NF-κB Dependent Gene Expression Research Articles

Inhibition of USP14 promotes TNFα-induced cell death in head and neck squamous cell carcinoma (HNSCC)

TNFα is a key mediator of immune, chemotherapy and radiotherapy-induced cytotoxicity, but several cancers, including head and neck squamous cell carcinomas (HNSCC), display resistance to TNFα due to activation of the canonical NFκB pro-survival pathway. However, direct targeting of this pathway is associated with significant toxicity; thus, it is vital to identify novel mechanism(s) contributing to NFκB activation and TNFα resistance in cancer cells. Here, we demonstrate that the expression of proteasome-associated deubiquitinase USP14 is significantly increased in HNSCC and correlates with worse progression free survival in Human Papillomavirus (HPV)- HNSCC. Inhibition or depletion of USP14 inhibited the proliferation and survival of HNSCC cells. Further, USP14 inhibition reduced both basal and TNFα-inducible NFκB activity, NFκB-dependent gene expression and the nuclear translocation of the NFκB subunit RELA. Mechanistically, USP14 bound to both RELA and IκBα and reduced IκBα K48-ubiquitination leading to the degradation of IκBα, a critical inhibitor of the canonical NFκB pathway. Furthermore, we demonstrated that b-AP15, an inhibitor of USP14 and UCHL5, sensitized HNSCC cells to TNFα-mediated cell death, as well as radiation-induced cell death in vitro. Finally, b-AP15 delayed tumor growth and enhanced survival, both as a monotherapy and in combination with radiation, in HNSCC tumor xenograft models in vivo, which could be significantly attenuated by TNFα depletion. These data offer new insights into the activation of NFκB signaling in HNSCC and demonstrate that small molecule inhibitors targeting the ubiquitin pathway warrant further investigation as a novel therapeutic avenue to sensitize these cancers to TNFα- and radiation-induced cytotoxicity.

Single-cell RNA sequencing uncovers the nuclear decoy lincRNA PIRAT as a regulator of systemic monocyte immunity during COVID-19

The systemic immune response to viral infection is shaped by master transcription factors, such as NF-κB, STAT1, or PU.1. Although long noncoding RNAs (lncRNAs) have been suggested as important regulators of transcription factor activity, their contributions to the systemic immunopathologies observed during SARS-CoV-2 infection have remained unknown. Here, we employed a targeted single-cell RNA sequencing approach to reveal lncRNAs differentially expressed in blood leukocytes during severe COVID-19. Our results uncover the lncRNA PIRAT (PU.1-induced regulator of alarmin transcription) as a major PU.1 feedback-regulator in monocytes, governing the production of the alarmins S100A8/A9, key drivers of COVID-19 pathogenesis. Knockout and transgene expression, combined with chromatin-occupancy profiling, characterized PIRAT as a nuclear decoy RNA, keeping PU.1 from binding to alarmin promoters and promoting its binding to pseudogenes in naïve monocytes. NF-κB-dependent PIRAT down-regulation during COVID-19 consequently releases a transcriptional brake, fueling alarmin production. Alarmin expression is additionally enhanced by the up-regulation of the lncRNA LUCAT1, which promotes NF-κB-dependent gene expression at the expense of targets of the JAK-STAT pathway. Our results suggest a major role of nuclear noncoding RNA networks in systemic antiviral responses to SARS-CoV-2 in humans.

Herpes Simplex Virus Type 2 Glycoprotein D Inhibits NF-κB Activation by Interacting with p65.

NF-κB plays a crucial role in regulating cell proliferation, inflammation, apoptosis, and immune responses. HSV type 2 (HSV-2) is one of the most predominant sexually transmitted pathogens worldwide, and its infection increases the risk of HIV type 1 (HIV-1) acquisition and transmission. HSV-2 glycoprotein D (gD), highly homologous to HSV-1 gD, is essential for viral adhesion, fusion, entry, and spread. It is known that HSV-1 gD can bind herpesvirus entry mediator (HVEM) to trigger NF-κB activation and thereby facilitate viral replication at the early stage of infection. In this study, we found that purified HSV-2 gD triggered NF-κB activation at the early stage of infection, whereas ectopic expression of HSV-2 gD significantly downregulated TNF-α-induced NF-κB activity as well as TNF-α-induced IL-6 and IL-8 expression. Mechanistically, HSV-2 gD inhibited NF-κB, but not IFN-regulatory factor 3 (IRF3), activation and suppressed NF-κB activation mediated by overexpression of TNFR-associated factor 2 (TRAF2), IκB kinase α (IKKα), IKKβ, or p65. Coimmunoprecipitation and binding kinetic analyses demonstrated that HSV-2 gD directly bound to the NF-κB subunit p65 and abolished the nuclear translocation of p65 upon TNF-α stimulation. Mutational analyses further revealed that HSV-2 gD interacted with the region spanning aa 19-187 of p65. Findings in this study together demonstrate that HSV-2 gD interacts with p65 to regulate p65 subcellular localization and thereby prevents NF-κB-dependent gene expression, which may contribute to HSV-2 immune evasion and pathogenesis.

Inhibitory effect of Salvia coccinea on inflammatory responses through NF-κB signaling pathways in THP-1 cells and acute rat diabetes mellitus

Hyperglycemia-induced oxidative stress has been implicated in diabetes and its complications. Medicinal plants possessing antioxidant activity may decrease oxidative stress by scavenging radicals and reducing power activity and would be a promising strategy for the treatment of inflammatory disorders like diabetes. This study was designed to evaluate the antioxidant effect of Aqueous Extract of S.coccinea leaf (AESL) in HG treated THP-1 cells and streptozotocin (STZ)-induced diabetic Wistar rats. AESL and the standard antidiabetic drug glibenclamide were administered orally by intragastric tube for 14 days and pre-treated HG grown THP-1 cells. AESL treatment reduced HG induced increase in ROS production, NF-κB dependent proinflammatory gene expression by influencing NF-κB nuclear translocation in THP-1 cells. Oral administration of AESL inhibited STZ-induced increase in serum lipid peroxidation, aspartate transaminase, alanine transaminase, and Lactate dehydrogenase of diabetic rats. Significant increase in activity of superoxide dismutase, catalase and glutathione peroxidase, and a reduced level of glutathione, were observed in AESL treatment. The results demonstrate that AESL is useful in controlling blood glucose and also has antioxidant potential to influence the translocation of NF-κB, protect damage caused by hyperglycemia-induced inflammation.

CD5 regulates the heterogenous depot of NFκB available for T cell activation

Abstract T cells bearing receptors to the same antigen often differ considerably in their ability to respond. We hypothesized that variations in intracellular signaling molecules in T cells may underlie these cell-to-cell heterogeneities. We found that the expression levels of PLCγ and IκBα, varied considerably between individual peripheral T cells. The ~1000-fold variation in IκBα levels, but that not of PLCγ, strictly correlated with surface levels of CD5. CD5 is typically used as a marker for T cell self-reactivity and differences in responses between CD5-high and -low cells is attributed to the self-awareness of the TCR. Our data suggested that CD5 directly regulates the NFκB pathway to modulate T cell responses. Accordingly, ablating CD5 using germline or conditional knock-out mice resulted in a significant reduction in IκBα. This regulation was independent of SHP-1 and casein kinase II and not evident in mRNA levels, suggesting a previously unknown post-translational mechanism. Overexpression of CD5 in TCR-deficient cells also increased basal IκBα levels indicating independence from TCR signaling. Intriguingly, cells with higher CD5 and IκBα expression maintained a proportionally greater amount of NFκBp65 in a cellular reservoir. This suggests that CD5-high T cells with a greater NFκB reservoir might allow for a more robust NFκB-dependent gene expression. Consistent with this postulate, we found that CD5-high thymocytes survived ex vivo better than CD5-low ones, but only when NFκB signaling was intact. These data suggest a key role for CD5 in the control of T cell heterogeneity, independent of the self-reactivity of a TCR itself.

Neonatal dysbiosis has long‐lasting detrimental effects on the microbiota‐gut‐brain axis

The microbiota‐gut‐brain (MGB) axis is essential in maintaining overall health and well‐being. Colonization of the gut microbiota begins at birth and plays a crucial role in the development of the immune system and neural development. Thus, dysbiosis of the developing microbiome can detrimentally impact the developing gut, immune system and brain. Broad spectrum antibiotics (Abx) are commonly prescribed for infants and toddlers whom are more prone to infection with bacterial pathogens than adults. Neonatal Abx administration has been shown to disrupt the gut microbiota and cause metabolic defects, increasing the risk for obesity in adulthood. However, it is not currently known whether neonatal Abx‐induced dysbiosis affects the development of the microbiota‐gut‐brain axis, including the brain and behavior. Clinically, Abx treatment within the first year of life is associated with detrimental effects on children including behavioural difficulties and depression.HypothesisWe hypothesize that neonatal Abx‐induced dysbiosis alters the development of the MGB axis.MethodsNeonatal C57BL/6 mice were treated from post‐natal day 7 (P7) until weaning (P21) with an Abx cocktail of vancomycin (0.5mg/ml), neomycin (1mg/ml) and ampicillin (1mg/ml) by oral gavage (water; vehicle control) in order to induce dysbiosis. Behavior was assessed using (1) light/dark box test (anxiety‐like behavior); (2) novel object recognition (NOR) task (recognition memory); and (3) general locomotor activity (open field test [OFT]) in adult mice (7 weeks). The impact of dysbiosis on intestinal physiology was assessed using Ussing chambers. The microbiota was assessed using 16S sequencing. Gene expression in the hippocampus and pre‐frontal cortex was assessed by qPCR and neurogenesis was accessed using immunohistochemistry (Ki67). The impact of antibiotics on the immune response was assayed using a splenocyte ex vivo model followed by qPCR.ResultsAdult mice neonatally treated with Abx have deficits in cognition and display reduced anxiety‐like behavior compared to vehicle treated controls. Neonatal Abx administration causes persistent intestinal dysbiosis characterized by decreased alpha diversity and increased abundance of Firmicutes. Gut physiology in adult Abx mice was altered with increased conductance, secretory state and FITC flux observed in the ileum alongside increased conductance in the colon. Expression of genes related to neurogenesis (BDNF and ARC) were altered in the hippocampus and PFC of adult Abx brains. Immunohistochemistry indicated that Ki67, a marker for proliferating cells, was increased in the hippocampus of adult Abx mice suggesting that neurogenesis in the hippocampus is altered. Splenocytes isolated from adult Abx mice had reduced NFκB‐dependent gene expression post LPS stimulation suggesting an impaired immune response.ConclusionTaken together this study identifies a long‐lasting impact of neonatal Abx administration on the MGB axis. Persistent dysbiosis leading to impaired intestinal physiology may consequently impact the brain and behaviour, well after completion of the Abx therapy.Support or Funding InformationNIH 1R01AT009365‐01

Anthocyanins reduce inflammation and improve glucose and lipid metabolism associated with inhibiting nuclear factor-kappaB activation and increasing PPAR-γ gene expression in metabolic syndrome subjects

Anthocyanins exhibit antioxidant and anti-inflammatory activities via a multitude of biochemical mechanisms. However, the signaling pathways involved in the actions of anthocyanins against chronic inflammation are not fully understood.The effects of berry-rich anthocyanin supplements (320 mg/day) for four weeks were examined on features of metabolic syndrome components and the expression of PPAR-γ, Nrf2, and NF-κB dependent genes in MetS and healthy subjects. Total RNA was isolated from whole blood with the PAXgene proprietary blood collection system.Four weeks anthocyanin consumption significantly decreased fasting blood glucose (15.7% vs 3.2%), TG (18.2% vs -1.39%), cholesterol (33.5% vs 1.56%) and LDL (28.4% vs -15.6%) in the MetS compared to Control group (P-value < 0.05, 95% CI). There was a significant up regulation in the expression PPAR-γ gene associated with the lipid and glucose metabolism in MetS subjects which negatively correlated (P-value < 0.01) with the change in the FBG (r = -0.488), Cholesterol (r = -0.496), TG (r = -0.513) and LDL (r = -0.519). Moreover, anthocyanin supplementation decreases serum hs-CRP (-36.3% vs 6.25%) in MetS in compared to Control group (P-value < 0.05). Anthocyanin supplementation also down-regulated the expression of NF-κB dependent genes including TNF-α (-28% and -15%), IL-6 (-16.1% and -13.6%), IL-1A (-21.5% and -12.9%), PCAM-1 (-15% and -17.5%), and COX-2(-26% and -27%) in both MetS and Control group respectively (P-value < 0.05).The study results suggested that berry supplements improved selected features of metabolic syndrome and related cardiovascular risk factors. These benefits may be due to the inhibition of NF-κB dependent gene expression and enhancement of PPAR-γ.

N-Substituted Pyrido-1,4-Oxazin-3-Ones Induce Apoptosis of Hepatocellular Carcinoma Cells by Targeting NF-κB Signaling Pathway.

Hepatocellular carcinoma (HCC) is a fatal disease and ranked fifth in cancer related mortality. Persistent activation of NF-κB is responsible for the oncogenesis, metastasis, tumor evasion, anti-apoptosis, angiogenesis and proliferation in HCC. Therefore, designing of chemically novel, biologically potent small molecules that target NF-κB signaling cascade have gained prominent clinical interest. Herein we synthesized a novel class of 4-(substituted)-2H-pyrido[3,2-b][1,4]oxazin-3(4H)-one by reacting 2H-pyrido[3,2-b][1,4]oxazin-3(4H)-one with various alkyl halides by using combustion derived bismuth oxide. We evaluated the antiproliferative efficacy of newly synthesized compounds against HCC cells and identified 4-(4-nitrobenzyl)-2H-pyrido[3,2-b][1,4]oxazin-3(4H)-one (NPO) as lead anticancer agent. In addition, we investigated the effect of NPO on the DNA binding ability of NF-κB and NF-κB regulated luciferase expression in HCC cells. The results demonstrated that NPO can induce significant growth inhibitory effects in HepG2, HCCLM3 and Huh-7 cells in dose and time-dependent manner. Interestingly, NPO induced significant downregulation in p65 DNA binding ability, p65 phosphorylation and subsequent expression of NF-κB dependent luciferase gene expression in diverse HCC cell lines. Further, in silico docking analysis suggested that NPO can show direct physical interaction with NF-κB. Finally, NPO was found to significantly abrogate tumor growth at a dose of 50 mg/kg in an orthotopic mouse model. Thus, we report the potential anticancer effects of NPO as a novel inhibitor of NF-κB signaling pathway in HCC.

Expression analysis of Akirin-2, NFκB-p65 and β-catenin proteins in imatinib resistance of chronic myeloid leukemia

ABSTRACTObjective: Chronic myleoid leukemia (CML) is a myeloproliferative disorder characterized with the constitutive activation of Bcr-Abl tyrosine kinase which is a target for imatinib, the first line treatment option for CML. Constitutive activation of NFκB and β-catenin signaling promotes cellular proliferation and survival and resistance to Imatinib therapy in CML. Akirin-2 is a nuclear protein which is required for NFκB dependent gene expression as a cofactor and has been linked to Wnt/beta-catenin pathway. The purpose of this study is to examine Akirin-2, NFκB and β-catenin in imatinib resistance of CML and to test if any direct physical protein–protein interaction exists between NFkB and both β-catenin and Akirin-2.Methods: RT–PCR and western blot were performed to determine Akirin-2, NFκB-p65 and β-catenin gene and protein expressions, Co-immunoprecipitation and chromatin immunoprecipitation analysis were carried out to detect the direct physical interactions and binding of NFκB-p65 and β-catenin proteins to MDR1 promoter region, respectively.Results: β-catenin and NFκB-p65 proteins bound to DNA promoter regions of MDR1 in imatinib-sensitive and resistant CML cells, whereas any direct protein–protein interaction could not be found between NFκB-p65 and Akirin-2 or β-catenin proteins. Nuclear β-catenin and NFκB-p65 levels increased in imatinib resistance. Moreover, increased Akirin-2 protein accumulation in the nucleus was shown for the first time in imatinib resistant CML cells.Discussion: We show for the first time that Akirin-2 can be a novel biomarker in imatinib resistance. Targeting Akirin-2, NFκB and β-catenin genes may provide an opportunity to overcome imatinib resistance in CML.

A comparative study of three akirin genes from big belly seahorse Hippocampus abdominalis: Molecular, transcriptional and functional characterization

Akirins, members of the NF-κB signaling pathway, are highly conserved nuclear proteins, which regulate gene expression in many physiological processes, including immunity, myogenesis, carcinogenesis, and embryogenesis. The akirin family in teleost fish consists of two to three genes. In the present study, three akirin genes from Hippocampus abdominalis were identified from a transcriptome database and designated as HaAkirin1, HaAkirin2(1), and HaAkirin2(2). The nuclear localization of HaAkirin1 and HaAkirin2(1) was confirmed by subcellular localization analysis. In contrast, diffused localization of HaAkirin2(2) was identified in the nucleus and cytoplasm that confirmed the aberrant nature of the nuclear localization signal. Phylogenetic analysis revealed a closer relationship of HaAkirins with other known teleost akirins. All three HaAkirin transcripts were ubiquitously expressed in all examined tissues with higher expression in ovary tissue. Immune challenge with LPS, poly I:C, and Streptococcus iniae exhibited a significant increase in the expression of all three HaAkirins in kidney and liver tissues. NF-κB luciferase assays revealed that relative luciferase activity was significantly higher for all three HaAkirin genes than mock controls. These results suggest that HaAkirin genes might play a role in regulating NF-κB dependent immune gene expression and their expression could be induced by bacterial and viral pathogen recognition molecular patterns.

Expression of PIM kinases in Reed-Sternberg cells fosters immune privilege and tumor cell survival in Hodgkin lymphoma

AbstractReed-Sternberg (RS) cells of classical Hodgkin lymphoma (cHL) express multiple immunoregulatory proteins that shape the cHL microenvironment and allow tumor cells to evade immune surveillance. Expression of certain immunoregulatory proteins is modulated by prosurvival transcription factors, such as NFκB and STATs. Because these factors also induce expression of the oncogenic PIM1/2/3 serine/threonine kinases, and as PIMs modulate transcriptional activity of NFκB and STATs, we hypothesized that these kinases support RS cell survival and foster their immune privilege. Here, we investigated PIM1/2/3 expression in cHL and assessed their role in developing RS cell immune privilege and survival. PIM1/2/3 were ubiquitously expressed in primary and cultured RS cells, and their expression was driven by JAK-STAT and NFκB activity. Genetic or chemical PIM inhibition with a newly developed pan-PIM inhibitor, SEL24-B489, induced RS cell apoptosis. PIM inhibition decreased cap-dependent protein translation, blocked JAK-STAT signaling, and markedly attenuated NFκB-dependent gene expression. In a cHL xenograft model, SEL24-B489 delayed tumor growth by 95.8% (P = .0002). Furthermore, SEL24-B489 decreased the expression of multiple molecules engaged in developing the immunosuppressive microenvironment, including galectin-1 and PD-L1/2. In coculture experiments, T cells incubated with SEL24-B489-treated RS cells exhibited higher expression of activation markers than T cells coincubated with control RS cells. Taken together, our data indicate that PIM kinases in cHL exhibit pleiotropic effects, orchestrating tumor immune escape and supporting RS cell survival. Inhibition of PIM kinases decreases RS cell viability and disrupts signaling circuits that link these cells with their niches. Thus, PIM kinases are promising therapeutic targets in cHL.

EXPRESSION OF PIM KINASES IN REED‐STERNBERG CELLS FOSTERS IMMUNE PRIVILEGE AND TUMOR CELL SURVIVAL IN HODGKIN LYMPHOMA

Reed-Sternberg (RS) cells of classical Hodgkin lymphoma (cHL) express multiple immunoregulatory proteins that shape the cHL microenvironment and allow tumor cells to evade immune surveillance. Expression of certain immunoregulatory proteins is modulated by prosurvival transcription factors, such as NFκB and STATs. Because these factors also induce expression of the oncogenic PIM1/2/3 serine/threonine kinases, and as PIMs modulate transcriptional activity of NFκB and STATs, we hypothesized that these kinases support RS cell survival and foster their immune privilege. Here, we investigated PIM1/2/3 expression in cHL and assessed their role in developing RS cell immune privilege and survival. PIM1/2/3 were ubiquitously expressed in primary and cultured RS cells, and their expression was driven by JAK-STAT and NFκB activity. Genetic or chemical PIM inhibition with a newly developed pan-PIM inhibitor, SEL24-B489, induced RS cell apoptosis. PIM inhibition decreased cap-dependent protein translation, blocked JAK-STAT signaling, and markedly attenuated NFκB-dependent gene expression. In a cHL xenograft model, SEL24-B489 delayed tumor growth by 95.8% (P = .0002). Furthermore, SEL24-B489 decreased the expression of multiple molecules engaged in developing the immunosuppressive microenvironment, including galectin-1 and PD-L1/2. In coculture experiments, T cells incubated with SEL24-B489-treated RS cells exhibited higher expression of activation markers than T cells coincubated with control RS cells. Taken together, our data indicate that PIM kinases in cHL exhibit pleiotropic effects, orchestrating tumor immune escape and supporting RS cell survival. Inhibition of PIM kinases decreases RS cell viability and disrupts signaling circuits that link these cells with their niches. Thus, PIM kinases are promising therapeutic targets in cHL.

Green Tea Extract Protects Against Hepatic NFκB Activation Along The Gut‐Liver Axis in Diet‐Induced Obese Mice With Nonalcoholic Steatohepatitis By Reducing Endotoxin and TLR4/MyD88 Signaling

Green tea extract (GTE) reduces NFκB‐mediated inflammation during nonalcoholic steatohepatitis (NASH). We hypothesized that its antiinflammatory activities would be mediated along the gut‐liver axis in a Toll‐like receptor‐4 (TLR4)‐dependent manner. Wild‐type (WT) and loss‐of‐function TLR4‐mutant (TLR4m) mice were fed a high‐fat diet containing 0 or 2% (w/w) GTE for 8 wk before assessing NASH, NFκB‐mediated inflammation, TLR4 adaptor proteins [myeloid differentiation 88 (MyD88) and TIR‐domain‐containing adapter‐inducing interferon‐β (TRIF)], circulating endotoxin, and intestinal tight junction proteins. In WT mice, phosphorylation of the NFκB p65 subunit and NFκB‐dependent gene expression (MCP‐1, iNOS, MPO) were lower in those provided GTE, and were not different from the lowered levels in TLR4m mice regardless of GTE. Compared with WT controls, TLR4m mice had lower TLR4 mRNA expression, and its expression was lowered by GTE in both genotypes. Protein expression of TRIF, which mediates MyD88‐independent TLR4 signaling, was unaffected by genotype and GTE. In contrast, MyD88 expression was lower in mice fed GTE regardless of genotype. Serum endotoxin was not exacerbated in TLR4m mice, but was lowered by GTE in both genotypes. Similarly, duodenal and jejunal claudin‐1 (CLDN‐1) mRNA expression were not affected in TLR4m mice, but GTE increased their expression in both genotypes. Serum endotoxin was inversely correlated with duodenal and jejunal CLDN‐1 (rp= −0.58 to −0.43, p&lt;0.05), and positively correlated with expression levels of TLR4 and phosphorylated‐p65 (rp= 0.46–0.57; p&lt;0.05). Thus, GTE protects against inflammation during NASH, likely by limiting gut‐derived endotoxin translocation and TLR4/MyD88‐dependent NFκB activation.Support or Funding InformationSupported by USDA‐NIFA.

OGG1-DNA interactions facilitate NF-\u03baB binding to DNA targets

DNA repair protein counteracting oxidative promoter lesions may modulate gene expression. Oxidative DNA bases modified by reactive oxygen species (ROS), primarily as 7, 8-dihydro-8-oxo-2′-deoxyguanosine (8-oxoG), which is repaired by 8-oxoguanine DNA glycosylase1 (OGG1) during base excision repair (BER) pathway. Because cellular response to oxidative challenge is accompanied by DNA damage repair, we tested whether the repair by OGG1 is compatible with transcription factor binding and gene expression. We performed electrophoretic mobility shift assay (EMSA) using wild-type sequence deriving from Cxcl2 gene promoter and the same sequence bearing a single synthetic 8-oxoG at defined 5′ or 3′ guanine in runs of guanines to mimic oxidative effects. We showed that DNA occupancy of NF-κB present in nuclear extracts from tumour necrosis factor alpha (TNFα) exposed cells is OGG1 and 8-oxoG position dependent, importantly, OGG1 counteracting 8-oxoG outside consensus motif had a profound influence on purified NF-κB binding to DNA. Furthermore, OGG1 is essential for NF-κB dependent gene expression, prior to 8-oxoG excised from DNA. These observations imply that pre-excision step(s) during OGG1 initiated BER evoked by ROS facilitates NF-κB DNA occupancy and gene expression.

Mitochondrial ubiquitin ligase activator of NF-κB regulates NF-κB signaling in cells subjected to ER stress.

The nuclear factor-κB (NF-κB) transcription factor family members control various biological processes, such as apoptosis and proliferation. The endoplasmic reticulum(ER) has emerged as a major site of cellular homeostasis regulation. The accumulation of misfolded protein in the ER causes stress and ER stress-induced NF-κB activation to protect cells from apoptosis. In this study, we found a putative ER stress-response element(ERSE) on the promoter of mitochondrial ubiquitin ligase activator of NF-κB(MULAN), and that MULAN expression was upregulated by ER stress. MULAN specifically activated NF-κB dependent gene expression in an E3ligase activity-dependent manner. The ectopic expression of MULAN induced the nuclear translocation of endogenous p65 and the degradation of IκB. Binding assay revealed that MULAN was associated with transforming growth factorβ-activated kinase(TAK1). The knockdown of MULAN using siRNA inhibited the activation of NF-κB in the cells subjected to ER stress. The findings of our study indicate that MULAN is an E3ligase that regulates NF-κB activation to protect cells from ER stress-induced apoptosis.

Up-regulation of Store-operated Ca2+ Entry and Nuclear Factor of Activated T Cells Promote the Acinar Phenotype of the Primary Human Salivary Gland Cells

The signaling pathways involved in the generation and maintenance of exocrine gland acinar cells have not yet been established. Primary human salivary gland epithelial cells, derived from salivary gland biopsies, acquired an acinar-like phenotype when the [Ca(2+)] in the serum-free medium (keratinocyte growth medium, KGM) was increased from 0.05 mm (KGM-L) to 1.2 mm (KGM-H). Here we examined the mechanism underlying this Ca(2+)-dependent generation of the acinar cell phenotype. Compared with cells in KGM-L, those in KGM-H display enhancement of Orai1, STIM1, STIM2, and nuclear factor of activated T cells 1 (NFAT1) expression together with an increase in store-operated Ca(2+) entry (SOCE), SOCE-dependent nuclear translocation of pGFP-NFAT1, and NFAT-dependent but not NFκB-dependent gene expression. Importantly, AQP5, an acinar-specific protein critical for function, is up-regulated in KGM-H via SOCE/NFAT-dependent gene expression. We identified critical NFAT binding motifs in the AQP5 promoter that are involved in Ca(2+)-dependent up-regulation of AQP5. These important findings reveal that the Ca(2+)-induced switch of salivary epithelial cells to an acinar-like phenotype involves remodeling of SOCE and NFAT signaling, which together control the expression of proteins critically relevant for acinar cell function. Our data provide a novel strategy for generating and maintaining acinar cells in culture.

Alteration of cytokine profile following hemorrhagic shock

Hemorrhage is one of the leading causes of death in patients with trauma. We recently demonstrated that resveratrol can improve cardiac function and prolong life following severe hemorrhagic injury (HI) in a rat model. The present work is focused on determining changes in NF-κB dependent gene expression in the heart and the systemic cytokine milieu following HI and the effect of resveratrol treatment. The results indicate an increase in phosphorylated NF-κB in the heart with a concomitant increase in the expression of NF-κB dependent genes following HI. There was also a significant increase of systemic cytokine levels, both pro and anti-inflammatory, following HI and resolution when treated with resveratrol. This study demonstrates the potential role NF-κB has in the physiological response to HI and the effectiveness of resveratrol in reducing immune activation.

Tubular Epithelial NF-κB Activity Regulates Ischemic AKI.

NF-κB is a key regulator of innate and adaptive immunity and is implicated in the pathogenesis of AKI. The cell type-specific functions of NF-κB in the kidney are unknown; however, the pathway serves distinct functions in immune and tissue parenchymal cells. We analyzed tubular epithelial-specific NF-κB signaling in a mouse model of ischemia-reperfusion injury (IRI)-induced AKI. NF-κB reporter activity and nuclear localization of phosphorylated NF-κB subunit p65 analyses in mice revealed that IRI induced widespread NF-κB activation in renal tubular epithelia and in interstitial cells that peaked 2-3 days after injury. To genetically antagonize tubular epithelial NF-κB activity, we generated mice expressing the human NF-κB super-repressor IκBαΔN in renal proximal, distal, and collecting duct epithelial cells. Compared with control mice, these mice exhibited improved renal function, reduced tubular apoptosis, and attenuated neutrophil and macrophage infiltration after IRI-induced AKI. Furthermore, tubular NF-κB-dependent gene expression profiles revealed temporally distinct functional gene clusters for apoptosis, chemotaxis, and morphogenesis. Primary proximal tubular cells isolated from IκBαΔN-expressing mice and exposed to hypoxia-mimetic agent cobalt chloride exhibited less apoptosis and expressed lower levels of chemokines than cells from control mice did. Our results indicate that postischemic NF-κB activation in renal tubular epithelia aggravates tubular injury and exacerbates a maladaptive inflammatory response.

Expression of PIM Kinases in Reed-Sternberg Cells Fosters Immune Privilege and Tumor Cell Survival in Classical Hodgkin Lymphoma

The classical Hodgkin lymphoma (cHL) is characterized by a presence of rare malignant Reed-Sternberg (RS) cells surrounded by abundant reactive infiltrate. Tumor RS cells express multiple cytokines, chemokines and immunoregulatory proteins, such as PD-L1 or galectin-1, that enhance recruitment of the infiltrating cells and allow malignant cells to escape from host immune surveillance. Since targeting these immunomodulatory molecules is a highly promising therapeutic strategy in cHL, identification of pathways and mechanisms orchestrating tumor immune evasion and supporting RS cell survival can further reveal targetable vulnerabilities of cHL. Expression of immunoregulatory proteins in cHL is modulated by tumor-specific activity of certain pro-survival transcription factors, such as NFκB and STATs. Since the activity of these transcription factors is modulated by oncogenic PIM1/2/3 serine/threonine kinases, we hypothesized that PIMs may support RS cell survival and foster their immune privilege. For these reasons, we investigated PIM1/2/3 expression in cHL and determined mechanisms underlying their expression. Furthermore, we assessed impact of PIM inhibition on expression of key factors engaged in development of the immunosuppressive microenvironment and HRS cell survival.Our analyses revealed that PIM1/2/3 are ubiquitously expressed in primary and cultured RS cell lines. At least one PIM isoform was expressed in each cell line and in 97% of 67 primary cHL biopsies. RS cells treated with JAK1/2/3 inhibitor exhibited reduced PIM1 and PIM2 levels. Genetic inhibition of canonical NFkB activity with IkB super-repressor or shRNA-mediated ablation of alternative NFkB pathway led to decrease of PIM2 and PIM3, suggesting that PIM-1/2/3 expresion in cHL depends at least in part on JAK-STAT and NFkB activity.To assess the role of PIM kinases in cell viability, we silenced expression of each PIM isoform (individually or simultaneously) in HDLM-2 cells. Knockdowns of individual PIM isoforms were associated with marked increased in remaining isoforms expression and were not associated with toxicity. In marked contrast, downregulation of all three isoforms increased cellular apoptosis by 17%. For this reason, for subsequent studies we used a newly developed pan-PIM inhibitor (SEL24-B489). The inhibitor was toxic to all cells with IC50 ranging from 3-5 µM. To determine mechanisms underlying toxicity, we assessed the activities of specific PIM substrates: 4EBP1, S6, and p65 (RelA). SEL24-B489 rapidly decreased PIM-dependent phosphorylation of these molecules in all tested cell lines. Furthermore, it reduced DNA binding activity of the NFκB-p65 complexes, indicating that PIM kinases modulate NFκB activity in cHL. For this reason, we next assessed the consequences of PIM inhibition on NFκB-dependent transcription. SEL24-B489 significantly downregulated mRNA of NFkB target genes associated with HRS cell survival and proliferation, such as Bfl-1, RelB and CD40. In cells treated with PIM inhibitor SEL24-B489 we also found markedly decreased expression NFkB-dependent cytokines and chemokines specifically shaping pro-tumoral microenvironment, such as IL-8, CCL5 and IL-13. In addition, cell lines exposed to SEL24-B489 treatment exhibited decreased expression of immunomodulatory PD-L1 and Gal-1 proteins. Finally, we investigated the efficacy of SEL24-B489 in vivo in the murine xenograft model using HDLM-2 cells. In contrast to animals exposed to vehicle alone, we observed inhibition of tumor growth by 95,8% in SEL24-B489-treated animals (p=0,0002). Consistent with the in vitro data, we observed strong downregulation of phospho-S6, GAL-1 and PD-L1 proteins in tumor sections from PIM inhibitor-treated animals.Taken together, we demonstrated that the oncogenic PIM-1/2/3 kinases are expressed in RS cells and their activity can be specifically blocked using a pan-PIM inhibitor SEL24-B489. PIM inhibition significantly reduced activity of specific PIM substrates and decreased the expression of NFκB-dependent pro-survival genes and key immunomodulatory proteins. These results highlight the pleiotropic activity of SEL24-B489 and indicate that PIM kinases are promising therapeutic targets in cHL. DisclosuresCzardybon:Selvita S.A.: Employment. Galezowski:Selvita S.A.: Employment. Windak:Selvita S.A.: Employment. Brzozka:Selvita S.A.: Employment.

Overexpression of Sirtuin 6 suppresses cellular senescence and NF-κB mediated inflammatory responses in osteoarthritis development.

The aim of our study was to evaluate if Sirt6, a NAD + dependent histone deacetylase, plays a protective role in cartilage degeneration by suppressing cellular senescence and inflammatory responses. The expression level of sirt6 in normal and OA human knee articular cartilage was compared by immunofluorescence and western blotting. The effect of sirt6 overexpression on replicative senescence of chondrocytes and NF-κB target genes expression was evaluated. Histological assessment of OA mice knee joint was carried out to assess the in vivo effects of sirt6 overexpression on mice chondrocytes. We found sirt6 level was significantly decreased in the articular chondrocytes of OA patients compare to normal human. SA-β-gal staining revealed that overexpression of sirt6 suppressed replicative senescence of chondrocytes. Meanwhile, the expression of NF-κB dependent genes were significantly attenuated by sirt6 overxpression. Safranin-O staining and OARSI score of knee joint cartilage in OA mice revealed that Lenti-Sirt6 intraarticular injection could protect mice chondrocytes from degeneration. These data strongly suggest that overexpression of Sirt6 can prevent OA development by reducing both the inflammatory response and chondrocytes senescence. Therefore, the development of specific activators of Sirt6 may have therapeutic potential for the treatment of OA.