Altered subcellular distribution of MSK1 induced by glucocorticoids contributes to NF-kappaB inhibition.
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ABSTRACT:
SUBMITTER: Beck IM
PROVIDER: S-EPMC2435130 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Acting via the glucocorticoid receptor (GR), glucocorticoids exert potent anti-inflammatory effects partly by repressing inflammatory gene transcription occurring via factors such as NF-kappaB. In the present study, the synthetic glucocorticoid, dexamethasone, induces expression of MKP-1 (mitogen-activated protein kinase (MAPK) phosphatase-1) in human bronchial epithelial (BEAS-2B) and pulmonary (A549) cells. This correlates with reduced TNFalpha-stimulated p38 MAPK phosphorylation. Since NF-kappaB-dependent transcription and IL-8 protein, mRNA, and unspliced RNA (a surrogate of transcription rate) are sensitive to p38 MAPK inhibitors (SB203580 and SB239063), we explored the role of MKP-1 in repression of these outputs. Repression of TNFalpha-induced p38 MAPK phosphorylation, NF-kappaB-dependent transcription, and IL-8 expression by dexamethasone are sensitive to transcriptional or translational inhibitors. This indicates a role for de novo gene synthesis. Adenoviral expression of MKP-1 profoundly reduces p38 MAPK phosphorylation and IL-8 expression. Similarly, NF-kappaB-dependent transcription is significantly reduced to levels consistent with maximal p38 MAPK inhibition. Thus, MKP-1 attenuates TNFalpha-dependent activation of p38 MAPK, induction of IL-8 expression, and NF-kappaB-dependent transcription. Small interfering RNA knockdown of dexamethasone-induced MKP-1 expression partially reverses the repression of TNFalpha-activated p38 MAPK, demonstrating that MKP-1 participates in the dexamethasone-dependent repression of this pathway. In the presence of MKK6 (MAPK kinase 6), a p38 MAPK activator, dexamethasone dramatically represses TNFalpha-induced NF-kappaB-dependent transcription, and this is significantly reversed by MKP-1-targeting small interfering RNA. This reveals an important and novel role for transcriptional activation (transactivation) of MKP-1 in the repression of NF-kappaB-dependent transcription by glucocorticoids. We conclude that GR transactivation is essential to the anti-inflammatory properties of GR ligands.
| S-EPMC2785369 | biostudies-literature
Project description:Tumor necrosis factor-alpha (TNF-alpha) is a cytokine that has an important role in immunity and inflammation by inducing cellular responses such as apoptosis. The transcription factor nuclear factor-kappaB (NF-kappaB) can paradoxically suppress and promote apoptosis in response to TNF-alpha. In this study, we found that p53 upregulated modulator of apoptosis (PUMA), a p53 downstream target and a BH3-only Bcl-2 family member, is directly regulated by NF-kappaB in response to TNF-alpha. TNF-alpha treatment led to increases in PUMA mRNA and protein levels in human colon cancer cells. The induction of PUMA was p53 independent, and mediated by the p65 component of NF-kappaB through a kappaB site in the PUMA promoter. The apoptotic effect of PUMA induction by TNF-alpha was unmasked by depleting the antiapoptotic protein Bcl-X(L). In mice, PUMA was also induced by TNF-alpha in an NF-kappaB-dependent manner. TNF-alpha-induced apoptosis in a variety of tissues and cell types, including small intestinal epithelial cells, hepatocytes, and thymocytes, was markedly reduced in PUMA-deficient mice. Collectively, these results demonstrated that PUMA is a direct target of NF-kappaB and mediates TNF-alpha-induced apoptosis in vitro and in vivo.
| S-EPMC2872087 | biostudies-literature
Project description:Nuclear factor kappaB (NF-kappaB) is one of the key regulators of transcription of a variety of genes involved in immune and inflammatory responses. NF-kappaB activity has long been thought to be regulated mainly by IkappaB family members, which keep the transcription factor complex in an inactive form in the cytoplasm by masking the nuclear localization signal. Nowadays, the importance of additional mechanisms controlling the nuclear transcription potential of NF-kappaB is generally accepted. We show that the mitogen-activated protein kinase inhibitors SB203580 and PD98059 or U0126, as well as a potent mitogen- and stress- activated protein kinase-1 (MSK1) inhibitor H89, counteract tumor necrosis factor (TNF)-mediated stimulation of p65 transactivation capacity. Mutational analysis of p65 revealed Ser276 as a target for phosphorylation and transactivation in response to TNF. Moreover, we identified MSK1 as a nuclear kinase for p65, since MSK1 associates with p65 in a stimulus-dependent way and phosphorylates p65 at Ser276. This effect represents, together with phosphorylation of nucleosome components such as histone H3, an essential step leading to selective transcriptional activation of NF-kappaB-dependent gene expression.
| S-EPMC151081 | biostudies-literature
Project description:AimsInflammatory molecules and their transcription factor, nuclear factor kappa-B (NF-kappaB), are thought to play important roles in diabetes-induced cardiac dysfunction. Here, we investigated the effects of pyrrolidine dithiocarbamate (PDTC), a NF-kappaB inhibitor, in diabetic mice.Methods and resultsObese db/db mice and heterozygous lean mice (n = 8) were allowed free access to drinking water (control) or water containing PDTC (100 mg/kg) for 20 weeks. Left ventricular (LV) function was measured using echocardiography at baseline and at study end. Mice were sacrificed and LV removed for gene expression, biochemical, immunofluorescence, and mitochondrial assays. LV and mitochondrial reactive oxygen species (ROS), superoxide and peroxynitrite were measured using electron spin resonance spectroscopy. Enhanced NF-kappaB activity in db/db mice was associated with increased oxidative stress as demonstrated by increased ROS, superoxide, and peroxynitrite production, and increased NF-kappaB, gp91phox, and Nox1 expression; PDTC ameliorated these effects. Mitochondrial free radical production and structural damage were higher in the db/db group than in the control, db/db PDTC, and PDTC-treated heterozygous animal groups.ConclusionThis study demonstrates that NF-kappaB blockade with PDTC mitigates oxidative stress and improves mitochondrial structural integrity directly, through down-regulation of increased oxygen-free radicals, thereby increasing ATP synthesis and thus restoring cardiac function in type II diabetes.
| S-EPMC2860708 | biostudies-literature
Project description:We have mapped the chromosomal binding site distribution of a transcription factor in human cells. The NF-kappaB family of transcription factors plays an essential role in regulating the induction of genes involved in several physiological processes, including apoptosis, immunity, and inflammation. The binding sites of the NF-kappaB family member p65 were determined by using chromatin immunoprecipitation and a genomic microarray of human chromosome 22 DNA. Sites of binding were observed along the entire chromosome in both coding and noncoding regions, with an enrichment at the 5' end of genes. Strikingly, a significant proportion of binding was seen in intronic regions, demonstrating that transcription factor binding is not restricted to promoter regions. NF-kappaB binding was also found at genes whose expression was regulated by tumor necrosis factor alpha, a known inducer of NF-kappaB-dependent gene expression, as well as adjacent to genes whose expression is not affected by tumor necrosis factor alpha. Many of these latter genes are either known to be activated by NF-kappaB under other conditions or are consistent with NF-kappaB's role in the immune and apoptotic responses. Our results suggest that binding is not restricted to promoter regions and that NF-kappaB binding occurs at a significant number of genes whose expression is not altered, thereby suggesting that binding alone is not sufficient for gene activation.
| S-EPMC218744 | biostudies-literature
Project description:BackgroundSilicosis is a complex lung disease for which no successful treatment is available and therefore lung transplantation is a potential alternative. Tumor necrosis factor alpha (TNFalpha) plays a central role in the pathogenesis of silicosis. TNFalpha signaling is mediated by the transcription factor, Nuclear Factor (NF)-kappaB, which regulates genes controlling several physiological processes including the innate immune responses, cell death, and inflammation. Therefore, inhibition of NF-kappaB activation represents a potential therapeutic strategy for silicosis.Methods/findingsIn the present work we evaluated the lung transplant database (May 1986-July 2007) at the University of Pittsburgh to study the efficacy of lung transplantation in patients with silicosis (n = 11). We contrasted the overall survival and rate of graft rejection in these patients to that of patients with idiopathic pulmonary fibrosis (IPF, n = 79) that was selected as a control group because survival benefit of lung transplantation has been identified for these patients. At the time of lung transplantation, we found the lungs of silica-exposed subjects to contain multiple foci of inflammatory cells and silicotic nodules with proximal TNFalpha expressing macrophage and NF-kappaB activation in epithelial cells. Patients with silicosis had poor survival (median survival 2.4 yr; confidence interval (CI): 0.16-7.88 yr) compared to IPF patients (5.3 yr; CI: 2.8-15 yr; p = 0.07), and experienced early rejection of their lung grafts (0.9 yr; CI: 0.22-0.9 yr) following lung transplantation (2.4 yr; CI:1.5-3.6 yr; p<0.05). Using a mouse experimental model in which the endotracheal instillation of silica reproduces the silica-induced lung injury observed in humans we found that systemic inhibition of NF-kappaB activation with a pharmacologic inhibitor (BAY 11-7085) of IkappaB alpha phosphorylation decreased silica-induced inflammation and collagen deposition. In contrast, transgenic mice expressing a dominant negative IkappaB alpha mutant protein under the control of epithelial cell specific promoters demonstrate enhanced apoptosis and collagen deposition in their lungs in response to silica.ConclusionsAlthough limited by its size, our data support that patients with silicosis appear to have poor outcome following lung transplantation. Experimental data indicate that while the systemic inhibition of NF-kappaB protects from silica-induced lung injury, epithelial cell specific NF-kappaB inhibition appears to aggravate the outcome of experimental silicosis.
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Project description:c-Abl is a non-receptor tyrosine kinase that participates in multiple signaling pathways linking the cell surface, cytoskeleton, and the nucleus. Recent in vitro studies have also linked c-Abl to amyloid-beta-induced toxicity and tau phosphorylation. To further characterize a potential role of c-Abl in Alzheimer's disease (AD), we examined the expression and distribution of total and phosphorylated forms of c-Abl in the hippocampus of AD and control subjects. Laser scanning confocal microscopy was used to examine the colocalization of c-Abl with AD pathology. Our results demonstrate alterations in the presence and distribution of c-Abl and phosphorylated isoforms of c-Abl within the hippocampus during AD. Total unphosphorylated c-Abl was highest in non-demented control hippocampus. Activated isoforms of c-Abl were most abundant in AD hippocampus and co-localized with AD pathology, including granulovacuolar degeneration bodies, c-Abl interacts with phosphorylated tau in AD brain and may contribute to the formation of tau pathology. These studies demonstrate altered activation and distribution of c-Abl during AD, suggesting a role for c-Abl in Abeta signal transduction and generation of tau pathology in AD.
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Project description:Optineurin is a ubiquitously expressed multifunctional cytoplasmic protein encoded by OPTN gene. The expression of optineurin is induced by various cytokines. Here we have investigated the molecular mechanisms which regulate optineurin gene expression and the relationship between optineurin and nuclear factor kappaB (NF-kappaB). We cloned and characterized human optineurin promoter. Optineurin promoter was activated upon treatment of HeLa and A549 cells with tumor necrosis factor alpha (TNFalpha). Mutation of a putative NF-kappaB-binding site present in the core promoter resulted in loss of basal as well as TNFalpha-induced activity. Overexpression of p65 subunit of NF-kappaB activated this promoter through NF-kappaB site. Oligonucleotides corresponding to this putative NF-kappaB-binding site showed binding to NF-kappaB. TNFalpha-induced optineurin promoter activity was inhibited by expression of inhibitor of NF-kappaB (IkappaBalpha) super-repressor. Blocking of NF-kappaB activation resulted in inhibition of TNFalpha-induced optineurin gene expression. Overexpressed optineurin partly inhibited TNFalpha-induced NF-kappaB activation in Hela cells. Downregulation of optineurin by shRNA resulted in an increase in TNFalpha-induced as well as basal NF-kappaB activity. These results show that optineurin promoter activity and gene expression are regulated by NF-kappaB pathway in response to TNFalpha. In addition these results suggest that there is a negative feedback loop in which TNFalpha-induced NF-kappaB activity mediates expression of optineurin, which itself functions as a negative regulator of NF-kappaB.
| S-EPMC2660438 | biostudies-literature
Project description:The ability of the transcription factor NF-kappaB to upregulate anti-apoptotic proteins has been linked to the chemoresistance of solid tumors to standard chemotherapy. In contrast, recent studies have proposed that, in response to doxorubicin, NF-kappaB can be pro-apoptotic through repression of anti-apoptotic target genes. However, there is little evidence analyzing the outcome of NF-kappaB inhibition on the cytotoxicity of doxorubicin in studies describing pro-apoptotic NF-kappaB activity. In this study, we further characterize the activation of NF-kappaB in response to doxorubicin and evaluate its role in chemotherapy-induced cell death in sarcoma cells where NF-kappaB is reported to be pro-apoptotic. Doxorubicin treatment in U2OS cells induced canonical NF-kappaB activity as evidenced by increased nuclear accumulation of phosphorylated p65 at serine 536 and increased DNA-binding activity. Co-treatment with a small molecule IKKbeta inhibitor, Compound A, abrogated this response. RT-PCR evaluation of anti-apoptotic gene expression revealed that doxorubicin-induced transcription of cIAP2 was inhibited by Compound A, while doxorubicin-induced repression of other anti-apoptotic genes was unaffected by Compound A or siRNA to p65. Furthermore, the combination of doxorubicin and canonical NF-kappaB inhibition with Compound A or siRNA to p65 resulted in decreased cell viability measured by trypan blue staining and MTS assay and increased apoptosis measured by cleaved poly (ADP-ribose) polymerase and cleaved caspase 3 when compared to doxorubicin alone. Our results demonstrate that doxorubicin-induced canonical NF-kappaB activity associated with phosphorylated p65 is anti-apoptotic in its function and that doxorubicin-induced repression of anti-apoptotic genes occurs independent of p65. Therefore, combination therapies incorporating NF-kappaB inhibitors together with standard chemotherapies remains a viable method to improve the clinical outcomes in patients with advanced stage malignancies.
| S-EPMC2734988 | biostudies-literature
Project description:Proteasome inhibition has become synonymous with inhibition of NF-kappaB activity. However, hyperactive NF-kappaB responses often accompany physiological conditions marked by proteasomal defects, i.e. advancing age, geriatric diseases, and bortezomib resistance. These paradoxical NF-kappaB responses are likely to be impervious to proteasomal defects because they stem from atypical NF-kappaB signaling induced by upstream mechanisms which are proteasome-independent. While this atypical pathway does not require proteasome for NF-kappaB nuclear translocation, a role for proteasome in regulating nuclear NF-kappaB remains unexplored. We now demonstrate that proteasome stringently controls transcription of inflammatory mediators regulated by this atypical NF-kappaB pathway. Proteolytic activity of the proteasome mediates the removal of the NF-kappaB subunit, p65/RelA, from inflammatory genes, thereby terminating atypical NF-kappaB-dependent transcriptional responses. For the first time, we demonstrate that both 19S and 20S components of the 26S proteasome complex are recruited to an inflammatory gene promoter; additionally, the 19S and 20S complexes appear to play distinct roles in the negative regulation of NF-kappaB-dependent transcription. By demonstrating that proteasome regulates the termination of atypical NF-kappaB-dependent transcriptional responses, these studies clearly indicate a novel, regulatory role for proteasome in atypical NF-kappaB signaling. Moreover, these results signal a potential interplay between lowered proteasomal function and increased inflammation and may explain why inflammation accompanies physiological conditions under which proteasomal function is compromised, such as during advancing age or following bortezomib treatment. Given this role for proteasome in inflammation resolution, restoration of proteasome function may constitute a novel mechanism for intervening in chronic inflammatory diseases.
| S-EPMC2812621 | biostudies-literature