Activator Protein-1 Binding Activity Research Articles

Echinochrome A inhibits HMGB1-induced vascular smooth muscle cell migration by suppressing osteopontin expression.

Echinochrome A (Ech A) isolated from marine organisms is a therapeutic effector for various cardiovascular diseases, but its precise mechanisms are unclear. This study identified the role and mechanisms mediating the effects of Ech A on the migration of vascular smooth muscle cells (VSMCs) induced by high-mobility group box 1 (HMGB1). Compared to the control cells, the migration of VSMCs stimulated with HMGB1 (100 ng/ml) was markedly increased, which was significantly attenuated in cells pretreated with MPIIIB10 (100 ng/ml), a neutralizing monoclonal antibody for osteopontin (OPN). In VSMCs stimulated with HMGB1, the increased expression of OPN mRNA and protein was accompanied by an increased OPN promoter activity. In reporter gene assays using OPN promoter-luciferase constructs, the promoter region 538-234 bp of the transcription start site containing the binding sites for activator protein 1 (AP-1) was shown to be responsible for the increased transcriptional activity by HMGB1. In addition, the binding activity of AP-1 was increased in HMGB1-stimulated cells, highlighting the pivotal role of AP-1 on OPN expression in HMGB1-stimulated VSMCs. An examination of the vascular effects of Ech A showed that the increased AP-1 binding/promoter activities and OPN expression induced by HMGB1 were attenuated in cells pretreated with Ech A (3 or 10 μM). Similarly, Ech A inhibited HMGB1-induced VSMC migration in a concentration-dependent manner. These findings suggest that Ech A inhibits VSMC migration by suppressing OPN expression. Hence, Ech A is suggested as a potential therapeutic strategy for vascular remodeling in the injured vasculatures.

Shizukaol B, an active sesquiterpene from Chloranthus henryi, attenuates LPS-induced inflammatory responses in BV2 microglial cells.

The objective of the current study was to evaluate the anti-inflammatory effects of shizukaol B, a lindenane-type dimeric sesquiterpene isolated from the whole plant of Chloranthus henryi, on lipopolysaccharide (LPS)-induced activation of BV2 microglial cells in vitro. Our data showed that shizukaol B concentration-dependently suppressed expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), production of nitric oxide (NO), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) in LPS-stimulated BV2 microglia. Meanwhile, shizukaol B concentration- and time-dependently inhibited LPS-mediated c-Jun N-terminal kinase 1/2 (JNK) activation, but had little effect on extracellular signal-regulated kinase 1/2 or p38 phosphorylation. Furthermore, shizukaol B significantly blocked LPS-induced activator protein-1 (AP-1) activation, evidenced by reduced phosphorylation and nuclear translocation of c-Jun and DNA binding activity of AP-1. Taken together, our findings suggest that shizukaol B exerts anti-inflammatory effects in LPS-activated microglia partly by modulating JNK-AP-1 signaling pathway.

SH3GL1 inhibition reverses multidrug resistance in colorectal cancer cells by downregulation of MDR1/P-glycoprotein via EGFR/ERK/AP-1 pathway.

Multidrug resistance is one of the major reasons colorectal cancer (CRC) chemotherapy-based treatments fail, and novel biologically based therapies are urgently needed. Src homology 3 (SH3)-domain GRB2-like protein 1 (SH3GL1) is a membrane-bound protein which was found to be involved in tumor formation, progression, and metastasis. In this study, immunohistochemistry staining, quantitative real-time polymerase chain reaction (qRT-PCR), and western blot analysis revealed a high expression of SH3GL1 in human CRC tumor specimens and several CRC cells resistant to chemotherapeutics. Cell Counting Kit-8 (CCK-8) assay showed that transfection of pCDNA3.1(+)-SH3GL1 increased while transfection of SH3GL1 siRNA decreased cell viability in response to 5-fluorouracil (5-FU) treatment (P<0.05). Further studies indicated that transfection of SH3GL1 siRNA significantly downregulated multidrug resistance protein 1 (MDR1)/P-glycoprotein expression (P<0.05), decreased MDR1 promoter activity and activator protein-1 (AP-1) binding activity (P<0.05), and inhibited the activation of epidermal growth factor receptor (EGFR) and extracellular signal-regulated kinases 1/2 (ERK1/2) signaling (P<0.05) in CRC cells resistant to chemotherapeutics. Transfection of pCDNA3.1(+)-SH3GL1 caused the opposite effect. Additionally, pre-treatment with either EGFR kinase inhibitor PD153035 or ERK1/2 kinase inhibitor PD98059 in HCT116/5-FU cells partly inhibits P-glycoprotein expression and AP-1 binding activity (P<0.05). In conclusion, we confirmed that inhibition of SH3GL1 reverses multidrug resistance through declining P-glycoprotein expression via the EGFR/ERK/AP-1 pathway.

SIRT3 Mediates the Antioxidant Effect of Hydrogen Sulfide in Endothelial Cells.

Oxidative stress is a key contributor to endothelial dysfunction and associated cardiovascular pathogenesis. Hydrogen sulfide (H2S) is an antioxidant gasotransmitter that protects endothelial cells against oxidative stress. Sirtuin3 (SIRT3), which belongs to the silent information regulator 2 (SIR2) family, is an important deacetylase under oxidative stress. H2S is able to regulate the activity of several sirtuins. The present study aims to investigate the role of SIRT3 in the antioxidant effect of H2S in endothelial cells. Cultured EA.hy926 endothelial cells were exposed to hydrogen peroxide (H2O2) as a model of oxidative stress-induced cell injury. GYY4137, a slow-releasing H2S donor, improved cell viability, reduced oxidative stress and apoptosis, and improved mitochondrial function following H2O2 treatment. H2S reversed the stimulation of MAPK phosphorylation, downregulation of SIRT3 mRNA and reduction of the superoxide dismutase 2 and isocitrate dehydrogenase 2 expression which were induced by H2O2. H2S also increased activator protein 1 (AP-1) binding activity with SIRT3 promoter and this effect was absent in the presence of the specific AP-1 inhibitor, SR11302 or curcumin. Paraquat administration to mice induced a defected endothelium-dependent aortic vasodilatation and increased oxidative stress in both mouse aorta and small mesenteric artery, which were alleviated by GYY4137 treatment. This vasoprotective effect of H2S was absent in SIRT3 knockout mice. The present results highlight a novel role for SIRT3 in the protective effect of H2S against oxidant damage in the endothelium both in vitro and in vivo. H2S enhances AP-1 binding activity with the SIRT3 promoter, thereby upregulating SIRT3 expression and ultimately reducing oxidant-provoked vascular endothelial dysfunction. Antioxid. Redox Signal. 24, 329-343.

Abstract 508: Identification of FRA-1 as a potential driver of pro-invasive properties in pancreatic cancer in conjunction with MUC1

Abstract The transmembrane mucin, MUC1, contributes to the progression of pancreatic cancer, in part by conducting signals from the cell surface that regulate expression of molecules associated with aggressive tumor behavior. We investigated transcriptional co-regulatory effects mediated by interactions of the MUC1 cytoplasmic tail with activator protein 1 (AP-1). Previous results suggest that MUC1 regulates the capacity of AP-1 to bind specific promoter elements and regulate genes that contribute to tumor progression. The DNA binding activity of AP-1 is controlled in several ways including composition of the protein dimer. The precise mechanism by which MUC1 alters transcriptional complexes remains unknown. We sought to determine how MUC1 influences the formation of AP-1 complexes and potential downstream effects. We found MUC1 increased c-Jun protein levels, the major component of AP-1, in a manner largely independent of transcriptional upregulation (mRNA upregulation was observed in only one cell line of several analyzed). Formation of the AP-1 dimer was assessed by quantitative and semi-quantitative assays. MUC1 promoted the association of c-Jun with the Fos protein, FRA-1. This association was promoted by enhanced ERK2 signaling in MUC1 expressing cell lines. In breast cancer, FRA-1 drives an invasive and metastatic phenotype. Overexpression of FRA-1 in pancreatic cancer cell lines promoted migratory and invasive behaviors in vitro. Blocking FRA-1 activity by shRNA knockdown or ERK inhibition decreased migration and invasion. Analysis of gene expression in pancreatic tumor samples and normal, uninvolved tissue [mined from the Gene Expression Omnibus (GEO)] revealed that tumor samples had significantly increased levels of FRA-1 mRNA. A subset of these samples exhibited gene expression patterns consistent with a FRA-1:EMT phenotype previously observed in colorectal cancer cells. We evaluated expression of FRA-1 protein in primary pancreatic tumors, liver metastases, and uninvolved non-malignant pancreas. Robust nuclear localization of FRA-1 was observed in primary and metastatic tumor cells whereas uninvolved pancreas showed lower levels of diffuse cytoplasmic staining. Based on these results, we hypothesize that FRA-1 plays an important role in a subset of pancreatic cancers by altering the binding profile of AP-1. Due to a dependence on ERK activity for FRA-1 function, we propose that cancers with activating mutations in the RAS-RAF-MEK-ERK cascade are the most likely candidates for utilization of FRA-1. Citation Format: Ryan L. Hanson, Michael A. Hollingsworth. Identification of FRA-1 as a potential driver of pro-invasive properties in pancreatic cancer in conjunction with MUC1. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 508. doi:10.1158/1538-7445.AM2015-508

Ameliorative Effects of Neurolytic Celiac Plexus Block on Stress and Inflammation in Rats with Partial Hepatectomy

Purpose: To investigate effects of neurolytic celiac plexus block (NCPB) on stress and inflammation in rats with partial hepatectomy (PH).Methods: A model of PH rat was established, and serum C-reactive protein (CRP); corticosterone (GC); adrenocorticotropin (ACTH); noradrenaline (NA); adrenalin (AD); aspartate transaminase (AST); alanine transaminase (ALT); as well as tumor necrosis factor-α (TNF-α); interleukin (IL)-1β and IL-6; high mobility group box1 (HMGB1); and nitric oxide (NO) concentrations in serum assessed after PH. Additionally, Western blotting was performed to determine the effect of NCPB on expressions of glucocorticoid receptors (GR), inhibitor of nuclear factor kappa B (IκB), p65, c-Jun and inducible nitric oxide synthase (iNOS) of PH rats, as well as assay effects of NCPB on nuclear translocation of GR, c- Jun and p65. DNA binding activities of nuclear factor kappa B (NF-κB) and activator protein 1 (AP-1) were also determined.Results: NCPB reduced AST and ALT (P &lt; 0.05), decreased secretion of inflammatory cytokines and NO (P &lt; 0.05), as well as decreased CRP, GC, ACTH, NA and AD after PH (p &lt; 0.05). NCPB increased expressions of GR and IκB, but expressions of p65, c-Jun, and iNOS (p &lt; 0.05). Additionally, NCPB increased nuclear translocation of GR (p &lt; 0.01), but decreased nuclear translocation of p65 and c-Jun after PH (p &lt; 0.05). Additionally, DNA binding activity of NF-κB and AP-1 was decreased by NCPB (p &lt; 0.05).Conclusion: The results indicate that NCPB treatment can significantly inhibit stress and inflammation in PH rats.Keywords: Neurolytic celiac plexus block, Cytokine, Nuclear translocation, Partial hepatectomy, Stress, Inflammation

Puerarin Suppresses Angiotensin II-Induced Cardiac Hypertrophy by Inhibiting NADPH Oxidase Activation and Oxidative Stress-Triggered AP-1 Signaling Pathways.

To examine the effects of puerarin (Pue) on angiotensin II (AngII)-induced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation and oxidative stress-related signaling pathways in the hypertrophic response of cardiomyocytes. Primary cardiomyocytes of neonatal C57BL/6J mice were pretreated with Pue (50, 100 μmol/L) and were then stimulated with AngII 1 μmol/L. NADPH oxidase activity and reactive oxygen species (ROS) levels were measured by lucigenin-enhanced chemiluminescence assay and flow cytometry. Western blotting was used to detect the distribution of the oxidase subunits, extracellular signal-regulated kinase (ERK1/2) and c-jun N-terminal kinase (JNK1/2) activation, and an electrophoretic mobility shift assay (EMSA) was performed to analyze the DNA binding activity of activator protein-1 (AP-1). Adult C57BL/6J mice were infused with AngII and were administered with Pue (100, 200 mg·kg-1·d-1) for 15 d. After the treatment, systolic blood pressure (SBP) and left ventricular wall thickness were examined. The ratios of heart weight to body weight (HW/BW) and left ventricular weight to body weight (LVW/BW) were measured, and heart morphometry was assessed. In vitro, Pue dose-dependently blocked the phosphorylation of ERK1/2 and JNK1/2 and eventually abolished AP-1 binding activity through the inhibition of ROS production. Further studies revealed that AngII treatment resulted in increased NADPH oxidase activity, which was suppressed by Pue via the disruption of Rac1 activation and membrane translocation of oxidase subunits. In vivo, Pue attenuated cardiac hypertrophy, as evaluated by decreased HW/BW, LVW/BW, myocyte surface area, and left ventricular wall thickness. The anti-hypertrophic mechanism of Pue occurred by blocking Rac1-dependent NADPH oxidase activation and downstream redox-sensitive AP-1 signaling pathways.

Silibinin Inhibits the Invasion of IL-6-Stimulated Colon Cancer Cells via Selective JNK/AP-1/MMP-2 Modulation in Vitro

Silibinin is a flavonoid with antihepatotoxic properties and pleiotropic anticancer capabilities. This study investigated silibinin inhibition of cell invasion by down-regulating matrix metalloproteinase-2 (MMP-2) expression, via attenuation of activator protein-1 (AP-1) in IL-6-stimulated LoVo colon cancer cells. Western blot data showed that the expression of MMP-2 protein was reduced 1.6- or 1.7-fold over the control by treatment with silibinin or JNK inhibitor in the models. Similar results were revealed in zymography and confocal microscopy. Pretreatment with silibinin also abolished the binding activity of AP-1 and MMP-2 promoter activity via AP-1 binding, as observed by EMSA and luciferase assay. Finally, a [(3)H]-thymidine incorporation proliferation assay and cell migration assay demonstrated that silibinin inhibited IL-6-stimulated LoVo cell proliferation and invasion. Taken together, these data indicated that silibinin inhibits LoVo cell invasion with the reduction of MMP-2 presentation by attenuating AP-1 binding activity, suggesting a novel antimetastatic application for silibinin in colon cancer chemoprevention.

Aldose Reductase Inhibition Prevents Colon Cancer Growth by Restoring Phosphatase and Tensin Homolog Through Modulation of miR-21 and FOXO3a

We have shown earlier that inhibition of aldose reductase (AR), an oxidative stress-response protein, prevents colon cancer cell growth in vitro and in vivo. Changes in microribonucleic acid (miR) expression can contribute to cancer by modulating the functional expression of critical genes involved in cancer growth and metastasis. However, the molecular mechanisms by which AR regulates miR expression and their dependent mitogenic effects in cancer cells are not known. Therefore, we investigated how AR regulates growth factor-induced expression of miRs and growth of colon cancer cells. Inhibition of AR significantly downregulated growth factor-induced miR-21 expression in human colon cancer cells, HT29, SW480, and Caco-2. Further, AR inhibition also increased phosphatase and tensin homolog (PTEN) (a direct target of miR-21) and forkhead box O3A (FOXO3a) in colon cancer cells. Our results obtained with HT29 cells ablated with FOXO3a siRNA showed increased activator protein-1 (AP-1) activation and miR-21 expression, indicating that FOXO3a represses miR-21 via AP-1 inactivation. Inhibition of AR also prevented the epidermal growth factor-induced phosphorylation of phosphatidylinositol 3-kinase (PI3K), serine/threonine kinase (AKT), c-Jun, c-Fos, PTEN, and FOXO3a, and deoxyribonucleic acid (DNA)-binding activity of AP-1. More importantly, in human colon adenocarcinoma xenograft tissues, miR-21 expression was lower, and PTEN and FOXO3a levels were significantly higher in AR inhibitor-treated mice compared to controls. These findings demonstrate a novel role of AR in the regulation of miR-21 and its target PTEN in growth factor-induced colon cancer cell growth. Collectively, these results show a novel role of AR in mediation of growth factor-induced colon cancer growth by modulating miR-21, PTEN, and FOXO3a expression through reactive oxygen species (ROS)/PI3K/AKT/AP-1.

The chloroethylating anticancer drug ACNU induces FRA1 that is involved in drug resistance of glioma cells

FRA1 belongs, together with c-Fos and FosB, to the family of Fos proteins that form with members of the ATF and Jun family the transcription factor AP-1 (activator protein 1). Previously we showed that c-Fos protects mouse embryonic fibroblasts against the cytotoxic effects of ultraviolet (UV) light by induction of the endonuclease XPF, leading to enhanced nucleotide excision repair (NER) activity. Here, we analyzed the regulation of FRA1 in glioma cells treated with the anticancer drug nimustine (ACNU) and its role in ACNU-induced toxicity. We show that FRA1 is upregulated in glioblastoma cells following ACNU on mRNA and protein levels. Knockdown of FRA1 by either siRNA or shRNA clearly sensitized glioma cells towards ACNU-induced cell death. Despite decreased AP-1 binding activity upon FRA1 knockdown, this effect is independent on regulation of the AP-1 target genes fasL, ercc1 and xpf. In addition, FRA1 knockdown does not affect DNA repair capacity. However, lack of FRA1 attenuated the ACNU-induced phosphorylation of CHK1 and led to a reduced arrest of cells in G2/M and, thereby, presumably leads to enhanced cell death in the subsequent cell cycle.

Abstract 5704: Curcumin-folate conjugate for increased bioavailability and targeted delivery of curcumin to cancer cells

Abstract Specific types of high risk Human Papillomavirus (HR-HPV) types, particularly the HPV type 16 and HPV 18 are known to cause cervical cancer. The expression of two viral oncogenes E6 and E7 responsible for tumorigenic transformation, is mainly dependent on specific host-cell transcription factor, Activator Protein 1 (AP-1) which acts as a signaling epicenter for cervical cancer. Although recently two prophylactic HPV vaccines have been developed, there is no therapeutic molecule available for the treatment of cervical lesions. We demonstrate that Curcumin (Diferuloylmethane), a yellow pigment, used in traditional medicines and present in the dietary spice turmeric (Curcuma longa), can selectively down regulate HPV 18 transcription as well as AP-1 binding activity. Curcumin can also reverse the expression dynamics of c-fos and fra-1 in tumorigenic HeLa cells by mimicking their expression pattern in normal cells. But the bioavailability of curcumin is extremely poor due to its hydrophobicity, rapid metabolism and lack of specificity in targeting cancer cells. Therefore, a folic acid-curcumin conjugate for mutation has been developed by attaching one molecule of curcumin with two molecules of folic acid (Cur-2FA) to make curcumin hydrophilic, enhance its bioavailability and to target only cancer cells which specifically express high level of folate receptors. The effect of both native curcumin and Cur-2FA on HPV and AP-1 has been analyzed in HPV positive cervical cancer and HPV negative breast cancer cell lines, by band shift assay, confocal microscopy, flow cytometry and western blotting including their cytotoxic potential and targeted cellular uptake of curcumin. In comparison to native curcumin, curcumin-2FA has been found to be atleast two times more effective in inducing down regulation of HPV transcription, AP-1 activity and expression of its components particularly c-fos and fra-1. Curcumin-2FA was found to be more potent than curcumin in inducing apoptosis, inhibits tumor cell proliferation and Cur-2FA conjugate was specifically taken up by cancer cells. In mice, curcumin-2FA was more bioavailable and had a longer half-life than curcumin. We demonstrate that this nontoxic low molecular weight curcumin-2Folic acid conjugate formulation specifically targets cancer cells which overexpress high affinity folate receptor and enhanced cellular uptake facilitating increased bioavailability and targeted delivery to cancer cells. 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 5704. doi:1538-7445.AM2012-5704

Histone deacetylase inhibitors suppress rheumatoid arthritis fibroblast-like synoviocyte and macrophage IL-6 production by accelerating mRNA decay

BackgroundHistone deacetylase inhibitors (HDACi) display potent therapeutic efficacy in animal models of arthritis and suppress inflammatory cytokine production in rheumatoid arthritis (RA) synovial macrophages and tissue.ObjectivesTo determine the molecular mechanisms...

Aspirin-triggered lipoxin A4attenuates LPS-induced pro-inflammatory responses by inhibiting activation of NF-κB and MAPKs in BV-2 microglial cells

BackgroundMicroglial activation plays an important role in neurodegenerative diseases through production of nitric oxide (NO) and several pro-inflammatory cytokines. Lipoxins (LXs) and aspirin-triggered LXs (ATLs) are considered to act as 'braking signals' in inflammation. In the present study, we investigated the effect of aspirin-triggered LXA4 (ATL) on infiammatory responses induced by lipopolysaccharide (LPS) in murine microglial BV-2 cells.MethodsBV-2 cells were treated with ATL prior to LPS exposure, and the effects of such treatment production of nitric oxide (NO), inducible nitric oxide synthase (iNOS), interleukin-1β (IL-1β) and tumour necrosis factor-α (TNF-α) were analysed by Griess reaction, ELISA, western blotting and quantitative RT-PCR. Moreover, we investigated the effects of ATL on LPS-induced nuclear factor-κB (NF-κB) activation, phosphorylation of mitogen-activated protein kinases (MAPKs) and activator protein-1 (AP-1) activation.ResultsATL inhibited LPS-induced production of NO, IL-1β and TNF-α in a concentration-dependent manner. mRNA expressions for iNOS, IL-1β and TNF-α in response to LPS were also decreased by ATL. These effects were inhibited by Boc-2 (a LXA4 receptor antagonist). ATL significantly reduced nuclear translocation of NF-κB p65, degradation of the inhibitor IκB-α, and phosphorylation of extracellular signal-regulated kinase (ERK) and p38 MAPK in BV-2 cells activated with LPS. Furthermore, the DNA binding activity of NF-κB and AP-1 was blocked by ATL.ConclusionsThis study indicates that ATL inhibits NO and pro-inflammatory cytokine production at least in part via NF-κB, ERK, p38 MAPK and AP-1 signaling pathways in LPS-activated microglia. Therefore, ATL may have therapeutic potential for various neurodegenerative diseases.

MAPKs, activator protein-1 and nuclear factor-κB mediate production of interleukin-1β-stimulated cytokines, prostaglandin E2 and MMP-1 in human periodontal ligament cells

Determination of the interleukin-1 (IL-1) signaling cascades that lead to the production of various inflammatory mediators and catabolic factors may clarify attractive targets for therapeutic intervention for periodontitis. We comprehensively assessed the involvement of MAPKs, activator protein-1 (AP-1) and nuclear factor-κB (NF-κB) in IL-1β-induced production of interleukin-6 (IL-6), interleukin-8 (IL-8), prostaglandin E(2) (PGE(2) ) and MMP-1 in human periodontal ligament cells. Human periodontal ligament cells were pretreated with an inhibitor for each of the MAPKs or NF-κB and subsequently treated with IL-1β. Following treatment, phosphorylation of three types of MAPK (ERK, p38 MAPK and c-Jun N-terminal kinase), IκB kinase (IKK) α/β/γ and IκB-α, as well as the DNA binding activity of AP-1 and NF-κB and the production of IL-6, IL-8, PGE(2) and MMP-1, were determined by western blotting, a gel mobility shift assay and ELISA, respectively. The three MAPKs, simultaneously activated by IL-1β, mediated the subsequent DNA binding of AP-1 at various magnitudes, while IKKα/β/γ, IκB-α and NF-κB were also involved in the IL-1 signaling cascade. Furthermore, IL-1β stimulated the production of IL-6, IL-8, PGE(2) and MMP-1 via activation of the three MAPKs and NF-κB, because inhibitors of these significantly suppressed the IL-1β-stimulated production of these factors. Our results strongly suggest that MAPK, AP-1 and NF-κB mediate the IL-1β-stimulated synthesis of IL-6, IL-8, PGE(2) and MMP-1 in human periodontal ligament cells. Therefore, inhibition of activation of MAPK, AP-1 and/or NF-κB may lead to therapeutic effects on progression of periodontitis.

Suppression of vascular smooth muscle cell responses induced by TNF-α in GM3 synthase gene transfected cells

The natural accumulation of ganglioside GM3 (N-glycolylneuraminic acid) on atherosclerotic lesions is a common theory. The present study is the first to examine the effects of the GM3 synthase gene on the responses of vascular smooth muscle cells (VSMC) to tumor necrosis factor-α (TNF-α). We found that overexpression of the GM3 synthase gene inhibited DNA synthesis and ERK1/2 activity induced by TNF-α in VSMC, whereas the basal levels of DNA synthesis and ERK1/2 activity remained unchanged. In addition, GM3 synthase gene transfectants significantly reduced the migration and invasion of VSMC following TNF-α treatment, compared with empty vector transfectants. Furthermore, TNF-α-induced matrix metalloproteinase-9 (MMP-9) expression and promoter activity were also decreased in GM3 synthase gene transfectants. GM3 synthase gene expression markedly suppressed the TNF-α-stimulated transcriptional activity of activator protein-1 (AP-1) and nuclear factor-κB (NF-κB), which are the controlling factors of MMP-9 expression. Consistent with these results, the addition of anti-GM3 antibody into the GM3 synthase gene transfectants blocked inhibition of DNA synthesis, ERK1/2 activity, migration and invasion. Finally, GM3 synthase gene transfectants treated with anti-GM3 antibody reversed the suppression of MMP-9 expression by reducing AP-1 and NF-κB binding activity. These results suggest regulatory roles for the GM3 synthase gene in VSMC proliferation and migration during the formation of atherosclerotic lesions.

Regulatory mechanism of activator protein-1 on the expression of MUC5AC induced by cigarette smoke extract

To investigate the mechanism of activator protein-1 (AP-1) on cigarette smoke-induced airway mucous hypersecretion and to explore the possible signal transduction pathway that activates AP-1. The airway epithelial cell line (BEAS-2B) was cultured in vivo and treated with cigarette smoke extract (CSE). The DNA binding activity of AP-1 was blocked by the transfection of c-Jun dominant negative mutant TAM67 into the cells. SP600125 and PD98059 were used to block the activation of c-Jun terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) respectively. MUC5AC protein was detected by enzyme-linked immunosorbent assay, MUC5AC mRNA level was analyzed by RT-PCR, while the protein contents of p-JNK, p-ERK and p-P38 were detected by Western blot, and the DNA binding activity of AP-1 was determined by electrophoretic mobility shift assay. The MUC5AC protein production and mRNA expression in the CSE group were significantly higher than those in the control group, and the DNA binding activity of AP-1 was also higher than that in the control group (P<0.01). The protein contents of p-ERK and p-JNK in the CSE group were higher than those in the control group (P<0.01), but the p-P38 level was not significantly different from that in the control group (P>0.05). After the transfection of TAM67 into the cells, the expression levels of MUC5AC protein and mRNA and the binding activity of AP-1 decreased significantly (P<0.01). The DNA binding activity of AP-1 and the expression levels of MUC5AC protein and mRNA were lower in the SP600125 group and in the PD98059 group than those in the CSE group (P<0.05). After being activated by JNK and ERK which are phosphorylated by cigarette smoke, AP-1 binds to its DNA binding elements on the promoter of MUC5AC gene and up-regulates the MUC5AC expression at the transcriptional level.

Effects of Cigarette Smoke on the Activation of Oxidative Stress-Related Transcription Factors in Female A/J Mouse Lung

Cigarette smoke contains a high concentration of free radicals and induces oxidative stress in the lung and other tissues. Several transcription factors are known to be activated by oxidative stress, including nuclear factor-κB (NF-κB), activator protein-1 (AP-1), and hypoxia-inducible factor (HIF). Studies were therefore undertaken to examine whether cigarette smoke could activate these transcription factors, as well as other transcription factors that may be important in lung carcinogenesis. Female A/J mice were exposed to cigarette smoke for 2, 5, 10, 15, 20, 42, or 56 d (6 hr/d, 5 d/wk). Cigarette smoke did not increase NF-κB activation at any of these times, but NF-κB DNA binding activity was lower after 15 d and 56 d of smoke exposure. The DNA binding activity of AP-1 was lower after 10 d and 56 d but was not changed after 42 d of smoke exposure. The DNA binding activity of HIF was quantitatively increased after 42 d of smoke exposure but decreased after 56 d. Whether the activation of other transcription factors in the lung could be altered after exposure to cigarette smoke was subsequently examined. The DNA binding activities of FoxF2, myc-CF1, RORE, and p53 were examined after 10 d of smoke exposure. The DNA binding activities of FoxF2 and p53 were quantitatively increased, but those of myc-CF1 and RORE were unaffected. These studies show that cigarette smoke exposure leads to quantitative increases in DNA binding activities of FoxF2 and p53, while the activations of NF-κB, AP-1, and HIF are largely unaffected or reduced.

Epigallocatechin-3-gallate (EGCG) downregulates gelatinase-B (MMP-9) by involvement of FAK/ERK/NFκB and AP-1 in the human breast cancer cell line MDA-MB-231

Epigallocatechin-3-gallate (EGCG) is effective against the initiation, progression, and invasion of carcinogenesis.Matrix-metalloproteinases (MMPs) are a family of endopeptidases that hydrolyze the majority of extracellular proteins. MMP-9 is one of the most important members of the family and we observed the effect of EGCG on MMP-9 in the human breast cancer cell line, MDA-MB-231.The effect of EGCG on MMP-9 was studied by gelatin zymography, western blot, quantitative and semiquantitative real-time RT-PCR, immunoflourescence, cell adhesion assay, enzyme-linked immunosorbent assay,and electrophoretic mobility shift assay. EGCG treatment reduced the activity, protein, and mRNA expression ofMMP-9 and enhanced the expression of the tissue inhibitor of MMP 1 (TIMP-1). EGCG downregulated the activation of focal adhesion kinase (FAK) and extracellular regulated kinase (ERK), reduced the adhesion of MDA-MB-231 cells to fibronectin and vitronectin, and reduced the mRNA expression of the integrin receptors alpha5beta1 and alphavbeta3. The expression of the nuclear factor kappa B (NFjB), and the DNA binding activity of NFjB and activator protein 1 (AP1)to MMP-9 promoter were noticeably reduced on EGCG treatment. Upregulation of TIMP-1 and disruption of the functional status of integrin receptors may indicate decreased MMP-9 activation; inhibition of FAK andERK activation might indicate disruption in the FAK/ERK-induced MMP-9 secretion and induction. Decreased DNA binding activity of NFjB and AP1 to MMP-9 promoter might indicate transcriptional deregulation of MMP-9 gene on EGCG treatment. We propose EGCG as a potential inhibitor of the expression and activity of MMP-9 by a process involving FAK/ERK and transcription factorsin MDA-MB-231.

Transcriptional regulation of SP-B gene expression by nitric oxide in H441 lung epithelial cells.

Surfactant protein B (SP-B) is essential for the surface tension-lowering function of pulmonary surfactant. Surfactant dysfunction and reduced SP-B levels are associated with elevated nitric oxide (NO) in inflammatory lung diseases, such as acute respiratory distress syndrome. We previously found that NO donors decreased SP-B expression in H441 and MLE-12 lung epithelial cells by reducing SP-B promoter activity. In this study, we determined the roles of DNA elements and interacting transcription factors necessary for NO inhibition of SP-B promoter activity in H441 cells. We found that the NO donor diethylenetriamine-nitric oxide adduct (DETA-NO) decreased SP-B promoter thyroid transcription factor 1 (TTF-1), hepatocyte nuclear factor 3 (HNF-3), and Sp1 binding activities but increased activator protein 1 (AP-1) binding activity. DETA-NO decreased TTF-1, but not Sp1, levels, suggesting that reduced TTF-1 expression contributes to reduced TTF-1 binding activity. Lack of effect on Sp1 levels suggested that DETA-NO inhibits Sp1 binding activity per se. Overexpression of Sp1, but not TTF-1, blocked DETA-NO inhibition of SP-B promoter activity. DETA-NO inhibited SP-B promoter induction by exogenous TTF-1 without altering TTF-1 levels. DETA-NO decreased TTF-1 mRNA levels and gene transcription rate, indicating that DETA-NO inhibits TTF-1 expression at the transcriptional level. We conclude that NO inhibits SP-B promoter by decreasing TTF-1, Sp1, and HNF-3 binding activities and increasing AP-1 binding activity. NO inhibits TTF-1 levels and activity to decrease SP-B expression. NO inhibition of SP-B expression could be a mechanism by which surfactant dysfunction occurs in inflammatory lung diseases.

Acacetin Inhibits TPA‐Induced MMP‐2 and u‐PA Expressions of Human Lung Cancer Cells Through Inactivating JNK Signaling Pathway and Reducing Binding Activities of NF‐κB and AP‐1

Acacetin (5,7-dihydroxy-4'-methoxyflavone), a flavonoid compound, has antiperoxidative and antiinflammatory effects. The effect of acacetin on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced MMPs and u-PA expressions in human lung cancer A549 cells was investigated. First, the result demonstrated acacetin could inhibit TPA-induced the abilities of the adhesion, invasion, and migration by cell-matrix adhesion assay and Boyden chamber assay. Data also showed acacetin could inhibit phosphorylation of c-Jun N-terminal kinase 1 and 2 (JNK1/2) involved in the down-regulating protein expressions and transcriptions of matrix metalloproteinase-2 (MMP-2) and urokinase-type plasminogen activator (u-PA) induced by TPA. Next, acacetin also strongly inhibited TPA-stimulated the nuclear levels of nuclear factor kappa B (NF-kappaB), c-Fos, and c-Jun. Also, a dose-dependent inhibition on the binding abilities of NF-kappaB and activator protein-1 (AP-1) by acacetin treatment was further observed. Further, the treatment of specific inhibitor for JNK (SP600125) to A549 cells could inhibit TPA-induced MMP-2 and u-PA expressions along with an inhibition on cell invasion and migration. Taken together, these results suggest the antimetastatic effects of acacetin on the TPA-induced A549 cells might be by reducing MMP-2 and u-PA expressions through inhibiting phosphorylation of JNK and reducing NF-kappaB and AP-1 binding activities.