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Role of ascorbate in the activation of NF-kappaB by tumour necrosis factor-alpha in T-cells.

ABSTRACT: The first product of ascorbate oxidation, the ascorbate free radical (AFR), acts in biological systems mainly as an oxidant, and through its role in the plasma membrane redox system exerts different effects on the cell. We have investigated the role of ascorbate, AFR and dehydroascorbate (DHA) in the activation of the NF-kappaB transcription factor in Jurkat T-cells stimulated by tumour necrosis factor-alpha (TNF-alpha). Here we show, by electrophoretic mobility shift assays, that ascorbate increases the binding of NF-kappaB to DNA in TNF-alpha-stimulated Jurkat cells. The ability of ascorbate to enhance cytoplasmic inhibitory IkBalpha protein degradation correlates completely with its capacity to induce NF-kappaB binding to DNA and to potentiate NF-kappaB-mediated transactivation of the HIV-1 long terminal repeat promoter in TNF-alpha-stimulated Jurkat cells but not in cells stimulated with PMA plus ionomycin. AFR behaves like ascorbate, while DHA and ascorbate phosphate do not affect TNF-alpha-mediated NF-kappaB activation. These results provide new evidence for a possible relationship between the activation of the electron-transport system at the plasma membrane by ascorbate or its free radical and redox-dependent gene transcription in T-cells.

SUBMITTER: Munoz E

PROVIDER: S-EPMC1218524 | biostudies-other | 1997 Jul

REPOSITORIES: biostudies-other

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Autophagosomal IkappaB alpha degradation plays a role in the long term control of tumor necrosis factor-alpha-induced nuclear factor-kappaB (NF-kappaB) activity.

Project description:Transcription factor NF-?B is persistently activated in many chronic inflammatory diseases and cancers. The short term regulation of NF-?B is well understood, but little is known about the mechanisms of its long term activation. We studied the effect of a single application of TNF-? on NF-?B activity for up to 48 h in intestinal epithelial cells. Results show that NF-?B remained persistently activated up to 48 h after TNF-? and that the long term activation of NF-?B was accompanied by a biphasic degradation of I?B?. The first phase of I?B? degradation was proteasome-dependent, but the second was not. Further investigation showed that TNF-? stimulated formation of autophagosomes in intestinal epithelial cells and that I?B? co-localized with autophagosomal vesicles. Pharmacological or genetic blockade of autophagosome formation or the inhibition of lysosomal proteases decreased TNF-?-induced degradation of I?B? and lowered NF-?B target gene expression. Together, these findings indicate a role of autophagy in the control of long term NF-?B activity. Because abnormalities in autophagy have been linked to ineffective innate immunity, we propose that alterations in NF-?B may mediate this effect.

| S-EPMC3123056 | biostudies-literature

NF-kappaB mediates tumor necrosis factor alpha-induced expression of optineurin, a negative regulator of NF-kappaB.

Project description:Optineurin is a ubiquitously expressed multifunctional cytoplasmic protein encoded by OPTN gene. The expression of optineurin is induced by various cytokines. Here we have investigated the molecular mechanisms which regulate optineurin gene expression and the relationship between optineurin and nuclear factor kappaB (NF-kappaB). We cloned and characterized human optineurin promoter. Optineurin promoter was activated upon treatment of HeLa and A549 cells with tumor necrosis factor alpha (TNFalpha). Mutation of a putative NF-kappaB-binding site present in the core promoter resulted in loss of basal as well as TNFalpha-induced activity. Overexpression of p65 subunit of NF-kappaB activated this promoter through NF-kappaB site. Oligonucleotides corresponding to this putative NF-kappaB-binding site showed binding to NF-kappaB. TNFalpha-induced optineurin promoter activity was inhibited by expression of inhibitor of NF-kappaB (IkappaBalpha) super-repressor. Blocking of NF-kappaB activation resulted in inhibition of TNFalpha-induced optineurin gene expression. Overexpressed optineurin partly inhibited TNFalpha-induced NF-kappaB activation in Hela cells. Downregulation of optineurin by shRNA resulted in an increase in TNFalpha-induced as well as basal NF-kappaB activity. These results show that optineurin promoter activity and gene expression are regulated by NF-kappaB pathway in response to TNFalpha. In addition these results suggest that there is a negative feedback loop in which TNFalpha-induced NF-kappaB activity mediates expression of optineurin, which itself functions as a negative regulator of NF-kappaB.

| S-EPMC2660438 | biostudies-literature

Tumour necrosis factor alpha induces co-ordinated activation of rat GSH synthetic enzymes via nuclear factor kappaB and activator protein-1.

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| S-EPMC1276939 | biostudies-literature

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| S-EPMC1221688 | biostudies-other

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c-IAP1 and c-IAP2 are critical mediators of tumor necrosis factor alpha (TNFalpha)-induced NF-kappaB activation.

Project description:The inhibitor of apoptosis (IAP) proteins are a family of anti-apoptotic regulators found in viruses and metazoans. c-IAP1 and c-IAP2 are recruited to tumor necrosis factor receptor 1 (TNFR1)-associated complexes where they can regulate receptor-mediated signaling. Both c-IAP1 and c-IAP2 have been implicated in TNFalpha-stimulated NF-kappaB activation. However, individual c-IAP1 and c-IAP2 gene knock-outs in mice did not reveal changes in TNF signaling pathways, and the phenotype of a combined deficiency of c-IAPs has yet to be reported. Here we investigate the role of c-IAP1 and c-IAP2 in TNFalpha-stimulated activation of NF-kappaB. We demonstrate that TNFalpha-induced NF-kappaB activation is severely diminished in the absence of both c-IAP proteins. In addition, combined absence of c-IAP1 and c-IAP2 rendered cells sensitive to TNFalpha-induced cell death. Using cells with genetic ablation of c-IAP1 or cells where the c-IAP proteins were eliminated using IAP antagonists, we show that TNFalpha-induced RIP1 ubiquitination is abrogated in the absence of c-IAPs. Furthermore, we reconstitute the ubiquitination process with purified components in vitro and demonstrate that c-IAP1, in collaboration with the ubiquitin conjugating enzyme (E2) enzyme UbcH5a, mediates polymerization of Lys-63-linked chains on RIP1. Therefore, c-IAP1 and c-IAP2 are required for TNFalpha-stimulated RIP1 ubiquitination and NF-kappaB activation.

| S-EPMC3259840 | biostudies-other

TRUSS, a novel tumor necrosis factor receptor 1 scaffolding protein that mediates activation of the transcription factor NF-kappaB.

Project description:We describe the cloning and characterization of tumor necrosis factor receptor (TNF-R)-associated ubiquitous scaffolding and signaling protein (TRUSS), a novel TNF-R1-interacting protein of 90.7 kDa. TRUSS mRNA was ubiquitously expressed in mouse tissues but was enriched in heart, liver, and testis. Co-immunoprecipitation experiments showed that TRUSS was constitutively associated with unligated TNF-R1 and that the complex was relatively insensitive to stimulation with TNF-alpha. Deletion mutagenesis of TNF-R1 indicated that TRUSS interacts with both the membrane-proximal region and the death domain of TNF-R1. In addition, the N-terminal region of TRUSS (residues 1 to 440) contains sequences that permit association with the cytoplasmic domain of TNF-R1. Transient overexpression of TRUSS activated NF-kappaB and increased NF-kappaB activation in response to ligation of TNF-R1. In contrast, a COOH-terminal-deletion mutant of TRUSS (TRUSS(1-723)) was found to inhibit NF-kappaB activation by TNF-alpha. Co-precipitation and co-immunoprecipitation assays revealed that TRUSS can interact with TRADD, TRAF2, and components of the IKK complex. These findings suggest that TRUSS may serve as a scaffolding protein that interacts with TNF-R1 signaling proteins and may link TNF-R1 to the activation of IKK.

| S-EPMC262424 | biostudies-literature

TNFR1 promotes tumor necrosis factor-mediated mouse colon epithelial cell survival through RAF activation of NF-kappaB.

Project description:Tumor necrosis factor (TNF) is a therapeutic target in the treatment of inflammatory bowel disease; however, the exact role of TNF signaling in the colon epithelium remains unclear. We demonstrate that TNF activation of TNF receptor (R)1 stimulates both pro- and anti-apoptotic signaling pathways in the colon epithelium; however, TNFR1 protects against colon epithelial cell apoptosis following TNF exposure. To investigate anti-apoptotic signaling pathways downstream of TNFR1, we generated an intestinal epithelium-specific Raf knock-out mouse and identified Raf kinase as a key regulator of colon epithelial cell survival in response to TNF. Surprisingly, Raf promotes NF-kappaB p65 phosphorylation, independent of MEK signaling, to support cell survival. Taken together, these data demonstrate a novel pathway in which Raf promotes colon epithelial cell survival through NF-kappaB downstream of TNFR1 activation. Thus, further understanding of colon epithelial cell-specific TNFR signaling may result in the identification of new targets for inflammatory bowel disease treatment and define novel mediators of colitis-associated cancer.

| S-EPMC2570867 | biostudies-literature

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