Effect of S534A substitution in the p65 subunit of Nfkb on response to 1mg LPS
Ontology highlight
ABSTRACT:
INSTRUMENT(S): [MoGene-1_0-st] Affymetrix Mouse Gene 1.0 ST Array [transcript (gene) version]
SUBMITTER: 
Robert F Schwabe
PROVIDER: GSE67178 | GEO | 2017-04-05
SECONDARY ACCESSION(S): PRJNA279183
REPOSITORIES: GEO
Dataset's files
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Publications
Negative regulation of NF-κB p65 activity by serine 536 phosphorylation.
Pradère Jean-Philippe JP Hernandez Céline C Koppe Christiane C Friedman Richard A RA Luedde Tom T Schwabe Robert F RF
Science signaling 20160823 442
Nuclear factor κB (NF-κB) is a master regulator of inflammation and cell death. Whereas most of the activity of NF-κB is regulated through the inhibitor of κB (IκB) kinase (IKK)-dependent degradation of IκB, IKK also phosphorylates subunits of NF-κB. We investigated the contribution of the phosphorylation of the NF-κB subunit p65 at the IKK phosphorylation site serine 536 (Ser(536)) in humans, which is thought to be required for the activation and nuclear translocation of NF-κB. Through experime ...[more]
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Project description:WT mice and Nfkb/p65 S534A were exposed to 1µg/kg LPS and their gene expression measured. Affymetrix Mouse 1.0ST chips were used to measure gene expression Overall design: Gene expression of the following mouse livers were characterized WT untreated (n=4). S534A treated with 1µg/kg LPS (n=4). WT treated with 1µg/kg LPS (n=4).
2017-04-05 | GSE67072 | GEO
Project description:It is increasingly recognized that vitamin D3 (VitD3) has an anti-inflammatory activity. The present study investigated the effects of maternal VitD3 supplementation during pregnancy on LPS-induced placental inflammation and fetal intrauterine growth restriction (IUGR). All pregnant mice except controls were intraperitoneally injected with LPS (100 μg/kg) daily from gestational day (GD)15-17. In VitD3 + LPS group, pregnant mice were orally administered with VitD3 (25 μg/kg) before LPS injection. As expected, maternal LPS exposure caused placental inflammation and fetal IUGR. Interestingly, pretreatment with VitD3 repressed placental inflammation and protected against LPS-induced fetal IUGR. Further analysis showed that pretreatment with VitD3, which activated placental vitamin D receptor (VDR) signaling, specifically suppressed LPS-induced activation of nuclear factor kappa B (NF-κB) and significantly blocked nuclear translocation of NF-κB p65 subunit in trophoblast gaint cells of the labyrinth layer. Conversely, LPS, which activated placental NF-κB signaling, suppressed placental VDR activation and its target gene expression. Moreover, VitD3 reinforced physical interaction between placental VDR and NF-κB p65 subunit. The further study demonstrates that VitD3 inhibits placental NF-κB signaling in VDR-dependent manner. These results provide a mechanistic explanation for VitD3-mediated anti-inflammatory activity. Overall, the present study provides evidence for roles of VDR as a key regulator of placental inflammation.
2015-01-01 | S-EPMC4464284 | BioStudies
Project description:BACKGROUND:It has been confirmed that NF-?B p65 signaling pathway is involved in the regulation of alveolar hypercoagulation and fibrinolysis inhibition in acute respiratory distress syndrome (ARDS). Whether SN50, a NF-?B cell permeable inhibitor, could attenuate alveolar hypercoagulation and fibrinolysis inhibition in ARDS remains to be elucidated. PURPOSE:We explored the efficacy and potential mechanism of SN50 on alveolar hypercoagulation and fibrinolysis inhibition in ARDS in mice. MATERIALS AND METHODS:Mouse ARDS was made by 50??l of lipopolysaccharide (LPS) (4?mg/ml) inhalation. Male BALB/c mice were intraperitoneally injected with different does of SN50 1?h before LPS inhalation. Lung tissues were collected for hematoxylin-eosin (HE) staining, wet/dry ratio. Pulmonary expressions of tissue factor (TF), plasminogen activator inhibitor-1 (PAI-1), collagen III, as well as phosphorylated p65 (p-p65), p65 in nucleus (p'-p65), I?B? and IKK?/? were measured. Bronchoalveolar lavage fluid (BALF) was gathered to test the concentrations of TF, PAI-1, activated protein C (APC) and thrombinantithrombin complex (TAT). DNA binding activity of NF-?B p65 was also determined. RESULTS:After LPS stimulation, pulmonary edema and exudation and alveolar collapse occured. LPS also stimulated higher expressions of TF and PAI-1 in lung tissues, and higher secretions of TF, PAI-1, TAT and low level of APC in BALF. Pulmonary collagen III expression was obviously enhanced after LPS inhalation. At same time, NF-?B signaling pathway was activated with LPS injury, shown by higher expressions of p-p65, p'-p65, p-IKK?/?, p-I?? in pulmonary tissue and higher level p65 DNA binding activity. SN50 dose-dependently inhibited TF, PAI-1 and collagen IIIexpressions, and decreased TF, PAI-1, TAT but increased APC in BALF. SN50 treatment attenuated pulmonary edema, exudation and reduced lung tissue damage as well. SN50 application significantly reduced p'-p65 expression and weakened p65 DNA binding activity, but expressions of p-p65, p-IKK?/?, p-I?? in cytoplasm of pulmonary tissue were not affected. CONCLUSIONS:SN 50 attenuates alveolar hypercoagulation and fibrinolysis inhibition in ARDS via inhibition of NF-?B p65 translocation. Our data demonstrates that NF-?B p65 pathway is a viable new therapeutic target for ARDS treatment.
2020-01-01 | S-EPMC7251840 | BioStudies
Project description:Compared to adults, neonates are at increased risk of infection. There is a growing recognition that dynamic qualitative and quantitative differences in immunity over development contribute to these observations. The liver plays a key role as an immunologic organ, but whether its contribution to the acute innate immune response changes over lifetime is unknown. We hypothesized that the liver would activate a developmentally-regulated acute innate immune response to intraperitoneal lipopolysaccharide (LPS). We first assessed the hepatic expression and activity of the NF-?B, a key regulator of the innate immune response, at different developmental ages (p0, p3, p7, p35, and adult). Ontogeny of the NF-?B subunits (p65/p50) revealed a reduction in Rela (p65) and Nfkb1 (p105, precursor to p50) gene expression (p0) and p65 subunit protein levels (p0 and p3) vs. older ages. The acute hepatic innate immune response to LPS was associated by the degradation of the NF-?B inhibitory proteins (I?B? and I?B?), and nuclear translocation of the NF-?B subunit p50 in all ages, whereas nuclear translocation of the NF-?B subunit p65 was only observed in the p35 and adult mouse. Consistent with these findings, we detected NF-?B subunit p65 nuclear staining exclusively in the LPS-exposed adult liver compared with p7 mouse. We next interrogated the LPS-induced hepatic expression of pro-inflammatory genes (Tnf, Icam1, Ccl3, and Traf1), and observed a gradually increase in gene expression starting from p0. Confirming our results, hepatic NF-?B subunit p65 nuclear translocation was associated with up-regulation of the Icam1 gene in the adult, and was not detected in the p7 mouse. Thus, an inflammatory challenge induces an NF-?B-mediated hepatic innate immune response activation across all developmental ages, but nuclear translocation of the NF-?B subunit p65 and associated induction of pro-inflammatory genes occurred only after the first month of life. Our results demonstrate that the LPS-induced hepatic innate immune response is developmentally regulated by the NF-?B subunit p65 in the mouse.
2020-01-01 | S-EPMC7472845 | BioStudies
Project description:Reactive oxygen species, produced by oxidative stress, initiate and promote many metabolic diseases through activation/suppression of redox-sensitive transcription factors. NF-?B and Nrf2 are important regulators of oxidation resistance and contribute to the pathogenesis of many diseases. We identified MafK, a novel transcriptional regulator that modulates NF-?B activity. MafK knockdown reduced NF-?B activation, whereas MafK overexpression enhanced NF-?B function. MafK mediated p65 acetylation by CBP upon LPS stimulation, thereby facilitating recruitment of p65 to NF-?B promoters such as IL-8 and TNF?. Consistent with these results, MafK-depleted mice showed prolonged survival with a reduced hepatic inflammatory response after LPS and D-GalN injection. Thus, our findings reveal a novel mechanism by which MafK controls NF-?B activity via CBP-mediated p65 acetylation.
1000-01-01 | S-EPMC3832860 | BioStudies
Project description:Nuclear factor ?B (NF-?B) is a master regulator of inflammation and cell death. Whereas most of the activity of NF-?B is regulated through the inhibitor of ?B (I?B) kinase (IKK)-dependent degradation of I?B, IKK also phosphorylates subunits of NF-?B. We investigated the contribution of the phosphorylation of the NF-?B subunit p65 at the IKK phosphorylation site serine 536 (Ser(536)) in humans, which is thought to be required for the activation and nuclear translocation of NF-?B. Through experiments with knock-in mice (S534A mice) expressing a mutant p65 with an alanine-to-serine substitution at position 534 (the murine homolog of human Ser(536)), we observed increased expression of NF-?B-dependent genes after injection of mice with the inflammatory stimulus lipopolysaccharide (LPS) or exposure to gamma irradiation, and the enhanced gene expression was most pronounced at late time points. Compared to wild-type mice, S534A mice displayed increased mortality after injection with LPS. Increased NF-?B signaling in the S534A mice was at least in part explained by the increased stability of the S534A p65 protein compared to that of the Ser(534)-phosphorylated wild-type protein. Together, our results suggest that Ser(534) phosphorylation of p65 in mice (and, by extension, Ser(536) phosphorylation of human p65) is not required for its nuclear translocation, but instead inhibits NF-?B signaling to prevent deleterious inflammation.
2016-01-01 | S-EPMC5327697 | BioStudies
Project description:BACKGROUND:Acute respiratory distress syndrome (ARDS) is a severe form of acute lung injury that has a high mortality rate and leads to substantial healthcare costs. MicroRNA-124-3p (miR-124-3p) helps to suppress inflammation during a pulmonary injury. However, its mechanism of action is largely unknown, and its role in ARDS remains to be determined. METHODS:Mice and NR8383 cells were exposed to lipopolysaccharides (LPS) to induce ARDS, and their miR-124-3p levels were determined. After a miRNA agomir was administrated to the mice, their pulmonary injuries were evaluated by H&E staining and assays for peripheral inflammatory cytokine levels. The direct interaction between miR-124-3p and p65 was predicted, and then confirmed by a luciferase activity assay. The role played by miRNA-124-3p in regulating p65 expression was further examined by transfection with its agomir, and its role in cell apoptosis was investigated by observing the effects of miRNA overexpression in vitro and in vivo. RESULTS:After exposure to LPS, there was a consistent decrease in miR-124-3p expression in the lungs of mice and in NR8383 cells. After treatment with the miR-124-3p agomir, the degrees of pulmonary injury (e.g. alveolar hemorrhage and interstitial edema), and the increases in IL-1?, IL-6, and TNF-? levels induced by LPS were significantly attenuated. Overexpression of miR-124-3p in NC8383 cells and lung tissues significantly suppressed LPS-induced p65 expression and cell apoptosis. CONCLUSIONS:These results suggest that miR-124-3p directly targeted p65, and thereby decreased the levels of inflammation and pulmonary injury in a mouse model of ARDS.
2020-01-01 | S-EPMC7253404 | BioStudies
Project description:Selective expression of dominant negative (DN) peroxisome proliferator-activated receptor ? (PPAR?) in vascular smooth muscle cells (SMC) results in hypertension, atherosclerosis, and increased nuclear factor-?B (NF-?B) target gene expression. Mesenteric SMC were cultured from mice designed to conditionally express wild-type (WT) or DN-PPAR? in response to Cre recombinase to determine how SMC PPAR? regulates expression of NF-?B target inflammatory genes. SMC-specific overexpression of WT-PPAR? or agonist-induced activation of endogenous PPAR? blunted tumor necrosis factor ? (TNF-?)-induced NF-?B target gene expression and activity of an NF-?B-responsive promoter. TNF-?-induced gene expression responses were enhanced by DN-PPAR? in SMC. Although expression of NF-?B p65 was unchanged, nuclear export of p65 was accelerated by WT-PPAR? and prevented by DN-PPAR? in SMC. Leptomycin B, a nuclear export inhibitor, blocked p65 nuclear export and inhibited the anti-inflammatory action of PPAR?. Consistent with a role in facilitating p65 nuclear export, WT-PPAR? coimmunoprecipitated with p65, and WT-PPAR? was also exported from the nucleus after TNF-? treatment. Conversely, DN-PPAR? does not bind to p65 and was retained in the nucleus after TNF-? treatment. Transgenic mice expressing WT-PPAR? or DN-PPAR? specifically in SMC (S-WT or S-DN) were bred with mice expressing luciferase controlled by an NF-?B-responsive promoter to assess effects on NF-?B activity in whole tissue. TNF-?-induced NF-?B activity was decreased in aorta and carotid artery from S-WT but was increased in vessels from S-DN mice. We conclude that SMC PPAR? blunts expression of proinflammatory genes by inhibition of NF-?B activity through a mechanism promoting nuclear export of p65, which is abolished by DN mutation in PPAR?.
2017-01-01 | S-EPMC5472224 | BioStudies
Project description:Milk fat globule-epidermal growth factor factor 8 (MFG-E8) regulates innate immune function by modulating cellular signaling, which is less understood. Herein, we aimed to investigate the direct anti-inflammatory role of MFG-E8 in macrophages by pre-treatment with recombinant murine MFG-E8 (rmMFG-E8) followed by stimulation with LPS in RAW264.7 cells and in peritoneal macrophages, isolated from wild-type (WT) or MFG-E8(-/-) mice. RAW264.7 cells and mouse peritoneal macrophages treated with rmMFG-E8 significantly downregulated LPS-induced TNF-α mRNA by 25% and 24%, and protein levels by 29% and 23%, respectively (P<0.05). Conversely, peritoneal macrophages isolated from MFG-E8(-/-) mice produced 28% higher levels of TNF-α, as compared to WT mice when treated with LPS. In in vivo, endotoxemia induced by intraperitoneal injection of LPS (5 mg/kg BW), at 4 h after induction, serum level of TNF-α was significantly higher in MFG-E8(-/-) mice (837 pg/mL) than that of WT (570 pg/mL, P<0.05). To elucidate the direct anti-inflammatory effect of MFG-E8, we examined STAT3 and its target gene, SOCS3. Treatment with rmMGF-E8 significantly induced pSTAT3 and SOCS3 in macrophages. Similar results were observed in in vivo treatment of rmMFG-E8 in peritoneal cells and splenic tissues. Pre-treatment with rmMFG-E8 significantly reduced LPS-induced NF-κB p65 contents. These data clearly indicated that rmMFG-E8 upregulated SOCS3 which in turn interacted with NF-κB p65, facilitating negative regulation of TLR4 signaling for LPS-induced TNF-α production. Our findings strongly suggest that MFG-E8 is a direct anti-inflammatory molecule, and that it could be developed as a therapy in attenuating inflammation and tissue injury.
2011-01-01 | S-EPMC3217009 | BioStudies
Project description:Background:Numerous studies have demonstrated that the inflammatory response is involved in the progression of lipopolysaccharide- (LPS-) induced myocardial cell apoptosis. Accumulating evidence has shown that thyroxine participates in diseases by downregulating the inflammatory response. This study aimed at investigating whether thyroxine alleviates LPS-induced myocardial cell apoptosis. Methods:Bone marrow-derived macrophages (Mø) were treated with LPS and thyroxine, and Mø differentiation and Mø-related cytokine expression were measured. The effect of Mø differentiation on mouse cardiomyocyte (MCM) apoptosis was also detected in vitro. In addition, C57BL/6 mice underwent thyroidectomy and were treated with LPS 35 days later; subsequently, Mø differentiation and myocardial cell apoptosis in hearts were analyzed. To determine whether the nuclear factor-kappa B (NF-?B) p65 pathway mediates the effect of thyroxine on Mø differentiation and myocardial cell apoptosis, the specific NF-?B p65 pathway inhibitor JSH-23 was administered to mice that underwent a thyroidectomy. Results:Levothyroxine treatment significantly reduced the activation of the NF-?B p65 pathway, decreased M1 macrophage (Mø1) differentiation and Mø1-related cytokine mRNA levels in LPS-treated Mø, and increased M2 macrophage (Mø2) differentiation and Mø2-related cytokine mRNA expression. The protective effects of levothyroxine on MCM apoptosis mediated by LPS-treated Mø were alleviated by JSH-23. In mice, thyroidectomy aggravated LPS-induced cardiac injury and cardiac dysfunction, further promoted NF-?B p65 activation, and increased cardiac Mø1 expression and myocardial cell apoptosis but decreased cardiac Mø2 expression. JSH-23 treatment significantly ameliorated the thyroidectomy-induced increases in myocardial cell apoptosis and Mø differentiation. Conclusions:Thyroxine alleviated the Mø1/Mø2 imbalance, reduced the inflammatory response, decreased myocardial cell apoptosis, and protected against cardiac injury and cardiac dysfunction in LPS-treated mice. Thyroxine may be a novel therapeutic strategy to prevent and treat LPS-induced cardiac injury.
2019-01-01 | S-EPMC6537024 | BioStudies