Inhibitor Of NF-κB Kinase Subunit Research Articles

Inhibitor of NF-κB Kinase Subunit ε Contributes to Neuropsychiatric Manifestations in Lupus-Prone Mice Through Microglial Activation.

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by multiorgan dysfunction. Neuropsychiatric SLE (NPSLE) occurs in 30-40% of lupus patients and is the most severe presentation of SLE, frequently resulting in limitation of daily life. Recent studies have shown that microglia, tissue-resident macrophages in the central nervous system, are involved in the pathogenesis of NPSLE. This study was undertaken to explore new therapeutic targets for NPSLE focusing on microglia. RNA sequencing of microglia in MRL/lpr, lupus-prone mice, as well as that of microglia cultured in vitro with cytokines were performed. A candidate gene, which could be a therapeutic target for NPSLE, was identified, and its role in microglial activation and phagocytosis was investigated using specific inhibitors and small interfering RNA. The effect of intracerebroventricular administration of the inhibitor on the behavioral abnormalities of MRL/lpr was also evaluated. Transcriptome analysis revealed the up-regulation of Ikbke, which encodes the inhibitor of NF-κB kinase subunit ɛ (IKBKε) in both microglia from MRL/lpr mice and cytokine-stimulated microglia in vitro. Intracerebroventricular administration of an IKBKε inhibitor ameliorated cognitive function and suppressed microglial activation in MRL/lpr mice. Mechanistically, IKBKε inhibition reduced glycolysis, which dampened microglial activation and phagocytosis. These findings suggest that IKBKε plays a vital role in the pathogenesis of NPSLE via microglial activation, and it could serve as a therapeutic target for NPSLE.

Salvianolic Acid B Reduces the Inflammation of Fat Grafts by Inhibiting the NF-Kb Signalling Pathway in Macrophages.

Autologous fat grafting is a common method for soft tissue defect repair. However, the high absorption rate of transplanted fat is currently a bottleneck in the process. Excessive inflammation is one of the main reasons for poor fat transplantation. Salvianolic acid B (Sal-B) is a herbal medicine that shows promise for improving the effectiveness of fat transplantation. The aim of this study was to improve fat graft survival by injecting Sal-B into fat grafts locally. In vivo, 0.2 mL of Coleman fat was transplanted into nude mice along with Sal-B. The grafts were evaluated by histologic analysis at 2, 4, and 12 weeks posttransplantation and by microcomputed tomography at 4 weeks posttransplantation. In vitro ribonucleic acid sequencing, cell proliferation assays, anti-inflammatory activity assays, molecular docking studies, and kinase activity assays were performed in RAW264.7 cells to detect the potential mechanism. Sal-B significantly improved fat graft survival and attenuated adipose tissue fibrosis and inflammation. Sal-B also inhibited the polarization of M1 macrophages in fat grafts. In vitro, Sal-B inhibited the proliferation and activation of inflammatory pathways in RAW264.7 cells. In addition, Sal-B had an inhibitory effect on NF-κB (nuclear factor κ light polypeptide gene enhancer in B cells) signaling. This bioactivity of Sal-B may result from its selective binding to the kinase domain of the inhibitor of NF-κB kinase subunit β. Sal-B could serve as a promising agent for improving the effect of fat transplantation by inhibiting the polarization of M1 macrophages through NF-κB signaling.

TRAF6 Phosphorylation Prevents Its Autophagic Degradation and Re-Shapes LPS-Triggered Signaling Networks.

Simple SummaryHere, we reveal that basal turnover and autophagy-induced decay of the ubiquitin E3 ligase TRAF6 is antagonized by IKKε-mediated phosphorylation at five serines. Phosphoproteomic experiments show that TRAF6 and its phosphorylation contribute to the remodeling of LPS- and autophagyinduced signaling networks, revealing an intricate link between inflammatory and metabolic processes that are frequently dysregulated in cancer.The ubiquitin E3 ligase TNF Receptor Associated Factor 6 (TRAF6) participates in a large number of different biological processes including innate immunity, differentiation and cell survival, raising the need to specify and shape the signaling output. Here, we identify a lipopolysaccharide (LPS)-dependent increase in TRAF6 association with the kinase IKKε (inhibitor of NF-κB kinase subunit ε) and IKKε-mediated TRAF6 phosphorylation at five residues. The reconstitution of TRAF6-deficient cells, with TRAF6 mutants representing phosphorylation-defective or phospho-mimetic TRAF6 variants, showed that the phospho-mimetic TRAF6 variant was largely protected from basal ubiquitin/proteasome-mediated degradation, and also from autophagy-mediated decay in autolysosomes induced by metabolic perturbation. In addition, phosphorylation of TRAF6 and its E3 ligase function differentially shape basal and LPS-triggered signaling networks, as revealed by phosphoproteome analysis. Changes in LPS-triggered phosphorylation networks of cells that had experienced autophagy are partially dependent on TRAF6 and its phosphorylation status, suggesting an involvement of this E3 ligase in the interplay between metabolic and inflammatory circuits.

Hepatic NF-κB-Inducing Kinase and Inhibitor of NF-κB Kinase Subunit α Promote Liver Oxidative Stress, Ferroptosis, and Liver Injury.

Drug‐induced hepatotoxicity limits development of new effective medications. Drugs and numerous endogenous/exogenous agents are metabolized/detoxified by hepatocytes, during which reactive oxygen species (ROS) are generated as a by‐product. ROS has broad adverse effects on liver function and integrity, including damaging hepatocyte proteins, lipids, and DNA and promoting liver inflammation and fibrosis. ROS in concert with iron overload drives ferroptosis. Hepatic nuclear factor kappa B (NF‐κB)‐inducing kinase (NIK) is aberrantly activated in a broad spectrum of liver disease. NIK phosphorylates and activates inhibitor of NF‐κB kinase subunit alpha (IKKα), and the hepatic NIK/IKKα cascade suppresses liver regeneration. However, the NIK/IKKα pathway has not been explored in drug‐induced liver injury. Here, we identify hepatic NIK as a previously unrecognized mediator for acetaminophen (APAP)‐induced acute liver failure. APAP treatment increased both NIK transcription and NIK protein stability in primary hepatocytes as well as in liver in mice. Hepatocyte‐specific overexpression of NIK augmented APAP‐induced liver oxidative stress in mice and increased hepatocyte death and mortality in a ROS‐dependent manner. Conversely, hepatocyte‐specific ablation of NIK or IKKα mitigated APAP‐elicited hepatotoxicity and mortality. NIK increased lipid peroxidation and cell death in APAP‐stimulated primary hepatocytes. Pretreatment with antioxidants or ferroptosis inhibitors blocked NIK/APAP‐induced hepatocyte death. Conclusion: We unravel a previously unrecognized NIK/IKKα/ROS/ferroptosis axis engaged in liver disease progression.

Investigating the impact of local inflammation on granulosa cells and follicular development in women with ovarian endometriosis

To investigate the possible impact of local inflammation on granulosa cells (GCs) and follicular development in endometriosis patients. Prospective study with related paired design. Reproductive medicine center. A total of 80 endometriosis patients and 104 controls, with cultured GCs collected from control participants younger than 35years. Tumor necrosis factor-α (TNF-α) and nuclear factor κB (NF-κB) inhibitor. Intrafollicular concentrations of cytokines measured with ELISA, NF-κB binding levels with electrophoretic mobility shift assay (EMSA), and telomerase activity (TA) with quantitative-telomeric repeat amplification protocol (Q-TRAP) assay, and protein and mRNA expression with Western blot and polymerase chain reaction analyses, respectively. Patients with endometriosis exhibited a statistically significantly lower antral follicle count (11.48 ± 8.11 vs. 15.68 ± 8.56), lower number of retrieved oocytes (8.28 ± 6.69 vs. 10.87 ± 6.26), and lower number of mature oocytes (6.67 ± 6.09 vs. 8.53 ± 5.69). The GCs from endometriosis patients showed higher NF-κB binding activity and increased expression of inhibitor of NF-κB kinase subunit β (IKKβ, 2.743-fold) and NF-κB inhibitor α (IκBα, 5.017-fold). Their NF-κB p65 expression was negatively associated with mature oocytes (bNF-κB' = -0.304, R2 = 0.195, R = 0.442) but positively associated with intrafollicular TNF-α (r = 0.37); TA showed a negative relationship with NF-κB binding levels (r = -0.667). Tumor necrosis factor-α induced expression of IκBα (5.408-fold) and NF-κB p65 (1.400-fold) but lowered human telomerase reverse transcriptase (hTERT) and TA levels (0.0009 vs. 0.5619) in cultured GCs. However, inhibiting NF-κB obviously increased hTERT expression (1.988-fold). Endometriosis showed activated NF-κB pathways in GCs, which might negatively affect TA and oocyte quality. Intrafollicular TNF-α might down-regulate TA and hTERT via NF-κB pathway, but further studies are required.

Prognostic values of inhibitory κB kinases mRNA expression in human gastric cancer.

Background: Inhibitory κB kinases (IKKs) play a key role in modulating proinflammatory and growth stimulating signals through their regulation of the nuclear factor κB (NF-κB) cascade. Therefore, the level of expression of IKKs represents a viable prognostic predictor with regard to various pathological processes. The prognostic value of IKKs expression in gastric cancer remains unclear. Methods: We used the ‘Kaplan–Meier plotter’ (KM plotter) online database, to explore the predictive prognostic value of individual IKKs members’ mRNA expression to overall survival (OS) in different clinical data including pathological staging, histology, and therapies employed. Results: Our results revealed that a higher mRNA expression of inhibitor of NF-κB kinase subunit α (IKKα) was correlated to better OS, whereas higher mRNA expression of IKKβ, inhibitor of NF-κB kinase subunit γ (IKKγ), inhibitor of NF-κB kinase subunit ε (IKKε), and suppressor of IKKε (SIKE) were generally correlated to unfavorable OS in gastric cancer. Increased mRNA expression of IKKε also showed better outcomes in stage IV gastric cancer. Further a correlation between elevated levels of mRNA expression of both IKKε and SIKE was found to have favorable OS in diffuse type gastric cancer. It was also revealed that high expression of SIKE had favorable OS when treated with other adjuvant therapies, while worse OS when treated only with 5FU therapy. Conclusion: Our results suggest that mRNA expression of individual IKKs and SIKE are associated with unique prognostic significance and may act as valuable prognostic biomarkers and potential targets for future therapeutic interventions in gastric cancer.

Simultaneous stimulation with tumor necrosis factor-α and transforming growth factor-β1 induces epithelial-mesenchymal transition in colon cancer cells via the NF-κB pathway.

Epithelial-mesenchymal transition (EMT) is critical in the progression of numerous types of carcinoma, and endows invasive and metastatic properties upon cancer cells. The tumor microenvironment facilitates tumor metastasis to distant organs. Various signaling pathways contribute to this process. In the present study, SW480 colon adenocarcinoma cells were treated with transforming growth factor-β1 (TGF-β1; 10 ng/ml) and tumor necrosis factor-α (TNF-α; 20 ng/ml), alone or in combination, for 72 h, and EMT was assessed using immunofluorescence, western blot analysis and migration assays. The functions of p38 mitogen-activated protein kinase, extracellular signal-regulated kinase (ERK) and nuclear factor-κB (NF-κB) pathways in EMT were examined. It was demonstrated that the cooperation of TGF-β1 and TNF-α signaling promoted the morphological conversion of the SW480 cells from an epithelial to a mesenchymal phenotype. Furthermore, simultaneous exposure to TNF-α and TGF-β1 downregulated the expression of E-cadherin (an epithelial marker) and increased the expression of N-cadherin and vimentin (mesenchymal markers). Additionally, the migratory capacity of the SW480 cells increased. The inhibition of p38 and ERK signaling exhibited no effect on EMT, whereas the inhibition of inhibitor of NF-κB kinase subunit β blocked the EMT induced by TGF-β1 and TNF-α. In conclusion, the results of the present study demonstrated that TNF-α and TGF-β1 synergistically promoted EMT in SW480 cells via the NF-κB pathway, independent of p38 activation and ERK1/2 signaling. These results suggest a novel function of TGF-β1 and TNF-α during EMT in colon carcinoma and, thus, provide insights into potential therapeutic interventions.

The anti-inflammatory activity of curcumin is mediated by its oxidative metabolites

The spice turmeric, with its active polyphenol curcumin, has been used as anti-inflammatory remedy in traditional Asian medicine for centuries. Many cellular targets of curcumin have been identified, but how such a wide range of targets can be affected by a single compound is unclear. Here, we identified curcumin as a pro-drug that requires oxidative activation into reactive metabolites to exert anti-inflammatory activities. Synthetic curcumin analogs that undergo oxidative transformation potently inhibited the pro-inflammatory transcription factor nuclear factor κB (NF-κB), whereas stable, non-oxidizable analogs were less active, with a correlation coefficient (R2) of IC50versus log of autoxidation rate of 0.75. Inhibition of glutathione biosynthesis, which protects cells from reactive metabolites, increased the potency of curcumin and decreased the amount of curcumin-glutathione adducts in cells. Oxidative metabolites of curcumin adducted to and inhibited the inhibitor of NF-κB kinase subunit β (IKKβ), an activating kinase upstream of NF-κB. An unstable, alkynyl-tagged curcumin analog yielded abundant adducts with cellular protein that were decreased by pretreatment with curcumin or an unstable analog but not by a stable analog. Bioactivation of curcumin occurred readily in vitro, which may explain the wide range of cellular targets, but if bioactivation is insufficient in vivo, it may also help explain the inconclusive results in human studies with curcumin so far. We conclude that the paradigm of metabolic bioactivation uncovered here should be considered for the evaluation and design of clinical trials of curcumin and other polyphenols of medicinal interest.

Berberine inhibits the ischemia-reperfusion injury induced inflammatory response and apoptosis of myocardial cells through the phosphoinositide 3-kinase/RAC-α serine/threonine-protein kinase and nuclear factor-κB signaling pathways.

Myocardial ischemia-reperfusion injury is one of the most common cardiovascular diseases, and can lead to serious damage and dysfunction of the myocardial tissue. Previous studies have demonstrated that berberine exhibits ameliorative effects on cardiovascular disease. The present study further investigated the efficacy and potential mechanism underlying the effects of berberine on ischemia-reperfusion injury in a mouse model. Inflammatory markers were measured in the serum and levels of inflammatory proteins in myocardial cells were investigated after treatment with berberine. In addition, the apoptosis of myocardial cells was investigated after berberine treatment. Apoptosis-associated gene expression levels and apoptotic signaling pathways were analyzed in myocardial cells after treatment with berberine. The phosphoinositide 3-kinase (PI3K)/RAC-α serine/threonine-protein kinase (AKT) and nuclear factor (NF)-κB signaling pathways were also analyzed in myocardial cells after treatment with berberine. Histological analysis was used to analyze the potential benefits of berberine in ischemia-reperfusion injury. The present study identified that inflammatory responses and inflammatory factors were decreased in the myocardial cells of the mouse model of ischemia-reperfusion injury. Mechanism analysis demonstrated that berberine inhibited apoptotic protease-activating factor 1, caspase-3 and caspase-9 expression in myocardial cells. The expression of Bcl2-associated agonist of cell death, Bcl-2-like protein 1 and cellular tumor antigen p53 was upregulated. Expression of NF-κB p65, inhibitor of NF-κB kinase subunit β (IKK-β), NF-κB inhibitor α (IκBα), and NF-κB activity, were inhibited in myocardial cells in the mouse model of ischemia-reperfusion injury. In conclusion, the results of the present study indicate that berberine inhibits inflammatory responses through the NF-κB signaling pathway and suppresses the apoptosis of myocardial cells via the PI3K/AKT signaling pathway in a mouse model of ischemia-reperfusion injury. These results suggest that berberine is a potential drug for the treatment of patients with ischemia-reperfusion injury.

IKKβ Activation in the Fetal Lung Mesenchyme Alters Lung Vascular Development but Not Airway Morphogenesis

In the immature lung, inflammation and injury disrupt the epithelial-mesenchymal interactions required for normal development. Innate immune signaling and NF-κB activation disrupt the normal expression of multiple mesenchymal genes that play a key role in airway branching and alveolar formation. To test the role of the NF-κB pathway specifically in lung mesenchyme, we utilized the mesenchymal Twist2-Cre to drive expression of a constitutively active inhibitor of NF-κB kinase subunit β (IKKβca) mutant in developing mice. Embryonic Twist2-IKKβca mice were generated in expected numbers and appeared grossly normal. Airway branching also appeared normal in Twist2-IKKβca embryos, with airway morphometry, elastin staining, and saccular branching similar to those in control littermates. While Twist2-IKKβca lungs did not contain increased levels of Il1b, we did measure an increased expression of the chemokine-encoding gene Ccl2. Twist2-IKKβca lungs had increased staining for the vascular marker platelet endothelial cell adhesion molecule 1. In addition, type I alveolar epithelial differentiation appeared to be diminished in Twist2-IKKβca lungs. The normal airway branching and lack of Il1b expression may have been due to the inability of the Twist2-IKKβca transgene to induce inflammasome activity. While Twist2-IKKβca lungs had an increased number of macrophages, inflammasome expression remained restricted to macrophages without evidence of spontaneous inflammasome activity. These results emphasize the importance of cellular niche inconsidering how inflammatory signaling influences fetal lung development.

BRCA1-Associated Protein Increases Invasiveness of Esophageal Squamous Cell Carcinoma

We performed a screen for genes whose expression correlates with invasiveness of esophageal squamous cell carcinoma (ESCC) cells. We studied the effects of overexpression and knockdown of these genes in cell lines and expression levels in patient samples. We selected genes for analysis from 11 loci associated with risk of ESCC. Weanalyzed the effects of knocking down expression of 47 of these genes using RNA interference on-chip analysis in ESCC cells and HeLa cells. Cells with gene overexpression and knockdown were analyzed in migration and invasion assays or injected into nude mice and metastasis of xenograft tumors was quantified. We collected ESCC and non-tumor esophageal tissues from 94 individuals who underwent surgery in China from 2010 and 2014; clinical information was collected and survival time was measured from the date of diagnosis to the date of last follow-up or death. Levels of messenger RNAs (mRNAs) were quantified by RNA sequencing, and levels of proteins were determined from immunoblot analyses. Patient survival was compared with mRNA levels using Kaplan-Meier methods and hazard ratios were calculated by Cox models. We identified 8 genes whose disruption increased migration and 10 genes whose disruption reduced migration. Knockdown of BRCA1-associated protein gene (BRAP) significantly reduced migration of KYSE30, KYSE150, and HeLa cells. In patient tumors, 90% of ESCCs examined had higher levels of BRAP protein than paired non-tumor tissues, and 63.8% had gains in BRAP DNA copy number. Levels of BRAP mRNA in ESCC tissues correlated with patient survival time, and high expression increased risk of death 2.4-fold compared with low expression. ESCCs that had metastasized to lymph node had significantly higher levels of BRAP mRNA than tumors without metastases. Knockdown of BRAP in ESCC and HeLa cell lines significantly reduced migration and invasiveness; these cell lines formed less metastases in mice than control cells. Nuclear translocation of the nuclear factor-κB (NF-κB) P65 subunit and phosphorylation of inhibitor of NF-κB kinase subunit β (IKBKB or IKKβ) increased in cells that overexpressed BRAP and decreased in cells with BRAP knockdown. In immunoprecipitation assays, BRAP interacted directly with IKKβ. Expression of matrix metalloproteinase 9 and vascular epithelial growth factor C, which are regulated by NF-κB, was significantly reduced in cells with knockdown of BRAP and significantly increased in cells that overexpressed BRAP. Expression of BRAP is increased in ESCC samples compared with non-tumor esophageal tissues; increased expression correlates with reduced patient survival time and promotes metastasis of xenograft tumors in mice. BRAP overexpression leads to increased activity of NF-κB and expression of matrix metalloproteinase 9 and vascular epithelial growth factor C.

A-Kinase-Anchoring Protein–Lbc Anchors IκB Kinase β To Support Interleukin-6-Mediated Cardiomyocyte Hypertrophy

In response to stress, the heart undergoes a pathological remodeling process associated with hypertrophy and the reexpression of a fetal gene program that ultimately causes cardiac dysfunction and heart failure. In this study, we show that A-kinase-anchoring protein (AKAP)-Lbc and the inhibitor of NF-κB kinase subunit β (IKKβ) form a transduction complex in cardiomyocytes that controls the production of proinflammatory cytokines mediating cardiomyocyte hypertrophy. In particular, we can show that activation of IKKβ within the AKAP-Lbc complex promotes NF-κB-dependent production of interleukin-6 (IL-6), which in turn enhances fetal gene expression and cardiomyocyte growth. These findings provide a new mechanistic hypothesis explaining how hypertrophic signals are coordinated and conveyed to interleukin-mediated transcriptional reprogramming events in cardiomyocytes.

Hypothalamic dysfunction in obesity

A growing number of studies have shown that a diet high in long chain SFA and/or obesity cause profound changes to the energy balance centres of the hypothalamus which results in the loss of central leptin and insulin sensitivity. Insensitivity to these important anorexigenic messengers of nutritional status perpetuates the development of both obesity and peripheral insulin insensitivity. A high-fat diet induces changes in the hypothalamus that include an increase in markers of oxidative stress, inflammation, endoplasmic reticulum (ER) stress, autophagy defect and changes in the rate of apoptosis and neuronal regeneration. In addition, a number of mechanisms have recently come to light that are important in the hypothalamic control of energy balance, which could play a role in perpetuating the effect of a high-fat diet on hypothalamic dysfunction. These include: reactive oxygen species as an important second messenger, lipid metabolism, autophagy and neuronal and synaptic plasticity. The importance of nutritional activation of the Toll-like receptor 4 and the inhibitor of NF-κB kinase subunit β/NK-κB and c-Jun amino-terminal kinase 1 inflammatory pathways in linking a high-fat diet to obesity and insulin insensitivity via the hypothalamus is now widely recognised. All of the hypothalamic changes induced by a high-fat diet appear to be causally linked and inhibitors of inflammation, ER stress and autophagy defect can prevent or reverse the development of obesity pointing to potential drug targets in the prevention of obesity and metabolic dysfunction.

Vasoactive Intestinal Peptide Downregulates Proinflammatory TLRs While Upregulating Anti-Inflammatory TLRs in the Infected Cornea

TLRs recognize microbial pathogens and trigger an immune response, but their regulation by neuropeptides, such as vasoactive intestinal peptide (VIP), during Pseudomonas aeruginosa corneal infection remains unexplored. Therefore, C57BL/6 (B6) mice were injected i.p. with VIP, and mRNA, protein, and immunostaining assays were performed. After VIP treatment, PCR array and real-time RT-PCR demonstrated that proinflammatory TLRs (conserved helix-loop-helix ubiquitous kinase, IRAK1, TLR1, TLR4, TLR6, TLR8, TLR9, and TNFR-associated factor 6) were downregulated, whereas anti-inflammatory TLRs (single Ig IL-1-related receptor [SIGIRR] and ST2) were upregulated. ELISA showed that VIP modestly downregulated phosphorylated inhibitor of NF-κB kinase subunit α but upregulated ST2 ~2-fold. SIGIRR was also upregulated, whereas TLR4 immunostaining was reduced in cornea; all confirmed the mRNA data. To determine whether VIP effects were cAMP dependent, mice were injected with small interfering RNA for type 7 adenylate cyclase (AC7), with or without VIP treatment. After silencing AC7, changes in mRNA levels of TLR1, TNFR-associated factor 6, and ST2 were seen and unchanged with addition of VIP, indicating that their regulation was cAMP dependent. In contrast, changes were seen in mRNA levels of conserved helix-loop-helix ubiquitous kinase, IRAK1, 2, TLR4, 9 and SIGIRR following AC7 silencing alone; these were modified by VIP addition, indicating their cAMP independence. In vitro studies assessed the effects of VIP on TLR regulation in macrophages and Langerhans cells. VIP downregulated mRNA expression of proinflammatory TLRs while upregulating anti-inflammatory TLRs in both cell types. Collectively, the data provide evidence that VIP downregulates proinflammatory TLRs and upregulates anti-inflammatory TLRs and that this regulation is both cAMP dependent and independent and involves immune cell types found in the infected cornea.