Activator Protein-1 Activation Research Articles

Interplay of transcription factors STAT3, STAT1 and AP-1 mediates activity of the matrix metallo-proteinase-1 promoter in colorectal carcinoma cells.

Signal Transducers (STATs) 1 and 3 and Activator Protein 1 (AP-1) are transcription factors involved in the development of malignancy in colorectal carcinoma (CRC). Matrix Metalloproteinase 1 (MMP-1) is a protease frequently dysregulated in de-differentiated and invasive cancer cells. Its expression is influenced by STAT and AP-1 transcription factors. We studied their contributions to transcriptional regulation of MMP-1 in colorectal carcinoma (CRC) cells. Both STAT3 and AP-1 contribute individual expression-inducing and additive effects and interact with the MMP-1 promoter. DNA binding of AP-1 protein c-Jun is stimulation-independent but modulated by STAT3 and a STAT recognition DNA element. Activated STAT3 showed a suppressive effect on AP-1-mediated MMP-1 mRNA upregulation as shown by STAT3 knockdown. Surprisingly, activated STAT1 overcame STAT3-dependent repression of AP-1-driven MMP-1 expression. Moreover, combined STAT3, STAT1 and AP-1 activities evoked maximal MMP-1 mRNA levels in a synergistic manner. Our results suggest a dominant role of AP-1 in transcriptional upregulation of MMP-1 in CRC cells which is modulated by joint functions of STAT3 and STAT1. The individual and combinatorial activity of these factors is of diagnostic and prognostic interest.

Herpes simplex virus type 2 infection triggers AP-1 transcription activity through TLR4 signaling in genital epithelial cells

BackgroundThe pattern recognition receptors (PPRs) are the earliest phase of the host defense against pathogens in genital epithelium, and toll-like receptors (TLRs) are best characterized PPRs mediating innate immune responses. Herpes simplex virus type 2 (HSV-2), a member of herpesviridae family, causes one of the most prevalent sexually transmitted infection in the world. In this paper, we described that HSV-2 infection would induce activator protein 1 (AP-1) via TLR4-MyD88/TRIF pathway in human genital epithelial cell.MethodsTLRs expression profiles and changes was investigated in HSV-2-infected cells. The effect of TLR4-MyD88/TRIF on HSV-2-induced AP-1 activation and viral replication was also evaluated. The TLR4 translocation change was examined after viral infection. Finally, viral ICP0 effect on TLR4 signaling and TLR4-promoter regulation were primarily studied.ResultsHSV-2-induced AP-1 activation was dependent on TLR4 and downstream adaptor molecules MyD88 and TRIF. And also, TLR4, MyD88 and TRIF was proved to affect HSV-2 replication. AP-1 activation would also be enhanced via overexpression of myeloid differentiation protein 2 (MD2), implicating that it might be a necessary accessory for TLR4 to sense HSV-2 infection. Protein quantification of cytoplasmic and membrane-associated TLR4 revealed that HSV-2 infection increased membrane-anchoring TLR4 level, but not cytoplasmic ones. Viral ICP0 could augment cellular AP-1, TLR4 promoter activation and TLR4 expression level. The specific inhibitor treatment and transcription factor binding site scanning in TLR4 promoter region showed that AP-1 activity was essential for TLR4-promoter activation.ConclusionsTaken together, HSV-2 infection could stimulate AP-1 activation via TLR4-MyD88/TRIF axis, and then feedback to up-regulate TLR4 expression in human genital epithelial cells.

Effect of thiol antioxidants on lipopolysaccharide-induced cyclooxygenase-2 expression in pulmonary epithelial cells.

Cyclooxygenase-2 (COX-2) plays an important role in pulmonary inflammatory response, and its expression is regulated by several transcription factors including nuclear factor kappa-B (NF-κB), activator protein-1 (AP-1), and signal transducer and activator of transcription-3 (STAT-3), which are activated by oxidative stress. Glutathione (GSH) and N-acetyl cysteine (NAC) are thiol antioxidants that scavenge reactive oxygen species (ROS). The present study investigated whether lipopolysaccharide (LPS) induces COX-2 expression through ROS generation and the activation of oxidant-sensitive transcription factors such as NF-κB, AP-1, and STAT-3 in pulmonary epithelial A549 cells. The cells were pretreated with GSH or NAC for 1 hour prior to LPS stimulation. Intracellular ROS levels, DNA-binding activities of NF-κB, AP-1, and STAT-3, and mRNA and protein levels of COX-2 were determined. Our results showed that LPS increased ROS levels that peaked at 2 hours. LPS activated NF-κB, AP-1, and STAT-3 and induced the expression of COX-2 in A549 cells in a time-dependent manner. Pretreatment of thiol antioxidants GSH and NAC reduced ROS levels and attenuated the increase in ROS, the activation of NF-κB, AP-1, and STAT-3, and the expression of COX-2 in LPS-treated A549 cells. In conclusion, GSH and NAC suppress COX-2 expression by reducing ROS levels and inhibiting the activation of NF-κB, AP-1, and STAT-3 in pulmonary epithelial A549 cells exposed to LPS. Pretreatment with thiol antioxidants GSH and NAC may be beneficial for the treatment of pulmonary inflammation associated with oxidative stress.

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.

Association of NF-κB and AP-1 with MMP-9 Overexpression in 2-Chloroethanol Exposed Rat Astrocytes.

Subacute poisoning of 1,2-dichloroethane (1,2-DCE) has become a serious occupational problem in China, and brain edema is its main pathological consequence, but little is known about the underlying mechanisms. As the metabolite of 1,2-DCE, 2-chloroethanol (2-CE) is more reactive, and might play an important role in the toxic effects of 1,2-DCE. In our previous studies, we found that matrix metalloproteinases-9 (MMP-9) expression was enhanced in mouse brains upon treatment with 1,2-DCE, and in rat astrocytes exposed to 2-CE. In the present study, we analyzed the association of nuclear factor kappa B (NF-κB) and activator protein-1 (AP-1) with MMP-9 overexpression in astrocytes treated with 2-CE. MMP-9, p65, c-Jun, and c-Fos were significantly upregulated by 2-CE treatment, which also enhanced phosphorylation of c-Jun, c-Fos and inhibitor of κBα (IκBα), and nuclear translocation of p65. Furthermore, inhibition of IκBα phosphorylation and AP-1 activity with the specific inhibitors could attenuate MMP-9 overexpression in the cells. On the other hand, inhibition of p38 mitogen-activated protein kinase (p38 MAPK) signaling pathway suppressed the activation of both NF-κB and AP-1 in 2-CE-treated astrocytes. In conclusion, MMP-9 overexpression induced by 2-CE in astrocytes could be mediated at least in part through the p38 signaling pathway via activation of both NF-κB and AP-1. This study might provide novel clues for clarifying the mechanisms underlying 1,2-DCE associated cerebral edema.

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.

O-Methylbulbocapnine and Dicentrine Suppress LPS-Induced Inflammatory Response by Blocking NF-κB and AP-1 Activation through Inhibiting MAPKs and Akt Signaling in RAW264.7 Macrophages.

The natural aporphine alkaloids including crebanine (CN), O-methylbulbocapnine (OMP), and dicentrine (DC), and protoberberine alkaloids, tetrahydropalmatine (THP) and N-methyl tetrahydropalmatine (NTHP), have been found in Stephania venosa. Previous reports demonstrated CN and THP exhibited anti-inflammatory properties. In this study, we investigated anti-inflammatory effect of CN analogs including OMP, DC, THP, and NTHP in RAW264.7 macrophages. The pre-treatment of macrophages with CN, OMP and DC suppressed lipopolysaccharide (LPS)-induced pro-inflammatory cytokines and mediators including interleukin-6 (IL-6), tumor necrosis factor alpha, prostaglandin E2 and nitric oxide, in which the rank-order of inhibitory potency was DC>CN≥OMP. Whereas, high dose THP (30-40 µg/mL) reduced LPS-induced IL-6 production in RAW264.7 cells but NTHP did not effect. Moreover, CN, OMP and DC inhibited the LPS-induced expression of inducible nitric oxide synthase and cyclooxygenase-2. OMP and DC inhibited LPS-induced nuclear factor kappa B (NF-κB) activation by suppressing the phosphorylation of NF-κB at Ser536, but not the nucleus translocation and inhibitor of kappaB (IκB)-α degradation. In addition, OMP and DC also reduced the phosphorylation and nucleus translocation of activator protein-1 (AP-1). Furthermore, OMP and DC suppressed the LPS-activated myeloid differentiation factor 88 (MyD88), Akt and mitogen-activated protein kinases (MAPKs) signaling pathway, which were the upstream signaling regulators of AP-1 and NF-κB. Collectively, OMP and DC have an anti-inflammatory effect on RAW264.7 macrophages by the suppression of pro-inflammatory cytokines and mediators. The inhibitory property of OMP and DC is mediated by blockage the activation of MyD88, MAPKs, Akt, NF-κB and AP-1 signaling molecules.

Urolithin A attenuates pro-inflammatory mediator production by suppressing PI3-K/Akt/NF-κB and JNK/AP-1 signaling pathways in lipopolysaccharide-stimulated RAW264 macrophages: Possible involvement of NADPH oxidase-derived reactive oxygen species

Urolithin A, a gut microbial metabolite of ellagic acid, is reported to exert anti-inflammatory effects in vitro and in vivo. However, complete mechanisms underlying the regulation of inflammatory responses by urolithin A remain unclear. This study aimed to evaluate the anti-inflammatory potential of urolithin A and its underlying mechanisms in lipopolysaccharide (LPS)-stimulated RAW264 macrophages. Urolithin A significantly attenuated the pro-inflammatory mediator production in LPS-stimulated RAW264 and mouse peritoneal macrophages. This compound significantly suppressed the LPS-elicited nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) activation. The phosphorylation of Akt and c-Jun N-terminal kinase (JNK) was also inhibited by the treatment with urolithin A. Through experiments using kinase inhibitors, urolithin A abolished the LPS-induced phosphatidylinositol 3-kinase (PI3-K)/Akt/NF-κB and JNK/AP-1 signaling pathways, resulting in suppression of pro-inflammatory mediator production. Furthermore, treatment with this compound significantly reduced the intracellular accumulation of reactive oxygen species, which are known to act as secondary messengers in the activation of redox-sensitive transcription factors NF-κB and AP-1. Urolithin A treatment also diminished the LPS-evoked activation of NADPH oxidase (NOX), which is the main source of reactive oxygen species in activated macrophages. The inhibition of this activity by urolithin A led to the prevention of LPS-elicited NF-κB and AP-1 activation as well as Akt and JNK phosphorylation, resulting in the reduction of pro-inflammatory mediator production. Collectively, these results indicate that urolithin A treatment attenuates pro-inflammatory mediator production by suppressing NOX-derived reactive oxygen species-mediated PI3-K/Akt/NF-κB and JNK/AP-1 signaling pathways in LPS-stimulated macrophages.

PAGE4 and Conformational Switching: Insights from Molecular Dynamics Simulations and Implications for Prostate Cancer

Prostate-associated gene 4 (PAGE4) is an intrinsically disordered protein implicated in prostate cancer. Thestress-response kinase homeodomain-interacting protein kinase 1 (HIPK1) phosphorylates two residues in PAGE4, serine 9 and threonine 51. Phosphorylation of these two residues facilitates the interaction of PAGE4 with activator protein-1 (AP-1) transcription factor complex to potentiate AP-1's activity. In contrast, hyperphosphorylation of PAGE4 by CDC-like kinase 2 (CLK2) attenuates this interaction with AP-1. Small-angleX-ray scattering and single-molecule fluorescence resonance energy transfer measurements have shown that PAGE4 expands upon hyperphosphorylation and that this expansion is localized to its N-terminal half. To understand the interactions underlying this structural transition, we performed molecular dynamics simulations using Atomistic AWSEM, a multi-scale molecular model that combines atomistic and coarse-grained simulation approaches. Our simulations show that electrostatic interactions drive transient formation of an N-terminal loop, the destabilization of which accounts for the dramatic change in size upon hyperphosphorylation. Phosphorylation also changes the preference of secondary structure formation of the PAGE4 ensemble, which leads to a transition between states that display different degrees of disorder. Finally, we construct a mechanism-based mathematical model that allows us to capture the interactions ofdifferent phosphoforms of PAGE4 with AP-1 and its downstream target, the androgen receptor (AR)—a key therapeutic target in prostate cancer. Our model predicts intracellular oscillatory dynamics of HIPK1-PAGE4, CLK2-PAGE4, and AR activity, indicating phenotypic heterogeneity in an isogenic cell population. Thus, conformational switching of PAGE4 may potentially affect the efficiency of therapeutically targeting AR activity.

YAP/TAZ regulates TGF-\u03b2/Smad3 signaling by induction of Smad7 via AP-1 in human skin dermal fibroblasts

BackgroundTranscription factors YAP and TAZ function as the primary mediators of the Hippo pathway. Yet, crosstalk of YAP and TAZ with other signaling pathways remains relatively unexplored. We have explored the impact of YAP and TAZ levels on the TGF-β/Smad signaling pathway in human skin dermal fibroblasts.MethodsYAP and TAZ levels in dermal fibroblasts were reduced in dermal fibroblasts by siRNA-mediated knockdown. The effects of YAP and TAZ reduction on TGF-β/Smad signaling were examined by quantitative real-time PCR, Western analysis, and immunostaining. Luciferase reporter assays and electrophoretic mobility shift assays were conducted to investigate the transcription factor DNA-binding and transcriptional activities.ResultsKnockdown of both YAP and TAZ (YAP/TAZ), but not either separately, impaired TGF-β1-induced Smad3 phosphorylation and Smad3 transcriptional activity, thereby inhibiting the expression of TGF-β target genes. This reduction by reduced levels of YAP/TAZ results from induction of inhibitory Smad7, which inhibits Smad3 phosphorylation and activity by TGF-β1. Conversely, prevention of Smad7 induction restores Smad3 phosphorylation and Smad3 transcriptional activity in fibroblasts that have reduced YAP/TAZ. In agreement with these findings, inhibition of YAP/TAZ transcriptional activity, similar to the reduction of YAP/TAZ levels, also significantly induced Smad7 and impaired TGF-β/Smad signaling. Further investigations revealed that reduced levels of YAP/TAZ led to induction of activator protein-1 (AP-1) activity, Activated AP-1 bound to DNA sequences in the Smad7 gene promoter, and deletion of these AP-1 binding sequences substantially reduced Smad7 promoter reporter activity.ConclusionYAP/TAZ functions in concert with transcription factor AP-1 and Smad7 to regulate TGF-β signaling, in human dermal fibroblasts. Reduction of YAP/TAZ levels leads to activation of AP-1 activity, which induces Smad7. Smad7 suppresses the TGF-β pathway.

Inhibition of GSK‐3β induces AP‐1‐mediated osteopontin expression to promote cholestatic liver fibrosis

The contribution of glycogen synthase kinase-3β (GSK-3β) to cholestatic liver disease (CLD) remains unknown. We investigated the role and mechanism of GSK-3β in vivo in liver tissues of patients with CLD and the bile duct ligation (BDL) mouse model and in vitro using a hepatic progenitor cell (HPC) and hepatic stellate cell (HSC) coculture system. In liver tissues of patients with CLD, expression of the inactive form of GSK-3β, phospho-GSK-3β(Ser9), was increased in HPCs. GSK-3β inhibition by SB216763 treatment aggravated liver fibrosis and elevated the expression of osteopontin (OPN) in the BDL mouse model. OPN was significantly overexpressed in liver tissues and serum from patients with CLD. In an HPC and HSC coculture system, inhibition of GSK-3β induced OPN production, which activated HSCs in a cholestatic environment. The expression of activator protein 1 (AP-1), an important downstream transcription factor of GSK-3β, was significantly increased in liver tissues of patients with CLD and SB216763-treated BDL mice. Finally, OPN expression was directly modulated by AP-1. These observations indicate that GSK-3β inhibition up-regulates OPN expression via AP-1 activation, which accelerates the progression of cholestatic liver fibrosis in patients with CLD and in BDL mice.-Zhuang, S., Hua, X., He, K., Zhou, T., Zhang, J., Wu, H., Ma, X., Xia, Q., Zhang, J. Inhibition of GSK-3β induces AP-1-mediated osteopontin expression to promote cholestatic liver fibrosis.

Anti-inflammatory activity of Khayandirobilide A from Khaya senegalensis via NF-κB, AP-1 and p38 MAPK/Nrf2/HO-1 signaling pathways in lipopolysaccharide-stimulated RAW 264.7 and BV-2 cells

BackgroundImmunocytes-involved inflammation is considered to modulate the damage in various diseases. Herein, novel therapeutics suppressing over-activation of immunocytes could prove an effective strategy to prevent inflammation-related diseases. PurposeThe objective of this study is to evaluate the anti-inflammatory activity of Khayandirobilide A (KLA), a new andirobin-type limonoid with modified furan ring isolated from the Khaya senegalensis (Desr.) A. Juss., and to explore its potential underlying mechanisms in LPS-stimulated inflammatory models. MethodsThe structure of KLA was elucidated on the basis of 1D- and 2D-NMR spectroscopic data as well as HR-ESI-MS. As for its anti-inflammatory effect, the production of pro-inflammatory mediators and cytokines in LPS-stimulated RAW 264.7 and BV-2 cells were measured by Griess reagent, ELISA and qRT-PCR. The relevant proteins including nuclear factor κB (NF-κB), p-AKT, p-p38 and Nrf2/HO-1 were investigated by western blot. Nuclear localisations of NF-κB, activator protein-1 (AP-1) and Nrf2 were also examined by western blot and immunofluorescence. ResultsKLA could inhibit the production of LPS-induced NO with IC50 values of 5.04 ± 0.14 µM and 4.97 ± 0.5 µM in RAW 264.7 and BV-2 cells, respectively. KLA also attenuated interleukin-6 (IL-6), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) at the protein and mRNA levels. Further mechanistic studies demonstrated the activation of NF-κB and AP-1 were reduced by KLA. Moreover, KLA elevated expression of heme oxygenase-1(HO-1) via inducing Keap1 autophagic degradation and promoting Nrf2 nuclear translocation. Despite KLA induced the phosphorylation of mitogen-activated protein kinases (MAPKs) family, inhibiting the phosphorylation of p38 by its specific inhibitor SB203580 attenuated the degradation of KLA-induced Keap1, and then reduced KLA-induced Nrf2 nuclear translocation and HO-1 expression. Furthermore, SB203580, Brusatol (a Nrf2 specific inhibitor) and ZnPP (a HO-1 specific inhibitor) could partly reverse the suppressive effects of KLA on LPS-induced NO production and mRNA levels of pro-inflammatory genes. ConclusionThese data displayed that KLA possessed anti-inflammatory activity, which was attributed to inhibit the release of LPS-stimulated inflammatory mediators via suppressing the activation of NF-κB, AP-1, and upregulating the induction of p38 MAPK/Nrf2-mediated HO-1.

Penehyclidine hydrochloride inhibits TLR4 signaling and inflammation, and attenuates blunt chest trauma and hemorrhagic shock-induced acute lung injury in rats.

Blunt chest trauma with hemorrhagic shock (THS) frequently induces pulmonary inflammation that leads to acute lung injury (ALI). Penehyclidine hydrochloride (PHC) possesses anti-inflammatory properties that may attenuate the systemic inflammatory response. The present study aimed to evaluate the molecular mechanism of PHC in modifying THS-induced ALI in rats. Rats underwent either THS or a sham procedure. At 6 h subsequent to blunt chest trauma, arterial blood was drawn for blood gas and pro-inflammatory factors analyses, and lung tissue samples were collected to examine pulmonary histopathological alterations, the wet/dry weight ratio, myeloperoxidase activity, and the protein expression levels of Toll-like receptor 4 (TLR4), phosphorylated (p-)p38 mitogen-activated protein kinase (MAPK), nuclear factor (NF)-κB and activator protein-1 (AP-1). THS caused significant reductions in heart rate and mean arterial blood pressure, and was associated with significant increases in tumor necrosis factor-α, interleukin (IL)-6, IL-1β, p-p38MAPK, NF-κB and AP-1 activation, in addition to TLR4 expression, in the lung. PHC effectively attenuated THS-induced ALI, and inhibited TLR4 expression, reduced the activation of p-p38MAPK, NF-κB and AP-1, and downregulated the expression of pro-inflammatory mediators. In conclusion, the results of the present study demonstrated that PHC may exert an anti-inflammatory effect and attenuate THS-induced ALI by inhibiting the TLR4 signaling pathway. These preclinical findings may offer a novel therapeutic strategy to restrict TLR4 overactivation in ALI.

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.

Staphylococcus aureus induces TGF-β1 and bFGF expression through the activation of AP-1 and NF-κB transcription factors in bovine mammary epithelial cells

Staphylococcus aureus is a common Gram-positive pathogen that causes bovine mastitis, a persistent infection of the bovine mammary gland. Bovine mammary epithelial cells (BMEC) are important parenchymal cells of the bovine mammary gland. To better understand the importance of BMEC and the roles of the TLR-NF-κBand TLR-AP-1 signaling pathways in the regulation of S. aureus-associated mastitis and mammary fibrosis, BMEC cultured in vitro were stimulated with different concentrations of heat-inactivated S. aureus to analyze the gene and protein expression and production of toll-like receptor 2 (TLR2), toll-like receptor 4 (TLR4), transforming growth factor beta 1 (TGF-β1), basic fibroblast growth factor (bFGF) as well as the protein expression of nuclear factor-kappa B (NF-κB) and activation protein-1 (AP-1) by means of quantitative polymerase chain reaction (qPCR) and western blotting, respectively. Specific NF-κB and AP-1 inhibitors were also used to investigate their effects on the regulation of TGF-β1 and bFGF expression. The results indicated that, in addition to increasing mRNA expression and secretion of TLR2 and TLR4, S. aureus could also upregulate TGF-β1 and bFGF mRNA expression and secretion through the activation of NF-κB and AP-1. The increase in TGF-β1 and bFGF expression was shown to be inhibited by AP-1- and NF-κB-specific inhibitors. Taken together, S. aureus induces TGF-β1 and bFGF expression through the activation of AP-1 and NF-κB in BMECs. This information offers new potential targets for the treatment of bovine mammary fibrosis.

Exposure to DBP and High Iodine Aggravates Autoimmune Thyroid Disease Through Increasing the Levels of IL-17 and Thyroid-Binding Globulin in Wistar Rats

Autoimmune thyroid disease (AITD) is the most common autoimmune disease that causes hypothyroidism. High iodine is a well-known factor that can induce thyroid disorders, including Hashimoto's thyroiditis, one of the main types of AITD. Recent epidemiological studies have indicated that phthalates, especially di-n-butyl phthalate (DBP) may induce thyroid disease. In this study, we aim to determine the effects and underlying mechanisms of high iodine and/or DBP exposure on AITD. Female Wistar rats were modeled with thyroglobulin and exposed to high iodine and/or DBP. We investigated histopathological changes in the thyroid and measured thyroid hormone levels in serum to assess thyroid function. In the thyroid and liver, we detected oxidative stress, proinflammatory factors (IL-1β, IL-6, and IL-17) and the activation of activator protein 1 (AP-1), a transcription factor that is related to the synthesis of the thyroxine-binding globulin (TBG) and the activation of Th17. After blocking AP-1 with SP600125, we detected TBG and the Th17 related cytokines (IL-6 and IL-17). The data showed that thyroid damage and the alteration of thyroid hormones were greater when the rats were exposed to both high iodine and DBP. Coexposure to DBP and high iodine enhanced the activation of AP-1 in the liver and thyroid, and induced an increase in the levels of TBG in serum and IL-17 in the thyroid. Blocking AP-1 activation prevented the increase of TBG and IL-17. The results indicate that high iodine and/or DBP exposure exacerbated AITD through altering TBG levels in serum and aggravating IL-17 in the thyroid.

Anti-Inflammatory Potential of Carpomitra costata Ethanolic Extracts via Inhibition of NF-κB and AP-1 Activation in LPS-Stimulated RAW264.7 Macrophages

Marine algae have valuable health and dietary benefits. The present study aimed to investigate whether an ethanol extract of Carpomitra costata (CCE) could inhibit the inflammatory response to LPS. CCE attenuated the production of proinflammatory mediators, such as prostaglandin E2 (PGE2) and nitric oxide (NO), by inhibiting inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression in LPS-induced RAW264.7 macrophages. CCE also inhibited the expression of proinflammatory cytokines such as IL-1β, TNF-α, and IL-6. CCE suppressed the LPS-induced DNA-binding activity of (NF-κB) and activator protein-1 (AP-1). In addition, CCE attenuated the LPS-stimulated phosphorylation of c-Jun N-terminal kinase/stress-activated protein kinase (JNK) and phosphatidylinositol 3′-kinase/Akt (PI3K/Akt). Functional aspects of the JNK and Akt signaling pathways were analyzed using specific inhibitors, which attenuated the LPS-induced production of proinflammatory cytokines, and NO and PGE2 expression by suppressing AP-1 and NF-κB activity. In particular, the AP-1 signaling pathway is not involved in the production of inflammatory cytokines, such as IL-6, TNF-α, and IL-1β. These results suggested that CCE might exert its anti-inflammatory action by downregulating transcriptional factors (NF-κB and AP-1) through JNK and Akt signaling pathways. The current study suggested that CCE might be a valuable candidate for the treatment of inflammatory disorders.

Berberine Suppresses Fibronectin Expression through Inhibition of c-Jun Phosphorylation in Breast Cancer Cells

PurposeThe exact mechanism regulating fibronectin (FN) expression in breast cancer cells has not been fully elucidated. In this study, we investigated the pharmacological mechanism of berberine (BBR) with respect to FN expression in triple-negative breast cancer (TNBC) cells.MethodsThe clinical significance of FN mRNA expression was analyzed using the Kaplan-Meier plotter database (http://kmplot.com/breast). FN mRNA and protein expression levels were analyzed by real-time polymerase chain reaction and western blotting, respectively.ResultsUsing publicly available clinical data, we observed that high FN expression was associated with poor prognosis in patients with breast cancer. FN mRNA and protein expression was increased in TNBC cells compared with non-TNBC cells. As expected, recombinant human FN significantly induced cell spreading and adhesion in MDA-MB231 TNBC cells. We also investigated the regulatory mechanism underlying FN expression. Basal levels of FN mRNA and protein expression were downregulated by a specific activator protein-1 (AP-1) inhibitor, SR11302. Interestingly, FN expression in TNBC cells was dose-dependently decreased by BBR treatment. The level of c-Jun phosphorylation was also decreased by BBR treatment.ConclusionOur findings demonstrate that FN expression is regulated via an AP-1–dependent mechanism, and that BBR suppresses FN expression in TNBC cells through inhibition of AP-1 activity.

6]-Shogaol attenuates inflammation, cell proliferation via modulate NF-κB and AP-1 oncogenic signaling in 7,12-dimethylbenz[a]anthracene induced oral carcinogenesis

Nuclear factor-kappaB (NF-κB) and activator protein 1 (AP-1) is a major transcription factor which regulates many biological and pathological processes such as inflammation and cell proliferation, which are major implicates in cancer progression.[6]-Shogaol ([6]-SHO) is a major constituent of ginger, exhibits various biological properties such as anti-oxidants, anti-inflammation and anti-tumor. Recently, we proven that [6]-SHO prevents oral squamous cell carcinoma by activating proapoptotic factors in in vitro and in vivo experimental model. However, the preventive efficacy of [6]-SHO in 7,12-dimethylbenz[a]anthracene (DMBA) induced hamster buccal pouch carcinogenesis (HBP) has not been fully elucidated, so far. Hence, we aimed to investigate the effect of [6]-SHO on inflammation and cell proliferation by inhibiting the translocation of NF-κB and AP-1 in DMBA induced HBP carcinogenesis. In this study, we observed upregulation of inflammatory markers (COX-2, iNOS, TNF-α, interleukin-1 and -6), cell proliferative markers (Cyclin D1, PCNA and Ki-67) and aberrant activation of NF-κB, AP-1, IKKβ, c-jun, c-fos and decreased IκB-α in DMBA induced hamsters. Conversely, oral administration of [6]-SHO strongly inhibited constitutive phosphorylation and degradation of IκB and inhibit phosphorylation of c-jun, c-fos, resulting in inhibition of nuclear translocation of NF-κBp65 and AP-1. Thus, inhibition of NF-κB and AP-1 activation by [6]-SHO attenuates inflammation and cell proliferative response in DMBA induced hamsters. Our finding suggested that [6]-SHO is a novel functional agent capable of preventing DMBA induced inflammation and cell proliferation associated tumorigenesis by modulating multiple signalling molecules.

Syk and Src-targeted anti-inflammatory activity of aripiprazole, an atypical antipsychotic

Aripiprazole (ARP) is a partial agonist of dopamine D2 receptors that is commonly prescribed to treat schizophrenia and bipolar disorder. The anti-inflammatory effect of ARP was recently documented in a few studies, but its molecular mechanisms have not been fully elucidated. In this study, peptidoglycan (PGN)-treated macrophages (RAW264.7 cells), reporter gene assay, an overexpression strategy, immunoprecipitation, and immunoblotting analysis were employed to clarify the anti-inflammatory mechanism of ARP. ARP was found to dose-dependently inhibit production of nitric oxide (NO) and prostaglandin E2 (PGE2) without exhibiting cytotoxicity. In agreement with this result, ARP was found to suppress the mRNA expression levels of inflammatory genes such as cyclooxygenase-2 (COX-2), inducible NO synthase (iNOS), and tumor necrosis factor (TNF)-α. Luciferase assay and immunoblotting analysis with nuclear fractions showed that activator protein-1 (AP-1) and nuclear factor (NF)-κB are targeted by ARP. Similar to these data, c-Jun N-terminal kinase (JNK), mitogen-activated protein kinase kinase 4 (MKK4), MKK7, and transforming growth factor beta-activated kinase 1 (TAK1) for AP-1 activation, and inhibitor of κBα (IκBα), IκBαkinase α/β (IKKα/β), AKT, phosphatidylinositide 3-kinases (PI3K), spleen tyrosine kinase (Syk), and Src for NF-κB activation were revealed to be inhibited by ARP treatment. These results suggest that ARP can suppress inflammatory responses triggered by Gram positive bacteria through suppression of both AP-1 and NF-κB pathways.