X-ray structure of a NF-kappaB p50/RelB/DNA complex reveals assembly of multiple dimers on tandem kappaB sites

We describe here the X-ray crystal structure of NF-kappaB p50/RelB heterodimer bound to a kappaB DNA. Although the global modes of subunit association and kappaB DNA recognition are similar to other NF-kappaB/DNA complexes, this complex reveals distinctive features not observed for non-RelB complexes. For example, Lys274 of RelB is removed from the protein-DNA interface whereas the corresponding residues in all other subunits make base-specific contacts. This mode of binding suggests that RelB may allow the recognition of more diverse kappaB sequences. Complementary surfaces on RelB and p50, as revealed by the crystal contacts, are highly suggestive of assembly of multiple p50/RelB heterodimers on tandem kappaB sites in solution. Consistent with this model our in vitro binding experiments reveal optimal assembly of two wild-type p50/RelB heterodimers on tandem HIV kappaB DNA with 2 bp spacing but not by a mutant heterodimer where one of the RelB packing surface is altered. We suggest that multiple NF-kappaB dimers assemble at diverse kappaB promoters through direct interactions utilizing unique protein-protein interaction surfaces.     

RelB is required for Peyer's patch development: differential regulation of p52-RelB by lymphotoxin and TNF

Targeted disruption of the Rel/NF-kappaB family members NF-kappaB2, encoding p100/p52, and RelB in mice results in anatomical defects of secondary lymphoid tissues. Here, we report that development of Peyer's patch (PP)-organizing centers is impaired in both NF-kappaB2- and RelB-deficient animals. IL-7-induced expression of lymphotoxin (LT) in intestinal cells, a crucial step in PP development, is not impaired in RelB-deficient embryos. LTbeta receptor (LTbetaR)-deficient mice also lack PPs, and we demonstrate that LTbetaR signaling induces p52-RelB and classical p50-RelA heterodimers, while tumor necrosis factor (TNF) activates only RelA. LTbetaR-induced binding of p52-RelB requires the degradation of the inhibitory p52 precursor, p100, which is mediated by the NF-kappaB-inducing kinase (NIK) and the IkappaB kinase (IKK) complex subunit IKKalpha, but not IKKbeta or IKKgamma. Activation of RelA requires all three IKK subunits, but is independent of NIK. Finally, we show that TNF increases p100 levels, resulting in the specific inhibition of RelB DNA binding via the C-terminus of p100. Our data indicate an important role of p52-RelB heterodimers in lymphoid organ development downstream of LTbetaR, NIK and IKKalpha.     

Gene expression profiling in conjunction with physiological rescues of IKKalpha-null cells with wild type or mutant IKKalpha reveals distinct classes of IKKalpha/NF-kappaB-dependent genes

Cellular responses to stress-like stimuli require the IkappaB kinase (IKK) signalsome (IKKalpha, IKKbeta, and NEMO/IKKgamma) to activate NF-kappaB-dependent genes. IKKbeta and NEMO/IKKgamma are required to release NF-kappaB p65/p50 heterodimers from IkappaBalpha, resulting in their nuclear migration and sequence-specific DNA binding; but IKKalpha was found to be dispensable for this initial phase of canonical NF-kappaB activation. Nevertheless, IKKalpha-/- mouse embryonic fibroblasts (MEFs) fail to express NF-kappaB targets in response to proinflammatory stimuli, uncovering a nuclear role for IKKalpha in NF-kappaB activation. However, it remains unknown whether the global defect in NF-kappaB-dependent gene expression of IKKalpha-/- cells is caused by the absence of IKKalpha kinase activity. We show by gene expression profiling that rescue of near physiological levels of wild type IKKalpha in IKKalpha-/- MEFs globally restores expression of their canonical NF-kappaB target genes. To prove that the kinase activity of IKKalpha was required on a genomic scale, the same physiological rescue was performed with a kinase-dead, ATP binding domain IKKalpha mutant (IKKalpha(K44M)). Remarkably, the IKKalpha(K44M) protein rescued approximately 28% of these genes, albeit in a largely stimulus-independent manner with the notable exception of several genes that also acquired tumor necrosis factor-alpha responsiveness. Thus the IKKalpha-containing signalsome unexpectedly functions in the presence and absence of extracellular signals in both kinase-dependent and -independent modes to differentially modulate the expression of five distinct classes of IKKalpha/NF-kappaB-dependent genes.     

RelB/p50 dimers are differentially regulated by tumor necrosis factor-alpha and lymphotoxin-beta receptor activation: critical roles for p100

Tumor necrosis factor-alpha (TNF-alpha) and lymphotoxin-beta receptor (LTbetaR) signaling both play important roles in inflammatory and immune responses through activation of NF-kappaB. Using various deficient mouse embryonic fibroblast cells, we have compared the signaling pathways leading to NF-kappaB induction in response to TNF-alpha and LTbetaR activation. We demonstrate that LTbetaR ligation induces not only RelA/p50 dimers but also RelB/p50 dimers, whereas TNF-alpha induces only RelA/p50 dimers. LTbetaR-induced binding of RelB/p50 requires processing of p100 that is mediated by IKKalpha but is independent of IKKbeta, NEMO/IKKgamma, and RelA. Moreover, we show that RelB, p50, and p100 can associate in the same complex and that TNF-alpha but not LTbeta 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-kappaB 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.     

Neutrality of the canonical NF-kappaB-dependent pathway for human and murine cytomegalovirus transcription and replication in vitro

Cytomegalovirus (CMV) is known to rapidly induce activation of nuclear factor kappaB (NF-kappaB) after infection of fibroblast and macrophage cells. NF-kappaB response elements are present in the enhancer region of the CMV major immediate-early promoter (MIEP), and activity of the MIEP is strongly upregulated by NF-kappaB in transient-transfection assays. Here we investigate whether the NF-kappaB-dependent pathway is required for initiating or potentiating human and murine CMV replication in vitro. We show that expression of a dominant negative mutant of the inhibitor of NF-kappaB-alpha (IkappaBalphaM) does not alter the replication kinetics of human or mouse CMV in cultured cells. In addition, mouse embryo fibroblasts genetically deficient for p65/RelA actually showed elevated levels of MCMV replication. Mutation of all NF-kappaB response elements within the enhancer of the MIEP in a recombinant mouse CMV containing the human MIEP (hMCMV-ES), which we have previously shown to replicate in murine fibroblasts with kinetics equivalent to that of wild-type mouse CMV, did not negatively affect replication in fibroblasts. Taken together, these data show that, for CMV replication in cultured fibroblasts activation of the canonical NF-kappaB pathway and binding of NF-kappaB to the MIEP are dispensable, and in the case of p65 may even interfere, thus uncovering a previously unrecognized level of complexity in the host regulatory network governing MIE gene expression in the context of a viral infection.     

Activation of Phosphatidylinositol 3-Kinase in Response to Interleukin-1 Leads to Phosphorylation and Activation of the NF-κB p65/RelA Subunit

The work of Reddy et al. (S. A. Reddy, J. A. Huang, and W. S. Liao, J. Biol. Chem. 272:29167-29173, 1997) reveals that phosphatidylinositol 3-kinase (PI3K) plays a role in transducing a signal from the occupied interleukin-1 (IL-1) receptor to nuclear factor kappaB (NF-kappaB), but the underlying mechanism remains to be determined. We have found that IL-1 stimulates interaction of the IL-1 receptor accessory protein with the p85 regulatory subunit of PI3K, leading to the activation of the p110 catalytic subunit. Specific PI3K inhibitors strongly inhibit both PI3K activation and NF-kappaB-dependent gene expression but have no effect on the IL-1-stimulated degradation of IkappaBalpha, the nuclear translocation of NF-kappaB, or the ability of NF-kappaB to bind to DNA. In contrast, PI3K inhibitors block the IL-1-stimulated phosphorylation of NF-kappaB itself, especially the p65/RelA subunit. Furthermore, by using a fusion protein containing the p65/RelA transactivation domain, we found that overexpression of the p110 catalytic subunit of PI3K induces p65/RelA-mediated transactivation and that the specific PI3K inhibitor LY294,002 represses this process. Additionally, the expression of a constitutively activated form of either p110 or the PI3K-activated protein kinase Akt also induces p65/RelA-mediated transactivation. Therefore, IL-1 stimulates the PI3K-dependent phosphorylation and transactivation of NF-kappaB, a process quite distinct from the liberation of NF-kappaB from its cytoplasmic inhibitor IkappaB.     

NF-kappa B RelA (p65) is essential for TNF-alpha-induced fas expression but dispensable for both TCR-induced expression and activation-induced cell death

The Fas death receptor plays a key role in the killing of target cells by NK cells and CTLs and in activation-induced cell death of mature T lymphocytes. These cytotoxic pathways are dependent on induction of Fas expression by cytokines such as TNF-alpha and IFN-gamma or by signals generated after TCR engagement. Although much of our knowledge of the Fas death pathway has been generated from murine studies, little is known about regulatory mechanisms important for murine Fas expression. To this end, we have molecularly cloned a region of the murine Fas promoter that is responsible for mediating TNF-alpha and PMA/PHA-induced expression. We demonstrate here that induction of Fas expression by both stimuli is critically dependent on two sites that associate with RelA-containing NF-kappaB complexes. To determine whether RelA and/or other NF-kappaB subunits are also important for regulating Fas expression in primary T cells, we used CD4 T cells from RelA(-/-), c-Rel(-/-), and p50(-/-) mice. Although proliferative responses were significantly impaired, expression of Fas and activation-induced cell death was unaffected in T cells obtained from these different mice. Importantly, we show that unlike fibroblasts, which consist primarily of RelA-containing NF-kappaB complexes, T cells have high levels of both RelA and c-Rel complexes, suggesting that Fas expression in T cells may be dependent on redundant functions of these NF-kappaB subunits.     

Lymphotoxin beta receptor induces sequential activation of distinct NF-kappa B factors via separate signaling pathways

Lymphotoxin beta receptor (LTbetaR)-induced activation of NF-kappaB in mouse embryo fibroblasts was mediated by the classical pathway and by an alternative or second pathway. The classical pathway involved the IkappaB kinase (IKK)beta- and IKKgamma-dependent degradation of IkappaBalpha and resulted in the rapid but transient activation of primarily RelA-containing NF-kappaB dimers. The alternative or second pathway proceeded via NF-kappaB-inducing kinase (NIK)-, IKKalpha-, and protein synthesis-dependent processing of the inhibitory NF-kappaB2 p100 precursor protein to the p52 form and resulted in a delayed but sustained activation of primarily RelB-containing NF-kappaB dimers. This second pathway was independent of the classical IKK complex, which is governed by its central IKKgamma regulatory subunit. The sequential engagement of two distinct pathways, coupled with the negative feedback inhibition of RelA complexes by NF-kappaB-induced resynthesis of IkappaBalpha, resulted in a pronounced temporal change in the nature of the NF-kappaB activity during the course of stimulation. Initially dominant RelA complexes were replaced with time by RelB complexes. Therefore, the alternative activation path mediated by processing of p100 was necessary for sustained NF-kappaB activity in mouse embryo fibroblasts in response to LTbetaR stimulation. Based on the phenotype of mice deficient in various components of the LTbetaR-induced activation of p100 processing, we conclude that this pathway is critically involved in the function of stromal cells during the generation of secondary lymphoid organ microarchitectures.