Nuclear Localization Of NF-κB Research Articles

Role of lamin A/C on dendritic cell function in antiviral immunity

Dendritic cells (DCs) play a crucial role in orchestrating immune responses, particularly in promoting IFNγ-producing-CD8 cytotoxic T lymphocytes (CTLs) and IFNγ-producing-CD4 T helper 1 (Th1) cells, which are essential for defending against viral infections. Additionally, the nuclear envelope protein lamin A/C has been implicated in T cell immunity. Nevertheless, the intricate interplay between innate and adaptive immunity in response to viral infections, particularly the role of lamin A/C in DC functions within this context, remains poorly understood. In this study, we demonstrate that mice lacking lamin A/C in myeloid LysM promoter-expressing cells exhibit a reduced capacity to induce Th1 and CD8 CTL responses, leading to impaired clearance of acute primary Vaccinia virus (VACV) infection. Remarkably, in vitro-generated granulocyte macrophage colony-stimulating factor bone marrow-derived DCs (GM-CSF BMDCs) show high levels of lamin A/C. Lamin A/C absence on GM-CSF BMDCs does not affect the expression of costimulatory molecules on the cell membrane but it reduces the cellular ability to form immunological synapses with naïve CD4 T cells. Lamin A/C deletion induces alterations in NFκB nuclear localization, thereby influencing NF-κB-dependent transcription. Furthermore, lamin A/C ablation modifies the gene accessibility of BMDCs, predisposing these cells to mount a less effective antiviral response upon TLR stimulation. This study highlights the critical role of DCs in interacting with CD4 T cells during antiviral responses and proposes some mechanisms through which lamin A/C may modulate DC function via gene accessibility and transcriptional regulation.

Unravelling bisphenol A-induced hepatotoxicity: Insights into oxidative stress, inflammation, and energy dysregulation

Bisphenol A (BPA), a prevalent plastic monomer and endocrine disruptor, negatively impacts metabolic functions. This study examines the chronic effects of eco-relevant BPA concentrations on hepatotoxicity, focusing on redox balance, inflammatory response, cellular energy sensors, and metabolic homeostasis in male Swiss albino mice. Chronic BPA exposure resulted in reactive oxygen species (ROS) accumulation, altered hepatic antioxidant defense, lipid peroxidation, and NOX4 expression, leading to reduced cell viability. Additionally, BPA exposure significantly upregulated hepatic pro-inflammatory cytokine genes (Tnf-α, Il-1β, Il-6), NOS2, and arginase II, correlating with increased TLR4 expression, NF-κB phosphorylation, and a dose-dependent decrease in IκBα levels. BPA-induced NF-κB nuclear localization and inflammasome activation (NLRP3, cleaved caspase-1, IL-1β) established an inflammatory milieu. Perturbations in hepatic AMPKα phosphorylation, SIRT1, and PGC-1α, along with elevated p38 MAPK phosphorylation and ERα expression, indicated BPA-induced energy dysregulation. Furthermore, increased PLA2G4A, COX1, COX2, and PTGES2 expression in BPA-treated liver correlated with hyperlipidemia, hepatic FASN expression, steatosis, and visceral adiposity, likely due to disrupted energy sensors, oxidative stress, and inflammasome activation. Elevated liver enzymes (ALP, AST, ALT) and apoptotic markers indicated liver damage. Notably, N-acetylcysteine (NAC) priming reversed BPA-induced hepatocellular ROS accumulation, NF-κB-inflammasome activation, and intracellular lipid accumulation, while upregulating cellular energy sensors and attenuating ERα expression, suggesting NAC's protective effects against BPA-induced hepatotoxicity. Pharmacological inhibition of the NF-κB/NLRP3 cascade in BAY11-7082 pretreated, or NLRP3 immunodepleted hepatocytes reversed BPA's negative impact on SIRT1/p-AMPKα/PGC-1α and intracellular lipid accumulation, providing mechanistic insights into BPA-induced metabolic disruption.

REDD1 Deletion Suppresses NF-κB Signaling in Cardiomyocytes and Prevents Deficits in Cardiac Function in Diabetic Mice.

Activation of the transcription factor NF-κB in cardiomyocytes has been implicated in the development of cardiac function deficits caused by diabetes. NF-κB controls the expression of an array of pro-inflammatory cytokines and chemokines. We recently discovered that the stress response protein regulated in development and DNA damage response 1 (REDD1) was required for increased pro-inflammatory cytokine expression in the hearts of diabetic mice. The studies herein were designed to extend the prior report by investigating the role of REDD1 in NF-κB signaling in cardiomyocytes. REDD1 genetic deletion suppressed NF-κB signaling and nuclear localization of the transcription factor in human AC16 cardiomyocyte cultures exposed to TNFα or hyperglycemic conditions. A similar suppressive effect on NF-κB activation and pro-inflammatory cytokine expression was also seen in cardiomyocytes by knocking down the expression of GSK3β. NF-κB activity was restored in REDD1-deficient cardiomyocytes exposed to hyperglycemic conditions by expression of a constitutively active GSK3β variant. In the hearts of diabetic mice, REDD1 was required for reduced inhibitory phosphorylation of GSK3β at S9 and upregulation of IL-1β and CCL2. Diabetic REDD1+/+ mice developed systolic functional deficits evidenced by reduced ejection fraction. By contrast, REDD1-/- mice did not exhibit a diabetes-induced deficit in ejection fraction and left ventricular chamber dilatation was reduced in diabetic REDD1-/- mice, as compared to diabetic REDD1+/+ mice. Overall, the results support a role for REDD1 in promoting GSK3β-dependent NF-κB signaling in cardiomyocytes and in the development of cardiac function deficits in diabetic mice.

Senescent Astrocytes Derived from Human Pluripotent Stem Cells Reveal Age-Related Changes and Implications for Neurodegeneration.

Astrocytes play a crucial role in maintaining brain homeostasis by regulating synaptic activity, providing metabolic support to neurons, and modulating immune responses in the central nervous system (CNS). During aging, astrocytes undergo senescence with various changes that affect their function and frequently lead to neurodegeneration. This study presents the first evidence of senescent astrocytes derived from human pluripotent stem cells (hPSCs). These senescent hPSC-derived astrocytes exhibited altered cellular and nuclear morphologies, along with increased expression of senescence-associated markers. Additionally, nuclear localization of NFκB, telomere shortening, and frequent signs of DNA damage were observed in these cells. Furthermore, senescent astrocytes showed defects in various critical functions necessary for maintaining a healthy CNS environment, including a reduced ability to support neuronal survival and clear neurotransmitters, synaptic debris, and toxic protein aggregates. Altered structural dynamics and reduced mitochondrial function were also observed in senescent astrocytes. Notably, treating hPSC-derived senescent astrocytes with chemicals targeting reactive oxygen species or an enzyme that regulates mitochondrial function can reverse senescence phenotypes. Thus, this study offers a valuable cellular model that can be utilized to investigate the mechanisms of brain aging and may present new avenues for discovering innovative therapeutic approaches for neurodegenerative diseases.

Catalpol reduced LPS induced BV2 immunoreactivity through NF-κB/NLRP3 pathways: an in Vitro and in silico study.

Background: Ischemic Stroke (IS) stands as one of the primary cerebrovascular diseases profoundly linked with inflammation. In the context of neuroinflammation, an excessive activation of microglia has been observed. Consequently, regulating microglial activation emerges as a vital target for neuroinflammation treatment. Catalpol (CAT), a natural compound known for its anti-inflammatory properties, holds promise in this regard. However, its potential to modulate neuroinflammatory responses in the brain, especially on microglial cells, requires comprehensive exploration. Methods: In our study, we investigated into the potential anti-inflammatory effects of catalpol using lipopolysaccharide (LPS)-stimulated BV2 microglial cells as an experimental model. The production of nitric oxide (NO) by LPS-activated BV2 cells was quantified using the Griess reaction. Immunofluorescence was employed to measure glial cell activation markers. RT-qPCR was utilized to assess mRNA levels of various inflammatory markers. Western blot analysis examined protein expression in LPS-activated BV2 cells. NF-κB nuclear localization was detected by immunofluorescent staining. Additionally, molecular docking and molecular dynamics simulations (MDs) were conducted to explore the binding affinity of catalpol with key targets. Results: Catalpol effectively suppressed the production of nitric oxide (NO) induced by LPS and reduced the expression of microglial cell activation markers, including Iba-1. Furthermore, we observed that catalpol downregulated the mRNA expression of proinflammatory cytokines such as IL-6, TNF-α, and IL-1β, as well as key molecules involved in the NLRP3 inflammasome and NF-κB pathway, including NLRP3, NF-κB, caspase-1, and ASC. Our mechanistic investigations shed light on how catalpol operates against neuroinflammation. It was evident that catalpol significantly inhibited the phosphorylation of NF-κB and NLRP3 inflammasome activation, both of which serve as upstream regulators of the inflammatory cascade. Molecular docking and MDs showed strong binding interactions between catalpol and key targets such as NF-κB, NLRP3, and IL-1β. Conclusion: Our findings support the idea that catalpol holds the potential to alleviate neuroinflammation, and it is achieved by inhibiting the activation of NLRP3 inflammasome and NF-κB, ultimately leading to the downregulation of pro-inflammatory cytokines. Catalpol emerges as a promising candidate for the treatment of neuroinflammatory conditions.

Moderate mechanical stimulation antagonizes inflammation of annulus fibrosus cells through YAP-mediated suppression of NF-κB signaling.

Intervertebral disc degeneration (IDD) is a leading cause of low back pain. The inflammatory responses caused by aberrant mechanical loading are one of the major factors leading to annulus fibrosus (AF) degeneration and IDD. Previous studies have suggested that moderate cyclic tensile strain (CTS) can regulate anti-inflammatory activities of AF cells (AFCs), and Yes-associated protein (YAP) as a mechanosensitive coactivator senses diverse types of biomechanical stimuli and translates them into biochemical signals controlling cell behaviors. However, it remains poorly understood whether and how YAP mediates the effect of mechanical stimuli on AFCs. In this study, we aimed to investigate the exact effects of different CTS on AFCs as well as the role of YAP signaling involving in it. Our results found that 5% CTS inhibited the inflammatory response and promoted cell growth through inhibiting the phosphorylation of YAP and nuclear localization of NF-κB, while 12% CTS had a significant proinflammatory effect with the inactivation of YAP activity and the activation of NF-κB signaling in AFCs. Furthermore, moderate mechanical stimulation may alleviate the inflammatory reaction of intervertebral discs through YAP-mediated suppression of NF-κB signaling in vivo. Therefore, moderate mechanical stimulation may serve as a promising therapeutic approach for the prevention and treatment of IDD.

Targeted blockade of interleukin-8 negates metastasis and chemoresistance via Akt/Erk-NFκB axis in oral cancer

BackgroundThe tumor microenvironment plays a significant role in tumor growth, metastasis and chemoresistance via dysregulated signaling pathways. Toward this, an inflammatory chemokine, interleukin-8 (IL-8), is overexpressed in various cancers and is involved in tumor progression and chemoresistance. However, the mechanistic role of IL-8 in mediating metastasis and chemoresistance in oral squamous cell carcinoma (OSCC) is not known. Methods and resultsIn the present study, we evaluated the effect of IL-8 in regulating metastasis as well as chemoresistance in OSCC cell lines. For this, IL-8 was blocked exogenously using neutralizing IL-8 monoclonal antibody and IL-8 levels were enhanced by exogenous supply of recombinant human IL-8 (rhIL-8) to OSCC cells. The epithelial-to-mesenchymal transition (EMT) was evaluated using qPCR, migration by scratch/wound healing assay and invasion ability using transwell assay. rIL-8 induced chemoresistance was studied by apoptosis assay and the nuclear localization of NFκB using immunocytochemistry. IL-8 was significantly overexpressed in OSCC patients and cell lines. While exogenous blockade of IL-8 significantly reduced EMT, migration and invasion potential in OSCC cells, IL-8 overexpression upregulated these cellular traits thereby confirming the role of IL-8 in OSCC metastasis. Exogenous blockade of IL-8 also reversed chemoresistance in cisplatin resistant OSCC subline via NFκB signaling. ConclusionIL-8 plays a crucial role in OSCC metastasis and its targeted blockade can help in management of cisplatin resistance.

Maslinic acid attenuates UVB-induced oxidative damage in HFF-1 cells.

Oxidative damage is one of the major mechanisms of ultraviolet B (UVB)-induced damage to the skin. Maslinic acid (MA) is a natural compound of pentacyclic triterpene acids. It has been proved to have anti-inflammatory and antioxidant properties. This study aimed to explore the effects of MA on oxidative damage in human foreskin fibroblast cells (HFF-1) and the potential molecular mechanisms. A specific dose of UVB radiation was used to induce oxidative damage in HFF-1. Based on this, we performed measurements of cell proliferation, reactive oxygen species (ROS) levels, antioxidant enzyme activity, inflammation-related mediators, and NF-κB nuclear localization with or without the addition of MA. MA significantly promoted cell proliferation viability at 10 and 20 μM. The addition of MA 24 h before UVB irradiation was more effective at enhancing cell proliferation and also produced lower ROS levels compared to co-cultured fibroblasts and MA for 24 h after irradiation. However, there was no statistically significant difference between groups at concentrations of 10 and 20 μM. The pretreatment group with MA had elevated superoxide dismutase and catalase activities, decreased IL-6 generation, and lowered mRNA levels of IL-6, TNF-α and MMP3 in comparison with the UVB-irradiated group without additional MA. Meanwhile, the nuclear translocation of NF-κB and the degradation of IκB were inhibited by MA pretreatment. Taken together, these findings suggest that MA may alleviate UVB-induced oxidative damage in HFF-1 by inhibiting the nuclear translocation of NF-κB.

Sichen Formula Ameliorates Lipopolysaccharide-Induced Acute Lung Injury via Blocking the TLR4 Signaling Pathways.

Sichen (SC) formula is a classic prescription of Tibetan medicine. Due to its potential anti-inflammatory effect, the SC formula has been clinically used to treat respiratory diseases for many years in the Chinese Tibet region. The present study aimed to investigate the anti-inflammatory effect of SC and explore the underlying mechanisms. SC formula was characterized by HPLC analysis. The acute lung injury (ALI) mouse model was induced by direct intratracheal lipopolysaccharide (LPS) instillation, and bronchoalveolar lavage fluid (BALF) and lung tissues were collected. Meanwhile, RAW264.7 macrophages were stimulated by LPS. The contents of inflammatory mediators in the culture medium were determined by ELISA. Protein levels were determined by immunohistochemical staining or Western blotting. Nuclear localization of NF-κB, AP-1, and IRF3 was performed using immunofluorescence and Western blotting. In the LPS-induced ALI mouse model, SC treatment suppressed the secretion of inflammatory mediators (TNF-α, IL-6, IL-1β, MCP-1, MIP-1α, and RANTES) in BALF. SC treatment hindered the recruitment of macrophages. SC treatment also inhibited the expression of CD68, p-p65, and TLR4 in the lung tissue. In the LPS-exposed RAW264.7 cells, the cell viability was not changed up to 400 μg/mL of SC. SC concentration-dependently suppressed the production of nitric oxide, prostaglandin E2, TNF-α, IL-6, MCP-1, MIP-1α, and RANTES in LPS-challenged RAW264.7 cells. The expression levels of iNOS, COX-2, p-p38, p-JNK, p-ERK, p-TBK1, p-IKKα/β, p-IκB, p-p65, p-c-Jun, and p-IRF3 were decreased after SC treatment. Moreover, the nuclear translocation of p65, c-Jun, and IRF3 was also blocked by SC treatment. SC treatment inhibited the inflammatory responses in LPS-induced ALI mouse model/RAW264.7 macrophages. The underlying mechanism of this action may be closely associated with the suppression of TLR4 signaling pathways. These research findings provide further pharmacological justifications for the medicinal use of SC in the management of respiratory diseases.

Abstract 6162: Histone deacetylase inhibitor modulates T cell recruitment to metastatic pancreatic neuroendocrine tumors by CCR5 regulation

Abstract Pancreatic neuroendocrine tumors (PNETS) represent the second most common pancreatic neoplasm with surgical resection serving as the mainstay of treatment for localized PNETs (lPNETs) > 2 cm in size; however, therapeutic options for managing metastatic PNETs (mPNETs) remain limited. Recently, immunotherapy with checkpoint inhibitors has emerged as effective therapy across a range of malignancies but has shown limited efficacy in PNET patients partly due to what has been reported as a “cold” tumor microenvironment (TME). Here, we aimed to characterize the TME among both localized and mPNETs and identify mechanisms to increase tumor immunogenicity and T cell recruitment among these patients. We analyzed tumor-infiltrating and peripheral T cells from patients with lPNETs and mPNETs by flow cytometry and found higher expression of CD69+ (activation), CCR5+ (recruitment), and PD1+ (exhaustion) in T cells isolated from TME compared to peripheral blood. Further, we identified a more profound degree of CD3+ and CD8+ T cell infiltration among mPNETs compared to lPNETs by immunohistochemistry analysis, suggesting that mPNETs are at least moderately enriched with activated T cells. RNA-sequencing performed on 22 sporadic, well-differentiated primary PNETs revealed distinct transcriptional signatures dependent upon metastatic potential. These divergent transcriptomes were analyzed by an L1000 computational algorithm, which classifies landmark gene signatures perturbed by drug treatments. We identified the histone deacetylase inhibitor vorinostat as the drug most likely to perturb the PNET gene signature. A KEGG pathway analysis revealed that these vorinostat-target genes were commonly involved in both Th1, Th2, and Th17 cell differentiation and in the NF-κB pathway. Vorinostat treatment of patient-derived mPNET resulted in increased T cell migration towards metastatic tumors across a semi-permeable membrane; cotreatment with a competitive CCR5 antagonist abrogated this effect. In addition, vorinostat treatment of BON-1 and QGP-1, two PNET cell lines, elevated both NF-κB activity and CCR5 expression, with induction reduced following inhibition of NF-κB nuclear localization. Our study demonstrates that T cell recruitment toward mPNET tissue is dependent upon CCR5 expression. These results reveal that PNET TME is regulatable by vorinostat and opens an avenue of potential sensitization to immune checkpoint blockade among patients with PNETs. Citation Format: Jacques A. Greenberg, Jessica N. Limberg, Akanksha Verma, David Kim, Maureen D. Moore, Timothy M. Ullmann, Jessica W. Thiesmeyer, Zachary Loewenstein, Kevin J. Chen, Caitlin E. Egan, Dessislava I. Stefanova, Rohan Bareja, Rasa Zarnegar, Brendan M. Finnerty, Theresa Scognamiglio, Olivier Elemento, Thomas J. Fahey, Irene M. Min. Histone deacetylase inhibitor modulates T cell recruitment to metastatic pancreatic neuroendocrine tumors by CCR5 regulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6162.

Endurance exercise training suppresses myostatin upregulation and nuclear factor-kappa B activation in a mouse model of Parkinson's disease.

Background and Aim:Muscle atrophy is common in Parkinson’s disease (PD). Although myostatin has been implicated in muscle atrophy, its expression in PD skeletal muscle has not been investigated. Therefore, this study aimed to elucidate the influence of PD induction and exercise training on myostatin expression in the gastrocnemius skeletal muscle.Materials and Methods:Thirty albino mice were randomly selected and separated into three groups of 10 mice each: Sedentary control, sedentary PD (SPD), and exercised PD (EPD). 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine and probenecid were used to induce chronic parkinsonism in the PD groups. Immunohistochemistry was used to investigate the expression of myostatin and nuclear factor kappa B (NF-kB) in gastrocnemius muscles of all three groups.Results:Myostatin expression and NF-kB nuclear localization, indicative of its activation, were significantly (p<0.01) higher in gastrocnemius skeletal muscle in the SPD group than in the control and EPD groups. Concomitantly, the average cross-sectional area of gastrocnemius muscle fibers in the SPD albino mice was significantly smaller (p<0.01) than in the control and EPD groups, indicating muscle atrophy.Conclusion:The present data are the first to indicate a correlation between PD induction and myostatin overexpression and NF-kB activation in the gastrocnemius muscle, potentially promoting the muscle atrophy commonly seen in PD. Additionally, the current data are the first to indicate the beneficial effects of exercise training on PD-associated myostatin overexpression, NF-κB activation, and muscle atrophy. Thus, our data are the first to suggest that myostatin and NF-κB might be regarded as potential therapeutic targets in an attempt to ameliorate skeletal muscle abnormalities commonly observed in PD.

HSP60 regulates the cigarette smoke-induced activation of TLR4-NF-κB-MyD88 signalling pathway and NLRP3 inflammasome

Chronic obstructive pulmonary disease (COPD) is characterized by increased cellular stress and inflammation. Heat shock protein 60 (HSP60) is a highly conserved stress protein that acts as a cellular “danger” signal for immune reactions. In this study, we investigated the role of HSP60 in COPD and explored the underlying mechanisms. Expression levels of HSP60 in patients with acute exacerbation of COPD (AECOPD), stable COPD, and healthy people were detected by Western blotting and enzyme-linked immunosorbent assay (ELISA). Moreover, the effect and molecular mechanism of HSP60 in COPD were studied in cigarette smoke (CS)-treated C57BL/6 mice and macrophages. The results showed significant upregulation of HSP60 expression in the peripheral blood mononuclear cells (PBMCs) and sera of patients with AECOPD compared to those with stable COPD or healthy people. CS induced the expression of HSP60 in the COPD mouse model, accelerated the activation of toll-like receptor 4 (TLR4) and NLR family pyrin domain containing 3 (NLRP3) signalling pathways, promoted the increase of inflammatory cells in alveolar lavage fluid and serum inflammatory factors, and induced destruction of lung tissue structure. Furthermore, HSP60 knockdown affected TLR4 and MyD88 expression, IκBα degradation, and nuclear localization of NF-κB and NLRP3 inflammasome activity. Our study revealed that CS stimulates the expression of HSP60, activating the TLR4-MyD88-NF-κB signalling pathway and the NLRP3 inflammasome.

Mitochondrial STAT3 exacerbates LPS-induced sepsis by driving CPT1a-mediated fatty acid oxidation.

Rationale: We found that a subset of signal transducer and activator of transcription 3 (STAT3) translocated into mitochondria in phagocytes, including macrophages isolated from individuals with sepsis. However, the role of mitochondrial STAT3 in macrophages remains unclear.Method: To investigate the function of mitochondrial STAT3 in vivo, we generated inducible mitochondrial STAT3 knock-in mice. A cytokine array analysis, a CBA analysis, flow cytometry, immunofluorescence staining and quantification and metabolic analyses in vivo were subsequently performed in an LPS-induced sepsis model. Single-cell RNA sequencing, a microarray analysis, metabolic assays, mass spectrometry and ChIP assays were utilized to gain insight into the mechanisms of mitochondrial STAT3 in metabolic reprogramming in LPS-induced sepsis.Results: We found that mitochondrial STAT3 induced NF-κB nuclear localization and exacerbated LPS-induced sepsis in parallel with a metabolic switch from mainly using glucose to an increased reliance on fatty acid oxidation (FAO). Moreover, mitochondrial STAT3 abrogated carnitine palmitoyl transferase 1a (CPT1a) ubiquitination and degradation in LPS-treated macrophages. Meanwhile, an interaction between CPT1a and ubiquitin-specific peptidase 50 (USP50) was observed. In contrast, knocking down USP50 decreased CPT1a expression and FAO mediated by mitochondrial STAT3. The ChIP assays revealed that NF-κB bound the USP50 promoter. Curcumin alleviated LPS-mediated sepsis by suppressing the activities of mitochondrial STAT3 and NF-κB.Conclusion: Our findings reveal that mitochondrial STAT3 could trigger FAO by inducing CPT1a stabilization mediated by USP50 in macrophages, at least partially.

δPKC-Mediated DRP1 Phosphorylation Impacts Macrophage Mitochondrial Function and Inflammatory Response to Endotoxin.

Recent studies have demonstrated that alterations in mitochondrial dynamics can impact innate immune function. However, the upstream mechanisms that link mitochondrial dynamics to innate immune phenotypes have not been completely elucidated. This study asks if Protein Kinase C, subunit delta (δPKC)-mediated phosphorylation of dynamin-related protein 1 (Drp1), a key driver of mitochondrial fission, impacts macrophage pro-inflammatory response following bacterial-derived lipopolysaccharide (LPS) stimulation. Using RAW 264.7 cells, bone marrow-derived macrophages from C57BL/6J mice, as well as human monocyte-derived macrophages, we first characterized changes in δPKC-mediated phosphorylation of Drp1 following LPS stimulation. Next, using rationally designed peptides that inhibit δPKC activation (δV1-1) and δPKC-Drp1 interaction (ψDrp1), we determined whether δPKC-mediated phosphorylation of Drp1 impacts LPS-induced changes in mitochondrial morphology, mitochondrial function, and inflammatory response. Our results demonstrated that δPKC-dependent Drp1 activation is associated with increased mitochondrial fission, impaired cellular respiration, and increased mitochondrial reactive oxygen species in LPS-treated macrophages. This is reversed using a rationally designed peptide that selectively inhibits δPKC phosphorylation of Drp1 (ψDrp1). Interestingly, limiting excessive mitochondrial fission using ψDrp1 reduced LPS-triggered pro-inflammatory response, including a decrease in NF-κB nuclear localization, decreased iNOS induction, and a reduction in pro-inflammatory cytokines (IL-1β, TNFα, IL-6). These data suggest that inhibiting Drp1 phosphorylation by δPKC abates the excessive mitochondrial fragmentation and mitochondrial dysfunction that is seen following LPS treatment. Furthermore, these data suggest that limiting δPKC-dependent Drp1 activation decreases the pro-inflammatory response following LPS treatment. Altogether, δPKC-dependent Drp1 phosphorylation might be an upstream mechanistic link between alterations in mitochondrial dynamics and innate immune phenotypes, and may have therapeutic potential.

Isoorientin Attenuated the Pyroptotic Hepatocyte Damage Induced by Benzo[a]pyrene via ROS/NF-κB/NLRP3/Caspase-1 Signaling Pathway.

Isoorientin (Iso), a natural bioactive flavonoid, possesses significant anti-tumor and anti-oxidant activities. Benzo[a]pyrene (BaP) is a food processing injurant with carcinogenicity, teratogenicity, and genotoxicity. Our preliminary study demonstrates that Iso attenuated the pyroptotic hepatocyte damage induced by BaP; however, the molecular mechanism remains unknown. The present study showed that Iso reduced the increase caused by BaP in the overflow of LDH, NO, and the electrical conductivity and the protein expressions of GSDMD-N, IL-18, and IL-1β, further showing that Iso could reduced the pyroptotic damage in HL-7702 cells induced by BaP. Caspase-1 inhibitor (Z-VAD-FMK) inhibited the characteristic pyroptosis protein expressions of Caspase-1, GSDMD-N, IL-18, and IL-1β, showing that the classic pyroptosis pathway depending on Caspase-1 was caused by BaP in HL-7702 cells. Consistent with the effects of the NLRP3 inhibitor (MCC950), NF-κB inhibitor (PDTC), ROS, and mtROS inhibitor (NAC and Mito-TEMPO), Iso weakened the stimulatory effects of BaP on the levels of ROS, the nuclear localization of NF-κB, and the activation of NLRP3 inflammasome and the characteristic indices of pyroptosis, demonstrating that Iso could alleviate the BaP-induced pyroptotic hepatocytes injury through inhibiting the ROS/NF-κB/NLRP3/Caspase-1 signaling pathway, which provides a new perspective and strategy to prevent liver injury induced by BaP.

Zinc Deficiency Drives Renal NFkB Activation

Background Zn2+ deficiency (ZnD) is a worldwide problem. In the United States, 14% of Americans are Zn2+ deficient, which represents 1 out of 7 people. In ZnD populations, the prevalence of hypertension is higher. In our recent studies, we demonstrated that ZnD induces hypertension by promoting renal Na+ reabsorption by the sodium chloride co-transporter (NCC). However, the exact molecular mechanisms involved in NCC upregulation were undefined. Nuclear factor-κB (NFκB) is a transcription factor found to play a role in ZnD-mediated detrimental effects throughout the body. Hypothesis As such, we hypothesized that ZnD drives renal NFκB activation. Experimental Design To examine the effects of ZnD on renal NFκB activation, adult, male C57Bl/6 mice were fed a ZnA- or ZnD-diet for 6 weeks. NFκB expression and nuclear translocation were examined by immunohistochemistry. To confirm the role of Zn2+ in NFκB regulation, mouse distal convoluted tubular (mDCT) cells were treated with the Zn2+ chelator - N,N,Nʹ,Nʹ-tetrakis(2-pyridylmethyl)ethane-1,2-diamine (TPEN) or vehicle ± Zn2+ supplementation. Results In ZnD mice, NFκB protein expression and nuclear localization were increased compared to ZnA mice. Consistently, in mDCT cells, TPEN-induced ZnD stimulated NFκB expression and nuclear translocation. However, Zn2+ supplementation reversed TPEN-induced NFκB upregulation. Conclusion These results indicate that 1) NFκB is a Zn2+-regulated nuclear transcription factor, and 2) ZnD drives renal NFkB activation. Significance NFκB represents a potential mediator that drives ZnD-induced NCC upregulation and consequently renal Na+ reabsorption and hypertension.

Zinc Deficiency‐Induced NFκB Activation Promotes Hypertension by Driving Renal Na + Reabsorption

Background : Zinc deficiency (ZnD) is a common comorbidity with chronic disorders including diabetes and kidney disease. Patients with these diseases also have a higher incidence of hypertension. Recently, we reported that a zinc deficient diet is sufficient to induce hypertension in mice. Changes in blood pressure were accompanied by increased Na+ reabsorption via the renal sodium chloride cotransporter (NCC). Although our novel results indicate that zinc plays a critical role in NCC regulation and its downstream effects on blood pressure, the specific mechanisms involved are unknown. Hypothesis : Since nuclear factor-κB (NFκB) is a key transcription factor implicated in ZnD-mediated effects, we tested the hypothesis that ZnD stimulates renal NFκB activation which drives NCC upregulation and thereby promotes hypertension. Experimental Design : Adult, male C57BL/6 mice were fed a zinc adequate (ZnA; 50ppm) or a ZnD (1ppm) diet for 6 weeks. To examine the role of NFκB in ZnD-induced hypertension, mice were switched from a ZnA-diet to a ZnD-diet for 5 weeks. After, ZnD mice were administered caffeic acid phenethyl ester (CAPE), an NFκB inhibitor, for 1 week. In mice, NCC expression (qRT-PCR) and blood pressure (tail-cuff) were measured. To further investigate the role of NFκB in NCC expression (qRT-PCR and Western blot), mouse distal convoluted tubule (mDCT) cells were treated with TPEN (a zinc chelator) or vehicle (DMSO) ± CAPE. Results : Compared to ZnA mice, NCC mRNA abundance was increased in ZnD mice (7.625 ± 1.747 vs 1.0 ± 0.127) and was accompanied by enhanced renal NFκB nuclear localization. In mDCT cells, TPEN-induced ZnD stimulated NCC mRNA expression (3.152 ± 0.966 vs 1.0 ± 0.174) and NFκB upregulation (1.573 ± 0.117 vs 1.0 ± 0). However, NFκB inhibition by CAPE prevented TPEN-induced NCC mRNA (0.636 ± 0.178 vs 3.152 ± 0.966) and protein (1.039 ± 0.018 vs 2.335 ± 0.062) expression in mDCT cells. Finally, CAPE administration lowered blood pressure (154.5 ± 5.42 vs 138.1 ± 1.97 mm Hg) in ZnD mice. Summary : These results demonstrate that 1) renal NFκB is a Zn2+-regulated transcription factor, 2) NFκB is involved in NCC regulation and 3) NFκB mediates ZnD-induced blood pressure increases. Significance : Taken together, these data indicate that ZnD stimulates NFκB activation thereby driving NCC upregulation and hypertension.

Analysis of Calcium Control of Canonical NF-κB Signaling in B Lymphocytes.

The central role of calcium (Ca2+) signaling in lymphocyte development and acquisition of functional immunity and tolerance is well established. Ca2+ signals are initiated upon antigen binding to cognate receptors on lymphocytes that trigger store operated Ca2+ entry (SOCE). The underlying mechanism of SOCE in lymphocytes involves TCR and BCR mediated activation of Stromal Interaction Molecule 1 and 2 (STIM1/2) embedded in the ER membrane. Once activated, STIM proteins oligomerize and re-localize to ER domains juxtaposed to the plasma membrane where they activate Orai channels to allow Ca2+ to enter the cell across the plasma membrane. Importantly, STIM/Orai-dependent Ca2+ signals guide antigen induced lymphocyte development and function principally by regulating the activity of transcription factors.The most widely studied of these transcription factors is the Nuclear Factor of Activated T cells (NFAT). NFAT is expressed ubiquitously and the mechanism by which Ca2+ regulates NFAT activation and signaling is well known. By contrast, a mechanistic understanding of how Ca2+ signals also shape the activation and specificity of NF-κB to control the expression of pro-inflammatory genes has lagged. Here we discuss the methodology used to investigate Ca2+ dependent mechanisms of NF-κB activation in lymphocytes. Our approach focuses on three main areas of signal transduction and signaling: (1) antigen receptor engagement and Ca2+ dependent initiation of NF-kB signaling, (2) Ca2+ dependent induction of NF-κB heterodimer activation and nuclear localization, and (3) and how Ca2+ regulates NF-κB dependent expression of target genes and proteins.

HSP60 Regulates Monosodium Urate Crystal-Induced Inflammation by Activating the TLR4-NF-κB-MyD88 Signaling Pathway and Disrupting Mitochondrial Function.

Acute gout is an inflammatory response induced by monosodium urate (MSU) crystals. HSP60 is a highly conserved stress protein that acts as a cellular “danger” signal for immune reactions. In this study, we aimed to investigate the role and molecular mechanism of HSP60 in gout. HSP60 expression was detected in peripheral blood mononuclear cells (PBMCs) and plasma of gout patients. The effect and molecular mechanism of HSP60 in gout were studied in MSU crystals treatment macrophages and C57BL/6 mice. JC-1 probe and MitoSOX Red were used to measure the mitochondrial membrane potential (MMP) and mitochondrial reactive oxygen species (mtROS). HSP60 expression was significantly upregulated in the PBMCs and sera of patients with acute gout (AG) compared to those with intercritical gout (IG) or healthy controls (HCs). MSU crystals induced the expression and secretion of HSP60 in the macrophages. HSP60 knockdown or overexpression affects TLR4 and MyD88 expression, IκBα degradation, and the nuclear localization of NF-κB in MSU crystal-stimulated inflammation. Further, HSP60 facilitates MMP collapse and mtROS production and activates the NLRP3 inflammasome in MSU crystal-stimulated macrophages. In MSU crystal-induced arthritis mouse models pretreated with HSP60 vivo-morpholino, paw swelling, myeloperoxidase (MPO) activity, and inflammatory cell infiltration significantly decreased. Our study reveals that MSU crystal stimulates the expression of HSP60, which accelerates the TLR4-MyD88-NF-κB signaling pathway and exacerbates mitochondrial dysfunction.

NF-κB and STAT3 co-operation enhances high glucose induced aggressiveness of cholangiocarcinoma cells

AimsThe present report aimed to investigate the underlying genes and pathways of high glucose driving cholangiocarcinoma (CCA) aggressiveness. Main methodsWe screened and compared the gene expression profiles obtained by RNA sequencing, of CCA cells cultured in high and normal glucose. Results from the transcriptomic analysis were confirmed in additional cell lines using in vitro migration-invasion assay, Western blotting and immunocytofluorescence. Key findingsData indicated that high glucose increased the expression of interleukin-1β (IL-1β), an upstream regulator of nuclear factor-κB (NF-κB) pathway, through the nuclear localization of NF-κB. High glucose-induced NF-κB increased the migration and invasion of CCA cells and the expression of downstream NF-κB targeted genes associated with aggressiveness, including interleukin-6, a potent triggering signal of the signal transducer and activator of transcription 3 (STAT3) pathway. Such effects were reversed by inhibiting NF-κB nuclear translocation which additionally reduced the phosphorylation of STAT3 at Y705. SignificanceThese results indicate that NF-κB is activated by high glucose and they suggest that NF-κB interaction with STAT3 enhances CCA aggressiveness. Therefore, targeting multiple pathways such as STAT3 and NF-κB might improve CCA treatment outcome especially in condition such as hyperglycemia.