Co-expression of anti-NFkappaB RNA aptamers and siRNAs leads to maximal suppression of NFkappaB activity in mammalian cells

The specific down-regulation of gene expression in cells is a powerful method for elucidating a gene's function. A common method for suppressing gene expression is the elimination of mRNA by RNAi or antisense. Alternatively, oligonucleotide-derived aptamers have been used as protein-directed agents for the specific knock-down of both intracellular and extracellular protein activity. Protein-directed methods offer the advantage of more closely mimicking small molecule therapeutics' mechanism of activity. Furthermore, protein-directed methods may synergize with RNA-directed methods since the two methods attack gene expression at different levels. Here we have knocked down a well-characterized intracellular protein's activity, NFkappaB, by expressing either aptamers or small interfering RNAs (siRNAs). Both methods can diminish NFkappaB's activity to similar levels (from 29 to 64%). Interestingly, expression of both aptamers and siRNAs simultaneously, suppressed NFkappaB activity better than either method alone (up to 90%). These results demonstrate that the expression of intracellular aptamers is a viable alternative to siRNA knock-down. Furthermore, for the first time, we show that the use of aptamers and siRNA together can be the most effective way to achieve maximal knock-down of protein activity.     

An incoherent feedforward loop interprets NFκB/RelA dynamics to determine TNF‐induced necroptosis decisions

Balancing cell death is essential to maintain healthy tissue homeostasis and prevent disease. Tumor necrosis factor (TNF) not only activates nuclear factor κB (NFκB), which coordinates the cellular response to inflammation, but may also trigger necroptosis, a pro‐inflammatory form of cell death. Whether TNF‐induced NFκB affects the fate decision to undergo TNF‐induced necroptosis is unclear. Live‐cell microscopy and model‐aided analysis of death kinetics identified a molecular circuit that interprets TNF‐induced NFκB/RelA dynamics to control necroptosis decisions. Inducible expression of TNFAIP3/A20 forms an incoherent feedforward loop to interfere with the RIPK3‐containing necrosome complex and protect a fraction of cells from transient, but not long‐term TNF exposure. Furthermore, dysregulated NFκB dynamics often associated with disease diminish TNF‐induced necroptosis. Our results suggest that TNF''s dual roles in either coordinating cellular responses to inflammation, or further amplifying inflammation are determined by a dynamic NFκB‐A20‐RIPK3 circuit, that could be targeted to treat inflammation and cancer.     

DNA-Dependent Protein Kinase Phosphorylation of IκBα and IκBβ Regulates NF-κB DNA Binding Properties

Regulation of the IκBα and IκBβ proteins is critical for modulating NF-κB-directed gene expression. Both IκBα and IκBβ are substrates for cellular kinases that phosphorylate the amino and carboxy termini of these proteins and regulate their function. In this study, we utilized a biochemical fractionation scheme to purify a kinase activity which phosphorylates residues in the amino and carboxy termini of both IκBα and IκBβ. Peptide microsequence analysis by capillary high-performance liquid chromatography ion trap mass spectroscopy revealed that this kinase was the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). DNA-PK phosphorylates serine residue 36 but not serine residue 32 in the amino terminus of IκBα and also phosphorylates threonine residue 273 in the carboxy terminus of this protein. To determine the biological relevance of DNA-PK phosphorylation of IκBα, murine severe combined immunodeficiency (SCID) cell lines which lack the DNA-PKcs gene were analyzed. Gel retardation analysis using extract prepared from these cells demonstrated constitutive nuclear NF-κB DNA binding activity, which was not detected in extracts prepared from SCID cells complemented with the human DNA-PKcs gene. Furthermore, IκBα that was phosphorylated by DNA-PK was a more potent inhibitor of NF-κB binding than nonphosphorylated IκBα. These results suggest that DNA-PK phosphorylation of IκBα increases its interaction with NF-κB to reduce NF-κB DNA binding properties.     

Genetic signature of human longevity in PKC and NF‐κB signaling

Gene variants associated with longevity are also associated with protection against cognitive decline, dementia and Alzheimer''s disease, suggesting that common physiologic pathways act at the interface of longevity and cognitive function. To test the hypothesis that variants in genes implicated in cognitive function may promote exceptional longevity, we performed a comprehensive 3‐stage study to identify functional longevity‐associated variants in ~700 candidate genes in up to 450 centenarians and 500 controls by target capture sequencing analysis. We found an enrichment of longevity‐associated genes in the nPKC and NF‐κB signaling pathways by gene‐based association analyses. Functional analysis of the top three gene variants (NFKBIA, CLU, PRKCH) suggests that non‐coding variants modulate the expression of cognate genes, thereby reducing signaling through the nPKC and NF‐κB. This matches genetic studies in multiple model organisms, suggesting that the evolutionary conservation of reduced PKC and NF‐κB signaling pathways in exceptional longevity may include humans.     

Scaffold compound L971 exhibits anti‐inflammatory activities through inhibition of JAK/STAT and NFκB signalling pathways

JAK/STAT and NFκB signalling pathways play essential roles in regulating inflammatory responses, which are important pathogenic factors of various serious immune‐related diseases, and function individually or synergistically. To find prodrugs that can treat inflammation, we performed a preliminary high‐throughput screening of 18 840 small molecular compounds and identified scaffold compound L971 which significantly inhibited JAK/STAT and NFκB driven luciferase activities. L971 could inhibit the constitutive and stimuli‐dependent activation of STAT1, STAT3 and IκBα and could significantly down‐regulate the proinflammatory gene expression in mouse peritoneal macrophages stimulated by LPS. Gene expression profiles upon L971 treatment were determined using high‐throughput RNA sequencing, and significant differentially up‐regulated and down‐regulated genes were identified by DESeq analysis. The bioinformatic studies confirmed the anti‐inflammatory effects of L971. Finally, L971 anti‐inflammatory character was further verified in LPS‐induced sepsis shock mouse model in vivo. Taken together, these data indicated that L971 could down‐regulate both JAK/STAT and NFκB signalling activities and has the potential to treat inflammatory diseases such as sepsis shock.     

Molecular profile of the NF‐κB signalling pathway in human colorectal cancer

The development and progression of colorectal cancer (CRC) have been associated with inflammation processes that involve the overactivation of the NF‐κB signalling pathway. The characterization of the NF‐κB expression profile in CRC is an important topic since the suppression of NF‐κB represents a potential therapeutic approach. In this study, we assessed the expression levels of 84 NF‐κB‐related genes in paired tumoral (T) and peritumoral (PT) tissues from 18 CRC patients and 18 normal colonic mucosae, and the expression levels of three miRNAs targeting the most dysregulated genes revealed by the case–control analysis. Comparing the gene expression profile of T and controls, 60 genes were dysregulated. The comparison of T and PT revealed 17 dysregulated genes in the tumoral tissues, with IL1B, CXCL8, IL1A, and CSF2 being the most upregulated. Notably, through a bioinformatics analysis, the differential gene expression of 11 out of the 17 genes was validated on a larger cohort of 308 CRC patients compared with 41 controls. Moreover, a decrease in the levels of RELA, NOD1, CASP8, BCL2L1, ELK1, and IKBKB was identified in poorly differentiated tumours compared to moderately differentiated tumours. The analysis of the three miRNAs targeting IL1B, CXCL8, IL1A, and CSF2 showed that miR‐182‐5p was upregulated in T compared with PT, whereas miR‐10b‐5p was downregulated in T compared with PT and control tissues. Our results may contribute to the design of new experimental therapeutic strategies based on endogenous molecules, such as miRNAs, to target the genetic key players of the NF‐ κB pathway.     

Pseudolaric acid B ameliorates synovial inflammation and vessel formation by stabilizing PPARγ to inhibit NF‐κB signalling pathway

Synovial macrophage polarization and inflammation are essential for osteoarthritis (OA) development, yet the molecular mechanisms and regulation responsible for the pathogenesis are still poorly understood. Here, we report that pseudolaric acid B (PAB) attenuated articular cartilage degeneration and synovitis during OA. PAB, a diterpene acid, specifically inhibited NF‐κB signalling and reduced the production of pro‐inflammatory cytokines, which further decreased M1 polarization and vessel formation. We further provide in vivo and in vitro evidences that PAB suppressed NF‐κB signalling by stabilizing PPARγ. Using PPARγ antagonist could abolish anti‐inflammatory effect of PAB and rescue the activation of NF‐κB signalling during OA. Our findings identify a previously unrecognized role of PAB in the regulation of OA and provide mechanisms by which PAB regulates NF‐κB signalling through PPARγ, which further suggest targeting synovial inflammation or inhibiting vessel formation at early stage could be an effective preventive strategy for OA.     

TIFA has dual functions in Helicobacter pylori‐induced classical and alternative NF‐κB pathways

Helicobacter pylori infection constitutes one of the major risk factors for the development of gastric diseases including gastric cancer. The activation of nuclear factor‐kappa‐light‐chain‐enhancer of activated B cells (NF‐κB) via classical and alternative pathways is a hallmark of H. pylori infection leading to inflammation in gastric epithelial cells. Tumor necrosis factor receptor‐associated factor (TRAF)‐interacting protein with forkhead‐associated domain (TIFA) was previously suggested to trigger classical NF‐κB activation, but its role in alternative NF‐κB activation remains unexplored. Here, we identify TRAF6 and TRAF2 as binding partners of TIFA, contributing to the formation of TIFAsomes upon H. pylori infection. Importantly, the TIFA/TRAF6 interaction enables binding of TGFβ‐activated kinase 1 (TAK1), leading to the activation of classical NF‐κB signaling, while the TIFA/TRAF2 interaction causes the transient displacement of cellular inhibitor of apoptosis 1 (cIAP1) from TRAF2, and proteasomal degradation of cIAP1, to facilitate the activation of the alternative NF‐κB pathway. Our findings therefore establish a dual function of TIFA in the activation of classical and alternative NF‐κB signaling in H. pylori‐infected gastric epithelial cells.     

Anisomycin protects against sepsis by attenuating IκB kinase-dependent NF-κB activation and inflammatory gene expression

Anisomycin is known to inhibit eukaryotic protein synthesis and has been established as an antibiotic and anticancer drug. However, the molecular targets of anisomycin and its mechanism of action have not been explained in macrophages. Here, we demonstrated the anti-inflammatory effects of anisomycin both in vivo and in vitro. We found that anisomycin decreased the mortality rate of macrophages in cecal ligation and puncture (CLP)- and lipopolysaccharide (LPS)-induced acute sepsis. It also declined the gene expression of proinflammatory mediators such as inducible nitric oxide synthase, tumor necrosis factor-α, and interleukin-1β as well as the nitric oxide and proinflammatory cytokines production in macrophages subjected to LPS-induced acute sepsis. Furthermore, anisomycin attenuated nuclear factor (NF)-κB activation in LPS-induced macrophages, which correlated with the inhibition of phosphorylation of NF-κB-inducing kinase and IκB kinase, phosphorylation and IκBα proteolytic degradation, and NF-κB p65 subunit nuclear translocation. These results suggest that anisomycin prevented acute inflammation by inhibiting NF-κB-related inflammatory gene expression and could be a potential therapeutic candidate for sepsis.     

Butyrate inhibits IL-1β-induced inflammatory gene expression by suppression of NF-κB activity in pancreatic beta cells

Cytokine-induced beta cell dysfunction is a hallmark of type 2 diabetes (T2D). Chronic exposure of beta cells to inflammatory cytokines affects gene expression and impairs insulin secretion. Thus, identification of anti-inflammatory factors that preserve beta cell function represents an opportunity to prevent or treat T2D. Butyrate is a gut microbial metabolite with anti-inflammatory properties for which we recently showed a role in preventing interleukin-1β (IL-1β)-induced beta cell dysfunction, but how prevention is accomplished is unclear. Here, we investigated the mechanisms by which butyrate exerts anti-inflammatory activity in beta cells. We exposed mouse islets and INS-1E cells to a low dose of IL-1β and/or butyrate and measured expression of inflammatory genes and nitric oxide (NO) production. Additionally, we explored the molecular mechanisms underlying butyrate activity by dissecting the activation of the nuclear factor-κB (NF-κB) pathway. We found that butyrate suppressed IL-1β-induced expression of inflammatory genes, such as Nos2, Cxcl1, and Ptgs2, and reduced NO production. Butyrate did not inhibit IκBα degradation nor NF-κB p65 nuclear translocation. Furthermore, butyrate did not affect binding of NF-κB p65 to target sequences in synthetic DNA but inhibited NF-κB p65 binding and RNA polymerase II recruitment to inflammatory gene promoters in the context of native DNA. We found this was concurrent with increased acetylation of NF-κB p65 and histone H4, suggesting butyrate affects NF-κB activity via inhibition of histone deacetylases. Together, our results show butyrate inhibits IL-1β-induced inflammatory gene expression and NO production through suppression of NF-κB activation and thereby possibly preserves beta cell function.     

The UVB-Stimulated Expression of Transglutaminase 1 Is Mediated Predominantly via the NFκB Signaling Pathway: New Evidence of Its Significant Attenuation through the Specific Interruption of the p38/MSK1/NFκBp65 Ser276 Axis

The influence of ultraviolet B (UVB) radiation on transglutaminase 1 (TGase 1), a major factor that regulates skin keratinization, has not been sufficiently characterized especially at the gene or protein level. Thus, we determined whether UVB affects the expression of TGase 1 in human keratinocytes and clarified the intracellular stress signaling mechanism(s) involved. Exposure of human keratinocytes to UVB significantly up-regulated the expression of TGase 1 at the gene and protein levels. Treatment with inhibitors of p38, MEK, JNK or NFκB significantly abolished the UVB-stimulated protein expression of TGase 1. Treatment with astaxanthin immediately after UVB irradiation did not attenuate the increased phosphorylation of Ser536/Ser468NFκBp65, c-Jun, ATK-2 and CK2, and did not abrogate the increased or diminished protein levels of c-Jun/c-Fos or I-κBα, respectively. However, the same treatment with astaxanthin significantly abolished the UVB-stimulated expression of TGase 1 protein, which was accompanied by the attenuated phosphorylation of Thr565/Ser376/Ser360MSK1, Ser276NFκBp65 and Ser133CREB. The MSK1 inhibitor H89 significantly down-regulated the increased protein expression of TGase 1 in UVB-exposed human keratinocytes, which was accompanied by an abrogating effect on the increased phosphorylation of Ser276NFκBp65 and Ser133CREB but not Thr565/Ser376/Ser360MSK1. Transfection of human keratinocytes with MSK1 siRNA suppressed the UVB-stimulated protein expression of TGase 1. These findings suggest that the UVB-stimulated expression of TGase 1 is mediated predominantly via the NFκB pathway and can be attenuated through a specific interruption of the p38/MSK1/NFκBp65Ser276 axis.     

Novel small molecule inhibition of IKK/NF‐κB activation reduces markers of senescence and improves healthspan in mouse models of aging

Constitutive NF‐κB activation is associated with cellular senescence and stem cell dysfunction and rare variants in NF‐κB family members are enriched in centenarians. We recently identified a novel small molecule (SR12343) that inhibits IKK/NF‐κB activation by disrupting the association between IKKβ and NEMO. Here we investigated the therapeutic effects of SR12343 on senescence and aging in three different mouse models. SR12343 reduced senescence‐associated beta‐galactosidase (SA‐β‐gal) activity in oxidative stress‐induced senescent mouse embryonic fibroblasts as well as in etoposide‐induced senescent human IMR90 cells. Chronic administration of SR12343 to the Ercc1 ^{−/ ∆} and Zmpste24 ^−/− mouse models of accelerated aging reduced markers of cellular senescence and SASP and improved multiple parameters of aging. SR12343 also reduced markers of senescence and increased muscle fiber size in 2‐year‐old WT mice. Taken together, these results demonstrate that IKK/NF‐κB signaling pathway represents a promising target for reducing markers of cellular senescence, extending healthspan and treating age‐related diseases.     

LUBAC assembles a ubiquitin signaling platform at mitochondria for signal amplification and transport of NF‐κB to the nucleus

Mitochondria are increasingly recognized as cellular hubs to orchestrate signaling pathways that regulate metabolism, redox homeostasis, and cell fate decisions. Recent research revealed a role of mitochondria also in innate immune signaling; however, the mechanisms of how mitochondria affect signal transduction are poorly understood. Here, we show that the NF‐κB pathway activated by TNF employs mitochondria as a platform for signal amplification and shuttling of activated NF‐κB to the nucleus. TNF treatment induces the recruitment of HOIP, the catalytic component of the linear ubiquitin chain assembly complex (LUBAC), and its substrate NEMO to the outer mitochondrial membrane, where M1‐ and K63‐linked ubiquitin chains are generated. NF‐κB is locally activated and transported to the nucleus by mitochondria, leading to an increase in mitochondria‐nucleus contact sites in a HOIP‐dependent manner. Notably, TNF‐induced stabilization of the mitochondrial kinase PINK1 furthermore contributes to signal amplification by antagonizing the M1‐ubiquitin‐specific deubiquitinase OTULIN. Overall, our study reveals a role for mitochondria in amplifying TNF‐mediated NF‐κB activation, both serving as a signaling platform, as well as a transport mode for activated NF‐κB to the nuclear.     

NF-κB Mediates αvβ3 Integrin-induced Endothelial Cell Survival

The α_vβ₃ integrin plays a fundamental role during the angiogenesis process by inhibiting endothelial cell apoptosis. However, the mechanism of inhibition is unknown. In this report, we show that integrin-mediated cell survival involves regulation of nuclear factor-kappa B (NF-κB) activity. Different extracellular matrix molecules were able to protect rat aorta- derived endothelial cells from apoptosis induced by serum withdrawal. Osteopontin and β₃ integrin ligation rapidly increased NF-κB activity as measured by gel shift and reporter activity. The p65 and p50 subunits were present in the shifted complex. In contrast, collagen type I (a β₁-integrin ligand) did not induce NF-κB activity. The α_vβ₃ integrin was most important for osteopontin-mediated NF-κB induction and survival, since adding a neutralizing anti-β₃ integrin antibody blocked NF-κB activity and induced endothelial cell death when cells were plated on osteopontin. NF-κB was required for osteopontin- and vitronectin-induced survival since inhibition of NF-κB activity with nonphosphorylatable IκB completely blocked the protective effect of osteopontin and vitronectin. In contrast, NF-κB was not required for fibronectin, laminin, and collagen type I–induced survival. Activation of NF-κB by osteopontin depended on the small GTP-binding protein Ras and the tyrosine kinase Src, since NF-κB reporter activity was inhibited by Ras and Src dominant-negative mutants. In contrast, inhibition of MEK and PI3-kinase did not affect osteopontin-induced NF-κB activation. These studies identify NF-κB as an important signaling molecule in α_vβ₃ integrin-mediated endothelial cell survival.     

TRIM22 activates NF-κB signaling in glioblastoma by accelerating the degradation of IκBα

NF-κB signaling plays a critical role in tumor growth and treatment resistance in GBM as in many other cancers. However, the molecular mechanisms underlying high, constitutive NF-κB activity in GBM remains to be elucidated. Here, we screened a panel of tripartite motif (TRIM) family proteins and identified TRIM22 as a potential activator of NF-κB using an NF-κB driven luciferase reporter construct in GBM cell lines. Knockout of TRIM22 using Cas9-sgRNAs led to reduced GBM cell proliferation, while TRIM22 overexpression enhanced proliferation of cell populations, in vitro and in an orthotopic xenograft model. However, two TRIM22 mutants, one with a critical RING-finger domain deletion and the other with amino acid changes at two active sites of RING E3 ligase (C15/18A), were both unable to promote GBM cell proliferation over controls, thus implicating E3 ligase activity in the growth-promoting properties of TRIM22. Co-immunoprecipitations demonstrated that TRIM22 bound a negative regulator of NF-κB, NF-κB inhibitor alpha (IκBα), and accelerated its degradation by inducing K48-linked ubiquitination. TRIM22 also formed a complex with the NF-κB upstream regulator IKKγ and promoted K63-linked ubiquitination, which led to the phosphorylation of both IKKα/β and IκBα. Expression of a non-phosphorylation mutant, srIκBα, inhibited the growth-promoting properties of TRIM22 in GBM cell lines. Finally, TRIM22 was increased in a cohort of primary GBM samples on a tissue microarray, and high expression of TRIM22 correlated with other clinical parameters associated with progressive gliomas, such as wild-type IDH1 status. In summary, our study revealed that TRIM22 activated NF-κB signaling through posttranslational modification of two critical regulators of NF-κB signaling in GBM cells.Subject terms: CNS cancer, Oncogenes, Ubiquitin ligases     

Aneuploid senescent cells activate NF‐κB to promote their immune clearance by NK cells

The immune system plays a major role in the protection against cancer. Identifying and characterizing the pathways mediating this immune surveillance are thus critical for understanding how cancer cells are recognized and eliminated. Aneuploidy is a hallmark of cancer, and we previously found that untransformed cells that had undergone senescence due to highly abnormal karyotypes are eliminated by natural killer (NK) cells in vitro. However, the mechanisms underlying this process remained elusive. Here, using an in vitro NK cell killing system, we show that non‐cell‐autonomous mechanisms in aneuploid cells predominantly mediate their clearance by NK cells. Our data indicate that in untransformed aneuploid cells, NF‐κB signaling upregulation is central to elicit this immune response. Inactivating NF‐κB abolishes NK cell‐mediated clearance of untransformed aneuploid cells. In cancer cell lines, NF‐κB upregulation also correlates with the degree of aneuploidy. However, such upregulation in cancer cells is not sufficient to trigger NK cell‐mediated clearance, suggesting that additional mechanisms might be at play during cancer evolution to counteract NF‐κB‐mediated immunogenicity.