RESUMEN
The mechanisms that regulate the induction of term or preterm delivery (PTD) are not fully understood. Infection is known to play a role in the induction of pro-inflammatory cascades in uteroplacental tissues associated with preterm pathological parturition. Similar but not identical cascades are evident in term labour. In the current study, we used a mouse model to evaluate the role of prokineticins in term and preterm parturition. Prokineticins are multi-functioning secreted proteins that signal through G-protein-coupled receptors to induce gene expression, including genes important in inflammatory responses. Expression of prokineticins (Prok1 and Prok2) was quantified in murine uteroplacental tissues by QPCR in the days preceding labour (days 16-19). Prok1 mRNA expression increased significantly on D18 in fetal membranes (compared with D16) but not in uterus or placenta. Intrauterine injection of PROK1 on D17 induced fetal membrane mRNA expression of the pro-inflammatory mediators Il6, Il1b, Tnf, Cxcl2 and Cxcl5, which are not normally up-regulated until D19 of pregnancy. However, intrauterine injection of PROK1 did not result in PTD. As expected, injection of lipopolysaccharide (LPS) induced PTD, but this was not associated with changes in expression of Prok1 or its receptor (Prokr1) in fetal membranes. These results suggest that although Prok1 exhibits dynamic mRNA regulation in fetal membranes preceding labour and induces a pro-inflammatory response when injected into the uterus on D17, it is insufficient to induce PTD. Additionally, prokineticin up-regulation appears not to be part of the LPS-induced inflammatory response in mouse fetal membranes.
Asunto(s)
Membranas Extraembrionarias/efectos de los fármacos , Inflamación/inducido químicamente , Factor de Crecimiento Endotelial Vascular Derivado de Glándula Endocrina/farmacología , Animales , Membranas Extraembrionarias/inmunología , Membranas Extraembrionarias/metabolismo , Femenino , Inflamación/genética , Inflamación/metabolismo , Mediadores de Inflamación/metabolismo , Inyecciones , Lipopolisacáridos/farmacología , Masculino , Ratones , Embarazo , Nacimiento Prematuro , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Útero , Factor de Crecimiento Endotelial Vascular Derivado de Glándula Endocrina/genética , Factor de Crecimiento Endotelial Vascular Derivado de Glándula Endocrina/metabolismoRESUMEN
The prostaglandin endoperoxide synthase (PTGS) pathway is a potent driver of tumour development in humans by enhancing the biosynthesis and signalling of prostaglandin (PG) E(2). PTGS2 expression and PGE(2) biosynthesis is elevated in endometrial adenocarcinoma, however the mechanism whereby PTGS and PGE(2) regulate endometrial tumour growth is unknown. Here we investigated (a) the expression profile of the PGE synthase enzymes (PTGES, PTGES-2, PTGES-3) and PGE receptors (PTGER1-4) in endometrial adenocarcinomas compared with normal endometrium and (b) the role of PTGER4 in endometrial tumorigenesis in vivo. We found elevated expression of PTGES2 and PTGER4 and suppression of PTGER1 and PTGER3 in endometrial adenocarcinomas compared with normal endometrium. Using WT Ishikawa endometrial adenocarcinoma cells and Ishikawa cells stably transfected with the full length PTGER4 cDNA (PTGER4 cells) xenografted in the dorsal flanks of nude mice, we show that PTGER4 rapidly and significantly enhances tumour growth rate. Coincident with enhanced PTGER4-mediated tumour growth we found elevated expression of PTGS2 in PTGER4 xenografts compared with WT xenografts. Furthermore we found that the augmented growth rate of the PTGER4 xenografts was not due to enhanced angiogenesis, but regulated by an increased proliferation index and hypoxia. In vitro, we found that PGE(2) and hypoxia independently induce expression of PTGER4 indicating two independent pathways regulating prostanoid receptor expression. Finally we have shown that PGE(2) and hypoxia synergise to promote cellular proliferation of endometrial adenocarcinoma cells.
Asunto(s)
Adenocarcinoma/metabolismo , Neoplasias Endometriales/metabolismo , Hipoxia/fisiopatología , Subtipo EP4 de Receptores de Prostaglandina E/metabolismo , Transducción de Señal , Adenocarcinoma/patología , Línea Celular Tumoral , Neoplasias Endometriales/patología , Femenino , Perfilación de la Expresión Génica , Humanos , Inmunohistoquímica , Microscopía Confocal , Subtipo EP4 de Receptores de Prostaglandina E/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Prostanoids are well-described primary mediators of inflammatory processes and are essential for the normal physiological function of the female reproductive system. The aim of this study was to determine the temporal expression of the prostanoid biosynthetic enzymes (PTGS1, PTGS2, PTGES, PTGES2, PTGES3, AKR1B1, AKR1C3, CBR1, HPGDS, PTGDS, PTGIS, TBXAS1 and HPGD) and the prostanoid receptors (PTGER1, PTGER2, PTGER3, PTGER4, PTGFR, PTGDR, GPR44, PTGIR and TBXA2R) in the human endometrium throughout the menstrual cycle. The analysis identified PTGFR to have a distinct expression profile compared with other components of the prostanoid system, as expression is maximal during the proliferative phase. Immunohistochemical analysis for PTGER1 suggests a dual function for this receptor depending on its temporal (proliferative versus secretory) and spatial (nuclear versus cell membrane) expression. The expression profiles of the PGF(2α) synthases identified AKR1B1 and CBR1 as the likely regulators of PGF(2α) production during the menstrual phase. Immunohistochemical analysis for AKR1B1, CBR1 and AKR1C3 suggest expression to be in the glandular epithelium and vasculature. This study represents the first comprehensive analysis of the components of prostanoid biosynthetic and signalling pathway in the human endometrium. The expression profiles described have the potential to identify specific prostanoid components that may be dysregulated in inflammatory-associated disorders of the endometrium.
Asunto(s)
Endometrio/metabolismo , Ciclo Menstrual/metabolismo , Prostaglandinas/metabolismo , Receptores de Prostaglandina/metabolismo , Adulto , Oxidorreductasas de Alcohol/genética , Oxidorreductasas de Alcohol/metabolismo , Aldehído Reductasa , Aldo-Ceto Reductasas , Endometrio/enzimología , Femenino , Humanos , Prostaglandinas/análisis , Prostaglandinas/genética , ARN Mensajero/metabolismo , Receptores de Prostaglandina/análisis , Receptores de Prostaglandina/genéticaRESUMEN
Pro-inflammatory mediators, like prostaglandin (PG) and chemokines, promote tumourigenesis by enhancing cell proliferation, migration of immune cells and recruitment of blood vessels. Recently we showed elevated expression of the chemokine (C-X-C motif) receptor 2 (CXCR2) in endometrial adenocarcinomas localized to neutrophils and neoplastic epithelial and vascular cells. Furthermore we found that PGF(2alpha)-F-prostanoid (FP) receptor regulates the expression of the CXCR2 ligand CXCL1, to promote neutrophil chemotaxis in endometrial adenocarcinomas. In the present study we identified another CXCR2 ligand, CXCL8 as a target for PGF(2alpha)-FP receptor signalling which enhances epithelial cell proliferation in endometrial adenocarcinoma cells in vitro and in nude mice in vivo. We found that PGF(2alpha)-FP receptor interaction induces CXCL8 expression in endometrial adenocarcinoma cells via the protein kinase C-calcium-calcineurin-NFAT signaling pathway. Promoter analysis revealed that CXCL8 transcriptional activation by PGF(2alpha) signaling is mediated by cooperative interactions between the AP1 and NFAT binding sites. Furthermore, PGF(2alpha) via the FP receptor induced the expression of the regulator of calcineurin 1 isoform 4 (RCAN1-4) via the calcineurin/NFAT pathway in a reciprocal manner to CXCL8. Using an adenovirus to overexpress RCAN1-4, we found that RCAN1-4 is a negative regulator of CXCL8 expression in endometrial adenocarcinoma cells. Taken together our data have elucidated the molecular and cellular mechanism whereby PGF(2alpha) regulates CXCL8 expression via the FP receptor in endometrial adenocarcinomas and have highlighted RCAN1-4 as a negative regulator of CXCL8 expression which may be exploited therapeutically to inhibit CXCL8-mediated tumour development.
Asunto(s)
Adenocarcinoma/metabolismo , Calcineurina/metabolismo , Calcio/metabolismo , Neoplasias Endometriales/metabolismo , Regulación Neoplásica de la Expresión Génica , Interleucina-8/biosíntesis , Factores de Transcripción NFATC/metabolismo , Proteínas de Neoplasias/metabolismo , Receptores de Prostaglandina/metabolismo , Transducción de Señal , Animales , Línea Celular Tumoral , Dinoprost/metabolismo , Femenino , Humanos , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Proteína Quinasa C/metabolismo , Elementos de Respuesta , Trasplante HeterólogoRESUMEN
The prostaglandin F(2alpha) (PGF(2alpha)) receptor (FP) is elevated in endometrial adenocarcinoma. This study found that PGF(2alpha) signaling via FP regulates expression of chemokine (C-X-C motif) ligand 1 (CXCL1) in endometrial adenocarcinoma cells. Expression of CXCL1 and its receptor, CXCR2, are elevated in cancer tissue compared with normal endometrium and localized to glandular epithelium, endothelium, and stroma. Treatment of Ishikawa cells stably transfected with the FP receptor (FPS cells) with 100 nmol/L PGF(2alpha) increased CXCL1 promoter activity, mRNA, and protein expression, and these effects were abolished by cotreatment of cells with FP antagonist or chemical inhibitors of Gq, epidermal growth factor receptor, and extracellular signal-regulated kinase. Similarly, CXCL1 was elevated in response to 100 nmol/L PGF(2alpha) in endometrial adenocarcinoma explant tissue. CXCL1 is a potent neutrophil chemoattractant. The expression of CXCR2 colocalized to neutrophils in endometrial adenocarcinoma and increased neutrophils were present in endometrial adenocarcinoma compared with normal endometrium. Conditioned media from PGF(2alpha)-treated FPS cells stimulated neutrophil chemotaxis, which could be abolished by CXCL1 protein immunoneutralization of the conditioned media or antagonism of CXCR2. Finally, xenograft tumors in nude mice arising from inoculation with FPS cells showed increased neutrophil infiltration compared with tumors arising from wild-type cells or following treatment of mice bearing FPS tumors with CXCL1-neutralizing antibody. In conclusion, our results show a novel PGF(2alpha)-FP pathway that may regulate the inflammatory microenvironment in endometrial adenocarcinoma via neutrophil chemotaxis.
Asunto(s)
Adenocarcinoma/patología , Quimiocina CXCL1/metabolismo , Neoplasias Endometriales/patología , Neutrófilos/metabolismo , Receptores de Prostaglandina/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Animales , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Quimiocina CXCL1/genética , Quimiotaxis de Leucocito/efectos de los fármacos , Dinoprost/farmacología , Neoplasias Endometriales/genética , Neoplasias Endometriales/metabolismo , Endometrio/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Inmunohistoquímica , Ratones , Ratones Desnudos , Microscopía Fluorescente , Trasplante de Neoplasias , Neoplasias Experimentales/genética , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Neutrófilos/citología , Receptores de Interleucina-8B/genética , Receptores de Interleucina-8B/metabolismo , Receptores de Prostaglandina/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Trasplante HeterólogoRESUMEN
BACKGROUND: Particulate air pollution (PM(10)) consists of a mixture of components, including nanoparticles and metals. Studies from our laboratory have demonstrated that transition metals can potentiate the ability of nanoparticles to induce lung inflammation and that the zinc content of PM(10) was largely responsible for their potential to induce inflammation. These results are also relevant to zinc-containing engineered nanoparticles. OBJECTIVES: To investigate the potential of ZnCl(2) and FeCl(3) to interact with nanoparticle carbon black in cell-free and biological systems to generate ROS, express pro-inflammatory mediators and cytotoxic ability. METHODS: ROS production was examined using DCFH-DA. J774 cells were treated for 4 h with 14 nm CB and/or ZnCl(2) before measuring TNF-alpha by ELISA. Cytoskeletal changes were investigated using confocal microscopy. Flow cytometry was used to examine apoptotic/necrotic cells and phagocytic ability. RESULTS: In a cell-free system the particles generated significant ROS, whereas ZnCl(2) did not. Treatment of cells with 100 microM ZnCl(2), but not FeCl(3), increased TNF-alpha. Treatment with 14 nm CB alone induced TNF-alpha, which was synergistically enhanced by ZnCl(2). No interactions were observed in cells treated with 14 nm CB and FeCl(3). Cytoskeletal changes were observed with increasing concentrations of ZnCl(2). These results were confirmed by flow cytometry indicating that ZnCl(2) induced markers of apoptosis and necrosis. The phagocytic ability of cells was also significantly decreased. Nanoparticle carbon black alone did not induce changes in apoptosis/necrosis or the phagocytosis activity of the cells. CONCLUSION: Despite an inability to induce ROS production, ZnCl(2) stimulated TNF-alpha production which was synergistically enhanced by 14 nm carbon black. The ability of zinc to induce morphological changes and cell death was not altered by nanoparticle treatment.
Asunto(s)
Contaminantes Atmosféricos/toxicidad , Cloruros/toxicidad , Compuestos Férricos/toxicidad , Nanopartículas/toxicidad , Hollín/toxicidad , Compuestos de Zinc/toxicidad , Animales , Apoptosis/efectos de los fármacos , Línea Celular , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Sinergismo Farmacológico , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Inflamación/inducido químicamente , Mediadores de Inflamación/metabolismo , Macrófagos , Ratones , Microscopía Confocal , Necrosis/metabolismo , Fagocitosis/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Factor de Necrosis Tumoral alfa/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Hill races usually include large downhill running sections, which can induce significant degrees of muscle damage in a field setting. This study examined the link between muscle damage, oxidative stress, and immune perturbations following a 7-km mountainous hill race with 457 m of ascent and 457 m of descent. Venous blood samples were taken from 7 club level runners before, immediately after, and 48 hrs postrace. Samples were analysed for total and differential leukocyte counts, markers of muscle damage (CK), lipid peroxidation (MDA), and acute phase proteins (CRP; fibrinogen; alpha-1-ACT). The total antioxidant status (TEAC) and plasma levels of the proinflammatory cytokines IL-6, IL-8, and TNF-alpha were also determined. Subjective pain reports, and plasma activities of CK, MDA, and circulatory monocytes reached peak values at 48 hrs postrace (p < 0.05). TEAC and the cytokine IL-8 increased immediately after the race (p < 0.05). Plasma TNF-alpha remained unchanged (p > 0.05). Despite the reports of muscle damage and soreness, no evidence of an acute phase response was observed (p > 0.05), which may be explained by the failure of the race to induce a plasma TNF-alpha response. Future studies should examine the link between muscle damage, oxidative stress, and the acute phase response following hill races of longer duration with larger eccentric components.
Asunto(s)
Proteínas de Fase Aguda/análisis , Peroxidación de Lípido , Músculo Esquelético/patología , Carrera/fisiología , Proteínas de Fase Aguda/fisiología , Adulto , Creatina Quinasa/sangre , Citocinas/sangre , Humanos , Peroxidación de Lípido/fisiología , Malondialdehído/sangre , Factor de Necrosis Tumoral alfa/análisisRESUMEN
The adverse health effects of elevated exposures to PM(10) (particulate matter collected through a size selective inlet with an efficiency of 50% for particles with an aerodynamic diameter of 10 µm) in relation to morbidity and mortality, especially in susceptible individuals, are now well recognised. PM(10) consists of a variable cocktail of components differing in chemical composition and size. Epidemiological and toxicological data suggest that transition metals and ultrafine particles are both able to drive the cellular and molecular changes that underlie PM(10)-induced inflammation and so worsen disease status. Toxicological evidence also suggest roles for the biological components of PM(10) including volatile organic compounds (VOC's), allergens and bacterial-derived endotoxin. Many of these components, in particular transition metals, ultrafine particles, endotoxin and VOC's induce a cellular oxidative stress which initiates an intracellular signaling cascade involving the activation of phosphatase and kinase enzymes as well as transcription factors such as nuclear factor kappa B. Activation of these signaling mechanisms results in an increase in the expression of proinflammatory mediators, and hence enhanced inflammation. Given that many of the components of PM(10) stimulate similar or even identical intracellular signaling pathways, it is conceivable that this will result in synergistic or additive interactions so that the biological response induced by PM(10) exposure is a response to the composition rather than the mass alone. A small number of studies suggest that synergistic interactions occur between ultrafine particles and transition metals, between particles and allergens, and between particles and VOC's. Elucidation of the consequences of interaction between the components of PM(10) in relation to their biological activity implies huge consequences for the methods used to monitor and to legislate pollution exposure in the future, and may drive a move from mass based measurements to composition.
RESUMEN
Both the ultrafine particle and transition metal components of particulate air pollution (PM(10)) have been hypothesized to be important factors in determining toxicity and potential adverse health effects. In this study we aimed to investigate interactions between transition metal salts and a surrogate environmental particle-ultrafine carbon black (ufCB). In all experimental systems employed, the ufCB was found to be more reactive than its fine counterpart (CB). Incubation of ufCB with the reactive oxygen species (ROS)-sensitive probe dichlorofluorescin in the absence of cells generated significantly more ROS than CB. With addition of either cupric sulfate (CuSO(4)), ferrous sulfate (FeSO(4)), or ferric chloride (FeCl(3)), the ROS generation in the presence of ufCB was enhanced in a potentiative manner. In Mono Mac 6 macrophages, ufCB again produced more ROS than CB. However, addition of iron salts had no additive effect over and above that induced in the macrophages by ufCB. In the mouse macrophage cell line J774, ufCB decreased the cellular content of GSH and ATP. Addition of iron further decreased both GSH and ATP and a potentiative interaction between ufCB and FeSO(4) was observed, but only at the highest iron concentrations tested. A concentration-dependent increase in tumor necrosis factor-alpha production by J774 cells was also observed following exposure to ufCB, which was not further enhanced by the addition of iron. J774 cells were also found to sequester or chelate iron without inducing toxicity. In the rat lung ufCB induced a significant neutrophil influx and this inflammatory effect was potentiativelly enhanced by the addition of FeCl(3) (100 microM). These findings suggest that (1) ultrafine particles and metals interact by chemical potentiation in a cell-free environment to generate ROS, (2) potentiation between ultrafine particles and metal salts is not observed in the presence of macrophages as iron is sequestered or chelated by the cells, (3) in the lung, ultrafine particles and iron salts interact in a potentiative manner to generate inflammation.
Asunto(s)
Contaminantes Atmosféricos , Carbono/toxicidad , Cobre/toxicidad , Hierro/toxicidad , Pulmón/efectos de los fármacos , Macrófagos/efectos de los fármacos , Adenosina Trifosfato/metabolismo , Animales , Carbono/administración & dosificación , Línea Celular , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Femenino , Glutatión/metabolismo , Humanos , Intubación Intratraqueal , Hierro/metabolismo , Pulmón/metabolismo , Pulmón/patología , Macrófagos/metabolismo , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Estrés Oxidativo , Tamaño de la Partícula , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
It has been known for some time that antibodies raised against ZP3, the major component of the glycoprotein shell that surrounds all mammalian oocytes, can successfully inhibit sperm-egg interaction in vitro. In our own studies using the non-human primate Callithrix jacchus, active immunisation was successfully achieved when homologous or heterologous ZP3 was used as an immunogen. However this long-term suppression of fertility was at the expense of ovarian function. An ovarian pathology was observed which was characterised by a disruption of folliculogenesis and depletion of the primordial follicle pool. Adverse auto-immune reactions have also been observed in mice following induction of immunity to mouse ZP3. Following careful selection of B-cell epitopes on mouse ZP3, peptide vaccines were formulated which could circumvent these adverse side effects and induce reversible infertility in actively immunised mice. To identify similar epitopes on primate ZP3, epitope mapping studies were performed and several candidate regions of the molecule were identified. These were incorporated into chimeric peptide vaccines and administered as single or triple peptide vaccines. Active immunisation successfully induced antibodies that bound exclusively to the zona pellucida of marmoset and human ovarian sections. These antibodies were able to suppress human sperm-egg binding by up to 60% in vitro. Encouragingly, no adverse side effects on ovarian function were observed following long-term immunisation however, no loss of fertility was consistently observed in vivo. Thus considerable research is still required to identify a combination of ZP epitopes that will induce reversible infertility in the absence of any ovarian dysfunction.