NF-[kappa]B is a pleiotropic transcription factor, which is instrumental in regulating diverse cellular processes such as inflammation and cell survival. Enhanced NF- [kappa]B activity, due either to prolonged NF-[kappa]B activation upon exposure to cellular stress or misregulation of NF-[kappa]B, can lead to many neurodegenerative diseases as well as cancer. Thus, in this study, biological and biochemical approaches were used to characterize how NF-[kappa]B regulates the cellular stress response and how NF-[kappa]B activity itself is regulated. Chapter 1 introduces oxidative stress, which is the specific cellular stress used in this study, and reviews how oxidative stress affects all cellular components, including the proteasome and NF-[kappa]B. Chapter 2 characterizes the mechanism of NF-[kappa]B's cell death promoting function in response to oxidative stress and shows that NF-[kappa]B signaling actually promotes cell death. Furthermore, we suggest that NF- [kappa]B promotes cell death through the repression of pro -survival genes and induction of pro-death genes. Chapter 3 focuses on whether oxidized 20S proteasome enhances the ubiquitin independent degradation of NF-[kappa]B inhibitor molecule I[kappa]B[gamma]. We clearly show that the 20S proteasome is able to degrade I[kappa]B[gamma] in an ubiquitin independent manner. Preliminary LC-MS/MS data suggests that the 20S proteasome, which displays enhanced activity towards I[kappa]B[gamma], can undergo cysteic acid modification. We suggest that mild oxidative stress can enhance proteasome activity, while severe oxidative stress impairs proteasomal activity. Chapter 4 provides strong evidence suggesting that PA28[alpha][beta] bound 20S proteasome is responsible for the ubiquitin independent degradation of another NF-[kappa]B inhibitor molecule, free I[kappa]B[alpha]. All together, this work delineates how NF-[kappa]B signaling can mediate cell death in response to oxidative stress, how oxidative stress can potentially increase the ubiquitin independent degradation of I[kappa]B[gamma] by enhancing the proteolytic activity of the 20S proteasome, and how PA28[alpha][beta] bound 20S proteasome is responsible for the ubiquitin independent degradation of free I[kappa]B[alpha]

Background

The pro-survival activity of NF-κB in response to a variety of stimuli has been extensively characterized. Although there have been a few reports addressing the pro-cell death role of NF-κB, the precise mechanism of NF-κB's pro-cell death function still remains elusive.

Methodology/principal findings

In the present study, we investigated the role of NF-κB in cell death induced by chronic insult with hydrogen peroxide (H(2)O(2)). Here, we show that NF-κB promotes H(2)O(2) induced caspase independent but PARP dependent fibroblast cell death. The pro-death activity of NF-κB is due to the DNA binding activity of RelA, which is induced through IKK- mediated IκBα degradation. NF-κB dependent pro-survival genes, Bcl-2 and XIAP, were significantly repressed, while NF-κB dependent pro-death genes, TNFα and Fas Ligand, were induced in response to H(2)O(2).

Conclusions/significance

We discovered an unexpected function of NF-κB, in that it potentiates chronic H(2)O(2) exposure induced cell death, and suggest that NF-κB mediates cell death through the repression of pro-survival genes and induction of pro-death genes. Since unremitting exposure of tissues to H(2)O(2) and other reactive oxygen species can lead to several degenerative disorders and diseases, our results have important implications for the use of NF-κB inhibitors in therapeutic drug design.

Nuclear factor kappa B (NFκB) is a transcription factor that controls inflammation and cell survival. In clinical histology, elevated NFκB activity is a hallmark of poor prognosis in inflammatory disease and cancer, and may be the result of a combination of diverse micro-environmental constituents. While previous quantitative studies of NFκB focused on its signaling dynamics in single cells, we address here how multiple stimuli may combine to control tissue level NFκB activity. We present a novel, simplified model of NFκB (SiMoN) that functions as an NFκB activity calculator. We demonstrate its utility by exploring how type I and type II interferons modulate NFκB activity in macrophages. Whereas, type I IFNs potentiate NFκB activity by inhibiting translation of IκBα and by elevating viral RNA sensor (RIG-I) expression, type II IFN amplifies NFκB activity by increasing the degradation of free IκB through transcriptional induction of proteasomal cap components (PA28). Both cross-regulatory mechanisms amplify NFκB activation in response to weaker (viral) inducers, while responses to stronger (bacterial or cytokine) inducers remain largely unaffected. Our work demonstrates how the NFκB calculator can reveal distinct mechanisms of crosstalk on NFκB activity in interferon-containing microenvironments.