Oxidative stress interference with the nuclear factor-kappa B activation pathways.

While intracellular redox balance is tightly controlled in many cell types, its modification leads to important cellular changes derived, in part, from a modification of the pattern of gene expression. This modification relies on many transcription factors whose activities are either increased or reduced by a disbalance of the redox environment. Among these transcription factors, nuclear factor-kappa B (NF-kappa B) plays a pivotal role in inducing genes involved in the control of the immune system as well as in the response to injury and infection. Because NF-kappa B can be induced in many cells by a diverse set of stimulating agents, it has been proposed that agents activating it do so by increasing oxidative stress within the cell. However, this model was not found to be universal, since the dependence between NF-kappa B activation and intracellular reactive oxygen species (ROS) generation was only detected in certain cell lines. The origin of this dependency is still unknown, but could very well be situated in a particular kinase or in adaptator molecules of the signaling cascade, leading to inhibitor kappa B alpha (I kappa B alpha phosphorylation. On the other hand, NF-kappa B can be activated by oxidants in many cell types, but this activation is well characterized only in lymphocytes. This activation is distinct from that of classical activators such as proinflammatory cytokines and phorbol esters, because the activation mechanisms appear to converge on a particular tyrosine residue of I kappa B-alpha instead of the two classical N-terminal serines. The nature of the protein kinases or protein phosphatases involved in this process is still undetermined. It will be a challenge in the future to identify the kinases/phosphatases activated by oxidants and to discover why ROS are required in some cells to turn on the transduction pathway leading to NF-kappa B activation by physiological stimuli.

Regulation of interleukin-6 gene expression by pro-inflammatory cytokines in a colon cancer cell line.

The two carcinoma cell lines HeLa and HTM-29 show different behaviour in terms of interleukin-6 (IL-6) production. Analyses of secreted IL-6 by ELISA and of IL-6 mRNA by reverse transcription-PCR revealed that, whereas HeLa cells produced high levels of IL-6 in response to tumour necrosis factor-alpha (TNF-alpha) and IL-1beta, the HTM-29 cell line failed to produce both IL-6 protein and mRNA. Nevertheless, the transcription factors nuclear factor-kappaB (NF-kappaB) and NF-IL6, the main factors involved in IL-6 gene transcriptional activation by cytokines, were activated in both cell lines after treatment with TNF-alpha or IL-1beta. In order to verify that the lack of IL-6 expression in HTM-29 cells was not due to an endogenous IL-6 gene deficiency or to IL-6 mRNA instability, we carried out transient transfection assays with an IL-6 promoter-reporter construct. Strong activation of the IL-6 promoter by cytokines could be observed in HeLa cells, whereas no induction could be detected in cytokine-treated HTM-29 cells. These cytokines induced a very strong stimulation of NF-kappaB-mediated transcription in HeLa cells transfected with a kappaB luceriferase reporter construct, whereas no induction could be detected in cytokine-stimulated HTM-29 cells. Thus IL-6 promoter repression in HTM-29 cells probably results from a failure of cytokine-activated NF-kappaB to exert its transactivating activities. Western blotting experiments demonstrated that the lack of NF-kappaB-mediated transcription was not due to increased expression of IkappaB (inhibitor of NF-kappaB) proteins in HTM-29 cells. Co-transfection experiments with the kappaB Luc reporter construct and the CBP [CREB (cAMP response element binding protein) binding protein] expression vector showed that the impairment in NF-kappaB-dependent transcription did not result from a deficiency in the co-activator CBP. Interestingly, both NF-kappaB-mediated transcription and IL-6 promoter activation could be restored in HTM-29 cells by transfection with RelA. Furthermore, CBP could have a significant synergistic effect on exogenous RelA-mediated transcription. Since sequencing of the endogenous relA gene did not reveal any mutation, it is likely that repression of NF-kappaB-mediated transcription results from negative cross-talk between NF-kappaB and another nuclear factor specifically expressed or regulated by TNF-alpha in HTM-29 cells.

Crucial role of the amino-terminal tyrosine residue 42 and the carboxyl-terminal PEST domain of I kappa B alpha in NF-kappa B activation by an oxidative stress.

Activation of transcription factor NF-kappa B involves the signal-dependent degradation of basally phosphorylated inhibitors such as I kappa B alpha. In response to proinflammatory cytokines or mitogens, the transduction machinery has recently been characterized, but the activation mechanism upon oxidative stress remains unknown. In the present work, we provide several lines of evidence that NF-kappa B activation in a T lymphocytic cell line (EL4) by hydrogen peroxide (H2O2) did not involve phosphorylation of the serine residues 32 and 36 in the amino-terminal part of I kappa B alpha. Indeed, mutation of Ser32 and Ser36 blocked IL-1 beta- or PMA-induced NF-kappa B activation, but had no effect on its activation by H2O2. Although I kappa B alpha was phosphorylated upon exposure to H2O2, tyrosine residue 42 and the C-terminal PEST (proline-glutamic acid-serine-threonine) domain played an important role. Indeed, mutation of tyrosine 42 or serine/threonine residues of the PEST domain abolished NF-kappa B activation by H2O2, while it had no effect on activation by IL-1 beta or PMA-ionomycin. This H2O2-inducible phosphorylation was not dependent on I kappa B kinase activation, but could involve casein kinase II, because an inhibitor of this enzyme (5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole) blocks NF-kappa B activation. H2O2-induced I kappa B alpha phosphorylation was followed by its degradation by calpain proteases or through the proteasome. Taken together, our findings suggest that NF-kappa B activation by H2O2 involves a new mechanism that is totally distinct from those triggered by proinflammatory cytokines or mitogens.

The ATM protein is required for sustained activation of NF-kappaB following DNA damage.

Cells lacking an intact ATM gene are hypersensitive to ionizing radiation and show multiple defects in the cell cycle-coupled checkpoints. DNA damage usually triggers cell cycle arrest through, among other things, the activation of p53. Another DNA-damage responsive factor is NF-kappaB. It is activated by various stress situations, including oxidative stress, and by DNA-damaging compounds such as topoisomerase poisons. We found that cells from Ataxia Telangiectasia patients exhibit a defect in NF-kappaB activation in response to treatment with camptothecin, a topoisomerase I poison. In AT cells, this activation is shortened or suppressed, compared to that observed in normal cells. Ectopic expression of the ATM protein in AT cells increases the activation of NF-kappaB in response to camptothecin. MO59J glioblastoma cells that do not express the DNA-PK catalytic subunit respond normally to camptothecin. These results support the hypothesis that NF-kappaB is a DNA damage-responsive transcription factor and that its activation pathway by DNA damage shares some components with the one leading to p53 activation.

Reactive oxygen intermediate-dependent NF-kappaB activation by interleukin-1beta requires 5-lipoxygenase or NADPH oxidase activity.

We previously reported that the role of reactive oxygen intermediates (ROIs) in NF-kappaB activation by proinflammatory cytokines was cell specific. However, the sources for ROIs in various cell types are yet to be determined and might include 5-lipoxygenase (5-LOX) and NADPH oxidase. 5-LOX and 5-LOX activating protein (FLAP) are coexpressed in lymphoid cells but not in monocytic or epithelial cells. Stimulation of lymphoid cells with interleukin-1beta (IL-1beta) led to ROI production and NF-kappaB activation, which could both be blocked by antioxidants or FLAP inhibitors, confirming that 5-LOX was the source of ROIs and was required for NF-kappaB activation in these cells. IL-1beta stimulation of epithelial cells did not generate any ROIs and NF-kappaB induction was not influenced by 5-LOX inhibitors. However, reintroduction of a functional 5-LOX system in these cells allowed ROI production and 5-LOX-dependent NF-kappaB activation. In monocytic cells, IL-1beta treatment led to a production of ROIs which is independent of the 5-LOX enzyme but requires the NADPH oxidase activity. This pathway involves the Rac1 and Cdc42 GTPases, two enzymes which are not required for NF-kappaB activation by IL-1beta in epithelial cells. In conclusion, three different cell-specific pathways lead to NF-kappaB activation by IL-1beta: a pathway dependent on ROI production by 5-LOX in lymphoid cells, an ROI- and 5-LOX-independent pathway in epithelial cells, and a pathway requiring ROI production by NADPH oxidase in monocytic cells.

Role of the protein kinase C lambda/iota isoform in nuclear factor-kappaB activation by interleukin-1beta or tumor necrosis factor-alpha: cell type specificities.

It has previously been reported that distinct signaling pathways can lead to nuclear factor (NF)-kappaB activation following stimulation of different cell types with inflammatory cytokines. As the role of atypical protein kinase C (PKC) isoforms in NF-kappaB activation remains a matter of controversy, we investigated whether this role might be cell type-dependent. Immunoblots detected atypical PKC expression in all the analyzed cell lines. The PKC inhibitor calphostin C inhibited NF-kappaB activation by tumor necrosis factor (TNF)-alpha or interleukin (IL)-1beta in Jurkat or NIH3T3 cells but not in MCF7 A/Z cells. Cell transfections with a PKC lambda/iota dominant negative mutant abolished TNF-alpha-induced NF-kappaB-dependent transcription in NIH3T3 and Jurkat cells but not in MCF7 A/Z cells. Similarly, the same mutant blocked NF-kappaB-dependent transactivation after IL-1beta stimulation of NIH3T3 cells, but was ineffective after IL-1beta treatment of MCF7 A/Z cells. In MCF7 A/Z cells, however, the PKC lambda/iota dominant negative mutant could abolish transactivation of an AP-1-dependent reporter plasmid after stimulation with TNF-alpha but not with IL-1beta. These data thus confirm that transduction pathways for NF-kappaB activation after cell stimulation with TNF-alpha or IL-1beta are cell-type specific and that atypical PKC isoforms participate in this pathway in NIH3T3 and Jurkat cells.

Nf-kappa B: an important transcription factor in photobiology.

Increased gene expression as a consequence of environmental stress is typically observed in mammalian cells. In the past few years the cis- and trans-acting genetic elements responsible for gene induction by radiation (from UV-C to visible light) started to be well characterized. The molecular mechanisms involved in the cell response to radiation reveal that an important control occurs at the transcriptional level and is coordinated by various transcription factors. Among these transcription factors, the well-known Rel/NF-kappa B family of vertebrate transcription factors plays a pivotal role as it controls both the inflammatory and immune responses. The NF-kappa B family comprises a number of structurally related, interacting proteins that bind DNA as dimers and whose activity is regulated by subcellular location. This family includes many members (p50, p52, RelA, RelB, c-Rel, ...), most of which can form DNA-binding homo- or heterodimers. Nuclear expression and consequent biological action of the eukaryotic NF-kappa B transcription factor complex are tightly regulated through its cytoplasmic retention by ankyrin-rich inhibitory proteins known as I kappa B. In the best-characterized example, I kappa B-alpha interacts with a p50/RelA (NF-kappa B) heterodimer to retain the complex in the cytoplasm and inhibit its DNA-binding activity. Upon receiving a variety of signals, many of which are probably mediated by the generation of reactive oxygen species (ROS), I kappa B-alpha undergoes phosphorylation, is then ubiquitinated at nearby lysine residues and finally degraded by the proteasome, while still complexed with NF- kappa B. Removal of I kappa B-alpha uncovers the nuclear localization signals on subunits of NF-kappa B, allowing the complex to enter the nucleus, bind to DNA and affect gene expression. In this paper, we shall show that molecular mechanisms leading to NF-kappa B activation by UV or by photosensitization are initiated by oxidative damage at the membrane level or by the induction of DNA alterations. While the exact nature of the transduction intermediates is still unknown, we shall show that NF-kappa B activation by radiation follows different pathways from those used by pro-inflammatory cytokines.

Involvement of different transduction pathways in NF-kappa B activation by several inducers.

Double-stimulation was used to demonstrate that, in a T lymphocytic cell line (CEM), phorbol myristate acetate (PMA) rapidly induced NF-kappa B through a signaling pathway which did not involve reactive oxygen species (ROS) and was different from the activation triggered by either H2O2 or tumor necrosis factor-alpha (TNF-alpha). Since these latter compounds were known to activate NF-kappa B translocation in a redox-sensitive way, we have demonstrated that NF-kappa B activation by PMA was resistant to antioxidant N-acetyl-L-cysteine (NAC) and sensitive to kinase inhibitors staurosporine and H7 while activation by H2O2 or TNF-alpha were not.

Activation of the NF-kappaB transcription factor in a T-lymphocytic cell line by hypochlorous acid.

Reactive oxygen species (ROS) such as hydrogen peroxide serve as second messengers in the induction of the transcription factor NF-kappaB, and hence in the activation and replication of human immunodeficiency virus type 1 (HIV-1) in human cells. During inflammatory reactions, many oxidative species are produced, one of which is hypochlorous acid (HOCl), which is responsible for the microbicidal effects of activated human polymorphonuclear leukocytes. Treatment of a T-lymphocytic cell line with micromolar concentrations of HOCl promoted the appearance of transcription factor NF-kappaB (the heterodimer p50/p65) in the nucleus of the cells, even in the absence of de novo protein synthesis. Western blot analysis of the NF-kappaB inhibitory subunits (IkappaB) demonstrated that both IkappaB-alpha proteolysis and p105 processing were induced by the treatment. NF-kappaB activation was very effective when cells were subjected to hyperthermia before being treated with HOCl. Various antioxidants, such as pyrrolidine dithiocarbamate, p-bromophenacyl-bromide and nordihydroguaiaretic acid could strongly reduce NF-kappaB translocation, demonstrating the importance of oxidative species in the transduction mechanism. Moreover, ACH-2 cells treated with HOCl or H2O2 released tumour necrosis factor-alpha (TNF-alpha) in the supernatants. The importance of TNF-alpha release in NF-kappaB induction by HOCl or H2O2 was demonstrated by the fact that: (1) the nuclear appearance of NF-kappaB was promoted in untreated cells; and (2) synergism between TNF-alpha and HOCl was detected. Collectively, these results suggest that HOCl should be considered as an oxidative species capable of inducing NF-kappaB in a T-lymphocytic cell line through a transduction mechanism involving ROS, and having a long-distance effect through subsequent TNF-alpha release.

Multiple redox regulation in NF-kappaB transcription factor activation.

The well-known Rel/NF-kappaB family of vertebrate transcription factors comprises a number of structurally related, interacting proteins that bind DNA as dimers and whose activity is regulated by subcellular location. This family includes many members (p50, p52, RelA, RelB, c-Rel, ...), most of which can form DNA-binding homo- or hetero-dimers. All Rel proteins contain a highly conserved domain of approximately 300 amino-acids, called the Rel homology domain (RH), which contains sequences necessary for the formation of dimers, nuclear localization, DNA binding and IkappaB binding. Nuclear expression and consequent biological action of the eukaryotic NF-kappaB transcription factor complex are tightly regulated through its cytoplasmic retention by ankyrin-rich inhibitory proteins known as IkappaB. The IkappaB proteins include a group of related proteins that interact with Rel dimers and regulate their activities. The interaction of a given IkappaB protein with a Rel complex can affect the Rel complex in distinct ways. In the best characterized example, IkappaB-alpha interacts with a p50/RelA (NF-kappaB) heterodimer to retain the complex in the cytoplasm and inhibit its DNA-binding activity. The NF-kappaB/IkappaB-alpha complex is located in the cytoplasm of most resting cells, but can be rapidly induced to enter the cell nucleus. Upon receiving a variety of signals, many of which are probably mediated by the generation of reactive oxygen species (ROS), IkappaB-alpha undergoes phosphorylation at serine residues by a ubiquitin-dependent protein kinase, is then ubiquitinated at nearby lysine residues and finally degraded by the proteasome, probably while still complexed with NF-kappaB. Removal of IkappaB-alpha uncovers the nuclear localization signals on subunits of NF-kappaB, allowing the complex to enter the nucleus, bind to DNA and affect gene expression. Like proinflammatory cytokines (e.g. IL-1, TNF), various ROS (peroxides, singlet oxygen, ...) as well as UV (C to A) light are capable of mediating NF-kappaB nuclear translocation, while the sensor molecules which are sensitive to these agents and trigger IkappaB-alpha proteolysis are still unidentified. We also show that a ROS-independent mechanism is activated by IL-1beta in epithelial cells and seems to involve the acidic sphingomyelinase/ceramide transduction pathway.

Lessons to be learned from varicella-zoster virus.

Varicella-zoster virus (VZV) is an alphaherpesvirus responsible for two human diseases: chicken pox and shingles. The virus has a respiratory port of entry. After two successive viremias, it reaches the skin where it causes typical lesions. There, it penetrates the peripheral nervous system and it remains latent in dorsal root ganglia. It is still debatable whether VZV persists in neurons or in satellite cells. During latency, VZV expresses a limited set of transcripts of its immediate early (IE) and early (E) genes but no protein has been detected. Mechanisms of reactivation from ganglia have not been identified. However, dysfunction of the cellular immune system appears to be involved in this process. The cell-associated nature of VZV has made it difficult to identify a temporal order of gene expression, but there appears to be a cascade mechanism as for HSV-1. The lack of high titre cell-free virions or recombination mutants has hindered so far the understanding of VZV gene functions. Five genes, ORFs 4, 10, 61, 62, and 63 that encode regulatory proteins could be involved in VZV latency. ORF4p activates gene promoters with basal activities. ORF10p seems to activate the ORF 62 promoter. ORF61p has trans-activating and trans-repressing activities. The major IE protein ORF62p, a virion component, has DNA-binding and regulatory functions, transactivates many VZV promoters and even regulates its own expression. ORF63p is a nuclear IE protein of yet unclear regulatory functions, abundantly expressed very early in infection. We have established an animal model of VZV latency in the rat nervous system, enabling us to study the expression of viral mRNA and protein expression during latency, and yielding results similar to those found in humans. This model is beginning to shed light on the molecular events in VZV persistent infection and on the regulatory mechanisms that maintain the virus in a latent stage in nerve cells.

Enhancement of varicella-zoster virus infection in cell lines expressing ORF4- or ORF62-encoded proteins.

Varicella-Zoster virus (VZV) open reading frames 4 (ORF4) and 62 (ORF62) encode putative immediate early proteins (ORF4p and ORF62p, respectively) which are strong transactivators of other VZV genes and are involved in the very early stages of viral infection. ORF4p and ORF62p transactivate immediate-early and early gene promoters but have little or no effect on late gene promoters. To investigate the effect of ORF4p or ORF62p overexpression on the viral replication cycle, we constructed Vero cell lines expressing those genes under the control of the human cytomegalovirus major immediate-early promoter. VZV OKA infection of these stably transformed cell lines was followed-up using VZV glycoprotein E (gE) antigen quantification and virus titration. Upon serial passaging of infection in these cell lines expressing functionally active ORF4p or ORF62p, a 5- to 10-fold increase in viral gE antigen production was observed. Viral titers also demonstrated a 2- to 5-fold increase in viral production in these transformed cell lines. These results emphasize the role that both ORF4p and ORF62p play in enhancing the VZV replicative cycle.

Intracellular distribution of the ORF4 gene product of varicella-zoster virus is influenced by the IE62 protein.

Varicella-zoster virus (VZV) open reading frame 4-encoded protein (IE4) possesses transactivating properties for VZV genes as well as for genes of heterologous viruses. The major regulatory immediate-early protein of VZV (IE62) is a transactivator of VZV gene expression. In transfection assays, IE4 has been shown to enhance activation induced by IE62. To investigate the functional interactions underlying this observation, indirect immunofluorescence studies were undertaken to determine whether IE62 could influence IE4 intracellular localization in transfected cells. In single transfections, IE4 was predominantly found in cytoplasm. In cotransfection with IE62, the IE4 localization pattern was altered, with nuclear staining predominating over cytoplasmic staining. This effect was specific to the IE62 protein since the gene products of ORF63 and ORF61, which are also regulatory proteins, did not influence IE4 distribution. The use of IE62 mutants indicated that IE62 influence is independent of its transactivation function and that the integrity of regions 3 and 4 is required. IE62 remained nuclear whether IE4 was present or not. These observations underline differences in the regulation of gene expression between VZV proteins and their herpes simplex virus type 1 homologues. In infected cells, IE4 was only sometimes found to colocalize with IE62 in nuclei. This observation suggests that when all VZV proteins are present, complex interactions probably occur which could diminish the influence of IE62.

Varicella-zoster virus induces apoptosis in cell culture.

Apoptosis is an active mechanism of cell death which can be initiated in response to various stimuli including virus infections. In this work, we demonstrate that lytic infection by varicella-zoster virus (VZV), a human herpesvirus, is characterized by nuclear fragmentation of DNA into oligonucleosomal fragments and by chromatin condensation. In vitro, VZV-induced cell death is actually mediated by apoptosis. The mechanisms developed by cells to protect themselves against apoptosis could be one of the parameters allowing the establishment of virus latency. In the case of VZV, which can remain latent in sensory ganglia, we have not yet identified a cellular or viral protein which could play this protective role, since the observed apoptosis mechanism seems to be independent from Bcl-2, the most frequently described inhibitor of apoptosis.

Mutational analysis of varicella-zoster virus major immediate-early protein IE62.

The varicella-zoster virus (VZV) open reading frame 62 encodes an immediate-early protein (IE62) that transactivates expression of various VZV promoters and autoregulates its own expression in transient expression assays. In Vero cells, IE62 was shown to transactivate the expression of all putative immediate-early (IE) and early (E) genes of VZV with an up-regulating effect at low intracellular concentrations. To define the functional domains involved in the regulatory properties of IE62, a large number of in-frame insertions and deletions were introduced into a plasmid-borne copy of the gene encoding IE62. Studies of the regulatory activities of the resultant mutant polypeptides in transient expression assays allowed to delineate protein regions important for repression of its own promoter and for transactivation of a VZV putative immediate-early gene (ORF61) promoter and an early gene (ORF29) promoter. This mutational analysis resulted in the identification of a new functional domain situated at the border between regions 4 and 5 which plays a crucial role in the IE62 regulatory functions. This domain turned out to be very well conserved amongst homologous alphaherpesvirus regulatory proteins and appeared to be rich in bulky hydrophobic and proline residues, similar to the proline-rich region of the CAAT box binding protein CTF-1. By immunofluorescence, a nuclear localization signal has been mapped in region 3.

Cytochemical distinction of various nucleolar components in insect cells.

The fine structure of the insect Sf9 cell nucleolus has been investigated by means of different cytochemical and immunocytochemical techniques at the electron microscope level. Apart from a few perinucleolar condensed chromatin clumps, the insect cell nucleolus comprises two compartments. The first of these consists of a roundish compact zone formed of fibrillar material. The other is composed of fibrillar and granular structures organized into a network separated by interstitial spaces. But, unlike mammalian cell nucleoli, any fibrillar center has been observed in the Sf9 cell nucleolus, even after actinomycin D treatment. We also show that the compact fibrillar zone of Sf9 cell nucleoli contains silver-stainable material and DNA. In actinomycin D-treated cells, a preferential contact of this compact fibrillar zone with condensed chromatin has been visualized. Finally, silver-stainable material has been found to persist throughout the whole mitosis. These results suggest that the compact fibrillar zone at the insect Sf9 cell nucleolus should, at least partly, correspond to the fibrillar center of mammalian cell nucleoli.