Emmanuel Dejardin

The Lymphotoxin-β Receptor Induces Different Patterns of Gene Expression via Two NF-κB Pathways

The lymphotoxin-beta receptor (LTbetaR) plays critical roles in inflammation and lymphoid organogenesis through activation of NF-kappaB. In addition to activation of the classical NF-kappaB, ligation of this receptor induces the processing of the cytosolic NF-kappaB2/p100 precursor to yield the mature p52 subunit, followed by translocation of p52 to the nucleus. This activation of NF-kappaB2 requires NIK and IKKalpha, while NEMO/IKKgamma is dispensable for p100 processing. IKKbeta-dependent activation of canonical NF-kappaB is required for the expression but not processing of p100 and for the expression of proinflammatory molecules including VCAM-1, MIP-1beta, and MIP-2 in response to LTbetaR ligation. In contrast, IKKalpha controls the induction by LTbetaR ligation of chemokines and cytokines involved in lymphoid organogenesis, including SLC, BLC, ELC, SDF1, and BAFF.

Specific and nonhepatotoxic degradation of nuclear hepatitis B virus cccDNA.

Clearance of Chronic Virus The family of mRNA-editing enzymes, APOBEC, restricts hepatitis B virus (HBV) replication. Lucifora et al. (p. 1221, published online 20 February; see the Perspective by Shlomai and Rice) provide evidence that specific APOBECs mediate the anti-HBV effects of host cytokines, which in turn apparently induce nuclear deaminase activity without damaging host cells. Thus, there may be potential in these findings for developing a therapeutic route to curing chronic HBV infection. Cytokine induction renders viral DNA vulnerable and eliminates infection. Current antiviral agents can control but not eliminate hepatitis B virus (HBV), because HBV establishes a stable nuclear covalently closed circular DNA (cccDNA). Interferon-α treatment can clear HBV but is limited by systemic side effects. We describe how interferon-α can induce specific degradation of the nuclear viral DNA without hepatotoxicity and propose lymphotoxin-β receptor activation as a therapeutic alternative. Interferon-α and lymphotoxin-β receptor activation up-regulated APOBEC3A and APOBEC3B cytidine deaminases, respectively, in HBV-infected cells, primary hepatocytes, and human liver needle biopsies. HBV core protein mediated the interaction with nuclear cccDNA, resulting in cytidine deamination, apurinic/apyrimidinic site formation, and finally cccDNA degradation that prevented HBV reactivation. Genomic DNA was not affected. Thus, inducing nuclear deaminases—for example, by lymphotoxin-β receptor activation—allows the development of new therapeutics that, in combination with existing antivirals, may cure hepatitis B.

The NF-kappa B transcription factor and cancer: high expression of NF-kappa B- and I kappa B-related proteins in tumor cell lines.

NF-kappa B is a pleiotropic transcription factor which controls the expression of many genes and viruses. To date, there is good evidence, but no definitive proof, for its role in tumor formation and development of metastasis. To investigate the possibility that members of the NF-kappa B family could participate in the molecular control of the transformed and invasive phenotype, we examined the expression of these proteins in a variety of human tumor cell lines. The expression of p50, p65, p52 and I kappa B was quantified at the protein level using western immunoblot and mobility shift assay and at the RNA level by northern blot. We observed high expression of the NF-kappa B inhibitor I kappa B in the ovarian carcinoma cell line OVCAR-3 together with constitutive nuclear NF-kappa B activity. We also studied the colon carcinoma cell line HT-29 and its metastatic counterpart HTM-29 and we observed specific expression of the p52 NF-kappa B-related protein in the metastatic cells. Our data confirm that NF-kappa B could be involved in the genesis of a variety of cancers including solid tumors and provide us with interesting models to explore the exact role of these transcription factors in cancer.

RelB/p50 Dimers Are Differentially Regulated by Tumor Necrosis Factor-α and Lymphotoxin-β Receptor Activation CRITICAL ROLES FOR p100

Tumor necrosis factor-α (TNF-α) and lymphotoxin-β receptor (LTβR) signaling both play important roles in inflammatory and immune responses through activation of NF-κB. Using various deficient mouse embryonic fibroblast cells, we have compared the signaling pathways leading to NF-κB induction in response to TNF-α and LTβR activation. We demonstrate that LTβR ligation induces not only RelA/p50 dimers but also RelB/p50 dimers, whereas TNF-α induces only RelA/p50 dimers. LTβR-induced binding of RelB/p50 requires processing of p100 that is mediated by IKKα but is independent of IKKβ, NEMO/IKKγ, and RelA. Moreover, we show that RelB, p50, and p100 can associate in the same complex and that TNF-α but not LTβ signaling increases the association of p100 with RelB/p50 dimers in the nucleus, leading to the specific inhibition of RelB DNA binding. These results suggest that the alternative NF-κB pathway based on p100 processing may account not only for the activation of RelB/p52 dimers but also for that of RelB/p50 dimers and that p100 regulates the binding activity of RelB/p50 dimers via at least two distinct mechanisms depending on the signaling pathway involved.

RIPK3 contributes to TNFR1-mediated RIPK1 kinase-dependent apoptosis in conditions of cIAP1/2 depletion or TAK1 kinase inhibition

Receptor-interacting protein kinase (RIPK) 1 and RIPK3 have emerged as essential kinases mediating a regulated form of necrosis, known as necroptosis, that can be induced by tumor necrosis factor (TNF) signaling. As a consequence, inhibiting RIPK1 kinase activity and repressing RIPK3 expression levels have become commonly used approaches to estimate the contribution of necroptosis to specific phenotypes. Here, we report that RIPK1 kinase activity and RIPK3 also contribute to TNF-induced apoptosis in conditions of cellular inhibitor of apoptosis 1 and 2 (cIAP1/2) depletion or TGF-β-activated kinase 1 (TAK1) kinase inhibition, implying that inhibition of RIPK1 kinase activity or depletion of RIPK3 under cell death conditions is not always a prerequisite to conclude on the involvement of necroptosis. Moreover, we found that, contrary to cIAP1/2 depletion, TAK1 kinase inhibition induces assembly of the cytosolic RIPK1/Fas-associated protein with death domain/caspase-8 apoptotic TNF receptor 1 (TNFR1) complex IIb without affecting the RIPK1 ubiquitylation status at the level of TNFR1 complex I. These results indicate that the recruitment of TAK1 to the ubiquitin (Ub) chains, and not the Ub chains per se, regulates the contribution of RIPK1 to the apoptotic death trigger. In line with this, we found that cylindromatosis repression only provided protection to TNF-mediated RIPK1-dependent apoptosis in condition of reduced RIPK1 ubiquitylation obtained by cIAP1/2 depletion but not upon TAK1 kinase inhibition, again arguing for a role of TAK1 in preventing RIPK1-dependent apoptosis downstream of RIPK1 ubiquitylation. Importantly, we found that this function of TAK1 was independent of its known role in canonical nuclear factor-κB (NF-κB) activation. Our study therefore reports a new function of TAK1 in regulating an early NF-κB-independent cell death checkpoint in the TNFR1 apoptotic pathway. In both TNF-induced RIPK1 kinase-dependent apoptotic models, we found that RIPK3 contributes to full caspase-8 activation independently of its kinase activity or intact RHIM domain. In contrast, RIPK3 participates in caspase-8 activation by acting downstream of the cytosolic death complex assembly, possibly via reactive oxygen species generation.

Inhibition of the Nf-Kappa B Transcription Factor Increases Bax Expression in Cancer Cell Lines

The NF-κB transcription factor has been shown to inhibit apoptosis in several experimental systems. We therefore investigated whether the expression of the Bax proapoptotic protein could be influenced by NF-κB activity. Increased Bax protein expression was detected in HCT116, OVCAR-3 and MCF7 cells stably expressing a mutated unresponsive IκB-α inhibitory protein that blocks NF-κB activity. Northern blots showed that bax mRNA expression was increased as a consequence of mutated IκB-α expression in HCT116 cells. A careful examination of the human bax gene promoter sequence showed three putative binding sites for NF-κB, and the κB2 site at position -687 could indeed bind NF-κB complexes in vitro. Transient transfection of a bax promoter luciferase construct in HCT116 cells showed that NF-κB proteins could partially inhibit the transactivation of the bax promoter by p53. Mutations or deletions of the κB sites, including κB2, indicated that this NF-κB-dependent inhibitory effect did not require NF-κB DNA-binding, and was thus an indirect effect. However, cotransfection of expression vectors for several known cofactors failed to identify a competition between p53 and NF-κB for a transcription coactivator. Our findings thus demonstrate for the first time that NF-κB regulates, through an indirect pathway, the bax gene expression.

Regulation of NF-kappaB activity by I kappaB-related proteins in adenocarcinoma cells.

Constitutive NF-κB activity varies widely among cancer cell lines. In this report, we studied the expression and the role of different IκB inhibitors in adenocarcinoma cell lines. High constitutive NF-κB activity and low IκB-α expression was found in a number of these cell lines. Moreover, some of these cells showed a high p100 expression, responsible for the cytoplasmic sequestration of most of p65 complexes. Treatment of these cells with TNF-α or other NF-κB activating agents induced only weakly nuclear NF-κB activity without significant p100 processing and led to a very weak transcription of NF-κB-dependent reporter gene. Induction of NF-κB activity can be restored by expression of the Tax protein or by treatment with antisense p100 oligonucleotides. In MCF7 A/Z cells stably transfected with a p100 expression vector, p65 complexes were sequestered in the cytoplasm by p100. These cells showed a reduced nuclear NF-κB induction and NF-κB-dependent gene transcription following TNF-α stimulation. As a consequence of a competition between IκB-α and p100, cells expressing high levels of p100 respond poorly to NF-κB activating stimuli as TNF-α.

NF-κB-Independent Role of IKKα/IKKβ in Preventing RIPK1 Kinase-Dependent Apoptotic and Necroptotic Cell Death during TNF Signaling

TNF is a master pro-inflammatory cytokine. Activation of TNFR1 by TNF can result in both RIPK1-independent apoptosis and RIPK1 kinase-dependent apoptosis or necroptosis. These cell death outcomes are regulated by two distinct checkpoints during TNFR1 signaling. TNF-mediated NF-κB-dependent induction of pro-survival or anti-apoptotic molecules is a well-known late checkpoint in the pathway, protecting cells from RIPK1-independent death. On the other hand, the molecular mechanism regulating the contribution of RIPK1 to cell death is far less understood. We demonstrate here that the IKK complex phosphorylates RIPK1 at TNFR1 complex I and protects cells from RIPK1 kinase-dependent death, independent of its function in NF-κB activation. We provide in vitro and in vivo evidence that inhibition of IKKα/IKKβ or its upstream activators sensitizes cells to death by inducing RIPK1 kinase-dependent apoptosis or necroptosis. We therefore report on an unexpected, NF-κB-independent role for the IKK complex in protecting cells from RIPK1-dependent death downstream of TNFR1.

RelB/p50 dimers are differentially regulated by TNF-α and lymphotoxin-β receptor activation: critical roles for p100

Tumor necrosis factor-α (TNF-α) and lymphotoxin-β receptor (LTβR) signaling both play important roles in inflammatory and immune responses through activation of NF-κB. Using various deficient mouse embryonic fibroblast cells, we have compared the signaling pathways leading to NF-κB induction in response to TNF-α and LTβR activation. We demonstrate that LTβR ligation induces not only RelA/p50 dimers but also RelB/p50 dimers, whereas TNF-α induces only RelA/p50 dimers. LTβR-induced binding of RelB/p50 requires processing of p100 that is mediated by IKKα but is independent of IKKβ, NEMO/IKKγ, and RelA. Moreover, we show that RelB, p50, and p100 can associate in the same complex and that TNF-α but not LTβ signaling increases the association of p100 with RelB/p50 dimers in the nucleus, leading to the specific inhibition of RelB DNA binding. These results suggest that the alternative NF-κB pathway based on p100 processing may account not only for the activation of RelB/p52 dimers but also for that of RelB/p50 dimers and that p100 regulates the binding activity of RelB/p50 dimers via at least two distinct mechanisms depending on the signaling pathway involved.

Egr Family Members Regulate Nonlymphoid Expression of Fas Ligand, TRAIL, and Tumor Necrosis Factor during Immune Responses

ABSTRACT The Fas ligand (FasL)/Fas pathway is crucial for homeostasis of the immune system and peripheral tolerance. Peripheral lymphocyte deletion involves FasL/Fas in at least two ways: coexpression of both Fas and its ligand on T cells, leading to activation-induced cell death, and expression of FasL by nonlymphoid cells, such as intestinal epithelial cells (IEC), that kill Fas-positive T cells. We demonstrate here that superantigen Staphylococcus enterotoxin B (SEB) induced a dramatic upregulation of FasL, TRAIL, and TNF mRNA expression and function in IEC from BALB/c and C57BL/6 mice. Using adoptive transfer in which CD4+ T cells from OT-2 T-cell receptor transgenic mice were transferred into recipients, we observed an induction in IEC of FasL, TRAIL, and TNF mRNA after administration of antigen. Specific Egr-binding sites have been identified in the 5′ promoter region of the FasL gene, and Egr-1, Egr-2, and Egr-3 mRNA in IEC from mice treated with SEB and from transgenic OT-2 mice after administration of antigen was upregulated. Overexpression of Egr-2 and Egr-3 induced endogenous ligand upregulation that was inhibited by overexpression of Egr-specific inhibitor Nab1. These results support a role for Egr family members in nonlymphoid expression of FasL, TRAIL, and TNF.