Transcription Factor Activator Protein-1 Research Articles

Evolutionary and functional analysis of Cyclina sinensis c-Jun AP-1 gene in response to LPS stimulation

The transcription factor activator protein-1 (AP-1) plays an essential and critical role in the regulation of numerous downstream genes involved in various physiological and chemical responses. In this study, we identified a full-length cDNA of the c-Jun AP-1 gene (termed Csc-Jun) from the transcriptome library in Cyclina sinensis. The cDNA contains an 825-bp open reading frame that encodes a 274-amino acid protein sequence, including a characteristic Jun transcription factor domain and a highly conserved basic leucine zipper (bZIP) signature that shares 90% identity to that of Ruditapes philippinarum. Furthermore, a phylogenetic analysis using MrBayes and PhyML software (with Bayesian and maximum likelihood approaches, respectively) revealed that the c-Jun AP-1 family genes might be involved in adapting to various environments in different invertebrates. We implemented the PAML software with the maximum likelihood method to further select and verify the positive selection sites (PSSs) in the Mollusca c-Jun AP-1 genes, and we detected four PSSs located in the Jun transcription factor domain. In addition, a spatial expression analysis showed that the Csc-Jun cDNA transcript was ubiquitously expressed in all of the tested tissues and was strongly expressed in the hepatopancreas and weakly expressed in the tissues of the hemocytes, gill filaments, mantle and adductor muscle. Quantitative real-time PCR showed that the expression profiles of Csc-Jun were significantly upregulated at different times in all of the tested tissues when challenged with lipopolysaccharide (LPS). Furthermore, knockdown of Csc-Jun by RNA interference resulted in a higher mortality of C. sinensis following LPS exposure. Finally, we explored the function of the TLR13-MyD88 signaling pathway in the innate immunity of C. sinensis by RNA interference and immune challenges. The results revealed that the mRNA expression levels of Csc-Jun were all decreased (P < 0.01) in normal and stimulated C. sinensis hemocytes. These data collectively indicated that the c-Jun AP-1 gene might play vital roles in innate immunity and provide new evidence for the evolutionary patterns of innate immune genes in Mollusca.

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.

Modulation of miR-139-5p on chronic morphine-induced, naloxone-precipitated cAMP overshoot in vitro.

Chronic exposure to morphine can produce tolerance, dependence and addiction, but the underlying neurobiological basis is still incompletely understood. c-Jun, as an important component of the activator protein-1 transcription factor, is supposed to take part in regulating gene expression in AC/cAMP/PKA signaling. MicroRNA (miRNA) has emerged as a critical regulator of neuronal functions. Although a number of miRNAs have been reported to regulate the μ-opioid receptor expression, there has been no report about miRNAs to regulate chronic morphine-induced, naloxone-precipitated cAMP overshoot. Our results showed that chronic morphine pretreatment induced naloxone-precipitated cAMP overshoot in concentration- and time-dependent manners in HEK 293/μ cells. Chronic morphine pretreatment alone elevated both c-Jun protein and miR-139-5p expression levels, while dramatically artificial elevation of miR-139-5p inhibited c-Jun at the translational level. Furthermore, dramatically artificial upregulation of intracellular miR-139-5p limited chronic morphine-induced, naloxone-precipitated cAMP overshoot. These findings suggested that miR-139-5p was involved in regulating chronic morphine-induced, naloxone-precipitated cAMP overshoot in a negative feedback manner through its target c-Jun, which extends our understanding of neurobiological mechanisms underlying morphine dependence and addiction.

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.

Targeting Inflammatory T Helper Cells via Retinoic Acid-Related Orphan Receptor Gamma t Is Ineffective to Prevent Allo-Response-Driven Colitis.

Intestinal graft-versus-host disease (GvHD) is a life-threatening, inflammatory donor T cell-mediated complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT). In the light of the reported efficacy of interleukin-23 (IL-23)-blockade to mitigate syngeneic intestinal inflammation in inflammatory bowel disease patients, targeting IL-23 and thereby interleukin-17a (IL-17a) producing T helper (Th17) cells as the T cell subset assumed to be mostly regulated by IL-23, has emerged as a putatively general concept to harness immune-mediated mucosal inflammation irrespective of the underlying trigger. However, the role of Th17 cells during allo-response driven colitis remains ambiguous due to a series of studies with inconclusive results. Interestingly, we recently identified granulocyte-macrophage colony-stimulating factor (GM-CSF+) T cells to be promoted by interleukin-7 (IL-7) signaling and controlled by the activating protein-1 transcription factor family member basic leucine zipper transcription factor ATF-like (BATF) as critical mediators of intestinal GvHD in mice. Given the dual role of BATF, the contribution of IL-23-mediated signaling within donor T cells and bona fide Th17 cells remains to be delineated from the regulation of GM-CSF+ T cells in the absence of BATF. Here, we found in a complete MHC class I-mismatched model that genetic inactivation of the IL-23 receptor (IL-23R) or the transcription factor retinoic acid-related orphan receptor gamma t (RORγt) within donor T cells similarly ablated Th17 cell formation in vivo but preserved the T cells’ ability to induce intestinal GvHD in a compared to wild-type controls indistinguishable manner. Importantly, RORγt-independent manifestation of intestinal GvHD was completely dependent on BATF-regulated GM-CSF+ T cells as BATF/RORγt double-deficient T cells failed to induce colitis and the antibody-mediated blockage of IL-7/IL-7R interaction and GM-CSF significantly diminished signs of intestinal GvHD elicited by RORγt-deficient donor T cells. Finally, in analogy to our murine studies, colonic RORC expression levels inversely correlated with the presence of GvHD in allo-HSCT patients. Together, this study provides a crucial example of a BATF-dependent, however, IL-23R signaling- and RORγt-, i.e., Th17 fate-independent regulation of a colitogenic T cell population critically impacting the current understanding of intestinal GvHD.

AP-1 Takes Centre Stage in Enhancer Chromatin Dynamics.

Recent studies involving induced pluripotent stem cell (iPSC) reprogramming in mice and monocyte-to-macrophage differentiation in humans have revealed a role for the transcription factor (TF) activator protein 1 (AP-1) in chromatin accessibility. Enhancer selection may be determined not only by cell type-specific sets of TFs but also by broadly expressed ones like AP-1.

The secreted Theileria annulata Ta9 protein contributes to activation of the AP-1 transcription factor.

Theileria annulata is an obligate intracellular protozoan parasite of the phylum Apicomplexa. Theileria sporozoites invade bovine leukocytes and develop into a multinucleate syncytial macroschizont that causes uncontrolled proliferation and dissemination of infected and transformed leukocytes. Activator protein 1 (AP-1) is a transcription factor driving expression of genes involved in proliferation and dissemination and is therefore a key player in Theileria-induced leukocytes transformation. Ta9 possesses a signal peptide allowing it to be secreted into the infected leukocyte cytosol and be presented to CD8 T cells in the context of MHC class I. First, we confirmed that Ta9 is secreted into the infected leukocyte cytosol, and then we generated truncated versions of GFP-tagged Ta9 and tested their ability to activate AP-1 in non-infected HEK293T human kidney embryo cells. The ability to activate AP-1-driven transcription was found to reside in the C-terminal 100 amino acids of Ta9 distant to the N-terminally located epitopes recognised by CD8+ T cells. Secreted Ta9 has therefore, not only the ability to stimulate CD8+ T cells, but also the potential to activate AP-1-driven transcription and contribute to T. annulata-induced leukocyte transformation.

MicroRNA 199a-5p induces apoptosis by targeting JunB

MicroRNAs participate in a variety of physiological and pathophysiological processes in various organs including the heart. Our previous work revealed that the level of miR-199a-5p was significantly higher in failing hearts than in control hearts. However, whether it is associated with the progression of heart failure (HF) and mediates cardiomyocyte apoptosis remained unclear. In the present study, we used various biochemical and molecular biological approaches to investigate the changes in miR-199a-5p levels in failing hearts in a rat model induced by acute myocardial infarction. We found that miR-199a-5p levels in the heart increased with the progression of HF, and overexpression of miR-199a-5p significantly increased apoptosis in untreated H9C2 cells and potentiated angiotensin II-induced apoptosis. Thus, our results indicate that miR-199a-5p is involved in the progression of HF and mediates cardiomyocyte apoptosis. We also confirmed that JunB, a member of the activator protein-1 transcription factor family, is one of direct targets of miR-199a-5p via a dual-luciferase reporter assay and mutagenesis on the 3′ untranslated region of the JunB gene. Consistent with the above findings, overexpression of JunB in H9c2 cells suppressed cell apoptosis. Based on our findings, miR-199a-5p induces apoptosis by targeting JunB.

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.

AP1 mediates uPA/uPAR induced FUT4 expression and trophoblast invasion.

Trophoblast invasion is crucial for embryo implantation and successful pregnancy. Urokinase-type plasminogen activator (uPA)/urokinase-type plasminogen activator receptor (uPAR) are expressed on trophoblasts and involved in trophoblast invasion. The transcription factor activator protein 1 (AP1) (c-Fos and cJun) and fucosyltransferase IV (FUT4) have been found to be involved in this process. However, the relationship of uPA/uPAR, AP1 and FUT4 is unclear. The current study aimed to investigate the role of AP1 in uPA/uPAR induced FUT4 expression and trophoblast invasion. We found that p-c-Fos and p-c-Jun were decreased in abortion patients compared to that in normal pregnant women. Employing human trophoblastic cells, we then demonstrated that uPA/uPAR induced the expression of p-c-Fos and p-c-Jun. Applying an electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP), we further proved that transcription factor AP1 bound to FUT4 promoter that could increase FUT4 transcriptional activity, further promoting trophoblast cell migration and invasion through JNK MAPK signaling pathway. Taken together, these results suggest that uPA/uPAR induces FUT4 expression, and trophoblast cell invasion mediated by AP1 transcription factor (c-Fos and c-Jun). Our findings provide novel insights into the relationship between AP1 and abortion.

The MAPK-activator protein-1 signaling regulates changes in lung tissue of rat exposed to hypobaric hypoxia.

This study reports the role of MAPKs (JNK, ERK, and p38), and activator protein-1 (AP-1) transcription factor in the hypobaric hypoxia induced change in lung tissue. Healthy male Sprague-Dawley rats were exposed to hypobaric hypoxia for 6, 12, 24, 48, 72, and 120 hr. Hypoxia resulted in significant increase in reactive oxygen species (ROS), vascular endothelial growth factor (VEGF) and decreased nitric oxide (NO), these act as signaling molecules for activation of MAPK and also contribute in development of vascular leakage (an indicator of pulmonary edema) as confirmed by histological studies. Our results confirmed JNK activation as an immediate early response (peaked at 6-48 hr), activation of ERKs (peaked at 24-72 hr) and p38 (peaked at 72-120 hr) as a secondary response to hypoxia. The MAPK pathway up regulated its downstream targets phospho c-Jun (peaked at 6-120 hr), JunB (peaked at 24-120 hr) however, decreased c-Fos, and JunD levels. DNA binding activity also confirmed activation of AP-1 transcription factor in lung tissue under hypobaric hypoxia. Further, we analyzed the proliferative and inflammatory genes regulated by different subunits of AP-1 to explore its role in vascular leakage. Increased expression of cyclin D1 (peaked at 12-72 hr) and p16 level (peaked at 48-120 hr) were correlated to the activation of c-jun, c-Fos and JunB. Administration of NFκB inhibitor caffeic acid phenethyl ester (CAPE) and SP600125 (JNK inhibitor) had no effect on increased levels of Interferon-γ (IFN-γ), Interleukin-1 (IL-1), and Tumor Necrosis Factor-α (TNF-α) thereby confirming the involvement of AP-1 as well as NFκB in inflammation. Expression of c-jun, c-Fos were correlated with activation of proliferative genes and JunB, Fra-1 with pro-inflammatory cytokines. In conclusion immediate response to hypobaric hypoxia induced c-Jun:c-Fos subunits of AP-1; responsible for proliferation that might cause inhomogeneous vasoconstriction leading to vascular leakage and inflammation at increased duration of hypobaric hypoxia exposure.

Glycyrrhetinic Acid Antagonizes Pressure-Induced Venous Remodeling in Mice.

Development of spider veins is caused by the remodeling of veins located in the upper dermis and promoted by risk factors such as obesity or pregnancy that chronically increase venous pressure. We have repeatedly shown that the pressure-induced increase in biomechanical wall stress is sufficient to evoke the formation of enlarged corkscrew-like superficial veins in mice. Subsequent experimental approaches revealed that interference with endothelial- and/or smooth muscle cell (SMC) activation counteracts this remodeling process. Here, we investigate whether the herbal agent glycyrrhetinic acid (GA) is a suitable candidate for that purpose given its anti-proliferative as well as anti-oxidative properties. While basic abilities of cultured venous SMCs such as migration and proliferation were not influenced by GA, it inhibited proliferation but not angiogenic sprouting of human venous endothelial cells (ECs). Further analyses of biomechanically stimulated ECs revealed that GA inhibits the DNA binding capacity of the mechanosensitive transcription factor activator protein-1 (AP-1) which, however, had only a minor impact on the endothelial transcriptome. Nevertheless, by decreasing gelatinase activity in ECs or mouse veins exposed to biomechanical stress, GA diminished a crucial cellular response in the context of venous remodeling. In line with the observed inhibitory effects, local transdermal application of GA attenuated pressure-mediated enlargement of veins in the mouse auricle. In summary, our data identifies GA as an inhibitor of EC proliferation, gelatinase activity and venous remodeling. It may thus have the capacity to attenuate spider vein formation and remodeling in humans.

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.

Association between the rs7583431 single nucleotide polymorphism close to the activating transcription factor 2 gene and the analgesic effect of fentanyl in the cold pain test

BackgroundActivating transcription factor 2 (ATF2) is a member of the leucine zipper family of DNA binding proteins and is widely distributed in tissues. Several recent studies have demonstrated that this protein is involved in mechanisms that are related to pain and inflammation. However, unclear is whether polymorphisms of the ATF2 gene, which encodes the human ATF2 protein, influence pain or analgesic sensitivity. This study examined associations between the analgesic effect of fentanyl in the cold pressor‐induced pain test and polymorphisms in the ATF2 gene in 355 Japanese subjects.ResultsIn this study, 33 single nucleotide polymorphisms (SNPs) were selected, and a total of 2 linkage disequilibrium blocks with 6 Tag SNPs (rs1153702, rs7583431, rs2302663, rs3845744, rs268214, and rs1982235) were observed in the region within and around the ATF2 gene. We further analyzed associations between these Tag SNPs and clinical data. Even after multiple testing with Bonferroni adjustments, an increase in the analgesic effect of fentanyl in the cold pressor‐induced pain test was significantly associated with a greater number of the A allele of the rs7583431 SNP (linear regression, P = .001).ConclusionsThe present findings may contribute to adequate pain relief in individual patients. Although more research on the genetic factors that influence opioid sensitivity is needed, analgesic requirements may be predicted by analyzing ATF2 SNPs, together with other polymorphisms of genes that are reportedly associated with opioid sensitivity, such as CREB1,OPRM1, and GIRK2.

Regulation of Krüppel-like factor 6 via activating transcription factor 4 pathway to apoptosis of human lens epithelial cells

Objective To investigate the regulating effects of Kruppel-like factor 6 (KLF6) on the apoptosis of human lens epithelial cells (HLECs) by activating transcription factor 4 (ATF4) pathway and explore the bio-molecular mechanism of KLF6/ATF4-induced HLECs apoptosis. Methods HLECs (HLE-B3) were cultured using high glucose DMEM medium.The eukaryotic expression plasmid pEGFP-C2-ATF4 was transfected into the cells by liposome 2000 in the ATF4-transfected group, and pEGFP-C2 was transfected in the empty plasmid group.Then the cells were exposed to 20 mJ/cm2 ultraviolet ray B (UVB) for 200 seconds, The morphological changes of the cells were observed by hematoxylin & eosin staining and Hoechst33258 fluorescein staining.Cultured cells were transfected using pEGFP-C2-KLF6 and pEGFP-C2 plasmid and pSilencer-KLF6 (siKLF6) and pSilencer plasmid, respectively, and the expression of ATF4 protein in the cells was detected by Western blot assay.Culture cells were divided into four groups.pEGFP-C2 and pSilencer plasmids were co-transfected into the cells in the empty plasmid group; pEGFP-C2-KLF6 and pSilencer empty plasmid were co-transfected into the cells of the KLF6+ pSilencer group; pEGFP-C2 empty plasmid and pSilencer-ATF4 were co-transfected in the cells of the siATF4+ pEGFP-C2 group; pEGFP-C2-KLF6 and pSilencer-ATF4 plasmids were co-transfected in the cells of the KLF6+ siATF4 group, and then the cells were exposed to UVB.The apoptosis of the cells were detected by ELISA assay. Results Cultured cells grew well in the normal control group with the uniform morphology and regular arrangement.The karyopyknosis, karyorrhexis and enlargement of intercellular space were found in the cells exposed to UVB.In the ATF4 transfected group, the number of cells was decreased.The relative expression level of the ATF4 protein in the cells was 0.99±0.06 and 0.13±0.02 in the UVB+ ATF4 transfected group and UVB+ pEGFP-C2 plasmid group, respectively, with a significant difference between them (t=23.13, P<0.01). The relative expression levels of KLF6 and ATF4 proteins in the KLF6 transfected group were higher than those in the empty plasmid group, and the relative expression levels of KLF6 and ATF4 proteins in the siKLF6 group were significantly lower than those in the empty plasmid group (all at P<0.01). ELISA assay showed that the apoptotic rate in the ATF4 transfected group was 1.37±0.11, which was significantly higher than 0.31±0.11 in the normal control group (t=8.034, P=0.001); the apoptotic rate of the cells was increased in the KLF6+ pSilencer group and decreased in the siATF4+ pEGFP-C2 group in comparison with the empty plasmid group (P<0.01, P=0.02). In addition, the apoptotic rate in the KLF6+ siATF4 group was remarkably lower than that in the KLF6+ pSilencer group (P<0.01). Conclusions KLF6 promotes the apoptosis of HLECs induced by UVB radiation.Silence of ATF4 gene reduces the apoptotic rate of the cells.ATF4 is probably a target factor in the regulating pathway of KLF6 to apoptosis. Key words: Kruppel-like factor 6; Kruppel-like transcription factors/metabolism; Activating transcription factor 4; Epithelial cells, lens; Cell line, crystalline; Humans; Apoptosis

Inhibition of DREAM-ATF6 interaction delays onset of cognition deficit in a mouse model of Huntington\u2019s disease

The transcriptional repressor DREAM (downstream regulatory element antagonist modulator) is a multifunctional neuronal calcium sensor (NCS) that controls Ca2+ and protein homeostasis through gene regulation and protein-protein interactions. Downregulation of DREAM is part of an endogenous neuroprotective mechanism that improves ATF6 (activating transcription factor 6) processing, neuronal survival in the striatum, and motor coordination in R6/2 mice, a model of Huntington’s disease (HD). Whether modulation of DREAM activity can also ameliorate cognition deficits in HD mice has not been studied. Moreover, it is not known whether DREAM downregulation in HD is unique, or also occurs for other NCS family members. Using the novel object recognition test, we show that chronic administration of the DREAM-binding molecule repaglinide, or induced DREAM haplodeficiency delays onset of cognitive impairment in R6/1 mice, another HD model. The mechanism involves a notable rise in the levels of transcriptionally active ATF6 protein in the hippocampus after repaglinide administration. In addition, we show that reduction in DREAM protein in the hippocampus of HD patients was not accompanied by downregulation of other NCS family members. Our results indicate that DREAM inhibition markedly improves ATF6 processing in the hippocampus and that it might contribute to a delay in memory decline in HD mice. The mechanism of neuroprotection through DREAM silencing in HD does not apply to other NCS family members.

Brain-Derived Neurotrophic Factor Elevates Activating Transcription Factor 4 (ATF4) in Neurons and Promotes ATF4-Dependent Induction of Sesn2.

Activating transcription factor 4 (ATF4) plays important physiologic roles in the brain including regulation of learning and memory as well as neuronal survival and death. Yet, outside of translational regulation by the eIF2α-dependent stress response pathway, there is little information about how its levels are controlled in neurons. Here, we show that brain-derived neurotrophic factor (BDNF) promotes a rapid and sustained increase in neuronal ATF4 transcripts and protein levels. This increase is dependent on tropomyosin receptor kinase (TrkB) signaling, but independent of levels of phosphorylated eIF2α. The elevation in ATF4 protein occurs both in nuclei and processes. Transcriptome analysis revealed that ATF4 mediates BDNF-promoted induction of Sesn2 which encodes Sestrin2, a protector against oxidative and genotoxic stresses and a mTor complex 1 inhibitor. In contrast, BDNF-elevated ATF4 did not affect expression of a number of other known ATF4 targets including several with pro-apoptotic activity. The capacity of BDNF to elevate neuronal ATF4 may thus represent a means to maintain this transcription factor at levels that provide neuroprotection and optimal brain function without risk of triggering neurodegeneration.

Mitogen-activated protein kinase signaling is involved in nonylphenol-induced proinflammatory cytokines secretion by BV2 microglia.

Microglia (MG) are the key cells involved in the innate immune response in the central nervous system, and their activation has been linked to inflammation and neurotoxicity by the production of proinflammatory cytokines. Recently, researchers have found that nonylphenol (NP), a ubiquitous endocrine disrupting chemical, could impair neurodevelopment and cognitive memory performance. However, whether NP affects the inflammatory responses of MG remains to be elucidated. The aim of this study was to explore the effects of NP on the inflammatory responses of BV2 MG and the underlying mechanisms. Our results showed that NP increased the secretion of interleukin (IL)-6 and IL-1β in BV2 MG. Increased phosphorylation of Akt, JNK and p38 mitogen-activated protein kinase and decreased phosphorylation of ERK were observed in NP-treated MG. The inflammatory transcription factor activator protein 1 was also activated in NP-treated BV2 MG. These results suggest that NP may activate Akt/mitogen-activated protein kinase/activator protein 1 signaling in MG and subsequently increase IL-6 and IL-1β secretion.

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 &amp;gt;3 times that of EndoR lines (p&amp;lt;0.001); clonogenic % inhibition at 100nM = 54 in P and &amp;gt;85 in EndoR lines (p&amp;lt;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.