Vibration activates the actin/NF‐κB axis and upregulates IL‐6 and IL‐8 expression in human periodontal ligament cells

We previously reported that mechanical vibration‐induced proinflammatory cytokines, interleukin‐6 (IL‐6) and IL‐8, expression in human periodontal ligament (hPDL) cells, however, the underlying mechanism remained unclear. Mechanical stimuli are able to activate cellular responses by inducing the activation of several signaling pathways including cytoskeletal changes and inflammation. The actin cytoskeleton is a highly dynamic network and plays many important roles in intracellular events. Here, we aimed to investigate the involvement of a pivotal mediator of inflammatory responses, nuclear factor‐κB (NF‐κB), and actin polymerization in vibration‐induced upregulation of IL‐6 and IL‐8 expression in hPDL cells. hPDL cells were pretreated with the NF‐κB inhibitor BAY 11‐7082 or cytochalasin D, respectively, before exposure to vibration. IL‐6 and IL‐8 messenger RNA (mRNA) and protein expression were quantified by quantitative polymerase chain reaction and enzyme‐linked immunosorbent assays, respectively. Subcellular localization of the NF‐κB p65 subunit was visualized by immunofluorescent staining. We found an increase in NF‐κB nuclear translocation in vibrated cells compared with control cells. Pretreatment with BAY 11‐7082 significantly inhibited vibration‐induced IL‐6 and IL‐8 mRNA and protein expression in hPDL cells. Moreover, pretreatment with cytochalasin D inhibited NF‐κB nuclear translocation and attenuated upregulation of IL‐6 and IL‐8 mRNA and protein in vibrated cells. Therefore, modulation of actin cytoskeletal polymerization in response to vibration may activate the NF‐κB signaling pathway and subsequently upregulate IL‐6 and IL‐8 expression in hPDL cells.     

Gastrokine 1 regulates NF‐κB signaling pathway and cytokine expression in gastric cancers

Gastrokine 1 (GKN1) plays an important role in the gastric mucosal defense mechanism and also acts as a functional gastric tumor suppressor. In this study, we examined the effect of GKN1 on the expression of inflammatory mediators, including NF‐κB, COX‐2, and cytokines in GKN1‐transfected AGS cells and shGKN1‐transfected HFE‐145 cells. Lymphocyte migration and cell viability were also analyzed after treatment with GKN1 and inflammatory cytokines in AGS cells by transwell chemotaxis and an MTT assay, respectively. In GKN1‐transfected AGS cells, we observed inactivation and reduced expression of NF‐κB and COX‐2, whereas shGKN1‐transfected HFE‐145 cells showed activation and increased expression of NF‐κB and COX‐2. GKN1 expression induced production of inflammatory cytokines including IL‐8 and ‐17A, but decreased expression of IL‐6 and ‐10. We also found IL‐17A expression in 9 (13.6%) out of 166 gastric cancer tissues and its expression was closely associated with GKN1 expression. GKN1 also acted as a chemoattractant for the migration of Jurkat T cells and peripheral B lymphocytes in the transwell assay. In addition, GKN1 significantly reduced cell viability in both AGS and HFE‐145 cells. These data suggest that the GKN1 gene may inhibit progression of gastric epithelial cells to cancer cells by regulating NF‐κB signaling pathway and cytokine expression. J. Cell. Biochem. 114: 1800–1809, 2013. © 2013 Wiley Periodicals, Inc.     

NF‐κB regulates a cassette of immune/inflammatory genes in human pregnant myometrium at term

The onset of human labour resembles inflammation with increased synthesis of prostaglandins and cytokines. There is evidence from rodent models for an important role for nuclear factor‐κB (NF‐κB) activity in myometrium which both up‐regulates contraction‐associated proteins and antagonizes the relaxatory effects of progesterone. Here we show that in the human, although there are no differences in expression of NF‐κB p65, or IκB‐α between upper‐ or lower‐segment myometrium or before or after labour, there is nuclear localization of serine‐256‐phospho‐p65 and serine‐536‐phospho‐p65 in both upper‐ and lower‐segment myometrium both before and after the onset of labour at term. This shows that NF‐κB is active in both upper and lower segment prior to the onset of labour at term. To identify the range of genes regulated by NF‐κB we overexpressed p65 in myocytes in culture. This led to NF‐κB activation identical to that seen following interleukin (IL)‐1β stimulation, including phosphorylation and nuclear translocation of p65 and p50. cDNA microarray analysis showed that NF‐κB increased expression of 38 genes principally related to immunity and inflammation. IL‐1β stimulation also resulted in an increase in the expression of the same genes. Transfection with siRNA against p65 abolished the response to IL‐1β proving a central role for NF‐κB. We conclude that NF‐κB is active in myocytes in both the upper and lower segment of the uterus prior to the onset of labour at term and principally regulates a group of immune/inflammation associated genes, demonstrating that myocytes can act as immune as well as contractile cells.     

Reactive oxygen species are involved in the IFN‐γ‐stimulated production of Th2 chemokines in HaCaT keratinocytes

The increased generation of reactive oxygen species (ROS) induces inflammation in different cell types. However, it is unclear whether ROS play an essential role in the production of thymus and activation‐regulated chemokine (TARC/CCL17) and macrophage‐derived chemokine (MDC/CCL22) in keratinocytes. Here, we investigated the function of ROS in the production of these two Th2 chemokines in interferon‐gamma (IFN‐γ)‐treated HaCaT keratinocytes. We found that IFN‐γ‐induced production of both chemokines in parallel with the increased generation of intracellular ROS. A ROS scavenger, N‐acetyl cysteine (NAC), significantly inhibited the IFN‐γ‐induced production of chemokines as well as the activation of I kappa‐B (IκB)–nuclear factor‐kappa B (NF‐κB). Inhibitors of Janus family kinases (JAKs), p38 mitogen‐activated kinase (MAPK), and NF‐κB suppressed IFN‐γ‐induced production of TARC and MDC. NF‐κB activation was inhibited by both inhibitors of JAKs and p38 MAPK. Importantly, IFN‐γ‐stimulated phosphorylation of p38 MAPK was significantly suppressed by JAKs inhibitors, but not significantly affected by NAC or L ‐buthionine sulfoximine (L‐BSO). However, IFN‐γ‐stimulated activation of IκB and NF‐κB was suppressed by NAC but enhanced by BSO. Furthermore, inhibition of p38 MAPK and JAKs did not affect ROS generation in IFN‐γ‐stimulated HaCaT cells. These results indicate that intracellular ROS and JAKs/p38 MAPK both contribute independently to IFN‐γ‐stimulated production of TARC and MDC in HaCaT keratinocytes, by increasing NF‐κB activation. J. Cell. Physiol. 226: 58–65, 2010. © 2010 Wiley‐Liss, Inc.     

Simvastatin abolishes nitric oxide‐ and reactive oxygen species‐induced cyclooxygenase‐2 expression by blocking the nuclear factor κB pathway in rabbit articular chondrocytes

Nitric oxide (NO) and reactive oxygen species (ROS) have been shown to be linked with numerous diseases, including osteoarthritis (OA). Our study aimed to examine the effect of simvastatin on NO‐ or ROS‐induced cyclooxygenase‐2 (COX‐2) expression in OA. Simvastatin has attracted considerable attention since the discovery of its pharmacological effects on different pathogenic processes, including inflammation. Here, we report that simvastatin treatment blocked sodium nitroprusside (SNP)‐ and interleukin 1 beta (IL‐1β)‐induced COX‐2 production. In addition, simvastatin attenuated SNP‐induced NO production and IL‐1β‐induced ROS generation. Treatment with simvastatin prevented SNP‐ and IL‐1β‐induced nuclear factor kappa B (NF‐κB) activity. Inhibiting NO production and ROS generation using N‐acetylcysteine (NAC) and NG‐monomethyl‐ l ‐arginine ( l ‐NMMA), respectively, accelerated the influence of simvastatin on NF‐κB activity. In addition, NAC blocked SNP and simvastatin‐mediated COX‐2 production and NF‐κB activity but did not alter IL‐1β and simvastatin‐mediated COX‐2 expression. l ‐NMMA treatment also abolished IL‐1β‐mediated COX‐2 expression and NF‐κB activation, whereas SNP and simvastatin‐mediated COX‐2 expression were not altered compared with the levels in the SNP and simvastatin‐treated cells. Our findings suggested that simvastatin blocks COX‐2 expression by inhibiting SNP‐induced NO production and IL‐1β‐induced ROS generation by blocking the NF‐κB pathway.     

Naringin prevents ultraviolet‐B radiation‐induced oxidative damage and inflammation through activation of peroxisome proliferator‐activated receptor γ in mouse embryonic fibroblast (NIH‐3T3) cells

The present study, we investigate the preventive role of naringin, a dietary flavonoid, against ultraviolet‐B (UVB) radiation (280‐320 nm) induced oxidative damage and inflammatory responses in mouse embryonic fibroblast cell lines (NIH‐3T3). In this study, 20 mJ/cm ² of UVB radiation induces cell cytotoxicity, reactive oxygen species (ROS) generation, DNA damage, and antioxidants depletion in NIH‐3T3 cells. Treatment with naringin (60 µM) prior UVB exposure prevented the cell cytotoxicity, ROS generation, DNA damage, and antioxidants depletion in NIH‐3T3 cells. Furthermore, naringin prevents UVB‐induced mitogen‐activated protein kinase families and nuclear factor‐κB (NF‐κB)‐mediated activation of inflammatory factors, that is TNF‐α, IL‐6, IL‐10, and COX‐2 in NIH‐3T3 cells. Peroxisome proliferator‐activated receptor γ (PPARγ) is an anti‐inflammatory agent and it suppressed the UVB‐mediated oxidative and inflammatory responses. In this study, naringin activates PPARγ and prevents inflammatory biomarkers in NIH‐3T3 cells. Thus, naringin prevents UVB‐mediated inflammation and oxidative damage in NIH‐3T3 cells probably over controlling NF‐κB expression and activation of PPARγ.     

Effect of arachidonic acid on hypoxia‐induced IL‐6 production in mouse ES cells: Involvement of MAPKs,NF‐κB,and HIF‐1α

This study examined the role of arachidonic acid (AA) in hypoxia‐induced production of interleukin (IL)‐6 and its related signaling pathways in mouse embryonic stem (ES) cells. Hypoxia with AA induced IL‐6 production, which was mediated by reactive oxygen species (ROS). In addition, hypoxia increased the levels of p38 mitogen‐activated protein kinases (MAPKs) and stress‐activated protein kinase/c‐jun NH₂‐terminal kinase (SAPK/JNK) phosphorylation, which were blocked by antioxidant (vitamin C). Inhibition of p38 MAPK and SAPK/JNK blocked hypoxia‐ or hypoxia with AA‐induced nuclear factor‐kappa B (NF‐κB) activation. Furthermore, hypoxia‐induced increase in hypoxia‐inducible factor‐1α (HIF‐1α) expression was regulated by NF‐κB activation. Consequently, the increased HIF‐1α expression induced activation of matrix metalloproteinase (MMP)‐2 and MMP‐9. The expression of each signaling molecule stimulated an increase in IL‐6 production that was greater in hypoxic conditions with AA than with hypoxia alone. Finally, inhibition of IL‐6 production using IL‐6 antibody or soluble IL‐6 receptor attenuated the hypoxia‐induced increases in DNA synthesis of mouse ES cells. In conclusion, AA potentiates hypoxia‐induced IL‐6 production through the MAPKs, NF‐κB, and HIF‐1α pathways in mouse ES cells. J. Cell. Physiol. 222: 574–585, 2010. © 2009 Wiley‐Liss, Inc.     

Central role of PAFR signalling in ExoU‐induced NF‐κB activation

ExoU is an important virulence factor in acute Pseudomonas aeruginosa infections. Here, we unveiled the mechanisms of ExoU‐driven NF‐κB activation by using human airway cells and mice infected with P. aeruginosa strains. Several approaches showed that PAFR was crucially implicated in the activation of the canonical NF‐κB pathway. Confocal microscopy of lungs from infected mice revealed that PAFR‐dependent NF‐κB activation occurred mainly in respiratory epithelial cells, and reduced p65 nuclear translocation was detected in mice PAFR?/? or treated with the PAFR antagonist WEB 2086. Several evidences showed that ExoU‐induced NF‐κB activation regulated PAFR expression. First, ExoU increased p65 occupation of PAFR promoter, as assessed by ChIP. Second, luciferase assays in cultures transfected with different plasmid constructs revealed that ExoU promoted p65 binding to the three κB sites in PAFR promoter. Third, treatment of cell cultures with the NF‐κB inhibitor Bay 11–7082, or transfection with IκBα negative‐dominant, significantly decreased PAFR mRNA. Finally, reduction in PAFR expression was observed in mice treated with Bay 11–7082 or WEB 2086 prior to infection. Together, our data demonstrate that ExoU activates NF‐κB by PAFR signalling, which in turns enhances PAFR expression, highlighting an important mechanism of amplification of response to this P. aeruginosa toxin.     

The interplay of LncRNA ANRIL and miR‐181b on the inflammation‐relevant coronary artery disease through mediating NF‐κB signalling pathway

This study was designed to investigate whether ANRIL affected the aetiology of coronary artery disease (CAD) by acting on downstream miR‐181b and NF‐κB signalling. Altogether 327 CAD patients diagnosed by angiography were included, and mice models of CAD were established. Human coronary endothelial cells (HCAECs) and human umbilical vein endothelial cells (HUVECs) were also purchased. In addition, shRNA‐ANRIL, shRNA‐NC, pcDNA3.1‐ANRIL, miR‐181b mimic, miR‐181b inhibitor and miR‐NC were transfected into the cells. The lipopolysaccharides (LPS) and pyrrolidine dithiocarbamate (PDTC) were also added to activate or deactivate NF‐κB signalling. Both highly expressed ANRIL and lowly expressed miR‐181b were associated with CAD population aged over 60 years old, with smoking history, with hypertension and hyperlipidemia, with CHOL H 4.34 mmol/L, TG ≥ 1.93 mmol/L and Hcy ≥ 16.8 μmol/L (all P < 0.05). Besides, IL‐6, IL‐8, NF‐κB, TNF‐α, iNOS, ICAM‐1, VCAM‐1 and COX‐2 expressions observed within AD mice models were all beyond those within NC and sham‐operated groups (P < 0.05). Also VEGF and HSP 70 were highly expressed within AD mice models than within NC and sham‐operated mice (P < 0.05). Transfection of either pcDNA‐ANRIL or miR‐181b inhibitor could significantly fortify HCAECs’ viability and put on their survival rate. At the meantime, the inflammatory factors and vascular‐protective parameters were released to a greater level (P < 0.05). Finally, highly expressed ANRIL also notably bring down miR‐181b expression and raise p50/p65 expressions within HCAECs (P < 0.05). The joint role of ANRIL, miR‐181b and NF‐κB signalling could aid in further treating and diagnosing CAD.     

Synovial fibroblast‐targeting liposomes encapsulating an NF‐κB‐blocking peptide ameliorates zymosan‐induced synovial inflammation

Synovial fibroblasts (SFs) play a crucial role in the inflammatory process of rheumatoid arthritis (RA). The highly activated NF‐κB signal in SFs is responsible for most of the synovial inflammation associated with this disease. In this study, we have developed an SF‐targeting liposomal system that encapsulates the NF‐κB‐blocking peptide (NBD peptide) HAP‐lipo/NBD. HAP‐lipo/NBDs demonstrated efficient SF‐specific targeting in vitro and in vivo. Our study also showed a significant inhibitory effect of HAP‐lipo/NBD on NF‐κB activation, inflammatory cytokine release and SF migration capability after zymosan stimulation. Furthermore, the systemic administration of HAP‐lipo/NBDs significantly inhibited synovial inflammation and improved the pathological scores of arthritis induced by zymosan. Thus, these results suggest that an SF‐targeting NF‐κB‐blocking strategy is a potential approach for the development of alternative, targeted anti‐RA therapies.     

Interleukin‐17‐induced expression of monocyte chemoattractant protein‐1 in cardiac myocytes requires nuclear factor κB through the phosphorylation of p65

IL‐17 plays a key role in a variety of autoimmune diseases. MCP‐1 is involved in the infiltration of mononuclear cells of myocardium in VMC. However, the relationship between IL‐17 and MCP‐1 in myocardial injury remains unclear. In this study, expression of MCP‐1 mRNA and protein in cardiac myocytes was detected with qRT‐PCR and ELISA, respectively. It was found that IL‐17A induced MCP‐1 expression in a dose‐ and time‐dependent manner in cardiac myocytes, which could be blocked by IL‐17A and IL‐17RA neutralizing antibodies. NF‐κB p65 and p‐p65 protein expression in cardiac myocytes was studied with western blotting. Rates of p‐p65 in whole lysates and in nuclear lysates all increased in the first 15 min. Meanwhile, the amount of NF‐κB p65 in whole lysates did not change, but the amount of NF‐κB p65 in nuclear lysates increased in the first 15 min. Then the optimal sequence and concentration of NF‐κB p65 siRNAs was selected. After transfection of 10 nM siRNA‐2 of NF‐κB p65 into cardiac myocytes before stimulation by IL‐17A, expression of MCP‐1 mRNA and protein obviously decreased. In conclusion, expression of MCP‐1 induced by IL‐17 requires NF‐κB through the phosphorylation of p65 in cardiac myocytes, which is meaningful to study the onset of chronic viral myocarditis and will provide a new target for the treatment of viral myocarditis.