Activator Protein-1 DNA Binding Activity Research Articles

Attenuation of the upregulation of NF‑κB and AP‑1 DNA‑binding activities induced by tunicamycin or hypoxia/reoxygenation inneonatal rat cardiomyocytes by SERCA2a overexpression.

The present study aimed to investigate the effects of the overexpression of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2a) on endoplasmic reticulum (ER) stress (ERS)-associated inflammation in neonatal rat cardiomyocytes (NRCMs) induced by tunicamycin (TM) or hypoxia/reoxygenation (H/R). The optimal multiplicity of infection (MOI) was 2 pfu/cell. Neonatal Sprague-Dawley rat cardiomyocytes cultured in vitro were infected with adenoviral vectors carrying SERCA2a or enhanced green fluorescent protein genes, the latter used as a control. At 48 h following gene transfer, the NRCMs were treated with TM (10 µg/ml) or subjected to H/R to induce ERS. The results of electrophoretic mobility shift assay (EMSA) revealed that overexpression of SERCA2a attenuated the upregulation of nuclear factor (NF)-κB and activator protein-1 (AP-1) DNA-binding activities induced by TM or H/R. Western blot analysis and semi-quantitative RT-PCR revealed that the overexpression of SERCA2a attenuated the activation of the inositol-requiring 1α (IRE1α) signaling pathway and ERS-associated apoptosis induced by TM. The overexpression of SERCA2a also decreased the level of phospho-p65 (Ser536) in the nucleus, as assessed by western blot analysis. However, the overexpression of SERCA2a induced the further nuclear translocation of NF-κB p65 and higher levels of tumor necrosis factor (TNF)-α transcripts in the NRCMs, indicating the occurrence of the ER overload response (EOR). Therefore, the overexpression of SERCA2a has a 'double-edged sword' effect on ERS-associated inflammation. On the one hand, it attenuates ERS and the activation of the IRE1α signaling pathway induced by TM, resulting in the attenuation of the upregulation of NF-κB and AP-1 DNA-binding activities in the nucleus, and on the other hand, it induces EOR, leading to the further nuclear translocation of NF-κB and the transcription of TNF-α. The preceding EOR may precondition the NRCMs against subsequent ERS induced by TM. Further studies using adult rat cardiomyocytes are required to prevent the interference of EOR. The findings of the present study may enhance the current understanding of the role of SERCA2a in cardiomyocytes.

Mycobacterium tuberculosis induces connective tissue growth factor expression through the TLR2‐JNK‐AP‐1 pathway in human lung fibroblasts

Mycobacterium tuberculosis (M.tb) infection in lung causes pulmonary fibrosis, which leads to the irreversible reduction of pulmonary function. Fibrotic protein connective tissue growth factor (CTGF) expression has been confirmed to play a crucial role in lung fibrosis. However, the underlying signal pathway and effect of M.tb on CTGF expression in human lung fibroblasts are unclear. Our results revaled that M.tb caused time- and concentration-dependent increases in CTGF expression in human lung fibroblasts. A mechanistic investigation revealed that M.tb induced CTGF expression through TLR2 but not TLR4. The promoter activity assay indicated that M.tb-induced CTGF activity was mainly controlled by the promoter region at -747 to -184 bp, which contained signal transducer and activator of transcription 3 and activator protein 1 (AP-1) binding sites. Moreover, curcumin (AP-1 inhibitor) restrained M.tb-induced CTGF expression. M.tb also induced increases in AP-1 luciferase activity and DNA binding activity of c-Jun and c-Fos on the CTGF promoter. Furthermore, the knockdown of c-Jun by small interfering RNA attenuated M.tb-induced CTGF expression and AP-1 luciferase activity. A JNK inhibitor (SP600125) and a JNK dominant-negative mutant suppressed M.tb-induced CTGF expression. We also discovered that M.tb could induce the phosphorylation of JNK and c-Jun. Furthermore, SP600125 inhibited M.tb-induced c-Jun phosphorylation and AP-1- luciferase activity. M.tb-induced fibronectin expression was inhibited by anti-CTGF antibody. These results demonstrate that M.tb is activated through TLR2 to induce JNK activation, further increasing the DNA binding activity of c-Jun and c-Fos and finally inducing CTGF expression and extracellular matrix production.-Lee, H.-S., Hua, H.-S., Wang, C.-H., Yu, M.-C., Chen, B.-C., Lin, C.-H. Mycobacterium tuberculosis induces connective tissue growth factor expression through the TLR2-JNK-AP-1 pathway in human lung fibroblasts.

Involvement of the p38 MAPK signaling pathway in overexpression of matrix metalloproteinase-9 during the course of brain edema in 1,2-dichloroethane-intoxicated mice

Accumulated data have revealed that subacute poisoning of 1,2-dichloroethane (1,2-DCE), an industrial solvent used in some countries can cause encephalopathy, in which brain edema is the main pathological change. However, the underlying mechanisms are unclear. In the present study, we hypothesized that the p38 MAPK (p38) signaling pathway could be activated in 1,2-DCE-intoxicated mice, which in turn stimulates transcription factors, such as nuclear factor-κB (NF-κB) and activator protein-1 (AP-1), and then enhances the expression of proinflammatory factors, including matrix metalloproteinase-9 (MMP-9), finally leading to blood-brain barrier (BBB) disruption and brain edema formation. Our results revealed that brain water content and BBB permeability increased significantly in the intoxicated mice. Meanwhile, the levels of phosphorylated p38 (p-p38) and inhibitory κBα (p-IκB), as well as the expression levels of MMP-9, c-jun, c-fos, and p65, also increased markedly in the brains of intoxicated mice. Conversely, the protein levels of ZO-1, occludin and claudin-5 in these mice decreased markedly, but their JAM-1 protein levels increased dramatically. Our results revealed that p-p38 levels in the brains of intoxicated mice were suppressed by pretreatment with a p38 inhibitor. In response to suppressed p-p38 levels, the brain water contents and DNA binding activities of NF-κB and AP-1, as well as the expression levels of MMP-9, c-jun, c-fos, p65, p-IκB and JAM-1, decreased, whereas the protein levels of ZO-1, occludin and claudin-5 increased markedly. Taken together, our findings indicated that the p38 signaling pathway might be activated and involved in the course of brain edema in 1,2-DCE-intoxicated mice.

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.

Perilla frutescens leaves extract ameliorates ultraviolet radiation-induced extracellular matrix damage in human dermal fibroblasts and hairless mice skin

Ethnopharmacological relevancePerilla frutescens (L.) Britt. (Lamiaceae) is a traditional herb that is consumed in East Asian countries as a traditional medicine. This traditional herb has been documented for centuries to treat various diseases such as depression, allergies, inflammation and asthma. However, the effect of Perilla frutescens on skin has not been characterized well. Aim of the studyThe present study aimed to investigate the effect of Perilla frutescens leaves extract (PLE) on ultraviolet radiation-induced extracellular matrix damage in human dermal fibroblasts and hairless mice skin. Materials and methodsHuman dermal fibroblasts and Skh-1 hairless mice were irradiated with UV and treated with PLE. Protein and mRNA levels of various target molecules were analyzed by western blotting and quantitative RT-PCR, respectively. Histological changes of mouse skin were analyzed by H&E staining. To elucidate underlying mechanism of PLE, activator protein-1 (AP-1) DNA binding assay and the measurement of reactive oxygen species (ROS) were performed. ResultsPLE significantly inhibited basal and UV-induced MMP-1 and MMP-3 expression dose-dependently, and also decreased UV-induced phosphorylation of extracellular signal-regulated kinases and c-Jun N-terminal kinases. This inhibitory effects of PLE on MMP-1 and MMP-3 were mediated by reduction of ROS generation and AP-1 DNA binding activity induced by UV. Furthermore, PLE promoted type I procollagen production irrespective of UV irradiation. In the UV-irradiated animal model, PLE significantly reduced epidermal skin thickness and MMP-13 expression induced by UV. ConclusionOur results demonstrate that PLE has the protective effect against UV-induced dermal matrix damage. Therefore, we suggest that PLE can be a potential agent for prevention of skin aging.

Opposing effects of bile acids deoxycholic acid and ursodeoxycholic acid on signal transduction pathways in oesophageal cancer cells.

Ursodeoxycholic acid (UDCA) was reported to reduce bile acid toxicity, but the mechanisms underlying its cytoprotective effects are not fully understood. The aim of the present study was to examine the effects of UDCA on the modulation of deoxycholic acid (DCA)-induced signal transduction in oesophageal cancer cells. Nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) activity was assessed using a gel shift assay. NF-κB activation and translocation was performed using an ELISA-based assay and immunofluorescence analysis. COX-2 expression was analysed by western blotting and COX-2 promoter activity was assessed by luciferase assay. DCA induced NF-κB and AP-1 DNA-binding activities in SKGT-4 and OE33 cells. UDCA pretreatment inhibited DCA-induced NF-κB and AP-1 activation and NF-κB translocation. This inhibitory effect was coupled with a blockade of IκB-α degradation and inhibition of phosphorylation of IKK-α/β and ERK1/2. Moreover, UDCA pretreatment inhibited COX-2 upregulation. Using transient transfection of the COX-2 promoter, UDCA pretreatment abrogated DCA-induced COX-2 promoter activation. In addition, UDCA protected oesophageal cells from the apoptotic effects of deoxycholate. Our findings indicate that UDCA inhibits DCA-induced signalling pathways in oesophageal cancer cells. These data indicate a possible mechanistic role for the chemopreventive actions of UDCA in oesophageal carcinogenesis.

Abstract 2536: AP-1/Fra-1 as a target for therapy promotes chemoradiosensitivity of cancer and cancer stem cells

Abstract Transcription factor activator protein-1 (AP-1) super-family is known to modulate expression of array of genes during development of many cancers. It is also an indispensable factor for efficient expression of high risk HPV oncogenes E6/E7. Recent studies demonstrated the role of AP-1 in promoting stemness/quiescence of cancer stem cells. We have recently reported that HPVE6 oncogene controls stemness and self-renewal of cervical cancer stem cells through Hes1/NOTCH-1expression but little is known about factor(s) responsible for chemo- radioresistance of cancer stem cells. To understand this we have dissected the role of AP-1and its family members by using a herbal compound, curcumin and UV irradiation in cervical cancer. We isolated cervical cancer stem cells and enriched them by sequential gating from HPV+ve and HPV-ve human cervical cancer cell lines (SiHa and C33a) using a set of functional and phenotypic markers (ABCG2, CD49f, CD71, CD133). The isolated cervical cancer stem cells (SP+ve→CD49+veCD71−ve→CD133+ve) designated as CaCxSLCs displayed spheroid forming and self-renewal property with increased expression of HPVE6, AP-1 transcripts and a higher AP-1 DNA-binding activity compared to non-stem cancer cells (NSP−ve→CD49−veCD71+ve→ CD133−ve) or unsorted parental cells. Treatment with UV-radiation alone resulted in clonogenic survival of CaCxSLCs but not non-stem cancer cells with enhanced AP-1 expression and activation. In contrast, CaCxSLCs treated with UV in combination with curcumin, resulted in complete loss of AP-1 DNA-binding activity followed by decrease in sphere formation and percentage of SP cells but an increased expression of the fos-related antigen-1 (Fra-1) which has been earlier shown to us have tumor suppressor activity in cervical cancer. Our study demonstrates a critical role of AP-1/Fra-1 signaling in imparting radiosensitization of cervical cancer stem cells. Curcumin, which down regulates almost all signalling pathways, including all members of AP-1 except Fra-1 indicating its role in chemo-radiosensitization of cancer stem cells. Thus combining curcumin with conventional cancer drugs may make cancer treatment most effective. Citation Format: Bhudev C. Das, Abhishek Tyagi, Bal Gangadhar Roy, Alok C. Bharti. AP-1/Fra-1 as a target for therapy promotes chemoradiosensitivity of cancer and cancer stem cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2536.

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 < 0.05), decreased secretion of inflammatory cytokines and NO (P < 0.05), as well as decreased CRP, GC, ACTH, NA and AD after PH (p < 0.05). NCPB increased expressions of GR and IκB, but expressions of p65, c-Jun, and iNOS (p < 0.05). Additionally, NCPB increased nuclear translocation of GR (p < 0.01), but decreased nuclear translocation of p65 and c-Jun after PH (p < 0.05). Additionally, DNA binding activity of NF-κB and AP-1 was decreased by NCPB (p < 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

Modulation of both activator protein-1 and nuclear factor-kappa B signal transduction of human T cells by amiodarone.

Amiodarone, a common and effective antiarrhythmic drug, has been reported to have anti-inflammatory effects such as reducing the activation and movement of neutrophils. However, its effects on human T cells remain unclear. The aim of this study was to elucidate the effects and possible underlying mechanisms of amiodarone on human T cells. We isolated human primary T cells from the peripheral blood of healthy volunteers and performed enzyme-linked immunosorbent assay (ELISA), flow cytometry, electrophoretic mobility shift assay, luciferase assay, and Western blotting to evaluate the modulatory effects of amiodarone on human T cells. We found that amiodarone dose dependently inhibited the production of cytokines, including interleukin-2 (IL-2), IL-4, tumor necrosis factor-alpha, and interferon-gamma in activated human T cells. By flow cytometry, we demonstrated that amiodarone suppressed the expression of IL-2 receptor-alpha (CD25) and CD69, the cell surface markers of activated T cells. Moreover, molecular investigations revealed that amiodarone down-regulated activator protein-1 (AP-1) and nuclear factor kappa-B (NF-κB) DNA-binding activities in activated human T cells and also inhibited DNA binding and transcriptional activities of both AP-1 and NF-κB in Jurkat cells. Finally, by Western blotting, we showed that amiodarone reduced the activation of c-Jun NH(2)-terminal protein kinase and P38 mitogen-activated protein kinase, and suppressed stimuli-induced I-kappa B-alpha degradation in activated human T cells. Through regulation of AP-1 and NF-κB signaling, amiodarone inhibits cytokine production and T cell activation. These results show the pleiotropic effects of amiodarone on human T cells and suggest its therapeutic potential in inflammation-related cardiovascular disorders.

Electroacupuncture attenuates mechanical allodynia by suppressing the spinal JNK1/2 pathway in a rat model of inflammatory pain.

Electroacupuncture (EA) has a substantial analgesic effect on inflammatory pain induced by complete Freund's adjuvant (CFA). The activation of the c-Jun N-terminal kinase 1/2 (JNK1/2) signal transduction pathway in the spinal cord is associated with inflammatory pain. However, the relationship between EA's analgesic effect and the JNK1/2 signal transduction pathway in the inflammatory pain remain unclear. In the present study, we used the established rat model of CFA-induced inflammatory pain to investigate the role of the spinal JNK1/2 pathway in EA-mediated analgesia. We observed a decrease in paw withdrawal thresholds and an increase in paw edema at 1 and 3 days after injecting CFA into the right hindpaw. CFA, 3 days after injection, upregulated expression of phospho-c-Jun N-terminal kinase1/2 (p-JNK1/2) protein and its downstream targets, the transcriptional regulators p-c-Jun and activator protein-1 (AP-1), as well as cyclooxygenase-2 (COX-2) and the transient receptor potential vanilloid 1 (TRPV1). EA significantly alleviated CFA-induced inflammatory pain. In addition, EA reduced p-JNK1/2 protein levels and COX-2 mRNA expressions, a degree of down-regulated p-c-Jun protein level and AP-1 DNA binding activity in the spinal dorsal horn of CFA-administered animals, but it had no effect on TRPV1 mRNA expression. Furthermore, EA and the JNK inhibitor SP600125 synergistically inhibited CFA-induced hyperalgesia and suppressed the COX-2 mRNA expression in the spinal dorsal horn. Our findings indicate that EA alleviates inflammatory pain behavior, at least in part, by reducing COX-2 expression in the spinal cord via the JNK1/2 signaling pathway. Inactivation of the spinal JNK1/2 signal transduction pathway maybe the potential mechanism of EA's antinociception in the inflammatory pain model.

Abstract 2568: Hexane fraction of Naematoloma sublateritium extracts inhibits metastatic potential of MDA-MB-231 Breast cancer cells through modulation of the JNK and p38 pathway

Abstract Despite years of therapeutic development and research, metastasis is still the major driver of mortality in cancer patients. Breast cancer is a second leading cause of cancer deaths in woman world-wide. The prevalence of breast cancer continues to increase by approximately 2% each year. Breast cancer also presents as metastatic disease, which has spread beyond the original organ, breast to bone, liver, lung and brain. Recently, many studies have reported that natural products- dietary phytochemicals including mushroom are known to exhibit a variety of anti-cancer, anti-invasive and anti-metastatic activities. Naematolma spp. is a basidiomycete of the fungus that is known to produce anti-tumor compound, clavaric acid. In our previous studies, HFNS has been shown to significantly inhibit cell proliferation and induce apoptosis of human estrogen-nonresponsive MDA-MB-231 breast cancer cells. In this present study, we report for the first time that hexane fraction of Naematoloma sublateritium (HFNS) regulates TNF-alpha induced invasion and migration in MDA-MB-231 cells. We observed that non-cytotoxic concentrations (10-50 μg/mL) of HFNS markedly inhibited the invasion and migration of highly metastatic MDA-MB-231 cells as shown by a Matrigel invasion assay and wound healing analysis, respectively. The results of a gelatin zymography showed that HFNS suppressed the activity of matrix metalloproteinase (MMP)-9 and urokinase plasminogen activator (uPA). Western blot results demonstrated that treatment with HFNS had decreased level of MMP-9, and uPA, while the expression of the endogenous inhibitor proteins including tissue inhibitors of MMP (TIMP-1 and TIMP-2), and plasminogen activator inhibitor (PAI)-1, was up-regulated in a dose-dependent manner. Furthermore, HFNS suppressed the phosphorylation of p38, and JNK1/2 in MDA-MB-231 cells, where extracellular signal-regulated kinase (ERK) 1/2 were not affected. It was confirmed by pretreatment with SB203580 (p38 inhibitor), SP600125 (JNK inhibitor), and PD98059 (ERK inhibitor), respectively, before stimulation with TNF-alpha. Interestingly, the HFNS treatment also led to a dose-dependent inhibition on NF-κB and activator protein-1(AP-1) binding activity which are downstream targets of JNK and p38. The gel-shift assay results supported that HFNS treatment caused down-regulation of MMP-9 and uPA gene in mRNA level. Taken together, these data suggest the inhibitory effect of HFNS on the metastatic potential of MDA-MB-231 cells through inhibiting the phosphorylation of JNK/p38 and reducing NFκB and AP-1 DNA binding activities. In conclusion, we propose that HFNS may be a safe and potential therapeutic agent against metastasis of breast cancer. 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 2568. doi:1538-7445.AM2012-2568

Effects of electroacupuncture on interleukin -2 , interferon -gamma and extracellular-regulated protein kinase signaling pathways in the splenic T cells of traumatized rats

Objective To investigate effect of electroacupuncture (EA) on interleukin-2 (IL -2)and interferon-gamma(IFN-γ)protein and mRNA expression,as well as extracellular- regulated protein kinase1/2 (ERK1/2) signaling pathway,and evaluate the signaling regulatory mechanism of EA effect. Methods Thirty-two male Sprague-Dawley (SD) rats were divided randomly into four groups (Control,EA,Trauma,and T+EA group,n=8).Rats were anesthetized,and aseptically incised longitudinally to a length of 6 cm along the dorsal median line and 5 cm along the abdominal median line,and the abdominal viscera were exposed for 3 min.EA was applied at bilateral Zusanli and Lanwei acupoints.At postoperative day 3,splenic T cells were isolated using the nylon column method.IL-2 and interferon-gamma(IFN-γ) protein and mRNA expressions were assayed by ELISA and reverse transcriptionpolymerase chain reaction (RT-PCR),respectively.We examine the activity of ERK1/2 using Western-blot,and the DNA binding activity of nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) using Trans-AM ELISA-based kits. Results IL-2 and IFN-γ protein expression in splenic T cells of rats in Trauma group were markedly decreased 3 days after operation than Control group (37.4±2.0)vs (76.1±3.7) ng/L, (30.6±1.3) vs (49.9±2.6) ng/L,P＜0.01),IL-2 and IFN-γ mRNA expression in Trauma group were also decreased (P＜0.01).This was accompanied with a significant depression in the activity of ERK1/2,NF-κB and AP-1 (P＜0.01,Trauma group vs Control group).IL-2 and IFN-γ production in T+EA group were significantly increased compared with Trauma group (58.3±1.1) vs (37.4±2.0) ng/L, (39.2±1.5) vs (30.6±1.3) ng/L,P＜0.01),and IL-2 and IFN-γ mRNA expression,ERK1/2activation as well as NF-κB and AP-1 DNA binding activity were also increased (P＜0.01).Compared with Control group,the protein and mRNA expressions of IL-2 and IFN-γ,and the activity of ERK1/2,NF-κB and AP-1 in splenic T cells of T+EA group are not different.Conclusions EA may ameliorate impaired immune function following surgical trauma by regulating IL-2,IFN -γ protein and mRNA expression in splenic T cells and at least in part,involves the signaling pathways of ERK1/2 as well as NF-κB and AP-1. Key words: Electroacupuncture; Surgical trauma; Interleukin-2; Interferon-gamma; Extracellular- regulated protein kinase

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.

Magnesium Sulfate Inhibits Activator Protein-1 Upregulation in Endotoxin-activated Murine Macrophages

Objective The expression of inflammatory molecules is regulated by the transcription factor activator protein-1 (AP-1), a heterodimeric protein that consists of proteins from various families, including c-Jun and c-Fos. We sought to elucidate whether MgSO 4 regulates the activation of AP-1 in endotoxin-activated RAW264.7 cells, a murine macrophage-like cell line. The possible roles of the L-type calcium channels in this process were also elucidated. Materials and Methods RAW264.7 cells were treated with phosphate buffered saline, MgSO 4, lipopolysaccharide (LPS), LPS plus MgSO 4 (20 mM), or LPS plus MgSO 4 plus the L-type calcium channel activator BAY-K8644 (1 μM). After harvesting, expression of AP-1 was evaluated. Results LPS induced AP-1 activation based on the fact that the nuclear protein concentrations of AP-1 components, including c-Jun and c-Fos, as well as the AP-1 DNA-binding activity, were significantly increased in LPS-treated RAW264.7 cells. MgSO 4, in contrast, significantly inhibited LPS-induced AP-1 activation in activated RAW264.7 cells. Moreover, the effect of MgSO 4 on AP-1 was reversed by BAY-K8644. Conclusion MgSO 4 inhibited AP-1 activation in LPS-treated macrophages and the mechanism may involve the L-type calcium channels.

Transcription activator protein 1 (AP-1)-related study in basophils from patients with chronic idiopathic urticaria.

Objective To investigate the role of AP-1 in the pathogenesis of chronic idiopathic urticaria (CIU). Methods By using immunomagnetic separation technology, peripheral blood basophils were isolated from 10 CIU patients and 10 normal human controls followed by the extraction of nuclear protein from the basophils. TransAMTM AP-1 family kit was used to detect the DNA binding activity changes of AP-1 family transcription factors in basophils, and Western blotting to detect the expression of P-c-jun protein. Results There were some differences in the DNA binding activity of AP-1 family transcription factors in basophils between CIU patients and normal controls. The DNA binding activity of Phospho-c-jun, c-fos, Fos-B, Jun-B and Jun-D factors was increased in CIU patients compared with the controls, and the increase in that of P-c-jun and Jun-D was statistically significant (both P 0.05). The P-c-jun (Ser73) protein expression was higher in CIU patients than that in the controls (0.527 ± 0.312 vs. 0.435 ± 0.042, P < 0.05),whereas there was no significant difference in the P-c-jun (Ser63) protein expression level. Conclusion Some changes in DNA binding activity of AP-1 and overexpression of P-c-jun (Ser73) protein in basophils may be involved in the pathogenesis of CIU. Key words: Urticaria; Basophils; Activating transcription factor 1

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.

Expression of intercellular cell adhesion molecule-1, interleukin-10 and the activation of activator protein-1 in ventilator-induced lung injury in rabbits

To study the expression of intercellular cell adhesion molecule-1 (ICAM-1), Interleukin-10 (IL-10) and the activation of transcription factor activator protein-1 (AP-1) in a rabbit model of ventilator-induced lung injury (VILI) and therefore to explore their possible role in VILI. The VILI model was established by mechanical ventilation with a large tide volum (V(T)) of 40 ml/kg. Forty healthy male New Zealand rabbits were randomly divided into 5 groups: a control group without mechanical ventilation, a conventional ventilation group, and injurious ventilation with large V(T) for 1 h group, 2 h group and 4 h group. The concentrations of soluble ICAM-1 (sICAM-1) and IL-10 in lung homogenates were measured by enzyme-linked immunosorbent assay (ELISA). The level of mRNA was measured by semiquantitative transcription-polymerase chain reaction (RT-PCR). The DNA-binding activity of AP-1 was measured by electrophoretic mobility shift assay (EMSA). The partial arterial blood pressure of oxygen (PaO2), myeloperoxidase (MPO) in lung homogenate, wet lung weight to dry lung weight ratio (W/D) were observed. (1) The concentrations of sICAM-1 in large V(T) for 2 h group (23 ± 5) ng/L and 4 h group (35 ± 5) ng/L were higher than that in the conventional ventilation group (16 ± 4) ng/L (P all < 0.05), and that in the Large V(T) for 4 h group were higher than that in 1 h group (19 ± 4) ng/L and 2 h group (P all < 0.01). The concentrations of IL-10 in the large V(T) for 2 h group (24 ± 4) ng/L and 4 h group (26 ± 5) ng/L were higher than that in the conventional ventilation group (15 ± 2) ng/L (P all < 0.05), and that in the Large V(T) for 4 h group was higher than that in the 1h group (19 ± 4) ng/L(P < 0.05). The level of ICAM-1 mRNA in the large V(T) for 2 h group (1.18 ± 0.19) and 4 h group (1.29 ± 0.19) were higher than that in the conventional ventilation group (0.84 ± 0.13) (P all < 0.05), and that in Large V(T) for 4 h group was higher than that in 1 h group (0.96 ± 0.24) (P < 0.05). The level of IL-10 mRNA in the large V(T) for 4 h group (1.13 ± 0.17) was higher than that in the conventional ventilation group (0.84 ± 0.20) and Large V(T) for 1 h group (0.86 ± 0.12) (P all < 0.05). (2) The DNA-binding activity of AP-1 in the large V(T) for 2 h group (33.77 ± 8.23) and 4 h group (38 ± 9) were higher than that in the conventional ventilation group (23 ± 9) (P all < 0.01), and that in Large V(T) for 4 h group was higher than that in 1h group (25 ± 9) (P < 0.01). (3) Histopathological findings demonstrated that diffused alveolar damage induced by mechanical ventilation was worse with time, and after mechanical ventilation with large V(T) for 2 h, the level of MPO began to increase, and for 4 h the PaO2 reduced and the W/D increased. ICAM-1 and IL-10 took part in the inflammatory responses of VILI, and their up-regulation maybe due to the increase of their mRNA. Nuclear transcription factor AP-1 maybe involved in the transcriptional regulation mechanisms of these inflammatory mediators.