Resynthesis Of IkappaBalpha Research Articles

Identification of PP2A as a crucial regulator of the NF-κB feedback loop: its inhibition by UVB turns NF-κB into a pro-apoptotic factor

Although nuclear factor-kappaB (NF-kappaB) usually exerts anti-apoptotic activity, upon activation by interleukin-1 (IL-1) it enhances ultraviolet-B radiation (UVB)-induced apoptosis. This paradoxical effect is associated with NF-kappaB-dependent pronounced secretion of tumour necrosis factor-alpha (TNF) which activates TNF-R1 in an autocrine fashion to enhance UVB-induced apoptosis. We demonstrate that sustained TNF transcription in UVB+IL-1-treated cells involves complete abrogation of the negative feedback loop of NF-kappaB preventing IkappaBalpha resynthesis, hence allowing uncontrolled NF-kappaB activity. We show that IkappaBalpha is not transcriptionally inhibited but resynthesized protein is immediately marked for degradation due to persistent inhibitor of kappaB kinasebeta (IKKbeta) activity. Continuous IKKbeta phosphorylation and activation is caused by UVB-mediated inhibition of the phosphatase PP2A. This study demonstrates that the cellular response to different NF-kappaB activators may be converted to the opposite reaction when both stimuli act in concert. Our data shed new light on the significance of negative feedback regulation of NF-kappaB and identifies PP2A as the key regulator of this process.

Superinduction of IL-6 by cycloheximide is associated with mRNA stabilization and sustained activation of p38 map kinase and NF-kappaB in cultured caco-2 cells.

Protein synthesis inhibitors paradoxically increase the expression of early-gene products, including various cytokines, through a process known as superinduction. Superinduction is cell-specific and the mechanisms involved are not fully understood but are usually attributed to decreased mRNA degradation. There is, however, increasing evidence that activation of signaling cascades and increased transcriptional activation may be involved as well. Recent studies suggest that IL-6 production in the intestinal mucosa is particularly important due to its anti-inflammatory and protective effects. The effect of protein synthesis inhibitors on IL-6 production in enterocytes, however, is unknown. Treatment of Caco-2 cells with cycloheximide (10 microg/ml) increased IL-6 mRNA and protein levels in IL-1beta-treated cells and this was associated with increased mRNA stabilization. In addition, cycloheximide suppressed IkappaBalpha resynthesis and prolonged p38MAP kinase activation and these changes were associated with sustained activation of the transcription factor NF-kappaB. NF-kappaB activation, in turn, was prevented by the specific p38MAP kinase inhibitor SB208350. Our results suggest that superinduction of IL-6 by cycloheximide in enterocytes results from both increased mRNA stabilization and upregulated transcriptional activity mediated by prolonged activation of the p38 MAP kinase and NF-kappaB.

Lymphotoxin ॆ Receptor Induces Sequential Activation of Distinct NF-κB Factors via Separate Signaling Pathways

Lymphotoxin beta receptor (LTbetaR)-induced activation of NF-kappaB in mouse embryo fibroblasts was mediated by the classical pathway and by an alternative or second pathway. The classical pathway involved the IkappaB kinase (IKK)beta- and IKKgamma-dependent degradation of IkappaBalpha and resulted in the rapid but transient activation of primarily RelA-containing NF-kappaB dimers. The alternative or second pathway proceeded via NF-kappaB-inducing kinase (NIK)-, IKKalpha-, and protein synthesis-dependent processing of the inhibitory NF-kappaB2 p100 precursor protein to the p52 form and resulted in a delayed but sustained activation of primarily RelB-containing NF-kappaB dimers. This second pathway was independent of the classical IKK complex, which is governed by its central IKKgamma regulatory subunit. The sequential engagement of two distinct pathways, coupled with the negative feedback inhibition of RelA complexes by NF-kappaB-induced resynthesis of IkappaBalpha, resulted in a pronounced temporal change in the nature of the NF-kappaB activity during the course of stimulation. Initially dominant RelA complexes were replaced with time by RelB complexes. Therefore, the alternative activation path mediated by processing of p100 was necessary for sustained NF-kappaB activity in mouse embryo fibroblasts in response to LTbetaR stimulation. Based on the phenotype of mice deficient in various components of the LTbetaR-induced activation of p100 processing, we conclude that this pathway is critically involved in the function of stromal cells during the generation of secondary lymphoid organ microarchitectures.

Induced stabilization of IkappaBalpha can facilitate its re-synthesis and prevent sequential degradation.

The transcription factor NF-kappaB is responsible for regulating genes that can profoundly impact cell proliferation, apoptosis, inflammation, and immune responses. The NF-kappaB inhibitor IkappaBalpha is rapidly degraded and then re-synthesized after an NF-kappaB stimulus. We have found that the re-synthesis of IkappaBalpha in a human colon-derived cell line (HT-29) includes the post-translational stabilization of newly synthesized IkappaBalpha. The TNF-alpha-induced stabilization of newly synthesized IkappaBalpha involves the C-terminal PEST region of the protein: N-terminal deletion mutants (lacking the IkappaB kinase phosphorylation sites) were readily stabilized by TNF-alpha, whereas deletion of the C-terminus resulted in a constitutively stable protein. The role of the C-terminus in stabilization was further supported by the finding that fusion of the IkappaBalpha C-terminus to GFP generated a protein that could also be stabilized by TNF-alpha. The p38 mitogen-activated protein (MAP) kinase inhibitor SB203580 prevented stabilization of IkappaBalpha and delayed the re-emergence of IkappaBalpha following TNF-alpha-induced degradation. The IkappaBalpha stabilization pathway could prevent sequential rounds of IkappaBalpha degradation without preventing IkappaBalpha phosphorylation. Analysis of two other cell lines (SW480 and THP-1) revealed similarities and cell-specific differences in the regulation of IkappaBalpha stabilization. We propose that cytokine stabilization of newly synthesized IkappaBalpha in some cell types is a critical homeostatic mechanism that limits inflammatory gene expression.

Tumor Necrosis Factor-regulated Biphasic Activation of NF-κB Is Required for Cytokine-induced Loss of Skeletal Muscle Gene Products

NF-kappaB activation is classically defined as a transient response initiated by the degradation of IkappaB inhibitor proteins leading to nuclear import of NF-kappaB and culminating with the resynthesis of IkappaBalpha and subsequent inactivation of the transcription factor. Although this type of regulation is considered the paradigm for NF-kappaB activation, other regulatory profiles are known to exist. By far the most common of these is chronic or persistent activation of NF-kappaB. In comparison, regulation of NF-kappaB in a biphasic manner represents a profile that is scarcely documented and whose biological significance remains poorly understood. Here we show using differentiated skeletal muscle cells, that tumor necrosis factor (TNF) induces NF-kappaB activation in a biphasic manner. Unlike the first transient phase, which is terminated within 1 h of cytokine addition, the second phase persists for an additional 24-36 h. Biphasic activation is mediated at both the levels of NF-kappaB DNA binding and transactivation function, and both phases are dependent on the IKK/26 S proteasome pathway. We find that regulation of the first transient phase is mediated by the degradation and subsequent resynthesis of IkappaBalpha, as well as by a TNF-induced expression of A20. Second phase activity correlates with persistent down-regulation of both IkappaBalpha and IkappaBbeta proteins, derived from a continuous TNF signal. Finally, we demonstrate that inhibition of NF-kappaB prior to initiation of the second phase of activity inhibits cytokine-mediated loss of muscle proteins. We propose that the biphasic activation of NF-kappaB in response to TNF may play a key regulatory role in skeletal muscle wasting associated with cachexia.

Hyperoxia prolongs tumor necrosis factor-alpha-mediated activation of NF-kappaB: role of IkappaB kinase.

Hyperoxia and tumor necrosis factor-alpha (TNFalpha) are two canonical signals centrally involved in the pathophysiology of acute lung injury. We have attempted to elucidate the effects of these two stimuli on the signal transduction pathways of lung parenchymal cells. In cultured human lung epithelial cells, exposure to hyperoxia alone (95% oxygen) did not affect NF-kappaB activation or degradation of the NF-kappaB inhibitory protein, IkappaB alpha. Stimulation with TNFalpha alone increased NF-kappaB activation within 1 h and induced IkappaB alpha degradation within 0.5 h. After TNFalpha alone, NF-kappaB activation returned to baseline within 2 h and this corresponded with near complete IkappaB alpha resynthesis within 1 h of stimulation. In contrast, simultaneous exposure to hyperoxia and TNFalpha prolonged NF-kappaB activation up to 4 h, and IkappaB alpha degradation up to 2 h after stimulation. Hyperoxia did not affect TNFalpha-mediated resynthesis of IkappaB alpha mRNA. Hyperoxia alone did not induce IkappaB kinase (IKK) activity, but significantly prolonged TNFalpha-mediated activation of IKK activity. Hyperoxia alone did not activate the intercellular adhesion molecule-1 (ICAM-1) promoter, but augmented TNFalpha-mediated activation of the ICAM-1 promoter. These data demonstrate that while hyperoxia alone does not affect activation of NF-kappaB, hyperoxia prolongs TNFalpha-mediated activation of NF-kappaB. The mechanism of this effect involves, in part, prolonged degradation of IkappaB alpha resulting from prolonged activation of IKK.

Regulation of NF-κB and ICAM-1 expression in human airway epithelial cells

The aim of this study was to elucidate the redox regulation of cytokine-induced NF-kappaB activation and NF-kappaB mediated gene induction in A549 cells and primary cultures of human airway epithelial cells. In A549 cells, Western blot analysis showed transient depletion of IkappaBalpha after 15 min IL-1beta treatment followed by its reappearance after 60 min, indicating efficient NF-kappaB-driven gene induction. A similar pattern was observed in primary epithelial cells however, the kinetics were slower and depletion was less. In primary airway epithelial cells IkappaBalpha levels were 59.8+/-8.5% of control following 30 min treatment with IL-1beta and in A549 cells 29.1+/-8.5% of control following 15 min IL-1beta treatment. Cytokine-induced IkappaBalpha depletion was associated with NF-kappaB nuclear accumulation and subsequent resynthesis of IkappaBalpha and upregulation of ICAM-1 in both cell types. The antioxidant, NAC (20 mM) had no effect on the kinetics of cytokine-induced IkappaBalpha depletion or NF-kappaB p65 nuclear translocation in either cell type and failed to influence kappaB dependent IkappaBalpha resynthesis. H2O2 treatment alone or in combination with cytokines had no significant effects on IkappaBalpha depletion, NF-kappaB p65 nuclear translocation or ICAM-1 expression in either cell type but did cause significant activation of p38 MAPK. These results suggest that cytokine-induced NF-kappaB activation in cultured human airway epithelial cells does not involve an NAC-sensitive oxidant stress and that H2O2-induced oxidant stress does not result in effective NF-kappaB activation and NF-kappaB mediated gene induction.

Dynamic Shuttling of Nuclear Factor κB between the Nucleus and Cytoplasm as a Consequence of Inhibitor Dissociation

Activation of the nuclear factor kappaB (NFkappaB) transcription factor is intimately associated with its translocation from the cytoplasm to the nucleus. Using the nuclear export inhibitor leptomycin B, we demonstrate shuttling of the RELA subunit of NFkappaB and the inhibitory subunit IkappaBalpha between these two compartments in unstimulated cells. Determination of the kinetics of nuclear entry shows marked differences for the two components; the entry of IkappaBalpha occurs more rapidly than RELA. The shuttling is suggested to be a consequence of the cytoplasmic dissociation of the NFkappaB.IkappaB complex rather than its direct nuclear import or degradation and resynthesis of IkappaBalpha. Using previously published kinetic data, this proposition is born out by the deduction that 17% of NFkappaB is not complexed to IkappaBalpha in a resting cell. A numerical model is presented to validate the proposed regulation of NFkappaB subcellular localization consequent in part on the nuclear export function and in part on the cytoplasmic retention function of IkappaBalpha. We suggest that the non-saturated interaction of NFkappaB with the inhibitor may enhance the specificity of action of IkappaB proteins on different NFkappaB dimers and allow additional modes of regulation of IkappaB function.

Pervanadate-induced Nuclear Factor-κB Activation Requires Tyrosine Phosphorylation and Degradation of IκBα: COMPARISON WITH TUMOR NECROSIS FACTOR-α

Tumor necrosis factor activates nuclear transcription factor kappaB (NF-kappaB) by inducing serine phosphorylation of the inhibitory subunit of NF-kappaB (IkappaBalpha), which leads to its ubiquitination and degradation. In contrast, pervanadate (PV) activates NF-kappaB and induces tyrosine phosphorylation of IkappaBalpha (Singh, S., Darney, B. G., and Aggarwal, B. B. (1996) J. Biol. Chem. 271, 31049-31054; Imbert, V., Rupec, R. A., Antonia, L., Pahl, H. L., Traenckner, E. B.-M., Mueller-Dieckmann, C., Farahifar, D., Rossi, B., Auderger, P., Baeuerle, P. A., and Peyron, J.-F. (1996) Cell 86, 787-798). Whether PV also induces IkappaBalpha degradation and whether degradation is required for NF-kappaB activation are not understood. We investigated the effect of PV-induced tyrosine phosphorylation on IkappaBalpha degradation and NF-kappaB activation. PV activated NF-kappaB, as determined by DNA binding, NF-kappaB-dependent reporter gene expression, and phosphorylation and degradation of IkappaBalpha. Maximum degradation of IkappaBalpha occurred at 180 min, followed by NF-kappaB-dependent IkappaBalpha resynthesis. N-Acetylleucylleucylnorlucinal, a proteasome inhibitor, blocked both IkappaBalpha degradation and NF-kappaB activation, suggesting that the IkappaBalpha degradation is required for NF-kappaB activation. PV did not induce serine phosphorylation of IkappaBalpha but induced phosphorylation at tyrosine residue 42. Unlike tumor necrosis factor (TNF), PV did not induce ubiquitination of IkappaBalpha. Like TNF, however, PV induced phosphorylation and degradation of IkappaBalpha, and subsequent NF-kappaB activation, which could be blocked by N-tosyl-L-phenylalanine chloromethyl ketone, calpeptin, and pyrrolidine dithiocarbomate, suggesting a close link between PV-induced NF-kappaB activation and IkappaBalpha degradation. Overall, our studies demonstrate that PV activates NF-kappaB, which, unlike TNF, requires tyrosine phosphorylation of IkappaBalpha and its degradation.

Effects of systemic immunogenic insults and circulating proinflammatory cytokines on the transcription of the inhibitory factor kappaB alpha within specific cellular populations of the rat brain.

Expression of the inhibitory factor kappaB alpha (IkappaB alpha) reflects the activity of nuclear factor kappaB(NF-kappaB) and is a powerful tool to investigate the regulation of the transcription factor within the CNS. IkappaB alpha mRNA was evaluated in the rat brain by means of in situ hybridization following different immunogenic stimuli; i.e., intraperitoneal (i.p.) and intravenous (i.v.) lipopolysaccharide (LPS), i.v. recombinant rat interleukin (IL) 1beta, IL-6, or tumor necrosis factor-alpha (TNF-alpha), and intramuscular (i.m.) turpentine injection, used here as a model of systemic localized inflammatory insult. Systemic LPS, IL-1beta, and TNF-alpha caused a rapid and transient transcriptional activation of IkappaB alpha along the blood vessels of the entire brain; the signal was very intense 30-60 min after the i.v. injections and returned to undetectable levels from 2 to 12 h depending on the challenge. Double-labeling procedure provided the anatomical evidence that IkappaB alpha-expressing cells within the microvasculature were essentially of the endothelial type, as they were immunoreactive to the von Willebrand factor. Scattered small cells were also found across the brain of LPS-, IL-1beta-, and TNF-alpha-injected rats at time 1-3 h, and microglial (OX-42)-immunoreactive cells were positive for the transcript. Such expression within parenchymal microglia was nevertheless not observed in the brain following a localized and sterile inflammatory insult. Indeed, i.m. turpentine administration stimulated IkappaB alpha transcription quite uniquely within the endothelium of the brain capillaries, an effect that paralleled the swelling of the injection site and lasted up to 24 h after the aggression. In contrast to these immunogenic challenges, i.v. IL-6 injection failed to activate the gene encoding IkappaB alpha in the rat brain. These results indicate that NF-kappaB may play a crucial role in specific cellular populations of the CNS to trigger transcription of immune-related genes and that IkappaB alpha resynthesis may act as a dynamic intracellular inhibitory feedback to avoid exaggeration of the response. It is possible that IkappaB alpha expression in cells of the blood-brain barrier is a general mechanism that takes place during systemic inflammation, whereas the participation of NF-kappaB-related molecules within parenchymal cells of the CNS is solicited during more severe conditions such as blood sepsis and endotoxemia.

Nuclear Retention of IκBα Protects It from Signal-induced Degradation and Inhibits Nuclear Factor κB Transcriptional Activation

Transcriptional activation of nuclear factor kappaB (NF-kappaB) is mediated by signal-induced phosphorylation and degradation of its inhibitor, IkappaBalpha. However, NF-kappaB activation induces rapid resynthesis of IkappaBalpha, which is responsible for post-induction repression of transcription. Newly synthesized IkappaBalpha translocates to the nucleus, where it dissociates NF-kappaB from DNA and transports NF-kappaB from the nucleus to the cytoplasm in a nuclear export sequence-dependent process that is sensitive to leptomycin B (LMB). In the present study, LMB was used as a tool to inhibit nuclear export sequence-mediated nuclear protein export and evaluate the consequences for regulation of NF-kappaB-dependent transcriptional activity. Pretreatment of cells with LMB inhibits NF-kappaB-dependent transcriptional activation mediated by interleukin 1beta or tumor necrosis factor alpha. This is a consequence of the inhibition of signal-induced degradation of IkappaBalpha. Although LMB treatment does not affect the signal transduction pathway leading to IkappaBalpha degradation, it blocks IkappaBalpha nuclear export. IkappaBalpha is thus accumulated in the nucleus, and in this compartment it is resistant to signal-induced degradation. These results indicate that the signal-induced degradation of IkappaBalpha is mainly, if not exclusively, a cytoplasmic process. An efficient nuclear export of IkappaBalpha is therefore essential for maintaining a low level of IkappaBalpha in the nucleus and allowing NF-kappaB to be transcriptionally active upon cell stimulation.

Interleukin-1-induced Nuclear Factor-κB-IκBα Autoregulatory Feedback Loop in Hepatocytes: A ROLE FOR PROTEIN KINASE Cα IN POST-TRANSCRIPTIONAL REGULATION OF IκBα RESYNTHESIS

The IkappaB inhibitors regulate the activity of the potent transcription factor nuclear factor-kappaB (NF-kappaB). Following signal-induced IkappaB proteolysis, NF-kappaB translocates into the nucleus to activate transcription of target genes, including IkappaBalpha itself, initiating the "NF-kappaB-IkappaBalpha autoregulatory feedback loop." Upon IkappaBalpha resynthesis, NF-kappaB is subsequently inactivated and redistributed back into the cytoplasm. We have previously reported a robust NF-kappaB-IkappaBalpha autoregulatory feedback loop in HepG2 hepatocytes. Sixty minutes after tumor necrosis factor (TNF-alpha) stimulation, IkappaBalpha is resynthesized to approximately 2-fold greater level than in control cells and completely inhibits NF-kappaB binding. Here we investigate the mechanism for IkappaBalpha resynthesis comparing the effect of stimulation of TNF-alpha with that of interleukin-1 (IL-1alpha). Although either TNF-alpha or IL-1alpha stimulation of protein kinase C (PKC)-down-regulated cells equivalently induces NF-kappaB translocation, the kinetics of IkappaBalpha resynthesis is slowed. Moreover, pretreatment with selective calcium-dependent PKC inhibitors selectively slowed the kinetics of the IL-1alpha-induced overshoot without affecting that produced by TNF-alpha. Down-regulation of PKCalpha by antisense phosphorothioate oligonucleotides and expression vectors selectively blocked the IL-1alpha-induced IkappaBalpha overshoot. In the absence of PKCalpha, although IL-1alpha induced similar amounts of IkappaBalpha transcription and changes in steady-state mRNA, a greater component of IkappaBalpha mRNA was retained in the nucleus. These data indicate a selective role for PKCalpha in IL-1alpha-induced IkappaBalpha resynthesis, which is mediated, at least in part, by post-transcriptional control of mRNA export.

Inhibition of Endothelial Vascular Cell Adhesion Molecule-1 Expression by Nitric Oxide Involves the Induction and Nuclear Translocation of IκBα

The induction of vascular cell adhesion molecule-1 (VCAM-1) expression by tumor necrosis factor (TNF)-alpha requires the activation of nuclear factor-kappaB (NF-kappaB) via a process involving the phosphorylation and degradation of its cytoplasmic inhibitor, IkappaBalpha. We have shown that nitric oxide (NO) decreases VCAM-1 expression via inhibition of NF-kappaB activation. To determine how NO inhibits NF-kappaB, we studied the fate of IkappaBalpha following TNF-alpha stimulation in the presence of NO donors S-nitrosoglutathione and sodium nitroprusside. Activation of NF-kappaB by TNF-alpha occurred within 15 min and coincided with rapid degradation of IkappaBalpha. Co-treatment with NO donors did not prevent IkappaBalpha phosphorylation or degradation. However, after 2 h of TNF-alpha stimulation, NO donors inhibited NF-kappaB activation and augmented IkappaBalpha resynthesis and nuclear translocation by 2.5- and 3-fold, respectively. This correlated with a 75% reduction in TNF-alpha-induced VCAM-1 expression. In a time-dependent manner, NO donors alone caused the nuclear translocation of IkappaBalpha. To confirm that NO donors have similar effects as endogenously derived NO, murine macrophage-like cells, RAW264.7, were co-cultured with endothelial cells. Induction of RAW264.7-derived NO inhibited lipopolysaccharide-induced endothelial VCAM-1 expression, which was reversed by the NO synthase inhibitor Nomega-monomethyl-L-arginine. These findings indicate that NO inhibits NF-kappaB activation and VCAM-1 expression by increasing the expression and nuclear translocation of IkappaBalpha.

Mechanism for Biphasic Rel A· NF-κB1 Nuclear Translocation in Tumor Necrosis Factor α-stimulated Hepatocytes

The proinflammatory cytokine, tumor necrosis factor alpha (TNFalpha), is a potent activator of angiotensinogen gene transcription in hepatocytes by activation of latent nuclear factor-kappaB (NF-kappaB) DNA binding activity. In this study, we examine the kinetics of TNFalpha-activated translocation of the 65-kDa (Rel A) and 50-kDa (NF-kappaB1) NF-kappaB subunits mediated by inhibitor (IkappaB) proteolysis in HepG2 hepatoblastoma cells. HepG2 cells express the IkappaB members IkappaBalpha, IkappaBbeta, and IkappaBgamma. In response to TNFalpha, Rel A.NF-kappaB1 translocation and DNA binding activity follows a biphasic profile, with an "early" induction (15-30 min), followed by a nadir to control levels at 60 min, and a "late" induction (>120 min). The early phase of Rel A.NF-kappaB1 translocation depends on simultaneous proteolysis of both IkappaBalpha and IkappaBbeta isoforms; IkappaBgamma is inert to TNFalpha treatment. The 60-min nadir is due to a rapid IkappaBalpha resynthesis that reassociates with Rel A and completely inhibits its DNA binding activity; the 60-min nadir is not observed when IkappaBalpha resynthesis is prevented by cycloheximide treatment. By contrast, selective inhibition of IkappaBbeta proteolysis by pretreatment of HepG2 cells with the peptide aldehyde N-acetyl-Leu-Leu-norleucinal completely blocks the late phase of Rel A.NF-kappaB1 translocation. These studies indicate the presence of inducible and constitutive cytoplasmic NF-kappaB pools in hepatocytes. TNFalpha induces a coordinated proteolysis and resynthesis of IkappaB isoforms to produce dynamic changes in NF-kappaB nuclear abundance.