RESUMO
In eukaryotes, clathrin-coated vesicles (CCVs) facilitate the internalization of material from the cell surface as well as the movement of cargo in post-Golgi trafficking pathways. This diversity of functions is partially provided by multiple monomeric and multimeric clathrin adaptor complexes that provide compartment and cargo selectivity. The adaptor-protein assembly polypeptide-1 (AP-1) complex operates as part of the secretory pathway at the trans-Golgi network (TGN), while the AP-2 complex and the TPLATE complex jointly operate at the plasma membrane to execute clathrin-mediated endocytosis. Key to our further understanding of clathrin-mediated trafficking in plants will be the comprehensive identification and characterization of the network of evolutionarily conserved and plant-specific core and accessory machinery involved in the formation and targeting of CCVs. To facilitate these studies, we have analyzed the proteome of enriched TGN/early endosome-derived and endocytic CCVs isolated from dividing and expanding suspension-cultured Arabidopsis (Arabidopsis thaliana) cells. Tandem mass spectrometry analysis results were validated by differential chemical labeling experiments to identify proteins co-enriching with CCVs. Proteins enriched in CCVs included previously characterized CCV components and cargos such as the vacuolar sorting receptors in addition to conserved and plant-specific components whose function in clathrin-mediated trafficking has not been previously defined. Notably, in addition to AP-1 and AP-2, all subunits of the AP-4 complex, but not AP-3 or AP-5, were found to be in high abundance in the CCV proteome. The association of AP-4 with suspension-cultured Arabidopsis CCVs is further supported via additional biochemical data.
Assuntos
Arabidopsis , Vesículas Revestidas por Clatrina , Arabidopsis/genética , Arabidopsis/metabolismo , Clatrina/metabolismo , Vesículas Revestidas por Clatrina/química , Vesículas Revestidas por Clatrina/metabolismo , Endocitose , Proteoma/metabolismo , Proteômica , Fator de Transcrição AP-1/análise , Fator de Transcrição AP-1/metabolismoRESUMO
Extensive phytochemical analysis of the CHCl3-soluble part of an ethanolic extract of branches and twigs of Broussonetia papyrifera led to the isolation of fourteen compounds, including a novel 5,11-dioxabenzo[b]fluoren-10-one derivative named broussofluorenone C (12). The isolated compounds 1-14 were characterized based on their NMR and HRMS data, and examined for their anti-inflammatory activities in LPS-stimulated THP-1 cells as well as for their cellular antioxidant effects. Compounds 7-10 and 12 showed inhibitory effects on NF-κB/AP-1 activation and compounds 7-9 were subsequently confirmed to suppress the secretion of both IL-1ß and TNF-α in LPS-stimulated THP-1 cells more significantly than the prednisone used as a positive control. In the CAA assay, compound 10 exhibited the greatest antioxidant effect, greater than that of the quercetin used as a positive control. The results show possible beneficial effects and utilization of B. papyrifera wood in the treatment of inflammatory diseases as well as oxidative stress.
Assuntos
Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Broussonetia/química , Anti-Inflamatórios/química , Anti-Inflamatórios/isolamento & purificação , Antioxidantes/química , Antioxidantes/isolamento & purificação , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Humanos , Interleucina-1beta/antagonistas & inibidores , Interleucina-1beta/biossíntese , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Estrutura Molecular , NF-kappa B/análise , NF-kappa B/antagonistas & inibidores , NF-kappa B/biossíntese , Estresse Oxidativo/efeitos dos fármacos , Relação Estrutura-Atividade , Células THP-1 , Fator de Transcrição AP-1/análise , Fator de Transcrição AP-1/antagonistas & inibidores , Fator de Transcrição AP-1/biossíntese , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/biossínteseRESUMO
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.
Assuntos
Câncer Papilífero da Tireoide/química , Neoplasias da Glândula Tireoide/química , Fator de Transcrição AP-1/análise , Adolescente , Adulto , Idoso , Feminino , Humanos , Imuno-Histoquímica , Proteínas Quinases JNK Ativadas por Mitógeno/fisiologia , Masculino , Metaloproteinase 9 da Matriz/análise , Pessoa de Meia-Idade , Câncer Papilífero da Tireoide/patologia , Neoplasias da Glândula Tireoide/patologia , Fator de Transcrição AP-1/fisiologia , Adulto JovemRESUMO
BACKGROUND: The objective of the present study was to evaluate the role of the TGFß/PDCD4/AP-1 pathway in nasopharyngeal carcinoma (NPC) and its relationship to NPC prognosis. METHODS: NPC tissues collected from 66 NPC patients were compared to 17 nasopharyngeal mucosa biopsy specimens collected as normal tissues. Immunohistochemical staining was performed to assess expression of transforming growth factor-ß receptor I (TGFßRI), programmed cell death 4 (PDCD4) and activator protein-1 (AP-1). The Kaplan-Meier method was applied to evaluate NPC patient overall survival (OS) and progression-free-survival (PFS). Cox regression analysis was used to estimate independent prognostic factors for NPC. The human NPC cell line CNE2 was selected and treated with SB431542, an inhibitor of TGFßRI; expression of TGFßRI and PDCD4 in CNE2 cells was determined by western blotting. NPC tissues showed higher expression of TGFßRI and AP-1 but lower expression of PDCD4 than normal tissues (all P < 0.05). RESULTS: The results of Kaplan-Meier analysis showed that TGFßRI-positive patients and AP-1-positive patients had shorter OS and PFS than TGFßRI-negative patients and AP-1-negative patients; additionally, PDCD4-positive patients had higher OS and PFS than PDCD4-negative patients. Cox regression analysis revealed that advanced tumor stage, overexpression of TGFßRI and AP-1, and low expression of PDCD4 were unfavorable factors influencing OS and PFS in NPC patients. Compared with the control group, expression of TGFßRI decreased and that of PDCD4 increased significantly in CNE2 cells treated with the inhibitor (all P < 0.05). These findings indicate that the TGFß/PDCD4/AP-1 pathway may be associated with NPC development and progression. CONCLUSION: High expression of TGFßRI and AP-1 and low expression of PDCD4 may be unfavorable prognostic factors for NPC.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Carcinoma/diagnóstico , Carcinoma/metabolismo , Neoplasias Nasofaríngeas/diagnóstico , Neoplasias Nasofaríngeas/metabolismo , Nasofaringe/patologia , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Fator de Transcrição AP-1/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Adulto , Idoso , Proteínas Reguladoras de Apoptose/análise , Carcinoma/patologia , Linhagem Celular Tumoral , Intervalo Livre de Doença , Feminino , Seguimentos , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas/patologia , Nasofaringe/metabolismo , Prognóstico , Proteínas de Ligação a RNA/análise , Fator de Transcrição AP-1/análise , Fator de Crescimento Transformador beta/análiseRESUMO
Engagement of the T cell receptor complex reprograms T cells for proliferation, cytokine production and differentiation towards effector cells. This process depends on activating costimulatory signals and is counteracted by coinhibitory molecules. Three transcription factors, namely NF-κB, NFAT and AP-1, have a major role in inducing the transcriptional program that is required for T cell activation and differentiation. Here we describe the generation of a triple parameter reporter based on the human Jurkat T cell line, where response elements for NF-κB, NFAT and AP-1 drive the expression of the fluorescent proteins CFP, eGFP and mCherry, respectively. The emission spectra of these proteins allow simultaneous assessment of NF-κB, NFAT and AP-1 activity in response to stimulation. Ligation of the TCR complex induced moderate reporter activity, which was strongly enhanced upon coengagement of the costimulatory receptors CD2 or CD28. Moreover, we have generated and tested triple parameter reporter cells that harbor costimulatory and inhibitory receptors not endogenously expressed in the Jurkat cells. In these experiments we could show that engagement of the costimulatory molecule 4-1BB enhances NF-κB and AP-1 activity, whereas coinhibition via PD-1 or BTLA strongly reduced the activation of NF-κB and NFAT. Engagement of BTLA significantly inhibited AP-1, whereas PD-1 had little effect on the activation of this transcription factor. Our triple parameter reporter T cell line is an excellent tool to assess the effect of costimulatory and coinhibitory receptors on NF-κB, NFAT and AP-1 activity and has a wide range of applications beyond the evaluation of costimulatory pathways.
Assuntos
Ativação Linfocitária , NF-kappa B/análise , Fatores de Transcrição NFATC/análise , Fator de Transcrição AP-1/análise , Antígenos CD2/fisiologia , Antígenos CD28 , Complexo CD3/metabolismo , Genes Reporter , Humanos , Células Jurkat , Proteínas Luminescentes , NF-kappa B/genética , NF-kappa B/metabolismo , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/metabolismo , Transdução de Sinais , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismo , Proteína Vermelha FluorescenteRESUMO
The c-Fos and c-Jun transcription factors, members of the activator protein 1 (AP-1) complex, form heterodimers and bind to DNA via a basic leucine zipper and regulate the cell cycle, apoptosis, differentiation, etc. Purified c-Jun leucine zipper fragments could also form stable homodimers, whereas c-Fos leucine zipper homodimers were found to be much less stable in earlier in vitro studies. The importance of c-Fos overexpression in tumors and the controversy in the literature concerning c-Fos homodimerization prompted us to investigate Fos homodimerization. Förster resonance energy transfer (FRET) and molecular brightness analysis of fluorescence correlation spectroscopy data from live HeLa cells transfected with fluorescent-protein-tagged c-Fos indicated that c-Fos formed homodimers. We developed a method to determine the absolute concentrations of transfected and endogenous c-Fos and c-Jun, which allowed us to determine dissociation constants of c-Fos homodimers (Kd = 6.7 ± 1.7 µM) and c-Fos-c-Jun heterodimers (on the order of 10 to 100 nM) from FRET titrations. Imaging fluorescence cross-correlation spectroscopy (SPIM-FCCS) and molecular dynamics modeling confirmed that c-Fos homodimers were stably associated and could bind to the chromatin. Our results establish c-Fos homodimers as a novel form of the AP-1 complex that may be an autonomous transcription factor in c-Fos-overexpressing tissues and could contribute to tumor development.
Assuntos
Simulação de Dinâmica Molecular , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Cromatina/metabolismo , DNA/metabolismo , Transferência Ressonante de Energia de Fluorescência , Células HeLa , Humanos , Microscopia de Fluorescência , Simulação de Acoplamento Molecular , Ligação Proteica , Multimerização Proteica , Proteínas Proto-Oncogênicas c-fos/análise , Proteínas Proto-Oncogênicas c-jun/análise , Fator de Transcrição AP-1/análise , Fator de Transcrição AP-1/metabolismoRESUMO
OBJECTIVES: The aims of this study were (i) to determine the localisation of activator protein (AP)-1 family members (cFos, FosB, cJun, JunB and JunD) in human myometrium; and (ii) to determine the effect of human term labour on the expression of AP-1 family of transcription factors in myometrium. STUDY DESIGN: This localised the AP-1 family members cFos, FosB, cJun, JunB and JunD in human myometrium was performed by immunohistochemistry. The effect of term labour on the expression of these family members at the mRNA and protein level was assessed by qRT-PCR and Western blotting, respectively. The effect of pro-inflammatory stimuli on AP-1 transcriptional activity was assessed using a luciferase assay in primary human myometrial cells. RESULTS: Immunohistochemical expression of cFos, FosB, cJun, JunB and JunD were all present in human myometrial tissue and displayed cytoplasmic staining. FosB and JunD also displayed nuclear staining. Term labour was associated with an increase in cFos and JunB mRNA and protein expression. On the other hand, JunD mRNA and protein expression was decreased with labour. FosB mRNA was increased with labour, but there was no change at the protein level. There was no change in cJun mRNA or protein expression. AP-1 transcriptional activity was increased in human myometrial cells by the pro-inflammatory cytokine TNF-α. There was, however, no effect of the bacterial products lipopolysaccharide (LPS; TLR4 ligand), iE-DAP (NOD1 ligand), MDP (NOD2 ligand), FSL-1 (TLR2 ligand) or flagellin (TLR5 ligand) on AP-1 transcriptional activity. CONCLUSION: This study shows that human labour is associated with changes in AP-1 family members. Further studies are required to determine the exact role of the AP-1 family members in myometrium.
Assuntos
Miométrio/metabolismo , Fator de Transcrição AP-1/metabolismo , Núcleo Celular/química , Células Cultivadas , Citoplasma/química , Ácido Diaminopimélico/análogos & derivados , Ácido Diaminopimélico/farmacologia , Diglicerídeos/farmacologia , Feminino , Flagelina/farmacologia , Humanos , Início do Trabalho de Parto , Lipopolissacarídeos/farmacologia , Miométrio/química , Oligopeptídeos/farmacologia , Gravidez , Proteínas Proto-Oncogênicas c-fos/análise , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/análise , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , RNA Mensageiro/metabolismo , Nascimento a Termo , Fator de Transcrição AP-1/análise , Fator de Transcrição AP-1/genética , Fatores de Transcrição/análise , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Asthma is a disease of airway inflammation characterized by airway hyperresponsiveness, eosinophilic inflammation, and hypersecretion of mucus. Ellagic acid, a compound derived from medicinal plants and fruits, has shown anti-inflammatory activity in several experimental disease models. We used the classical experimental model, in BALB/c mice, of sensibilization with ovalbumin to determine the effect of ellagic acid (10 mg/kg; oral route) in the resolution of allergic airways response. Dexamethasone (1 mg/kg; subcutaneous route) was used as a positive control. The control group consisted of nonimmunized mice that received challenge with ovalbumin. Ellagic acid and dexamethasone or vehicle (water) were administered before or after intranasal allergen challenge. Ellagic acid accelerated the resolution of airways inflammation by decreasing total leukocytes and eosinophils numbers in the bronchoalveolar lavage fluid (BALF), the mucus production and lung inflammation in part by reducing IL-5 concentration, eosinophil peroxidase (EPO) activity, and P-selectin expression, but not activator protein 1 (AP-1) and nuclear factor kappa B (NF-κB) pathways. In addition, ellagic acid enhanced alveolar macrophage phagocytosis of IgG-OVA-coated beads ex vivo, a new proresolving mechanism for the clearance of allergen from the airways. Together, these findings identify ellagic acid as a potential therapeutic agent for accelerating the resolution of allergic airways inflammation.
Assuntos
Asma/tratamento farmacológico , Ácido Elágico/uso terapêutico , Animais , Asma/imunologia , Asma/patologia , Feminino , Interleucina-5/análise , Leucócitos/efeitos dos fármacos , Leucócitos/fisiologia , Macrófagos/imunologia , Metaplasia , Camundongos , Camundongos Endogâmicos BALB C , NF-kappa B/análise , Selectina-P/análise , Fagocitose/efeitos dos fármacos , Fator de Transcrição AP-1/análiseRESUMO
Obesity has been shown to be associated with the risk of colorectal cancer (CRC). Adipokines produced by the adipose tissue are linked to some malignancies, including CRC. Visfatin is an adipokine shown to be a biomarker of CRC malignant potential. In addition, the stromal cell-derived factor-1 (SDF-1) has been reported to play a role in CRC progression. Although the relationship between visfatin and CRC has been established, the underlying mechanism has not been clarified. We investigated the molecular mechanism governing the interaction between visfatin stimulation and SDF-1 expression in human CRC cell lines. We found that visfatin stimulation led to an increase in the expression and secretion of SDF-1 in CRC DLD-1 and SW48 cells. Experiments involving specific inhibitors and small interfering RNA demonstrated that the activation of ERK and p38 mitogen-activated protein kinase (MAPK) pathways are critical for visfatin-induced SDF-1 expression. Analysis of transcription factor binding using ELISA and luciferase reporter assays revealed that visfatin increased NF-κB- and AP-1-DNA-binding activities in DLD-1 cells. Inhibition of NF-κB and AP-1 activation blocked the visfatin-induced expression and activity of the SDF-1 promoter. The effect of visfatin on DLD-1 signaling and SDF-1 expression was mediated by ß1 integrin. In summary, these findings provide novel insights pertaining to the pathophysiological role of visfatin in CRC.
Assuntos
Quimiocina CXCL12 , Neoplasias Colorretais , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Integrina beta1 , Nicotinamida Fosforribosiltransferase/farmacologia , Adipocinas/metabolismo , Tecido Adiposo/metabolismo , Linhagem Celular Tumoral , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Humanos , Integrina beta1/genética , Integrina beta1/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , NF-kappa B , Nicotinamida Fosforribosiltransferase/metabolismo , Fator de Transcrição AP-1/análiseRESUMO
Many studies have indicated that activation of matrix metalloproteinase (MMP)-9 and smooth muscle cell (SMC) migration are involved in neointimal formation and atherosclerosis. In this study, we revealed that pimaric acid (PiMA) purified from Aralia cordata had an inhibitory effect on MMP-9 production and migration of human aortic smooth muscle cells (HASMCs) induced by tumor necrosis factor (TNF)-α. Down-regulated MMP-9 mRNA transcription was detected in PiMA-treated cells using RT-PCR and the luciferase-tagged MMP-9 promoter assay. Results of an electrophoretic mobility shift assay indicated that PiMA-treated HASMCs showed decreased binding activity of nuclear factor (NF)-κB and activator protein-1 transcription factors. A Western-blot analysis using nuclear extract demonstrated that PiMA reduced the levels of NF-κB p65, c-Fos, p-c-Jun, Jun-D, and p-ATF2 proteins in the nucleus. In addition, TNF-α stimulated mitogen activated protein kinase (MAPK) containing extracellular signal regulated kinase 1 and 2, p38, and c-Jun N-terminal kinase was inhibited by PiMA. Using the Transwell system, we found that PiMA inhibited TNF-α stimulated HASMC migration/invasion in a dose-dependent manner. To confirm whether MAPK mediated MMP-9 expression, we used MAPK inhibitors including U0126, SB253580, and SP600125 and found that those inhibitors reduced MMP-9 expression and HASMC migration/invasion. These results suggest that PiMA has potent anti-atherosclerotic activity with inhibitory action on MMP-9 production and cell migration in TNF-α-induced HASMCs.
Assuntos
Anti-Inflamatórios/farmacologia , Aralia/química , Movimento Celular/efeitos dos fármacos , Diterpenos/farmacologia , Metaloproteinase 9 da Matriz/imunologia , Miócitos de Músculo Liso/efeitos dos fármacos , Fator de Necrose Tumoral alfa/imunologia , Anti-Inflamatórios/isolamento & purificação , Aorta/citologia , Linhagem Celular , Diterpenos/isolamento & purificação , Regulação para Baixo/efeitos dos fármacos , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Metaloproteinase 9 da Matriz/genética , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/imunologia , Miócitos de Músculo Liso/metabolismo , NF-kappa B/análise , NF-kappa B/imunologia , Fosforilação/efeitos dos fármacos , Fator de Transcrição AP-1/análise , Fator de Transcrição AP-1/imunologiaRESUMO
The present study reports the histopathological, ultrastructural changes and modulation of NF-kappaB, AP-1, and STAT 1 during skin carcinogenesis in LACA mice and its intervention with Azadirachta indica. Skin tumors were induced by topical application of 7,12-dimethylbenz(a)anthracene (DMBA) (500 nmol/100 microl for 2 weeks) followed by 12-O-tetradecanoylphorbol-13-acetate (TPA) (1.7 nmol/100 microl of acetone, twice weekly) as a promoter. Male LACA mice were divided into four groups: Control, DMBA/TPA, aqueous Azadirachta indica leaf extract (AAILE), and AAILE + DMBA/TPA. AAILE was administered orally at a dose level of 300 mg/kg body weight three times a week for 20 weeks. Topical application of DMBA/ TPA to the skin resulted in well-developed squamous cell carcinomas characterized by hyperproliferation, hyperkeratosis, and corrugation of the epidermis. Degenerative changes were observed in the tumors of AAILE + DMBA/TPA-treated animals. Scanning electron microscopy revealed surface disruptions and certain rounded structures on the skin tumors of DMBA/TPA-treated animals. Topographical changes were also observed in the tumors of AAILE + DMBA/TPA-treated animals, which resembled regions of degeneration. Tumors obtained in DMBA/TPA group were associated with enhanced expression of NF-kappaB and AP-1 when compared to the control counterparts. Inhibition in tumorigenesis in response to A. indica treatment was accompanied by an overexpression of STAT 1 and AP-1 and decrease in NF-kappaB expression. The results of the present study provide a basis for the chemopreventive potential of A. indica against murine skin carcinogenesis.
Assuntos
Anticarcinógenos/farmacologia , Azadirachta , NF-kappa B/fisiologia , Extratos Vegetais/farmacologia , Fator de Transcrição STAT1/fisiologia , Neoplasias Cutâneas/prevenção & controle , Fator de Transcrição AP-1/fisiologia , 9,10-Dimetil-1,2-benzantraceno , Animais , Masculino , Camundongos , NF-kappa B/análise , NF-kappa B/genética , RNA Mensageiro/análise , Fator de Transcrição STAT1/análise , Fator de Transcrição STAT1/genética , Acetato de Tetradecanoilforbol , Fator de Transcrição AP-1/análise , Fator de Transcrição AP-1/genéticaRESUMO
Diosgenin is a precursor of steroid hormones, which can be found in several plant species. Diosgenin has been shown to have a variety of biological activities including anti-inflammatory activity, but through a mechanism that is unclear. Especially, the effect of this agent on macrophage function has not been characterized in detail. In the present study, we examined the effects of diosgenin on the production of inflammatory mediators in lipopolysaccharide (LPS)/interferon gamma (IFN-gamma)-activated murine macrophage. Macrophages pre-exposed to diosgenin (0.1-10 microM) were stimulated with LPS/IFN-gamma. Pretreatment with diosgenin resulted in the inhibition of NO production and inducible nitric oxide synthase (iNOS) protein and mRNA expression in a concentration-dependent manner. In addition, diosgenin inhibits production of reactive oxygen species (ROS), interleukin-1 (IL-1), and IL-6, but not that of tumor necrosis factor-alpha (TNF-alpha). Inhibition of these inflammatory mediators appears to be at the transcriptional level, since diosgenin decreased LPS/IFN-gamma-induced NF-kappaB and AP-1 activity. Diosgenin blocked CK2 activation and phosphorylation of c-Jun NH(2)-terminal kinase (JNK), but not that of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase 1/2 (ERK 1/2). These results indicate that the inhibition of these signaling molecules expression was correlated with the reduced production of inflammatory mediators in macrophages. Taken together the present data suggest that diosgenin reduces the production of inflammatory meditators by inhibiting LPS/IFN-gamma-triggered CK2, JNK, NF-kappaB and AP-1 activation, thereby implicating a mechanism by which diosgenin may exert its immunosuppressive effects.
Assuntos
Anti-Inflamatórios/farmacologia , Diosgenina/farmacologia , Mediadores da Inflamação/antagonistas & inibidores , Macrófagos Peritoneais/efeitos dos fármacos , Animais , Caseína Quinase II/análise , Caseína Quinase II/antagonistas & inibidores , Técnicas de Cultura de Células , Regulação para Baixo , Mediadores da Inflamação/metabolismo , Interferon gama/farmacologia , Interleucina-1/análise , Interleucina-6/análise , Proteínas Quinases JNK Ativadas por Mitógeno/análise , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Macrófagos Peritoneais/enzimologia , Masculino , Camundongos , NF-kappa B/análise , NF-kappa B/antagonistas & inibidores , Óxido Nítrico/antagonistas & inibidores , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo II/análise , Espécies Reativas de Oxigênio/análise , Espécies Reativas de Oxigênio/metabolismo , Fator de Transcrição AP-1/análise , Fator de Transcrição AP-1/antagonistas & inibidores , Transcrição Gênica/efeitos dos fármacosRESUMO
BACKGROUND: Hemorrhagic shock and resuscitation (HSR) induces pulmonary inflammation that leads to acute lung injury. Carbon monoxide (CO), a by-product of heme catalysis, was shown to have potent cytoprotective and anti-inflammatory effects. The aim of this study was to examine the effects of CO inhalation at low concentration on lung injury induced by HSR in rats. METHODS: Rats were subjected to HSR by bleeding to achieve mean arterial pressure of 30 mm Hg for 60 minutes followed by resuscitation with shed blood and saline as needed to restore blood pressure. HSR animals were either maintained in room air or were exposed to CO at 250 ppm for 1 hour before and 3 hours after HSR. RESULTS: HSR caused an increase in the DNA binding activity of nuclear factor-kappaB and activator protein-1 in the lung followed by the up-regulation of pulmonary gene expression of tumor necrosis factor-alpha, inducible nitric oxide synthase, and interleukin (IL)-10. HSR also resulted in an increase in myeloperoxidase activity and wet weight to dry weight ratio in the lung, and more prominent histopathologic changes including congestion, edema, cellular infiltration, and hemorrhage. In contrast, CO inhalation significantly ameliorated these inflammatory events as judged by fewer histologic changes, less up-regulation of inflammatory mediators, and less activation of nuclear factor-kappaB and activator protein-1. Interestingly, the protective effects against lung injury afforded by CO were associated with further increases in mRNA expression of IL-10 in the lung. CONCLUSIONS: These findings suggest that inhaled CO at a low concentration ameliorated HSR-induced lung injury and attenuated inflammatory cascades by up-regulation of anti-inflammatory IL-10.
Assuntos
Lesão Pulmonar Aguda/prevenção & controle , Monóxido de Carbono/uso terapêutico , Choque Hemorrágico/prevenção & controle , Lesão Pulmonar Aguda/patologia , Administração por Inalação , Animais , Monóxido de Carbono/análise , Carboxihemoglobina/análise , Modelos Animais de Doenças , Pulmão/química , Pulmão/patologia , Masculino , Óxido Nítrico Sintase Tipo II/análise , Peroxidase/análise , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição AP-1/análise , Fator de Necrose Tumoral alfa/análiseRESUMO
OBJECTIVE: There is remarkable controversy over the processes driving abdominal aneurysm growth. The inherent limitations of animal and human studies hamper elucidation of the key inflammatory and proteolytic processes. Human data are largely derived from surgical specimens that typically reflect the final stages of the disease process and thus do not allow distinction between primary and secondary processes. Clear epidemiologic and genetic associations between abdominal aortic aneurysm (AAA) and popliteal artery aneurysms (PAA) suggest that that these two pathologies share common grounds. On this basis, we reasoned that information of corresponding and discordant processes in these aneurysms might provide critical clues on the processes that are crucial for aneurysm progression. METHODS: Messenger RNA (semi-quantitative real-time polymerase chain reaction) and protein analysis (enzyme-linked immunosorbent assay, multiplex, Western blotting), and histology were performed on aneurysm wall samples obtained during elective PAA and AAA repair. Nonaneurysmal aorta tissue from organ donors was included as reference. RESULTS: Messenger RNA and protein analysis showed that PAA and AAA are both characterized by a marked activation of nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1) proinflammatory transcription factors, and hyperexpression of interleukin (IL)-6 and IL-8. Discordant findings were found for other inflammatory markers such as interferon-gamma, interferon-inducible protein 10, tumor necrosis factor-alpha, monocyte chemotactic protein-1, and macrophage inflammatory protein 1alpha and beta, which were all lower in PAA. On the cellular level, both pathologies exhibited profuse infiltration of macrophages, neutrophils, and T-helper cells. Results for B cells, plasma cells, and cytotoxic T cells were discordant, with minimal infiltration of these cell types in PAA. Evaluation of protease expression and activation showed that both conditions are dominated by increased matrix metalloproteinase 8 and 9, and cathepsin K, L and S expression and activation. CONCLUSION: This explorative study characterizes degenerative aneurysmal disease general inflammatory conditions that are dominated by profound activation of the NF-kappaB and AP-1 pathways, hyperexpression of IL-6 and IL-8, and neutrophil involvement. Discordant findings for interferon gamma, cytotoxic T cells, B cells, and plasma cells challenge a critical role for these factors in the process of aneurysm growth. Pharmaceutic strategies targeting the common components in AAA and PAA may prove effective for the stabilization of AAA.
Assuntos
Aneurisma/fisiopatologia , Aorta Abdominal/fisiopatologia , Aneurisma da Aorta Abdominal/fisiopatologia , Artéria Poplítea/fisiopatologia , Idoso , Idoso de 80 Anos ou mais , Aneurisma/genética , Aneurisma/metabolismo , Aneurisma/patologia , Aorta Abdominal/química , Aorta Abdominal/patologia , Aneurisma da Aorta Abdominal/genética , Aneurisma da Aorta Abdominal/metabolismo , Aneurisma da Aorta Abdominal/patologia , Western Blotting , Catepsinas/análise , Colagenases/análise , Citocinas/análise , Progressão da Doença , Ensaio de Imunoadsorção Enzimática , Feminino , Regulação da Expressão Gênica , Humanos , Mediadores da Inflamação/análise , Macrófagos/patologia , Masculino , Pessoa de Meia-Idade , NF-kappa B/análise , Infiltração de Neutrófilos , Artéria Poplítea/química , Artéria Poplítea/patologia , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T Auxiliares-Indutores/patologia , Fator de Transcrição AP-1/análiseRESUMO
In this study, a novel member of BTB-kelch proteins, named KBTBD7, was cloned from a human embryonic heart cDNA library. The cDNA of KBTBD7 is 3,008 bp long and encodes a protein product of 684 amino acids (77.2 kD). This protein is highly conserved in evolution across different species. Western blot analysis indicates that a 77 kD protein specific for KBTBD7 is wildly expressed in all embryonic tissues examined. In COS-7 cells, KBTBD7 proteins are localized to the cytoplasm. KBTBD7 is a transcription activator when fused to GAL4 DNA-binding domain. Deletion analysis indicates that the BTB domain and kelch repeat motif are main regions for transcriptional activation. Overexpression of KBTBD7 in MCF-7 cells activates the transcriptional activities of activator protein-1 (AP-1) and serum response element (SRE), which can be relieved by siRNA. These results suggest that KBTBD7 proteins may act as a new transcriptional activator in mitogen-activated protein kinase (MAPK) signaling. [BMB reports 2010; 43(1): 17-22].
Assuntos
Elemento de Resposta Sérica , Transativadores/metabolismo , Fator de Transcrição AP-1/metabolismo , Ativação Transcricional , Sequência de Aminoácidos , Animais , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Evolução Molecular , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Dados de Sequência Molecular , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Transativadores/análise , Transativadores/genética , Fator de Transcrição AP-1/análise , Fator de Transcrição AP-1/genéticaRESUMO
Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) and lymphocyte-rich classical Hodgkin lymphoma (LRCHL), although clinically and morphologically similar, differ biologically and in prognosis. Immunolabeling of Reed-Sternberg (RS) cells in LRCHL and lymphocytic and/or histiocytic variants (L&H cells) in NLPHL is often required to help distinguish between the 2 variants. Our aim was to evaluate fascin (a distinct 55-kd actin-bundling protein) and JunB (an activator protein-1 family transcription factor) to differentiate NLPHL from LRCHL. A total of 35 archival cases of NLPHL (n = 24) and LRCHL (n = 11) from adults and children were studied. Slides were reviewed for all cases and clinical, morphologic, and immunohistochemical features were evaluated. Each case was immunostained for fascin and JunB, and immunoreactivity of RS cells, L&H cells, and background lymphocytes were recorded. Whereas occasional L&H cells were weakly positive for fascin in 3 out of 24 (12.5%) cases of NLPHL, RS cells in LRCHL were positive for fascin in 11 out of 11 (100%) cases with a strong cytoplasmic staining pattern. JunB was positive in 10 out of 24 (41.7%) of NLPHL cases, and 11 out of 11 (100%) of LRCHL cases, showing a stippled and/or diffuse nuclear staining pattern. In addition to L & H Cells, JunB also stained small background lymphocytes, particularly in areas of progressively transformed germinal centers of NLPHL. Either stains when tested alone, if negative, or with rare L&H cell weak positivity for fascin, is indicative of NLPHL. The L&H cells of NLPHL cases were negative for concomitant staining in 24 out of 24 (100%) cases. Concomitant positive staining of classic RS cells for fascin and JunB was found in 11 out of 11 (100%) of LRCHL cases. Although fascin positivity alone supports the diagnosis of LRCHL, concomitant positivity offers stronger support and is less likely to lead to a false conclusion if aberrant fascin staining were to be encountered in a case of NLPHL. Staining for fascin and JunB provides a basis for distinguishing NLPHL from LRCHL and offers an alternative to other antibody profiles.
Assuntos
Proteínas de Transporte/análise , Doença de Hodgkin/diagnóstico , Imuno-Histoquímica/métodos , Proteínas dos Microfilamentos/análise , Valor Preditivo dos Testes , Fator de Transcrição AP-1/análise , Biomarcadores Tumorais/análise , Criança , Diagnóstico Diferencial , Feminino , Doença de Hodgkin/classificação , Doença de Hodgkin/patologia , Humanos , Linfócitos/patologia , Masculino , Pessoa de Meia-Idade , Células de Reed-Sternberg/patologiaRESUMO
To identify the molecular background of esophageal cancer, we conducted a proteomics study using an antibody microarray consisting of 725 antibodies and surgical specimens from three cases. The microarray analysis identified 24 proteins with aberrant expression in esophageal cancer compared with the corresponding normal mucosa. The overexpression of 14 of the 24 proteins was validated by western blotting analysis of the same samples. These 14 proteins were examined by immunohistochemistry, in which nine proteins showed consistent results with those obtained by western blotting. Among the nine proteins, seven were localized in tumor cells, and two in infiltrating cells. The former included proteins associated with mitotic checkpoint control and the nuclear factor (NF)-kappaB pathway. Although mitotic checkpoint gene products (budding uninhibited by benzidazoles 1 homolog beta (BubR1) and mitotic arrest deficient-like 1 (Mad2)) have previously been reported to be involved in esophageal cancer, the association of NF-kappaB-activating kinase, caspase 10, and activator protein-1 with esophageal cancer has not been previously reported. These proteins play a key role in the NF-kappaB pathway, and NF-kappaB is a signal transduction factor that has emerged as an important modulator of altered gene programs and malignant phenotype in the development of cancer. The association of these proteins with esophageal cancer may indicate that mitotic checkpoint gene products and NF-kappaB play an important part in the carcinogenesis of esophageal cancer.
Assuntos
Carcinoma de Células Escamosas/química , Proteínas de Ciclo Celular/análise , Neoplasias Esofágicas/química , Esôfago/metabolismo , NF-kappa B/análise , Proteínas de Neoplasias/análise , Análise Serial de Proteínas , Western Blotting , Proteínas de Ligação ao Cálcio/análise , Caspase 10/análise , Esôfago/patologia , Feminino , Perfilação da Expressão Gênica , Humanos , Técnicas Imunoenzimáticas , Proteínas Mad2 , Masculino , Pessoa de Meia-Idade , NF-kappa B/genética , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Serina-Treonina Quinases/análise , Proteômica/métodos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Repressoras/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição AP-1/análiseRESUMO
BACKGROUND AND PURPOSE: alpha-Humulene and trans-caryophyllene are plant sesquiterpenes with pronounced anti-inflammatory properties. Here, we evaluated the effects of these compounds in an experimental model of airways allergic inflammation. EXPERIMENTAL APPROACH: Female BALB/c mice, sensitized to and challenged with ovalbumin received daily alpha-humulene or trans-caryophyllene (50 mg.kg(-1), orally) or alpha-humulene (1 mg.mL(-1), by aerosol) as either a preventive (for 22 days) or therapeutic (from the 18th to the 22nd day) treatment. Dexamethasone or budesonide was used as a positive control drug. Inflammation was determined on day 22 post-immunization by leukocyte recruitment, interleukin-5 (IL-5), CCL11, interferon-gamma (IFN-gamma) and leukotriene (LT)B(4) levels in bronchoalveolar lavage fluid (BALF). In addition, transcription factors [nuclear factor kappaB (NF-kappaB), activator protein 1 (AP-1)] and P-selectin in lung tissue were measured by immunohistochemistry and mucus secretion by histochemistry. KEY RESULTS: Preventive or therapeutic treatments with alpha-humulene, but not with trans-caryophyllene, significantly reduced the eosinophil recruitment to the BALF. In addition, alpha-humulene recovery INF-gamma and reduced the IL-5, CCL11 and LTB(4) levels in BALF, as well as the IL-5 production in mediastinal lymph nodes (in vitro assay). Furthermore, alpha-humulene decreased the NF-kB and the AP-1 activation, the expression of P-selectin and the increased mucus secretion in the lung. CONCLUSIONS AND IMPLICATIONS: alpha-Humulene, given either orally or by aerosol, exhibited marked anti-inflammatory properties in a murine model of airways allergic inflammation, an effect that seemed to be mediated via reduction of inflammatory mediators, adhesion molecule expression and transcription factors activation.
Assuntos
Anti-Inflamatórios/farmacologia , Pulmão/efeitos dos fármacos , Hipersensibilidade Respiratória/tratamento farmacológico , Sesquiterpenos/farmacologia , Administração Oral , Aerossóis , Animais , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/uso terapêutico , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/imunologia , Quimiocina CCL11/análise , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Eosinófilos/efeitos dos fármacos , Feminino , Imuno-Histoquímica , Interferon gama/análise , Interleucina-5/análise , Leucotrieno B4/análise , Pulmão/metabolismo , Pulmão/patologia , Camundongos , Camundongos Endogâmicos BALB C , Sesquiterpenos Monocíclicos , Monócitos/efeitos dos fármacos , NF-kappa B/análise , NF-kappa B/metabolismo , Neutrófilos/efeitos dos fármacos , Ovalbumina/administração & dosagem , Ovalbumina/imunologia , Hipersensibilidade Respiratória/patologia , Sesquiterpenos/administração & dosagem , Sesquiterpenos/uso terapêutico , Fator de Transcrição AP-1/análise , Fator de Transcrição AP-1/metabolismoRESUMO
Pharmaceuticals are regularly released into the environment; in particular non-steroidal anti-inflammatory drugs (NSAIDs) and antibiotics. Erythromycin, naproxen, furosemide and atenolol are reported to be stable for up to 1 year in the environment, which increases the risk for accumulation. In the present study we have measured the occurrence and concentration of pharmaceuticals in river Viskan (Jössabron) downstream of a sewage treatment plant in Borås, Sweden. Pharmaceuticals and water samples were tested for potential human risk by evaluating inflammatory responses (NF-kappaB and AP-1) using human T24 bladder epithelial cells and Jurkat T-cells. NF-kappaB activity in T24 cells was significantly reduced by all NSAIDs analysed (diclofenac, ketoprofen, naproxen, ibuprophen and dextropropoxyphene), but also by trimethoprim, using environmentally relevant concentrations. NF-kappaB and AP-1 activation was further analysed in response to water samples collected from different locations in Sweden. Dose-dependent down-regulation of AP-1 activity in Jurkat cells was observed at all locations. At two locations (Jössabron and Almenäs) down-regulation of NF-kappaB was observed. In contrast, the NF-kappaB response was potentiated by exposure to water from both locations following activation of NF-kappaB by treatment with heat-killed Escherichia coli. To determine the involvement of pharmaceuticals in the responses, T24 cells were exposed to the pharmaceutical mixture, based on the determined levels at Jössabron. This resulted in reduction of the NF-kappaB response following exposure to the pharmaceutical mixture alone while no potentiation was observed when cells were co-exposed to heat killed E. coli and pharmaceuticals. The obtained results demonstrate that the identified pharmaceuticals affect the inflammatory responses and furthermore indicate the presence of unknown substance(s) with the ability to potentiate inflammatory responses.
Assuntos
NF-kappa B/análise , Preparações Farmacêuticas/análise , Fator de Transcrição AP-1/análise , Poluentes Químicos da Água/farmacologia , Xenobióticos/farmacologia , Linhagem Celular Tumoral , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Inflamação/induzido quimicamente , Inflamação/metabolismo , Células Jurkat , Rios , Extração em Fase Sólida , Suécia , Poluentes Químicos da Água/análise , Xenobióticos/análiseRESUMO
Resveratrol (3,5,4'-trihydroxystilbene) is a natural polyphenol that presents various physiological activities. It has been reported that the methylated derivatives of resveratrol show better potential antifungal and antiproliferative activities than resveratrol. In the present study, we investigated the inhibitory effect of 3,5,4'-trimethoxy-trans-stilbene (MR-3), a methylated derivative of resveratrol, on the invasion of A549 cells (a human lung adenocarcinoma cell line). We found that treatment with MR-3 at the concentration of 5 muM resulted in antiadhesive, antimigratory, and antiinvasive activities on A549 cells through the suppression of matrix metalloproteinase (MMP)-2 protein expression and transcriptional levels in a time-dependent manner. The suppression of MMP-2 expression by MR-3 led to an inhibition of A549 cell invasion by inactivating phosphorylation of SAPK/c-Jun N-terminal kinase (JNK) and p38 MAPK signaling pathways. A time-dependent inhibition of protein levels for p65, c-Jun, and c-Fos in the nucleus by MR-3 treatment was also observed. In conclusion, our data demonstrate that the antiinvasive effects of MR-3 on A549 cells are likely mediated through the inhibition of phosphorylation of JNK and p38, as well as a reduction in the protein levels of nuclear factor-kappa B (NF-kappaB) and activator protein-1 (AP-1) in the nucleus, ultimately leading to downregulation of MMP-2 expression.