Cytokine-induced activation of nuclear factor-kappa B is inhibited by hydrogen peroxide through oxidative inactivation of IkappaB kinase

Rapid activation of the IkappaB kinase (IKK) complex is considered an obligatory step in the activation of nuclear factor-kappaB (NF-kappaB) in response to diverse stimuli. Since oxidants have been implicated in the regulation of NF-kappaB, the focus of the present study was the activation of IKK by tumor necrosis factor alpha (TNFalpha) in the presence or absence of hydrogen peroxide (H(2)O(2)). Exposure of mouse alveolar epithelial cells to H(2)O(2) was not sufficient to activate IKK, degrade IkappaBalpha, or activate NF-kappaB. In contrast, TNFalpha induced IKK activity rapidly and transiently resulting in IkappaBalpha degradation and NF-kappaB activation. Importantly, in the presence of H(2)O(2), the ability of TNFalpha to induce IKK activity was markedly decreased and resulted in prevention of IkappaBalpha degradation and NF-kappaB activation. Neither tyrosine kinases nor phosphatidylinositol 3-kinases, known regulators of NF-kappaB by oxidants, were involved in IKK inhibition by H(2)O(2). Direct addition of H(2)O(2) to the immunoprecipitated IKK complex inhibited enzyme activity. Inhibition of IKK activity by H(2)O(2) was associated with direct oxidation of cysteine residues present in the IKK complex and occurred only in enzymatically active IKK. In contrast to previously published observations, our findings demonstrate that the oxidant H(2)O(2) reduces NF-kappaB activation by inhibiting activated IKK activity.     

Activation of IKK by TNFalpha requires site-specific ubiquitination of RIP1 and polyubiquitin binding by NEMO

The receptor interacting protein kinase 1 (RIP1) is essential for the activation of nuclear factor kappaB (NF-kappaB) by tumor necrosis factor alpha (TNFalpha). Here, we present evidence that TNFalpha induces the polyubiquitination of RIP1 at Lys-377 and that this polyubiquitination is required for the activation of IkappaB kinase (IKK) and NF-kappaB. A point mutation of RIP1 at Lys-377 (K377R) abolishes its polyubiquitination as well as its ability to restore IKK activation in a RIP1-deficient cell line. The K377R mutation of RIP1 also prevents the recruitment of TAK1 and IKK complexes to TNF receptor. Interestingly, polyubiquitinated RIP1 recruits IKK through the binding between the polyubiquitin chains and NEMO, a regulatory subunit of the IKK complex. Mutations of NEMO that disrupt its polyubiquitin binding also abolish IKK activation. These results reveal the biochemical mechanism underlying the essential signaling function of NEMO and provide direct evidence that signal-induced site-specific ubiquitination of RIP1 is required for IKK activation.     

Dynein light chain LC8 negatively regulates NF-kappaB through the redox-dependent interaction with IkappaBalpha

Redox regulation of nuclear factor kappaB (NF-kappaB) has been described, but the molecular mechanism underlying such regulation has remained unclear. We recently showed that a novel disulfide reductase, TRP14, inhibits tumor necrosis factor alpha (TNFalpha)-induced NF-kappaB activation, and we identified the dynein light chain LC8, which interacts with the NF-kappaB inhibitor IkappaBalpha, as a potential substrate of TRP14. We now show the molecular mechanism by which NF-kappaB activation is redox-dependently regulated through LC8. LC8 inhibited TNFalpha-induced NF-kappaB activation in HeLa cells by interacting with IkappaBalpha and thereby preventing its phosphorylation by IkappaB kinase (IKK), without affecting the activity of IKK itself. TNFalpha induced the production of reactive oxygen species, which oxidized LC8 to a homodimer linked by the reversible formation of a disulfide bond between the Cys(2) residues of each subunit and thereby resulted in its dissociation from IkappaBalpha. Butylated hydroxyanisol, an antioxidant, and diphenyleneiodonium, an inhibitor of NADPH oxidase, attenuated the phosphorylation and degradation of IkappaBalpha by TNFalpha stimulation. In addition LC8 inhibited NF-kappaB activation by other stimuli including interleukin-1beta and lipopolysaccharide, both of which generated reactive oxygen species. Furthermore, TRP14 catalyzed reduction of oxidized LC8. Together, our results indicate that LC8 binds IkappaBalpha in a redox-dependent manner and thereby prevents its phosphorylation by IKK. TRP14 contributes to this inhibitory activity by maintaining LC8 in a reduced state.     

Activation of the Ikappa B kinases by RIP via IKKgamma /NEMO-mediated oligomerization

To understand the mechanism of activation of the IkappaB kinase (IKK) complex in the tumor necrosis factor (TNF) receptor 1 pathway, we examined the possibility that oligomerization of the IKK complex triggered by ligand-induced trimerization of the TNF receptor 1 complex is responsible for activation of the IKKs. Gel filtration analysis of the IKK complex revealed that TNFalpha stimulation induces a large increase in the size of this complex, suggesting oligomerization. Substitution of the C-terminal region of IKKgamma, which interacts with RIP, with a truncated DR4 lacking its cytoplasmic death domain, produced a molecule that could induce IKK and NF-kappaB activation in cells in response to TRAIL. Enforced oligomerization of the N terminus of IKKgamma or truncated IKKalpha or IKKbeta lacking their serine-cluster domains can also induce IKK and NF-kappaB activation. These data suggest that IKKgamma functions as a signaling adaptor between the upstream regulators such as RIP and the IKKs and that oligomerization of the IKK complex by upstream regulators is a critical step in activation of this complex.     

Signaling role of intracellular iron in NF-kappaB activation

Iron chelators inhibit endotoxin-induced NF-kappaB activation in hepatic macrophages (HMs), suggesting a role for the intracellular chelatable pool of iron in NF-kappaB activation. The present study tested this hypothesis. Analysis of Fe(59)-loaded HMs stimulated with lipopolysaccharide (LPS), revealed a previously unreported, transient rise in intracellular low molecular weight (LMW).Fe(59) complex ([LMW.Fe](i)) at /=15 min) and NF-kappaB (>/=30 min) activation. Iron chelators (1,2-dimethyl-3-hydroxypyridin-4-one and N,N'-bis-2-hydroxybenzylethylenediamine-N,N'-diacetic acid) abrogated the [LMW.Fe](i) response and IKK and NF-kappaB activation. The [LMW.Fe](i) response was also observed in tumor necrosis factor alpha (TNFalpha)-stimulated HMs and RAW264.7 cells treated with LPS and interferon-gamma but not in primary rat hepatocytes or myofibroblastic cells exposed to LPS or TNFalpha. Both [LMW.Fe](i) response and IKK activation in LPS-stimulated HMs were inhibited by diphenylene iodonium (nonspecific inhibitor for flavin-containing oxidases), l-N(6)-(1-iminoethyl)lysine (selective iNOS inhibitor), and adenoviral-mediated expression of a dominant negative mutant of Rac1 or Cu,Zn-superoxide dismutase, suggesting the role of (.)NO and O(2)() in mediating the iron signaling. In fact, this inhibition was recapitulated by a cell-permeable scavenger of ONOO(-), 5,10,15,20-tetrakis (4-sulfonatophenyl)porphyrinato iron (III) chloride. Conversely, ONOO(-) alone induced both [LMW.Fe](i) response and IKK activation. Finally, direct addition of ferrous iron to cultured HMs activated IKK and NF-kappaB. These results support a novel signaling role for [LMW.Fe](i) in IKK activation, which appears to be induced by ONOO(-) and selectively operative in macrophages.     

NF-kappaB activation by double-stranded-RNA-activated protein kinase (PKR) is mediated through NF-kappaB-inducing kinase and IkappaB kinase

The interferon (IFN)-inducible double-stranded-RNA (dsRNA)-activated serine-threonine protein kinase (PKR) is a major mediator of the antiviral and antiproliferative activities of IFNs. PKR has been implicated in different stress-induced signaling pathways including dsRNA signaling to nuclear factor kappa B (NF-kappaB). The mechanism by which PKR mediates activation of NF-kappaB is unknown. Here we show that in response to poly(rI). poly(rC) (pIC), PKR activates IkappaB kinase (IKK), leading to the degradation of the inhibitors IkappaBalpha and IkappaBbeta and the concomitant release of NF-kappaB. The results of kinetic studies revealed that pIC induced a slow and prolonged activation of IKK, which was preceded by PKR activation. In PKR null cell lines, pIC failed to stimulate IKK activity compared to cells from an isogenic background wild type for PKR in accord with the inability of PKR null cells to induce NF-kappaB in response to pIC. Moreover, PKR was required to establish a sustained response to tumor necrosis factor alpha (TNF-alpha) and to potentiate activation of NF-kappaB by cotreatment with TNF-alpha and IFN-gamma. By coimmunoprecipitation, PKR was shown to be physically associated with the IKK complex. Transient expression of a dominant negative mutant of IKKbeta or the NF-kappaB-inducing kinase (NIK) inhibited pIC-induced gene expression from an NF-kappaB-dependent reporter construct. Taken together, these results demonstrate that PKR-dependent dsRNA induction of NF-kappaB is mediated by NIK and IKK activation.     

TNAP, a novel repressor of NF-kappaB-inducing kinase, suppresses NF-kappaB activation

NF-kappaB-inducing kinase (NIK) has been implicated as an essential component of NF-kappaB activation. However, the regulatory mechanism of NIK signaling remains elusive. We have identified a novel NIK interacting protein, TNAP (for TRAFs and NIK-associated protein). In mammalian cells, TNAP physically interacts with NIK, TRAF2, and TRAF3 but not IKK1 or IKK2. TNAP specifically inhibits NF-kappaB activation induced by tumor necrosis factor (TNF)-alpha, TNF receptor 1, TRADD, RIP, TRAF2, and NIK but does not affect IKK1- and IKK2-mediated NF-kappaB activation. Knockdown of TNAP by lentiviral-mediated small interference RNA potentiates TNF-alpha-induced NF-kappaB activation. TNAP suppresses NIK kinase activity and subsequently reduces p100 processing, p65 phosphorylation, and IkappaBalpha degradation. These data suggest that TNAP is a repressor of NIK activity and regulates both the classical and alternative NF-kappaB signaling pathways.     

A fourth IkappaB protein within the NF-kappaB signaling module

Inflammatory NF-kappaB/RelA activation is mediated by the three canonical inhibitors, IkappaBalpha, -beta, and -varepsilon. We report here the characterization of a fourth inhibitor, nfkappab2/p100, that forms two distinct inhibitory complexes with RelA, one of which mediates developmental NF-kappaB activation. Our genetic evidence confirms that p100 is required and sufficient as a fourth IkappaB protein for noncanonical NF-kappaB signaling downstream of NIK and IKK1. We develop a mathematical model of the four-IkappaB-containing NF-kappaB signaling module to account for NF-kappaB/RelA:p50 activation in response to inflammatory and developmental stimuli and find signaling crosstalk between them that determines gene-expression programs. Further combined computational and experimental studies reveal that mutant cells with altered balances between canonical and noncanonical IkappaB proteins may exhibit inappropriate inflammatory gene expression in response to developmental signals. Our results have important implications for physiological and pathological scenarios in which inflammatory and developmental signals converge.     

ATM and the catalytic subunit of DNA-dependent protein kinase activate NF-kappaB through a common MEK/extracellular signal-regulated kinase/p90(rsk) signaling pathway in response to distinct forms of DNA damage

We have identified a novel pathway of ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PK) signaling that results in nuclear factor kappaB (NF-kappaB) activation and chemoresistance in response to DNA damage. We show that the anthracycline doxorubicin (DOX) and its congener N-benzyladriamycin (AD 288) selectively activate ATM and DNA-PK, respectively. Both ATM and DNA-PK promote sequential activation of the mitogen-activated protein kinase (MAPK)/p90(rsk) signaling cascade in a p53-independent fashion. In turn, p90(rsk) interacts with the IkappaB kinase 2 (IKK-2) catalytic subunit of IKK, thereby inducing NF-kappaB activity and cell survival. Collectively, our findings suggest that distinct members of the phosphatidylinositol kinase family activate a common prosurvival MAPK/IKK/NF-kappaB pathway that opposes the apoptotic response following DNA damage.     

E1A sensitizes cells to tumor necrosis factor-induced apoptosis through inhibition of IkappaB kinases and nuclear factor kappaB activities.

The adenovirus E1A protein has been implicated in increasing cellular susceptibility to apoptosis induced by tumor necrosis factor (TNF); however, its mechanism of action is still unknown. Since activation of nuclear factor kappaB (NF-kappaB) has been shown to play an anti-apoptotic role in TNF-induced apoptosis, we examined apoptotic susceptibility and NF-kappaB activation induced by TNF in the E1A transfectants and their parental cells. Here, we reported that E1A inhibited activation of NF-kappaB and rendered cells more sensitive to TNF-induced apoptosis. We further showed that this inhibition was through suppression of IkappaB kinase (IKK) activity and IkappaB phosphorylation. Moreover, deletion of the p300 and Rb binding domains of E1A abolished its function in blocking IKK activity and IkappaB phosphorylation, suggesting that these domains are essential for the E1A function in down-regulating IKK activity and NF-kappaB signaling. However, the role of E1A in inhibiting IKK activity might be indirect. Nevertheless, our results suggest that inhibition of IKK activity by E1A is an important mechanism for the E1A-mediated sensitization of TNF-induced apoptosis.