Bridging the gap between in silico and cell-based analysis of the nuclear factor-kappaB signaling pathway by in vitro studies of IKK2

Previously, we have shown by sensitivity analysis, that the oscillatory behavior of nuclear factor (NF-kappaB) is coupled to free IkappaB kinase-2 (IKK2) and IkappaBalpha(IkappaBalpha), and that the phosphorylation of IkappaBalpha by IKK influences the amplitude of NF-kappaB oscillations. We have performed further analyses of the behavior of NF-kappaB and its signal transduction network to understand the dynamics of this system. A time lapse study of NF-kappaB translocation in 10,000 cells showed discernible oscillations in levels of nuclear NF-kappaB amongst cells when stimulated with interleukin (IL-1alpha), which suggests a small degree of synchronization amongst the cell population. When the kinetics for the phosphorylation of IkappaBalpha by IKK were measured, we found that the values for the affinity and catalytic efficiency of IKK2 for IkappaBalpha were dependent on assay conditions. The application of these kinetic parameters in our computational model of the NF-kappaB pathway resulted in significant differences in the oscillatory patterns of NF-kappaB depending on the rate constant value used. Hence, interpretation of in silico models should be made in the context of this uncertainty.     

IkappaB kinase-independent IkappaBalpha degradation pathway: functional NF-kappaB activity and implications for cancer therapy

Antiapoptotic activity of NF-kappaB in tumors contributes to acquisition of resistance to chemotherapy. Degradation of IkappaB is a seminal step in activation of NF-kappaB. The IkappaB kinases, IKK1 and IKK2, have been implicated in both IkappaB degradation and subsequent modifications of NFkappaB. Using mouse embryo fibroblasts (MEFs) devoid of both IKK1 and IKK2 genes (IKK1/2(-/-)), we document a novel IkappaB degradation mechanism. We show that this degradation induced by a chemotherapeutic agent, doxorubicin (DoxR), does not require the classical serine 32 and 36 phosphorylation or the PEST domain of IkappaBalpha. Degradation of IkappaBalpha is partially blocked by phosphatidylinositol 3-kinase inhibitor LY294002 and is mediated by the proteasome. Free NF-kappaB generated by DoxR-induced IkappaB degradation in IKK1/2(-/-) cells is able to activate chromatin based NF-kappaB reporter gene and expression of the endogenous target gene, IkappaBalpha. These results also imply that modification of NF-kappaB by IKK1 or IKK2 either prior or subsequent to its release from IkappaB is not essential for NF-kappaB-mediated gene expression at least in response to DNA damage. In addition, DoxR-induced cell death in IKK1/2(-/-) MEFs is enhanced by simultaneous inhibition of NF-kappaB activation by blocking the proteasome activity. These results reveal an additional pathway of activating NF-kappaB during the course of anticancer therapy and provide a mechanistic basis for the observation that proteasome inhibitors could be used as adjuvants in chemotherapy.     

Interleukin-1-induced NF-kappaB activation is NEMO-dependent but does not require IKKbeta

Activation of NF-kappaB by the pro-inflammatory cytokines tumor necrosis factor (TNF) and interleukin-1 (IL-1) requires the IkappaB kinase (IKK) complex, which contains two kinases named IKKalpha and IKKbeta and a critical regulatory subunit named NEMO. Although we have previously demonstrated that NEMO associates with both IKKs, genetic studies reveal that only its interaction with IKKbeta is required for TNF-induced NF-kappaB activation. To determine whether NEMO and IKKalpha can form a functional IKK complex capable of activating the classical NF-kappaB pathway in the absence of IKKbeta, we utilized a panel of mouse embryonic fibroblasts (MEFs) lacking each of the IKK complex subunits. This confirmed that TNF-induced IkappaBalpha degradation absolutely requires NEMO and IKKbeta. In contrast, we consistently observed intact IkappaBalpha degradation and NF-kappaB activation in response to IL-1 in two separate cell lines lacking IKKbeta. Furthermore, exogenously expressed, catalytically inactive IKKbeta blocked TNF- but not IL-1-induced IkappaBalpha degradation in wild-type MEFs, and reconstitution of IKKalpha/beta double knockout cells with IKKalpha rescued IL-1- but not TNF-induced NF-kappaB activation. Finally, we have shown that incubation of IKKbeta-deficient MEFs with a cell-permeable peptide that blocks the interaction of NEMO with the IKKs inhibits IL-1-induced NF-kappaB activation. Our results therefore demonstrate that NEMO and IKKalpha can form a functional IKK complex that activates the classical NF-kappaB pathway in response to IL-1 but not TNF. These findings further suggest NEMO differentially regulates the fidelity of the IKK subunits activated by distinct upstream signaling pathways.     

Heat shock protein 70 negatively regulates the heat-shock-induced suppression of the IkappaB/NF-kappaB cascade by facilitating IkappaB kinase renaturation and blocking its further denaturation

Heat shock (HS) treatment has been previously shown to suppress the IkappaB/nuclear factor-kappaB (NF-kappaB) cascade by denaturing, and thus inactivating IkappaB kinase (IKK). HS is characterized by the induction of a group of heat shock proteins (HSPs). However, their role in the HS-induced suppression of the IkappaB/NF-kappaB cascade is unclear. Adenovirus-mediated HSP70 overexpression was found not to suppress the TNF-alpha-induced activation of the IkappaB/NF-kappaB pathway, thus suggesting that HSP70 is unlikely to suppress this pathway. When TNF-alpha-induced activation of the IkappaB/NF-kappaB pathway was regained 24 h after HS, HSP70 was found to be highly up-regulated. Moreover, blocking HSP70 induction delayed TNF-alpha-induced IkappaBalpha degradation and the resolubilization of IKK. In addition, HSP70 associated physically with IKK, suggesting that HSP70 is involved in the recovery process via molecular chaperone effect. Adenovirus-mediated HSP70 overexpression prior to HS blocked the IkappaBalpha stabilizing effect of HS by suppressing IKK insolubilization. Moreover, the up-regulation of endogenous HSP70 by preheating, suppressed this subsequent HS-induced IKK insolubilization, and this effect was abrogated by blocking HSP70 induction. These findings indicate that HSP70 accumulates during HS and negatively regulates the HS-induced suppression of the IkappaB/NF-kappaB cascade by facilitating the renaturation of IKK and blocking its further denaturation.     

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.     

The heat-shock-induced suppression of the IkappaB/NF-kappaB cascade is due to inactivation of upstream regulators of IkappaBalpha through insolubilization

Heat shock (HS) was found to suppress the IkappaB/NF-kappaB cascade via the inhibition of IkappaB kinase (IKK) activity; however, the mechanism has not been clear. This study was undertaken to elucidate the detail of the mechanism involved. TNF-alpha-induced activation of IKK was suppressed by HS in human bronchial epithelial cells, and this was associated with the absence of IKK in the immunoprecipitates. It was not due to a degradation of IKK, but due to a conversion of IKK from a soluble to an insoluble form. IKK lost its activity rapidly upon exposure to HS in vitro. The time course of the insolubilization of IKK coincided with the decrease in IKK activity. However, inhibition of IKK insolubilization by the induction of thermotolerance did not reverse the HS-induced suppression of IKK activation and IkappaBalpha degradation. Upstream activators of IKK, such as NF-kappaB-inducing kinase (NIK) and IL-1R-associated kinase (IRAK) were also insolubilized by HS. The HS-induced insolubilization of NIK was not blocked by the induction of thermotolerance. Overexpression of NIK resumed TNF-alpha-induced activation of IKK in thermotolerant cells. These results indicate that the loss of activity of NIK, IRAK, and IKK through insolubilization is responsible for the HS-induced suppression of IkappaB/NF-kappaB pathway.     

Successful therapy of lethal murine visceral leishmaniasis with cystatin involves up-regulation of nitric oxide and a favorable T cell response

The virulence of Leishmania donovani in mammals depends at least in part on cysteine proteases because they play a key role in CD4(+) T cell differentiation. A 6-fold increase in NO production was observed with 0.5 microM chicken cystatin, a natural cysteine protease inhibitor, in IFN-gamma-activated macrophages. In a 45-day BALB/c mouse model of visceral leishmaniasis, complete elimination of spleen parasite burden was achieved by cystatin in synergistic activation with a suboptimal dose of IFN-gamma. In contrast to the case with promastigotes, cystatin and IFN-gamma inhibited the growth of amastigotes in macrophages. Although in vitro cystatin treatment of macrophages did not induce any NO generation, significantly enhanced amounts of NO were generated by macrophages of cystatin-treated animals. Their splenocytes secreted soluble factors required for the induction of NO biosynthesis, and the increased NO production was paralleled by a concomitant increase in antileishmanial activity. Moreover, splenocyte supernatants treated with anti-IFN-gamma or anti-TNF-alpha Abs suppressed inducible NO generation, whereas i.v. administration of these anticytokine Abs along with combined therapy reversed protection against infection. mRNA expression and flow cytometric analysis of infected spleen cells suggested that cystatin and IFN-gamma treatment, in addition to greatly reducing parasite numbers, resulted in reduced levels of IL-4 but increased levels of IL-12 and inducible NO synthase. Not only was this treatment curative when administered 15 days postinfection, but it also imparted resistance to reinfection. These studies provide a promising alternative for protection against leishmaniasis with a switch of CD4(+) differentiation from Th2 to Th1, indicative of long-term resistance.     

TLR8-mediated NF-kappaB and JNK activation are TAK1-independent and MEKK3-dependent

TLR8-mediated NF-kappaB and IRF7 activation are abolished in human IRAK-deficient 293 cells and IRAK4-deficient fibroblast cells. Both wild-type and kinase-inactive mutants of IRAK and IRAK4, respectively, restored TLR8-mediated NF-kappaB and IRF7 activation in the IRAK- and IRAK4-deficient cells, indicating that the kinase activity of IRAK and IRAK4 is probably redundant for TLR8-mediated signaling. We recently found that TLR8 mediates a unique NF-kappaB activation pathway in human 293 cells and mouse embryonic fibroblasts, accompanied only by IkappaBalpha phosphorylation and not IkappaBalpha degradation, whereas interleukin (IL)-1 stimulation causes both IkappaBalpha phosphorylation and degradation. The intermediate signaling events mediated by IL-1 (including IRAK modifications and degradation and TAK1 activation) were not detected in cells stimulated by TLR8 ligands. TLR8 ligands trigger similar levels of IkappaBalpha phosphorylation and NF-kappaB and JNK activation in TAK1(-/-) mouse embryo fibroblasts (MEFs) as compared with wild-type MEFs, whereas lack of TAK1 results in reduced IL-1-mediated NF-kappaB activation and abolished IL-1-induced JNK activation. The above results indicate that although TLR8-mediated NF-kappaB and JNK activation are IRAK-dependent, they do not require IRAK modification and are TAK1-independent. On the other hand, TLR8-mediated IkappaBalpha phosphorylation, NF-kappaB, and JNK activation are completely abolished in MEKK3(-/-) MEFs, whereas IL-1-mediated signaling was only moderately reduced in these deficient MEFs as compared with wild-type cells. The differences between IL-1R- and TLR8-mediated NF-kappaB activation are also reflected at the level of IkappaB kinase (IKK) complex. TLR8 ligands induced IKKgamma phosphorylation, whereas IKKalpha/beta phosphorylation and IKKgamma ubiquitination that can be induced by IL-1 were not detected in cells treated with TLR8 ligands. We postulate that TLR8-mediated MEKK3-dependent IKKgamma phosphorylation might play an important role in the activation of IKK complex, leading to IkappaBalpha phosphorylation.     

Sequential DNA damage-independent and -dependent activation of NF-kappaB by UV.

NF-kappaB activation in response to UV irradiation of HeLa cells or of primary human skin fibroblasts occurs with two overlapping kinetics but totally different mechanisms. Although both mechanisms involve induced dissociation of NF-kappaB from IkappaBalpha and degradation of IkappaBalpha, targeting for degradation and signaling are different. Early IkappaBalpha degradation at 30 min to approximately 6 h is not initiated by UV-induced DNA damage. It does not require IkappaB kinase (IKK), as shown by introduction of a dominant-negative kinase subunit, and does not depend on the presence of the phosphorylatable substrate, IkappaBalpha, carrying serines at positions 32 and 36. Induced IkappaBalpha degradation requires, however, intact N- (positions 1-36) and C-terminal (positions 277-287) sequences. IkappaB degradation and NF-kappaB activation at late time points, 15-20 h after UV irradiation, is mediated through DNA damage-induced cleavage of IL-1alpha precursor, release of IL-1alpha and autocrine/paracrine action of IL-1alpha. Late-induced IkappaBalpha requires the presence of Ser32 and Ser36. The late mechanism indicates the existence of signal transfer from photoproducts in the nucleus to the cytoplasm. The release of the 'alarmone' IL-1alpha may account for some of the systemic effects of sunlight exposure.     

The IKK-2/Ikappa Balpha /NF-kappa B pathway plays a key role in the regulation of CCR3 and eotaxin-1 in fibroblasts. A critical link to dermatitis in Ikappa Balpha -deficient mice.

Tumor necrosis factor (TNF)-alpha-induced phosphorylation of the IkappaB proteins by the IkappaB kinase (IKK) complex containing IKK-2 and subsequent degradation of the IkappaB proteins are prerequisites for NF-kappaB activation, resulting in the stimulation of a variety of pro-inflammatory target genes. The C-C chemokine eotaxin-1 is a potent chemoattractant for eosinophils and Th2 lymphocytes, may play an important role in the pathogenesis of atopic dermatitis, and acts via binding to its receptor CCR3. To investigate the role of NF-kappaB signaling in the regulation of these genes, we stably expressed a transdominant mutant of IkappaBalpha and a constitutively active mutant of IKK-2 in mouse NIH3T3 fibroblasts. The transdominant IkappaBalpha mutant completely inhibited TNF-alpha-mediated induction of both eotaxin-1 and CCR3, whereas expression of constitutively active IKK-2 was sufficient to drive almost full expression of these two genes in the absence of TNF-alpha. Moreover, we observed elevated expression levels of CCR3 and eotaxin-1 protein levels in the skin of IkappaBalpha-deficient mice characterized by a widespread dermatitis. Finally, using dermal fibroblasts derived from IkappaBalpha-deficient mice, we observed elevated basal expression, enhanced inducibility by TNF-alpha, and attenuated down-regulation upon TNF-alpha withdrawal of both CCR3 and eotaxin-1 mRNA levels. These results demonstrate that the IKK-2/IkappaBalpha/NF-kappaB pathway plays a critical role for CCR3 and eotaxin-1 expression in fibroblasts and suggests a critical link to the pathogenesis of atopic dermatitis.     

PKA-dependent activation of PKC, p38 MAPK and IKK in macrophage: implication in the induction of inducible nitric oxide synthase and interleukin-6 by dibutyryl cAMP

In this study, we examined the signal transduction of dibutyryl cyclic adenosine monophosphate (dBcAMP) to stimulate the release of nitric oxide (NO) and interleukin-6 (IL-6) from J774 macrophages. These actions of dBcAMP were diminished by the presence of the inhibitors of protein kinase A (PKA), protein kinase C (PKC), p38 MAPK and nuclear factor-kappa B (NF-kappaB). In contrast, Go 6976 and PD98059 had no significant effects. Consistently, dBcAMP caused membrane translocation of PKCbetaII, delta, mu, lambda and zeta isoforms, and increased atypical protein kinase C (aPKC) and p38 MAPK activities. The nuclear translocation and DNA-binding study revealed that dBcAMP stimulated NF-kappaB, activator protein-1 (AP-1), and CAAT/enhancer-binding protein (c/EBPbeta). Via PKA, PKC and p38 MAPK-dependent signals, dBcAMP also induced inhibitory subunit of NF-kappaB (IkappaB) degradation, IkappaB kinase (IKK) activation, nuclear translocation of NF-kappaB subunit p65 and its association with the CREB-binding protein (CBP). These results illustrate that PKA activation in macrophages is able to stimulate PKC and p38 MAPK, which lead to IKK-dependent NF-kappaB activation and contribute to the induction of inducible nitric oxide synthase (iNOS) and IL-6 genes.     

Taurine chloramine inhibits inducible nitric oxide synthase and TNF-alpha gene expression in activated alveolar macrophages: decreased NF-kappaB activation and IkappaB kinase activity

Taurine prevents tissue damage in a variety of models that involve inflammation, including oxidant-induced lung damage. The mechanism of protection is uncertain, but is postulated to involve the actions of taurine chloramine (Tau-Cl) derived via halide-dependent myeloperoxidase associated with neutrophils. Understanding the influence of Tau-Cl on the production of inflammatory mediators by alveolar macrophages provides an opportunity for determining the mechanism of Tau-Cl action. The effects of Tau-Cl were evaluated on the production of NO and TNF-alpha in NR8383, a cloned cell line derived from rat alveolar macrophages (RAM), and in primary cultures of RAM. Production of NO and TNF-alpha, and expression of inducible NO synthase was inhibited by Tau-Cl in activated NR8383 cells as well as in RAM. Temporal (2, 4, 8, 24 h) expression of inducible NO synthase and TNF-alpha mRNAs was reduced by Tau-Cl in NR8383 cells. Tau-Cl depressed NF-kappaB migration into the nucleus of activated NR8383 cells and caused a more sustained presence of IkappaB in the cytoplasm. Stabilization of cytoplasmic IkappaB-alpha in Tau-Cl-treated cells resulted from decreased phosphorylation of IkappaB-alpha serine-32 and a lower activity of IkappaB kinase (IKK). Additional experiments demonstrated that Tau-Cl does not directly inhibit IKK activity. These results suggest that Tau-Cl exerts its effects at some level upstream of IKK in the signaling pathway and inhibits production of inflammatory mediators through a mechanism that, at least in part, involves inhibition of NF-kappaB activation.     

Stress-induced inhibition of the NF-kappaB signaling pathway results from the insolubilization of the IkappaB kinase complex following its dissociation from heat shock protein 90

Activation of the stress response attenuates proinflammatory responses by suppressing cytokine-stimulated activation of the NF-kappaB signaling pathway. In this study, we show that the activation of the cellular stress response, either by heat shock treatment or after exposure to sodium arsenite, leads to a transient inhibition of IkappaBalpha phosphorylation. Inhibition of IkappaBalpha phosphorylation after stress was associated with the detergent insolubilization of the upstream kinases, IkappaB kinase alpha (IKKalpha) and IkappaB kinase beta, components involved in IkappaBalpha phosphorylation. Pretreatment of cells with glycerol, a chemical chaperone that reduces the extent of stress-induced protein denaturation, reduced the stress-dependent detergent insolubility of the IKK complex and restored the cytokine-stimulated phosphorylation of IkappaB. The stress-dependent insolubility of the IKK complex appeared reversible; as the cells recovered from the heat shock treatment, the IKK complex reappeared within the soluble fraction of cells and was again capable of mediating the phosphorylation of IkappaBalpha in response to added cytokines. Treatment of cells with geldanamycin, an inhibitor of heat shock protein 90 (Hsp90) function, also resulted in IKK detergent insolubility and proteasome-mediated degradation of the IKK complex. Furthermore, while IKKalpha coprecipitated with Hsp90 in control cells, coprecipitation of the two proteins was greatly reduced in those cells early after stress or following exposure to geldanamycin. Stress-induced transient insolubilization of the IkappaB kinase complex following its dissociation from Hsp90 represents a novel mechanism by which the activation of the stress response inhibits the NF-kappaB signaling pathway in response to proinflammatory stimuli.     

Adiponectin enhances IL-6 production in human synovial fibroblast via an AdipoR1 receptor, AMPK, p38, and NF-kappa B pathway

Articular adipose tissue is a ubiquitous component of human joints, and adiponectin is a protein hormone secreted predominantly by differentiated adipocytes and involved in energy homeostasis. We investigated the signaling pathway involved in IL-6 production caused by adiponectin in both rheumatoid arthritis synovial fibroblasts and osteoarthritis synovial fibroblasts. Rheumatoid arthritis synovial fibroblasts and osteoarthritis synovial fibroblasts expressed the AdipoR1 and AdipoR2 isoforms of the adiponectin receptor. Adiponectin caused concentration- and time-dependent increases in IL-6 production. Adiponectin-mediated IL-6 production was attenuated by AdipoR1 and 5'-AMP-activated protein kinase (AMPK)alpha1 small interference RNA. Pretreatment with AMPK inhibitor (araA and compound C), p38 inhibitor (SB203580), NF-kappaB inhibitor, IkappaB protease inhibitor, and NF-kappaB inhibitor peptide also inhibited the potentiating action of adiponectin. Adiponectin increased the kinase activity and phosphorylation of AMPK and p38. Stimulation of synovial fibroblasts with adiponectin activated IkappaB kinase alpha/beta (IKK alpha/beta), IkappaBalpha phosphorylation, IkappaBalpha degradation, p65 phosphorylation at Ser (276), p65 and p50 translocation from the cytosol to the nucleus, and kappaB-luciferase activity. Adiponectin-mediated an increase of IKK alpha/beta activity, kappaB-luciferase activity, and p65 and p50 binding to the NF-kappaB element and was inhibited by compound C, SB203580 and AdipoR1 small interference RNA. Our results suggest that adiponectin increased IL-6 production in synovial fibroblasts via the AdipoR1 receptor/AMPK/p38/IKKalphabeta and NF-kappaB signaling pathway.     

Novel IKK inhibitors: beta-carbolines

Inhibitors of IkappaB kinase (IKK) have long been sought as specific regulators of NF-kappaB. A screening effort of the endogenous IKK complex allowed us to identify 5-bromo-6-methoxy-beta-carboline as a nonspecific IKK inhibitor. Optimization of this beta-carboline natural product derivative resulted in a novel class of selective IKK inhibitors with IC(50)s in the nanomolar range. In addition, we show that one of these beta-carboline analogues inhibits the phosphorylation of IkappaBalpha and subsequent activation of NF-kappaB in whole cells, as well as blocking TNF-alpha release in LPS-challenged mice.