NF-kappaB Activation In Cells Research Articles

Vanadate Induction of NF-κB Involves IκB Kinase β and SAPK/ERK Kinase 1 in Macrophages

The present studies investigated the signaling pathways of vanadate, a vanadium ion with +5 oxidation state, to activate NF-kappaB transcription factor, a pivotal regulator of inflammatory responses. Treatment of macrophages with vanadate results in the activation of both NF-kappaB and c-Jun N-terminal kinase (JNK). The activity of a recently identified cellular kinase, IkappaB kinase-beta (IKKbeta), was significantly elevated concomitant with the increased degradation of IkappaBalpha and enhanced NF-kappaB activity in cells exposed to vanadate. To determine whether the IKK pathway and JNK pathway are interconnected or bifurcate upon vanadate stimulation, cells were transfected with either a kinase inactive form of IKKbeta or a kinase inactive form of SAPK/ERK kinase 1 (SEK1). Inactive IKKbeta was able to block vanadate-induced degradation of IkappaBalpha, yet it was unable to influence the activation of JNK by vanadate. Conversely, blockage of JNK activation by transfection of a kinase-inactive form of SEK1 resulted in partially inhibition of vanadate-induced IkappaBalpha degradation. Both vanadate-induced degradation of IkappaBalpha and activation of JNK were potently inhibited by pretreatment of cells with N-acetylcysteine or dimercaprol. These results demonstrate that early activation of stress kinases or change of cellular redox states plays a key role in vanadate-induced activation of NF-kappaB and JNK.

Mutations in the IkBa gene in Hodgkin's disease suggest a tumour suppressor role for IkappaBalpha.

The NF-kappaB/Rel family of transcription factors regulates a wide variety of genes whose products play a fundamental role in inflammatory and immune responses. The implication of NF-kappaB/Rel proteins and their IkappaB regulatory subunits in the control of cellular growth and oncogenesis, was suggested by the induction of fatal lymphomas in birds by the v-rel oncoprotein, and the rearrangement and amplification of several genes encoding the NF-kappaB/Rel/IkappaB signal transduction factors in human malignancies, primarily of lymphoid origin. Hodgkin's disease (HD) is a lymphoma characterized by a low frequency of malignant Hodgkin and Reed-Sternberg (H/RS) cells in a reactive background of non-neoplastic cells. The peculiar activated phenotype of Hodgkin and Reed-Sternberg cells and their pattern of cytokine secretion are believed to be a consequence of constitutive activation of the NF-kappaB transcription factor. Here, we report the detection of mutations of the IkBa gene, in two HD-derived cell lines and in two out of eight biopsy samples from patients with relapsed Hodgkin's disease. The presence of defective IkappaBalpha is thus likely to explain the constitutive activation of NF-kappaB in these cells and suggests that IkappaBalpha is a tumour suppressor controlling the oncogenic activation of NF-kappaB in Hodgkin and Reed-Sternberg cells.

Acrolein Causes Inhibitor κB-independent Decreases in Nuclear Factor κB Activation in Human Lung Adenocarcinoma (A549) Cells

Acrolein is a highly electrophilic alpha,beta-unsaturated aldehyde to which humans are exposed in various situations. In the present study, the effects of sublethal doses of acrolein on nuclear factor kappaB (NF-kappaB) activation in A549 human lung adenocarcinoma cells were investigated. Immediately following a 30-min exposure to 45 fmol of acrolein/cell, glutathione (GSH) and DNA synthesis and NF-kappaB binding were reduced by more than 80%. All parameters returned to normal or supranormal levels by 8 h post-treatment. Pretreatment with acrolein completely blocked 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced activation of NF-kappaB. Cells treated for 1 h with 1 mM diethyl maleate (DEM) showed a 34 and 53% decrease in GSH and DNA synthesis, respectively. DEM also reduced NF-kappaB activation by 64% at 2 h post-treatment, with recovery to within 22% of control at 8 h. Both acrolein and DEM decreased NF-kappaB function approximately 50% at 2 h after treatment with TPA, as shown by a secreted alkaline phosphatase reporter assay. GSH returned to control levels by 8 h after DEM treatment, but proliferation remained significantly depressed for 24 h. Interestingly, DEM caused a profound decrease in NF-kappaB binding, even at doses as low as 0.125 mM that had little effect on GSH. Neither acrolein nor DEM had any effect on the levels of phosphorylated or nonphosphorylated inhibitor kappaB-alpha (IkappaB-alpha). Furthermore, acrolein decreased NF-kappaB activation in cells depleted of IkappaB-alpha by TPA stimulation in the presence of cycloheximide, demonstrating that the decrease in NF-kappaB activation was not the result of increased binding by the inhibitory protein. This conclusion was further supported by the finding that acrolein modified NF-kappaB in the cytosol prior to chemical dissociation from IkappaB with detergent. Together, these data support the conclusion that the inhibition of NF-kappaB activation by acrolein and DEM is IkappaB-independent. The mechanism appears to be related to direct modification of thiol groups in the NF-kappaB subunits.

Hyperglycemia-induced activation of nuclear transcription factor kappaB in vascular smooth muscle cells.

The transcriptional nuclear factor (NF)-kappaB can be activated by diverse stimuli such as cytokines, mitogens, oxidative stress, and lipids, leading to the transactivation of several genes that play important roles in the development of atherosclerosis. Because oxidative stress may play a key role in the pathogenesis of diabetic vascular disease, we have examined whether culture of porcine vascular smooth muscle cells (PVSMCs) under high glucose (HG) conditions (25 mmol/l) to simulate the diabetic state can lead to the activation of NF-kappaB, and also whether cytokine- or growth factor-induced NF-kappaB activation is altered by HG culture. We observed that PVSMCs cultured in HG showed significantly greater activation of NF-kappaB in the basal state compared with cells cultured in normal glucose (NG) (5.5 mmol/l). Treatment of the cells with cytokines, such as tumor necrosis factor (TNF)-alpha and interleukin-1beta, or with growth factors, such as platelet-derived growth factor, insulin-like growth factor-I, and epidermal growth factor, all led to NF-kappaB activation in cells cultured in both NG and HG. However, their effects were markedly greater in HG. The augmented TNF-alpha-induced NF-kappaB activation in HG was associated with increased TNF-alpha-mediated transcriptional activation of the vascular cell adhesion molecule-1 promoter. Immunoblotting with an antibody to the p65 subunit of NF-kappaB indicated that the levels of this protein were higher in the nuclear extracts from cells cultured in HG compared with NG. Cells cultured in HG also produced significantly greater amounts of the reactive oxygen species superoxide. HG-induced NF-kappaB activation was inhibited by a protein kinase C inhibitor, calphostin C. These results suggest that hyperglycemia-induced activation of NF-kappaB in VSMCs may be a key mechanism for the accelerated vascular disease observed in diabetes.

The role of hydroxyl radical as a messenger in the activation of nuclear transcription factor NF-kappaB.

Although it is generally believed that reactive oxygen species activate NF-kappaB, a primary oxidative stress-responsive transcription factor, it is unclear which one among these species causes NF-kappaB activation. Our hypothesis is that hydroxyl radical (*OH) functions as a messenger for the activation of NF-kappaB. Jurkat cells, macrophages and JB6 cells were used to test this hypothesis. Cr(VI), silica and ZnO were used as sources of *OH radicals. None of these *OH generating systems involves exogenous H2O2. Cr(VI) expressed enhanced activity in induction of NF-kappaB in Jurkat cells. This activation of NF-kappaB was decreased by a metal chelator, diethylene triaminepentaacetic acid or a H2O2 scavenger, catalase, but was increased by superoxide dismutase. Mn(II), which reacts with Cr(IV) to inhibit this metal ion-mediated *OH generation, decreased the NF-kappaB activation. Sodium formate, an *OH radical scavenger, also inhibited the NF-kappaB activation. Electron spin resonance measurements show that Cr(VI) was reduced by Jurket cells to Cr(IV) and Cr(V). During the reduction process, molecular oxygen was reduced to O2 and then to H2O2, which reacted with Cr(IV) and Cr(V) to generate *OH radical. The *OH generation correlated with the Cr(VI)-induced NF-kappaB activation. Similarly, silica caused NF-kappaB activation in macrophages via the *OH radical-mediated reaction. This radical was generated via metal mediated reaction from H2O2, which was generated by the reduction of molecular oxygen via O2- as an intermediate during the silica-stimulated 'respirable burst'. Silica particles did not cause *OH generation either in Jurket or in JB6 cells and thus did not cause any observable NF-kappaB activation in these cells. ZnO induced NF-kappaB activation in JB6 cells through the generation of *OH resulting from light irradiation of ZnO which was measured by electron spin resonance. The results thus show that *OH radical functions as a messenger for NF-kappaB activation. Antioxidants, which scavenge *OH radical or its precursors, inhibit NF-kappaB activation. Metal chelators, which make metal ions incapable of generating *OH from H2O2, inhibit activation of this transcription factor.

Secreted β-Amyloid Precursor Protein Counteracts the Proapoptotic Action of Mutant Presenilin-1 by Activation of NF-κB and Stabilization of Calcium Homeostasis

Mutations in the presenilin-1 (PS-1) gene account for approximately 50% of the cases of autosomal dominant, early onset, inherited forms of Alzheimer's disease (AD). PS-1 is an integral membrane protein expressed in neurons and is localized primarily in the endoplasmic reticulum (ER). PS-1 mutations may promote neuronal degeneration by altering the processing of the beta-amyloid precursor protein (APP) and/or by engaging apoptotic pathways. Alternative processing of APP in AD may increase production of neurotoxic amyloid beta-peptide (Abeta) and reduce production of the neuroprotective alpha-secretase-derived form of APP (sAPPalpha). In differentiated PC12 cells expressing an AD-linked PS-1 mutation (L286V), sAPPalpha activated the transcription factor NF-kappaB and prevented apoptosis induced by Abeta. Treatment of cells with kappaB decoy DNA blocked the antiapoptotic action of sAPPalpha, demonstrating the requirement for NF-kappaB activation in the cytoprotective action of sAPPalpha. Cells expressing mutant PS-1 exhibited an aberrant pattern of NF-kappaB activity following exposure to Abeta, which was characterized by enhanced early activation of NF-kappaB followed by a prolonged depression of activity. Blockade of NF-kappaB activity in cells expressing mutant PS-1 by kappaB decoy DNA was associated with enhanced Abeta-induced increases of [Ca2+]i and mitochondrial dysfunction. Treatment of cells with sAPPalpha stabilized [Ca2+]i and mitochondrial function and suppressed oxidative stress by a mechanism involving activation of NF-kappaB. Blockade of ER calcium release prevented (and stimulation of ER calcium release by thapsigargin induced) apoptosis in cells expressing mutant PS-1, suggesting a pivotal role for ER calcium release in the proapoptotic action of mutant PS-1. Finally, a role for NF-kappaB in preventing apoptosis induced by ER calcium release was demonstrated by data showing that sAPPalpha prevents thapsigargin-induced apoptosis, an effect blocked by kappaB decoy DNA. We conclude that sAPPalpha stabilizes cellular calcium homeostasis and protects neural cells against the proapoptotic action of mutant PS-1 by a mechanism involving activation of NF-kappaB. The data further suggest that PS-1 mutations result in aberrant NF-kappaB regulation that may render neurons vulnerable to apoptosis.