclear factor-κB subunits (RelA and p50), and increased κB consensus DNA-binding activity. Excretory–secretory product-induced nuclear factor-κB activation was markedly attenuated by preincubation with specific inhibitors of each free radical-producing enzyme or the antioxidant, N-acetylcysteine. Moreover, excretory–secretory products induced an increase in the mRNA and protein expression of the proinflammatory cytokines, IL-1β and IL-6, in an nuclear factor-κB-dependent manner, indicating that enzymatic production of free radicals in ESP-treated cells participates in nuclear factor-κB-mediated inflammation. These findings provide new insights into the pathophysiological role of C. sinensis excretory–secretory products in host chronic inflammatory processes, which are initial events in hepatobiliary diseases.

Anti-Myeloma Activity of LDC-000067, a Novel Selective Inhibitor of Cyclin Dependent Kinase 9: A Targeted Approach

Abstract 5110 Background:Cyclin-dependent kinase (CDK) inhibitors are reported to induce apoptosis in myeloma cells (Gojo et al., 2002; MacCallum et al., 2005). Here we investigated pro-apoptotic and anti-proliferative effects of LDC-000067, a novel selective CDK9-inhibitor, in three multiple myeloma (MM) cell lines (L-363; RPMI-8226; U-266). In addition, we studied consequences of selective CDK 9-inhibition regarding transcriptional activity (RNA-polymerase II phosphorylation) and related anti-apoptotic pathways (Mcl-1 and NFkB). Methods:L-363, RPMI-8226 and U-266 cells were exposed to either DMSO (vehicle control) or serial dilutions of LDC-000067 [0.5–20μM]. Apoptosis was quantified by flow cytometry analyses (FITC Annexin V / propidium iodide staining) after 24 hours in corresponding samples. To further investigate mechanisms of induced apoptosis by LDC-000067 we examined corresponding protein extracts by immunoblot analyses. Results:We found that LDC-000067 showed anti-myeloma activity in all myeloma cell lines tested. The proportion of induced apoptosis by exposure to LDC-000067 was equipotent (RPMI-8226) or higher (L-363 and U-266) compared to Seliciclib, a selective CDK 2-, 7- and 9-inhibitor. The IC50 for LDC-000067 was 4.0 μmol/L for L-363, 6.9 μmol/L for U-266 and 11.5 μmol/L for RPMI-8226. LDC-000067 inhibited phosphorylation of RNA polymerase II at Ser2 of the carboxyl-terminal domain in all MM lines tested. In addition, exposure to LDC-000067 resulted in a rapid decline of the protein levels of Mcl-1, known as an anti-apoptotic protein in MM. Furthermore, it is reported that in 40% of MM cell lines and 17% of MM patients the NFkB pathway is constitutionally activated by mutations (Demchenko et al., 2010) and inhibition of the NFkB activity causes induction of apoptosis (Hongyu et al., 2001). In our study we showed that LDC-000067 potently inhibited the NFkB pathway. Immunoblot analyses revealed a decreased phosphorylation and degradation of the NFkB inhibitor IkBa and an inhibition of phosphorylation of the NFkB subunit RelA (p65). Moreover, our data demonstrated that exposure to LDC-000067 caused a significant reduction of the protein levels of cyclin D1 and cyclin D2 strongly correlated to cell cycle arrest. Conclusion:In conclusion, our data corroborate that LDC-000067, a novel selective CDK 9-inhibitor, shows anti-myeloma activity by targeting three key pathways in the molecular pathogenesis of MM: firstly, it induces consecutive decline of Mcl-1 protein and inhibition of RNA polymerase II phosphorylation; secondly, it inhibits constitutively activated NFkB pathway; and thirdly, it reduces levels of D-type cyclins. Disclosures:No relevant conflicts of interest to declare.

Aryl hydrocarbon receptor in association with RelA modulates IL-6 expression in non-smoking lung cancer

Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is activated by cigarette smoke. Previously, we demonstrated that AhR is overexpressed in lung adenocarcinomas (ADs). In this study we observed that AhR expression is significantly correlated with nuclear RelA (a nuclear factor-κB (NFκB) subunit) and cytosolic interleukin-6 (IL-6) in 200 non-small cell lung cancer patients, especially among never smokers. Overexpression of AhR increased IL-6 expression in H1355 cells and immortalized human bronchial epithelial cells BEAS-2B. As NFκB inhibitor and knockdown RelA expression greatly reduced constitutive AhR-induced IL-6 expression, we hypothesized that AhR expression, in the absence of exogenous ligand, is able to modulate NFκB activity and subsequently upregulate IL-6 expression, thus promoting the development of lung AD. Specifically, AhR overexpression significantly increased NFκB activity, whereas interference with AhR expression significantly reduced NFκB activity and IL-6 expression in H1355 cells. We demonstrated that AhR associates with RelA in the cytosol and nucleus of human lung cells. Furthermore, AhR overexpression enhanced nuclear localization of AhR and RelA, and increased the association of AhR-RelA with the NFκB response element of the IL-6 promoter. However, p50 was not involved. Our results indicate that AhR, without exposure to a ligand, associates with RelA, which then positively modulates NFκB activity and then upregulates IL-6 expression in human lung cells. Thus we have identified a new mechanism for lung tumorigenesis in non-smokers.

Disruption of IκB Kinase (IKK)-mediated RelA Serine 536 Phosphorylation Sensitizes Human Multiple Myeloma Cells to Histone Deacetylase (HDAC) Inhibitors

Post-translational modifications of RelA play an important role in regulation of NF-κB activation. We previously demonstrated that in malignant hematopoietic cells, histone deacetylase inhibitors (HDACIs) induced RelA hyperacetylation and NF-κB activation, attenuating lethality. We now present evidence that IκB kinase (IKK) β-mediated RelA Ser-536 phosphorylation plays a significant functional role in promoting RelA acetylation, inducing NF-κB activation, and limiting HDACI lethality in human multiple myeloma (MM) cells. Immunoblot profiling revealed that although basal RelA phosphorylation varied in MM cells, Ser-536 phosphorylation correlated with IKK activity. Exposure to the pan-HDACIs vorinostat or LBH-589 induced phosphorylation of IKKα/β (Ser-180/Ser-181) and RelA (Ser-536) in MM cells, including cells expressing an IκBα "super-repressor," accompanied by increased RelA nuclear translocation, acetylation, DNA binding, and transactivation activity. These events were substantially blocked by either pan-IKK or IKKβ-selective inhibitors, resulting in marked apoptosis. Consistent with these events, inhibitory peptides targeting either the NF-κB essential modulator (NEMO) binding domain for IKK complex formation or RelA phosphorylation sites also significantly increased HDACI lethality. Moreover, IKKβ knockdown by shRNA prevented Ser-536 phosphorylation and significantly enhanced HDACI susceptibility. Finally, introduction of a nonphosphorylatable RelA mutant S536A, which failed to undergo acetylation in response to HDACIs, impaired NF-κB activation and increased cell death. These findings indicate that HDACIs induce Ser-536 phosphorylation of the NF-κB subunit RelA through an IKKβ-dependent mechanism, an action that is functionally involved in activation of the cytoprotective NF-κB signaling cascade primarily through facilitation of RelA acetylation rather than nuclear translocation.

Coordinated Post-transcriptional Regulation of Hsp70.3 Gene Expression by MicroRNA and Alternative Polyadenylation

Heat shock protein 70 (Hsp70) is well documented to possess general cytoprotective properties in protecting the cell against stressful and noxious stimuli. We have recently shown that expression of the stress-inducible Hsp70.3 gene in the myocardium in response to ischemic preconditioning is NF-κB-dependent and necessary for the resulting late phase cardioprotection against a subsequent ischemia/reperfusion injury. Here we show that the Hsp70.3 gene product is subject to post-transcriptional regulation through parallel regulatory processes involving microRNAs and alternative polyadenylation of the mRNA transcript. First, we show that cardiac ischemic preconditioning of the in vivo mouse heart results in decreased levels of two Hsp70.3-targeting microRNAs: miR-378* and miR-711. Furthermore, an ischemic or heat shock stimulus induces alternative polyadenylation of the expressed Hsp70.3 transcript that results in the accumulation of transcripts with a shortened 3'-UTR. This shortening of the 3'-UTR results in the loss of the binding site for the suppressive miR-378* and thus renders the alternatively polyadenylated transcript insusceptible to miR-378*-mediated suppression. Results also suggest that the alternative polyadenylation-mediated shortening of the Hsp70.3 3'-UTR relieves translational suppression observed in the long 3'-UTR variant, allowing for a more robust increase in protein expression. These results demonstrate alternative polyadenylation of Hsp70.3 in parallel with ischemic or heat shock-induced up-regulation of mRNA levels and implicate the importance of this process in post-transcriptional control of Hsp70.3 expression.

PLK1 Is Transcriptionally Activated by NF-κB during Cell Detachment and Enhances Anoikis Resistance through Inhibiting β-Catenin Degradation in Esophageal Squamous Cell Carcinoma

To investigate the molecular mechanisms through which polo-like kinase-1 (PLK1) takes part in anoikis resistance of esophageal squamous cell carcinoma (ESCC) cells. The role of PLK1 in cell anoikis resistance was examined by ectopic gene expression and siRNA-mediated knockdown. Glutathione S-transferase pull-down and co-immunoprecipitation assays were utilized to investigate PLK1-interacting proteins. Electrophoretic mobility shift assay, chromatin immunoprecipitation, and reporter gene assays were carried out to identify the transcription factors responsible for PLK1 expression during anoikis resistance. We found that detachment of ESCC cells triggers the upregulation of PLK1. Elevated PLK1 expression contributes to protection against anoikis in cancer cells through the regulation of β-catenin expression. Moreover, we showed that, through direct binding to the PLK1 promoter, the NF-κB subunit RelA transcriptionally activates PLK1, which inhibits the ubiquitination and degradation of β-catenin. Inhibition of the NF-κB pathway restores the sensitivity of cancer cells to anoikis by downregulating PLK1/β-catenin expression. In addition, RelA gene amplification and protein overexpression was significantly correlated with PLK1 expression in ESCC tissues. Our findings suggest that upregulation of PLK1 triggered by cell detachment is regulated by RelA at the transcriptional level. PLK1 protects esophageal carcinoma cells from anoikis through modulation of β-catenin protein levels by inhibiting their degradation. Taken together, this study reveals critical mechanisms involved in the role of RelA/PLK1/β-catenin in anoikis resistance of ESCC cells.

NF-κB Protects Cells from Gamma Interferon-Induced RIP1-Dependent Necroptosis

Interferons (IFNs) are cytokines with well-described immunomodulatory and antiviral properties, but less is known about the mechanisms by which they promote cell survival or cell death. Here, we show that IFN-γ induces RIP1 kinase-dependent necroptosis in mammalian cells deficient in NF-κB signaling. Induction of necroptosis by IFN-γ was found to depend on Jak1 and partially on STAT1. We also demonstrate that IFN-γ activates IκB kinase β (IKKβ)-dependent NF-κB to regulate a transcriptional program that protects cells from necroptosis. IFN-γ induced progressive accumulation of reactive oxygen species (ROS) and eventual loss of mitochondrial membrane potential in cells lacking the NF-κB subunit RelA. Whole-genome microarray analyses identified sod2, encoding the antioxidant enzyme manganese superoxide dismutase (MnSOD), as a RelA target and potential antinecroptotic gene. Overexpression of MnSOD inhibited IFN-γ-mediated ROS accumulation and partially rescued RelA-deficient cells from necroptosis, while RNA interference (RNAi)-mediated silencing of sod2 expression increased susceptibility to IFN-γ-induced cell death. Together, these studies demonstrate that NF-κB protects cells from IFN-γ-mediated necroptosis by transcriptionally activating a survival response that quenches ROS to preserve mitochondrial integrity.

Dynamic Chromatin Localization of Sirt6 Shapes Stress- and Aging-Related Transcriptional Networks

The sirtuin Sirt6 is a NAD-dependent histone deacetylase that is implicated in gene regulation and lifespan control. Sirt6 can interact with the stress-responsive transcription factor NF-κB and regulate some NF-κB target genes, but the full scope of Sirt6 target genes as well as dynamics of Sirt6 occupancy on chromatin are not known. Here we map Sirt6 occupancy on mouse promoters genome-wide and show that Sirt6 occupancy is highly dynamic in response to TNF-α. More than half of Sirt6 target genes are only revealed upon stress-signaling. The majority of genes bound by NF-κB subunit RelA recruit Sirt6, and dynamic Sirt6 relocalization is largely driven in a RelA-dependent manner. Integrative analysis with global gene expression patterns in wild-type, Sirt6−/−, and double Sirt6−/− RelA−/− cells reveals the epistatic relationships between Sirt6 and RelA in shaping diverse temporal patterns of gene expression. Genes under the direct joint control of Sirt6 and RelA include several with prominent roles in cell senescence and organismal aging. These data suggest dynamic chromatin relocalization of Sirt6 as a key output of NF-κB signaling in stress response and aging.

Regulation of NF-κB activity by competition between RelA acetylation and ubiquitination

The NF-κB transcription factor plays essential roles in inflammation and oncogenesis. Its ubiquitous RelA subunit is regulated by several post-translational modifications (PTMs) including phosphorylation, ubiquitination, and acetylation. Ubiquitination promotes the termination of RelA-dependent transcription, but its regulation is incompletely understood. Through mass spectrometry analysis of ubiquitinated RelA, we identified 7 lysines that were attached to degradative and non-degradative forms of polyubiquitin. Interestingly, lysines targeted for acetylation were among the residues identified as ubiquitin acceptor sites. Mutation of these particular sites resulted in decreased polyubiquitination. Acetylation and ubiquitination were found to inhibit each other consistent with their use of overlapping sites. Reconstitution of rela−/− fibroblasts with wild-type and mutant forms of RelA revealed that modifications at these residues can play activating and inhibitory functions depending on the target gene context. Altogether, this study elucidates that ubiquitination and acetylation can modulate each other and regulate nuclear NF-κB function in a gene-specific manner.

Cytoplasmic and nuclear events controlling Tax-mediated activation of the NF-κB pathway: involvement of TAB2, IKKgamma/NEMO and calreticulin

The Tax oncoprotein of HTLV-1 initiates T-cell transformation by dysregulating cell cycle progression and inhibiting DNA damage responses. The subsequent genomic instability might result in constitutive activation of the NF-κB pathway observed in HTLV-1-transformed T lymphocytes. Our previous results indicated that differential modifications of Tax by ubiquitination or sumoylation controlled its retention either in the cytoplasm or in the nucleus, respectively. Here we show that Tax is targeted to pre-existing punctate cytoplasmic structures which contain the TNF-receptor associated protein 2 (TAB2). Colocalization of Tax with TAB2 in these cytoplasmic structures induced the recruitment of additional components involved in the NF-κB activation cascade, including the regulatory subunit of the IKK complex, IKKgamma/NEMO, the RelA subunit of NF-κB and TAX1-BP1, which are Tax interacting partners. Overexpression of TAB2 strongly stimulated Tax-mediated activation of the NF-κB pathway indicating that the concentration of these NF-κB factors in Tax-containing cytoplasmic punctate structures is a step important for activation of the NF-κB pathway by Tax. Interestingly, calreticulin, a multifunctional calcium buffering chaperone previously involved in the nucleocytoplasmic transport of nuclear receptors such as glucocorticoid and thyroid receptors, was also recruited by Tax in these cytoplasmic structures. The fact that overexpressed IKKgamma concentrated in nuclear foci that included calreticulin and that co-expression of Tax and IKKgamma led to the redistribution of both IKKgamma and calreticulin from the nucleus to the cytoplasm suggested that calreticulin might be involved in the nuclear export of these factors and their recruitment in TAB2-containing cytoplasmic punctate structures. The possibility that Tax activates the NF-κB pathway via initiation of events in the nucleus leading to the transport of IKKgamma from the nucleus to the cytoplasm will be discussed.

Structural basis of SETD6-mediated regulation of the NF-kB network via methyl-lysine signaling

SET domain containing 6 (SETD6) monomethylates the RelA subunit of nuclear factor kappa B (NF-κB). The ankyrin repeats of G9a-like protein (GLP) recognizes RelA monomethylated at Lys310. Adjacent to Lys310 is Ser311, a known phosphorylation site of RelA. Ser311 phosphorylation inhibits Lys310 methylation by SETD6 as well as binding of Lys310me1 by GLP. The structure of SETD6 in complex with RelA peptide containing the methylation site, in the presence of S-adenosyl-l-methionine, reveals a V-like protein structure and suggests a model for NF-κB binding to SETD6. In addition, structural modeling of the GLP ankyrin repeats bound to Lys310me1 peptide provides insight into the molecular basis for inhibition of Lys310me1 binding by Ser311 phosphorylation. Together, these findings provide a structural explanation for a key cellular signaling pathway centered on RelA Lys310 methylation, which is generated by SETD6 and recognized by GLP, and incorporate a methylation–phosphorylation switch of adjacent lysine and serine residues. Finally, SETD6 is structurally similar to the Rubisco large subunit methyltransferase. Given the restriction of Rubisco to plant species, this particular appearance of the protein lysine methyltransferase has been evolutionarily well conserved.

Abstract 572: Suppression of urokinase plasminogen activator receptor blocks recruitment of endothelial cells by inhibiting irradiation-induced monocyte chemo-attractant protein-1 in meningioma

Abstract Chemokines, a common outcome of inflammatory reaction, play a vital role in recruiting various cell types during tissue repair. Radiation, a major therapeutic modality in cancer treatment, has been described to induce inflammatory response, which might lead to the expression of several chemokines. In the present study, we investigated the mechanisms underlying radiation-induced monocyte chemo-attractant protein (MCP-1) in meningioma cell lines and the paracrine effect on human microvascular endothelial cells (HMEC). Upon radiation, meningioma cell lines (IOMM Lee and SF 3061) showed an increased expression of MCP-1. In addition, irradiated meningioma cancer cell culture conditioned medium (CM) demonstrated an increased ability to attract HMEC and stimulated MCP1-induced protein (MCPIP), VEGF and angiogenin expression in HMEC as compared to HMEC grown in non-irradiated cancer cell CM. This chemotactic activity and angiogenic stimulator effect in HMEC was nearly abrogated by depleting MCP-1 from the irradiated cancer cell CM using a neutralizing anti-MCP-1 monoclonal antibody. Inhibition of ERK activation and NF-κB nuclear translocation hindered irradiation-induced MCP-1 expression in both meningioma cell lines. Considering that radiation induces uPA/uPAR expression and stimulates the ERK signaling pathway, we correlated the role of irradiation-induced uPA/uPAR in MCP-1 regulation. Supplementing cancer cells with exogenous ATF-uPA (with and without radiation) activated ERK phosphorylation, nuclear translocation of the NF-κB p65 sub-unit (Rel-A), and MCP-1 expression. Downregulation of uPA and uPAR by transfecting the cancer cells with a siRNA-expressing plasmid (pU) inhibited irradiation-induced ERK activation, nuclear translocation of Rel-A, NF-κB DNA binding activity, and MCP-1 expression. Further, pU-transfected cancer cells (with or with radiation) reduced the potential to attract and stimulate angiogenic signaling in HMEC under in vitro conditions. Under in vivo conditions, we observed that pU transfected cancer cells (with and without irradiation) showed a significant reduction in number of CD31+ endothelial cells and CD14+ monocytes present in the tumor regions compared to tumor formed from irradiated cancer cells. In conclusion, we demonstrated that blockade of uPA/uPAR expression inhibits irradiation-induced MCP-1, thereby inhibiting the chemo-attractant ability of cancer cells to recruit other cells during the inflammatory process induced by radiation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 572. doi:10.1158/1538-7445.AM2011-572

Pyrin-only protein 3: a pseudogene product in the human genome regulating NF-kB activity (157.4)

Abstract Pyrin-only proteins influence NF-kB and inflammasome signaling pathways. In viruses, POPs act as pathogenic factors by reducing innate immune responses and favoring viral survival within the host. In humans, two known POPs are thought to have a role in regulating innate immunity and inflammation. POP1 interferes with IKKα/β activation and prevents the downstream activation of NF-kB with no known effect on ASC-dependent inflammasomes. POP2 however, inhibits NF-kB signaling at the level of the p65 (RelA) subunit and down-modulates the formation and activation of a variety of pyrin domain/ASC-dependent inflammasomes. Here we describe a third human POP, POP3, the product of an NLRP pseudogene encoded on chromosome X that is expressed in a wide variety of tissues. Like its close genetic relative POP2, POP3 inhibits p65 activation downstream of IKK, but lacks the capacity of inhibit the NLRP3 inflammasome. Our data suggest that the human POP family has three members, all of which may play distinct biological roles during inflammation.

Characterization and functional study of CPAP-associated protein-1(CPAPAP-1) in NF-κB-mediated transcriptional activation (116.3)

Abstract Nuclear factor-kappaB (NF-κB) is a Rel transcription factor that controls the gene expression involved in immune response, inflammation, apoptosis, and proliferation. Centrosomal P4.1-associated protein (CPAP) is an associating partner of the p65 subunit (RelA) of NF-κB. Molecular biology assays and cell biology experiments showed that the role of CPAP is a coactivator of NF-κB-mediated transcription. The C-terminal region of CPAP appears to contribute NF-κB-mediated transcriptional activation. However, knowledge about the factors that interact with the C-terminal region of CPAP is still little. In a yeast two-hybrid screen using the C-terminal region of CPAP as bait, we isolated a novel associating partner named CPAP-associated protein-1 (CPAPAP-1, GenBank: BC114945). CPAPAP-1 has the effector domain (cAMP) of the CAP family of transcription factors. We confirmed that CPAPAP-1 associates with C-terminal region of CPAP by using Glutathione S-transferase pull-down assays and co-immunoprecipitation experiments. After treatment with TNFα, a portion of CPAPAP-1 was observed to accumulate in the nucleus. We also confirmed that CPAPAP-1 controlled activation of TNFα-induced NF-κB by using NF-κB-dependent reporter gene expression assay and RNA interference technique. These results suggest that CPAPAP-1 functions as a coactivator of NF-κB-mediated transcription.

Suppression of uPA and uPAR blocks radiation-induced MCP-1 mediated recruitment of endothelial cells in meningioma

Chemokines play a vital role in recruiting various cell types in the process of tissue repair. Radiation, a major therapeutic modality in cancer treatment, has been described to induce inflammatory response that might lead to the expression of several chemokines. In the present study, we investigated the mechanism of monocyte chemoattractant protein-1 (MCP-1) induction by radiation in meningioma cell lines and the paracrine effect on human microvascular endothelial cells (HMEC). After radiation, meningioma cell lines (IOMM Lee and SF-3061) showed an increased expression of MCP-1. In addition, irradiated meningioma cancer cell conditioned medium (CM) showed an increased ability to attract HMEC and to stimulate MCP-1-induced protein (MCPIP), VEGF and angiogenin expression in HMEC. This chemotactic activity and angiogenic stimulator effect on HMEC were almost abrogated by depleting MCP-1 from the irradiated cancer cell CM. Further, inhibition of either ERK activation/expression or NF-κB nuclear translocation hindered radiation-induced MCP-1 expression in both meningioma cell lines. Further, supplementing cancer cells with exogenous ATF-uPA (with and without radiation) activated ERK phosphorylation, nuclear translocation of the NF-κB p65 sub-unit (Rel-A), and MCP-1 expression. Downregulation of uPA and uPAR, simultaneously by transfecting the cancer cells with bi-cistronic siRNA-expressing plasmid (pU) inhibited radiation-induced ERK activation, nuclear translocation of Rel-A, NF-κB DNA binding activity, and MCP-1 expression. In addition, pU-transfected cancer cells (with or without radiation) reduced radiation-induced MCP-1 and blocked the recruitment of other cell types during the inflammatory process induced by radiation both in in vitro and in vivo conditions.

Differential utilization of NF-kappaB RELA and RELB in response to extracellular versus intracellular polyIC stimulation in HT1080 cells

BackgroundPattern recognition receptors (PRRs) for double-stranded RNA (dsRNA) are components of innate immunity that recognize the presence of viral infection and initiate efficient defense mechanisms. In addition to previously well-characterized signaling pathways that are mediated by PKR and TLR3, new intracellular dsRNA sensors, that are members of CARD and DExD/H box helicase family, have been identified. However, the molecular mechanisms involved in the signaling pathways mediated by these new dsRNA sensors have not been extensively characterized.ResultsHere, we studied an intracellular dsRNA pathway in the human fibrosarcoma cell line HT1080, which is distinct from the TLR3-mediated extracellular dsRNA pathway. Particularly, the NF-kB subunits RELA and RELB were differentially utilized by these two dsRNA signaling pathways. In TLR3-mediated dsRNA signaling, siRNA knock-down studies suggested a limited role for RELA on regulation of interferon beta and other cytokines whereas RELB appeared to have a negative regulatory role. By contrast, intracellular dsRNA signaling was dependent on RELA, but not RELB.ConclusionsOur study suggests that extracellular and intracellular dsRNA signaling pathways may utilize different NF-kB members, and particularly the differential utilization of RELB may be a key mechanism for powerful inductions of NF-kB regulated genes in the intracellular dsRNA signaling pathway.

Distinct Roles for the NF-κB RelA Subunit during Antiviral Innate Immune Responses

Production of type I interferons (IFNs; prominently, IFN-α/β) following virus infection is a pivotal antiviral innate immune response in higher vertebrates. The synthesis of IFN-β proceeds via the virus-induced assembly of the transcription factors IRF-3/7, ATF-2/c-Jun, and NF-κB on the ifnβ promoter. Surprisingly, recent data indicate that the NF-κB subunit RelA is not essential for virus-stimulated ifnβ expression. Here, we show that RelA instead sustains autocrine IFN-β signaling prior to infection. In the absence of RelA, virus infection results in significantly delayed ifnβ induction and consequently defective secondary antiviral gene expression. While RelA is not required for ifnβ expression after infection, it is nonetheless essential for fully one-fourth of double-stranded RNA (dsRNA)-activated genes, including several mediators of inflammation and immune cell recruitment. Further, RelA directly regulates a small subset of interferon-stimulated genes (ISGs). Finally, RelA also protects cells from dsRNA-triggered RIP1-dependent programmed necrosis. Taken together, our findings suggest distinct roles for RelA in antiviral innate immunity: RelA maintains autocrine IFN-β signaling in uninfected cells, facilitates inflammatory and adaptive immune responses following infection, and promotes infected-cell survival during this process.

Lysine methylation of the NF-κB subunit RelA by SETD6 couples activity of the histone methyltransferase GLP at chromatin to tonic repression of NF-κB signaling.

Protein lysine methylation signaling is implicated in diverse biological and disease processes. Yet the catalytic activity and substrate specificity are unknown for many human protein lysine methyltransferases (PKMTs). We screened over forty candidate PKMTs and identified SETD6 as a methyltransferase that monomethylates chromatin-associated NF-κB RelA at lysine 310 (RelAK310me1). SETD6-mediated methylation rendered RelA inert and attenuated RelA-driven transcriptional programs, including inflammatory responses in primary immune cells. RelAK310me1 was recognized by the ankryin repeat of GLP, which under basal conditions, promoted a repressed chromatin state at RelA target genes through GLP-mediated H3K9 methylation. NF-κB activation-linked phosphorylation of RelA by PKCζ at serine 311 blocked GLP binding to RelAK310me1 and relieved target gene repression. Our findings establish a new mechanism by which chromatin signaling regulates inflammation programs.

Activation of Phosphatidylinositol 3-Kinase/Akt and Impairment of Nuclear Factor-κB: Molecular Mechanisms Behind the Arrested Maturation/Activation State of Leishmania infantum-Infected Dendritic Cells

Understanding the complex interactions between Leishmania and dendritic cells (DCs) is central to the modulation of the outcome of this infection, given that an effective immune response against Leishmania is dependent on the successful activation and maturation of DCs. We report here that Leishmania infantum promastigotes successfully infect mouse bone marrow-derived DCs without triggering maturation, as shown by a failure in the up-regulation of CD40 and CD86 expression, and that parasites strongly counteract the lipopolysaccharide-triggered maturation of DCs. A small increase in interleukin (IL)-12 and IL-10 transcription and secretion and a decrease in IL-6 were observed in infected cells. This arrested DC maturation state is actively promoted by parasites because heat-killed or fixed parasites increased cytokine and costimulatory molecule expression. At a molecular level, L. infantum rapidly induced activation of phosphatidylinositol 3-kinase/Akt and extracellular signal-regulated kinase 1/2, whereas no effect was observed in the c-Jun N-terminal kinase and p38 mitogen-activated protein kinase proinflammatory pathways. Moreover, parasites actively promoted cleavage of the nuclear factor-κB p65(RelA) subunit, causing its impairment. The blockade of phosphatidylinositol 3-kinase/Akt by either treatment of bone marrow-derived DCs with wortmannin or transfection with an Akt dominant-negative mutant resulted in a strong decrease in infection rates, revealing for the first time a crucial role of this pathway on Leishmania engulfment by DCs. Overall, our data indicate that activation of Akt and impairment of nuclear factor-κB are responsible for immunogenicity subversion of L. infantum-infected DCs.