Activator Protein-1 Pathway Research Articles

A Novel Inhibitor of I-KappaB Kinase Beta Ameliorates Experimental Arthritis through Downregulation of Proinflammatory Cytokines in Arthritic Joints

Inhibitor of kappaB (IκB) kinase beta (IKKβ) plays a critical role in nuclear factor-kappaB (NF-κB) activation and production of proinflammatory cytokines in various inflammatory diseases including rheumatoid arthritis. We previously reported a novel IKKβ inhibitor Compound D, 4-[6-(cyclobutylamino)imidazo[1,2-b]pyridazin-3-yl]-2-fluoro-N-{[(2S,4R)-4-fluoropyrrolidin-2-yl]methyl}benzamide, which is efficacious in experimental arthritis models. In the present study, we characterized the pharmacological properties of Compound D and investigated the mechanisms of the anti-arthritic effect. Compound D inhibited IKKβ kinase activity with 160-fold selectivity against IKKα. The cellular analyses revealed that Compound D selectively blocked NF-κB promoter activity among major cellular signaling pathways, such as the activator protein-1 pathway, consistent with inhibition of the NF-κB signaling pathway including phosphorylation of IκBα. In addition, Compound D inhibited NF-κB-driven production of tumor necrosis factor alpha (TNFα) and interleukin-6 comparably. The correlation between inhibitory effect on TNFα production and plasma concentration of the compound was observed in vivo. Consecutive administration of Compound D decreased gene expression of proinflammatory cytokines and inflammatory mediators in the paws of arthritic mice with attenuation of paw swelling. Notably, Compound D was rapidly distributed to the arthritic paws, rather than healthy paws, and where it decreased the gene expression of proinflammatory cytokines by a single oral administration. Furthermore, Compound D completely inhibited arthritis progression even when treatment occurred after disease development. These data suggest that the downregulation of proinflammatory cytokines in local inflamed joints is one of the mechanisms underlying the anti-arthritic effect of the IKKβ inhibitor, Compound D.

Andrographolide suppresses high glucose-induced fibronectin expression in mesangial cells via inhibiting the AP-1 pathway

Mesangial cells (MCs) proliferation and accumulation of glomerular matrix proteins such as fibronectin (FN) are the early features of diabetic nephropathy, with MCs known to upregulate matrix protein synthesis in response to high glucose. Recently, it has been found that andrographolide has renoprotective effects on diabetic nephropathy. However, the molecular mechanism underlying these effects remains unclear. Cell viability and proliferation was evaluated by MTT. FN expression was examined by immunofluorescence and immunoblotting. Activator protein-1 (AP-1) activation was assessed by immunoblotting, luciferase reporter and electrophoretic mobility shift assays. Andrographolide significantly decreased high glucose-induced cell proliferation and FN expression in MCs. Exposure of MCs to high glucose markedly stimulated the expression of phosphorylated c-jun, whereas the stimulation was inhibited by andrographolide. Plasmid pAP-1-Luc luciferase reporter assay showed that andrographolide blocked high glucose-induced AP-1 transcriptional activity. EMSA assay demonstrated that increased AP-1 binding to an AP-1 binding site at -1,029 in the FN gene promoter upon high glucose stimulation, and the binding were disrupted by andrographolide treatment. These data indicate that andrographolide suppresses high glucose-induced FN expression by inhibiting AP-1-mediated pathway.

Connective tissue growth factor induces collagen I expression in human lung fibroblasts through the Rac1/MLK3/JNK/AP-1 pathway

Connective tissue growth factor (CTGF) plays an important role in lung fibrosis. In this study, we investigated the role of Rac1, mixed-lineage kinase 3 (MLK3), c-Jun N-terminal kinase (JNK), and activator protein-1 (AP-1) in CTGF-induced collagen I expression in human lung fibroblasts. CTGF caused concentration- and time-dependent increases in collagen I expression. CTGF-induced collagen I expression was inhibited by the dominant negative mutant (DN) of Rac1 (RacN17), MLK3DN, MLK3 inhibitor (K252a), JNK1DN, JNK2DN, a JNK inhibitor (SP600125), and an AP-1 inhibitor (curcumin). Treatment of cells with CTGF caused activation of Rac1, MLK3, JNK, and AP-1. The CTGF-induced increase in MLK3 phosphorylation was inhibited by RacN17. Treatment with RacN17 and the MLK3DN inhibited CTGF-induced JNK phosphorylation. CTGF caused increases in c-Jun phosphorylation and the recruitment of c-Jun and c-Fos to the collagen I promoter. Furthermore, stimulation of cells with the CTGF resulted in increases in AP-1-luciferase activity; this effect was inhibited by Rac1N17, MLK3DN, JNK1DN, and JNK2DN. Moreover, CTGF-induced α-smooth muscle actin (α-SMA) expression was inhibited by the procollagen I small interfering RNA (siRNA). These results suggest for the first time that CTGF acting through Rac1 activates the MLK3/JNK signaling pathway, which in turn initiates AP-1 activation and recruitment of c-Jun and c-Fos to the collagen I promoter and ultimately induces collagen I expression in human lung fibroblasts.

Anticancer effects of marine carotenoids, fucoxanthin and its deacetylated product, fucoxanthinol, on osteosarcoma

Survival of osteosarcoma patients hinges on prevention or treatment of recurrent and metastatic lesions. Therefore, novel chemotherapeutics for more effective treatment and prevention of this disease are required. Carotenoids are natural pigments and exhibit various biological functions. We evaluated the anti-osteosarcoma properties of several carotenoids. Among carotenoids, fucoxanthin and its metabolite fucoxanthinol, inhibited the cell viability of osteosarcoma cell lines. Fucoxanthinol induced G1 cell cycle arrest by reducing the expression of cyclin-dependent kinase 4, cyclin-dependent kinase 6 and cyclin E and apoptosis by reducing the expression of survivin, XIAP, Bcl-2 and Bcl-xL. Apoptosis was associated with activation of caspases-3, -8 and -9. In addition, fucoxanthinol inhibited the phosphorylation of phosphoinositide-dependent kinase 1 and Akt and the downstream glycogen synthase kinase 3β, resulting in downregulation of β-catenin. Fucoxanthinol inhibited the cell migration and invasion of osteosarcoma cells. It also reduced matrix metalloproteinase-1 expression and the activator protein-1 signal. Treatment of mice inoculated with osteosarcoma cells with fucoxanthin inhibited the development of osteosarcoma in mice. Fucoxanthin and fucoxanthinol inhibit cell growth, migration and invasion and induce apoptosis of osteosarcoma cells at least in part by inhibiting Akt and activator protein-1 pathways. Our findings provide a rationale for clinical evaluation of these novel agents in osteosarcoma.

Lung tumor growth-promoting function of peroxiredoxin 6

This study compared lung tumor growth in PRDX6-overexpressing transgenic (Tg) mice and normal mice. These mice expressed elevated levels of PRDX6 mRNA and protein in multiple tissues. In vivo, Tg mice displayed a greater increase in the growth of lung tumor compared with normal mice. Glutathione peroxidase and calcium-independent phospholipase 2 (iPLA2) activities in tumor tissues of Tg mice were much higher than in tumor tissues of normal mice. Higher tumor growth in PRDX6-overexpressing Tg mice was associated with an increase in activating protein-1 (AP-1) DNA-binding activity. Moreover, expression of proliferating cell nuclear antigen, Ki67, vascular endothelial growth factor, c-Jun, c-Fos, metalloproteinase-9, cyclin-dependent kinases, and cyclins was much higher in the tumor tissues of PRDX6-overexpressing Tg mice than in tumor tissues of normal mice. However, the expression of apoptotic regulatory proteins including caspase-3 and Bax was slightly less in the tumor tissues of normal mice. In tumor tissues of PRDX6-overexpressing Tg mice, activation of mitogen-activated protein kinases (MAPKs) was much higher than in normal mice. In cultured lung cancer cells, PRDX6 siRNA suppressed glutathione peroxidase and iPLA2 activities and cancer cell growth, but the enforced overexpression of PRDX6 increased cancer cell growth associated with their increased activities. In vitro, among the tested MAPK inhibitors, c-Jun NH2-terminal kinase (JNK) inhibitor clearly suppressed the growth of lung cancer cells and AP-1 DNA binding, glutathione peroxidase activity, and iPLA2 activity in normal and PRDX6-overexpressing lung cancer cells. These data indicate that overexpression of PRDX6 promotes lung tumor growth via increased glutathione peroxidase and iPLA2 activities through the upregulation of the AP-1 and JNK pathways.

Endothelin-1 enhances cell migration through COX-2 up-regulation in human chondrosarcoma

BackgroundChondrosarcoma is a type of highly malignant tumor with a potent capacity of local invasion and distant metastasis. The effect of endothelin-1 (ET-1) on migration activity in human chondrosarcoma cells is not clearly understood. Here, we found that ET-1 increased the migration and expression of cyclooxygenase (COX)-2 in human chondrosarcoma cells. MethodsET-1-mediated COX-2 expression was assessed by qPCR and Western blot analysis. The mechanisms of action of ET-1 in different signaling pathways were studied using Western blotting. Knockdown of proteins was achieved by transfection with siRNA. Chromatin immunoprecipitation assays were used to study in vivo binding of c-Jun to the COX-2 promoter. ResultsHuman chondrosarcoma tissues had significant expression levels of ET-1 and COX-2, which were higher than that in normal cartilage. Exogenous ET-1 increased cell migration and the expression of COX-2. In addition, COX-2 protein levels and cell migration ability were abolished by ET receptor antagonists. Activation of the mitogen-activated protein kinase (MAPK) and activator protein-1 (AP-1) pathways after ET-1 treatment was demonstrated, and ET-1-induced COX-2 expression and cell migration activity were inhibited by the specific inhibitor and mutant of MAPK and AP-1 cascades. ET-1 increased the binding of c-Jun to the AP-1 element on the COX-2 promoter. Furthermore, knockdown of ET-1 decreased cell metastasis in vitro and in vivo. ConclusionsOur results indicated that ET-1 enhances the cell migration of chondrosarcoma by increasing COX-2 expression through the ET receptors, MAPK, and AP-1 signal transduction pathway. General significanceWe link high ET-1 and COX-2 expression to chondrosarcoma.

Interleukin‐1β enhances cell migration through AP‐1 and NF‐κB pathway‐dependent FGF2 expression in human corneal endothelial cells

Interleukin (IL)-1β is a major pro-inflammatory cytokine that plays a crucial role in the regulation of inflammation and wound healing in the cornea. Elucidation of IL-1β signalling may help identify therapeutic targets for corneal wound healing; however, mechanisms such as cell migration, a component of IL-1β-induced wound healing response in human corneal endothelial cells (CEC), have not been well characterised. Stimulation of human CEC with IL-1β activated expression of fibroblast growth factor 2 (FGF2) and resulted in enhanced cell migration. This, in turn, was abolished by treatment with either IL-1 receptor antagonist or SU-5402, a pan-fibroblast growth factor signalling inhibitor. Phosphatidyl inositol (PI) 3-kinase or IL receptor-associated kinase 1/4 antagonists demonstrated that IL receptor-associated kinase 1/4 activates PI 3-kinase, which in turn phosphorylates p38 and inhibitor κB kinase α/β, leading to FGF2 expression through activation of activator protein 1 (AP-1) and nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB) in human CEC. Treatment of IL-1β-stimulated human CEC with either AP-1 or NF-κB antagonists decreased FGF2 expression and resulted in reduced IL-1β-enhanced cell migration. Co-treatment of IL-1β-stimulated human CEC with both inhibitors completely blocked FGF2 expression and IL-1β-enhanced cell migration. Chromatin immunoprecipitation assays demonstrated that AP-1 and NF-κB directly bind to the FGF2 promoter following IL-1β stimulation. The results show that binding of IL-1β to its receptor in human CEC leads to parallel activation of AP-1 and NF-κB pathways, leading, in turn, to FGF2 expression and enhanced cell migration.

Pioglitazone inhibits high glucose-induced synthesis of extracellular matrix by NF-κB and AP-1 pathways in rat peritoneal mesothelial cells

High glucose (HG) in peritoneal dialysates has been demonstrated to induce extracellular matrix (ECM) synthesis by peritoneal mesothelial cells (PMCs) and to contribute to peritoneal fibrosis during continuous ambulatory peritoneal dialysis (CAPD). In the present study, we investigated the effects of pioglitazone, a peroxisome proliferator-activated receptor γ (PPARγ) agonist, on HG-induced ECM accumulation and the underlying mechanism in rat PMCs (RPMCs). In cultured RPMCs, HG treatment increased the expression of fibronectin (FN), collagen I and plasminogen activation inhibitor-1 (PAI-1) at the mRNA and protein levels, while it downregulated the expression of PPARγ in a time- and concentration-dependent manner. Pretreatment with pioglitazone not only decreased the expression of PAI-1 and matrix proteins (FN and collagen I), but prevented the downregulation of PPARγ in RPMCs under HG conditions. HG treatment activated the nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) pathways. In addition, the NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC), and the AP-1 inhibitor, SP600125, decreased the protein levels of FN, collagen I and PAI-1, suggesting a role for the NF-κB and AP-1 pathways in the regulation of ECM accumulation induced by HG in RPMCs. Notably, we demonstrated that pretreatment with pioglitazone significantly inhibited HG-induced NF-κB and AP-1 activation. Collectively, these results suggest that pioglitazone inhibits HG-induced ECM accumulation in RPMCs by increasing PPARγ expression, and by inhibiting the NF-κB and AP-1 pathways.

Phyllostachys edulis Compounds Inhibit Palmitic Acid-Induced Monocyte Chemoattractant Protein 1 (MCP-1) Production

Background Phyllostachys edulis Carriere (Poaceae) is a bamboo species that is part of the traditional Chinese medicine pharmacopoeia. Compounds and extracts from this species have shown potential applications towards several diseases. One of many complications found in obesity and diabetes is the link between elevated circulatory free fatty acids (FFAs) and chronic inflammation. This study aims to present a possible application of P. edulis extract in relieving inflammation caused by FFAs. Monocyte chemoattractant protein 1 (MCP-1/CCL2) is a pro-inflammatory cytokine implicated in chronic inflammation. Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and activator protein 1 (AP-1) are transcription factors activated in response to inflammatory stimuli, and upregulate pro-inflammatory cytokines such as MCP-1. This study examines the effect of P. edulis extract on cellular production of MCP-1 and on the NF-κB and AP-1 pathways in response to treatment with palmitic acid (PA), a FFA.Methodology/Principal FindingsMCP-1 protein was measured by cytometric bead assay. NF-κB and AP-1 nuclear localization was detected by colorimetric DNA-binding ELISA. Relative MCP-1 mRNA was measured by real-time quantitative PCR. Murine cells were treated with PA to induce inflammation. PA increased expression of MCP-1 mRNA and protein, and increased nuclear localization of NF-κB and AP-1. Adding bamboo extract (BEX) inhibited the effects of PA, reduced MCP-1 production, and inhibited nuclear translocation of NF-κB and AP-1 subunits. Compounds isolated from BEX inhibited MCP-1 secretion with different potencies.Conclusions/SignificancePA induced MCP-1 production in murine adipose, muscle, and liver cells. BEX ameliorated PA-induced production of MCP-1 by inhibiting nuclear translocation of NF-κB and AP-1. Two O-methylated flavones were isolated from BEX with functional effects on MCP-1 production. These results may represent a possible therapeutic application of BEX and its compounds toward alleviating chronic inflammation caused by elevated circulatory FFAs.

Polyphenon-60 displays a therapeutic effect on acne by suppression of TLR2 and IL-8 expression via down-regulating the ERK1/2 pathway

Propionibacterium acnes (P. acnes) is a well-known acne-inducing factor which causes inflammatory skin lesions by enhancing cytokine production through toll-like receptor 2 (TLR2). Green tea extract catechin has been documented to possess anti-inflammatory effects. However, little is known about the mechanisms involved or any direct effect of green tea catechin on acne. The present study investigated the therapeutic effects and mechanism of polyphenon-60, also known as green tea catechin compound, on acne in vitro and in vivo. In a clinical study using topical polyphenon-60 treatment, acne patients showed symptomatic improvement with decrease in the number of comedos and pustules. To investigate the mechanism underlying the activity of polyphenon-60 in acne therapy, an in vitro study was performed. We found that polyphenon-60 reduced the levels of P. acnes-enhanced TLR2 and interleukin-8 (IL-8) in THP-1 cells, human monocyte cell line and human primary monocytes. Taken together, these data demonstrate that polyphenon-60 has a therapeutic effect on acne by suppressing inflammation, specifically by inhibiting TLR2 expression and IL-8 secretion via down-regulation of extracellular signal-regulated kinases 1/2 (ERK1/2) pathway and activator protein-1 (AP-1) pathway.

Up-regulation of IL-23 p19 expression in human periodontal ligament fibroblasts by IL-1β via concurrent activation of the NF-κB and MAPKs/AP-1 pathways

Interleukin (IL)-23 is an essential cytokine involved in the expansion of a novel CD4+ T helper subset known as Th17, which has been implicated in the pathogenesis of periodontitis recently. Our previous study first identified specialized human periodontal ligament fibroblasts (hPDLFs) as an important production source of IL-23. The present study was undertaken to investigate the effects of the pro-inflammatory and Th17-polarizing mediator IL-1β on hPDLFs-mediated IL-23 p19 production, and the molecular mechanism involved. IL-23 p19 expression was in situ detected in IL-1β-stimulated hPDLFs. IL-1β was capable of stimulating the expression of IL-23 p19 mRNA and protein in cultured hPDLFs, which was attenuated by IL-1 receptor antagonist (IL-1Ra) or myeloid differentiation primary response gene 88 (MyD88) inhibitor. Meanwhile, inhibitors of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK) 1/2, c-Jun-N-terminal kinase (JNK), activator protein-1 (AP-1), or nuclear factor-kappaB (NF-κB) significantly suppressed IL-23 p19 production from IL-1β-stimulated hPDLFs. Moreover, IL-1β-initiated AP-1 activation was blocked by p38 MAPK, ERK 1/2, or JNK inhibition, whereas NF-κB activity remained unaltered by all the above pathway specific inhibitors. Thus, these results provide evidence that Th17-polarizing mediator IL-1β up-regulated the expression of IL-23 p19 in hPDLFs via NF-κB signaling and MAPKs-dependent AP-1 pathways. Taken together, our findings indicate that IL-1Ra may be used therapeutically to inhibit Th17-driven inflammatory diseases including periodontitis.

Water extract of edible bird’s nest attenuated the oxidative stress-induced matrix metalloproteinase-1 by regulating the mitogen-activated protein kinase and activator protein-1 pathway in human keratinocytes

To better understand the molecular mechanisms underlying the anti-aging properties of water extract of edible bird’s nest (WEBN), the effect of WEBN on oxidative stress-induced matrix metalloproteinase-1 (MMP-1) was assessed in human HaCaT keratinocyte. Upon exposure to H2O2, WEBN inhibited H2O2-induced cytotoxicity and scavenged intracellular reactive oxygen species, down-regulated H2O2-induced MMP-1 mRNA expression, protein expression and activity, and lowered the activation of extracellular signal-regulated protein kinase (ERK) and c-Jun N-terminal kinases (JNK), which are the upstream of MMP-1 expression. Furthermore, WEBN inhibited the expression of c-Fos and phospho c-Jun, which are components of the transcription factor activator protein-1 (AP-1), AP-1 transcriptional activity, and AP-1 binding to the MMP-1 promoter. These data indicate that the anti-aging properties of WEBN involve the inhibition of MMP-1 expression via down-regulation of the ERK /JNK and AP-1 pathway.

Abstract 1810: Mechanistic studies of the dual Akt and JNK inhibitor Chaetoglobosin K: Effect on downstream pathways and AP-1 proteins in tumorigenic cells

Abstract We have previously shown that the anti-tumorigenic indolylcytochalasin Chaetoglobosin K (ChK) concomitantly down-regulates Akt and JNK activation in ras-transformed cells. This dual inhibition was found not be regulated by Rac1, a Rho protein that is involved in both signaling pathways, or MKK4, an upstream kinase of JNK. The purpose of this study was to determine: 1) the effect of ChK on the activation of downstream mediators of the PI3K/Akt (including mdm2), and JNK (including the activator protein-1 (AP-1) proteins and stat3) signaling pathways; 2) whether the upstream mitogen-activated protein kinase kinase 7 (MKK7) is involved in ChK's effect on JNK. The AP-1 transcription factor complex, composed of proteins of the JUN, FOS, and ATF families, has become a promising therapeutic target due to the proposed role of AP-1 activation in tumorigenesis. In these studies, ras-transformed rat liver epithelial cells were grown to confluence and treated with either ChK (5μM or 10μM) or vehicle (DMSO) for 24 hours. Cell proteins were extracted and separated using SDS-PAGE. Western blot analysis was performed using phosphorylation site-specific antibodies to monitor changes in MKK7, c-JUN, ATF-2, mdm2, and stat3 protein activation levels. The results of these studies indicate that ChK treatment decreased c-JUN and ATF-2 phosphorylation, but had no effect on stat3 phosphorylation. Additionally, ChK treatment decreased mdm2 phosphorylation. ChK did not alter MKK7 activation levels and, taking into consideration its lack of effect on MKK4, suggest ChK does not inhibit JNK via its archetypical MAPK signaling pathway. These studies indicate ChK may serve as a small molecule inhibitor of the AP-1 pathway proteins, as well as the Akt/mdm2 pathway. This data further supports ChK's potential as a unique targeted tumor therapy. This project was funded by NIH grant 1R15CA135415 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1810. doi:1538-7445.AM2012-1810

Varicella-Zoster Virus ORF12 Protein Triggers Phosphorylation of ERK1/2 and Inhibits Apoptosis

Mitogen-activated protein kinases (MAPKs) are a family of serine-threonine protein kinases involved in many cellular processes, including cell proliferation, differentiation, inflammation, and cell death. Activation of several MAPKs, including extracellular signal-regulated kinase 1 and 2 (ERK1/2), p38, and c-Jun N-terminal kinase (JNK), results in stimulation of activator protein 1 (AP-1), which promotes gene transcription. Previous studies have demonstrated that varicella-zoster virus (VZV) infection activates ERK1/2, p38, and JNK to promote viral replication, but the underlying mechanism(s) is unclear. To identify viral proteins responsible for the activation of MAPK, we used a proteomic approach to screen viral proteins for AP-1 promoter activation by an AP-1-luciferase reporter assay. We found that VZV ORF12 protein, located in the tegument of virions, enhances AP-1 reporter activity. This effect of ORF12 protein was markedly inhibited by a MAPK/ERK kinase 1 and 2 (MEK1/2) inhibitor (U0126), partially blocked by a p38 inhibitor (SB202190), but not inhibited by a JNK inhibitor (SP600125). Expression of VZV ORF12 protein in cells resulted in phosphorylation of ERK1/2 and p38 but not JNK. Infection of cells with a VZV ORF12 deletion mutant resulted in reduced levels of phosphorylated ERK1/2 (p-ERK1/2) compared to infection with wild-type VZV. Furthermore, deletion of ORF12 rendered VZV-infected cells more susceptible to staurosporine-induced apoptosis. In conclusion, VZV ORF12 protein activates the AP-1 pathway by selectively triggering the phosphorylation of ERK1/2 and p38. Cells infected with a VZV ORF12 deletion mutant have reduced levels of p-ERK1/2 and are more susceptible to apoptosis than cells infected with wild-type VZV.

EGFRvIII promotes glioma angiogenesis and growth through the NF-κB, interleukin-8 pathway

Sustaining a high growth rate requires tumors to exploit resources in their microenvironment. One example of this is the extensive angiogenesis that is a typical feature of high-grade gliomas. Here, we show that expression of the constitutively active mutant epidermal growth factor receptor, ΔEGFR (EGFRvIII, EGFR*, de2-7EGFR) is associated with significantly higher expression levels of the pro-angiogenic factor interleukin (IL)-8 in human glioma specimens and glioma stem cells. Furthermore, the ectopic expression of ΔEGFR in different glioma cell lines caused up to 60-fold increases in the secretion of IL-8. Xenografts of these cells exhibit increased neovascularization, which is not elicited by cells overexpressing wild-type (wt)EGFR or ΔEGFR with an additional kinase domain mutation. Analysis of the regulation of IL-8 by site-directed mutagenesis of its promoter showed that ΔEGFR regulates its expression through the transcription factors nuclear factor (NF)-κB, activator protein 1 (AP-1) and CCAAT/enhancer binding protein (C/EBP). Glioma cells overexpressing ΔEGFR showed constitutive activation and DNA binding of NF-κB, overexpression of c-Jun and activation of its upstream kinase c-Jun N-terminal kinase (JNK) and overexpression of C/EBPβ. Selective pharmacological or genetic targeting of the NF-κB or AP-1 pathways efficiently blocked promoter activity and secretion of IL-8. Moreover, RNA interference-mediated knock-down of either IL-8 or the NF-κB subunit p65, in ΔEGFR-expressing cells attenuated their ability to form tumors and to induce angiogenesis when injected subcutaneously into nude mice. On the contrary, the overexpression of IL-8 in glioma cells lacking ΔEGFR potently enhanced their tumorigenicity and produced highly vascularized tumors, suggesting the importance of this cytokine and its transcription regulators in promoting glioma angiogenesis and tumor growth.

Triterpenes from the Fungus Poria cocos and Their Inhibitory Activity on Nitric Oxide Production in Mouse Macrophages via Blockade of Activating Protein‐1 Pathway

Two new triterpenes, 29-hydroxydehydrotumulosic acid (1) and 29-hydroxydehydropachymic acid (2), together with six known compounds, dehydropachymic acid (3), dehydrotumulosic acid (4), 29-hydroxypolyporenic acid C (5), polyporenic acid C (6), tumulosic acid (7), and pachymic acid (8), were isolated from the dried sclerotia of Poria cocos. In the in vitro bioassays, these isolated compounds reduced, in a dose-dependent manner, nitric oxide (NO) production from lipopolysaccharide (LPS)-induced RAW 264.7 cells, with compounds 5 and 6, the IC(50) values of which were 16.8±2.7 and 18.2±3.3 μM, respectively, exhibiting the greatest inhibition activity. Further Western blot analysis conducted on cells pre-treated with compounds 5 and 6, and luciferase assays on activator protein 1-dependent gene expression revealed that the inhibited NO release was attributed to the reduced expression of iNOs (=inducible NO synthase) enzymes, which might be regulated via the blockade of activator protein-1 signaling pathway.

Involvement of activated transcriptional process in efficient gene transfection using unmodified and mannose-modified bubble lipoplexes with ultrasound exposure

Recently, our group developed ultrasound (US)-responsive and mannose-modified gene carriers (Man-PEG 2000 bubble lipoplexes), and successfully obtained a high level of gene expression in mannose receptor-expressing cells following gene transfection using Man-PEG 2000 bubble lipoplexes and US exposure. We also reported that large amounts of plasmid DNA (pDNA) were transferred into the cytoplasm of the targeted cells in the gene transfection using this method. In the present study, we investigated the involvement of transcriptional processes on enhanced gene expression obtained by unmodified and Man-PEG 2000 bubble lipoplexes with US exposure. The transcriptional process related to activator protein-1 (AP-1) and nuclear factor-κB (NFκB) was activated by US exposure, and was founded to be involved in enhanced gene expression obtained by gene transfection using unmodified and Man-PEG 2000 bubble lipoplexes with US exposure. On the other hand, activation of AP-1 and NFκB pathways followed by US exposure was hardly involved in the inflammatory responses in the gene transfection using this method. These findings suggest that activation of AP-1 and NFκB followed by US exposure is involved in the enhanced gene expression using unmodified and Man-PEG 2000 bubble lipoplexes with US exposure, and the selection of pDNAs activated by US exposure is important in this gene transfection method.

CSN1 inhibits c-Jun phosphorylation and down-regulates ectopic expression of JNK1

CSN1 is a component of the COP9 signalosome (CSN), a conserved protein complex with pleiotropic functions in many organs and cell types. CSN regulates ubiquitinproteasome dependent protein degradation via the deneddylation and the associated deubiquitination activities. In addition, CSN associates with protein kinases and modulates cell signaling, particularly the activator protein 1 (AP-1) pathway. We have shown previously that CSN1 suppresses AP-1 transcription activity and inhibits ultraviolet (UV) and serum activation of c-fos expression. Here we show that CSN1 can inhibit phosphorylation of proto-oncogene c-Jun product and repress c-Jun dependent transcription. Further, CSN1 dramatically downregulates ectopic expression of c-Jun N-terminal kinase 1 (JNK1) in cultured cells. The decline in JNK1 is not caused by excessive proteolysis or by 3' UTR-dependent mRNA instability, but by CSN1-dependent repression of one or multiple steps in transcriptional and posttranscriptional mechanisms. Thus, in contrast to CSN5/Jab1, which promotes AP-1 activity, CSN1 displays a negative effect on the AP-1 pathway. Finally, we discuss about the dynamic equilibrium of the CSN complexes in regulation of the AP-1 pathway.

Abstract 1390: Endothelin-1/ETBR axis promote COX-2 production and migration in human chondrosarcoma cells through MAPK and AP-1 pathways

Abstract Chondrosarcoma is a type of highly malignant tumor with a potent capacity to invade locally and cause distant metastasis. Endothelin-1 (ET-1) the most potent vasoconstrictor, plays a crucial role in migration and metastasis of human cancer cells. Cyclooxygenase (COX)-2 has been implicated in tumor metastasis. However, the effects of ET-1 in migration and COX-2 expression in chondrosarcoma cells are largely unknown. The aim of this study was to investigate whether ET-1 is associated with the motility of human chondrosarcoma cell. We found that treatment of human chondrosarcoma cells (JJ012 cells) with ET-1 increased migration and expression of COX-2. Activations of MAPK (p38, JNK and ERK) and activator protein-1 (AP-1) pathways after ET-1 treatment were demonstrated, and ET-1-induced expression of COX-2 and migration activity was inhibited by the specific inhibitor and mutant of Endothelin B receptor (ETBR), MAPK and AP-1 cascades. Moreover, ET-1 increased the binding of c-Jun to the AP-1 element on the COX-2 promoter. ETBR and MAPK inhibitor reduced ET-1 mediated binding activity and AP-1 luciferase activity. Taken together, our results indicated that ET-1 enhances the migration of chondrosarcoma cells by increasing COX-2 expression through the ETBR axis, MAPK and AP-1 signal transduction pathways. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1390. doi:10.1158/1538-7445.AM2011-1390

Abstract 1377: Identification and characterization of small molecule inhibitors of the activator protein-1 pathway

Abstract The activation of transcription factor activator protein-1 (AP-1) plays a critical role in multiple stages associated with carcinogenesis. The AP-1 complex is a dimeric transcription factor typically made up of proteins from JUN, FOS, ATF, and MAF families. Growth factors including EGF activate AP-1 via cell surface receptors such as EGF receptor (EGFR). EGFR activation of MAPK cascades lead to phosphorylation and nuclear translocation of AP-1 subunits. Discovery of selective inhibitors of the AP-1 pathway would lead to invaluable tools in the study, treatment and prevention of multiple cancers. To screen for AP-1 pathway inhibitors, a FRET-based AP-1 β-lactamase reporter was used to measure AP-1 activation in a ME-180 cervical cancer cell line. Approximately 75,000 compounds were screened, each at 7-15 concentrations, in a quantitative high-throughput screening (qHTS) format. Several novel structure series were identified from this screen. Hits selected based on a structure-activity relationship analysis were confirmed and further investigated for mechanism of action because EGF-stimulated AP-1 activation can be inhibited by multiple mechanisms in the pathway. Inhibitors mechanisms of action in the EFGR-mediated pathway were determined in follow-up studies including receptor inhibition, MAPK inhibition, and direct disruption of the AP-1 signaling complex. The identification and characterization of novel AP-1 inhibitors will lead to compounds that serve as probes of the AP-1 pathway and potential candidates for anticancer drug development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1377. doi:10.1158/1538-7445.AM2011-1377