Regulation of α(2B)-adrenergic receptor-mediated extracellular signal-regulated kinase 1/2 (ERK1/2) activation by ADP-ribosylation factor 1

A number of signaling molecules are involved in the activation of the mitogen-activated protein kinase (MAPK) pathway by G protein-coupled receptors. In this study, we have demonstrated that α(2B)-adrenergic receptor (α(2B)-AR) interacts with ADP-ribosylation factor 1 (ARF1), a small GTPase involved in vesicle-mediated trafficking, in an agonist activation-dependent manner and that the interaction is mediated through a unique double Trp motif in the third intracellular loop of the receptor. Interestingly, mutation of the double Trp motif and siRNA-mediated depletion of ARF1 attenuate α(2B)-AR-mediated activation of extracellular signal-regulated kinases 1/2 (ERK1/2) without altering receptor intracellular trafficking, whereas expression of the constitutively active mutant ARF1Q71L and ARNO, a GDP-GTP exchange factor of ARF1, markedly enhances the activation of Raf1, MEK1, and ERK1/2. These data strongly demonstrate that the small GTPase ARF1 modulates ERK1/2 activation by α(2B)-AR and provide the first evidence indicating a novel function for ARF1 in regulating the MAPK signaling pathway.     

Naringin lauroyl ester inhibits lipopolysaccharide-induced activation of nuclear factor κB signaling in macrophages

Naringin (Nar) has antioxidant and anti-inflammatory properties. It was recently reported that enzymatic modification of Nar enhanced its functions. Here, we acylated Nar with fatty acids of different sizes (C2–C18) using immobilized lipase from Rhizomucor miehei and investigated the anti-inflammatory effects of these molecules. Treatment of murine macrophage RAW264.7 cells with Nar alkyl esters inhibited lipopolysaccharide (LPS)-induced nitric oxide (NO) production, with Nar lauroyl ester (Nar-C12) showing the strongest effect. Furthermore, Nar-C12 suppressed the LPS-induced expression of inducible NO synthase by blocking the phosphorylation of inhibitor of nuclear factor (NF)-κB-α as well as the nuclear translocation of NF-κB subunit p65 in macrophage cells. Analysis of Nar-C12 uptake in macrophage cells revealed that Nar-C12 ester bond was partially degraded in the cell membrane and free Nar was translocated to the cytosol. These results indicate that Nar released from Nar-C12 exerts anti-inflammatory effects by suppressing NF-κB signaling pathway.     

Autophagy in the intestinal epithelium reduces endotoxin-induced inflammatory responses by inhibiting NF-κB activation

Autophagy is a lysosomal degradation pathway that is essential for survival, differentiation, development and homeostasis. There is growing evidence that impaired autophagy leads to the pathogenesis of diverse diseases. However, the role of autophagy in intestinal epithelium is not clearly understood, although previous studies have pointed out the possibility for the relationships of autophagy with bowel inflammation. In this study, we investigated the involvement of autophagy in intestinal epithelium with inflammatory responses. We generated the mice with a conditional deletion of Atg7, which is one of the autophagy regulated gene, in intestinal epithelium. In Atg7-deficient small intestinal epithelium, LPS-induced production of TNF-α and IL-1β mRNA was enhanced in comparison to the control small intestinal tissues. In addition, the degree of LPS-induced activation of NF-κB was promoted in Atg7-deficient intestinal epithelium. These results demonstrate that autophagy can attenuate endotoxin-induced inflammatory responses in intestinal epithelium resulting in the maintenance of intestinal homeostasis.     

α1A-adrenergic receptor induces activation of extracellular signal-regulated kinase 1/2 through endocytic pathway

G protein-coupled receptors (GPCRs) activate mitogen-activated protein kinases through a number of distinct pathways in cells. Increasing evidence has suggested that endosomal signaling has an important role in receptor signal transduction. Here we investigated the involvement of endocytosis in α(1A)-adrenergic receptor (α(1A)-AR)-induced activation of extracellular signal-regulated kinase 1/2 (ERK1/2). Agonist-mediated endocytic traffic of α(1A)-AR was assessed by real-time imaging of living, stably transfected human embryonic kidney 293A cells (HEK-293A). α(1A)-AR was internalized dynamically in cells with agonist stimulation, and actin filaments regulated the initial trafficking of α(1A)-AR. α(1A)-AR-induced activation of ERK1/2 but not p38 MAPK was sensitive to disruption of endocytosis, as demonstrated by 4°C chilling, dynamin mutation and treatment with cytochalasin D (actin depolymerizing agent). Activation of protein kinase C (PKC) and C-Raf by α(1A)-AR was not affected by 4°C chilling or cytochalasin D treatment. U73122 (a phospholipase C [PLC] inhibitor) and Ro 31-8220 (a PKC inhibitor) inhibited α(1B)-AR- but not α(1A)-AR-induced ERK1/2 activation. These data suggest that the endocytic pathway is involved in α(1A)-AR-induced ERK1/2 activation, which is independent of G(q)/PLC/PKC signaling.     

Salicortin suppresses lipopolysaccharide-stimulated inflammatory responses via blockade of NF-κB and JNK activation in RAW 264.7 macrophages

We isolated the phenolic glucoside salicortin from a Populus euramericana bark extract, and examined its ability to suppress inflammatory responses as well as the molecular mechanisms underlying these abilities, using lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Salicortin inhibited iNOS expression and the subsequent production of NO in a dose-dependent manner in the LPS-stimulated RAW 264.7 cells. Salicortin significantly suppressed LPS-induced signal cascades of NF-κB activation, such as IKK activation, IκBα phosphorylation and p65 phosphorylation in RAW 264.7 cells. In addition, salicortin inhibited the LPS-induced activation of JNK, but not ERK or p38 MAPK. Furthermore, salicortin significantly inhibited production of pro-inflammatory cytokines, such as TNF-α, IL-1β and IL-6 in the LPS-stimulated RAW 264.7 cells. These findings suggest that salicortin may show its anti-inflammatory activity by suppressing the LPS-induced expression of pro-inflammatory mediators through inhibition of NF-κB and JNK MAPK signaling cascades in macrophages. [BMB Reports 2014; 47(6): 318-323]     

Adriamycin induces myocardium apoptosis through activation of nuclear factor κB in rat

Adriamycin is one of the most effective and useful antineoplastic agents. Acute doxorubicin cardiotoxicity involved cardiomyocyte apoptosis. In this study, we investigated whether adriamycin induced myocardium apoptosis through activation of nuclear factor κB in rat. Forty male Wistar rats were randomly divided into five groups: control, ADR 5 mg/kg, ADR 10 mg/kg, ADR 15 mg/kg group and ADR + PDTC 200 mg/ml group. Myocardial apoptosis was detected by DNA fragmentation assay and TUNEL assay; Location and distribution of p-IκBα was observed by immunohistochemical assay; Myocardial expression of p-IκBα protein was assessed by Western blot analysis; Activity of NF-κB was evaluated by Electrophoretic Mobility Shift Assay. The myocardial apoptotic index, expression of p-IκBα, and binding activity of NF-κB increased significantly in ADR groups in dose-dependent manner. PDTC as a nonspecific inhibitor of NF-κB protected myocardium from apoptosis by inhibiting NF-κB activation. Adriamycin induces myocardium apoptosis through activation of nuclear factor κB in rat and NF-κB activation requires IκBα degradation.     

Tumor necrosis factor α-mediated induction of interleukin 17C in human keratinocytes is controlled by nuclear factor κB

IL-17C is a member of the IL-17 family of cytokines. The expression of IL-17C has been demonstrated to be strongly induced by TNFα in human keratinocytes, and recently the level of IL-17C was found to be increased in the inflammatory skin disease psoriasis. However, little is known about the molecular mechanisms involved in the regulation of IL-17C. Here, we show that pretreatment of cultured human keratinocytes with the inhibitor of κB kinase 2 inhibitor, SC-514, resulted in a significant reduction in both IL-17C mRNA and protein expression, indicating the significance of this pathway in the regulation of IL-17C. NF-κB binding sites were identified upstream from the IL-17C gene, and by electrophoretic mobility shift assay NF-κB was shown to bind to all three identified binding sites. Moreover, NF-κB binding to these sites was inducible by TNFα. Supershift analysis revealed binding of the NF-κB subunits p65 and p50 to all three NF-κB binding sites. To determine the contribution of NF-κB in IL-17C expression, we conducted luciferase gene reporter experiments and demonstrated that a 3204-bp promoter fragment of IL-17C containing three putative NF-κB binding sites was strongly activated by TNFα. Interestingly, mutations of the three NF-κB binding sites revealed that one specific NF-κB binding site was crucial for the TNFα-mediated IL-17C induction because mutation of this specific site completely abolished TNFα-induced IL-17C promoter activation. We conclude that the activation of NF-κB (p65/p50) is crucial for the TNFα-induced stimulation of IL-17C expression in human keratinocytes.     

Palmitate-induced PTP1B expression is mediated by ceramide-JNK and nuclear factor κB (NF-κB) activation

Palmitate induces PTP1B expression in skeletal muscle cells. The purpose of this study was to investigate the mechanisms responsible for palmitate-induced PTP1B expression in mouse skeletal muscle cells. Three truncated fragments of PTP1B promoter were cloned into PGL3-basic vector and the promoter activity of PTP1B was assessed in C2C12 cells exposed to palmitate either in the presence or in the absence of several inhibitors to study the biochemical pathways involved. EMSA was performed to examine binding of NF-κB to NF-κB consensus sequence and PTP1B oligonucelotides in the cells treated with palmitate. Lentiviral PTP1B-shRNA was used to knockdown PTP1B in myotubes. The phosphorylation and protein levels of IRS-1 and Akt were detected by western blot. 0.5mM palmitate induced PTP1B promoter activity in fragment -1715/+59 by 50% (p<0.01). Palmitate increased NF-κB binding to both NF-κB consensus sequence and one NF-κB sequence (-920 to -935) in PTP1B promoter. Incubation of C2C12 cells with different concentrations of C2-ceramide enhanced PTP1B promoter activity dose-dependently. Inhibitors of de novo ceramide synthesis prevented palmitate-induced PTP1B promoter activity in myotubes. In addition, inhibitor of JNK pathway prevented ceramide-induced PTP1B promoter activity in myotubes. Knockdown of PTP1B also prevented ceramide-reduced IRS-1 and Akt phosphorylations in the myotubes. Exposure of the cells to PMA and calphostin C, an inhibitor of PKC, did not affect the activity of PTP1B promoter. Our data provide the evidence that the mechanism by which palmitate increased the expression of PTP1B seems to be through a mechanism involving the activation of ceramide-JNK and NF-κB pathways.     

H<Subscript>2</Subscript> inhibits TNF-α-induced lectin-like oxidized LDL receptor-1 expression by inhibiting nuclear factor κB activation in endothelial cells

H₂ is a therapeutic antioxidant that can reduce oxidative stress. Oxidized low-density lipoprotein, which plays roles in atherosclerosis, may promote endothelial dysfunction by binding the cell-surface receptor LOX-1. LOX-1 expression can be upregulated by various stimuli, including TNF-α. Thus, we aimed to examine whether the upregulation of LOX-1 by different stimuli could be blocked by H₂ in endothelial cells. H₂ significantly abolished the upregulation of LOX-1 by different stimuli, including TNF-α, at the protein and mRNA levels. The TNF-α-induced upregulation of LOX-1 was also attenuated by the NF-κB inhibitor N-acetyl-l-cysteine. H₂ inhibited the TNF-α-induced activation of NF-κB and the phosphorylation of IκB-α. Furthermore, H₂ inhibited the expression of LOX-1 and the activation of NF-κB in apolipoprotein E knockout mice, an animal model of atherosclerosis. Thus, H₂ probably inhibits cytokine-induced LOX-1 gene expression by suppressing NF-κB activation.     

Decoy receptor 3 suppresses TLR2-mediated B cell activation by targeting NF-κB

Decoy receptor 3 (DcR3) is a soluble protein in the TNFR superfamily. Its known ligands include Fas ligand, homologous to lymphotoxin, showing inducible expression, and competing with HSV glycoprotein D for herpes virus entry mediator, a receptor expressed by T lymphocytes, TNF-like molecule 1A, and heparan sulfate proteoglycans. DcR3 has been reported to modulate the functions of T cells, dendritic cells, and macrophages; however, its role in regulating B cell activation is largely unknown. In this study, we found that the DcR3.Fc fusion protein bound to human and mouse B cells and suppressed the activation of B cells. DcR3.Fc attenuated Staphylococcus aureus, IgM-, Pam(3)CSK(4)-, and LPS-mediated B cell proliferation but did not affect cytokine-induced B cell growth. In the presence of these mitogens, DcR3.Fc did not induce B cell apoptosis, suggesting that DcR3 may inhibit the signal(s) important for B cell activation. Because the combination of Fas.Fc, LT-βR.Fc (homologous to lymphotoxin, showing inducible expression, and competing with HSV glycoprotein D for herpes virus entry mediator, a receptor expressed by T lymphocytes receptor), and DR3.Fc (TNF-like molecule 1A receptor) did not suppress B cell proliferation and because the biological effect of DcR3.Fc on B cells was not blocked by heparin, we hypothesize that a novel ligand(s) of DcR3 mediates its inhibitory activity on B cells. Moreover, we found that TLR2-stimulated NF-κB p65 activation and NF-κB-driven luciferase activity were attenuated by DcR3.Fc. The TLR2-induced cytokine production by B cells was consistently reduced by DcR3. These results imply that DcR3 may regulate B cell activation by suppressing the activation of NF-κB.     

Ubiquitin-like protein MNSFβ/endophilin II complex regulates Dectin-1-mediated phagocytosis and inflammatory responses in macrophages

Post-translational modification by monoclonal nonspecific suppressor factor β (MNSFβ) has been implicated in the regulation of a variety of cellular events. Previous studies have demonstrated that MNSFβ covalently binds to the intracellular pro-apoptotic protein Bcl-G in a macrophage cell line, Raw264.7, suggesting involvement of this ubiquitin-like protein in apoptosis. Most recently, we found that MNSFβ covalently conjugates to endophilin II, a member of the endophilin A family, and inhibits phagocytosis by macrophages. In this study, we further examined the mechanism of action of MNSFβ/endophilin II complex in the phagocytosis of zymosan. MNSFβ/endophilin II I mediated inhibition of phagocytosis in Raw264.7 cells was neutralized by anti-Decti-1, β-glucan receptor, mAb, indicating that MNSFβ/endophilin II is a mediator of Dectin-1 signaling in regulating phagocytosis. The β-glucan-dependent TNFα response to zymosan was significantly increased by the treatment with endophilin II siRNA and/or MNSFβ siRNA. Conversely, cotransfection of endophilin II and MNSFβ cDNAs inhibited the enhancement of zymosan-induced TNFα production. Interestingly, endophilin II siRNA did not affect Pam₃CSK₄ (TLR2 specific ligand)-induced TNFα production. Endophilin II and/or MNSFβ siRNA enhanced zymosan-induced IκBα degradation. Together, these results demonstrate that MNSFβ/endophilin II inhibits the signal pathway upstream of IKK activation, but not downstream of TLR2 signaling.     

Coumestrol suppresses hypoxia inducible factor 1α by inhibiting ROS mediated sphingosine kinase 1 in hypoxic PC-3 prostate cancer cells

Among many signals to regulate hypoxia inducible factor 1α (HIF-1α), sphingosine kinase 1 (SPHK1) is also involved in various biological activities such as cell growth, survival, invasion, angiogenesis, and carcinogenesis. Thus, in the present study, molecular mechanisms of coumestrol were investigated on the SPHK1 and HIF-1α signaling pathway in hypoxic PC-3 prostate cancer cells. Coumestrol significantly suppressed SPHK1 activity and accumulation of HIF-1α in a time- and concentration-dependent manner in hypoxic PC-3 cells. In addition, coumestrol inhibited the phosphorylation status of AKT and glycogen synthase kinase-3β (GSK 3β) signaling involved in cancer metabolism. Furthermore, SPHK1 siRNA transfection, sphigosine kinase inhibitor (SKI), reactive oxygen species (ROS) enhanced the inhibitory effect of coumestrol on the accumulation of HIF-1α and the expression of pAKT and pGSK 3β in hypoxic PC-3 cells by combination index. Overall, our findings suggest that coumestrol suppresses the accumulation of HIF-1α via suppression of SPHK1 pathway in hypoxic PC-3 cells.     

Suppression of LPS-induced inflammatory and NF-κB responses by anomalin in RAW 264.7 macrophages

The treatment of inflammatory diseases today is largely based on interrupting the synthesis or action of the mediators that drive the host's response to injury. It is on the basis of this concept that most of the anti-inflammatory drugs have been developed. In our continuous search for novel anti-inflammatory agents from traditional medicinal plants, Saposhnikovia divaricata has been a focus of our investigations. Anomalin, a pyranocoumarin constituent of S. divaricata, exhibits potent anti-inflammatory activity. To clarify the cellular signaling mechanisms underlying the anti-inflammatory action of anomalin, we investigated the effect of anomalin on the production of inflammatory molecules in LPS-stimulated murine macrophages. The anomalin dose-dependently inhibited inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) mRNA and protein expression in LPS-stimulated RAW 264.7 macrophage. Molecular analysis using quantitative real time polymerase chain reaction (qRT-PCR) revealed that several pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), were reduced by anomalin, and this reduction correlated with the down-regulation of the NF-κB signaling pathway. In addition, anomalin suppressed the LPS-induced phosphorylation and degradation of IκBα. To further study the mechanisms underlying its anti-inflammatory activity, an electrophoretic mobility shift assay (EMSA) using a (32) P-labeled NF-κB probe was conducted. LPS-induced NF-κB DNA binding was drastically abolished by anomalin. The present data suggest that anomalin is a major anti-inflammatory agent and may be a potential therapeutic candidate for the treatment of inflammatory disorders.     

Inhibition of nuclear factor κB regresses cardiac hypertrophy by modulating the expression of extracellular matrix and adhesion molecules

Myocardial remodeling denotes a chronic pathological condition of dysfunctional myocardium that occurs in cardiac hypertrophy (CH) and heart failure (HF). Reactive oxygen species (ROS) are major initiators of excessive collagen and fibronectin deposition in cardiac fibrosis. Increased production of ROS and nuclear factor κB (NF-κB) activation provide a strong link between oxidative stress and extracellular matrix (ECM) remodeling in cardiac hypertrophy. The protective inhibitory actions of pyrrolidine dithiocarbamate (PDTC), a pharmacological inhibitor of NF-κB and a potent antioxidant, make this a good agent to evaluate the role of inhibition of NF-κB and prevention of excessive ECM deposition in maladaptive cardiac remodeling during HF. In this report, we used a transgenic mouse model (Myo-Tg) that has cardiac-specific overexpression of myotrophin. This overexpression of myotrophin in the Myo-Tg model directs ECM deposition and increased NF-κB activity, which result in CH and ultimately HF. Using the Myo-Tg model, our data showed upregulation of profibrotic genes (including collagen types I and III, connective tissue growth factor, and fibronectin) in Myo-Tg mice, compared to wild-type mice, during the progression of CH. Pharmacological inhibition of NF-κB by PDTC in the Myo-Tg mice resulted in a significant reduction in cardiac mass, NF-κB activity, and profibrotic gene expression and improved cardiac function. To the best of our knowledge, this is the first report of ECM regulation by inhibition of NF-κB activation by PDTC. The study highlights the importance of the NF-κB signaling pathway and therapeutic benefits of PDTC treatment in cardiac remodeling.