SUMMARY Bacteria, such as Fusobacterium nucleatum, are present in the tumor microenvironment. However, the immunological consequences of intra-tumoral bacteria remain unclear. Here, we have shown that natural killer (NK) cell killing of various tumors is inhibited in the presence of various F. nucleatum strains. Our data support that this F. nucleatum-mediated inhibition is mediated by human, but not by mouse TIGIT, an inhibitory receptor present on all human NK cells and on various T cells. Using a library of F. nucleatum mutants, we found that the Fap2 protein of F. nucleatum directly interacted with TIGIT, leading to the inhibition of NK cell cytotoxicity. We have further demonstrated that tumor-infiltrating lymphocytes expressed TIGIT and that T cell activities were also inhibited by F. nucleatum via Fap2. Our results identify a bacterium-dependent, tumor-immune evasion mechanism in which tumors exploit the Fap2 protein of F. nucleatum to inhibit immune cell activity via TIGIT.
We aimed to investigate the dynamics of the NF-B signaling pathway in living cells using GFP variants of p65-NF-B, IB␣, tumor necrosis factor-receptor associated factor 2 (TRAF2), the NF-B inducing kinase (NIK) and IB kinases (IKK1 and IKK2). Detailed kinetic analysis of constitutive nucleocytoplasmic shuttling processes revealed that IB␣ enters the nucleus faster than p65. Examination of signaling molecules upstream of NF-B and IB␣ revealed a predominant cytoplasmic localization at steady state. However, after addition of leptomycin B, NIK rapidly accumulated in the nucleus, whereas we could not detect any significant effect on TRAF2 or IKK2. Using various truncation mutants of NIK, we identified a functional nuclear export signal within the COOH-terminal region 795-805, which counteracts the inherent NLS at amino acids 143-149. Prolonged incubation in the presence of LMB also leads to nuclear accumulation of IKK1, which was dependent on a lysine residue at position 44, which is also essential for kinase activity. Investigation of endogenous protein levels by immunofluorescence staining and Western blots verified the results obtained with GFP chimeras. We conclude that NF-B⅐IB complexes and the upstream signaling kinases NIK and IKK1 shuttle between cytoplasm and nucleus of nonactivated cells and that this process leads to a basal transcriptional activity of NF-B.
The transient expression of many different genes is mediated by the inducible transcription factor p50‐p65 NF kappa B, which in turn is regulated by complex formation with its inhibitor I kappa B alpha. We describe here that in porcine aortic endothelial cells, either IL‐1 alpha, TNF alpha or LPS upregulates an inhibitor of NF kappa B which we refer to as ECI‐6. ECI‐6 is by structural and functional criteria an I kappa B alpha protein, the porcine homologue of MAD‐3, pp40 and RL/IF‐1. We have studied the promoter of the ECI‐6/I kappa B alpha gene and provide three lines of evidence that its expression is directly regulated by NF kappa B. First, the 5′ regulatory region of ECI‐6/I kappa B alpha contains two sites that bind NF kappa B in electrophoretic mobility shift assays. Second, expression following transfection of an ECI‐6/I kappa B alpha promoter‐luciferase reporter construct is dependent on a co‐transfected NF kappa B‐p65 subunit. Third, pretreatment of endothelial cells with antioxidants, agents that inhibit activation of NF kappa B, inhibit the expression of ECI‐6/I kappa B alpha. We conclude that the regulated expression of ECI‐6/I kappa B alpha could represent a novel feedback mechanism by which NF kappa B downregulates its own activity after transient activation of target genes has been achieved.
Tissue factor (TF) has been shown to be up-regulated in endothelial cells by the inflammatory cytokine tumor necrosis factor alpha (TNF-alpha) as well as by the main angiogenic factor VEGF. Since both stimuli induce the transcription factor EGR-1, which is critically involved in TF gene regulation, we used EGR-1-dependent TF induction as a model to identify potential cross-talks between the various signal transduction cascades initiated by VEGF and TNF-alpha. The data show that at the MAP kinase level, VEGF mainly activates ERK1/2 and p38 MAP kinases in human endothelial cells. TNF-alpha is able to activate all three MAP kinase cascades as well as the classical inflammatory IkappaB/NFkappaB pathway. Furthermore, the MEK/ERK module of MAP kinases appears to act as the convergence point of VEGF- and TNF-alpha-initiated signaling cascades, which lead to the activation of EGR-1 and subsequent TF expression, whereas the upstream signals are distinct. We found that induction of TF by VEGF via EGR-1 is strongly PKC dependent. The TNF-alpha-initiated MEK/ERK cascade connected to EGR-1 and TF expression is clearly less sensitive to PKC inhibition. TNF-alpha-mediated activation of MEK/ERK and EGR-1 can be blocked by adenoviral expression of a dominant negative mutant of IKK2, whereas the VEGF signaling pathway is unaffected. Thus, our data demonstrate a new link between the classical inflammatory IKK/IkappaB and the MEK/ERK cascades triggered by TNF-alpha. The additional finding that EGF induces ERK and EGR-1 in a PKC-independent manner and that this signal is not sufficient to up-regulate TF emphasizes the importance of a VEGF-specific signaling pattern for the induction of TF.
IntroductionTissue factor (TF) is a cell surface receptor initiating blood coagulation, 1 thereby promoting thrombotic events in atherosclerosis, sepsis, and cancer. 2,3 Enhanced endothelial TF expression has been demonstrated in atherosclerotic plaques, 4,5 a process that may account for thrombotic events associated with early and advanced atherosclerosis. TF expression in endothelial cells (ECs) can be induced by a variety of agonists, including inflammatory cytokines, angiogenic growth factors, infectious agents, and minimally modified low-density lipoprotein (MM-LDL). 1,6 MM-LDL regulates TF expression at the level of transcription 5 ; however, the signaling pathways and transcription factors involved in this process are not known. Some of the effects of MM-LDL can be mimicked by oxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (PAPC). 7 Three biologically active components of oxidized PAPC (Ox-PAPC) have been structurally identified as 1-palmitoyl-2-oxovaleroyl-sn-glycero-3-phosphorylcholine (POVPC), 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine (PGPC), 8 and 1-palmitoyl-2-epoxyisoprostane-sn-glycero-3-phosphorylcholine (PEIPC). 9 Which of these components of MM-LDL is responsible for induction of TF is not known.In contrast to interleukin-1 (IL-1) or tumor necrosis factor ␣ (TNF-␣), MM-LDL neither up-regulates E-selectin, intercellular adhesion molecule 1 (ICAM-1), and vascular cell adhesion molecule 1 (VCAM-1), nor stimulates neutrophil binding to human ECs. 7 This suggests that classical inflammatory agonists and MM-LDL activate different signaling mechanisms. The major pathway induced by inflammatory cytokines activates transcription factors of the nuclear factor-B (NF-B) family. 10 Whether NF-B is activated by MM-LDL is a subject of controversy. 6,11 In fact, it was shown that MM-LDL and some of its components were capable of down-regulating NF-B-mediated transcription induced by inflammatory cytokines. 12 Thus, the role of the NF-B pathway in inflammatory activation of ECs by oxidized lipids requires further investigation.Apart from NF-B, 13 transcription of the TF gene can be promoted by early growth response factor 1 (EGR-1) and nuclear factor of activated T cells (NFAT). 14,15 Whereas inflammatory cytokines induce NF-B as well as EGR-1, vascular endothelial In the present study, we investigated signaling pathways and transcription factors mediating induction of TF expression in human ECs by biologically active oxidized phospholipids. We show that expression of TF is elevated by OxPAPC, and that this induction was mainly mediated by EGR-1-and NFAT-dependent transcription, but was independent of NF-B activation. Upstream mechanisms activated by OxPAPC were elevation of cytosolic Ca ϩϩ , activation of protein kinase C (PKC), and the mitogenactivated protein (MAP) kinase/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK MAP kinase cascade. Materials and methods MaterialsCyclosporin A was purchased from Novartis (Vienna, Austria); TNF-␣ from Genzyme (Cambridge...
Fumaric acid esters, mainly dimethylfumarate (DMF), have been successfully used to treat psoriasis. Based on previous observations that DMF inhibited expression of several TNF-induced genes in endothelial cells, we wished to explore the molecular basis of DMF function in greater detail. In first experiments we analyzed DMF effects on tissue factor expression in human endothelial cells in culture, because tissue factor is expressed by two independent sets of transcription factors, by NF-κB via TNF and by early gene response-1 transcription factor via vascular endothelial growth factor (VEGF). We show that DMF inhibits TNF-induced tissue factor mRNA and protein expression as well as TNF-induced DNA binding of NF-κB proteins, but not VEGF-induced tissue factor protein, mRNA expression, or VEGF-induced early gene response-1 transcription factor/DNA binding. To determine where DMF interferes with the TNF/NF-κB signaling cascade, we next analyzed DMF effects on IκB and on the subcellular distribution of NF-κB. DMF does not inhibit TNF-induced IκBα phosphorylation and IκB degradation; thus, NF-κB is properly released from IκB complexes even in the presence of DMF. Importantly, DMF inhibits the TNF-induced nuclear entry of NF-κB proteins, and this effect appears selective for NF-κB after the release from IκB, because the constitutive shuttling of inactive NF-κB/IκB complexes into and out from the nucleus is not blocked by DMF. Moreover, DMF does not block NF-κB/DNA binding. In conclusion, DMF appears to selectively prevent the nuclear entry of activated NF-κB, and this may be the basis of its beneficial effect in psoriasis.
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