The stimulation of arginine transport by TNFα in human endothelial cells depends on NF-κB activation

In human saphenous vein endothelial cells (HSVECs), tumor necrosis factor-α (TNFα) and bacterial lipopolysaccharide (LPS), but neither interferon γ (IFNγ) nor interleukin 1β (IL-1β), stimulate arginine transport. The effects of TNFα and LPS are due solely to the enhancement of system y⁺ activity, whereas system y⁺L is substantially unaffected. TNFα  causes an increased expression of SLC7A2/CAT-2B gene while SLC7A1/CAT-1 expression is not altered by the cytokine. The suppression of PKC-dependent transduction pathways, obtained with the inhibitor chelerytrhine, the inhibitor peptide of PKCζ isoform, or chronic exposure to phorbol esters, does not prevent TNFα effect on arginine transport. Likewise, ERK, JNK, and p38 MAP kinases are not involved in the cytokine effect, since arginine transport stimulation is unaffected by their specific inhibitors. On the contrary, inhibitors of NF-κB pathway hinder the increase in CAT2B mRNA and the stimulation of arginine uptake. These results indicate that in human endothelial cells the activation of NF-κB pathway mediates the TNFα effects on arginine transport.     

Tat-DJ-1 enhances cell survival by inhibition of oxidative stress,NF-κB and MAPK activation in HepG2 cells

Objectives

To identify the protective effect of DJ-1 protein against oxidative stress-induced HepG2 cell death, we used cell-permeable wild type (WT) and a mutant (C106A Tat-DJ-1) protein.

Results

By using western blotting and fluorescence microscopy, we observed WT and C106A Tat-DJ-1 proteins were efficiently transduced into HepG2 cells. Transduced WT Tat-DJ-1 proteins increased cell survival and protected against DNA fragmentation and intracellular ROS generation levels in H₂O₂-exposed HepG2 cells. At the same time, transduced WT Tat-DJ-1 protein significantly inhibited NF-κB and MAPK (JNK and p38) activation as well as regulated the Bcl-2 and Bax expression levels. However, C106A Tat-DJ-1 protein did not show any protective effect against cell death responses in H₂O₂-exposed HepG2 cells.

Conclusions

Oxidative stress-induced HepG2 cell death was significantly reduced by transduced WT Tat-DJ-1 protein, not by C106A Tat-DJ-1 protein. Thus, transduction of WT Tat-DJ-1 protein could be a novel strategy for promoting cell survival in situations of oxidative stress-induced HepG2 cell death.

     

Poly (ADP-ribose) polymerase inhibition synergizes with the NF-κB inhibitor DHMEQ to kill hepatocellular carcinoma cells

Poly (ADP-ribose) polymerase (PARP) enzymes play a key role in the cellular machinery responsible for DNA repair. Dehydroxymethylepoxyquinomicin (DHMEQ), a new inhibitor of NF-κB, induces oxidative stress and DNA damage. The effects of DHMEQ in combination with Olaparib (PARP inhibitor) were studied on hepatocellular carcinoma (HCC) cells. The DHMEQ–Olaparib combination synergistically inhibited cell viability, cell proliferation and colony formation of Hep3B, but had additive effects on Huh7 cells. The synergistic effects of the combination correlated with increased apoptosis, caspase 3/7 activity and PARP cleavage. There was an induction of an endoplasmic reticulum (ER) stress response with significant up-regulation of CHOP and TRB3 genes and splicing of XBP1 mRNA in Hep3B cells but not in Huh7 cells. Silencing of the TRB3 mRNA in Hep3B cells reversed the reduction in viability caused by DHMEQ–Olaparib treatment, while depletion of unspliced XBP1 mRNA in DHMEQ–Olaparib-treated Huh7 cells reduced viability. ROS production was increased after DHMEQ–Olaparib treatment of Hep3B, which caused DNA damage by an accumulation of γH2AX, increased AKT phosphorylation and reduced cell viability. The combination reduced Rad51 nuclear foci in Hep3B cells (not Huh7 cells), and silencing of Rad51 enhanced sensitivity of Huh7 cells to the DHMEQ–Olaparib combination. Knockdown of AKT in Hep3B cells restored the number of Rad51 nuclear foci after DHMEQ–Olaparib treatment. In summary, the DHMEQ–Olaparib combination induced ROS production, which killed HCC cells via DNA damage that could not be repaired by Rad51.SummaryPARPs and NF-κB are frequently deregulated in HCC. The DHMEQ–Olaparib combination exerted synergistic anti-tumour effects on HCC cells through ROS production via DNA damage that could not be repaired by Rad51. This suggested that the DHMEQ–Olaparib combination could be used to treat tumours that were resistant to Olaparib treatment.     

DA-9601 inhibits activation of the human mast cell line HMC-1 through inhibition of NF-κB

Mast cell-mediated allergic inflammation is involved in many diseases such as asthma, sinusitis, and rheumatoid arthritis. Mast cells induce synthesis and production of pro-inflammatory cytokines including tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6 with immune regulatory properties. The formulated ethanol extract of Artemisia asiatica Nakai (DA-9601) has been reported to have antioxidative and anti-inflammatory activities. In this report, we investigated the effect of DA-9601 on the expression of pro-inflammatory cytokines by the activated human mast cell line HMC-1 and studied its possible mechanisms of action. DA-9601 dose-dependently decreased the gene expression and production of TNF-α, IL-1β, and IL-6 on phorbol 12-myristate 13-acetate (PMA)- and calcium ionophore A23187-stimulated HMC-1 cells. In addition, DA-9601 attenuated PMA- and A23187-induced activation of NF-κB as indicated by inhibition of degradation of IκBα, nuclear translocation of NF-κB, NF-κB/DNA binding, and NF-κB-dependent gene reporter assay. Our in vitro studies provide evidence that DA-9601 might contribute to the treatment of mast cell-derived allergic inflammatory diseases.     

TRAF6 is functional in inhibition of TLR4-mediated NF-κB activation by resveratrol

Resveratrol was suggested to inhibit Toll-like receptor (TLR)4-mediated activation of nuclear factor-κB (NF-κB) and Toll/interleukin-1 receptor domain-containing adaptor inducing interferon-β (TRIF)–(TANK)-binding kinase 1, but the myeloid differentiation primary response gene 88–tumor necrosis factor receptor-associated factor 6 (TRAF6) pathway is not involved in this effect. However, involvement of TRAF6 in this process is still elusive since cross talk between TRIF and TRAF6 has been reported in lipopolysaccharide (LPS)-induced signaling. Using RAW 264.7 macrophages, we determined the effect of resveratrol on LPS-induced TRAF6 expression, ubiquitination as well as activation of mitogen-activated protein (MAP) kinases and Akt in order to elucidate its involvement in TLR4 signaling. LPS-induced transient elevation in TRAF6 mRNA and protein expressions is suppressed by resveratrol. LPS induces the ubiquitination of TRAF6, which has been reported to be essential for Akt activation and for transforming growth factor-β activated kinase-1–NAP kinase kinase 6 (MKK6)-mediated p38 and c-Jun N-terminal kinase (JNK) activation. We found that resveratrol diminishes the effect of LPS on TRAF6 ubiquitination and activation of JNK and p38 MAP kinases, while it has no effect on the activation of extracellular-signal-regulated kinase (ERK)1/2. The effect of resveratrol on MAP kinase inhibition is significant since TRAF6 activation was reported to induce activation of JNK and p38 MAP kinase while not affecting ERK1/2. Moreover, Akt was identified previously as a direct target of TRAF6, and we found that, similarly to MAPKs, phosphorylation pattern of Akt followed the activation of TRAF6, and it was inhibited by resveratrol at all time points. Here, we provide the first evidence that resveratrol, by suppressing LPS-induced TRAF6 expression and ubiquitination, attenuates the LPS-induced TLR4–TRAF6, MAP kinase and Akt pathways that can be significant in its anti-inflammatory effects.     

Varicella-zoster virus inhibition of the NF-κB pathway during infection of human dendritic cells: role for open reading frame 61 as a modulator of NF-κB activity

Dendritic cells (DC) are antigen-presenting cells essential for initiating primary immune responses and therefore an ideal target for viral immune evasion. Varicella-zoster virus (VZV) can productively infect immature human DCs and impair their function as immune effectors by inhibiting their maturation, as evidenced by the expression modulation of functionally important cell surface immune molecules CD80, CD86, CD83, and major histocompatibility complex I. The NF-κB pathway largely regulates the expression of these immune molecules, and therefore we sought to determine whether VZV infection of DCs modulates the NF-κB pathway. Nuclear localization of NF-κB p50 and p65 indicates pathway activation; however, immunofluorescence studies revealed cytoplasmic retention of these NF-κB subunits in VZV-infected DCs. Western blotting revealed phosphorylation of the inhibitor of κBα (IκBα) in VZV-infected DCs, indicating that the pathway is active at this point. We conclude that VZV infection of DC inhibits the NF-κB pathway following protein phosphorylation but before the translocation of NF-κB subunits into the nucleus. An NF-κB reporter assay identified VZV open reading frame 61 (ORF61) as an inhibitor of tumor necrosis factor alpha-induced NF-κB reporter activity. Mutational analysis of ORF61 identified the E3 ubiquitin ligase domain as a region required for NF-κB pathway inhibition. In summary, we provide evidence that VZV inhibits the NF-κB signaling pathway in human DCs and that the E3 ubiquitin ligase domain of ORF61 is required to modulate this pathway. Thus, this work identifies a mechanism by which VZV modulates host immune function.     

BCA3 contributes to the malignant progression of hepatocellular carcinoma through AKT activation and NF-κB translocation

Hepatocellular carcinoma (HCC) is one of the most lethal malignancies worldwide with elusive molecular mechanisms. The aim of this study is to investigate the clinical significance and biological roles of breast cancer-associated protein 3 (BCA3) in HCC. Our investigation demonstrated that BCA3 expression was up-regulated in primary HCC tissues, and BCA3 levels were positively correlated with tumor size, TNM stage, microvascular invasion and poor prognosis. BCA3 promoted tumor growth, metastasis and angiogenesis of HCC in vitro and in vivo. Moreover, we found that BCA3 induced aggressive behaviors were mediated by AKT activation, which in turn activated mTOR signalling pathway and induced cytoplasm-nuclear translocation of NF-κB p65. Blockage of AKT signalling pathway by a specific AKT inhibitor LY294002 impaired BCA3 mediated phenotypes. Collectively, our current study indicated the pleiotropic effects of BCA3 in HCC progression, and blockage of BCA3-AKT pathway might contribute to development of therapeutic measures for HCC.     

1,25-dihydroxyvitamin D3 impairs NF-κB activation in human naïve B cells

1α,25-dihydroxyvitamin D(3) (calcitriol), the bioactive metabolite of vitamin D, modulates the activation and inhibits IgE production of anti-CD40 and IL-4 stimulated human peripheral B cells. Engagement of CD40 results in NF-κB p50 activation, which is essential for the class switch to IgE. Herein, we investigated by which mechanism calcitriol modulates NF-κB mediated activation of human na?ve B cells. Na?ve B cells were predominantly targeted by calcitriol in comparison with memory B cells as shown by pronounced induction of the VDR target gene cyp24a1. Vitamin D receptor activation resulted in a strongly reduced p105/p50 protein and mRNA expression in human na?ve B cells. This effect is mediated by impaired nuclear translocation of p65 and consequently reduced binding of p65 to its binding site in the p105 promoter. Our data indicate that the vitamin D receptor reduces NF-κB activation by interference with NF-κB p65 and p105. Thus, the vitamin D receptor inhibits costimulatory signal transduction in na?ve B cells, namely by reducing CD40 signaling.