Role Of Activator Protein-1 Research Articles

The Role of Activator Protein-1 Complex in Diabetes-Associated Atherosclerosis: Insights From Single-Cell RNA Sequencing.

Despite advances in the treatment of atherosclerotic cardiovascular disease, it remains the leading cause of death in patients with diabetes. Even when risk factors are mitigated, the disease progresses, and thus newer targets need to be identified that directly inhibit the underlying pathobiology of atherosclerosis in diabetes. A single cell sequencing approach was utilised to distinguish the proatherogenic transcriptional profile in aortic cells in diabetes using a streptozotocin induced-diabetic Apoe-/- mouse model. Human carotid endarterectomy specimens from individuals with and without diabetes were also evaluated via immunohistochemical analysis. Further mechanistic studies were performed in human aortic endothelial cells and human THP-1 derived macrophages. We then performed a preclinical study using an AP-1 inhibitor in a diabetic Apoe-/- mouse model. Single cell RNA sequencing analysis identified the AP-1 complex as a novel target in diabetes-associated atherosclerosis. AP-1 levels were elevated in carotid endarterectomy specimens from diabetic when compared to non-diabetic individuals. AP-1 was validated as a mechanosensitive transcription factor via immunofluorescence staining for regional heterogeneity of endothelial cells of the aortic region exposed to turbulent blood flow and by performing microfluidics experiments in HAECs. AP-1 inhibition with T-5224 blunted endothelial cell activation as assessed by a monocyte adhesion assay and expression of genes relevant to endothelial function. Furthermore, AP-1 inhibition attenuated foam cell formation. Critically, treatment with T-5224 attenuated atherosclerosis development in diabetic Apoe-/- mice. This study has identified the AP-1 complex as a novel target, inhibition of which treats the underlying pathobiology of atherosclerosis in diabetes.

Activating Protein-1 (AP-1): A Promising Target for the Treatment of Fibrotic Diseases.

The fibrosis of tissues and organs occurs via an aberrant tissue remodeling process characterized by an excessive deposition of extracellular matrix, which can lead to organ dysfunction, organ failure, and death. Because the pathogenesis of fibrosis remains unclear and elusive, there is currently no medication to reverse it; hence, this process deserves further study. Activating protein-1 (AP-1)-comprising Jun (c-Jun, JunB, JunD), Fos (c-fos, FosB, Fra1, and Fra2), and activating transcription factor-is a versatile dimeric transcription factor. Numerous studies have demonstrated that AP-1 plays a crucial role in advancing tissue and organ fibrosis via induction of the expression of fibrotic molecules and activating fibroblasts. This review focuses on the role of AP-1 in a range of fibrotic disorders as well as on the antifibrotic effects of AP-1 inhibitors. It also discusses the potential of AP-1 as a new therapeutic target in conditions involving tissue and organ fibrosis.

The role of activator protein-1 (AP-1) transcription factors in diabetes associated atherosclerosis

Background and Aims : Atherosclerotic cardiovascular disease is the leading cause of death in individuals with diabetes. Endothelial activation and foam cell formation are crucial steps in the plaque development. Diabetes and blood flow-mediated shear stress are independent risk factors. Regions of turbulent blood flow where the blood flow represents low shear stress such as arterial bends are known to develop atherosclerosis. However, the mechanisms linking diabetes and shear stress to the atherosclerosis are not known.

Efficient Non-Epigenetic Activation of HIV Latency through the T-Cell Receptor Signalosome.

Human immunodeficiency virus type-1 (HIV-1) can either undergo a lytic pathway to cause productive systemic infections or enter a latent state in which the integrated provirus remains transcriptionally silent for decades. The ability to latently infect T-cells enables HIV-1 to establish persistent infections in resting memory CD4+ T-lymphocytes which become reactivated following the disruption or cessation of intensive drug therapy. The maintenance of viral latency occurs through epigenetic and non-epigenetic mechanisms. Epigenetic mechanisms of HIV latency regulation involve the deacetylation and methylation of histone proteins within nucleosome 1 (nuc-1) at the viral long terminal repeats (LTR) such that the inhibition of histone deacetyltransferase and histone lysine methyltransferase activities, respectively, reactivates HIV from latency. Non-epigenetic mechanisms involve the nuclear restriction of critical cellular transcription factors such as nuclear factor-kappa beta (NF-κB) or nuclear factor of activated T-cells (NFAT) which activate transcription from the viral LTR, limiting the nuclear levels of the viral transcription transactivator protein Tat and its cellular co-factor positive transcription elongation factor b (P-TEFb), which together regulate HIV transcriptional elongation. In this article, we review how T-cell receptor (TCR) activation efficiently induces NF-κB, NFAT, and activator protein 1 (AP-1) transcription factors through multiple signal pathways and how these factors efficiently regulate HIV LTR transcription through the non-epigenetic mechanism. We further discuss how elongation factor P-TEFb, induced through an extracellular signal-regulated kinase (ERK)-dependent mechanism, regulates HIV transcriptional elongation before new Tat is synthesized and the role of AP-1 in the modulation of HIV transcriptional elongation through functional synergy with NF-κB. Furthermore, we discuss how TCR signaling induces critical post-translational modifications of the cyclin-dependent kinase 9 (CDK9) subunit of P-TEFb which enhances interactions between P-TEFb and the viral Tat protein and the resultant enhancement of HIV transcriptional elongation.

Abstract 2652: Defining the role of AP1 in molecular adaptation to hypoxia in colorectal cancer

Abstract Background: The aim of this project was to investigate the role of Activator Protein 1 (AP1) in molecular adaptation to hypoxia in colorectal cancer. Colorectal cancer is the 3rd most common cancer worldwide. Low oxygen (hypoxia) found in 30-50% of colorectal tumors is associated with resistance to chemotherapy, radiotherapy and poor patient prognosis. Hypoxia stabilizes the transcription factors HIF1α and HIF2α which enable molecular adaptation to the hypoxic insult at a transcriptional level, in cooperation with additional transcription factors. To identify genes that regulate hypoxic survival in colorectal cancer, we carried out a lentiviral shRNA unbiased screen targeting 6142 genes, in HCT116 cells. We identified AP1 transcription factor subunits as key mediators of hypoxic cell viability that were required in hypoxia and in 3D spheroid culture in colorectal cancer Methods: Our experiments were conducted in a panel of cancer and normal colorectal cell lines (HCT116, LS174T, SW620, HT29 and CCD 841 CoN) in normoxia and hypoxia (1%O2) and in 3D spheroid cultures. The association of the different AP1 subunits and the impact of AP1 subunits on hypoxia-regulated expression and cell phenotypes was investigated using a variety of cell and molecular biology approaches. Results: Our results identify that AP1 subunits cJUN, JUNB, JUND, FOSL1 and FOSL2 are upregulated by hypoxia in colorectal cancer in a HIF independent manner. We observed that individual AP1 subunit knockdown in particular FOSL2 and JUND knockdown significantly decreased cancer cell survival in hypoxia. FOSL2 and JUND decreased the cell survival respectively by 40% and 60% in HCT116 and by 45% and 60% in LS174T (n=3, p<0.001). This effect was not observed when the cells were treated with T-5224 (10 µM), an AP-1 inhibitor suggesting a complex modulation of the transcriptional response by AP1 in hypoxia. In hypoxic conditions, by Co-IP studies, we established in HCT116 and LS174T that a selected pattern of AP1 heterodimers in particular FOSL2/JUNB and FOSL2/cJUN heterodimers were induced to mediate the transcriptional response to hypoxia. siRNA knockdown demonstrated that AP1 subunits modulate the expression of a specific set of hypoxia-regulated genes such as CA9 (Carbonic Anhydrase 9) and ANGPTL4 (n=3, p<0.05), important modulators of the hypoxic response. Conclusions: These data identify AP1 as a key mediator of the molecular adaptation to the hypoxic insult in the colorectal tumour microenvironment. Targeting AP1 subunits is a likely therapeutic approach for the treatment of the therapy resistant hypoxic regions of colorectal cancers. Citation Format: Eric Vancauwenberghe, Hannah Bolland, Christopher Carroll, Leonardo Da Motta, Anna Grabowska, Francesca Buffa, Adrian Harris, Alan McIntyre. Defining the role of AP1 in molecular adaptation to hypoxia in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2652.

The role of AP-1 in self-sufficient proliferation and migration of cancer cells and its potential impact on an autocrine/paracrine loop.

Activating protein-1 (AP-1) family members, especially Fra-1 and c-Jun, are highly expressed in invasive cancers and can mediate enhanced migration and proliferation. The aim of this study was to explore the significance of elevated levels of AP-1 family members under conditions that restrict growth. We observed that invasive MDA-MB-231 cells express high levels of Fra-1, c-Jun, and Jun-D during serum starvation and throughout the cell cycle compared to non-tumorigenic and non-invasive cell lines. We then analyzed Fra-1 levels in additional breast and other cancer cell lines. We found breast and lung cancer cells with higher levels of Fra-1 during serum starvation had relatively higher ability to proliferate and migrate under these conditions. Utilizing a dominant negative construct of AP-1, we demonstrated that proliferation and migration of MDA-MB-231 in the absence of serum requires AP-1 activity. Finally, we observed that MDA-MB-231 cells secrete factors(s) that induce Fra-1 expression and migration in non-tumorigenic and non-metastatic cells and that both the expression of and response to these factors require AP-1 activity. These results suggest the presence of an autocrine/paracrine loop that maintains high Fra-1 levels in aggressive cancer cells, enhancing their proliferative and metastatic ability and affecting neighbors to alter the tumor environment.

The Role of Activator Protein-1 (AP-1) Family Members in CD30-Positive Lymphomas.

The Activator Protein-1 (AP-1) transcription factor (TF) family, composed of a variety of members including c-JUN, c-FOS and ATF, is involved in mediating many biological processes such as proliferation, differentiation and cell death. Since their discovery, the role of AP-1 TFs in cancer development has been extensively analysed. Multiple in vitro and in vivo studies have highlighted the complexity of these TFs, mainly due to their cell-type specific homo- or hetero-dimerization resulting in diverse transcriptional response profiles. However, as a result of the increasing knowledge of the role of AP-1 TFs in disease, these TFs are being recognized as promising therapeutic targets for various malignancies. In this review, we focus on the impact of deregulated expression of AP-1 TFs in CD30-positive lymphomas including Classical Hodgkin Lymphoma and Anaplastic Large Cell Lymphoma.

Abstract P4-03-05: AP-1 as a potential mediator of resistance to the cyclin-dependent kinase (CDK) 4/6-inhibitor palbociclib in ER-positive endocrine-resistant breast cancer

Abstract Background: The CDK4/6-inhibitor palbociclib (Palbo) in combination with endocrine therapy (ET) substantially improves progression-free survival compared to ET alone. However, almost all initial responders eventually develop resistance and relapse. Delineating the early adaptive signaling and the mechanisms underlying resistance to CDK4/6 inhibition is therefore crucial to identify new biomarkers and therapeutic targets to enhance the efficacy of Palbo and improve patient outcome. Materials and Methods: MCF7 parental (P) cells and derivative lines made resistant (R) to tamoxifen (TamR), estrogen deprivation (EDR), or fulvestrant (FulR) were used. The MCF7P line and its endocrine-R (EndoR) derivatives were exposed to increasing concentrations of Palbo to generate acquired Palbo-R (PDR) models. The proteomic/signaling profiles of P and EndoR cells upon short-term Palbo treatment and as PDR develops were determined using reverse-phase protein arrays (RPPA). Transcriptional activity of the activator protein-1 (AP-1) transcription factor (TF) was measured by luciferase reporter assay. Global AP-1 blockade was achieved using a pINDUCER system to express doxycycline (Dox)-inducible dominant-negative (DN) c-Jun that lacks the transcriptional activation domain. Cell growth and colony formation were assessed using methylene blue staining and clonogenic assays, respectively. Levels of phosphorylated (p)-RB and CDK2 were assessed by Western Blot. Results: In P and all EndoR cell models, Palbo inhibited cell growth and colony formation in a dose-dependent manner, though the inhibitory effect was greater in the EndoR cells compared to P cells [IC50 value of P cells >3 times that of EndoR lines (p<0.001); clonogenic % inhibition at 100nM = 54 in P and >85 in EndoR lines (p<0.001)]. Across the P and all EndoR models, short-term Palbo treatment resulted in increased levels of several membrane and intracellular signaling molecules, TFs, and enzymes. Among these, the AP-1 TF components c-Jun and p-c-Jun showed the highest increase across all models, with the utmost change observed in the TamR model (Fold-change = 4.4, 4.0 for total and p-c-Jun, respectively). Since we also observed that acquired resistance to Palbo in the TamR model was associated with higher AP-1 transcriptional activity and increased total and p-c-Fos levels, we assessed the efficacy of combining Palbo with AP-1 blockade in the TamR model. In two independent TamR clones ectopically expressing inducible DN-c-Jun, AP-1 blockade (+Dox) in combination with Palbo was highly effective in inhibiting cell growth and reducing p-RB and CDK2 levels compared to single agent treatments. In addition, in both the TamR/DN-c-Jun-expressing clones, the combination of Palbo, AP-1 blockade, and fulvestrant resulted in cell death and a significantly greater cell growth inhibition compared to any dual or mono treatments. Conclusion: Our results suggest activation of AP-1 as a potential mechanism of resistance to Palbo in ER+ EndoR models. Transcriptomic profiling of the Palbo-sensitive and R cells, currently underway, will provide an in-depth understanding of the role of AP-1 as well as other key targets and associated molecular mechanisms in Palbo resistance. Citation Format: De Angelis C, Nardone A, Cataldo ML, Veeraraghavan J, Fu X, Giuliano M, Malorni L, Jeselsohn R, Osborne KC, Schiff R. AP-1 as a potential mediator of resistance to the cyclin-dependent kinase (CDK) 4/6-inhibitor palbociclib in ER-positive endocrine-resistant breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P4-03-05.

O32 Curcumin induces expression of 15-hydroxyprostaglandin dehydrogenase in normal gastric mucosa RGM-1 cells and mouse stomach in vivo: A potential role of AP-1

Overproduction of prostaglandin E 2 (PGE 2 ) has been reported to be implicated in carcinogenesis. The intracellular level of PGE 2 is regulated not only by its biosynthesis, but also by the degradation process. 15-Hydroxyprostaglandin dehydrogenase (15-PGDH) is the key enzyme that catalyzes the inactivation of PGE 2 . In the present study, we found that curcumin, a yellow coloring agent present in the rhizome of Curcuma longa Linn (Zingiberaceae), induced expression of 15-PGDH at both the protein and mRNA levels in normal rat gastric mucosa cells (RGM-1). By using deletion constructs of the 15-PGDH promoter, we have found that activator protein-1 (AP-1) is the most essential transcription factor responsible for curcumin-induced upregulation of 15-PGDH expression. In addition, oral administration of curcumin increased the expression of 15-PGDH in the mouse stomach. Curcumin enhanced the expression of c-Jun and c-Fos in the nuclear fraction of RGM-1 cells and the mouse stomach in vivo . Knockdown of c- jun , a gene encoding one of the major components of AP-1, suppressed curcumin-induced expression of 15-PGDH. In addition, curcumin increased phosphorylation of ERK1/2 and JNK. Curcumin-induced 15-PGDH expression was abrogated by the pharmacological inhibition of the aforementioned kinases. In contrast, tetrahydrocurcumin which lacks the α , β -unsaturated carbonyl group failed to induce expression of 15-PGDH, suggesting that the electrophilic α , β -unsaturated carbonyl group of curcumin is essential for its induction of 15-PGDH expression in RGM-1 cells. Taken together, our study suggests that curcumin-induced upregulation of 15-PGDH in RGM-1 cells and mouse stomach through activation of AP-1 may contribute to chemopreventive effects of this phytochemical on inflammation-associated gastric carcinogenesis.

Abstract 2912: The AP-1 transcription factor JunB promotes multiple myeloma cell proliferation, survival and drug resistance in the bone marrow microenvironment

Abstract Introduction: The activator protein-1 (AP-1) transcription factor has been implicated in a multitude of physiologic processes, but also tumorigenesis. In multiple myeloma (MM), the role of AP-1 is largely unknown. Experimental procedures: MM cell lines and primary tumor cells were co-cultured with primary bone marrow stromal cells (BMSCs) or BMSC lines. AP-1 expression was measured by western blot analysis and real-time PCR. AP-1 activation was determined using TransAM AP-1 assay kit. To investigate the upstream regulators of JunB, cytokine array and specific inhibitors were used followed by 3H-thymidine incorporation, western blot and TransAM AP-1 assays. To delineate the specific functional role of JunB in MM pathogenesis, we used pLKO.1- JunB shRNA (shJunB) and pLKO.1- scrambled shRNA (SCR) vectors for constitutive knockdown, as well as pMSCV-JunB-ER-IRES-GFP and empty vectors for inducible overexpression, together with 3H-thymidine incorporation, alamarBlue, flow cytometry and western blot analysis, as well as gene expression profiling (GEP). To evaluate the functional role of JunB in vivo, a MM xenograft mouse model was used. Results: Co-cultures of MM cells with BMSCs rapidly and strongly induced sustained expression and activation of JunB, but not of other AP-1 family members. Induction of JunB was predominantly mediated by soluble factors secreted by BMSCs rather than direct MM-BMSC contact. Indeed, IL-6 stimulation of MM.1S cells resulted in rapid and strong upregulation of JunB. Conversely, anti-IL-6 receptor antibody tocilizumab blocked BMSC-induced JunB expression and activation. Pharmacologic inhibition identified the requirement of the MEK/ERK and NF-κB pathways for BMSC-induced JunB expression. Functionally, significant inhibition of proliferation was observed in MM cells carrying pLKO.1- shJunB, but not pLKO.1-SCR. Importantly, knockdown of other AP-1 family members had minor effects on MM cell proliferation. Moreover, GEP performed on MM.1S- shJunB cells co-cultured with BMSCs as well as data analysis of a patient cohort using Gene Set Enrichment Analysis (GSEA) suggested a key role for JunB in the regulation of Mcl-1 and c-Myc expression. Furthermore, knockdown of JunB overcame resistance of MM cells to dexamethasone. Conversely, 4-OHT treatment of MM cell lines transduced with JunB-ER but not control vector induced significant JunB/AP-1 luciferase activity and protected MM cells against bortezomib-induced apoptosis and ER stress. Confirming our in vitro data, preliminary results show significant inhibition of tumor growth in a xenograft mouse model inoculated with inducible Tet-shJunB-GFP but not Tet-SCR-GFP MM.1S cells upon treatment with doxycycline. Conclusion: Taken together, our data demonstrate for the first time an important and surprising role of JunB/AP-1 in MM tumorigenesis and strongly propose it as a novel therapeutic target in MM. Citation Format: Fengjuan Fan, Sonia Vallet, Muhammad Hasan Bashari, Mostafa Jarahian, Eugenio Morelli, Dirk Hose, Latifa Bakiri, Claudia Ball, Hanno Glimm, Martin Sattler, Hartmut Goldschmidt, Giovanni Tonon, Pierfrancesco Tassone, Erwin F. Wagner, Dirk Jäger, Klaus Podar. The AP-1 transcription factor JunB promotes multiple myeloma cell proliferation, survival and drug resistance in the bone marrow microenvironment. [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 2912.

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.

Molecular Mechanism of Transcriptional Regulation of Matrix Metalloproteinase-9 in Diabetic Retinopathy.

Increase in matrix metalloproteinase-9 (MMP-9) is implicated in retinal capillary cell apoptosis, a phenomenon which precedes the development of diabetic retinopathy. MMP-9 promoter has multiple sites for binding the transcriptional factors, including two for activator protein 1 (AP-1). The binding of AP-1, a heterodimer of c-Jun and c-Fos, is regulated by posttranslational modifications, and in diabetes, deacetylating enzyme, Sirt1, is inhibited. Our aim, is to investigate the molecular mechanism of MMP-9 transcriptional regulation in diabetes. Binding of AP-1 (c-Jun, c-Fos) at the MMP-9 promoter, and AP-1 acetylation were analyzed in retinal endothelial cells incubated in normal or high glucose by chromatin-immunoprecipitation and co-immunoprecipitation respectively. Role of AP-1 in MMP-9 regulation was confirmed by c-Jun or c-Fos siRNAs, and that of its acetylation, by Sirt1 overexpression. In vitro results were validated in the retina from diabetic mice overexpressing Sirt1, and in the retinal microvessels from human donors with diabetic retinopathy. In experimental models, AP-1 binding was increased at the proximal and distal sites of the MMP-9 promoter, and similar phenomenon was confirmed in the retinal microvessels from human donors with diabetic retinopathy. Silencing of AP-1, or overexpression of Sirt1 ameliorated glucose-induced increase in MMP-9 expression and cell apoptosis. Thus, in diabetes, due to Sirt1 inhibition, AP-1 is hyperacetylated, which increases its binding at MMP-9 promoter, and hence, activation of Sirt1 could inhibit the development of diabetic retinopathy by impeding MMP-9-mediated mitochondrial damage. J. Cell. Physiol. 231: 1709-1718, 2016. © 2015 Wiley Periodicals, Inc.

Role of activator protein-1 in electro-acupuncture-induced up-regulation of heme oxygenase-1 expression in lung tissues in a rabbit model of endotoxic shock

Objective To evaluate the role of activator protein-1 （AP-1 ） in electro-acupuncture （EA ）-induced up-regulation of heme oxygenase-1 （HO-1 ） expression in lung tissues in a rabbit model of endotoxic shock .Methods Seventy healthy male New Zealand white rabbits ,aged 2 months ,weighing 1.5-2.0 kg ,were randomly divided into 7 groups （ n=10 each ） using a random number table ：normal control group （group C ） , endotoxic shock-induced acute lung injury （ALI ） group （group ALI ） , EA ＋ ALI group （group EL ） , non-acupoint＋EA＋ ALI group （group NEL ） , curcumin （HO-1 inhibitor ） group （group Cur ） , dimethyl sulfoxide group （group D ） ,and EA＋ALI＋curcumin group （group ELC ） .Bilateral 15 min EA stimulation of Zusanli and＆nbsp;Feishu （according to atlas of animals acupoints ） was performed （frequency 15Hz ） once a day for 5 consecutive days before lipopolysaccharide （LPS ） administration in EL and ELC groups ,while in group NEL ,EA stimulation was performed at non-acupoints located 0.5 cm lateral to Zusanli and Feishu acupoints with the same parameters . The animals were anesthetized with urethane and tracheostomized .The animals kept spontaneous breathing .Right internal carotid artery was cannulated for blood pressure monitoring . Ear vein was cannulated for drug administration .At 5 days after EA stimulation ,curcumin 25 mg/kg （in 0.5 ml of 0.1% dimethyl sulfoxide ） was injected in Cur and ELC groups ,0.1% dimethyl sulfoxide 0.5 ml was injected in D group ,and the equal volume of normal saline was given in the other groups .LPS 5 mg/kg （in 2 ml of 0.9% normal saline ） was injected intravenously at 30 min after administration in ALI ,EL ,NEL and ELC groups ,while the equal volume of normal saline was given in the other three groups .Endotoxic shock was confirmed by decrease in mean arterial pressure to 75% of the baseline value within 2 h after LPS injection .Blood samples were collected from the common carotid artery at 6 h after LPS or normal saline administration and the rabbits were then sacrificed .The lungs were removed for microscopic examination and the pathological changes were scored .The wet/dry lung weight ratio （W/D ratio ） was calculated .The malondialdehyde （MDA ） content and superoxide dismutase （SOD ） activity in lung tissues were detected ,and the expression of HO-1 mRNA ,HO-1 ,c-Jun mRNA and c-Jun in lung tissues was determined by Western blot .Results Compared with group C ,the pathological score ,W/D ratio and MDA content were significantly increased ,and the SOD activity was decreased in ALI ,EL ,NEL and ELC groups ,the expression of HO-1 mRNA ,HO-1 ,c-Jun mRNA and c-Jun was up-regulated in groups ALI ,EL and NEL （ P0.05） .There was no significant difference in the parameters mentioned above between Cur and D groups （ P〉0.05 ） .Compared with group ALI ,the pathological score ,W/D ratio and MDA content were significantly decreased ,and SOD activity was increased ,and the expression of HO-1 mRNA and HO-1 was up-regulated in EL and ELC groups （ P0.05） .The pathological score ,W/D ratio and MDA content were significantly higher ,and the SOD activity and expression of HO-1 mRNA ,HO-1 ,c-Jun mRNA and c-Jun were lower in group ELC than in group EL （ P〈0.05） .Conclusion EA up-regulates HO-1 expression through activating AP-1 during endotoxic shock-induced ALI in rabbits ,thus protecting the lung . Key words: Transcription factor AP-1; Electric stimulation therapy; Shock,septic; Lung; Heme oxygenase-1

Estrogen receptor-α mediates human multidrug resistance associated protein 3 induction by 17α-ethynylestradiol. Role of activator protein-1

Previously, we have demonstrated that 17α-ethynylestradiol (EE) induces rat multidrug-resistance associated protein 3 (Mrp3, Abcc3) expression transcriptionally through estrogen receptor-α (ER-α) activation. We explored the effect of EE on MRP3 expression of human origin. HepG2 cells were transfected with ER-α and incubated with EE (1–10–50μM) for 48h. MRP3 protein and mRNA levels were measured by Western blotting and Real time PCR, respectively. EE up-regulated MRP3 protein and mRNA at 50μM only in ER-α(+)-HepG2 cells. The in silico analysis of mrp3 promoter region demonstrated absence of estrogen response elements, but showed several Ap-1 binding sites. We further evaluated the potential involvement of the transcription factors c-JUN and c-FOS (members of Ap-1) in MRP3 up-regulation. ER-α(+) HepG2 cells were incubated with EE and c-FOS and c-JUN levels measured by Western blotting in nuclear extracts. EE up-regulated only c-JUN. Experiments of overexpression and knock-down of c-JUN by siRNA further demonstrated that this transcription factor is indeed implicated in MRP3 upregulation by EE. Co-immunoprecipitation assay demonstrated that EE induces c-JUN/ER-α interaction, and chromatin immunoprecipitation assay showed that this complex is recruited to the AP-1 binding consensus element present at the position (−1300/−1078bp) of human mrp3 promoter. We conclude that EE induces MRP3 expression through ER-α, with recruitment of ER-α in complex with c-JUN to the human mrp3 promoter.

TF/FVIIa/PAR2 promotes cell proliferation and migration via PKCα and ERK-dependent c-Jun/AP-1 pathway in colon cancer cell line SW620

Our previous study has demonstrated that tissue factor-factor VIIa (TF/FVIIa) complex promotes the proliferation and migration of colon cancer cell line SW620 through the activation of protease-activated receptor 2 (PAR2). In the current study, the underlying molecular mechanisms of TF/FVIIa/PAR2 signaling in SW620 cells were further explored, with the focus on the role of activator protein-1 (AP-1) subunit c-Jun. The results revealed that PAR2-AP and FVIIa could upregulate c-Jun expression and c-Jun phosphorylation in SW620 cells in a time-dependent manner. The effect of FVIIa was significantly blocked by anti-TF and anti-PAR2 antibodies. Protein kinase Cα (PKCα) inhibitor safingol and extracellular signal-regulated kinase 1 and 2 (ERK1/2) inhibitor U0126 abrogated the activation of c-Jun. In contrast, Ca(2+) chelators EGTA and thapsigargin, and p38MAPK inhibitor SB203580 had no effect. Suppression of c-Jun/AP-1 activation using a natural inhibitor curcumin decreased the expression of caspase-3, MMP-9, and TF, as well as the proliferation and migration of SW620 cells induced by PAR2-AP or FVIIa. Collectively, our findings suggest that c-Jun/AP-1 activation is required for TF/FVIIa/PAR2-induced SW620 cell proliferation and migration. PKCα and ERK1/2 are located upstream of c-Jun/AP-1 in this signaling pathway. Pharmacological inhibition of this pathway might be a novel strategy for colon cancer therapy.

Role of activator protein-1 in up-regulation of heme oxygenase-1 expression during lipopolysaccharide-induced acute lung injury in rats

Objective To evaluate the role of activator protein-1 (AP-1) in the up-regulation of heme oxygenase-1 (HO-1) expression during lipopolysaccharide (LPS)-induced acute lung injury (ALI) in rats.Methods Forty-eight healthy male Sprague-Dawley rats,weighing 200-220 g,aged 2.5-3.0 months,were randomly divided into 4 groups (n=12 each): normal control group (group C),ALI group,curcumin + ALI group (group Cur+ ALI),and curcumin group (group Cur).In groups C and ALI,normal saline 0.5 ml and LPS 10 mg/kg (0.5 ml) were injected intravenously,respectively,30 min after 0.1％ dimethyl sulfoxide (the vehicle for curcumin) 0.5 ml was injected intraperitoneally.In groups Cur+ ALl and Cur,curcumin 20 mg/kg (0.5 ml) was injected intraperitoneally,and 30 min later LPS 10 mg/kg and normal saline 0.5 ml were injected,respectively.The rats were then sacrificed at 6 h after injection of LPS.The lungs were removed for microscopic examination.The pathological changes of the lung were scored.The malondialdehyde (MDA) content,superoxide dismutase (SOD)activity and expression of HO-1,AP-1 and HO-1 mRNA in lung tissues were determined.Results Compared with group C,the pathological score and MDA content were significantly increased,the SOD activity was significantly decreased,and the expression of HO-1,AP-1 and HO-1 mRNA was up-regulated in groups ALl and Cur +AL(l) (P ＜ 0.05),and no significant change was found in the parameters mentioned above in group Cur (P ＞ 0.05).The pathological score and MDA content were significantly higher,and the SOD activity and expression of HO-1,AP-1 and HO-1 mRNA were significantly lower in group Cur + ALl than in group ALI(P ＜ 0.05).Conclusion Transcription factor AP-1 activation is involved in the up-regulation of HO-1 expression during LPS-induced ALI in rats. Key words: Activator protein-1; Endotoxemia; Heme oxygenase-1; Respiratory distress syndrome,adult

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

Genome-Wide Mapping of Estrogen Receptor-β–Binding Regions Reveals Extensive Cross-Talk with Transcription Factor Activator Protein-1

Estrogen signaling can occur through a nonclassical pathway involving the interaction of estrogen receptors (ER) with other transcription factors such as activator protein-1 (AP-1) and SP-1. However, there is little mechanistic understanding about this pathway, with conflicting results from in vitro investigations. In this study, we applied the ChIP-on-chip approach to identify ERbeta-binding sites on a genome-wide scale, identifying 1,457 high-confidence binding sites in ERbeta-overexpressing MCF7 breast cancer cells. Genes containing ERbeta-binding sites can be regulated by E2. Notably, approximately 60% of the genomic regions bound by ERbeta contained AP-1-like binding regions and estrogen response element-like sites, suggesting a functional association between AP-1 and ERbeta signaling. Chromatin immunoprecipitation (ChIP) analysis confirmed the association of AP-1, which is composed of the oncogenic transcription factors c-Fos and c-Jun, to ERbeta-bound DNA regions. Using a re-ChIP assay, we showed co-occupancy of ERbeta and AP-1 on chromatin. Short interfering RNA-mediated knockdown of c-Fos or c-Jun expression decreased ERbeta recruitment to chromatin, consistent with the role of AP-1 in mediating estrogen signaling in breast cancer cells. Additionally, ERalpha and ERbeta recruitment to AP-1/ERbeta target regions exhibited gene-dependent differences in response to antiestrogens. Together, our results broaden insights into ERbeta DNA-binding at the genomic level by revealing crosstalk with the AP-1 transcription factor.

Transactivation and expression patterns of Jun and Fos/AP‐1 super‐family proteins in human oral cancer

Transcription factor activator protein-1 (AP-1) super-family is known to modulate expression of array of genes during development of many cancers and considered as an important target for modern therapeutics. But the role of AP-1 during development of human oral cancers is still poorly understood. Because oral cancer is one of the most common cancers in India and south-east Asia, we studied the activation and expression pattern of AP-1 family of proteins and mRNA in different stages of oral carcinogenesis. Gel-shift assay, western blotting, immunohistochemistry and northern blotting have been used to assess the binding activity and expression pattern of AP-1 family (c-Jun, JunB, JunD, c-Fos, FosB, Fra-1 and Fra-2) proteins and mRNA transcripts in a total of 100 fresh oral tissue specimens comprising precancer (n = 40), cancer (n = 50) and healthy control (n = 10). Constitutive activation of AP-1 with concomitant upregulated expression of majority of AP-1 family of proteins and mRNA was observed in cancer cases. Interestingly, almost all precancerous cases showed JunD homodimers, whereas c-Fos/JunD was the most prevalent complex found in cancer tissues. The overexpression of EGFR mRNA, p50:p50/NF-kappaB homodimer formation, together with overexpression of pERK and c-Fos proteins in this study suggests an interesting cross talk between AP-1 and NF-kappaB pathways in oral cancers. Thus, this study demonstrates differential expression and activation of AP-1 super-family proteins in relation to severity of lesion and their crucial role in human oral carcinogenesis.

Differential involvement of PKC-dependent MAPKs activation in lipopolysaccharide-induced AP-1 expression in human tracheal smooth muscle cells

Lipopolysaccharide (LPS) has been shown to up-regulate the expression of vascular cell adhesion molecule (VCAM)-1 which contributes to the occurrence of airway inflammatory diseases. Genetic analysis reveals the existence of activator protein-1 (AP-1) binding site on VCAM-1 promoter region. However, the role of AP-1 in LPS-induced VCAM-1 expression in human tracheal smooth muscle cells (HTSMCs) is not known. Here, we show that LPS increased VCAM-1 expression and adhesiveness of HTSMCs through AP-1, since pretreatment with an AP-1 inhibitor tanshinone attenuated LPS-induced VCAM-1 expression and leukocytes adhesion. The implication of AP-1 in LPS-induced VCAM-1 expression was confirmed by animal studies showing that pretreatment of mice with tanshinone attenuated LPS-induced VCAM-1 mRNA expression in airway tissues and accumulation of leukocytes in bronchoalveolar lavage. By using the pharmacological inhibitors and transfection with siRNA of PKC, p42, p38, or JNK2, LPS-induced expression of c-Fos was mediated through protein kinase C (PKC), p42/p44 MAPK and p38 MAPK. While, c-Jun expression was mediated through PKC and mitogen-activated protein kinases (MAPKs, p42/p44 MAPK, p38 MAPK and JNK) in HTSMCs. Pretreatment with the inhibitors of PKCs or MAPKs attenuated LPS-stimulated nuclear translocation and VCAM-1 promoter binding abilities of AP-1, which attenuated promoter activity and gene expression of VCAM-1 and the adhesiveness between HTSMCs and leukocytes. These results indicated that differential regulation of AP-1 through PKCs-dependent MAPKs activation plays central roles in LPS-induced VCAM-1 expression. The altered modulation of this axis with inhibitors or siRNAs may contribute to the improvement of airway inflammatory diseases.