Dengue Virus Serotype 2 Blocks Extracellular Signal-Regulated Kinase and Nuclear Factor-κB Activation to Downregulate Cytokine Production

Background

Dengue virus (DENV) infection is the most common mosquito-borne viral disease threatening human health around the world. Type I interferon (IFN) and cytokine production are crucial in the innate immune system. We previously reported that DENV serotype 2 (DENV-2) induced low levels of interferon regulatory factor 3 and NF-κB activation, thus leading to reduced production of IFN-β in the early phase of infection. Here, we determined whether DENV infection not only hampers type I IFN activation but also cytokine production triggered by Toll-like receptor (TLR) signaling.

Methodology/Principal Findings

We used quantitative RT-PCR and found that only low levels of IFN-β and inflammatory cytokines such as interleukin 10 (IL-10), IL-12 and tumor necrosis factor α (TNFα) mRNA were detected in DENV-2–infected bone-marrow–derived dendritic cells. Furthermore, DENV-2 infection repressed cytokine production triggered by TLR signaling. To elucidate the molecular mechanisms underlying this suppression event, we measured NF-κB activation by p65 nuclear translocation and luciferase reporter assay and found that NF-κB activation triggered by TLR ligands was blocked by DENV-2 infection. As well, extracellular signal-regulated kinase (ERK) activity was suppressed by DENV-2 infection.

Conclusions/Significance

To downregulate the host innate immunity, DENV-2 by itself is a weak inducer of type I IFN and cytokines, furthermore DENV-2 can also block the TLR-triggered ERK–NF-κB activation and cytokine production.     

Mechanisms of the TRIF-induced interferon-stimulated response element and NF-kappaB activation and apoptosis pathways

Toll-like receptor-3 is critically involved in host defense against viruses through induction of type I interferons (IFNs). Recent studies suggest that a Toll/interleukin-1 receptor domain-containing adapter protein (TRIF) and two protein kinases (TANK-binding kinase-1 (TBK1) and IkappaB kinase (IKK)-epsilon) are critically involved in Toll-like receptor-3-mediated IFN-beta production through activation of IFN regulatory factor (IRF)-3 and IRF-7. In this study, we demonstrate that TRIF interacts with both IRF-7 and IRF-3. In addition to TBK1 and IKKepsilon, our results indicate that IKKbeta can also phosphorylate IRF-3 and activate the IFN-stimulated response element. TRIF-induced IRF-3 and IRF-7 activation was mediated by TBK1 and its downstream kinases IKKbeta and IKKepsilon. TRIF induced NF-kappaB activation through an IKKbeta- and tumor necrosis factor receptor-associated factor-6-dependent (but not TBK1- and IKKepsilon-dependent) pathway. In addition, TRIF also induced apoptosis through a RIP/FADD/caspase-8-dependent and mitochondrion-independent pathway. Furthermore, our results suggest that the TRIF-induced IFN-stimulated response element and NF-kappaB activation and apoptosis pathways are uncoupled and provide a molecular explanation for the divergent effects induced by the adapter protein TRIF.     

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.     

Alteration of subcellular redox equilibrium and the consequent oxidative modification of nuclear factor kappaB are critical for anticancer cytotoxicity by emodin, a reactive oxygen species-producing agent

We previously found that emodin produced reactive oxygen species (ROS) intracellularly. In various tumor cells at low doses it enhances the cytotoxicity of As₂O₃, and at higher doses it renders cytotoxicity independently in vitro and in vivo. The effects involve redox-mediated inhibition of NF-κB activation. In this study, we focus on the mechanisms by which emodin inhibits NF-κB activation. Results in HeLa cells demonstrated that emodin at high doses or in combination with As₂O₃, via generation of ROS especially in the nucleus, altered subcellular redox equilibrium and thus oxidized the redox-sensitive site on NF-κB and prevented its binding to the target DNA. In vivo study showed that tumors exposed to the arsenic/emodin cotreatment had dramatically smaller sizes and weaker antioxidant capacity, compared with arsenic alone. NF-κB binding and transactivation were inhibited in these tumors. These data help in the understanding of the mechanisms by which manipulation of cellular redox and NF-κB activation may enhance chemotherapy.     

Tumor Necrosis Factor �� Represses Bone Morphogenetic Protein (BMP) Signaling by Interfering with the DNA Binding of Smads through the Activation of NF-��B

Bone morphogenetic proteins (BMPs) induce not only bone formation in vivo but also osteoblast differentiation of mesenchymal cells in vitro. Tumor necrosis factor α (TNFα) inhibits both osteoblast differentiation and bone formation induced by BMPs. However, the molecular mechanisms of these inhibitions remain unknown. In this study, we found that TNFα inhibited the alkaline phosphatase activity and markedly reduced BMP2- and Smad-induced reporter activity in MC3T3-E1 cells. TNFα had no effect on the phosphorylation of Smad1, Smad5, and Smad8 or on the nuclear translocation of the Smad1-Smad4 complex. In p65-deficient mouse embryonic fibroblasts, overexpression of p65, a subunit of NF-κB, inhibited BMP2- and Smad-induced reporter activity in a dose-dependent manner. Furthermore, this p65-mediated inhibition of BMP2- and Smad-responsive promoter activity was restored after inhibition of NF-κB by the overexpression of the dominant negative IκBα. Although TNFα failed to affect receptor-dependent formation of the Smad1-Smad4 complex, p65 associated with the complex. Chromatin immunoprecipitation and electrophoresis mobility shift assays revealed that TNFα suppressed the DNA binding of Smad proteins to the target gene. Importantly, the specific NF-κB inhibitor, BAY11-7082, abolished these phenomena. These results suggest that TNFα inhibits BMP signaling by interfering with the DNA binding of Smads through the activation of NF-κB.     

Phosphorylation of Thr-516 and Ser-520 in the Kinase Activation Loop of MEKK3 Is Required for Lysophosphatidic Acid-mediated Optimal I��B Kinase �� (IKK��)/Nuclear Factor-��B (NF-��B) Activation

MEKK3 serves as a critical intermediate signaling molecule in lysophosphatidic acid-mediated nuclear factor-κB (NF-κB) activation. However, the precise regulation for MEKK3 activation at the molecular level is still not fully understood. Here we report the identification of two regulatory phosphorylation sites at Thr-516 and Ser-520 within the kinase activation loop that is essential for MEKK3-mediated IκB kinase β (IKKβ)/NF-κB activation. Substitution of these two residues with alanine abolished the ability of MEKK3 to activate IKKβ/NF-κB, whereas replacement with acidic residues rendered MEKK3 constitutively active. Furthermore, substitution of these two residues with alanine abolished the ability of MEKK3 to mediate lysophosphatidic acid-induced optimal IKKβ/NF-κB activation.     

Involvement of protein kinase C in the inhibition of lipopolysaccharide-induced nitric oxide production by thapsigargin in RAW 264.7 macrophages

This study explored the effects of inhibition of endoplasmic reticulum (ER) Ca²⁺-ATPase on lipopolysaccharide (LPS)-induced protein kinase C (PKC) activation, nuclear factor-κB (NF-κB) translocation, inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production in RAW 264.7 macrophages. Thapsigargin (TG) irreversibly inhibits ER Ca²⁺-ATPase and LPS-induced NO production is reduced even after washout. TG also attenuated LPS-stimulated iNOS expression by using immunoblot analysis. However, another distinct fully reversible ER Ca²⁺-ATPase inhibitor, 2,5-di-tert-butylhydroquinone (DBHQ), ionophore A23187 and ionomycin could exert a similar effect to TG in increasing intracellular calcium concentration; however, these agents could not mimic TG in reducing iNOS expression and NO production. LPS increased PKC- and -β activation, and TG pretreatment attenuated LPS-stimulated PKC activation. Not did pretreatment with DBHQ, A23187 and ionomycin reduce LPS-stimulated PKC activation. Furthermore, NF-κB-specific DNA–protein-binding activity in the nuclear extracts was enhanced by treatment with LPS, and TG pretreatment attenuated LPS-stimulated NF-κB activation. None of DBHQ, A23187 and ionomycin pretreatment reduced LPS-stimulated NF-κB activation. These data suggest that persistent inhibition of ER Ca²⁺-ATPase by TG would influence calcium release from ER Ca²⁺ pools that was stimulated by the LPS activated signal processes, and might be the main mechanism for attenuating PKC and NF-κB activation that induces iNOS expression and NO production.     

Alpha-tomatine induces apoptosis and inhibits nuclear factor-kappa B activation on human prostatic adenocarcinoma PC-3 cells

Background

Alpha-tomatine (α-tomatine) is the major saponin in tomato (Lycopersicon esculentum). This study investigates the chemopreventive potential of α-tomatine on androgen-independent human prostatic adenocarcinoma PC-3 cells.

Methodology/Principal Findings

Treatment of highly aggressive human prostate cancer PC-3 cells with α-tomatine resulted in a concentration-dependent inhibition of cell growth with a half-maximal efficient concentration (EC₅₀) value of 1.67±0.3 µM. It is also less cytotoxic to normal human liver WRL-68 cells and normal human prostate RWPE-1 cells. Assessment of real-time growth kinetics by cell impedance-based Real-Time Cell Analyzer (RTCA) showed that α-tomatine exhibited its cytotoxic effects against PC-3 cells as early as an hour after treatment. The inhibitory effect of α-tomatine on PC-3 cancer cell growth was mainly due to induction of apoptosis as evidenced by positive Annexin V staining and decreased in mitochondrial membrane potential but increased in nuclear condensation, polarization of F-actin, cell membrane permeability and cytochrome c expressions. Results also showed that α-tomatine induced activation of caspase-3, -8 and -9, suggesting that both intrinsic and extrinsic apoptosis pathways are involved. Furthermore, nuclear factor-kappa B (NF-κB) nuclear translocation was inhibited, which in turn resulted in significant decreased in NF-κB/p50 and NF-κB/p65 in the nuclear fraction of the treated cells compared to the control untreated cells. These results provide further insights into the molecular mechanism of the anti-proliferative actions of α-tomatine.

Conclusion/Significance

α-tomatine induces apoptosis and inhibits NF-κB activation on prostate cancer cells. These results suggest that α-tomatine may be beneficial for protection against prostate cancer development and progression.     

Tumor Necrosis Factor (TNF) Receptor-associated Factor (TRAF)-interacting Protein (TRIP) Negatively Regulates the TRAF2 Ubiquitin-dependent Pathway by Suppressing the TRAF2-Sphingosine 1-Phosphate (S1P) Interaction

The signaling pathway downstream of TNF receptor (TNFR) is involved in the induction of a wide range of cellular processes, including cell proliferation, activation, differentiation, and apoptosis. TNFR-associated factor 2 (TRAF2) is a key adaptor molecule in TNFR signaling complexes that promotes downstream signaling cascades, such as nuclear factor-κB (NF-κB) and mitogen-activated protein kinase activation. TRAF-interacting protein (TRIP) is a known cellular binding partner of TRAF2 and inhibits TNF-induced NF-κB activation. Recent findings that TRIP plays a multifunctional role in antiviral response, cell proliferation, apoptosis, and embryonic development have increased our interest in exploring how TRIP can affect the TNFR-signaling pathway on a molecular level. In our current study, we demonstrated that TRIP is negatively involved in the TNF-induced inflammatory response through the down-regulation of proinflammatory cytokine production. Here, we demonstrated that the TRAF2-TRIP interaction inhibits Lys⁶³-linked TRAF2 ubiquitination by inhibiting TRAF2 E3 ubiquitin (Ub) ligase activity. The TRAF2-TRIP interaction inhibited the binding of sphingosine 1-phosphate, which is a cofactor of TRAF2 E3 Ub ligase, to the TRAF2 RING domain. Finally, we demonstrated that TRIP functions as a negative regulator of proinflammatory cytokine production by inhibiting TNF-induced NF-κB activation. These results indicate that TRIP is an important cellular regulator of the TNF-induced inflammatory response.