Cobrotoxin inhibits NF-kappa B activation and target gene expression through reaction with NF-kappa B signal molecules

Cobrotoxin is known to bind with cysteine residues of biological molecules such as nicotine acetylcholine receptor. Cobrotoxin may modify IKKs and p50 through protein-protein interaction since cysteine residues are present in the kinase domains of IKKalpha and IKKbeta and in the p50 of NF-kappaB. Our surface plasmon resonance analysis showed that cobrotoxin directly binds to p50 (K(d) = 1.54 x 10(-)(5) M), IKKalpha (K(d) = 3.94 x 10(-)(9) M) and IKKbeta (K(d) = 3.4 x 10(-)(8) M) with high binding affinity. Moreover, these protein-protein interactions suppressed the lipopolysaccharide (LPS, 1 microg/mL)- and the sodium nitroprusside (SNP, 200 microM)-induced DNA binding activity of NF-kappaB and NF-kappaB-dependent luciferase activity in astrocytes and Raw 264.7 macrophages. These inhibitory effects were correlated with the inhibition of IkappaB release and p50 translocation. Inhibition of NF-kappaB by cobrotoxin resulted in reductions in the LPS-induced expressions of COX-2, iNOS, cPLA(2), IL-4, and TNF-alpha in astrocytes and in COX-2 expression induced by SNP, LPS, and TNF-alpha in astrocytes. Moreover, these inhibitory effects of cobrotoxin were reversed by adding reducing agents, dithiothreitol and glutathione. In addition, cobrotoxin did not have any inhibitory effect on NF-kappaB activity in cells carrying mutant p50 (C62S), IKKalpha (C178A), and IKKbeta (C179A), with the exception of IKKbeta (K44A) mutant plasmid. Confocal microscopic analysis showed that cobrotoxin is uptaken into the nucleus of cells. These results demonstrate that cobrotoxin directly binds to the sulfhydryl groups of p50 and IKKs, and that this results in reduced IkappaB release and the translocation of p50, thereby inhibiting the activation of NF-kappaB.     

Cell type-specific roles of Jak3 in IL-2-induced proliferative signal transduction

Binding of interleukin-2 (IL-2) to its specific receptor induces activation of two members of Jak family protein tyrosine kinases, Jak1 and Jak3. An IL-2 receptor (IL-2R)-reconstituted NIH 3T3 fibroblast cell line proliferates in response to IL-2 only when hematopoietic lineage-specific Jak3 is ectopically expressed. However, the mechanism of Jak3-dependent proliferation in the fibroblast cell line is not known. Here, I showed that Jak3 expression is dispensable for IL-2-induced activation of Jak1 and Stat proteins and expression of nuclear proto-oncogenes in the IL-2R-reconstituted fibroblast cell line. Jak3 expression markedly enhanced these IL-2-induced signaling events. In contrast, Jak3 expression was essential for induction of cyclin genes involved in the G1-S transition. These data suggest a critical role of Jak3 in IL-2 signaling in the fibroblast cell line and may provide further insight into the cell type-specific mechanism of cytokine signaling.     

IL-2 gene expression and NF-kappa B activation through CD28 requires reactive oxygen production by 5-lipoxygenase.

Activation of the CD28 surface receptor provides a major costimulatory signal for T cell activation resulting in enhanced production of interleukin-2 (IL-2) and cell proliferation. In primary T lymphocytes we show that CD28 ligation leads to the rapid intracellular formation of reactive oxygen intermediates (ROIs) which are required for CD28-mediated activation of the NF-kappa B/CD28-responsive complex and IL-2 expression. Delineation of the CD28 signaling cascade was found to involve protein tyrosine kinase activity, followed by the activation of phospholipase A2 and 5-lipoxygenase. Our data suggest that lipoxygenase metabolites activate ROI formation which then induce IL-2 expression via NF-kappa B activation. These findings should be useful for therapeutic strategies and the development of immunosuppressants targeting the CD28 costimulatory pathway.     

TNF-alpha and H2O2 induce IL-18 and IL-18R beta expression in cardiomyocytes via NF-kappa B activation

Myocardial ischemia/reperfusion is characterized by oxidative stress and induction of proinflammatory cytokines. Interleukin (IL)-18, a member of the IL-1 family, acts as a proinflammatory cytokine, and is induced during various immune and inflammatory disorders. Therefore, in the present study we investigated whether IL-18 expression is regulated by cytokines and oxidative stress in cardiomyocytes. TNF-alpha induced rapid and sustained activation of NF-kappaB whereas H(2)O(2) induced delayed and transient activation. Both TNF-alpha and H(2)O(2) induced IL-18 mRNA and precursor protein in cardiomyocytes, and IL-18 release into culture supernatants. However, only TNF-alpha led to sustained expression. Expression of IL-18Rbeta, but not alpha, was induced by both agonists. TNF-alpha and H(2)O(2) induced delayed expression of IL-18 BP. Pretreatment with PDTC attenuated TNF-alpha and H(2)O(2) induced IL-18 and IL-18Rbeta, but not basal expression of IL-18Ralpha. These results indicate that adult cardiomyocytes express IL-18 and its receptors, and proinflammatory cytokines and oxidative stress regulate their expression via activation of NF-kappaB. Presence of both ligand and receptors suggests IL-18 impacts myocardial biology through an autocrine pathway.     

IL-6 induces NF-kappa B activation in the intestinal epithelia

IL-6 is a potent proinflammatory cytokine that has been shown to play an important role in the pathogenesis of inflammatory bowel disease (IBD). It is classically known to activate gene expression via the STAT-3 pathway. Given the crucial role of IL-6 in the pathogenesis of chronic intestinal inflammation, it is not known whether IL-6 activates NF-kappaB, a central mediator of intestinal inflammation. The model intestinal epithelial cell line, Caco2-BBE, was used to study IL-6 signaling and to analyze whether suppressor of cytokine signaling 3 (SOCS-3) proteins play a role in the negative regulation of IL-6 signaling. We show that IL-6 receptors are present in intestinal epithelia in a polarized fashion. Basolateral IL-6 and, to a lesser extent, apical IL-6 induces the activation of the NF-kappaB pathway. Basolateral IL-6 stimulation results in a maximal induction of NF-kappaB activation and NF-kappaB nuclear translocation at 2 h. IL-6 induces polarized expression of ICAM-1, an adhesion molecule shown to be important in the neutrophil-epithelial interactions in IBD. Using various deletion constructs of ICAM-1 promoter, we show that ICAM-1 induction by IL-6 requires the activation of NF-kappaB. We also demonstrate that overexpression of SOCS-3, a protein known to inhibit STAT activation in response to IL-6, down-regulates IL-6-induced NF-kappaB activation and ICAM-1 expression. In summary, we demonstrate the activation of NF-kappaB by IL-6 in intestinal epithelia and the down-regulation of NF-kappaB induction by SOCS-3. These data may have mechanistic and therapeutic implications in diseases such as IBD and rheumatoid arthritis in which IL-6 plays an important role in the pathogenesis.     

The neuroprotective adenosine-activated signal transduction pathway involves activation of phospholipase C

We have demonstrated before that exposure of neuronal cultures to poisoning by iodoacetic acid (IAA) followed by "reperfusion" (IAA-R insult), results in severe cytotoxicity, which could be markedly attenuated by prior activation of the adenosine A1 receptors. We also have demonstrated that adenosine activates a signal transduction pathway (STP), which involves activation of PKC epsilon and opening of KATP channels. Here, we provide proof for the involvement also of phospholipase C (PLC) in the neuronal protective adenosine-activated STP. R-PIA, a specific A1 adenosine receptor agonist, was found to enhance neuronal PLC activity and protect against the IAA-R insult. The PLC inhibitor U73122, abrogated both R-PIA-induced effects. These results demonstrate that activation of PLC is a vital step in the neuronal protective adenosine-induced STP.     

Oncogene activation of HIV-LTR-driven expression via the NF-kappa B binding sites.

The Raf-1 proto-oncogene product is a highly regulated serine/threonine kinase that functions in signal transduction downstream from growth factor receptors and upstream from nuclear proto-oncogene products. Using a transient cotransfection assay we have found that activated Raf-1 activates expression from the HIV-LTR. Analysis of a series of 5' deletion and point mutations revealed the NF-kappa B motifs as the Raf-responsive element in the HIV-LTR. Moreover, Raf-BXB activated expression from heterologous promoters driven by the HIV NF-kappa B binding sites. In addition to Raf, we show that v-Src, v-H-Ras and v-Mos activate HIV-LTR expression through the NF-kappa B binding sites and v-H-Ras-induced HIV-LTR expression is mediated by Raf-1. These findings may have implications for the involvement of the cellular homologues of these oncogenes in the switch from latent to productive infection by HIV in response to T-cell activation.     

Functional role of c-Src in IL-1-induced NF-kappa B activation: c-Src is a component of the IKK complex

Interleukin-1 (IL-1) mediates numerous host responses through the rapid activation of nuclear factor-kappa B (NF-kappa B), but the signal pathways leading to NF-kappa B activation are regulated at multiple stages. Here, we propose a novel regulatory system for IL-1-induced NF-kappa B activation by a tyrosine kinase, c-Src. The kinase activity of c-Src increases in an IL-1-dependent manner and the ectopic expression of c-Src augments IL-1-induced NF-kappa B activation, suggesting the involvement of c-Src in IL-1 signaling. However, a Src family inhibitor, PP2 failed to inhibit IL-1-induced NF-kappa B activation, and the expression of a c-Src mutant lacking kinase activity (c-Src KD) augmented IL-1-induced NF-kappa B activation as well as wild type c-Src, indicating that the tyrosine kinase activity is not required for IL-1-induced NF-kappa B activation. Furthermore, a physiological interaction between c-Src and I kappa B kinase gamma (IKK gamma) was observed, implying the involvement of c-Src in the IKK-complex. While c-Src augmented IL-1-induced IKK activation independent of its kinase activity, the region comprising amino acids 361-440 in the c-Src kinase domain are required for NF-kappa B activation. The same region of c-Src is also required for IL-1-induced IKK activation and the association with IKK gamma. Taken together, our results suggest that c-Src plays a critical role in IL-1-induced NF-kappa B activation through the IKK complex.     

Herbimycin A blocks IL-1-induced NF-kappa B DNA-binding activity in lymphoid cell lines.

The mechanism by which interleukin-1 alpha (IL-1 alpha) activates NF-kappa B DNA-binding activity is not completely understood. While it is well established that protein kinase C can activate NF-kappa B, neither protein kinase C nor protein kinase A appears to be critical in the induction of NF-kappa B by IL-1 alpha. Since a number of growth factors signal via protein tyrosine kinase, in this study we examined a possible involvement of protein tyrosine kinase in the IL-1 alpha-induced NF-kappa B. The results showed that in the murine pre-B cell line 70Z/3 and in the murine T cell line EL-4 6.1 C10 IL-1 alpha-induced NF-kappa B was associated with transient increase in protein tyrosine kinase activity. Pre-treatment of these cell lines with herbimycin A, an inhibitor of tyrosine kinase activity, blocked the IL-1 alpha-enhanced protein tyrosine kinase activity and the IL-1 alpha-induced NF-kappa B DNA-binding activity. Herbimycin A at concentrations sufficient to block IL-1 alpha-induced NF-kappa B did not block the phorbol 12-myristate 13-acetate (PMA)-induced NF-kappa B. The data suggest that IL-1 alpha and PMA activate NF-kappa B by different pathways and that induction of NF-kappa B DNA-binding activity by IL-1 might be dependent on protein tyrosine phosphorylation.