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Intracellular detection of RNA virus infection is mediated by the RNA helicase RIG-I, which is recruited to mitochondria by the adaptor protein MAVS and triggers activation of the transcription factors NF-κB, IRF3 and IRF7. Here we demonstrate that virus-induced activation of IRF3 and IRF7 depended on the NF-κB modulator NEMO, which acted 'upstream' of the kinases TBK1 and IKKε. IRF3 phosphorylation, formation of IRF3 dimers and DNA binding, as well as IRF3-dependent gene expression, were abrogated in NEMO-deficient cells. IRF3 phosphorylation and interferon production were restored by ectopic expression of NEMO. Thus, NEMO, like MAVS, acts as an adaptor protein that allows RIG-I to activate both the NF-κB and IRF signaling pathways.

Transcriptional activation of the human interleukin-2 (IL-2) gene, like induction of the IL-2 receptor $\\alpha $ (IL-2R$\\alpha $) gene and the type 1 human immunodeficiency virus (HIV-1), is shown to be modulated by a $\\kappa $B-like enhancer element. Mutation of a $\\kappa $B core sequence identified in the IL-2 promoter (-206 to -195) partially inhibits both mitogen- and HTLV-I Tax-mediated activation of this transcription unit and blocks the specific binding of two inducible cellular factors. These $\\kappa $B-specific proteins (80 to 90 and 50 to 55 kilodaltons) similarly interact with the functional $\\kappa $B enhancer present in the IL-2R$\\alpha $ promoter. These data suggest that these $\\kappa $B-specific proteins have a role in the coordinate regulation of this growth factor-growth factor receptor gene system that controls T cell proliferation.

Biochemical coupling of transcription factor NF-κB to antigen and co-stimulatory receptors is required for the temporal control of T-cell proliferation. In contrast to its transitory activation during normal growth-signal transduction, NF-κB is constitutively deployed in T-cells transformed by the type 1 human T-cell leukemia virus (HTLV-1). This viral/host interaction is mediated by the HTLV-1-encoded Tax protein, which has potent oncogenic properties. As reviewed here, Tax activates NF-κB primarily via a pathway leading to the chronic phosphorylation and degradation of IκBα, a cytoplasmic inhibitor of NF-κB. To access this pathway, Tax associates stably with a cytokine-inducible IκB kinase (IKK), which contains both catalytic (IKKα and IKKβ) and noncatalytic (IKKγ) subunits. Unlike their transiently induced counterparts in cytokine-treated cells, Tax-associated forms of IKKα and IKKβ are persistently activated in HTLV-1-infected T cells. Acquisition of the deregulated IKK phenotype is contingent on the presence of IKKγ, which functions as a molecular adaptor in the assembly of pathologic Tax/IκB kinase complexes. These findings highlight a key mechanistic role for IKK in the Tax/NF-κB signaling axis and define new intracellular targets for the therapeutic control of HTLV-1-associated disease.

The eukaryotic transcription factor nuclear factor-kappa B (NF-κB) participates in many parts of the genetic program mediating T lymphocyte activation and growth. Nuclear expression of NF-κB occurs after its induced dissociation from its cytoplasmic inhibitor IκBα. Phorbol ester and tumor necrosis factor-alpha induction of nuclear NF-κB is associated with both the degradation of preformed IκBα and the activation of IκBα gene expression. Transfection studies indicate that the IκBα gene is specifically induced by the 65-kilodalton transactivating subunit of NF-κB. Association of the newly synthesized IκBα with p65 restores intracellular inhibition of NF-κB DNA binding activity and prolongs the survival of this labile inhibitor. Together, these results show that NF-κB controls the expression of IκBα by means of an inducible autoregulatory pathway.

Oncogenic mutations in ras lead to constitutive activation of downstream signaling pathways that modulate the activities of transcription factors. In turn, these factors control the expression of a subset of genes responsible for neoplastic cell transformation. Recent studies suggest that transcription factor NF-kappa B contributes to cell transformation by inhibiting the cell death signal activated by oncogenic Ras. In this study, inhibition of NF-kappa B activity by forced expression of a super-repressor form of I kappa B alpha, the major inhibitor of NF-kappa B, markedly decreased the growth rate, saturation density and tumorigenicity of oncogenic H-Ras transformed rat embryo fibroblasts. Such clonally isolated cells overexpressing I kappa B alpha super-repressor not only were viable but also exhibited no sign of spontaneous apoptosis. Inhibition of NF-kappa B in these cells was functionally demonstrated by both the loss of cytokine induced DNA binding activity and a profoundly increased sensitivity to cell death in response to TNF-alpha treatment. In contrast, inhibition of NF-kappa B activity in non-transformed fibroblasts had minimal effect on growth, but rendered the cells resistant to a subsequent transformation by H-ras oncogene. Similar results were also obtained with rat intestinal epithelial cells harboring an inducible ras oncogene. Taken together, these findings suggest that NF-kappa B activity is essential for abnormal cell proliferation and tumorigenicity activated by the ras oncogene and highlight an alternative functional role for NF-kappa B in oncogenic Ras-mediated cell transformation that is distinct from its anti-apoptotic activity. Oncogene (2000) 19, 841 - 849.

Members of the NF-κ B/Rel and inhibitor of apoptosis (IAP) protein families have been implicated in signal transduction programs that prevent cell death elicited by the cytokine tumor necrosis factor α (TNF). Although NF-κ B appears to stimulate the expression of specific protective genes, neither the identities of these genes nor the precise role of IAP proteins in this anti-apoptotic process are known. We demonstrate here that NF-κ B is required for TNF-mediated induction of the gene encoding human c-IAP2. When overexpressed in mammalian cells, c-IAP2 activates NF-κ B and suppresses TNF cytotoxicity. Both of these c-IAP2 activities are blocked in vivo by coexpressing a dominant form of Iκ B that is resistant to TNF-induced degradation. In contrast to wild-type c-IAP2, a mutant lacking the C-terminal RING domain inhibits NF-κ B induction by TNF and enhances TNF killing. These findings suggest that c-IAP2 is critically involved in TNF signaling and exerts positive feedback control on NF-κ B via an Iκ B targeting mechanism. Functional coupling of NF-κ B and c-IAP2 during the TNF response may provide a signal amplification loop that promotes cell survival rather than death.

The long terminal repeat (LTR) of the type 1 human immunodeficiency virus (HIV-1) and the 5' regulatory region of the gene encoding the interleukin 2 receptor α subunit (IL-2Rα) share functional κB enhancer elements involved in the regulation of these inducible transcription units during T-cell activation. These κB enhancer elements are recognized by a structurally related family of interactive proteins that includes p50, p65, and the product of the c-rel protooncogene (c-Rel). Recent biochemical studies have shown that p65 and p50 form the prototypical NF-κB complex, which is rapidly translocated from the cytoplasm to the nucleus during T-cell activation. This intracellular signaling complex potently stimulates κB-directed transcription from either the HIV-1 LTR or the IL-2Rα promoter via the strong transactivation domain present in p65. We now demonstrate that nuclear expression of human c-Rel, which is induced by either phorbol ester or tumor necrosis factor α with delayed kinetics relative to p65, markedly represses p65-mediated activation of these transcription units. These inhibitory effects of c-Rel correlate with its DNA-binding activity but not with its ability to heterodimerize with p50, suggesting that c-Rel inhibition involves competition with p50/p65 for occupancy of the κB enhancer element. Together, these findings suggest that one function of c-Rel is as a physiologic repressor of the HIV-1 LTR and IL-2Rα promoters, serving to efficiently counter the strong transcriptional activating effects of p65.

Intracellular detection of RNA virus infection is mediated by the RNA helicase RIG-I, which is recruited to mitochondria by the adaptor protein MAVS and triggers activation of the transcription factors NF-kappaB, IRF3 and IRF7. Here we demonstrate that virus-induced activation of IRF3 and IRF7 depended on the NF-kappaB modulator NEMO, which acted 'upstream' of the kinases TBK1 and IKKepsilon. IRF3 phosphorylation, formation of IRF3 dimers and DNA binding, as well as IRF3-dependent gene expression, were abrogated in NEMO-deficient cells. IRF3 phosphorylation and interferon production were restored by ectopic expression of NEMO. Thus, NEMO, like MAVS, acts as an adaptor protein that allows RIG-I to activate both the NF-kappaB and IRF signaling pathways.

RelA (p65) functions as the critical transactivating component of the heterodimeric p50-p65 NF-κB complex and contains a high-affinity binding site for its cytoplasmic inhibitor, IκBα. After cellular activation, IκBα is rapidly degraded in concert with the induced nuclear translocation of NF-κB. The present study demonstrates that tumor necrosis factor α-induced degradation of IκBα in human T cells is preceded by its rapid phosphorylation in vivo. However, these effects on IκBα result in nuclear mobilization of only a fraction of the entire cytoplasmic pool of RelA. Subsequent studies have revealed that (i) cytoplasmic RelA is stably associated not only with IκBα but also with other ankyrin motifrich proteins including the products of the NF-κB2 (p100) and NF-κB1 (p105) genes; (ii) in contrast to RelA-IκBα, RelA-p100 cytoplasmic complexes are not dissociated following tumor necrosis factor α activation; (iii) p100 functions as a potent inhibitor of RelA-mediated transcription in vivo; (iv) the interaction of RelA and p100 involves the conserved Rel homology domain of both proteins but not the nuclear localization signal of RelA, which is required for IκBα binding; (v) p100 inhibition of RelA function requires the C-terminal ankyrin motif domain, which mediates cytoplasmic retention of RelA; and (vi) as observed with IκBα, nuclear RelA stimulates p100 mRNA and protein expression. These findings thus reveal the presence of a second inducible autoregulated inhibitory pathway that helps ensure the rapid but transient action of nuclear NF-κB.

Molecular cloning of the polypeptide component of the Rel-related human p75 nucleoprotein complex has revealed its identity with the 65-kDa (p65) subunit of NF-κB. Functional analyses of chimeric proteins composed of NF-κB p65 C-terminal sequences linked to the DNA-binding domain of the yeast GAL4 polypeptide have indicated that the final 101 amino acids of NF-κB p65 comprise a potent transcriptional activation domain. Transient transfection of human T cells with an expression vector encoding NF-κB p65, but not NF-κB p50, produced marked transcriptional activation of a basal promoter containing duplicated κB enhancer motifs from the long terminal repeat of type 1 human immunodeficiency virus. These stimulatory effects of NF-κB p65 were synergistically enhanced by coexpression of NF-κB p50 but were completely inhibited by coexpression of the v-rel oncogene product. Together, these functional studies demonstrate that NF-κB p65 is a transactivating subunit of the heterodimeric NF-κB complex and serves as one cellular target for v-Rel-mediated transcriptional repression.