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Nuclear factor-?B (NF-?B) inhibitory protein I?B? determines apoptotic cell death following exposure to oxidative stress.

ABSTRACT: The transcription factor NF-?B regulates the cellular response to inflammatory and oxidant stress. Although many studies have evaluated NF-?B activity following exposure to oxidative stress, the role of the I?B family of inhibitory proteins in modulating this activity remains unclear. Specifically, the function of I?B? in mediating the cellular response to oxidative stress has not been evaluated. We hypothesized that blocking oxidative stress-induced NF-?B signaling through I?B? would prevent apoptotic cell death. Using I?B? knock-in mice (AKBI), in which the I?B? gene is replaced with the I?B? cDNA, we show that I?B? overexpression prevented oxidative stress-induced apoptotic cell death. This was associated with retention of NF-?B subunits in the nucleus and maintenance of NF-?B activity. Furthermore, the up-regulation of pro-apoptotic genes in WT murine embryonic fibroblasts (MEFs) exposed to serum starvation was abrogated in AKBI MEFs. Inhibition of apoptosis was observed in WT MEFs overexpressing I?B? with simultaneous I?B? knockdown, whereas I?B? overexpression alone did not produce this effect. These findings represent a necessary but not sufficient role of I?B? in preventing oxidant stress-induced cell death.

SUBMITTER: Wright CJ

PROVIDER: S-EPMC3307281 | biostudies-literature | 2012 Feb

REPOSITORIES: biostudies-literature

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Nuclear factor-κB (NF-κB) inhibitory protein IκBβ determines apoptotic cell death following exposure to oxidative stress.

Wright Clyde J CJ Agboke Fadeke F Muthu Manasa M Michaelis Katherine A KA Mundy Miles A MA La Ping P Yang Guang G Dennery Phyllis A PA

The Journal of biological chemistry 20120105 9

The transcription factor NF-κB regulates the cellular response to inflammatory and oxidant stress. Although many studies have evaluated NF-κB activity following exposure to oxidative stress, the role of the IκB family of inhibitory proteins in modulating this activity remains unclear. Specifically, the function of IκBβ in mediating the cellular response to oxidative stress has not been evaluated. We hypothesized that blocking oxidative stress-induced NF-κB signaling through IκBβ would prevent ap ...[more]

PMID: 22223647

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Project description:Cells, particularly mechano-sensitive musculoskeletal cells such as tenocytes, routinely encounter oxidative stress. Oxidative stress can not only stimulate tissue repair, but also cause damage leading to tissue degeneration. As diabetes is associated with increased oxidative damage as well as increased risk of tendon degeneration, the aim of this study was to determine if extracellular glucose levels alter the response of tendon cells to oxidative stress. Primary human tenocytes were cultured in either high (17.5?mM) or low (5?mM) glucose and treated with 100??M hydrogen peroxide. In low glucose, peroxide-treated cells remained fully viable and collagen synthesis was increased, suggesting an anabolic response. In high glucose, however, peroxide treatment led to increased bim-mediated apoptosis. The activities of both forkhead box O (FOXO1) and p53 were required for upregulation of bim RNA expression in high glucose. We found that both p53-mediated inhibition of the bim repressor micro RNA (miR17-92) and FOXO1-mediated upregulation of bim transcription were required to permit accumulation of bim RNA. High glucose coupled with oxidative stress resulted in upregulation of miR28-5p, which directly inhibited expression of the p53 deacetylase sirtuin 3, resulting in increased levels of acetylated p53. In peroxide-treated cells in both high and low glucose, protein levels of acetylated FOXO1 as well as HIF1? (hypoxia-inducible factor 1?) were increased. However, under low-glucose conditions, peroxide treatment resulted in activation of p38, which inhibited FOXO1-mediated but promoted HIF1?-mediated transcriptional activity. In low glucose, HIF1? upregulated expression of sox9 and scleraxis, two critical transcription factors involved in establishing the tenocyte phenotype, and increased collagen synthesis. The switch from FOXO1-mediated (proapoptosis) to HIF1?-mediated (prodifferentiation) transcription occurred at an extracellular glucose concentration of 7?mM, a concentration equivalent to the maximum normal blood glucose concentration. Extracellular glucose has a profound effect on the cellular response to oxidative stress. A level of oxidative stress normally anabolic may be pathological in high glucose.

Project description:We previously demonstrated that protein kinase C? (PKC?; PKC delta) is an oxidative stress-sensitive kinase that plays a causal role in apoptotic cell death in neuronal cells. Although PKC? activation has been extensively studied, relatively little is known about the molecular mechanisms controlling PKC? expression. To characterize the regulation of PKC? expression, we cloned an ?2-kbp 5'-promoter segment of the mouse Prkcd gene. Deletion analysis indicated that the noncoding exon 1 region contained multiple Sp sites, including four GC boxes and one CACCC box, which directed the highest levels of transcription in neuronal cells. In addition, an upstream regulatory region containing adjacent repressive and anti-repressive elements with opposing regulatory activities was identified within the region -712 to -560. Detailed mutagenesis studies revealed that each Sp site made a positive contribution to PKC? promoter expression. Overexpression of Sp family proteins markedly stimulated PKC? promoter activity without any synergistic transactivating effect. Furthermore, experiments in Sp-deficient SL2 cells indicated long isoform Sp3 as the essential activator of PKC? transcription. Importantly, both PKC? promoter activity and endogenous PKC? expression in NIE115 cells and primary striatal cultures were inhibited by mithramycin A. The results from chromatin immunoprecipitation and gel shift assays further confirmed the functional binding of Sp proteins to the PKC? promoter. Additionally, we demonstrated that overexpression of p300 or CREB-binding protein increases the PKC? promoter activity. This stimulatory effect requires intact Sp-binding sites and is independent of p300 histone acetyltransferase activity. Finally, modulation of Sp transcriptional activity or protein level profoundly altered the cell death induced by oxidative insult, demonstrating the functional significance of Sp-dependent PKC? gene expression. Collectively, our findings may have implications for development of new translational strategies against oxidative damage.

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Nuclear factor-?B (NF-?B) inhibitory protein I?B? determines apoptotic cell death following exposure to oxidative stress.

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Nuclear factor-κB (NF-κB) inhibitory protein IκBβ determines apoptotic cell death following exposure to oxidative stress.

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