RESUMEN
Chondrosarcoma is a malignant bone tumor that is characterized by high metastatic potential and marked resistance to radiation and chemotherapy. The knowledge that adipokines facilitate the initiation, progression, metastasis, and treatment resistance of various tumors has driven several in vitro and in vivo investigations into the effects of adipokines resistin, leptin, and adiponectin upon the development and progression of chondrosarcomas. Another adipokine, visfatin, is known to regulate tumor progression and metastasis, although how this molecule may affect chondrosarcoma metastasis is unclear. Here, we found that visfatin facilitated cellular migration via matrix metalloproteinase-2 (MMP-2) production in human chondrosarcoma cells and overexpression of visfatin enhanced lung metastasis in a mouse model of chondrosarcoma. Visfatin-induced stimulation of MMP-2 synthesis and activation of the AP-1 transcription factor facilitated chondrosarcoma cell migration via the ERK, p38, and JNK signaling pathways. This evidence suggests that visfatin is worth targeting in the treatment of metastatic chondrosarcoma.
Asunto(s)
Neoplasias Óseas/patología , Condrosarcoma/patología , Citocinas/fisiología , Metaloproteinasa 2 de la Matriz/genética , Nicotinamida Fosforribosiltransferasa/fisiología , Animales , Neoplasias Óseas/genética , Neoplasias Óseas/metabolismo , Condrosarcoma/genética , Condrosarcoma/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Sistema de Señalización de MAP Quinasas/genética , Masculino , Metaloproteinasa 2 de la Matriz/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Metástasis de la Neoplasia , Factor de Transcripción AP-1/metabolismo , Factor de Transcripción AP-1/fisiología , Células Tumorales CultivadasRESUMEN
Neutrophils are required for host resistance against Streptococcus pneumoniae, but their function declines with age. We previously found that CD73, an enzyme required for antimicrobial activity, is downregulated in neutrophils (also known as polymorphonuclear leukocytes [PMNs]) from aged mice. This study explored transcriptional changes in neutrophils induced by S. pneumoniae to identify pathways controlled by CD73 and dysregulated with age. Pure bone marrow-derived neutrophils isolated from wild-type (WT) young and old and CD73 knockout (CD73KO) young mice were mock challenged or infected with S. pneumoniae ex vivo. RNA sequencing (RNA-Seq) was performed to identify differentially expressed genes (DEGs). We found that infection triggered distinct global transcriptional changes across hosts that were strongest in CD73KO neutrophils. Surprisingly, there were more downregulated than upregulated genes in all groups upon infection. Downregulated DEGs indicated a dampening of immune responses in old and CD73KO hosts. Further analysis revealed that CD73KO neutrophils expressed higher numbers of long noncoding RNAs (lncRNAs) than those in WT controls. Predicted network analysis indicated that CD73KO-specific lncRNAs control several signaling pathways. We found that genes in the c-Jun N-terminal kinase (JNK)-mitogen-activated protein kinase (MAPK) pathway were upregulated upon infection in CD73KO mice and in WT old mice, but not in WT young mice. This corresponded to functional differences, as phosphorylation of the downstream AP-1 transcription factor component c-Jun was significantly higher in neutrophils from infected CD73KO mice and old mice. Importantly, inhibition of JNK/AP-1 rescued the ability of these neutrophils to kill S. pneumoniae. Together, our findings revealed that the ability of neutrophils to modify their gene expression to better adapt to bacterial infection is in part regulated by CD73 and declines with age.
Asunto(s)
5'-Nucleotidasa/fisiología , Perfilación de la Expresión Génica , Neutrófilos/inmunología , Streptococcus pneumoniae/inmunología , Factores de Edad , Animales , Sistema de Señalización de MAP Quinasas/fisiología , Ratones , Ratones Endogámicos C57BL , Neutrófilos/metabolismo , ARN Largo no Codificante/análisis , ARN Mensajero/análisis , Factor de Transcripción AP-1/fisiologíaRESUMEN
FOS, a subunit of the activator protein-1 (AP-1) transcription factor, has been implicated in various cellular changes. In the human ovary, the expression of FOS and its heterodimeric binding partners JUN, JUNB, and JUND increases in periovulatory follicles. However, the specific role of the FOS/AP-1 remains elusive. The present study determined the regulatory mechanisms driving the expression of FOS and its partners and functions of FOS using primary human granulosa/lutein cells (hGLCs). Human chorionic gonadotropin (hCG) induced a biphasic increase in the expression of FOS, peaking at 1 to 3 hours and 12 hours. The levels of JUN proteins were also increased by hCG, with varying expression patterns. Coimmunoprecipitation analyses revealed that FOS is present as heterodimers with all JUN proteins. hCG immediately activated protein kinase A and p42/44MAPK signaling pathways, and inhibitors for these pathways abolished hCG-induced increases in the levels of FOS, JUN, and JUNB. To identify the genes regulated by FOS, high-throughput RNA sequencing was performed using hGLC treated with hCG ± T-5224 (FOS inhibitor). Sequencing data analysis revealed that FOS inhibition affects the expression of numerous genes, including a cluster of genes involved in the periovulatory process such as matrix remodeling, prostaglandin synthesis, glycolysis, and cholesterol biosynthesis. Quantitative PCR analysis verified hCG-induced, T-5224-regulated expression of a selection of genes involved in these processes. Consistently, hCG-induced increases in metabolic activities and cholesterol levels were suppressed by T-5224. This study unveiled potential downstream target genes of and a role for the FOS/AP-1 complex in metabolic changes and cholesterol biosynthesis in granulosa/lutein cells of human periovulatory follicles.
Asunto(s)
Colesterol/biosíntesis , Metabolismo Energético/genética , Células de la Granulosa/metabolismo , Proteínas Proto-Oncogénicas c-fos/fisiología , Células Cultivadas , Gonadotropina Coriónica/farmacología , Metabolismo Energético/efectos de los fármacos , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Células de la Granulosa/efectos de los fármacos , Humanos , Ovulación/efectos de los fármacos , Ovulación/genética , Ovulación/metabolismo , Proteínas Proto-Oncogénicas c-fos/efectos de los fármacos , Proteínas Proto-Oncogénicas c-jun/efectos de los fármacos , Proteínas Proto-Oncogénicas c-jun/genética , Proteínas Proto-Oncogénicas c-jun/metabolismo , Factores de Tiempo , Factor de Transcripción AP-1/efectos de los fármacos , Factor de Transcripción AP-1/fisiologíaRESUMEN
The AP-1 member Fra-1 is overexpressed in TNBC and plays crucial roles in tumor progression and treatment resistance. In a previous large-scale screen, we identified PARP1 to be among 118 proteins that interact with endogenous chromatin-bound Fra-1 in TNBC cells. PARP1 inhibitor (olaparib) is currently in clinical use for treatment of BRCA-mutated TNBC breast cancer. Here, we demonstrate that the Fra-1-PARP1 interaction impacts the efficacy of olaparib treatment. We show that PARP1 interacts with and downregulates Fra-1, thereby reducing AP-1 transcriptional activity. Olaparib treatment, or silencing of PARP1, consequently, increases Fra-1 levels and enhances its transcriptional activity. Increased Fra-1 can have adverse effect, including treatment resistance. We also found that a large fraction of PARP1-regulated genes was dependent on Fra-1. We show that by inhibiting Fra-1/AP-1, non-BRCA-mutated TNBC cells can become sensitized to olaparib treatment. We identify that high PARP1 expression is indicative of a poor clinical outcome in breast cancer patients overall (P = 0.01), but not for HER-2 positive patients. In conclusion, by exploring the functionality of the Fra-1 and PARP1 interaction, we propose that targeting Fra-1 could serve as a combinatory therapeutic approach to improve olaparib treatment outcome for TNBC patients.
Asunto(s)
Poli(ADP-Ribosa) Polimerasa-1/fisiología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Proteínas Proto-Oncogénicas c-fos/fisiología , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Antígeno B7-H1/fisiología , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ftalazinas/farmacología , Piperazinas/farmacología , Proteínas Proto-Oncogénicas c-fos/antagonistas & inhibidores , Factor de Transcripción AP-1/fisiologíaRESUMEN
A growing body of experimental evidence strongly suggests that cannabidiolic acid (CBDA), a major component of the fiber-type cannabis plant, exerts a variety of biological activities. We have reported that CBDA can abrogate cyclooxygenase-2 (COX-2) expression and its enzymatic activity. It is established that aberrant expression of COX-2 correlates with the degree of malignancy in breast cancer. Although the reduction of COX-2 expression by CBDA offers an attractive medicinal application, the molecular mechanisms underlying these effects have not fully been established. It has been reported that COX-2 expression is positively controlled by peroxisome proliferator-activated receptor ß/δ (PPARß/δ) in some cancerous cells, although there is "no" modulatory element for PPARß/δ on the COX-2 promoter. No previous studies have examined whether an interaction between PPARß/δ-mediated signaling and COX-2 expression exists in MDA-MB-231 cells. We confirmed, for the first time, that COX-2 expression is positively modulated by PPARß/δ-mediated signaling in MDA-MB-231 cells. CBDA inhibits PPARß/δ-mediated transcriptional activation stimulated by the PPARß/δ-specific agonist, GW501516. Furthermore, the disappearance of cellular actin stress fibers, a hallmark of PPARß/δ and COX-2 pathway activation, as evoked by the GW501516, was effectively reversed by CBDA. Activator protein-1 (AP-1)-driven transcriptional activity directly involved in the regulation of COX-2 was abrogated by the PPARß/δ-specific inverse agonists (GSK0660/ST-247). Thus, it is implicated that there is positive interaction between PPARß/δ and AP-1 in regulation of COX-2. These data support the concept that CBDA is a functional down-regulator of COX-2 through the abrogation of PPARß/δ-related signaling, at least in part, in MDA-MB-231 cells.
Asunto(s)
Neoplasias de la Mama/enzimología , Cannabinoides/farmacología , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Expresión Génica/genética , PPAR delta/fisiología , Femenino , Humanos , PPAR delta/agonistas , Transducción de Señal/genética , Transducción de Señal/fisiología , Sulfonas/farmacología , Tiazoles/farmacología , Tiofenos/farmacología , Factor de Transcripción AP-1/fisiología , Células Tumorales CultivadasRESUMEN
Amphetamine (AMPH), an appetite suppressant, alters expression levels of neuropeptide Y (NPY) and cocaine- and amphetamine-regulated transcript (CART) in the hypothalamus. This study explored the potential role of cJun-N-terminal kinases (JNK) in appetite control, mediated by reactive oxygen species (ROS) and activator protein-1 (AP-1) in AMPH-treated rats. Rats were given AMPH daily for 4 days. Changes in feeding behavior and expression levels of hypothalamic NPY, CART, cFos, cJun, phosphorylated JNK (pJNK), as well as those of anti-oxidative enzymes, including superoxide dismutase (SOD), glutathione peroxidase (GP) and glutathione S-transferase (GST), were examined and compared. Following AMPH treatment, food intake and NPY expression decreased, whereas the other proteins expression and AP-1/DNA binding activity increased. Both cerebral cJun inhibition and ROS inhibition attenuated AMPH anorexia and modified detected protein, revealing a crucial role for AP-1 and ROS in regulating AMPH-induced appetite control. Moreover, both pJNK/CART and SOD/CART activities detected by double immunofluorescent staining increased in hypothalamic arcuate nucleus in AMPH-treated rats. The results suggested that pJNK/AP-1 signaling and endogenous anti-oxidants participated in regulating NPY/CART-mediated appetite control in rats treated with AMPH. These findings advance understanding of the molecular mechanism underlying the role of pJNK/AP-1 and oxidative stress in NPY/CART-mediated appetite suppression in AMPH-treated rats.
Asunto(s)
Regulación del Apetito/fisiología , Proteínas Quinasas JNK Activadas por Mitógenos/fisiología , Neuropéptido Y/fisiología , Estrés Oxidativo/fisiología , Especies Reactivas de Oxígeno/metabolismo , Factor de Transcripción AP-1/fisiología , Anfetamina/farmacología , Animales , Antracenos/administración & dosificación , Antracenos/farmacología , Antioxidantes/metabolismo , Regulación del Apetito/efectos de los fármacos , Conducta Alimentaria/efectos de los fármacos , Técnica del Anticuerpo Fluorescente , Hipotálamo/metabolismo , Hipotálamo/fisiología , Infusiones Intraventriculares , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Masculino , Proteínas del Tejido Nervioso/metabolismo , Neuropéptido Y/biosíntesis , Ratas , Transducción de Señal/fisiología , Factor de Transcripción AP-1/metabolismoRESUMEN
Background: Inflammation is associated with marked changes in cellular thyroid hormone (TH) metabolism in triiodothyronine (T3) target organs. In the hypothalamus, type 2 deiodinase (D2), the main T3 producing enzyme, increases upon inflammation, leading to an increase in local T3 availability, which in turn decreases thyrotropin releasing hormone expression in the paraventricular nucleus. Type 3 deiodinase (D3), the T3 inactivating enzyme, decreases during inflammation, which might also contribute to the increased T3 availability in the hypothalamus. While it is known that D2 is regulated by nuclear factor κB (NF-κB) during inflammation, the underlying mechanisms of D3 regulation are unknown. Therefore, the aim of the present study was to investigate inflammation-induced D3 regulation using in vivo and in vitro models. Methods: Mice were injected with a sublethal dose of bacterial endotoxin (lipopolysaccharide [LPS]) to induce a systemic acute-phase response. A human neuroblastoma (SK-N-AS) cell line was used to test the involvement of the thyroid hormone receptor alpha 1 (TRα1) as well as the activator protein-1 (AP-1) and NF-κB inflammatory pathways in the inflammation-induced decrease of D3. Results: D3 expression in the hypothalamus was decreased 24 hours after LPS injection in mice. This decrease was similar in mice lacking the TRα. Incubation of SK-N-AS cells with LPS robustly decreased both D3 mRNA expression and activity. This led to increased intracellular T3 concentrations. The D3 decrease was prevented when NF-κB or AP-1 was inhibited. TRα1 mRNA expression decreased in SK-N-AS cells incubated with LPS, but knockdown of the TRα in SK-N-AS cells did not prevent the LPS-induced D3 decrease. Conclusions: We conclude that the inflammation-induced D3 decrease in the hypothalamus is mediated by the inflammatory pathways NF-κB and AP-1, but not TRα1. Furthermore, the observed decrease modulates intracellular T3 concentrations. Our results suggest a concerted action of inflammatory modulators to regulate both hypothalamic D2 and D3 activities to increase the local TH concentrations.
Asunto(s)
Hipotálamo/enzimología , Inflamación/metabolismo , Yoduro Peroxidasa/genética , Animales , Línea Celular Tumoral , Regulación hacia Abajo , Femenino , Humanos , Yoduro Peroxidasa/fisiología , Lipopolisacáridos , Masculino , Ratones , FN-kappa B/fisiología , ARN Mensajero/análisis , Transducción de Señal , Receptores alfa de Hormona Tiroidea/fisiología , Factor de Transcripción AP-1/fisiología , Yodotironina Deyodinasa Tipo IIRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: The quality control of Traditional Chinese medicine (TCM) is a scientific problem and an industrial issue, which hampers the development of evidence based TCM. The concept of quality markers (Q-markers) is proposed and has been applied to the quality evaluation of TCM based on its clinical efficacy. However, more specific methods are needed to put this idea into practice. The standard decoction is a representative of decoction used in clinical practice and it can be used for the discovery of Q-markers related to the clinical efficacy of TCM. AIM OF THE STUDY: In this study, a systemic strategy was established to discover Q-markers related to the clinical efficacy of TCM Ephedrae Herba (EH), dried stem of Ephedra sinica Stapf. The different processed materials of EH have different clinical applications, though originating from the same medicinal herb. MATERIALS AND METHODS: The standard decoction of each of the processed materials was prepared and a 1HNMR metabolomics approach and total polysaccharide analysis were used to identify potential Q-markers related to the different clinical applications of EH. Correlation analysis was made of the measured biological activity and the holistic chemical profile. RESULTS: The results showed that total polysaccharides and alkaloids were Q-markers for EH preparations. CONCLUSION: This study demonstrates that the standard decoction is a reasonable research objective to explore chemical markers that correlate with the clinical efficacy of TCM.
Asunto(s)
Ephedra , Extractos Vegetales/farmacología , Tallos de la Planta , Control de Calidad , Células HEK293 , Humanos , Luciferasas/fisiología , Metabolómica , Regiones Promotoras Genéticas , Receptor Toll-Like 2/fisiología , Factor de Transcripción AP-1/fisiología , Resultado del TratamientoRESUMEN
BACKGROUND: The abnormal expression of activator protein-1(AP-1) has recently been investigated in a variety of tumors. While the relationship between AP-1 and thyroid cancer is poorly studied, our study was to evaluate the protein expression and clinical value of AP-1 in papillary thyroid carcinoma (PTC). METHODS: The expression of AP-1 was examined by immunohistochemistry on paraffin-embedded tissues obtained from PTC and correspondent paracancerous tissues of 82 patients. RESULTS: Compared with paracancerous tissues, AP-1 expression was significantly elevated in PTC tissues and the positive rate was 79.3% (65/82). Our study found a linear trend relationship between the expression of AP-1 and tumor size. However, the differences in AP-1 expression among gender, age, lymph node metastasis, number of lesions, location of the lesion, and extrathyroid invasion are not statistically significant. CONCLUSIONS: The expression of AP-1 plays an important role in the proliferation process of PTC.
Asunto(s)
Cáncer Papilar Tiroideo/química , Neoplasias de la Tiroides/química , Factor de Transcripción AP-1/análisis , Adolescente , Adulto , Anciano , Femenino , Humanos , Inmunohistoquímica , Proteínas Quinasas JNK Activadas por Mitógenos/fisiología , Masculino , Metaloproteinasa 9 de la Matriz/análisis , Persona de Mediana Edad , Cáncer Papilar Tiroideo/patología , Neoplasias de la Tiroides/patología , Factor de Transcripción AP-1/fisiología , Adulto JovenRESUMEN
Although NFE2L2 transcription factor is considered to make the most significant contribution to the NFE2L2/AP-1-pathway-dependent antioxidants regulation in the human cell, AP-1 has the potential to provide significant backup and even play an equal role in the cell. Considering this, the present study is focused on revealing how JUN, an AP-1 component, and NFE2L2 contribute to regulation of four target genes containing AREs with embedded TREs-SQSTM1, FTH1, HMOX1 and CBR3 and to cellular oxidative status in general in basal conditions and under pro-oxidative influence. NFE2L2 and JUN were down-regulated in HeLa cells using siRNA-mediated knockdown approach. These cells were subsequently exposed to 400 µM hydrogen peroxide in the medium or equal volume of sterile water. They revealed some evidence of both backup functioning and competing between the two factors. Importantly, JUN demonstrated a high level of participation (inc. as a negative regulator) in functioning of the classic NFE2L2 targets and in cellular oxidative status establishment in general. One of the key findings was a dramatic increase in JUN expression following NFE2L2 knockdown in basal conditions. The both AP-1 and NFE2L2 sub-pathways equally determine the outcome of the NFE2L2/AP-1 pathway activation induced by various stimuli, and the outcome is stimulus type- and stimulus-intensity-specific and results from either of the two eventually dominating sub-pathways.
Asunto(s)
Factor 2 Relacionado con NF-E2/fisiología , Estrés Oxidativo/genética , Proteínas Proto-Oncogénicas c-jun/fisiología , Antioxidantes/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Células Epiteliales/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen/métodos , Células HeLa , Hemo-Oxigenasa 1/genética , Humanos , Peróxido de Hidrógeno/metabolismo , Factor 2 Relacionado con NF-E2/genética , Oxidación-Reducción , Estrés Oxidativo/efectos de los fármacos , Proteínas Proto-Oncogénicas c-jun/genética , Factor de Transcripción AP-1/genética , Factor de Transcripción AP-1/fisiologíaRESUMEN
Endothelium-derived colony stimulating factor (CSF1) plays a key role in a range of human pathologies. Angiotensin II (Ang II) has been documented to stimulate CSF1 transcription although the underlying epigenetic mechanism remains unclear. Here we report that induction of CSF1 transcription by Ang II in vascular endothelial cells paralleled alterations of signature histone modifications surrounding the CSF1 promoter. Specifically, ChIP assays indicated that there was a simultaneous up-regulation of both acetylated H3 and trimethylated H3K4, indicative of transcriptional activation, and down-regulation of dimethyl H3K9, implicated in transcriptional repression, surrounding the proximal CSF1 promoter. Further analysis revealed that silencing of brahma related gene 1 (BRG1), a chromatin remodeling protein, abrogated CSF1 induction by Ang II. In the meantime, BRG1 silencing erased H3 acetylation and H3K4 trimethylation and restored H3K9 dimethylation. Mechanistically, BRG1 interacted with and recruited SET1A, a histone H3K4 methyltransferase, and JMJD1A, a histone H3K9 demethylase, to the CSF1 promoter to alter chromatin structure thereby promoting CSF1 trans-activation in response to Ang II stimulation. Knockdown of either SET1A or JMJD1A blocked CSF1 induction by Ang II. Finally, we demonstrate that the crosstalk between BRG1 and histone modifying enzymes was mediated by the transcription factor AP-1. In conclusion, our data unveil a novel epigenetic mechanism whereby a BRG1-centered complex mediates transcriptional activation of CSF1 by Ang II in vascular endothelial cells.
Asunto(s)
Angiotensina II/fisiología , Células Endoteliales/metabolismo , Epigénesis Genética , Factor Estimulante de Colonias de Macrófagos/genética , ADN Helicasas/fisiología , Código de Histonas/genética , Humanos , Proteínas Nucleares/fisiología , Receptor Cross-Talk , Factor de Transcripción AP-1/fisiología , Factores de Transcripción/fisiología , Transcripción Genética , Activación TranscripcionalRESUMEN
Purpose: Systemic hypertension is a risk factor of neovascular age-related macular degeneration; consumption of dietary salt resulting in extracellular hyperosmolarity is a main cause of hypertension. Extracellular hyperosmolarity was shown to induce expression of angiogenic growth factors, such as vascular endothelial growth factor (VEGF) and placental growth factor (PlGF), in RPE cells. The aim of the present study was to determine whether the hyperosmotic expression of growth factor genes in RPE cells is mediated by activator protein-1 (AP-1), and whether c-Fos and c-Jun genes are regulated by extracellular osmolarity. Methods: Hyperosmotic media were made up with the addition of NaCl or sucrose. Gene expression was quantified with real-time reverse transcription (RT)-PCR, and protein secretion was investigated with enzyme-linked immunosorbent assay (ELISA). Nuclear factor of activated T cell 5 (NFAT5) was depleted with siRNA. DNA binding of AP-1 protein was evaluated with electrophoretic mobility shift assay (EMSA). Results: High NaCl and the addition of sucrose triggered expression of the c-Fos gene, but not of the c-Jun gene. High NaCl also increased the levels of c-Fos and phosphorylated c-Jun proteins and the level of DNA binding of AP-1. Hypoosmolarity decreased the expression of the c-Fos and c-Jun genes. NaCl-induced expression of the c-Fos gene was in part mediated by NFAT5. Autocrine/paracrine activation of fibroblast growth factor and adenosine A1 receptors is involved in mediating NaCl-induced expression of the c-Fos gene. Pharmacological inhibition of the AP-1 activity decreased the NaCl-induced expression of the HIF-1α, NFAT5, VEGF, PlGF, and TGF-ß2 genes, and prevented the NaCl-induced secretion of PlGF but not of VEGF. Conclusions: The data indicate that AP-1 is activated in RPE cells in response to extracellular hyperosmolarity and mediates in part via the NaCl-induced expression of VEGF and PlGF, and secretion of PlGF. It is suggested that high consumption of dietary salt may exacerbate the angiogenic response of RPE cells in part via activation of AP-1.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Factor de Crecimiento Placentario/genética , Epitelio Pigmentado de la Retina/efectos de los fármacos , Cloruro de Sodio/farmacología , Factor de Transcripción AP-1/fisiología , Factor A de Crecimiento Endotelial Vascular/genética , Western Blotting , Células Cultivadas , Ensayo de Cambio de Movilidad Electroforética , Ensayo de Inmunoadsorción Enzimática , Genes fos/fisiología , Genes jun/fisiología , Humanos , Fosforilación , Factor de Crecimiento Placentario/metabolismo , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Epitelio Pigmentado de la Retina/metabolismo , Transducción de Señal/efectos de los fármacos , Factor de Transcripción AP-1/antagonistas & inhibidores , Transfección , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
AIM: The airway epithelium of smokers exhibits upregulated SPRR3, an indicator of pathogenic keratinization. The mechanisms underlying this phenomenon require investigation. PATIENTS & METHODS: Human bronchial epithelial (HBE) SPRR3 expression was analyzed by smoking status. Primary HBE cells were exposed to cigarette smoke (CS). SPRR3 expression, SPRR3 promoter activity, AP-1 factor binding and AP-1 factors' effects were analyzed. RESULTS: Current smokers display SPRR3 upregulation relative to never smokers. CS upregulates SPRR3 transcription in an exposure-dependent manner. CS promotes c-Jun and Fra1 binding to the SPRR3-AP-1/TRE site. Wild-type c-Jun and Fra1 upregulate, whereas c-Jun and Fra1, dominant-negative mutants, suppress SPRR3 promoter activity. CONCLUSION: CS induces SPRR3 upregulation in HBE cells by promoting aberrant c-Jun/Fra1 dimerization.
Asunto(s)
Bronquios/metabolismo , Proteínas Ricas en Prolina del Estrato Córneo/genética , Multimerización de Proteína , Proteínas Proto-Oncogénicas c-fos/química , Proteínas Proto-Oncogénicas c-jun/química , Fumar Tabaco/efectos adversos , Anciano , Anciano de 80 o más Años , Células Cultivadas , Células Epiteliales/metabolismo , Femenino , Regulación de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Proteína Quinasa 7 Activada por Mitógenos/fisiología , Proteína Quinasa 8 Activada por Mitógenos/fisiología , Regiones Promotoras Genéticas , Factor de Transcripción AP-1/fisiología , Regulación hacia ArribaRESUMEN
The HIV accessory protein Nef counteracts immune defenses by subverting coated vesicle pathways. The 3.7 Å cryo-EM structure of a closed trimer of the clathrin adaptor AP-1, the small GTPase Arf1, HIV-1 Nef, and the cytosolic tail of the restriction factor tetherin suggested a mechanism for inactivating tetherin by Golgi retention. The 4.3 Å structure of a mutant Nef-induced dimer of AP-1 showed how the closed trimer is regulated by the dileucine loop of Nef. HDX-MS and mutational analysis were used to show how cargo dynamics leads to alternative Arf1 trimerization, directing Nef targets to be either retained at the trans-Golgi or sorted to lysosomes. Phosphorylation of the NL4-3 M-Nef was shown to regulate AP-1 trimerization, explaining how O-Nefs lacking this phosphosite counteract tetherin but most M-Nefs do not. These observations show how the higher-order organization of a vesicular coat can be allosterically modulated to direct cargoes to distinct fates.
Asunto(s)
Factor de Transcripción AP-1/ultraestructura , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismo , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/ultraestructura , Factor 1 de Ribosilacion-ADP/metabolismo , Factor 1 de Ribosilacion-ADP/ultraestructura , Proteínas Adaptadoras del Transporte Vesicular , Antígeno 2 del Estroma de la Médula Ósea/metabolismo , Antígeno 2 del Estroma de la Médula Ósea/ultraestructura , Clatrina , Aparato de Golgi , Células HEK293 , VIH-1 , Humanos , Transporte de Proteínas/fisiología , Factor de Transcripción AP-1/metabolismo , Factor de Transcripción AP-1/fisiología , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/fisiologíaRESUMEN
OBJECTIVE: In Alzheimer's disease (AD), astrocytes are generally found in the surrounding of senile plaques participating in the production of phagocytosis and the removal of toxic compounds such as Aß. This study aimed at investigating the effect of Aß1-42 on astrocytes. MATERIALS AND METHODS: Cellular viability of primary cultured astrocytes was analyzed using CCK-8 assay. Quantitative Real-time PCR was used to assess the mRNA expression of JNK and AP-1. The proteins of JNK/AP-1 pathway were investigated using Western blot. RESULTS: Our findings showed that Aß1-42 inhibited cell viability and promoted apoptosis in astrocytes in primary culture. Additionally, Aß1-42 increased the mRNA expression level of AP-1, but had no effect on the expression of JNK. Furthermore, Aß1-42 increased the protein expression of p-JNK, p-c-jun and Fra-1 and the ratio of p-c-jun/c-jun and p-JNK/JNK. CONCLUSIONS: We showed that Aß1-42 promoted cell apoptosis in astrocytes in primary culture. Furthermore, Aß1-42 activated JNK/AP-1 pathway through promoting the phosphorylation of JNK, c-jun and Fra-1 expression, then inducing cell apoptosis.
Asunto(s)
Péptidos beta-Amiloides/farmacología , Astrocitos/efectos de los fármacos , Proteínas Quinasas JNK Activadas por Mitógenos/fisiología , Fragmentos de Péptidos/farmacología , Factor de Transcripción AP-1/fisiología , Astrocitos/metabolismo , Células Cultivadas , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-fos/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
The AP-1 Adaptor Complex assists clathrin-coated vesicle assembly in the trans-Golgi network (TGN) of eukaryotic cells. However, the role of AP-1 in the protozoan Trypanosoma cruzi-the Chagas disease parasite-has not been addressed. Here, we studied the function and localization of AP-1 in different T. cruzi life cycle forms, by generating a gene knockout of the large AP-1 subunit gamma adaptin (TcAP1-γ), and raising a monoclonal antibody against TcAP1-γ. Co-localization with a Golgi marker and with the clathrin light chain showed that TcAP1-γ is located in the Golgi, and it may interact with clathrin in vivo, at the TGN. Epimastigote (insect form) parasites lacking TcAP1-γ (TcγKO) have reduced proliferation and differentiation into infective metacyclic trypomastigotes (compared with wild-type parasites). TcγKO parasites have also displayed significantly reduced infectivity towards mammalian cells. Importantly, TcAP1-γ knockout impaired maturation and transport to lysosome-related organelles (reservosomes) of a key cargo-the major cysteine protease cruzipain, which is important for parasite nutrition, differentiation and infection. In conclusion, the defective processing and transport of cruzipain upon AP-1 ablation may underlie the phenotype of TcγKO parasites.
Asunto(s)
Enfermedad de Chagas/parasitología , Cisteína Endopeptidasas/química , Factor de Transcripción AP-1/genética , Factor de Transcripción AP-1/fisiología , Trypanosoma cruzi/genética , Animales , Animales Modificados Genéticamente , Anticuerpos Monoclonales/química , Vesículas Cubiertas por Clatrina , Endocitosis , Prueba de Complementación Genética , Aparato de Golgi/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Orgánulos , Plásmidos/metabolismo , Proteínas Protozoarias , Proteínas Recombinantes/química , Red trans-Golgi/metabolismoRESUMEN
SCOPE: Indole-3-carbinol (I3C) and 3,3'-diindolylmethane (DIM) from Brassica plants are regarded as promising anticancer phytochemicals. The enzyme telomerase is a very attractive target for cancer therapeutics; in normal cells such as lymphocytes, it plays a decisive role for cell maintenance. The effect of I3C and DIM on telomerase in normal human immune cells (PBMC) was studied compared to leukaemia cells (HL-60). Signalling of telomerase regulation via estrogen receptor (ER) was addressed. METHODS AND RESULTS: Short-term treatment with I3C and DIM inhibited telomerase activity in leukaemia cells (>30 µM I3C; >3 µM DIM). In CD3/CD28 activated PBMC, inhibition was stronger, though (>3 µM I3C; >1 µM DIM). DIM long-term treatment resulted in DNA damage induction and proliferation inhibition in PBMC as determined by the comet assay and CFSE staining, respectively. A relevance of ERα/ß-AP1 signaling for telomerase inhibition on enzyme activity, but not transcription level became evident indicating a nonclassical mode for ER regulation of telomerase by DIM. CONCLUSION: Although desired in cancer cells, this study identified a potential adverse impact of I3C and DIM on telomerase action in normal human immune cells, partly mediated by an ER-dependent mechanism. These new findings should be considered for potential chronic high-dose chemoprevention strategies using these compounds.
Asunto(s)
Brassica/química , Receptor alfa de Estrógeno/fisiología , Receptor beta de Estrógeno/fisiología , Indoles/farmacología , Leucocitos Mononucleares/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Telomerasa/antagonistas & inhibidores , Factor de Transcripción AP-1/fisiología , Daño del ADN , Células Hep G2 , HumanosRESUMEN
Vagus nerve stimulation through alpha7 nicotine acetylcholine receptors (α7-nAChR) signaling had been demonstrated attenuation of inflammation. This study aimed to determine whether PNU-282987, a selective α7-nAChR agonist, affected activities of matrix metalloproteinase (MMP) and inflammatory cytokines in nicotine-treatment RAW264.7 and MOVAS cells and to assess the underlying molecular mechanisms. RAW264.7 and MOVAS cells were treated with nicotine at different concentrations (0, 1, 10, and 100 ng/ml) for 0-120 min. Nicotine markedly stimulated the phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2) and c-Jun in RAW264.7 cells. Pretreatment with U0126 significantly suppressed phosphorylation of ERK1/2 and further attenuated nicotine-induced activation of c-Jun and upregulation of MMP-2, MMP-9, monocyte chemotactic protein- (MCP-) 1, and regulated upon activation normal T cell expressed and secreted (RANTES). Similarly, nicotine treatment also increased phosphorylation of c-Jun and expressions of MMP-2, MMP-9, MCP-1, and RANTES in MOVAS cells. When cells were pretreated with PNU-282987, nicotine-induced activations of ERK1/2 and c-Jun in RAW264.7 cells and c-Jun in MOVAS cells were effectively inhibited. Furthermore, nicotine-induced secretions of MMP-2, MMP-9, MCP-1, and RANTES were remarkably downregulated. Treatment with α7-nAChR agonist inhibits nicotine-induced upregulation of MMP and inflammatory cytokines through modulating ERK1/2/AP-1 signaling in RAW264.7 cells and AP-1 in MOVAS cells, providing a new therapeutic for abdominal aortic aneurysm.
Asunto(s)
Quimiocina CCL2/genética , Quimiocina CCL5/genética , Quinasas MAP Reguladas por Señal Extracelular/fisiología , Metaloproteinasas de la Matriz/genética , Nicotina/farmacología , Transducción de Señal/fisiología , Factor de Transcripción AP-1/fisiología , Receptor Nicotínico de Acetilcolina alfa 7/fisiología , Animales , Benzamidas/farmacología , Compuestos Bicíclicos con Puentes/farmacología , Butadienos/farmacología , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Ratones , Nitrilos/farmacología , Fosforilación , Células RAW 264.7 , Regulación hacia Arriba/fisiología , Receptor Nicotínico de Acetilcolina alfa 7/agonistasRESUMEN
Cutaneous exposure to solar ultraviolet (UV) radiation is a major causative factor in skin carcinogenesis, and improved molecular strategies for efficacious chemoprevention of nonmelanoma skin cancer (NMSC) are urgently needed. Toll-like receptor 4 (TLR4) signaling has been shown to drive skin inflammation, photoimmunosuppression, and chemical carcinogenesis. Here we have examined the feasibility of genetic and pharmacological antagonism targeting cutaneous TLR4 for the suppression of UV-induced NF-κB and AP-1 signaling in keratinocytes and mouse skin. Using immunohistochemical and proteomic microarray analysis of human skin, we demonstrate for the first time that a significant increase in expression of TLR4 occurs in keratinocytes during the progression from normal skin to actinic keratosis, also detectible during further progression to squamous cell carcinoma. Next, we demonstrate that siRNA-based genetic TLR4 inhibition blocks UV-induced stress signaling in cultured keratinocytes. Importantly, we observed that resatorvid (TAK-242), a molecularly targeted clinical TLR4 antagonist, blocks UV-induced NF-κB and MAP kinase/AP-1 activity and cytokine expression (Il-6, Il-8, and Il-10) in cultured keratinocytes and in topically treated murine skin. Taken together, our data reveal that pharmacological TLR4 antagonism can suppress UV-induced cutaneous signaling, and future experiments will explore the potential of TLR4-directed strategies for prevention of NMSC.
Asunto(s)
Queratinocitos/efectos de los fármacos , FN-kappa B/fisiología , Transducción de Señal/efectos de los fármacos , Piel/efectos de los fármacos , Sulfonamidas/farmacología , Receptor Toll-Like 4/antagonistas & inhibidores , Factor de Transcripción AP-1/fisiología , Animales , Humanos , Queratinocitos/metabolismo , Ratones , Protectores contra Radiación/farmacología , Transducción de Señal/efectos de la radiación , Rayos UltravioletaRESUMEN
A coordinated and faithful DNA damage response is of central importance for maintaining genomic integrity and survival. Here, we show that exposure of human cells to benzo(a)pyrene 9,10-diol-7,8-epoxide (BPDE), the active metabolite of benzo(a)pyrene (B(a)P), which represents a most important carcinogen formed during food preparation at high temperature, smoking and by incomplete combustion processes, causes a prompt and sustained upregulation of the DNA repair genes DDB2, XPC, XPF, XPG and POLH. Induction of these repair factors on RNA and protein level enhanced the removal of BPDE adducts from DNA and protected cells against subsequent BPDE exposure. However, through the induction of POLH the mutation frequency in the surviving cells was enhanced. Activation of these adaptive DNA repair genes was also observed upon B(a)P treatment of MCF7 cells and in buccal cells of human volunteers after cigarette smoking. Our data provide a rational basis for an adaptive response to polycyclic aromatic hydrocarbons, which occurs however at the expense of mutations that may drive cancer formation.