Ubiquitin-specific Peptidase 21 Inhibits Tumor Necrosis Factor ��-induced Nuclear Factor ��B Activation via Binding to and Deubiquitinating Receptor-interacting Protein 1

Ubiquitination and deubiquitination of receptor-interacting protein 1 (RIP1) play an important role in the positive and negative regulation of the tumor necrosis factor α (TNFα)-induced nuclear factor κB (NF-κB) activation. Using a combination of functional genomic and proteomic approaches, we have identified ubiquitin-specific peptidase 21 (USP21) as a deubiquitinase for RIP1. USP21 is constitutively associated with RIP1 and deubiquitinates RIP1 in vitro and in vivo. Notably, knockdown of USP21 in HeLa cells enhances TNFα-induced RIP1 ubiquitination, IκB kinase β (IKKβ), and NF-κB phosphorylation, inhibitor of NF-κB α (IκBα) phosphorylation and ubiquitination, as well as NF-κB-dependent gene expression. Therefore, our results demonstrate that USP21 plays an important role in the down-regulation of TNFα-induced NF-κB activation through deubiquitinating RIP1.     

Inhibition of I��B Kinase by Vaccinia Virus Virulence Factor B14

The IκB kinase (IKK) complex is a key regulator of signal transduction pathways leading to the induction of NF-κB-dependent gene expression and production of pro-inflammatory cytokines. It therefore represents a major target for the development of anti-inflammatory therapeutic drugs and may be targeted by pathogens seeking to diminish the host response to infection. Previously, the vaccinia virus (VACV) strain Western Reserve B14 protein was characterised as an intracellular virulence factor that alters the inflammatory response to infection by an unknown mechanism. Here we demonstrate that ectopic expression of B14 inhibited NF-κB activation in response to TNFα, IL-1β, poly(I:C), and PMA. In cells infected with VACV lacking gene B14R (vΔB14) there was a higher level of phosphorylated IκBα but a similar level of IκBα compared to cells infected with control viruses expressing B14, suggesting B14 affects IKK activity. Direct evidence for this was obtained by showing that B14 co-purified and co-precipitated with the endogenous IKK complex from human and mouse cells and inhibited IKK complex enzymatic activity. Notably, the interaction between B14 and the IKK complex required IKKβ but not IKKα, suggesting the interaction occurs via IKKβ. B14 inhibited NF-κB activation induced by overexpression of IKKα, IKKβ, and a constitutively active mutant of IKKα, S176/180E, but did not inhibit a comparable mutant of IKKβ, S177/181E. This suggested that phosphorylation of these serine residues in the activation loop of IKKβ is targeted by B14, and this was confirmed using Ab specific for phospho-IKKβ.     

RelA/p65 functions to maintain cellular senescence by regulating genomic stability and DNA repair

Nuclear factor (NF)-κB is a positive regulator of tumour development and progression, but how it functions in normal cells leading to oncogenesis is not clear. As cellular senescence has proven to be an intrinsic tumour suppressor mechanism that cells must overcome to establish deregulated growth, we used primary fibroblasts to follow NF-κB function in cells transitioning from senescence to subsequent immortalization. Our findings show that RelA/p65^−/− murine fibroblasts immortalize at considerably faster rates than RelA/p65^+/+ cells. The ability of RelA/p65^−/− fibroblasts to escape senescence earlier is due to their genomic instability, characterized by high frequencies of DNA mutations, gene deletions and gross chromosomal translocations. This increase in genomic instability is closely related to a compromised DNA repair that occurs in both murine RelA/p65^−/− fibroblasts and tissues. Significantly, these results can also be duplicated in human fibroblasts lacking NF-κB. Altogether, our findings present a fresh perspective on the role of NF-κB as a tumour suppressor, which acts in pre-neoplastic cells to maintain cellular senescence by promoting DNA repair and genomic stability.     

NF-��B Functions in Stromal Fibroblasts to Regulate Early Postnatal Muscle Development

Classical NF-κB activity functions as an inhibitor of the skeletal muscle myogenic program. Recent findings reveal that even in newborn RelA/p65^−/− mice, myofiber numbers are increased over that of wild type mice, suggesting that NF-κB may be a contributing factor in early postnatal skeletal muscle development. Here we show that in addition to p65 deficiency, repression of NF-κB with the IκBα-SR transdominant inhibitor or with muscle-specific deletion of IKKβ resulted in similar increases in total fiber numbers as well as an up-regulation of myogenic gene products. Upon further characterization of early postnatal muscle, we observed that NF-κB activity progressively declines within the first few weeks of development. At birth, the majority of this activity is compartmentalized to muscle fibers, but by neonatal day 8 NF-κB activity from the myofibers diminishes, and instead, stromal fibroblasts become the main cellular compartment within the muscle that contains active NF-κB. We find that NF-κB functions in these fibroblasts to regulate inducible nitric-oxide synthase expression, which we show is important for myoblast fusion during the growth and maturation process of skeletal muscle. Together, these data broaden our understanding of NF-κB during development by showing that in addition to its role as a negative regulator of myogenesis, NF-κB also regulates nitric-oxide synthase expression within stromal fibroblasts to stimulate myoblast fusion and muscle hypertrophy.     

B-cell CLL/Lymphoma 10 (BCL10) Is Required for NF-��B Production by Both Canonical and Noncanonical Pathways and for NF-��B-inducing Kinase (NIK) Phosphorylation

B-cell CLL/lymphoma 10 (BCL10), the caspase recruitment domain (CARD)-containing protein involved in the etiology of the mucosa-associated lymphoid tissue (MALT) lymphomas, has been implicated in inflammatory processes in epithelial cells, as well as in immune cells. Experiments in this report indicate that BCL10 is required for activation of nuclear factor (NF)-κB by both canonical and noncanonical pathways, following stimulation by the sulfated polysaccharide carrageenan (CGN). In wild type and IκB-kinase (IKK)α^−/− mouse embryonic fibroblasts, increases in phospho-IκBα, nuclear NF-κB p65 (RelA) and p50, and KC, the mouse analog of human interleukin-8, were markedly reduced by silencing BCL10 or by exposure to the free radical scavenger Tempol. In IKKβ^−/− cells, BCL10 silencing, but not Tempol, reduced the CGN-induced increases in KC, phospho-NF-κB-inducing kinase (NIK), cytoplasmic NF-κB p100, and nuclear NF-κB p52 and RelB, suggesting a BCL10 requirement for activation of the noncanonical pathway. In NCM460 cells, derived from normal, human colonic epithelium, the CGN-induced increases in NF-κB family members, p65, p50, p52, and RelB, were inhibited by BCL10 silencing. Although enzyme-linked immunosorbent assay and confocal images demonstrated no change in total NIK following CGN, increases in phospho-NIK in the wild type, IKKβ^−/− and IKKα^−/− cells were inhibited by silencing BCL10. These findings indicate an upstream signaling role for BCL10, in addition to its effects on IKKγ, the regulatory component of the IKK signalosome, and a requirement for BCL10 in both canonical and noncanonical pathways of NF-κB activation. Also, the commonly used food additive carrageenan can be added to the short list of known activators of both pathways.     

Azadirachtin Interacts with the Tumor Necrosis Factor (TNF) Binding Domain of Its Receptors and Inhibits TNF-induced Biological Responses

The role of azadirachtin, an active component of a medicinal plant Neem (Azadirachta indica), on TNF-induced cell signaling in human cell lines was investigated. Azadirachtin blocks TNF-induced activation of nuclear factor κB (NF-κB) and also expression of NF-κB-dependent genes such as adhesion molecules and cyclooxygenase 2. Azadirachtin inhibits the inhibitory subunit of NF-κB (IκBα) phosphorylation and thereby its degradation and RelA (p65) nuclear translocation. It blocks IκBα kinase (IKK) activity ex vivo, but not in vitro. Surprisingly, azadirachtin blocks NF-κB DNA binding activity in transfected cells with TNF receptor-associated factor (TRAF)2, TNF receptor-associated death domain (TRADD), IKK, or p65, but not with TNFR, suggesting its effect is at the TNFR level. Azadirachtin blocks binding of TNF, but not IL-1, IL-4, IL-8, or TNF-related apoptosis-inducing ligand (TRAIL) with its respective receptors. Anti-TNFR antibody or TNF protects azadirachtin-mediated down-regulation of TNFRs. Further, in silico data suggest that azadirachtin strongly binds in the TNF binding site of TNFR. Overall, our data suggest that azadirachtin modulates cell surface TNFRs thereby decreasing TNF-induced biological responses. Thus, azadirachtin exerts an anti-inflammatory response by a novel pathway, which may be beneficial for anti-inflammatory therapy.     

Modulation of SCF��-TrCP-dependent I��B�� Ubiquitination by Hydrogen Peroxide

Reactive oxygen species are known to participate in the regulation of intracellular signaling pathways, including activation of NF-κB. Recent studies have indicated that increases in intracellular concentrations of hydrogen peroxide (H₂O₂) have anti-inflammatory effects in neutrophils, including inhibition of the degradation of IκBα after TLR4 engagement. In the present experiments, we found that culture of lipopolysaccharide-stimulated neutrophils and HEK 293 cells with H₂O₂ resulted in diminished ubiquitination of IκBα and decreased SCF^β-TrCP ubiquitin ligase activity. Exposure of neutrophils or HEK 293 cells to H₂O₂ was associated with reduced binding between phosphorylated IκBα and SCF^β-TrCP but no change in the composition of the SCF^β-TrCP complex. Lipopolysaccharide-induced SCF^β-TrCP ubiquitin ligase activity as well as binding of β-TrCP to phosphorylated IκBα was decreased in the lungs of acatalasemic mice and mice treated with the catalase inhibitor aminotriazole, situations in which intracellular concentrations of H₂O₂ are increased. Exposure to H₂O₂ resulted in oxidative modification of cysteine residues in β-TrCP. Cysteine 308 in Blade 1 of the β-TrCP β-propeller region was found to be required for maximal binding between β-TrCP and phosphorylated IκBα. These findings suggest that the anti-inflammatory effects of H₂O₂ may result from its ability to decrease ubiquitination as well as subsequent degradation of IκBα through inhibiting the association between IκBα and SCF^β-TrCP.     

Histone Deacetylase Inhibitors Activate NF-��B in Human Leukemia Cells through an ATM/NEMO-related Pathway

Mechanisms underlying histone deacetylase inhibitor (HDACI)-mediated NF-κB activation were investigated in human leukemia cells. Exposure of U937 and other leukemia cells to LBH-589 induced reactive oxygen species (ROS) followed by single strand (XRCC1) and double strand (γ-H2AX) DNA breaks. Notably, LBH-589 lethality was markedly attenuated by small interfering RNA (siRNA) knockdown of the DNA damage-linked histone, H1.2. LBH-589 triggered p65/RelA activation, NF-κB-dependent induction of Mn-SOD2, and ROS elimination. Interference with LBH-589-mediated NF-κB activation (e.g. in IκBα super-repressor transfected cells) diminished HDACI-mediated Mn-SOD2 induction and increased ROS accumulation, DNA damage, and apoptosis. The Mn-SOD2 mimetic TBAP (manganese(III)-tetrakis 4-benzoic acid porphyrin) prevented HDACI-induced ROS and NF-κB activation while dramatically attenuating DNA damage and cell death. In contrast, TRAF2 siRNA knockdown, targeting receptor-mediated NF-κB activation, blocked TNFα- but not HDACI-mediated NF-κB activation and lethality. Consistent with ROS-mediated DNA damage, LBH-589 exposure activated ATM (on serine 1981) and increased its association with NEMO. Significantly, siRNA NEMO or ATM knockdown blocked HDACI-mediated NF-κB activation, resulting in diminished MnSOD2 induction and enhanced oxidative DNA damage and cell death. In accord with the recently described DNA damage/ATM/NEMO pathway, SUMOylation site mutant NEMO (K277A or K309A) cells exposed to LBH-589 displayed diminished ATM/NEMO association, NEMO and p65/RelA nuclear localization/activation, and MnSOD2 up-regulation. These events were accompanied by increased ROS production, γ-H2AX formation, and cell death. Together, these findings indicate that in human leukemia cells, HDACIs activate the cytoprotective NF-κB pathway through an ATM/NEMO/SUMOylation-dependent process involving the induction of ROS and DNA damage and suggest that blocking NF-κB activation via the atypical ATM/NEMO nuclear pathway can enhance HDACI antileukemic activity.     

Natural proteasome inhibitor celastrol suppresses androgen-independent prostate cancer progression by modulating apoptotic proteins and NF-kappaB

Background

Celastrol is a natural proteasome inhibitor that exhibits promising anti-tumor effects in human malignancies, especially the androgen-independent prostate cancer (AIPC) with constitutive NF-κB activation. Celastrol induces apoptosis by means of proteasome inhibition and suppresses prostate tumor growth. However, the detailed mechanism of action remains elusive. In the current study, we aim to test the hypothesis that celastrol suppresses AIPC progression via inhibiting the constitutive NF-κB activity as well as modulating the Bcl-2 family proteins.

Methodology/Principal Findings

We examined the efficacy of celastrol both in vitro and in vivo, and evaluated the role of NF-κB in celastrol-mediated AIPC regression. We found that celastrol inhibited cell proliferation in all three AIPC cell lines (PC-3, DU145 and CL1), with IC₅₀ in the range of 1–2 µM. Celastrol also suppressed cell migration and invasion. Celastrol significantly induced apoptosis as evidenced by increased sub-G1 population, caspase activation and PARP cleavage. Moreover, celastrol promoted cleavage of the anti-apoptotic protein Mcl-1 and activated the pro-apoptotic protein Noxa. In addition, celastrol rapidly blocked cytosolic IκBα degradation and nuclear translocation of RelA. Likewise, celastrol inhibited the expression of multiple NF-κB target genes that are involved in proliferation, invasion and anti-apoptosis. Celastrol suppressed AIPC tumor progression by inhibiting proliferation, increasing apoptosis and decreasing angiogenesis, in PC-3 xenograft model in nude mouse. Furthermore, increased cellular IκBα and inhibited expression of various NF-κB target genes were observed in tumor tissues.

Conclusions/Significance

Our data suggest that, via targeting the proteasome, celastrol suppresses proliferation, invasion and angiogenesis by inducing the apoptotic machinery and attenuating constitutive NF-κB activity in AIPC both in vitro and in vivo. Celastrol as an active ingredient of traditional herbal medicine could thus be developed as a new therapeutic agent for hormone-refractory prostate cancer.