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Is NF-B the sensor of oxidative stress?

Laboratory of Gene Regulation and Signal Transduction, Department of Pharmacology, University of California, San Diego, La Jolla, California 92093-0636, USA

¹Correspondence: Laboratory of Gene Regulation and Signal Transduction, Department of Pharmacology, University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0636, USA. E-mail: karinoffice{at}ucsd.edu

	ABSTRACT

TOP ABSTRACT INTRODUCTION NF-{kappa}B ACTIVATION OXIDATIVE STRESS AND NF-{kappa}B... REFERENCES

 
NF-B is a dimeric transcription factor that is involved in the regulation of a large number of genes that control various aspects of the immune and inflammatory response. It is activated by a variety of stimuli ranging from cytokines, to various forms of radiation, to oxidative stress (such as exposure to H₂O₂). Recent studies have advanced our understanding of the signal transduction pathway leading to NF-B activation by cytokines and will provide insights for the mechanism by which NF-B is regulated by oxidative stress. An important question that is yet to be answered is whether reactive oxygen species play a physiological role in NF-B activation.—Li, N., Karin, M. Is NF-B the sensor of oxidative stress?

Key Words: IB degradation • IKK • reactive oxygen species • tumor necrosis factor • GSH

	INTRODUCTION

TOP ABSTRACT INTRODUCTION NF-{kappa}B ACTIVATION OXIDATIVE STRESS AND NF-{kappa}B... REFERENCES

 
IN RESPIRING CELLS, a small amount of the consumed oxygen is reduced in a specific way, yielding a variety of highly reactive chemical entities. These are collectively called reactive oxygen species (ROS)² or reactive oxidative intermediates (ROIs), and include nitric oxide radical (NO^.), superoxide anion (O₂^-), hydroxy radical (OH^.), and their by-products (e.g., hydrogen peroxide H₂O₂). ROS are capable of causing oxidative damage to macromolecules leading to lipid peroxidation, oxidation of amino acid side chains (especially cysteine), formation of protein-protein cross-links, oxidation of polypeptide backbones resulting in protein fragmentation, DNA damage, and DNA strand breaks (1 2 3) . High doses of ROS, which may be generated during chronic and acute inflammatory diseases or on environmental stresses, are cytotoxic. Small amounts of ROS, produced as a consequence of electron transfer reactions in mitochondria, peroxisomes, and cytosol, are scavenged by cellular defending systems including nonenzymatic and enzymatic antioxidants. A state of moderately increased levels of intracellular ROS is referred to as oxidative stress. Cells respond to these adverse conditions by modulation of their antioxidant levels, induction of new gene expression, and protein modification (1 , 2) . The homeostatic modulation of oxidant levels is a highly efficient mechanism that appeared early in evolution, allowing all cells to tightly control their redox status within a very narrow range.

The Rel/NF-B family of transcriptional factors regulate expression of numerous cellular and viral genes and play important roles in immune and stress responses, inflammation, and apoptosis (4 5 6 7) . It was suggested that NF-B activity is regulated by the intracellular ROS levels, but the molecular mechanism involved in this regulation remains to be elucidated (8 , 9) . In the past several years, our understanding of how NF-B is activated by inflammatory cytokines has been advanced tremendously, and this review focuses on the recent discoveries with the aim to explore the possible mechanism(s) leading to regulation of NF-B by oxidative stress. In addition, we critically examine the issue of whether ROS have a general signaling role in NF-B activation.

	NF-B ACTIVATION

TOP ABSTRACT INTRODUCTION NF-{kappa}B ACTIVATION OXIDATIVE STRESS AND NF-{kappa}B... REFERENCES

 
Activation of NF-B by cytokines
The NF-B transcriptional factors are composed of homodimers or heterodimers of Rel proteins, which are characterized by the presence of a Rel homology domain (RHD) (5 , 10 , 11) . The RHD is ~300 amino acids long and is required for sequence specific DNA binding, dimerization, nuclear localization, and interaction with inhibitory proteins, members of the IB family. As a consequence of binding to cytoplasmic IBs, the nuclear localization signal of the NF-B dimer is masked and NF-B is sequestered in the cytoplasm. The IB proteins contain multiple copies of the ankyrin repeat, which interact with the RHD of Rel/NF-B proteins (11 , 12) . IB, IBß, and IB are three IB proteins that are expressed ubiquitously. In response to proinflammatory cytokines such as tumor necrosis factor (TNF) and interleukin-1 (IL-1), bacterial lipopolysaccharide (LPS) or viral double-strand RNA (dsRNA), the IBs are rapidly phosphorylated at two specific serine residues located at their N₂-terminal region (Ser-32 and Ser-36 for IB, Ser-19 and Ser-23 for IBß, Ser-157 and Ser-161 for IB) and then undergo ubiquitination and proteolysis by the 26S proteasome, resulting in release and translocation of NF-B to the nucleus, where it activates transcription of specific target genes. The cytokine-induced phosphorylation of IB is a prerequisite for its degradation and subsequent NF-B activation because substitutions of those two serines with alanine residues render IB resistant to degradation, and the expression of such mutant forms of IB result in suppression of NF-B activation.

Recently, a large TNF-inducible cytoplasmic protein kinase complex that is able to phosphorylate IB on Ser-32 and Ser-36 and IBß on Ser-19 and Ser-23 was purified and the genes encoding several of its subunits were molecularly cloned (13 14 15 16) . The first two subunits to be identified are two related protein kinases of molecular mass 85,000 and 87,000, and are called IB kinase (IKK) (IKK1) and IKKß (IKK2), respectively (13 14 15) . IKK and IKKß have a similar overall structure and share ~51% identity. They contain a Ser/Thr kinase domain in their N₂-terminal portion and a leucine zipper as well as a helix-loop-helix protein interaction motif in their carboxyl-terminal region. IKK was also isolated via a yeast two-hybrid screen as a protein that interacts with a member of the MAP kinase kinase kinase (MAPKKK) family called NIK (NF-B-inducing kinase) (17) . Although NIK is not a direct IB kinase, its overexpression results in efficient NF-B activation (18) . Both IKK and IKKß are rapidly activated by cytokines, with kinetics matching those of IB phosphorylation and degradation. Expression of a catalytically inactive IKK or IKKß mutants blocks cytokine-induced IB degradation and NF-B activation, suggesting that IKK and IKKß are important for induction of NF-B activity by cytokines (13 14 15 , 19) . Further evidence that IKK or IKKß are indeed responsible for IB phosphorylation was provided by purified recombinant IKK and IKKß proteins produced in insect cells by a baculovirus expression system (20) . Although IKK and IKKß can form stable homodimers and heterodimers in vitro, most IKK complexes contain heterodimers; very little IKK or IKKß homodimeric complexes were found to exist in cells so far (E. Zandi, D. Rothwarf, and M. Karin, unpublished results). Mutations in the leucine zipper motif abolish the dimerization of IKK or IKKß and their kinase activity, indicating that dimerization is required for the formation of a functional IB kinase (20) . Mutations in the helix-loop-helix motif of IKK or IKKß strongly reduce the kinase activity, although they did not affect dimerization (20) .

IKK activity depends on its phosphorylation, as it is inactivated by protein phosphatase 2A (13) . Analysis of the protein kinase domains of IKK and IKKß reveals several potential phosphoacceptor sites in the T (activation) loop, a region conserved in all protein kinases. Although mutations at those sites abrogate the kinase activity (15 , 21) , so far these sites have not been shown to be phosphorylated in cytokine-stimulated cells or to be involved in cytokine-mediated IKK activation. It is not clear whether phosphorylation of these sites occurs through autophosphorylation, cross-phosphorylation by a partner IKK, or by a separate kinase. As IKK was also identified as an NIK-interacting protein, it was immediately suggested that NIK might be an upstream kinase for IKK. In fact, overexpression of NIK activates IKK in cells and NIK immunoprecipitated from cells is able to phosphorylate IKK in vitro (21 22 23) . But it remains to be determined whether NIK can directly phosphorylate and activate IKK and whether NIK activity is regulated by cytokine stimulation. Recently, NIK was reported to be a part of a large IL-1-inducible IKK complex together with IB, IKK, IKKß, and a 150 kDa protein called IKAP (IKK complex-associated protein) (24) . The interaction between IKAP and NIK was not affected by cytokine stimulation. IKAP could bind to NIK and IKKs through separate domains and its overexpression inhibited NF-B dependent gene expression induced by TNF and IL-1. It was therefore suggested that IKAP functions as a scaffolding protein to assemble kinases to form an active IKK complex. More biochemical analysis will be needed to confirm this hypothesis and it will be interesting to see whether IKAP assembles the same complex in response to other IKK-inducing stimuli. It should be noted, however, that in these experiments the IKK complex was not purified very extensively and there is little biochemical evidence that NIK or IKAP are indeed integral parts of this complex. A complete characterization of the composition of IKK complex and elucidation of the mechanisms that lead to its activation by a variety of stimuli are the focus of intensive research at present.

In addition to two protein kinase subunits, another bona fide subunit of the large IKK complex has been cloned through two different approaches (16 , 25) . The IKK complex was purified to homogeneity from TNF-stimulated cell lysates and found to contain two additional polypeptides (IKK1 and IKK2), which represent differently modified forms of the same protein IKK (16) . It should be noted that the purified IKK complex does not contain easily detectable amount of NIK or IKAP. The importance of IKK was revealed by the reduction in cytokine-induced activity of IKK and degradation of IB caused by expression of an antisense IKK construct, which reduced IKK expression by ~50%. IKK was also identified in a screen for genes that are able to complement a cell line that is unresponsive to multiple NF-B-activating stimuli including TNF, IL-1, LPS, phorbol 12-myristate 13-acetate (PMA), dsRNA, and the Tax transactivator protein of human T cell leukemia virus (HTLV) (25) . A cDNA was found to be able to reconstitute activation of NF-B by all of these inducers upon its expression. It was therefore named NEMO (NF-B essential modulator) and is the mouse homologue of IKK. Consistent with its essential role for NF-B activation, IKK/NEMO is stoichiometrically present in the large IKK complex that contains IKK/ß heterodimers. IKK/NEMO is rich in glutamine and contains a putative leucine zipper motif in its carboxyl-terminal region and several coiled-coil motifs. The carboxyl-terminal region of IKK is required for activation of IKK by various stimuli, as expression of a carboxyl-terminal truncation mutant IKK inhibited activation of IKK by TNF, IL-1, and other stimuli (16) . However, this truncation mutant is still able to bind to IKKß, and its expression did not alter dimerization between IKK and IKKß. Furthermore, a complex assembled with the IKK truncation mutant has the same hydrodynamic properties as the native IKK complex, but is refractory to activation (E. Zandi and M. Karin, unpublished results). Therefore, it is unlikely that IKK/NEMO functions as a chaperon or a structural component that stabilizes IKK-IKKß dimers. Instead, a likely function for IKK/NEMO is to physically link IKK complex to upstream activators through its carboxyl-terminal region. The definite functions for these three IKK subunits will wait the analysis of corresponding gene knockout mice.

Activation of NF-B by some stimuli does not involve IKK
As described above, NF-B-activating stimuli that are also able to induce IKK activity include cytokines (TNF and IL-1), PMA, LPS, dsRNA, the HTLV transactivator protein Tax, and ionizing radiation (26 , 27) . In certain cases, however, NF-B activation does not seem to involve IB phosphorylation by IKK or even IB degradation. Short-wavelength UV (UV-C) light activates NF-B in certain cell types concomitantly with IB degradation. Pretreatment of cells with proteasome inhibitors blocked IB degradation and NF-B activation induced by UV radiation, indicating that IB degradation is required (27) . However, neither IKK activation nor the phosphorylation of IB on Ser-32 and Ser-36 was observed to occur after UV-C irradiation (27 , 28) . Furthermore, even the IB mutant that contains alanines at positions 32 and 36 was still susceptible to UV-C induced degradation. Similar to UV-C radiation, treatment of cells with amino acid analogs also activates NF-B through IB degradation by the proteasome without apparent phosphorylation at Ser-32 and Ser-36 (29) . It was suggested that the aberrant protein conformation generated by the incorporation of amino acid analogs into newly synthesized IB may contribute to its proteolysis. However, it is more likely that exposure to amino acid analogs activates a stress response similar to the one triggered by UV-C radiation. Another pathway leading to NF-B activation was reported when cells were treated with tyrosine phosphatase inhibitors (e.g., pervanadate) or upon reoxygenation of hypoxic cells (30 , 31) . In these treatments, NF-B was activated through tyrosine phosphorylation of IB without its degradation. The phosphorylation site was identified as Tyr-42, and this site is present only in IB. It was shown that the tyrosine phosphorylation of IB led to its dissociation from NF-B (30) . The tyrosine phosphorylation has been found to protect IB from cytokine-induced serine phosphorylation and degradation (32 , 33) . Since Tyr-42 is in proximity with Ser-32 and Ser-36, it is possible that phosphorylation on Tyr-42 may inhibit recognition of the substrate site by IKK. It is also speculated that tyrosine phosphorylation may target IB to another docking protein (e.g., a SH2 domain containing protein), thereby dissociating it from NF-B. The interaction of tyrosine-phosphorylated IB with this docking protein may also prevent serine phosphorylation by IKK.

	OXIDATIVE STRESS AND NF-B ACTIVATION

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Activation of NF-B by ROS
The direct evidence that ROS level may be able to regulate NF-B was provided by frank exposure of cells to H₂O₂. In certain cell types, such as Wurzburg subclone of T cells, L6 skeletal muscle myotubes, human breast MCF-7, and 70Z/3 pre-B cells, H₂O₂ was shown to be an effective inducer of NF-B activation (9 , 34 , 35) . Several groups have reported that H₂O₂ can also induce NF-B activation in HeLa cells, albeit to quite a different extent (36 , 37) . We and others could not detect NF-B activation by H₂O₂ in HeLa, 293, fibroblast, or Jurkat T cells (N. Li, Y. Chen, and M. Karin, unpublished results) (38) . It is becoming clear from the available data that H₂O₂-induced NF-B activation is highly cell type dependent and therefore H₂O₂ is unlikely to be a general mediator of NF-B activation. Intracellular level of reduced glutathione (GSH), which may differ from one cell type to another, may be crucial for H₂O₂-induced NF-B response. GSH is the major intracellular thiol and ROI scavenger (39) . N-acetyl-L-cysteine (NAC) is a nontoxic compound that protects cells from oxidative damage (40) . It provides a precursor for GSH synthesis and can also react directly with ROI. Buthionine-sulfoximine (BSO) is a drug that causes GSH depletion since it selectively inhibits glutamylcysteine synthetase, the enzyme responsible for GSH synthesis (41) . Preincubation with NAC abolished H₂O₂-induced NF-B activation in the Wurzburg T cells (34) , while preincubation with BSO made otherwise undetectable H₂O₂-induced NF-B activation detectable in HeLa cells (Fig. 1 ). However, even in BSO-treated cells, whose GSH level is very low (our unpublished results), activation of NF-B by H₂O₂ is inefficient compared with physiological inducers such as TNF.

View larger version (63K): [in this window] [in a new window]   Figure 1. The effect of NAC and BSO on H2O2–induced NF-B DNA binding activity. HeLa cells were mock treated or incubated with 30 mM NAC or 100 µM BSO overnight before addition of H2O2 (lanes 1–3) or 500 µM H2O2 (lanes 4–6). After 3 h incubation with H2O2, cells were harvested and subjected to the electrophoretic mobility shift assay (EMSA) with 32P-labeled B oligonucleotide. As a control, the sample in lane 7 was from cells that were treated with TNF for 30 min.

The involvement of oxidative stress in NF-B activation
Several indirect lines of evidence suggest a role for ROIs as a common and critical intermediate for various NF-B-activating signals. This conclusion is based largely on the inhibition of NF-B activation by a variety of antioxidants (42) and by overexpression of antioxidant enzymes. These reagents have been reported to block NF-B activation in many instances, although the extent of inhibition appears to vary depending on cell type and stimulus. For example, the antioxidant pyrrolidinedithiocarbamate (PDTC) has been shown to be inhibitory for TNF, IL-1, PMA, LPS, amino acid analogs and amyloid ß peptides in Jurkat T cells, HeLa cells, 70Z/3 cells, and primary neurons (29 , 43 44 45 46) . Although TNF-induced NF-B activation was sensitive to PDTC in a transformed human endothelial cell line, it was insensitive in a primary endothelial cell line (47) . NAC could inhibit NF-B activation induced by TNF, LPS, and UV in Jurkat and HeLa cells (42 , 48 , 49) , but failed to inhibit okadaic acid-induced NF-B activation in Jurkat cells (49) . Overexpression of superoxide manganese dismutase (SMD-Mg) or glutathione peroxidase abolished NF-B activation induced by TNF, LPS, PMA, and H₂O₂ (35 , 50) . AP-1 activity, JNK activation, and apoptosis induced by TNF were also suppressed by overexpression of SMD-Mg (35) . A novel thioredoxin peroxidase named antioxidant enzyme AOE372 suppressed TPA-induced NF-B activation in HeLa cells upon its overexpression (51) . Additional support for the involvement of the oxidative stress derives from evidence showing elevated cellular levels for ROIs in response to TNF, IL-1, PMA, LPS, UV light, and ionizing radiation (42) . Despite these observations, lack of easy, fast, and sensitive assays to measure the changes in intracellular levels for ROIs after treatment of cells with NF-B-inducing agents hinders the biochemical study for oxidative stress. For instance, it is essential to show that production of ROIs precedes the increase in IKK activity, which occurs within 2 min after TNF or IL-1 stimulation. Besides, many of the antioxidants that were used have multiple targets. For example, PDTC is a metal chelator (52) and does not always inhibit NF-B activation. It does not have an anti-inflammatory activity (52) , which is expected to be associated with inhibition of NF-B activity or activation. Therefore, a direct function role for ROIs in signaling to NF-B still remains to be proved.

Which signaling step is affected by oxidative stress?
It has been shown that adding H₂O₂ to HeLa cells induced the appearance of a slow-migrating form of IB in SDS-polyacrylamide gel, which was rapidly degraded unless cells were treated with the proteasome inhibitor (29) . Addition of antioxidants (PDTC or NAC) or overexpression of peroxidases blocked IB degradation induced by TNF, PMA, and LPS (35 , 44 , 45 , 50) . PDTC also blocked the appearance of the slow-migrating form of IB after TNF stimulation, suggesting that IB phosphorylation was inhibited (45) . Similarly, it was shown by 2-dimensional immunoblot analysis that glutathione peroxidase overexpression abolished TNF-mediated transient accumulation of the more acidic and apparent higher molecular weight isoform of IB (50) . Taken together, these data suggested that IB phosphorylation and degradation might be the step that is responsive to oxidative stress. But it was not clear whether IKK activity is affected by oxidative stress.

To examine this, we treated HeLa cells with NAC before stimulation with TNF and then examined NF-B activation, IB degradation, and IKK activity. The results showed that NF-B DNA binding activity induced by TNF was decreased when cells were pretreated with NAC (Fig. 2A ). Immunoblot analysis indicated that IB degradation was also reduced by NAC, although the slow-migrating form of IB, which is indicative of its phosphorylation, still appeared, suggesting that IB still became phosphorylated (Fig. 2B ). Indeed, IKK activity induced by TNF was not affected by NAC treatment (Fig. 2C ). These results suggest that cytokine-induced degradation rather than phosphorylation of IB was inhibited by the antioxidant NAC in HeLa cells. This disagrees with the results mentioned above that NAC blocked phosphorylation of IB. The discrepancy could be due to the different HeLa cells used in the experiments and also to possible dephosphorylation of IB in cell extracts that do not contain proper amounts of phosphatase inhibitors.

View larger version (58K): [in this window] [in a new window]   Figure 2. The effect of NAC on TNF-induced NF-B activation. HeLa cells were mock treated or incubated with 30 mM NAC for 1 h before addition of TNF. After 15 min treatment with TNF, cells were harvested and subjected to the EMSA with 32P-labeled B oligonucleotide (A), immunoblotting with anti-IB (B), and immunokinase assay with anti-IKK and GST-IB 1–54 as the substrate (C). The positions of DNA–NF-B complex (A), IB and phosphorylated IB (p-IB) (B), and GST-IB 1–54 are indicated (C).

If NAC does not affect activation of IKK by TNF, the question immediately arises as to which step in the signaling pathway is affected. Although phosphorylation of IB is still detected as the slow-migrating form (Fig. 2B ), it is difficult to say whether phosphorylation still occurs with the same efficiency. For instance, NAC might affect recognition of IB by IKK complex to slow down the phosphorylation. It is certainly possible that NAC may inhibit ubiquitination or/and degradation of IB, which involve many proteins and are little understood. The formation of ubiquitin-protein conjugates involves three components that participate in a series of ubiquitin transfer reactions: a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin ligase (E3) (53) . The specificity in protein ubiquitination often derives from the E3 component. It has been demonstrated in yeast that members of F-box proteins are components of E3 ligase required for ubiquitination and degradation of cyclins (54) . A mammalian F-box protein, E3RS^IB, was identified recently as a component of E3 ligase for IB (55) . E3RS^IB specifically interacted with the IB that was phosphorylated at Ser-32 and Ser-36 to promote its ubiquitination. It is not yet clear how many components comprise the E3 for IB. The assembly of the E3 complex, its interaction with phosphorylated IB, or the entire polyubiquitination and proteasome degradation processes might be sensitive to oxidative stress and antioxidants. This may explain why oxidative stress has been implicated in NF-B activation by so many unrelated stimuli ranging from cytokines to UV radiation. Apparently, more work will be needed to determine which step is sensitive to oxidative stress. Nevertheless, it is now clear that the key regulatory step in NF-B activation by proinflammatory stimuli (TNF, IL-1, LPS, and dsRNA) and ionizing radiation is the activation of IKK. Once IBs are phosphorylated by IKK, NF-B activation proceeds via constitutively active components. Thus, unless shown to be directly and specifically involved in IKK activation, ROIs are unlikely to have a general signaling role (as second messengers) in NF-B activation.

Prospects and final remarks
Through the accumulated data, it is clear that certain cell types, but certainly not all, respond to oxidative stress by up-regulation of NF-B activity. However, the molecular basis for this regulation is largely unknown. As we learn more about the signaling pathways leading to NF-B activation, the questions such as to which step in the pathway is affected by oxidative stress and how it is affected should be addressed with higher molecular precision, and eventually such knowledge will help us to understand why the regulation of NF-B by oxidative stress is cell type specific. Oxidative stress has been implicated in apoptosis that occurs during normal aging as well as in various inflammatory diseases and neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease. NF-B activation, on the other hand, was shown to provide cells and organisms with potent antiapoptotic defense. A study of the modulation of the antiapoptotic gene expression program normally activated by NF-B in response to oxidative stress may provide additional insight into the molecular basis of many acute pathologies and degenerative diseases and aid in the development of potential drugs to control and prevent these disorders.

	ACKNOWLEDGMENTS

 
We thank Y. Chen, M. Delhase, G. Natoli, D. Rothwarf, and E. Zandi for sharing unpublished results and helpful discussions. N.L. is supported by a postdoctoral fellowship from the Cancer Research Institute. M.K. is the Frank and Else Schilling-American Cancer Society Research Professor. This work was supported by grants from NIEHS (ES 04151, ES 06376) and the Department of Energy (DE-FG03-86ER60429-A0).

	FOOTNOTES

 
² Abbreviations: BSO, buthionine-sulfoximine; dsRNA, double-strand RNA; GSH, reduced glutathione; HTLV, human T cell leukemia virus; IKAP, IKK complex-associated protein; IKK, IB kinase; IL, interleukin; LPS, lipopolysaccharide; NAC, N-acetyl-L-cysteine; NEMO, NF-B essential modulator; NIK, NF-B-inducing kinase; NO^., nitric oxide radical; O₂^-, superoxide anion; OH^., hydroxyl radical; PDTC, pyrrolidinedithiocarbamate; PMA, phorbol 12-myristate 13-acetate; ROIs, reactive oxidative intermediates; ROS, reactive oxygen species; SMD-Mg, superoxide manganese dismutase; TNF, tumor necrosis factor; UV-C, short-wavelength UV.

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