The Rel/NF-kappaB family of transcription factors is sequestered in the cytoplasm of most mammalian cells by inhibitor proteins belonging to the IkappaB family. Degradation of IkappaB by a phosphorylation-dependent ubiquitin-proteasome (inducible) pathway is believed to allow nuclear transport of active Rel/NF-kappaB dimers. Rel/NF-kappaB (a p50-c-Rel dimer) is constitutively nuclear in murine B cells, such as WEHI231 cells. In these cells, p50, c-Rel, and IkappaB alpha are synthesized at high levels but only IkappaB alpha is rapidly degraded. We have examined the mechanism of IkappaB alpha degradation and its relation to constitutive p50-c-Rel activation. We demonstrate that all IkappaB alpha is found complexed with c-Rel protein in the cytoplasm. Additionally, rapid IkappaB alpha proteolysis is independent of but coexistent with the inducible pathway and can be inhibited by calcium chelators and some calpain inhibitors. Conditions that prevent degradation of IkappaB alpha also inhibit nuclear p50-c-Rel activity. Furthermore, the half-life of nuclear c-Rel is much shorter than that of the cytoplasmic form, underscoring the necessity for its continuous nuclear transport to maintain constitutive p50-c-Rel activity. We observed that IkappaB beta, another NF-kappaB inhibitor, is also complexed with c-Rel but slowly degraded by a proteasome-dependent process in WEHI231 cells. In addition, IkappaB beta is basally phosphorylated and cytoplasmic. We thus suggest that calcium-dependent IkappaB alpha proteolysis maintains nuclear transport of a p50-c-Rel heterodimer which in turn activates the synthesis of IkappaB alpha, p50, and c-Rel to sustain this dynamic process in WEHI231 B cells.

Macrophages are known to be one of the first lines of defense against influenza virus infection. However, they may also contribute to severe disease caused by the highly pathogenic avian (HPAI) H5N1 influenza viruses. One reason for this may be the ability of certain influenza virus strains to productively replicate in macrophages. However, studies investigating the productive replication of influenza viruses in macrophages have been contradictory, and the results may depend on both the type of macrophages used and the specific viral strain. In this work, we investigated the ability of H1 to H16 viruses to productively replicate in primary murine alveolar macrophages and RAW264.7 macrophages. We show that only a subset of HPAI H5N1 viruses, those that cause high morbidity and mortality in mammals, can productively replicate in macrophages, as measured by the release of newly synthesized virus particles into the cell supernatant. Mechanistically, we found that these H5 strains can overcome a block early in the viral life cycle leading to efficient nuclear entry, viral transcription, translation, and ultimately replication. Studies with reassortant viruses demonstrated that expression of the hemagglutinin gene from an H5N1 virus rescued replication of H1N1 influenza virus in macrophages. This study is the first to characterize H5N1 influenza viruses as the only subtype of influenza virus capable of productive replication in macrophages and establishes the viral gene that is required for this characteristic. The ability to productively replicate in macrophages is unique to H5N1 influenza viruses and may contribute to their increased pathogenesis.

Activation of the transcription factor NF-B by extracellular signals involves its release from the inhibitor protein IB␣ in the cytoplasm and subsequent nuclear translocation. NF-B can also be activated by the anticancer agent camptothecin (CPT), which inhibits DNA topoisomerase (Topo) I activity and causes DNA doublestrand breaks during DNA replication to induce S phasedependent cytotoxicity. Here we show that CPT activates NF-B by a mechanism that is dependent on initial nuclear DNA damage followed by cytoplasmic signaling events. NF-B activation by CPT is dramatically diminished in cytoplasts and in CEM/C2 cells expressing a mutant Topo I protein that fails to bind CPT. This response is intensified in S phase cell populations and is prevented by the DNA polymerase inhibitor aphidicolin. In addition, CPT activation of NF-B involves degradation of cytoplasmic IB␣ by the ubiquitin-proteasome pathway in a manner that depends on the IB kinase complex. Finally, inhibition of NF-B activation augments CPT-induced apoptosis. These findings elucidate the progression of signaling events that initiates in the nucleus with CPT-Topo I interaction and continues in the cytoplasm resulting in degradation of IB␣ and nuclear translocation of NF-B to attenuate the apoptotic response.The NF-B/Rel family of transcription factors regulates expression of genes critical for multiple biological processes, including immune responses, inflammatory reactions, and apoptosis (1-3). In mammalian cells, NF-B exists as dimeric complexes composed of p50, p65 (RelA), c-Rel, RelB, or p52. These proteins share a conserved Rel homology domain that encodes dimerization, DNA binding, and nuclear localization functions. NF-B associates with members of the IB family of proteins, most notably IB␣, which masks the nuclear localization sequence of NF-B and retains it in the cytoplasm (4, 5). Dissociation from IB␣ is essential for NF-B to enter the nucleus and to activate gene expression. Several signaling cascades that control NF-B activation converge at an IB kinase (IKK) 1 complex, responsible for site-specific phosphorylation of IB␣ at serines 32 and 36 (6 -10). Phosphorylation of IB␣ induces multiubiquitination of IB␣ and its subsequent degradation by the ubiquitin-dependent 26 S proteasome (11,12). This sequence of events can be induced without de novo protein synthesis by multiple extracellular stimuli, including tumor necrosis factor ␣ (TNF␣), interleukin-1, phorbol ester (PMA), bacterial lipopolysaccharide (LPS), and others. However, NF-B activation can also be achieved through mechanisms that are distinct from the above IKK-dependent model. These include phosphorylation-independent yet proteasome-mediated IB␣ degradation induced by ultraviolet irradiation (13,14), calpain-dependent degradation of IB␣ by silica and TNF␣ (15, 16), and tyrosine phosphorylation-induced dissociation of IB␣ from NF-B following hypoxia and reoxygenation (17). Thus, depending on the stimuli, NF-B can be activated through multiple distinct regulatory pathways.Activation ...

The inactive transcription factor NF-jB is localized in the cytoplasm and rapidly responds to a variety of extracellular factors and intracellular stress conditions to initiate multiple cellular responses. While the knowledge regarding NF-jB signaling pathways initiated by extracellular ligands is rapidly expanding, the mechanisms of activation by intracellular stress conditions are not well understood. We recently described a critical role for a small ubiquitin-like modifier (SUMO) modification of NF-jB essential modulator (NEMO), the regulatory subunit of the IjB kinase, in response to certain genotoxic stress conditions. One important unanswered question is whether the role of this modification is limited to the genotoxic agents or some other signaling pathways also employ SUMOylation of NEMO to regulate NF-jB activation. Here, we report that a variety of other stress conditions, including oxidative stress, ethanol exposure, heat shock and electric shock, also induce NEMO SUMOylation, thus demonstrating that DNA damage per se is not necessary for this NEMO modification to occur. Moreover, combinations of certain SUMO stress and ATM (ataxia telangiectasia mutated) activation conditions lead to NF-jB activation without inducing DNA damage. Our study helps to conceptualize how individual or a combination of different stress conditions may funnel into this previously unappreciated signal transduction mechanism to regulate the activity of the ubiquitous NF-jB transcription factor.

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