No abstract

I kappa B-alpha inhibits transcription factor NF-kappa B by retaining it in the cytoplasm. Various stimuli, typically those associated with stress or pathogens, rapidly inactivate I kappa B-alpha. This liberates NF-kappa B to translocate to the nucleus and initiate transcription of genes important for the defense of the organism. Activation of NF-kappa B correlates with phosphorylation of I kappa B-alpha and requires the proteolysis of this inhibitor. When either serine-32 or serine-36 of I kappa B-alpha was mutated, the protein did not undergo signal-induced phosphorylation or degradation, and NF-kappa B could not be activated. These results suggest that phosphorylation at one or both of these residues is critical for activation of NF-kappa B.

NF-B is a family of related, dimeric transcription factors that are readily activated in cells by signals associated with stress or pathogens. These factors are critical to host defense, as demonstrated previously with mice deficient in individual subunits of NF-B. We have generated mice deficient in both the p50 and p52 subunits of NF-B to reveal critical functions that may be shared by these two highly homologous proteins. We now demonstrate that unlike the respective single knockout mice, the p50/p52 double knockout mice fail to generate mature osteoclasts and B cells, apparently because of defects that track with these lineages in adoptive transfer experiments. Furthermore, these mice present markedly impaired thymic and splenic architectures and impaired macrophage functions. The blocks in osteoclast and B-cell maturation were unexpected. Lack of mature osteoclasts caused severe osteopetrosis, a family of diseases characterized by impaired osteoclastic bone resorption. These findings now establish critical roles for NF-B in development and expand its repertoire of roles in the physiology of differentiated hematopoietic cells.

In addition to coordinating immune and inflammatory responses, NF-kappaB/Rel transcription factors control cell survival. Normally, NF-kappaB dimers are sequestered in the cytoplasm by binding to inhibitory IkappaB proteins, and can be activated rapidly by signals that induce the sequential phosphorylation and proteolysis of IkappaBs. Activation of NF-kappaB antagonizes apoptosis or programmed cell death by numerous triggers, including the ligand engagement of 'death receptors' such as tumour-necrosis factor (TNF) receptor. The anti-apoptotic activity of NF-kappaB is also crucial to oncogenesis and to chemo- and radio-resistance in cancer. Cytoprotection by NF-kappaB involves the activation of pro-survival genes; however, its basis remains poorly understood. Here we report that NF-kappaB complexes downregulate the c-Jun amino-terminal kinase (JNK) cascade, thus establishing a link between the NF-kappaB and the JNK pathways. This link involves the transcriptional upregulation of gadd45beta/myd118 (ref. 4), which downregulates JNK signalling induced by the TNF receptor (TNF-R). This NF-kappaB-dependent inhibition of the JNK pathway is central to the control of cell death. Our findings define a protective mechanism that is mediated by NF-kappaB complexes and establish a role for the persistent activation of JNK in the apoptotic response to TNF-alpha.

During inflammation, NF-kappaB transcription factors antagonize apoptosis induced by tumor necrosis factor (TNF)alpha. This antiapoptotic activity of NF-kappaB involves suppressing the accumulation of reactive oxygen species (ROS) and controlling the activation of the c-Jun N-terminal kinase (JNK) cascade. However, the mechanism(s) by which NF-kappaB inhibits ROS accumulation is unclear. We identify ferritin heavy chain (FHC)--the primary iron storage factor--as an essential mediator of the antioxidant and protective activities of NF-kappaB. FHC is induced downstream of NF-kappaB and is required to prevent sustained JNK activation and, thereby, apoptosis triggered by TNFalpha. FHC-mediated inhibition of JNK signaling depends on suppressing ROS accumulation and is achieved through iron sequestration. These findings establish a basis for the NF-kappaB-mediated control of ROS induction and identify a mechanism by which NF-kappaB suppresses proapoptotic JNK signaling. Our results suggest modulation of FHC or, more broadly, of iron metabolism as a potential approach for anti-inflammatory therapy.

The NF-#cB transcription factor complex is sequestered in the cytoplasm by the inhibitory protein I#cB-a (MAD-3). Various cellular stimuli relieve this inhibition by mechanisms largely unknown, leading to NF-KB nuclear localization and transactivation of its target genes. It is demonstrated here with human T lymphocytes and monocytes that different stimuli, including tumor necrosis factor a and phorbol 12-myristate 13-acetate, cause rapid degradation ofIOcB-a, with concomitant activation of NF-cB, followed by a dramatic increase in IKB-a mRNA and protein synthesis. Transfection studies reveal that the IKB-a mRNA and the encoded protein are potently induced by NF-cB and by homodimers of p65 and of c-Rel. We propose a model in which NF-.cB and IKB-a mutually regulate each other in a cycle: saturating amounts of the inhibitory IKB-a protein are destroyed upon stimulation, allowing rapid activation of NF-KB. Subsequently, IcB-a mRNA and protein levels are qulickly induced by the activated NF-cB. This resurgence of IKB-a protein acts to restore an equilibrium in which NF-KB is again inhibited.NF-KB is a dimeric transcription factor that binds and regulates gene expression through decameric cis-acting KB DNA motifs (reviewed in refs. 1 and 2). Although a p50/p65 heterodimer has traditionally been referred to as NF-KB and remains the prototypical and most abundant form, it has been recognized recently that several distinct but closely related homo-and heterodimeric factors are responsible for KB site-dependent DNA binding activity and regulation. The various dimeric factors are composed of members of the family of Rel-related polypeptides. One subclass of this family, distinguished by its proteolytic processing from precursor forms and lack of recognized activation domains, includes p50 (NFKB1) (3-6) and pSOB (NFKB2, pS2) (7-10), whereas the second subclass contains recognized activation domains and includes p65 (RelA) (11-13), RelB (14,15),18) Activation of the NF-KB transcription factor and various related forms can be initiated by a variety of agents, including tumor necrosis factor a (TNF-a) and phorbol 12-myristate 13-acetate (PMA) (1, 2). Activation proceeds through a post-translational event in which preformed cytoplasmic NF-KB is released from a cytoplasmic inhibitory protein, (20)(21)(22)(23). IKB-a inhibits transactivation of the p50/p65 heterodimer, by binding to the p65 component, blocking the dimer's translocation to the nucleus (20,21,23). IKB-a also inhibits complexes containing c-Rel or RelB (24,25). IKB-a blocks binding in vitro of various NF-KB dimers to KB binding sites in DNA (11,12,15,22,26,27). Because the latter effect requires nuclear IKB-a, its relevance, in vivo, is unknown. Although lKB-a is generally a cytoplasmic protein (21, 23), it and its chicken homolog (pp4O) have also been detected in the nucleus (refs. 28-30 and K.B., G.F., and U.S., unpublished results). In addition to the wellcharacterized and cloned IKB-a and its chicken and rat homologs (24, 31), another biochemically d...

Bcl-3 is an I kappa B-related protein with ankyrin repeat motifs. Its gene is located at a site of recurrent translocations in a subset of B cell chronic lymphocytic leukemias. Bcl-3 associates tightly with p50B (NFKB2, p52) homodimers in cells, and together these proteins form a ternary complex with DNA at kappa B sites. Such an association functionally leads to a novel and potent form of transactivation through the kappa B motif: the tethering of Bcl-3 to DNA via the p50B homodimers allows Bcl-3 to transactivate directly, while p50B homodimers alone cannot. Transactivation mediated by Bcl-3 requires two cooperating domains located amino- and carboxy-terminal to the ankyrin domain. Bcl-3 is localized to the nucleus, and a Bcl-3-p50B complex is detected in certain lymphoid cells. Our data reveal a novel role for Bcl-3, distinct from that of the inhibitor I kappa B. The results have implications for tumorigenesis.

NF-kappa B/Rel transcription factors control apoptosis, also known as programmed cell death. This control is crucial for oncogenesis, cancer chemo-resistance and for antagonizing tumour necrosis factor alpha (TNFalpha)-induced killing. With regard to TNFalpha, the anti-apoptotic activity of NF-kappa B involves suppression of the c-Jun N-terminal kinase (JNK) cascade. Using an unbiased screen, we have previously identified Gadd45 beta/Myd118, a member of the Gadd45 family of inducible factors, as a pivotal mediator of this suppressive activity of NF-kappa B. However, the mechanisms by which Gadd45 beta inhibits JNK signalling are not understood. Here, we identify MKK7/JNKK2--a specific and essential activator of JNK--as a target of Gadd45 beta, and in fact, of NF-kappa B itself. Gadd45 beta binds to MKK7 directly and blocks its catalytic activity, thereby providing a molecular link between the NF-kappa B and JNK pathways. Importantly, Gadd45 beta is required to antagonize TNFalpha-induced cytotoxicity, and peptides disrupting the Gadd45 beta/MKK7 interaction hinder the ability of Gadd45 beta, as well as of NF-kappa B, to suppress this cytotoxicity. These findings establish a basis for the NF-kappa B control of JNK activation and identify MKK7 as a potential target for anti-inflammatory and anti-cancer therapy.