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.     

HIV-1 Tat potentiates TNF-induced NF-kappa B activation and cytotoxicity by altering the cellular redox state.

This study demonstrates that human immunodeficiency virus type 1 (HIV-1) Tat protein amplifies the activity of tumor necrosis factor (TNF), a cytokine that stimulates HIV-1 replication through activation of NF-kappa B. In HeLa cells stably transfected with the HIV-1 tat gene (HeLa-tat cells), expression of the Tat protein enhanced both TNF-induced activation of NF-kappa B and TNF-mediated cytotoxicity. A similar potentiation of TNF effects was observed in Jurkat T cells and HeLa cells treated with soluble Tat protein. TNF-mediated activation of NF-kappa B and cytotoxicity involves the intracellular formation of reactive oxygen intermediates. Therefore, Tat-mediated effects on the cellular redox state were analyzed. In both T cells and HeLa cells HIV-1 Tat suppressed the expression of Mn-dependent superoxide dismutase (Mn-SOD), a mitochondrial enzyme that is part of the cellular defense system against oxidative stress. Thus, Mn-SOD RNA protein levels and activity were markedly reduced in the presence of Tat. Decreased Mn-SOD expression was associated with decreased levels of glutathione and a lower ratio of reduced:oxidized glutathione. A truncated Tat protein (Tat1-72), known to transactivate the HIV-1 long terminal repeat (LTR), no longer affected Mn-SOD expression, the cellular redox state or TNF-mediated cytotoxicity. Thus, our experiments demonstrate that the C-terminal region of HIV-1 Tat is required to suppress Mn-SOD expression and to induce pro-oxidative conditions reflected by a drop in reduced glutathione (GSH) and the GSH:oxidized GSH (GSSG) ratio.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of NF-kappa B activation by peptides targeting NF-kappa B essential modulator (nemo) oligomerization

NF-kappa B essential modulator/IKK-gamma (NEMO/IKK-gamma) plays a key role in the activation of the NF-kappa B pathway in response to proinflammatory stimuli. Previous studies suggested that the signal-dependent activation of the IKK complex involves the trimerization of NEMO. The minimal oligomerization domain of this protein consists of two coiled-coil subdomains named Coiled-coil 2 (CC2) and leucine zipper (LZ) (Agou, F., Traincard, F., Vinolo, E., Courtois, G., Yamaoka, S., Israel, A., and Veron, M. (2004) J. Biol. Chem. 279, 27861-27869). To search for drugs inhibiting NF-kappa B activation, we have rationally designed cell-permeable peptides corresponding to the CC2 and LZ subdomains that mimic the contact areas between NEMO subunits. The peptides were tagged with the Antennapedia/Penetratin motif and delivered to cells prior to stimulation with lipopolysaccharide. Peptide transduction was monitored by fluorescence-activated cell sorter, and their effect on lipopolysaccharide-induced NF-kappa B activation was quantified using an NF-kappa B-dependent beta-galactosidase assay in stably transfected pre-B 70Z/3 lymphocytes. We show that the peptides corresponding to the LZ and CC2 subdomains inhibit NF-kappa B activation with an IC(50) in the mum range. Control peptides, including mutated CC2 and LZ peptides and a heterologous coiled-coil peptide, had no inhibitory effect. The designed peptides are able to induce cell death in human retinoblastoma Y79 cells exhibiting constitutive NF-kappa B activity. Our results provide the "proof of concept" for a new and promising strategy for the inhibition of NF-kappa B pathway activation through targeting the oligomerization state of the NEMO protein.     

Inducible activation of IFI 16 results in suppression of telomerase activity,growth suppression and induction of cellular senescence

Expression of the human HIN‐200 family member IFI 16 has been reported to suppress cell growth and contribute to the onset of cellular senescence. However the molecular events involved in this process have not been fully characterised. We fused IFI 16 to the estrogen receptor ligand‐binding domain to establish an inducible model for studying the molecular events that cause these phenomena. In cells induced to express the ER‐IFI 16 within the nucleus there was a decrease in cellular proliferation and concomitant growth arrest in the G1 phase of the cell cycle. Unlike previous reports, this did not appear to involve the p53‐p21^WAF1/CIP1‐cdk2‐pRb pathway. Following nuclear expression of ER‐IFI 16 we noted senescence‐like morphological changes and expression of senescence‐associated β‐galactosidase in growth arrested cells. Importantly, we also found a marked reduction in telomerase activity in arrested cells compared to controls. Moreover, IFI 16 and hTERT co‐localised within the nucleus and these two proteins physically interacted in vivo and in vitro. Together, these data suggest that IFI 16 may act as an endogenous regulator of telomerase activity and, through its interaction with hTERT, contributes to the inhibition of proliferation and induces a senescence‐like state. J. Cell. Biochem. 109: 103–112, 2010. © 2009 Wiley‐Liss, Inc.     

Carboxylated glycans mediate colitis through activation of NF-kappa B

The role of carbohydrate modifications of glycoproteins in leukocyte trafficking is well established, but less is known concerning how glycans influence pathogenesis of inflammation. We previously identified a carboxylate modification of N-linked glycans that is recognized by S100A8, S100A9, and S100A12. The glycans are expressed on macrophages and dendritic cells of normal colonic lamina propria, and in inflammatory infiltrates in colon tissues from Crohn's disease patients. We assessed the contribution of these glycans to the development of colitis induced by CD4(+)CD45RB(high) T cell transfer to Rag1(-/-) mice. Administration of an anti-carboxylate glycan Ab markedly reduced clinical and histological disease in preventive and early therapeutic protocols. Ab treatment reduced accumulation of CD4(+) T cells in colon. This was accompanied by reduction in inflammatory cells, reduced expression of proinflammatory cytokines and of S100A8, S100A9, and receptor for advanced glycation end products. In vitro, the Ab inhibited expression of LPS-elicited cytokines and induced apoptosis of activated macrophages. It specifically blocked activation of NF-kappaB p65 in lamina propria cells of colitic mice and in activated macrophages. These results indicate that carboxylate-glycan-dependent pathways contribute to the early onset of colitis.     

Overexpression of thioredoxin reductase 1 regulates NF-kappa B activation

Thioredoxin reductase (TrxR) is a flavoprotein that contains a C-terminal penultimate selenocysteine (Sec) and has an ability to reduce thioredoxin (Trx), which regulates the activity of NF-kappa B. To date, three TrxR isozymes, TrxR1, TrxR2, and TrxR3, have been identified. In the present study, we found that among these isozymes only TrxR1 was induced by tumor necrosis factor-alpha (TNF alpha) in vascular endothelial cells. Furthermore, the overexpression of TrxR1 enhanced TNF alpha-induced DNA-binding activity of NF-kappa B and NF-kappa B-dependent gene expression. The catalytic Sec residue of TrxR1, which is essential for reducing Trx, was required for this NF-kappa B activation, and aurothiomalate, an inhibitor of TrxR, suppressed TNF alpha-induced activation of NF-kappa B and the expression of NF-kappa B-targeted proinflammatory genes such as E-selectin and cyclooxygenase-2. These results suggest that TrxR1 may act as a positive regulator of NF-kappa B and may play an important role in the cellular inflammatory response.     

TRAF family proteins link PKR with NF-kappa B activation

The double-stranded RNA (dsRNA)-dependent protein kinase PKR activates NF-kappa B via the I kappa B kinase (IKK) complex, but little is known about additional molecules that may be involved in this pathway. Analysis of the PKR sequence enabled us to identify two putative TRAF-interacting motifs. The viability of such an interaction was further suggested by computer modeling. Here, we present evidence of the colocalization and physical interaction between PKR and TRAF family proteins in vivo, as shown by immunoprecipitation and confocal microscopy experiments. This interaction is induced upon PKR dimerization. Most importantly, we show that the binding between PKR and TRAFs is functionally relevant, as observed by the absence of NF-kappa B activity upon PKR expression in cells genetically deficient in TRAF2 and TRAF5 or after expression of TRAF dominant negative molecules. On the basis of sequence information and mutational and computer docking analyses, we favored a TRAF-PKR interaction model in which the C-terminal domain of TRAF binds to a predicted TRAF interaction motif present in the PKR kinase domain. Altogether, our data suggest that TRAF family proteins are key components located downstream of PKR that have an important role in mediating activation of NF-kappa B by the dsRNA-dependent protein kinase.     

Curcumin (diferuloylmethane) inhibits receptor activator of NF-kappa B ligand-induced NF-kappa B activation in osteoclast precursors and suppresses osteoclastogenesis

Numerous studies have indicated that inflammatory cytokines play a major role in osteoclastogenesis, leading to the bone resorption that is frequently associated with cancers and other diseases. Gene deletion studies have shown that receptor activator of NF-kappaB ligand (RANKL) is one of the critical mediators of osteoclastogenesis. How RANKL mediates osteoclastogenesis is not fully understood, but an agent that suppresses RANKL signaling has potential to inhibit osteoclastogenesis. In this report, we examine the ability of curcumin (diferuloylmethane), a pigment derived from turmeric, to suppress RANKL signaling and osteoclastogenesis in RAW 264.7 cells, a murine monocytic cell line. Treatment of these cells with RANKL activated NF-kappaB, and preexposure of the cells to curcumin completely suppressed RANKL-induced NF-kappaB activation. Curcumin inhibited the pathway leading from activation of IkappaBalpha kinase and IkappaBalpha phosphorylation to IkappaBalpha degradation. RANKL induced osteoclastogenesis in these monocytic cells, and curcumin inhibited both RANKL- and TNF-induced osteoclastogenesis and pit formation. Curcumin suppressed osteoclastogenesis maximally when added together with RANKL and minimally when it was added 2 days after RANKL. Whether curcumin inhibits RANKL-induced osteoclastogenesis through suppression of NF-kappaB was also confirmed independently, as RANKL failed to activate NF-kappaB in cells stably transfected with a dominant-negative form of IkappaBalpha and concurrently failed to induce osteoclastogenesis. Thus overall these results indicate that RANKL induces osteoclastogenesis through the activation of NF-kappaB, and treatment with curcumin inhibits both the NF-kappaB activation and osteoclastogenesis induced by RANKL.     

In vivo imaging of NF-kappa B activity.

A wide range of human disorders involves inappropriate regulation of NF-kappaB, including cancers and numerous inflammatory conditions. Toward our goal to define mechanisms through which NF-kappaB leads to the development of disease, we have developed transgenic mice that express luciferase under the control of NF-kappaB, enabling real-time in vivo imaging of NF-kappaB activity in intact animals. We show that in the absence of extrinsic stimulation, strong luminescence is evident in lymph nodes in the neck region, thymus, and Peyer's patches. Treating mice with TNF-alpha, IL-1alpha, or LPS increased the luminescence in a tissue-specific manner, with the strongest activity observed in skin, lungs, spleen, Peyer's patches, and the wall of the small intestine. Liver, kidney, heart, muscle, and adipose tissue displayed less intense activities. Also, exposure of skin to a low dose of UV radiation increased luminescence in the exposed areas. Furthermore, induction of chronic inflammation resembling rheumatoid arthritis produced strong NF-kappaB activity in the affected joints, as revealed by in vivo imaging. Thus, we have developed a versatile model for monitoring NF-kappaB activation in vivo.     

FLASH coordinates NF-kappa B activity via TRAF2

FLASH is a protein recently shown to interact with the death effector domain of caspase-8 and is likely to be a component of the death-inducing signaling complex in receptor-mediated apoptosis. Here we show that antisense oligonucleotide-induced inhibition of FLASH expression abolished TNF-alpha-induced activation of NF-kappaB in HEK293 cells, as determined by luciferase reporter gene expression driven by a NF-kappaB responsive promoter. Conversely, overexpression of FLASH dose-dependently activated NF-kappaB, an effect suppressed by dominant negative mutants of TRAF2, NIK, and IKKalpha, and partially by those of TRAF5 and TRAF6. TRAF2 was co-immunoprecipitated with FLASH from the cell extracts of HEK293 cells or HeLa cells stably expressing exogenous FLASH (HeLa/HA-FLASH). Furthermore, serial deletion mapping demonstrated that a domain spanning the residues 856-1191 of FLASH activated NF-kappaB as efficiently as the full-length and could directly bind to TRAF2 in vitro and in the transfected cells. Taken together, these results suggest that FLASH coordinates downstream NF-kappaB activity via a TRAF2-dependent pathway in the TNF-alpha signaling.