RESUMEN

Growth Hormone (GH) plays crucial roles in mammary gland development and growth, and its upregulation has been associated with breast cancer promotion and/or progression. To ascertain how high GH levels could promote mammary tissue oncogenic transformation, morphological characteristics and the expression of receptors involved in mammary growth, development and cancer, and of mitogenic mediators were analyzed in the mammary gland of virgin adult transgenic mice that overexpress GH. Whole mounting and histologic analysis evidenced that transgenic mice exhibit increased epithelial ductal elongation and enlarged ducts along with deficient branching and reduced number of alveolar structures compared to wild type mice. The number of differentiated alveolar structures was diminished in transgenic mice while the amount of terminal end buds (TEBs) did not differ between both groups of mice. GH, insulin-like growth factor 1 (IGF1) and GH receptor mRNA levels were augmented in GH-overexpressing mice breast tissue, as well as IGF1 receptor protein content. However, GH receptor protein levels were decreased in transgenic mice. Fundamental receptors for breast growth and development like progesterone receptor and epidermal growth factor receptor were also increased in mammary tissue from transgenic animals. In turn, the levels of the proliferation marker Ki67, cFOS and Cyclin D1 were increased in GH-overexpressing mice, while cJUN expression was decreased and cMYC did not vary. In conclusion, prolonged exposure to high GH levels induces morphological and molecular alterations in the mammary gland that affects its normal development. While these effects would not be tumorigenic per se, they might predispose to oncogenic transformation.

Asunto(s)

Proteínas Portadoras/genética , Hormona del Crecimiento/genética , Factor I del Crecimiento Similar a la Insulina/genética , Glándulas Mamarias Animales/anomalías , Animales , Animales Modificados Genéticamente , Biomarcadores/metabolismo , Femenino , Hormona del Crecimiento/metabolismo , Glándulas Mamarias Animales/metabolismo , Ratones , Proteínas Proto-Oncogénicas c-jun/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo

RESUMEN

The prediction of regulatory single nucleotide polymorphisms (rSNPs) in proximal promoters of disease-related genes could be a useful tool for personalized medicine in both patient stratification and customized therapy. Using our previously reported method of rSNPs prediction (currently a software called SNPClinic v.1.0) as well as with PredictSNP tool, we performed in silico prediction of regulatory SNPs in the antimicrobial peptide human ß-defensin 1 gene in three human cell lines from 1,000 Genomes Project (1kGP), namely A549 (epithelial cell line), HL-60 (neutrophils) and TH 1 (lymphocytes). These predictions were run in a proximal pseudo-promoter comprising all common alleles on each polymorphic site according to the 1,000 Genomes Project data (1kGP: ALL). Plasmid vectors containing either the major or the minor allele of a putative rSNP rs5743417 (categorized as regulatory by SNPClinic and confirmed by PredictSNP) and a non-rSNP negative control were transfected to lung A549 human epithelial cell line. We assessed functionality of rSNPs by qPCR using the Pfaffl method. In A549 cells, minor allele of the SNP rs5743417 G→A showed a significant reduction in gene expression, diminishing DEFB1 transcription by 33% when compared with the G major allele (p-value = .03). SNP rs5743417 minor allele has high frequency in Gambians (8%, 1kGP population: GWD) and Afro-Americans (3.3%, 1kGP population: ASW). This SNP alters three transcription factors binding sites (TFBSs) comprising SREBP2 (sterols and haematopoietic pathways), CREB1 (cAMP, insulin and TNF pathways) and JUND (apoptosis, senescence and stress pathways) in the proximal promoter of DEFB1. Further in silico analysis reveals that this SNP also overlaps with GS1-24F4.2, a lincRNA gene complementary to the X Kell blood group related 5 (XKR5) mRNA. The potential clinical impact of the altered constitutive expression of DEFB1 caused by rSNP rs5743417 in DEFB1-associated diseases as tuberculosis, COPD, asthma, cystic fibrosis and cancer in African and Afro-American populations deserves further research.

Asunto(s)

Polimorfismo de Nucleótido Simple/genética , Proteínas Citotóxicas Formadoras de Poros/genética , Regiones Promotoras Genéticas/genética , beta-Defensinas/genética , Células A549 , Negro o Afroamericano/genética , Sitios de Unión , Población Negra/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Regulación de la Expresión Génica/genética , Humanos , Linfocitos/metabolismo , Neutrófilos/metabolismo , Proteínas Proto-Oncogénicas c-jun/genética , ARN Mensajero/genética , Proteína 2 de Unión a Elementos Reguladores de Esteroles/genética

RESUMEN

Macrophage galactose-C type lectin (MGL)1 receptor is involved in the recognition of Trypanosoma cruzi (T. cruzi) parasites and is important for the modulation of the innate and adaptive immune responses. However, the mechanism by which MGL1 promotes resistance to T. cruzi remains unclear. Here, we show that MGL1 knockout macrophages (MGL1-/- Mφ) infected in vitro with T. cruzi were heavily parasitized and showed decreased levels of reactive oxygen species (ROS), nitric oxide (NO), IL-12 and TNF-α compared to wild-type macrophages (WT Mφ). MGL1-/- Mφ stimulated in vitro with T. cruzi antigen (TcAg) showed low expression of TLR-2, TLR-4 and MHC-II, which resulted in deficient splenic cell activation compared with similar co-cultured WT Mφ. Importantly, the activation of p-ERK1/2, p-c-Jun and p-NF-κB p65 were significantly reduced in MGL1-/- Mφ exposed to TcAg. Similarly, procaspase 1, caspase 1 and NLRP3 inflammasome also displayed a reduced expression that was associated with low IL-ß production. Our data reveal a previously unappreciated role for MGL1 in Mφ activation through the modulation of ERK1/2, c-Jun, NF-κB and NLRP3 signaling pathways, and to the development of protective innate immunity against experimental T. cruzi infection.

Asunto(s)

Asialoglicoproteínas/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Lectinas Tipo C/metabolismo , Activación de Macrófagos , Proteínas de la Membrana/metabolismo , FN-kappa B/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Animales , Antígenos de Protozoos/metabolismo , Citocinas/biosíntesis , Mediadores de Inflamación/metabolismo , Linfocitos/inmunología , Masculino , Ratones Endogámicos C57BL , Óxido Nítrico/metabolismo , Parásitos/metabolismo , Proteínas Protozoarias/metabolismo , Estallido Respiratorio , Trypanosoma cruzi/inmunología

RESUMEN

Immediate early genes (IEGs) are a fundamental element in the way we respond and adapt to a variety of stimuli. We have recently reported that IEG response, as measured by c-Fos expression, is different between rodents and primates. Here, we further extend this analysis by assessing the expression of c-Jun, one of the main complements of c-Fos, under the same stimulation protocol. For this, we investigated the immunohistochemical expression of c-Jun (and compared with that previously shown for c-Fos) after stimulation with pentylenetetrazol in the cingulate gyrus, motor cortex, piriform cortex, inferior temporal cortex, and visual cortex of rats and marmosets (Callithrix jacchus), both male and female. Overall the immunohistochemical expression of c-Jun was more intense but remained elevated for a shorter duration in marmosets as compared to rats. These results are in contrast to what we had previously shown for c-Fos. Furthermore, in terms of the temporal profile, c-Fos and c-Jun expression occurred in a complementary manner in rats-the peak of c-Fos expression coincided with low levels of c-jun expression-and in a superimposed manner in marmosets-the peak of c-Fos expression coincided with the peak of c-Jun expression. Since Fos proteins may form dimers with Jun proteins and together control late gene expressions in the cell nucleus, this different expression profile between primates and rodents may bear meaningful impact for how the nervous system reacts and adapts to stimulation.

Asunto(s)

Encéfalo/metabolismo , Pentilenotetrazol/farmacología , Proteínas Proto-Oncogénicas c-jun/metabolismo , Animales , Callithrix , Femenino , Genes Inmediatos-Precoces , Giro del Cíngulo/metabolismo , Masculino , Proteínas Proto-Oncogénicas c-fos/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Wistar

RESUMEN

OBJECTIVE: Chronic stress-induced oxidative damage and protease synthesis cause a loss of extracellular matrix components promoting human skin ageing. The administration of antioxidant compounds, such as those observed in olive oil, may attenuate stress-induced ageing signs in human skin. Thus, the aim of this study was to investigate the effect of olive oil administration in ex vivo stressed human skin. METHODS: Explants of human skin were treated with high levels of epinephrine (as observed in stressed patients) and olive oil in medium for 13 days. Cultures treated with medium alone were used as controls. RESULTS: Olive oil reversed the high epinephrine level-induced reduction in epidermis and dermis thickness and collagen fibre content in ex vivo human skin. The increase in the production of reactive oxygen species (ROS) and malondialdehyde levels (an index of lipid peroxidation) promoted by high levels of epinephrine were also attenuated by olive oil in ex vivo human skin. Moreover, olive oil was able to reverse the high epinephrine level-induced increase in extracellular signal-related kinase 1/2 (ERK 1/2) and c-JUN (a major component of transcription factor AP-1) phosphorylation and protein matrix metalloproteinase-2 (MMP-2) expression in ex vivo human skin. CONCLUSION: Olive oil attenuates stress-induced ageing signs (thinner dermis and collagen fibre loss) in ex vivo human skin by reducing MMP-2 expression, ROS production, and ERK 1/2 and c-JUN phosphorylation.

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OBJECTIF: Le dommage oxydatif chronique induit par le stress et la synthèse de protéases entraînent une dégradation des composants de la matrice extracellulaire favorisant le vieillissement de la peau humaine. L'administration de composés antioxydants, tels que ceux observés dans l'huile d'olive, peut atténuer les signes de vieillissement induits par le stress sur la peau humaine. L'objectif de cette étude était donc d'étudier l'effet de l'administration d'huile d'olive sur une peau humaine stressée ex vivo. MÉTHODES: Des explants de peau humaine ont été traités avec des niveaux élevés d'épinéphrine (comme observé chez les patients stressés) et d'huile d'olive dans un milieu pendant 13 jours. Les cultures traitées avec le milieu seul ont été utilisées comme témoins. RÉSULTATS: L'huile d'olive a renversé la réduction de l'épaisseur de l'épiderme et du derme et des fibres de collagène, induite par le niveau élevé d'épinéphrine dans la peau humaine ex vivo. L'augmentation de la production d'espèces réactives de l'oxygène (ROS) et de malondialdéhyde (indice de péroxydation lipidique) favorisée par des taux élevés d'épinéphrine a également été atténuée par l'huile d'olive dans la peau humaine ex vivo. En outre, l'huile d'olive a pu inverser l'augmentation, induite par le niveau élevé d'épinéphrine, de la phosphorylation de la kinase liée au signal extracellulaire 1/2 (ERK 1/2) et c-JUN (un composant majeur du facteur de transcription AP-1) et de la expression de la métalloprotéinase matricielle protéique-2 (MMP-2) dans la peau humaine ex vivo. CONCLUSION: L'huile d'olive atténue les signes du vieillissement induit par le stress (mincissement du derme et perte de fibres de collagène) dans la peau humaine ex vivo en réduisant l'expression de MMP-2, la production de ROS et la phosphorylation de ERK 1/2 et de cJUN.

Asunto(s)

Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Aceite de Oliva/farmacología , Estrés Oxidativo , Proteínas Proto-Oncogénicas c-jun/antagonistas & inhibidores , Envejecimiento de la Piel/efectos de los fármacos , Piel/efectos de los fármacos , Enfermedad Crónica , Humanos , Técnicas In Vitro , Fosforilación , Piel/enzimología , Piel/metabolismo

RESUMEN

c-Jun is a member of the early mammalian transcriptional regulators belonging to the AP-1 family, which participates in a wide range of cellular processes such as proliferation, apoptosis, tumorigenesis, and differentiation. Despite its established role in cell survival upon stress, its participation in the stress response induced by bacterial infections has been poorly investigated. To study the potential role of c-Jun in this context we choose the widely studied α-toxin produced by Staphylococcus aureus, a pore-forming toxin that is a critical virulence factor in the pathogenesis of these bacteria. We analyzed the effect of α-toxin treatment in the activation, expression, and protein levels of c-Jun in A549 lung epithelial cells. Furthermore, we explored the role of c-Jun in the cellular fate after exposure to α-toxin. Our results show that staphylococcal α-toxin per se is able to activate c-Jun by inducing phosphorylation of its Serine 73 residue. Silencing of the JNK (c-Jun N-terminal Kinase) signaling pathway abrogated most of this activation. On the contrary, silencing of the ERK (Extracellular Signal-Regulated Kinase) pathway exacerbated this response. Intriguingly, while the exposure to α-toxin induced a marked increase in the levels of c-Jun transcripts, c-Jun protein levels noticeably decreased in the same time-frame as a consequence of active proteolytic degradation through the proteasome-dependent pathway. In addition, we established that c-Jun promoted cell survival when cells were challenged with α-toxin. Similarly, c-Jun phosphorylation was also induced in cells upon intoxication with the cytolysin produced by Vibrio cholerae in a JNK-dependent manner, suggesting that c-Jun-JNK axis would be a conserved responsive cellular pathway to pore-forming toxins. This study contributes to understanding the role of the multifaceted c-Jun proto-oncoprotein in cell response to bacterial pore-forming toxins, positioning it as a relevant component of the complex early machinery mounted to deal with staphylococcal infections.

Asunto(s)

Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/toxicidad , Células Epiteliales/efectos de los fármacos , Proteínas Hemolisinas/metabolismo , Proteínas Hemolisinas/toxicidad , Pulmón/efectos de los fármacos , Proteínas Proto-Oncogénicas c-jun/metabolismo , Células A549 , Anexina A5/farmacología , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Humanos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Perforina , Fosforilación , Propidio/farmacología , Complejo de la Endopetidasa Proteasomal/metabolismo , Transducción de Señal , Staphylococcus aureus/metabolismo , Vibrio cholerae/metabolismo

RESUMEN

Usurpation of the host's signalling pathways is a common strategy employed by viruses to promote their successful replication. Here we show that infection with the orthopoxvirus vaccinia virus (VACV) leads to sustained stimulation of c-Jun activity during the entire infective cycle. This stimulation is temporally regulated through MEK/ERK or MKK/JNK pathways, i.e. during the early/mid phase (1 to 6 hpi) and in the late phase (9 to 24 hpi) of the infective cycle, respectively. As a transcriptional regulator, upon infection with VACV, c-Jun is translocated from the cytoplasm to the nucleus, where it binds to the AP-1 DNA sequence found at the promoter region of its target genes. To investigate the role played by c-Jun during VACV replication cycle, we generated cell lines that stably express a c-Jun-dominant negative (DNc-Jun) mutation. Our data revealed that c-Jun is required during early infection to assist with viral DNA replication, as demonstrated by the decreased amount of viral DNA found in the DNc-Jun cells. We also demonstrated that c-Jun regulates the expression of the early growth response gene (egr-1), a gene previously shown to affect VACV replication mediated by MEK/ERK signalling. VACV-induced stimulation of the MKK/JNK/JUN pathway impacts viral dissemination, as we observed a significant reduction in both viral yield, during late stages of infection, and virus plaque size. Collectively, our data suggest that, by modulating the host's signalling pathways through a common target such as c-Jun, VACV temporally regulates its infective cycle in order to successfully replicate and subsequently spread.

Asunto(s)

Quinasas MAP Reguladas por Señal Extracelular/metabolismo , MAP Quinasa Quinasa 4/metabolismo , Quinasas Quinasa Quinasa PAM/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Virus Vaccinia/fisiología , Animales , Línea Celular , ADN Viral , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/genética , Fibroblastos/virología , Regulación Enzimológica de la Expresión Génica/fisiología , Regulación Viral de la Expresión Génica/fisiología , MAP Quinasa Quinasa 4/genética , Quinasas Quinasa Quinasa PAM/genética , Ratones , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Fosforilación , Proteínas Proto-Oncogénicas c-jun/genética , Replicación Viral

RESUMEN

Focal adhesion kinase (FAK) has emerged as a mediator of mechanotransduction in cardiomyocytes, regulating gene expression during hypertrophic remodeling. However, how FAK signaling is relayed onward to the nucleus is unclear. Here, we show that FAK interacts with and regulates myocyte enhancer factor 2 (MEF2), a master cardiac transcriptional regulator. In cardiomyocytes exposed to biomechanical stimulation, FAK accumulates in the nucleus, binds to and upregulates the transcriptional activity of MEF2 through an interaction with the FAK focal adhesion targeting (FAT) domain. In the crystal structure (2.9 Å resolution), FAT binds to a stably folded groove in the MEF2 dimer, known to interact with regulatory cofactors. FAK cooperates with MEF2 to enhance the expression of Jun in cardiomyocytes, an important component of hypertrophic response to mechanical stress. These findings underscore a connection between the mechanotransduction involving FAK and transcriptional regulation by MEF2, with potential relevance to the pathogenesis of cardiac disease.

Asunto(s)

Quinasa 1 de Adhesión Focal/química , Mecanotransducción Celular , Miocitos Cardíacos/metabolismo , Proteínas Proto-Oncogénicas c-jun/química , Transcripción Genética , Secuencias de Aminoácidos , Animales , Animales Recién Nacidos , Sitios de Unión , Línea Celular , Núcleo Celular/metabolismo , Clonación Molecular , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Quinasa 1 de Adhesión Focal/genética , Quinasa 1 de Adhesión Focal/metabolismo , Expresión Génica , Regulación de la Expresión Génica , Cinética , Factores de Transcripción MEF2/química , Factores de Transcripción MEF2/genética , Factores de Transcripción MEF2/metabolismo , Ratones , Modelos Moleculares , Miocitos Cardíacos/citología , Cultivo Primario de Células , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Proteínas Proto-Oncogénicas c-jun/genética , Proteínas Proto-Oncogénicas c-jun/metabolismo , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo

RESUMEN

OBJECTIVE: Oral cancer may be preceded by potentially malignant lesions, and smoking is a risk factor. Oral leukoplakia (OL), which is the most common among these lesions, is defined by the World Health Organization as "a white plaque of questionable risk having excluded known diseases or disorders that carry no increased risk for cancer." Thus, OL is a clinical diagnosis used to designate oral white lesions, which are histologically represented by hyperkeratosis associated or not associated with epithelial dysplasia. It is known that c-Jun and pc-Jun have a role in cell proliferation and that p27 is decreased during carcinogenesis; thus, the aim of this study was to investigate whether these proteins are differently expressed in OL in smokers and never-smokers. STUDY DESIGN: Seventy-three cases diagnosed as OL were selected and divided into four groups according to the presence or absence of dysplasia and patients' smoking status (smokers: 39 cases, 24 dysplastic; never-smokers: 34 cases, 20 dysplastic). The immunoexpressions of c-Jun, pc-Jun, and p27 were evaluated. RESULTS: A significant correlation between smoking condition and the percentages of c-Jun (P = .0356) and pc-Jun (P = .0216) was found and was more intense in cases that underwent malignant transformation (6/47). CONCLUSIONS: Smoking habits may be linked to the expression of proteins directly associated with cell cycle progression.

Asunto(s)

Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Leucoplasia Bucal/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Fumar/efectos adversos , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Factores de Riesgo

RESUMEN

The injured skin produces a number of mediators that directly or indirectly modulate cell chemotaxis, migration, proliferation, and angiogenesis. Components of the kinin pathway including the kinin B1 receptor (B1R) have been found to occur in the human skin, but information about its role on keratinocyte biology is still scarce. Our aim was to determine whether stimulation of B1R causes the secretion of IL-4 and/or VEGF from human keratinocytes and to evaluate the role of the B1R agonist Lys-des[Arg(9)]bradykinin and IL-4 on various stages of angiogenesis, such as cell migration, proliferation, and release of metalloproteases. By using ELISA and Western blotting, we showed that HaCaT keratinocytes stimulated with the B1R agonist release IL-4 and VEGF. Stimulation of B1R also caused transient c-JunN-terminal kinase phosphorylation and JunB nuclear translocation, transcription factor that regulates IL-4 expression. The 3D-angiogenesis assay, performed on spheroids of EA.hy923 endothelial cells embedded in a collagen matrix, showed that their cumulative sprout area increased significantly following stimulation with either IL-4 or B1R agonist. Furthermore, these ligands produced significant endothelial cell migration and release of metalloproteases 2 and 9, but did not increase endothelial cell proliferation as measured by 5-bromo-2'-deoxyuridine incorporation. Our results provide experimental evidence that establishes IL-4 and B1R agonist as important angiogenic factors of relevance for skin repair.

Asunto(s)

Células Endoteliales/metabolismo , Interleucina-4/metabolismo , Interleucina-4/farmacología , Queratinocitos/metabolismo , Neovascularización Fisiológica/fisiología , Receptor de Bradiquinina B1/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Proteínas Angiogénicas/farmacología , Línea Celular , Movimiento Celular , Proliferación Celular , Medios de Cultivo Condicionados/farmacología , Activación Enzimática , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Calidina/análogos & derivados , Calidina/farmacología , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-jun , Interferencia de ARN , ARN Interferente Pequeño/genética , Receptor de Bradiquinina B1/agonistas , Receptor de Bradiquinina B1/genética , Transducción de Señal/fisiología , Piel/citología , Piel/metabolismo , Esferoides Celulares

RESUMEN

The carcinogenic potential of urban particulate matter (PM) has been partly attributed to polycyclic aromatic hydrocarbons (PAHs) content, which activates the aryl hydrocarbon receptor (AhR). Here we report the effect of PM with an aerodynamic size of 10 µm (PM10) on the induction of AhR pathway in A549 cells, evaluating its downstream targets CYP1B1, IL-6, IL-8 and c-Jun. Significant increases in CYP1B1 protein and enzyme activity; IL-6 and IL-8 secretion and c-Jun protein were found in response to PM10. The formation of PAH-DNA adducts was also detected. The involvement of AhR pathway was confirmed with Resveratrol as AhR antagonist, which reversed CYP1B1 and c-Jun induction. Nevertheless, in IL-6 and IL-8 secretion, the Resveratrol was ineffective, suggesting an effect independent of this pathway. Considering the role of c-Jun in oncogenesis, its induction by PM may be contributing to its carcinogenic potential through induction of AhR pathway by PAHs present in PM10.

Asunto(s)

Contaminantes Atmosféricos/toxicidad , Material Particulado/toxicidad , Hidrocarburos Policíclicos Aromáticos/toxicidad , Proteínas Proto-Oncogénicas c-jun/metabolismo , Línea Celular Tumoral , Ciudades , Citocromo P-450 CYP1B1/metabolismo , Aductos de ADN/metabolismo , Humanos , Interleucina-6/metabolismo , Interleucina-8/metabolismo , México , Hidrocarburos Policíclicos Aromáticos/metabolismo , Receptores de Hidrocarburo de Aril/antagonistas & inhibidores , Receptores de Hidrocarburo de Aril/metabolismo , Resveratrol , Estilbenos/farmacología

RESUMEN

In this study, we investigated the molecular mechanism of doxorubicin (dxr)-induced cytotoxicity on Jurkat cells - a model cell of human acute lymphoblastic leukemia - under normoxic (20% O2) and hypoxic (5% O2) conditions. Using in-cell western analysis, immunofluorescence, flow cytometry analysis, and biochemical inhibitors, we evaluated several oxidative stress (OS) and cell death markers. It was found that dxr (5-100 µmol/l) induced apoptosis by OS mechanisms involving DNA fragmentation (8-48%), loss of mitochondrial membrane potential (ΔΨm, 33-92%), and H2O2 production (15-42%) under normoxia. In addition, dxr (10 µmol/l) induced activation and/or nuclei translocation of NF-κB (6.6, 1.6-fold increase), p53 (4.3, 3.1 f), c-Jun (9.5, 5.0 f), apoptosis-inducing factor (AIF) (1.9, 3.9 f), caspase-3 (3.7, 1.9 f), overexpression of Parkin (2.1, 1.2 f)/PINK-1 (2.1 f) proteins, and reduced DJ-1 levels by half compared with untreated cells under normoxia, according to immunofluorescence and in-cell western analysis, respectively. In contrast, dxr (10 µmol/l) could not induce apoptosis in Jurkat cells under hypoxia. Effectively, dxr significantly reduced DNA fragmentation (6%), expression levels of cell death (e.g. p53, c-Jun, caspase-3, AIF), and OS (e.g. Parkin) markers, whereas it increased ΔΨm, hypoxia-inducible factor 1-α (HIF-1α, 3.1, 2.3 f), NF-κB (6.8, 2.0 f), and DJ-1 (1.3, 1.0 f) levels. This investigation suggests that dxr might efficiently eliminate acute lymphoblastic leukemia cells by OS-induced apoptosis under normoxic conditions through a minimal completeness of cell death signaling (i.e. mitochondria-caspase-3/AIF-dependent pathways) and through a direct DNA damage process. However, hypoxic conditions may reduce the effectiveness of dxr toxicity.

Asunto(s)

Antibióticos Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Doxorrubicina/farmacología , Mitocondrias/efectos de los fármacos , Acetilcisteína/metabolismo , Factor Inductor de la Apoptosis/metabolismo , Caspasa 3/metabolismo , Hipoxia de la Célula , Humanos , Peróxido de Hidrógeno/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Células Jurkat , Potencial de la Membrana Mitocondrial , Mitocondrias/metabolismo , FN-kappa B/metabolismo , Proteínas Oncogénicas/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras , Proteína Desglicasa DJ-1 , Proteínas Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo

RESUMEN

El fenómeno de la transexualidad es un asunto en el que el peso social, en concreto de los colectivos transexuales, ha sido y sigue siendo crucial en muchos aspectos, desde la progresiva eliminación de la discriminación hasta la influencia para que el poder legislativo se pronuncie. En este artículo de investigación se tratará especialmente una de las reivindicaciones clásicas del colectivo, esto es, el tratamiento sanitario integral de la persona transexual dentro del Sistema Nacional de Salud. En este sentido, se observarán los avances en el desarrollo de un sistema sanitario adecuado para este colectivo, su tratamiento por parte de los distintos ordenamientos jurídicos en España, en general, y en alguna de sus comunidades autónomas con legislaciones más destacables (en especial Andalucía como comunidad autónoma pionera, el País Vasco y la Comunidad Foral de Navarra) y los retos pendientes, haciendo una especial investigación en torno a las sustanciales novedades que ha implantado en este ámbito la publicación de la quinta edición del Manual diagnóstico y estadístico de los trastornos mentales.

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The social weight of transsexual groups has been and continues to be crucial in many aspects regarding transsexuality, from the progressive elimination of discrimination to influence in the legislative branch. This paper especially discusses a classic demand of these groups, comprehensive medical treatment of transsexual people within the National Health System. Thus, progress in the development of an adequate healthcare system for these groups, their treatment in the legal systems of Spain in general and of some of its autonomous communities with more noteworthy laws (especially in Andalusia, an autonomous community that has been pioneering in this regard, as well as the Basque Country and Navarre) and remaining challenges will be observed in this work. The article will also take particular note of the substantial developments that the publication of the Fifth Edition of the Diagnostic and Statistical Manual of Mental Disorders has established in this area.

Asunto(s)

Humanos , Proteínas Proto-Oncogénicas c-jun/genética , Neoplasias Gástricas , Vitamina E/análogos & derivados , Vitamina E/farmacología , Western Blotting , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteínas Proto-Oncogénicas c-jun/análisis , ARN Mensajero/análisis , Tocoferoles , Células Tumorales Cultivadas

RESUMEN

c-Fos is a well-recognized member of the AP-1 (activator protein-1) family of transcription factors. In addition to this canonical activity, we previously showed that cytoplasmic c-Fos activates phospholipid synthesis through a mechanism independent of its genomic AP-1 activity. c-Fos associates with particular enzymes of the lipid synthesis pathway at the endoplasmic reticulum and increases the Vmax of the reactions without modifying the Km values. This lipid synthesis activation is associated with events of differentiation and proliferation that require high rates of membrane biogenesis. Since lipid synthesis also occurs in the nucleus, and different phospholipids have been assigned transcription regulatory functions, in the present study we examine if c-Fos also acts as a regulator of phospholipid synthesis in the nucleus. Furthermore, we examine if c-Fos modulates transcription through its phospholipid synthesis activator capacity. We show that nuclear-localized c-Fos associates with and activates PI4P5K (phosphatidylinositol-4-monophosphate 5-kinase), but not with PI4KIIIß (type IIIß phosphatidylinositol 4-kinase) thus promoting PtdIns(4,5)P2 (phosphatidylinositol 4,5-bisphosphate) formation, which, in turn, promotes transcriptional changes. We propose c-Fos as a key regulator of nuclear PtdIns(4,5)P2 synthesis in response to growth signals that results in c-Fos-dependent transcriptional changes promoted by the newly synthesized lipids.

Asunto(s)

Núcleo Celular/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Transcripción Genética , Regulación hacia Arriba , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Núcleo Celular/efectos de los fármacos , Núcleo Celular/enzimología , Núcleo Celular/ultraestructura , Tamaño del Núcleo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ratones , Células 3T3 NIH , Proteínas Asociadas a Matriz Nuclear/genética , Proteínas Asociadas a Matriz Nuclear/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Transporte de Proteínas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-fos/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-jun/genética , Proteínas Proto-Oncogénicas c-jun/metabolismo , Interferencia de ARN , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transcripción Genética/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos

RESUMEN

Genomic aberrations in the CREBBP (CREB-binding protein - CREBBP or CBP) gene such as point mutations, small insertions or exonic copy number changes are usually associated with Rubinstein-Taybi syndrome (RTs). In this study, the disruption of the CREBBP gene on chromosome 16p13.3, as revealed by CGH-array and FISH, suggests immune dysregulation in a patient with the Rubinstein Taybi syndrome (RTs) phenotype. Further investigation with Western blot techniques demonstrated decreased expression of CREB, NFκB, c-Jun, c-Fos, BCL2 and cMyc in peripheral blood mononuclear cells, thus indicating that the CREBBP gene is essential for the normal expression of these proteins and the regulation of immune responses.

Asunto(s)

Proteína de Unión a CREB/genética , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Inmunidad/genética , Proteína de Unión a CREB/metabolismo , Bandeo Cromosómico , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Variaciones en el Número de Copia de ADN , Proteínas de Unión al ADN/metabolismo , Femenino , Humanos , Hibridación Fluorescente in Situ , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-jun/genética , Proteínas Proto-Oncogénicas c-jun/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Factor de Transcripción ReIA/genética , Factor de Transcripción ReIA/metabolismo , Translocación Genética , Adulto Joven

RESUMEN

Multidrug resistance-associated protein 3 (Mrp3; Abcc3) expression and activity are up-regulated in rat liver after in vivo repeated administration of ethynylestradiol (EE), a cholestatic synthetic estrogen, whereas multidrug resistance-associated protein 2 (Mrp2) is down-regulated. This study was undertaken to determine whether Mrp3 induction results from a direct effect of EE, independent of accumulation of any endogenous common Mrp2/Mrp3 substrates resulting from cholestasis and the potential mediation of estrogen receptor (ER). In in vivo studies, male rats were given a single, noncholestatic dose of EE (5 mg/kg s.c.), and basal bile flow and the biliary excretion rate of bile salts and glutathione were measured 5 hours later. This treatment increased Mrp3 mRNA by 4-fold, detected by real-time polymerase chain reaction, despite the absence of cholestasis. Primary culture of rat hepatocytes incubated with EE (1-10 µM) for 5 hours exhibited a 3-fold increase in Mrp3 mRNA (10 µM), consistent with in vivo findings. The increase in Mrp3 mRNA by EE was prevented by actinomycin D, indicating transcriptional regulation. When hepatocytes were incubated with an ER antagonist [7α,17ß-[9-[(4,4,5,5,5-Pentafluoropentyl)sulfinyl]nonyl]estra-1,3,5(10)-triene-3,17-diol (ICI182/780), 1 µM], in addition to EE, induction of Mrp3 mRNA was abolished, implicating ER as a key mediator. EE induced an increase in ER-α phosphorylation at 30 minutes and expression of c-Jun, a well-known ER target gene, at 60 minutes, as detected by Western blotting of nuclear extracts. These increases were prevented by ICI182/780. In summary, EE increased the expression of hepatic Mrp3 transcriptionally and independently of any cholestatic manifestation and required participation of an ER, most likely ER-α, through its phosphorylation.

Asunto(s)

Colestasis/metabolismo , Receptor alfa de Estrógeno/agonistas , Estrógenos/farmacología , Etinilestradiol/farmacología , Hígado/efectos de los fármacos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/efectos de los fármacos , Animales , Bilis/metabolismo , Ácidos y Sales Biliares/metabolismo , Células Cultivadas , Colestasis/genética , Dactinomicina/farmacología , Estradiol/análogos & derivados , Estradiol/farmacología , Antagonistas de Estrógenos/farmacología , Receptor alfa de Estrógeno/antagonistas & inhibidores , Receptor alfa de Estrógeno/metabolismo , Fulvestrant , Glutatión/metabolismo , Hígado/metabolismo , Masculino , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Fosforilación , Cultivo Primario de Células , Proteínas Proto-Oncogénicas c-jun/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Reacción en Cadena en Tiempo Real de la Polimerasa , Factores de Tiempo , Regulación hacia Arriba

RESUMEN

A Na+, K+ ATPase (NKA) atua na manutenção do potencial de membrana dascélulas e em mecanismos de transdução de sinal. Alterações na atividade da NKAsão importantes em muitos processos biológicos e patológicos. A NKA pode serinibida pelo álcool perílico (POH), um monoterpeno utilizado no tratamento detumores, incluindo cerebrais. Neste trabalho, determinamos que o POH tambématua sobre cascatas de sinalização, moduladas via NKA, que controlam aproliferação e/ou morte celular. Foi avaliado o efeito do POH e do PA seu principalmetabólito, sobre a atividade da NKA em duas linhagens de células de glioblastoma(GBM) humano (U87 e U251), células não tumorais de astrócitos de camundongo eda linhagem (VERO). Nossos resultados, baseados na avaliação da atividade daNKA por incorporação do Rb+ , o qual mimetiza o K+ , mostraram uma sensibilidade àinibição pelo POH semelhante entre os quatro tipos de células (IC50 U87 2 mM;U251 1,8 mM; VERO 2,4 mM e astrócitos de camundongo 1,4 mM), enquanto o PAnão apresentou efeito. Sabe-se que nos GBMs existe uma superexpressão dasubunidade α1 da NKA, situada na estrutura das cavéolas que é provavelmenteresponsável pelo papel sinalizador atribuído a essa enzima, especialmente emrelação aos mecanismos apoptóticos. Comparamos a viabilidade celular,determinando a atividade da enzima lactato desidrogenase presente nosobrenadante das células tratadas por 24 h com POH e PA. O PA não alcançouefeito citotóxico igual ou superior a 30 por cento nas células mesmo na concentraçãoelevada de 4 mM. Já o POH reduziu, de maneira dependente da concentração, aviabilidade das células (IC50 U87 1,1 mM; U251 1,4 mM; VERO 0,9 mM e astrócitosde camundongo 1,4 mM). Na análise por western blot, 1,5 mM de POH ativou aproteína c-Jun N-terminal quinase (JNK), nas células U87, U251 e nos astrócitos decamundongo (incubação de 30 min)...

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The Na+, K+ ATPase (NKA) acts in keeping the cell membrane potential and in signaltransduction mechanisms. Modifications in the activity of this enzyme are important inphysiological and pathological processes. The NKA is inhibited by perillyl alcohol(POH), a monotherpene used in the treatment of tumors, including brain tumors. Inthis work, we also show that POH acts in signaling cascades associated to NKA,controlling cell proliferation and/or cellular death. We evaluated the effect of POH andof its main metabolite (perillic acid - PA) on the NKA activity in cultured glioblastomacells (GBM) U87 and U251 and on non-tumor cells (mouse astrocytes and VEROcells). NAK activity was measured by non-radioactive Rb+incorporation by cells (Rb+is a K+substitute). Our results showed a similar sensitivity for the four cells typestested (IC50 U87 – 2 mM; U251 - 1,8 mM; VERO - 2,4 mM and mouse astrocytes -1,4 mM). Perillic acid did not show any effect in any cell type. In GBMs, it is knownthat NKA α1 subunit is super expressed. This isoform is embedded in caveolarstructures and is probably responsible by the signaling properties of this enzyme inapoptosis mechanisms. Cell viability was measured by lactate dehydrogenase in cellsupernatants of POH treated cells. The maximum PA cytotoxic effect obtained was30 percent even at 4 mm. However, POH reduced dose dependently cell viability, (IC50 U87- 1,1 mM; U251 - 1,4 mM; VERO - 0,9 mM and mouse astrocyte - 1,4 mM).Considering the western blot analysis, 1,5 mM POH activated the c-Jun N-terminalKinase (JNK), on U87, U251 and in mouse astrocytes after 30min incubation...

Asunto(s)

Animales , Adenosina Trifosfatasas , Glicósidos Cardíacos/química , Monoterpenos , Proteínas Proto-Oncogénicas c-jun , Ensayo de Inmunoadsorción Enzimática , Neoplasias

RESUMEN

Systemic lupus erythematosus (SLE) is characterized by abnormalities in the function of T and B lymphocytes and in the signaling pathways induced through their receptors. Cbl-b is an intracellular adaptor protein that plays a key role in the negative regulation of lymphocyte activity. We explored the expression and function of Cbl-b in T lymphocytes from SLE patients. In addition, the possible association of SLE and a single nucleotide polymorphism (SNP) of the Cblb gene was determined. We studied 150 SLE patients, 163 healthy individuals, and 14 patients with rheumatoid arthritis (RA). The expression of Cbl-b was analyzed in the peripheral blood mononuclear cells, and the negative regulatory function of Cbl-b was assessed by analyzing actin polymerization and the phosphorylation of JNK and c-Jun induced through CD3. Furthermore, the 2126(A/G) SNP of the Cblb gene was detected by real-time polymerase chain reaction. We found a significant small reduction in the expression of Cbl-b as well as increased levels of activation of c-Jun and actin polymerization in T lymphocytes from patients with SLE compared with healthy controls or RA patients. In addition, a significant association between the 2126(A/G) SNP and SLE was detected. Our data suggest that Cbl-b may contribute to the deregulated activation of T lymphocytes observed in SLE.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales/genética , Lupus Eritematoso Sistémico/inmunología , Proteínas Proto-Oncogénicas c-cbl/genética , Linfocitos T/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/inmunología , Artritis Reumatoide/genética , Artritis Reumatoide/inmunología , Estudios de Casos y Controles , Humanos , Leucocitos Mononucleares/metabolismo , Lupus Eritematoso Sistémico/genética , México , Fosforilación , Reacción en Cadena de la Polimerasa , Polimerizacion , Polimorfismo de Nucleótido Simple , Proteínas Proto-Oncogénicas c-cbl/inmunología , Proteínas Proto-Oncogénicas c-jun/metabolismo

RESUMEN

An essential role for the Krüppel-like transcription factor family has been determined in the regulation of remarkable processes including cell proliferation, differentiation, signal transduction, oncogenesis, and cell death. A member of this group, Krüppel-like factor 6 (KLF6), identified on the basis of its ability to regulate a group of genes belonging to the carcinoembryonic antigen gene family, has been involved in human carcinogenesis. Early studies proposed a tumor suppressor function for KLF6 because of its ability to reduce cell proliferation through several biochemical mechanisms including regulation of cell cycle components, oncogene products, and apoptosis. Mutations within the klf6 gene, decreased expression and/or loss-of-heterozygosity were associated with the development of different human malignancies, and, hence, further supporting the tumor suppressor function of KLF6. This view has been challenged by other studies in distinct types of human cancers describing infrequent genetic alterations of klf6 gene or even enhanced expression in some tumors. The scenario about KLF6 function became still more complex as the description of oncogenic KLF6 splice variant 1 (SV1) with dominant negative activity against the wild type KLF6 (wtKLF6) protein. Additionally, increased evidence is suggesting that KLF6 is a bonafide target of several signaling cascades, which ultimate regulatory effect on this protein could drive decisions of cell life and death, facing the dilemma about how wtKLF6 could be involved in both processes. These apparently conflicting situations, emerged by apparently opposite effects mediated by wtKLF6, may be related, at least in part, to the biological cross-talk with the c-Jun oncoprotein. Depending on the stimulus received by the cell, wtKLF6 interaction with c-Jun determines different cell outcomes such as proliferation control or apoptosis. Thus, KLF6 responsiveness represents a kind of cell warning signal on receiving different stimuli, including oncogenic activation and microbial infections, orchestrating the implementation of proliferation and apoptotic programs.

Asunto(s)

Apoptosis , Proliferación Celular , Regulación del Desarrollo de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Factores de Transcripción de Tipo Kruppel , Proteínas Proto-Oncogénicas , Transducción de Señal , Animales , Apoptosis/genética , Diferenciación Celular/genética , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Genes Supresores de Tumor/fisiología , Humanos , Factor 6 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Pérdida de Heterocigocidad , Ratones , Ratones Noqueados , Mutación , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Ratas , Transducción de Señal/genética

RESUMEN

We have previously reported in C2C12 murine skeletal muscle cells that 10(-8)M 17ß-estradiol promotes MAPKs stimulation which in turn mediates the activation of CREB and Elk-1 transcription factors. In this work, we demonstrated that the hormone induces ERK2 phosphorylation (without affecting ERK1 activation) and also stimulates p38 MAPK, both in a dose-dependent manner. Moreover, estrogen receptors involvement in MAPKs activation by the estrogen was studied. The use of ICI182780 (1 µM), an antagonist of ERs, and specific siRNAs to block ERα and ERß expression, demonstrated that ERα mediates ERK2 activation but not p38 MAPK phosphorylation by 17ß-estradiol, and that ERß isoform is not implicated in MAPKs activation by the hormone. Furthermore, Src and PKC contribution in estrogen stimulation of the MAPKs was investigated. Compounds PP2 and Ro318220, Src and PKC family inhibitors, respectively abrogated ERK2 and p38 MAPK phosphorylation by 17ß-estradiol. Of interest, the hormone was able to induce Src and PKCδ activation. In addition, Ro318220 decreased estrogen-dependent Src modulation implicating PKC in hormone upregulation of Src. Accordingly, PP2 and Ro318220 suppressed CREB and Elk-1 phosphorylation as well as c-Fos and c-Jun oncoprotein levels induced by 17ß-estradiol. Altogether, these data indicate that 17ß-estradiol activates ERK2 through ERα and p38 MAPK in an ERα/ß-independent manner and that PKC and Src proteins are key upstream components on MAPKs activation in C2C12 skeletal muscle cells.

Asunto(s)

Estradiol/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Proteína Quinasa C/metabolismo , Receptores de Estrógenos/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Familia-src Quinasas/metabolismo , Animales , Línea Celular , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Transducción de Señal , Proteína Elk-1 con Dominio ets/metabolismo