Overexpression Of IκBα Research Articles

Lost and Found: The Family of NF-κB Inhibitors Is Larger than Assumed in Salmonid Fish.

NF-κB signalling is largely controlled by the family of 'inhibitors of NF-κB' (IκB). The relevant databases indicate that the genome of rainbow trout contains multiple gene copies coding for iκbα (nfkbia), iκbε (nfkbie), iκbδ (nkfbid), iκbζ (nfkbiz), and bcl3, but it lacks iκbβ (nfkbib) and iκbη (ankrd42). Strikingly, three nfkbia paralogs are apparently present in salmonid fish, two of which share a high sequence identity, while the third putative nfkbia gene is significantly less like its two paralogs. This particular nfkbia gene product, iκbα, clusters with the human IκBβ in a phylogenetic analysis, while the other two iκbα proteins from trout associate with their human IκBα counterpart. The transcript concentrations were significantly higher for the structurally more closely related nfkbia paralogs than for the structurally less similar paralog, suggesting that iκbβ probably has not been lost from the salmonid genomes but has been incorrectly designated as iκbα. In the present study, two gene variants coding for iκbα (nfkbia) and iκbε (nfkbie) were prominently expressed in the immune tissues and, particularly, in a cell fraction enriched with granulocytes, monocytes/macrophages, and dendritic cells from the head kidney of rainbow trout. Stimulation of salmonid CHSE-214 cells with zymosan significantly upregulated the iκbα-encoding gene while elevating the copy numbers of the inflammatory markers interleukin-1-beta and interleukin-8. Overexpression of iκbα and iκbε in CHSE-214 cells dose-dependently quenched both the basal and stimulated activity of an NF-κB promoter suggesting their involvement in immune-regulatory processes. This study provides the first functional data on iκbε-versus the well-researched iκbα factor-in a non-mammalian model species.

Overexpression of IκB⍺ modulates NF-κB activation of inflammatory target gene expression.

Cells respond to inflammatory stimuli such as cytokines by activation of the nuclear factor-κB (NF-κB) signalling pathway, resulting in oscillatory translocation of the transcription factor p65 between nucleus and cytoplasm in some cell types. We investigate the relationship between p65 and inhibitor-κB⍺ (IκBα) protein levels and dynamic properties of the system, and how this interaction impacts on the expression of key inflammatory genes. Using bacterial artificial chromosomes, we developed new cell models of IκB⍺-eGFP protein overexpression in a pseudo-native genomic context. We find that cells with high levels of the negative regulator IκBα remain responsive to inflammatory stimuli and maintain dynamics for both p65 and IκBα. In contrast, canonical target gene expression is dramatically reduced by overexpression of IκBα, but can be partially rescued by overexpression of p65. Treatment with leptomycin B to promote nuclear accumulation of IκB⍺ also suppresses canonical target gene expression, suggesting a mechanism in which nuclear IκB⍺ accumulation prevents productive p65 interaction with promoter binding sites. This causes reduced target promoter binding and gene transcription, which we validate by chromatin immunoprecipitation and in primary cells. Overall, we show how inflammatory gene transcription is modulated by the expression levels of both IκB⍺ and p65. This results in an anti-inflammatory effect on transcription, demonstrating a broad mechanism to modulate the strength of inflammatory response.

TRIM46 aggravated high glucose-induced hyper permeability and inflammatory response in human retinal capillary endothelial cells by promoting IκBα ubiquitination

BackgroundDiabetic retinopathy (DR) as a severe diabetic complication contributes to blindness. The increased permeability of retinal capillary endothelial cells (RCECs) as well as the production of inflammatory markers are closely related to DR occurrence. We recently revealed that TRIM46 promotes high glucose (HG)-caused ferroptosis in human RCECs (HRCECs). The current study aims to explore the molecular mechanism of how TRIM46 plays its role in DR progression.MethodsWestern blot was utilized to determine protein expression. The cell counting kit-8 assay was used to observe cell viability. The permeability of the cell layer was determined by measuring the transepithelial electrical resistance and fluorescein isothiocyanate (FITC)-dextran leak. Enzyme-linked immunosorbent assay was used to quantify the protein level of pro-inflammatory cytokines and co-immunoprecipitation was employed to verify the relationship between TRIM46 and IκBα.ResultsHG dramatically upregulated TRIM46 protein expression in a dose-dependent way. Silencing TRIM46 effectively reversed HG-induced cell growth inhibition, cell cycle arrest, hyper permeability and pro-inflammatory cytokines secretion in HRCECs, while overexpression of TRIM46 exhibited an opposite effect. Furthermore, TRIM46 was able to interact with IκBα and promote the ubiquitination and degradation of IκBα. IκBα overexpression recovered the effects of TRIM46 overexpression in HRCECs. Furthermore, inhibiting the activation of NF-κB partially recovered HG-induced HRCEC injury, whereas TRIM46 overexpression reversed these effects.ConclusionThis study demonstrates that TRIM46 interacts with IκBα to activate the NF-κB signaling pathway, thereby enhancing cell proliferation inhibition, hyper permeability and the inflammatory response of HRCECs in a HG state.

I\u03baB\u03b1 targeting promotes oxidative stress-dependent cell death

BackgroundOxidative stress is a hallmark of many cancers. The increment in reactive oxygen species (ROS), resulting from an increased mitochondrial respiration, is the major cause of oxidative stress. Cell fate is known to be intricately linked to the amount of ROS produced. The direct generation of ROS is also one of the mechanisms exploited by common anticancer therapies, such as chemotherapy.MethodsWe assessed the role of NFKBIA with various approaches, including in silico analyses, RNA-silencing and xenotransplantation. Western blot analyses, immunohistochemistry and RT-qPCR were used to detect the expression of specific proteins and genes. Immunoprecipitation and pull-down experiments were used to evaluate protein-protein interactions.ResultsHere, by using an in silico approach, following the identification of NFKBIA (the gene encoding IκBα) amplification in various cancers, we described an inverse correlation between IκBα, oxidative metabolism, and ROS production in lung cancer. Furthermore, we showed that novel IκBα targeting compounds combined with cisplatin treatment promote an increase in ROS beyond the tolerated threshold, thus causing death by oxytosis.ConclusionsNFKBIA amplification and IκBα overexpression identify a unique cancer subtype associated with specific expression profile and metabolic signatures. Through p65-NFKB regulation, IκBα overexpression favors metabolic rewiring of cancer cells and distinct susceptibility to cisplatin. Lastly, we have developed a novel approach to disrupt IκBα/p65 interaction, restoring p65-mediated apoptotic responses to cisplatin due to mitochondria deregulation and ROS-production.

Protein kinase D, ubiquitin and proteasome pathways are involved in adenosine receptor-stimulated NR4A expression in myeloid cells

Adenosine is a purine nucleoside pivotal for homeostasis in cells and tissues. Stimulation of the adenosine receptors (AR) has been shown to regulate the nuclear orphan receptor 4A (NR4A1-3) family, resulting in attenuation of hyper-inflammatory responses in myeloid cells. The NR4A1-3 orphan receptors are early immediate response genes and transcriptional regulators of cell and tissue homeostasis. The signal transduction and transcriptional mechanism(s) of how AR-stimulation promotes NR4A expression in myeloid cells is unknown and is the focus of this study. We confirm that adenosine and the stable analogue, 5′-N-Ethylcarboxamidoadenosine (NECA), enhance NR4A1-3 expression in THP-1 cells. Pharmacological approaches identified that protein kinase D (PKD) mediates AR-stimulated NR4A expression in myeloid cells and reveals no involvement of PKA nor PKC. The role of NF-κB, a principal regulator of NR4A expression in myeloid cells, was examined as a possible transcriptional regulator downstream of PKD. Utilising BAY11-7082 and MG-132, inhibitors of the respective ubiquitin and proteasome pathways essential for NF-κB activation, suggested a prospective role for NF-κB, or more specifically signalling via IKKα/β. However, biological interventional studies using overexpression of IκBα in myeloid cells and MEF cells lacking IKKα and IKKβ (IKKα/β−/-) revealed the NF-κB pathway is not utilised in mediating AR-stimulated NR4A expression. Thus, this study contributes mechanistic insight into how AR signalling modulates the expression of NR4A receptors, pivotal regulators of inflammatory responses in myeloid cells.

Study on Apoptosis of Prostate Cancer Cells Induced by IκBα Overexpression Synergistic with Poly (Lactic‐Co‐Glycolic Acid)‐Curcumin Nanoparticles

Objective. To investigate the synergistic effects of IκBα overexpression and poly (lactic‐co‐glycolic acid)‐curcumin nanoparticles (PLGA‐Cur‐NPs) on prostate cancer (PC) and reveal the underlying mechanisms of cooperative sensitization induced by curcumin. Methods. Proliferation and apoptosis rate in IκBα overexpressed and PLGA‐Cur‐NPs‐treated PC cells were detected by MTT and flow cytometry assay. The expression levels of IκBα, apoptosis‐related, and signaling proteins were measured by western blotting assay. Results. PC cell proliferation was significantly inhibited by the overexpression of IκBα. The apoptosis rate of PC cells was significantly increased at a high concentration of curcumin exposure, while the activation of NF‐κB pathway was obviously inhibited. In addition, PLGA‐Cur‐NPs treatment synergistic with IκBα overexpression enhanced the apoptosis of PC cells by suppressing the NF‐κB pathway activation. Conclusion. IκBα overexpression synergistic with PLGA‐Cur‐NPs could obviously inhibit proliferation, induce apoptosis, and suppress the activation of NF‐κB pathway in PC cells. These findings may provide an experimental basis to establish the tumor gene therapy combined with traditional Chinese medicine treatment, thus promoting the clinical application of both tumor gene therapy and anti‐tumor Chinese medicine.

Ubiquitin-Specific Protease 34 Inhibits Osteoclast Differentiation by Regulating NF-κB Signaling.

The ubiquitination and deubiquitination enzymes ensure the stability and proper function of most cellular proteins. Disturbance of either enzyme compromises tissue homeostasis. We recently have identified that the ubiquitin-specific protease 34 (USP34) contributes to bone formation by promoting osteogenic differentiation of mesenchymal stem cells. However, its role in bone resorption, which couples bone formation, remains unknown. Here we show that knockdown of Usp34 promotes osteoclast differentiation of RAW264.7 cells. Conditional knockout of Usp34 in bone marrow-derived macrophages (BMMs) or in osteoclasts leads to elevated osteoclast function and low bone mass. Mechanically, we identify that USP34 restrains NF-κB signaling by deubiquitinating and stabilizing the NF-κB inhibitor alpha (IκBα). Overexpression of IκBα represses osteoclastic hyperfunction of Usp34-deficient RAW264.7 cells. Collectively, our results show that USP34 inhibits osteoclastogenesis by regulating NF-κB signaling. © 2020 American Society for Bone and Mineral Research.

ZBTB20 promotes cell migration and invasion of gastric cancer by inhibiting IκBα to induce NF-κB activation

Zinc finger and BTB domain containing 20 (ZBTB20), a sequence-specific transcriptional repressor, has been found to be involved in tumorigenesis. However, its role(s) in gastric cancer and the molecular mechanisms involved are poorly investigated. Here, our data demonstrated that ZBTB20 expression was markedly upregulated in gastric cancer cell lines infected with Helicobacter pylori (H. pylori) and in gastric cancer tumor samples. Loss- and gain-of-function studies showed that ZBTB20 promoted cell proliferation, invasion and migration of gastric cancer cell lines. Mechanistically, the phosphorylation of NF-κBp65 and expression and activity of MMP-2 and MMP-9 were increased, while IκBα expression was decreased by ZBTB20 in gastric cancer cells. We further revealed that IκBα overexpression significantly inhibited NF-κB signaling as well as cell migration, invasion and proliferation in gastric cancer cell lines induced by ZBTB20 overexpression. Therefore, our findings emphasize an important role for ZBTB20 in controlling gastric cancer development, which is helpful to identify potential therapeutic targets for its treatment.

Pravastatin sodium attenuated TREM-1-mediated inflammation in human peripheral blood mononuclear cells

Pravastatin sodium on triggering receptor expressed on myeloid cell-1 (TREM-1)-mediated inflammation in human peripheral blood mononuclear cells (PBMCs) has been poorly investigated. In this study, we isolated PBMCs from the peripheral blood samples of patients with chronic obstructive pulmonary disease, treated the cells with pravastatin sodium, and determined a concentration at which more than 90% cells could survive. Then we treated cells with 10 ng/ml of lipopolysaccharide, added with 10, 50, 100 μM of pravastatin sodium combined with or without LR-12, a known TREM-1 inhibitor. The expression of TREM-1 was determined by quantitative RT-PCR. The levels of TREM-1, IL-6, and TNF-α in cell culture supernatant were measured with ELISA. Simultaneously, NF-κB signaling pathway-related protein p-p65 and p-IκBα were detected by Western blot assay. Results demonstrated that pravastatin sodium significantly mitigated lipopolysaccharide-stimulated TREM-1 over-expression at mRNA and protein levels dose-dependently. Elevated IL-6 and TNF-α levels changed synchronously. LR-12 inhibited the TREM-1 over-expression and inflammatory factor production but did not show extra synergistic effect to pravastatin. Lipopolysaccharide induced phospho-p65 and -IκBα over-expression was weakened significantly when cells were treated with pravastatin sodium. In conclusion, pravastatin could inhibit TREM-1-medieted inflammation and NF-κB signaling pathway was involved.

NF-\u03baB signaling regulates cell-autonomous regulation of CXCL10 in breast cancer 4T1 cells

The chemokine CXCL10 and its receptor CXCR3 play a role in breast cancer metastasis to bone and osteoclast activation. However, the mechanism of CXCL10/CXCR3-induced intracellular signaling has not been fully investigated. To evaluate CXCL10-induced cellular events in the mouse breast cancer cell line 4T1, we developed a new synthetic CXCR3 antagonist JN-2. In this study, we observed that secretion of CXCL10 in the supernatant of 4T1 cells was gradually increased during cell growth. JN-2 inhibited basal and CXCL10-induced CXCL10 expression and cell motility in 4T1 cells. Treatment of 4T1 cells with CXCL10 increased the expression of P65, a subunit of the NF-κB pathway, via activation of the NF-κB transcriptional activity. Ectopic overexpression of P65 increased CXCL10 secretion and blunted JN-2-induced suppression of CXCL10 secretion, whereas overexpression of IκBα suppressed CXCL10 secretion. These results indicate that the CXCL10/CXCR3 axis creates a positive feedback loop through the canonical NF-κB signaling pathway in 4T1 cells. In addition, treatment of osteoblasts with conditioned medium from JN-2-treated 4T1 cells inhibited the expression of RANKL, a crucial cytokine for osteoclast differentiation, which resulted in an inhibitory effect on osteoclast differentiation in the co-culture system of bone marrow-derived macrophages and osteoblasts. Direct intrafemoral injection of 4T1 cells induced severe bone destruction; however, this effect was suppressed by the CXCR3 antagonist via downregulation of P65 expression in an animal model. Collectively, these results suggest that the CXCL10/CXCR3-mediated NF-κB signaling pathway plays a role in the control of autonomous regulation of CXCL10 and malignant tumor properties in breast cancer 4T1 cells.

Intermittent hypoxia confers pro-metastatic gene expression selectively through NF-κB in inflammatory breast cancer cells

Inflammatory breast cancer (IBC) is the most aggressive form of breast cancer. Treatment options are limited and the mechanisms underlying its aggressiveness are poorly understood. Intermittent hypoxia (IH) causes oxidative stress and is emerging as important regulator of tumor metastasis. Vessels in IBC tumors have been shown to be immature, which is a primary cause of IH. We therefore investigated the relevance of IH for the modulation of gene expression in IBC cells in order to assess IH as potential regulator of IBC aggressiveness. Gene array analysis of IBC cells following chronic IH (45–60 days) demonstrated increased expression of pro-metastatic genes of the extracellular matrix, such as tenascin-C (TNC; an essential factor of the metastatic niche) and matrix metalloproteinase 9 (MMP9), and of pro-inflammatory processes, such as cyclooxygenase-2 (COX-2). Investigating the oxidative stress-dependent regulation of TNC, we found a gradual sensitivity on mRNA and protein levels. Oxidative stress activated NF-E2-related factor 2 (Nrf2), c-Jun N-terminal kinase (JNK), c-Jun and nuclear factor κB (NF-κB), but TNC upregulation was only dependent on NF-κB activation. Pharmacological inhibition of inhibitor of NF-κB α (IκBα) phosphorylation as well as overexpression of IκBα prevented TNC, MMP9 and COX-2 induction, whereas the pro-inflammatory cytokine interleukin-1β (IL-1β) increased their expression levels. Analysis of the gene array data showed NF-κB binding sites for 64% of all upregulated genes, linking NF-κB with IH-dependent regulation of pro-metastatic gene expression in IBC cells. Our results provide a first link between intermittent hypoxia and pro-metastatic gene expression in IBC cells, revealing a putative novel mechanism for the high metastatic potential of IBC.

Inhibition of Runx2 signaling by TNF-α in ST2 murine bone marrow stromal cells undergoing osteogenic differentiation.

Tumor necrosis factor-alpha (TNF-α) inhibits osteogenic differentiation of murine bone marrow stromal cells, and transcription factor Runx2 serves as an essential regulation target in the process. The underlying mechanism may involve the regulation of Runx2 expression and the Runx2 activity in downstream gene transcription, which has not been fully elucidated. In this study, ST2 murine bone marrow-derived stromal cells were treated with bone morphogenetic protein-2 (BMP-2) and/or TNF-α in osteogenic medium, and the expression of Runx2 was estimated. Cells were transfected with Runx2, p65, inhibitor of κBα (IκBα), 9.0kb bone sialoprotein (BSP) promoter-luciferase or osteoblast-specific cis-acting element 2 (OSE2)-luciferase reporter vectors, and then real time-PCR and dual luciferase analysis were used to investigate the effect of TNF-α on Runx2-activated osteogenic gene transcription and the molecular mechanism. We found that TNF-α inhibited BMP-2-induced osteogenic marker expression and both the spontaneous and BMP-2-induced Runx2 expression. TNF-α stimulation or overexpression of nuclear factor-kappa B (NF-κB) p65 subunit repressed the Runx2-activated BSP and osteocalcin (OC) transcriptions. The Runx2-induced 9.0kb BSP promoter activity was attenuated by TNF-α or p65, while the OSE2 activity was not affected. Besides, blockage of NF-κB by IκBα overexpression eliminated these inhibitory effects of TNF-α on Runx2 signaling. These results suggest that in murine bone marrow stromal cells undergoing osteogenic differentiation, TNF-α and it activated NF-κB pathway inhibit the expression of Runx2 gene, and suppress the Runx2-mediated osteogenic gene transcription via the 9.0kb BSP promoter.

Hypercapnic acidosis attenuates pulmonary epithelial stretch-induced injury via inhibition of the canonical NF-κB pathway.

BackgroundHypercapnia, with its associated acidosis (HCA), is a consequence of respiratory failure and is also seen in critically ill patients managed with conventional “protective” ventilation strategies. Nuclear factor kappa-B (NF-κB), a pivotal transcription factor, is activated in the setting of injury and repair and is central to innate immunity. We have previously established that HCA protects against ventilation-induced lung injury in vivo, potentially via a mechanism involving inhibition of NF-κB signaling. We wished to further elucidate the role and mechanism of HCA-mediated inhibition of the NF-κB pathway in attenuating stretch-induced injury in vitro.MethodsInitial experiments examined the effect of HCA on cyclic stretch-induced inflammation and injury in human bronchial and alveolar epithelial cells. Subsequent experiments examined the role of the canonical NF-κB pathway in mediating stretch-induced injury and the mechanism of action of HCA. The contribution of pH versus CO2 in mediating this effect of HCA was also examined.ResultsPulmonary epithelial high cyclic stretch (22 % equibiaxial strain) activated NF-κB, enhanced interleukin-8 (IL-8) production, caused cell injury, and reduced cell survival. In contrast, physiologic stretch (10 % strain) did not activate inflammation or cause cell injury. HCA reduced cyclic mechanical stretch-induced NF-κB activation, attenuated IL-8 production, reduced injury, and enhanced survival, in bronchial and alveolar epithelial cells, following shorter (24 h) and longer (120 h) cyclic mechanical stretch. Pre-conditioning with HCA was less effective than when HCA was applied after commencement of cell stretch. HCA prevented the stretch-induced breakdown of the NF-κB cytosolic inhibitor IκBα, while IκBα overexpression “occluded” the effect of HCA. These effects were mediated by a pH-dependent mechanism rather than via CO2 per se.ConclusionsHCA attenuates adverse mechanical stretch-induced epithelial injury and death, via a pH-dependent mechanism that inhibits the canonical NF-κB activation by preventing IκBα breakdown.Electronic supplementary materialThe online version of this article (doi:10.1186/s40635-016-0081-6) contains supplementary material, which is available to authorized users.

IκBα mediates prostate cancer cell death induced by combinatorial targeting of the androgen receptor.

BackgroundCombining different clinical agents to target multiple pathways in prostate cancer cells, including androgen receptor (AR) signaling, is potentially an effective strategy to improve outcomes for men with metastatic disease. We have previously demonstrated that sub-effective concentrations of an AR antagonist, bicalutamide, and the histone deacetylase inhibitor, vorinostat, act synergistically when combined to cause death of AR-dependent prostate cancer cells.MethodsIn this study, expression profiling of human prostate cancer cells treated with bicalutamide or vorinostat, alone or in combination, was employed to determine the molecular mechanisms underlying this synergistic action. Cell viability assays and quantitative real time PCR were used to validate identified candidate genes.ResultsA substantial proportion of the genes modulated by the combination of bicalutamide and vorinostat were androgen regulated. Independent pathway analysis identified further pathways and genes, most notably NFKBIA (encoding IκBα, an inhibitor of NF-κB and p53 signaling), as targets of this combinatorial treatment. Depletion of IκBα by siRNA knockdown enhanced apoptosis of prostate cancer cells, while ectopic overexpression of IκBα markedly suppressed cell death induced by the combination of bicalutamide and vorinostat.ConclusionThese findings implicate IκBα as a key mediator of the apoptotic action of this combinatorial AR targeting strategy and a promising new therapeutic target for prostate cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2188-2) contains supplementary material, which is available to authorized users.

LYSOPHOSPHATIDIC ACID ACTIVATES BMP2 GENE PROMOTER THROUGH A RHOA-PHOSPHORYLATED P65 SERINE 536 PATHWAY IN AORTIC VALVE INTERSTITIAL CELLS: IMPLICATION FOR CALCIFIC AORTIC VALVE DISEASE

Calcific aortic valve disease (CAVD) is the most common heart valve disorder. There is so far no medical treatment for CAVD that could prevent its progression. Recently, we demonstrated that lysophosphatidic acid (LPA) is produced by autotaxine enzyme (ATX) in mineralized aortic valves. However, no clear evidence has been given regarding the mechanism of action by which the LPA acts on mineralization and the inflammatory process within the aortic valve. By using different approaches in vitro we have investigated the effect of LPA on the osteogenic phenotype and its role as pro-mineralizing. We have also documented the NF-κB signaling pathway by analysis of promoter activity and of transcripts targets measurement by q - PCR. In cell culture we found that the expression of osteogenic genes (BMP2, RUNX2, BGLAP, and COL1A1), alkaline phosphatase (ALP) activity and VICs mineralization were increased by several-fold after treatment of cells with LPA. Also, we have shown that LPA-induced osteogenic response relied on RhoA pathway downstream of the LPA receptor-1 (LPAR1). In this regard, we found that RhoA is a regulator of the NF-κB pathway and promotes BMP2 expression. In addition, by using promoter luciferase assay we documented that NF-κB-p65 phosphorylation on Ser536 (phospho-Ser-536 p65) activates BMP2 promoter following treatment with LPA. We next showed by using chromatin immunoprecipitation assays (ChIP) that the binding of phospho-Ser-536 p65 to BMP2 promoter is not reversed by IκBα overexpression, an inhibitor of NF-κB pathway. Together, these results suggest that LPA induced VICs mineralization by a phospho-Ser-536 p65 pathway, which defines a mechanism of activation that does not respond to the negative feedback of IκBα. Hence, we documented a novel mechanism whereby LPAR1 and RhoA modulate the NF-κB pathway and its downstream target BMP-2, which is a strong promoter of VICs mineralization.

Central inhibition of IKKβ/NF-κB signaling attenuates high-fat diet-induced obesity and glucose intolerance.

Metabolic inflammation in the central nervous system might be causative for the development of overnutrition-induced metabolic syndrome and related disorders, such as obesity, leptin and insulin resistance, and type 2 diabetes. Here we investigated whether nutritive and genetic inhibition of the central IκB kinase β (IKKβ)/nuclear factor-κB (NF-κB) pathway in diet-induced obese (DIO) and leptin-deficient mice improves these metabolic impairments. A known prominent inhibitor of IKKβ/NF-κB signaling is the dietary flavonoid butein. We initially determined that oral, intraperitoneal, and intracerebroventricular administration of this flavonoid improved glucose tolerance and hypothalamic insulin signaling. The dose-dependent glucose-lowering capacity was profound regardless of whether obesity was caused by leptin deficiency or high-fat diet (HFD). To confirm the apparent central role of IKKβ/NF-κB signaling in the control of glucose and energy homeostasis, we genetically inhibited this pathway in neurons of the arcuate nucleus, one key center for control of energy homeostasis, via specific adeno-associated virus serotype 2-mediated overexpression of IκBα, which inhibits NF-κB nuclear translocation. This treatment attenuated HFD-induced body weight gain, body fat mass accumulation, increased energy expenditure, and reduced arcuate suppressor of cytokine signaling 3 expression, indicative for enhanced leptin signaling. These results reinforce a specific role of central proinflammatory IKKβ/NF-κB signaling in the development and potential treatment of DIO-induced comorbidities.

Pulmonary overexpression of inhibitor κBα decreases the severity of ventilator-induced lung injury in a rat model

Activation of the nuclear factor-κB (NF-κB) pathway is central to the pathogenesis of lung injury and inflammation. We determined whether targeted overexpression of inhibitor-κBα (IκBα) in the lung could decrease the severity of ventilator-induced lung injury (VILI). Anaesthetized adult male Sprague-Dawley rats were randomly allocated to undergo intratracheal instillation of: (i) vehicle alone (surfactant, n=10); (ii) 1×10(10) adeno-associated virus encoding IκBα (AAV-IκBα, n=10); (iii) 5×10(10) AAV-IκBα (n=10); and (iv) 1×10(10) AAV-Null (n=5). This was followed by 4 h of injurious mechanical ventilation. Subsequent experiments examined the effect of IκBα overexpression in animals undergoing 'protective' mechanical ventilation. IκBα overexpression increased survival duration at both the lower [3.8 h (0.4)] and higher [3.6 h (0.7)] doses compared with vehicle [2.7 h (1.0)] or the null transgene [2.2 h (0.8)]. IκBα overexpression reduced the alveolar-arterial oxygen gradient (kPa) at both the lower [53 (21)] and higher [52 (19)] doses compared with vehicle [75 (8.5)] or the null transgene [70 (15)], decreased alveolar neutrophil infiltration, and reduced alveolar concentrations of interleukin (IL)-1β and IL-10. The lower IκBα dose was as effective as the higher dose. IκBα overexpression had no effect in the setting of protective lung ventilation. Inhibition of pulmonary NF-κB activity by IκBα overexpression reduced the severity of VILI in a rat model.

Dengue Viral Protease Interaction with NF-κB Inhibitor α/β Results in Endothelial Cell Apoptosis and Hemorrhage Development

Hemorrhagic manifestations occur frequently accompanying a wide range of dengue disease syndromes. Much work has focused on the contribution of immune factors to the pathogenesis of hemorrhage, but how dengue virus (DENV) participates in the pathogenic process has never been explored. Although there is no consensus that apoptosis is the basis of vascular permeability in human dengue infections, we showed in dengue hemorrhage mouse model that endothelial cell apoptosis is important to hemorrhage development in mice. To explore the molecular basis of the contribution of DENV to endothelial cell death, we show in this study that DENV protease interacts with cellular IκBα and IκBβ and cleaves them. By inducing IκBα and IκBβ cleavage and IκB kinase activation, DENV protease activates NF-κB, which results in endothelial cell death. Intradermal inoculation of DENV protease packaged in adenovirus-associated virus-9 induces endothelial cell death and dermal hemorrhage in mice. Although the H51 activity site is not involved in the interaction between DENV protease and IκB-α/β, the enzymatic activity is critical to the ability of DENV protease to induce IκBα and IκBβ cleavage and trigger hemorrhage development. Moreover, overexpression of IκBα or IκBβ protects endothelial cells from DENV-induced apoptosis. In this study, we show that DENV protease participates in the pathogenesis of dengue hemorrhage and discover IκBα and IκBβ to be the new cellular targets that are cleaved by DENV protease.

Strategic targeting of the PI3K–NFκB axis in cisplatin-resistant NSCLC

Chemoresistance is a major therapeutic challenge to overcome in NSCLC, in order to improve the current survival rates of <15% at 5 years. We and others have shown increased PI3K signaling in NSCLC to be associated with a more aggressive disease, and a poorer prognosis. In this study, targeted inhibition of three strategic points of the PI3K–NFκB axis was performed with the aim of exploiting vulnerabilities in cisplatin-resistant NSCLC cells. Cisplatin-resistant cell lines were previously generated through prolonged exposure to the drug. Expression of PI3K and NFκB pathway-related genes were compared between cisplatin-resistant cells and their matched parent cells using a gene expression array, qRT-PCR, DNA sequencing, western blot, and immunofluorescence. Targeted inhibition was performed using GDC-0980, a dual PI3K–mTOR inhibitor currently in Phase II clinical trials in NSCLC, and DHMEQ, an inhibitor of NFκB translocation which has been used extensively both in vitro and in vivo. Effects of the two inhibitors were assessed by BrdU proliferation assay and multiparameter viability assay. NFKBIA was shown to be 12-fold overexpressed in cisplatin-resistant cells, with no mutations present in exons 3, 4, or 5 of the gene. Corresponding overexpression of IκBα was also observed. Treatment with DHMEQ (but not GDC-0980) led to significantly enhanced effects on viability and proliferation in cisplatin-resistant cells compared with parent cells. We conclude that NFκB inhibition represents a more promising strategy than PI3K–mTOR inhibition for treatment in the chemoresistance setting in NSCLC.

Low-Dose Rapamycin Unmasks the Protective Potential of Targeting Intragraft NF-κB for Islet Transplants

Islet grafts can contribute to their own destruction via the elaboration of proinflammatory genes, many of which are transcriptionally regulated by nuclear factor κ-light-chain-enhancer of activated B-cells (NF-κB). Thus, NF-κB constitutes an enticing gene therapy candidate to improve the success of islet transplantation. To test this hypothesis in vivo, we blocked NF-κB in BALB/c (H2(d)) to C57/BL6 (H2(b)) mouse islet allografts by genetically engineering islets to express the NF-κB superrepressor, IκBα. Here we show by microarray and RTqPCR that islets exhibit an intrinsic early immediate proinflammatory response, with the most highly upregulated proinflammatory genes comprising the chemokines Cxcl1, Cxcl2, Cxcl10, and Ccl2; the cytokines Tnf-α and Il-6; and the adhesion molecule Icam1. Overexpression of IκBα inhibited the expression of these genes by 50-95% in islets and MIN6 β-cells in vitro, by inhibiting NF-κB-dependent gene transcription. Histological and RTqPCR analysis at postoperative day (POD) 10 revealed that IκBα-transduced islet allografts exhibited improved islet architecture and strong insulin-labeling with decreased Ccl2 and Il-6 mRNA levels compared to the GFP-transduced control grafts. Despite these protective effects, NF-κB-blocked islet allografts were promptly rejected in our MHC-mismatched mouse model. However, IκBα-expressing grafts did harbor localized "pockets" of Foxp3(+) mononuclear cells not evident in the control grafts. This result suggested that the effect of the NF-κB blockade might synergize with regulatory T-cell-sparing rapamycin. Indeed, combining intragraft IκBα expression with low-dose rapamycin increased the mean survival time of islet allografts from 20 to 81 days, with 20% of the grafts surviving for greater than 100 days. In conclusion, rapamycin unmasks the protective potential of intragraft NF-κB blockade, which can, in some cases, permit permanent allograft survival without continuous systemic immunosuppression.