RESUMO
BACKGROUND: Inflamed environments are typically hypercellular, rich in pro-inflammatory cytokines, and profoundly hypoxic. While the effects of hypoxia on neutrophil longevity and function have been widely studied, little is known about the consequences of this stimulus on eosinophils. OBJECTIVE: We sought to investigate the effects of hypoxia on several key aspects of eosinophil biology, namely secretion, survival, and their sensitivity to glucocorticosteroids (GCS), agents that normally induce eosinophil apoptosis. METHODS: Eosinophils derived from patients with asthma/atopy or healthy controls were incubated under normoxia and hypoxia, with or without glucocorticoids. Activation was measured by flow cytometry, ELISA of cultured supernatants, and F-actin staining; apoptosis and efferocytosis by morphology and flow cytometry; and GCS efficacy by apoptosis assays and qPCR. RESULTS: Hypoxic incubation (3 kPa) caused (i) stabilization of HIF-2α and up-regulation of hypoxia-regulated genes including BNIP3 (BCL2/adenovirus E1B 19-kDa protein-interacting protein 3) and GLUT1 (glucose transporter 1); (ii) secretion of pre-formed IL-8, and Charcot Leyden crystal (CLC) formation, which was most evident in eosinophils derived from atopic and asthmatic donors; (iii) enhanced F-actin formation; (iv) marked prolongation of eosinophil lifespan (via a NF-κB and Class I PI3-kinase-dependent mechanism); and (v) complete abrogation of the normal pro-apoptotic effect of dexamethasone and fluticasone furoate. This latter effect was evident despite preservation of GCS-mediated gene transactivation under hypoxia. CONCLUSION AND CLINICAL RELEVANCE: These data indicate that hypoxia promotes an eosinophil pro-inflammatory phenotype by enhancing eosinophil secretory function, delaying constitutive apoptosis, and importantly, antagonizing the normal pro-apoptotic effect of GCS. As eosinophils typically accumulate at sites that are relatively hypoxic, particularly during periods of inflammation, these findings may have important implications to understanding the behaviour of these cells in vivo.
Assuntos
Hipóxia Celular/fisiologia , Eosinófilos/patologia , Interleucina-8/metabolismo , Anti-Inflamatórios/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Células Cultivadas , Dexametasona/farmacologia , Eosinófilos/efeitos dos fármacos , Eosinófilos/metabolismo , Glucocorticoides/farmacologia , Humanos , Corpos de Inclusão/patologia , Inflamação/imunologia , Inflamação/patologiaRESUMO
Macrolide antibiotics were discovered over 50 years ago and following their use as antimicrobials it became apparent that this group of antibiotics also possessed anti-inflammatory properties. Subsequent clinical trials showed benefits of macrolides as long-term adjuncts in the treatment of a spectrum of chronic inflammatory respiratory diseases, particularly diffuse panbronchiolitis, cystic fibrosis, post-transplant bronchiolitis obliterans and more recently chronic obstructive pulmonary disease (COPD). The evidence for efficacy of macrolides in the long-term treatment of chronic asthma and bronchiectasis is less well established. The mechanism(s) of action of macrolides in the treatment of these diseases remains unexplained, but may be due to their antibacterial and/or anti-inflammatory actions, which include reductions in interleukin-8 production, neutrophil migration and/or function. Macrolides have additional potentially beneficial properties including anti-viral actions and an ability to restore corticosteroid sensitivity. The increased prescribing of macrolides for long-term treatment could result in the development of microbial resistance and adverse drug effects. New macrolides have been developed which do not possess any antimicrobial activity and hence lack the ability to produce microbial resistance, but which still retain immunomodulatory effects. Potentially novel macrolides may overcome a significant barrier to the use of this type of drug for the long-term treatment of chronic inflammatory airway diseases.
Assuntos
Anti-Inflamatórios/uso terapêutico , Asma/tratamento farmacológico , Inflamação/tratamento farmacológico , Macrolídeos/uso terapêutico , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Doenças Respiratórias/tratamento farmacológico , Anti-Inflamatórios/efeitos adversos , Anti-Inflamatórios/química , Asma/imunologia , Doença Crônica , Ensaios Clínicos como Assunto , Humanos , Inflamação/imunologia , Macrolídeos/efeitos adversos , Macrolídeos/química , Doença Pulmonar Obstrutiva Crônica/imunologia , Doenças Respiratórias/imunologia , Resultado do TratamentoRESUMO
BACKGROUND: Eosinophils are pro-inflammatory cells implicated in the pathogenesis of asthma and atopy. Apoptosis has been proposed as a potential mechanism underlying the resolution of eosinophilic inflammation and studies have indicated the ability of interventions that induce human eosinophil apoptosis to promote the resolution of eosinophilic inflammation. Recently, the cyclin-dependent kinase (CDK) inhibitor R-roscovitine was shown to enhance neutrophil apoptosis and promote the resolution of neutrophilic inflammation. OBJECTIVE: The purpose of this study was to examine the expression of CDKs in human blood eosinophils, the effects of R-roscovitine on eosinophil survival in vitro and whether R-roscovitine could influence eosinophilic lung inflammation in vivo. METHODS: Eosinophils were isolated from human peripheral blood and the effects of R-roscovitine on apoptosis, degranulation and phagocytic uptake examined in vitro. The effects of R-roscovitine on eosinophilic lung inflammation in vivo were also assessed using an ovalbumin mouse model. RESULTS: Our data demonstrate that human eosinophils express five known targets for R-roscovitine: CDK1, -2, -5, -7 and -9. R-roscovitine induced eosinophil apoptosis in a time- and concentration-dependent manner but also accelerated transition to secondary necrosis as assessed by microscopy, flow cytometry and caspase activation. In addition, we show that R-roscovitine can override the anti-apoptotic signals of GM-CSF and IL-5. We report that the pro-apoptotic effect of R-roscovitine is associated with suppression of Mcl-1L expression and that this compound enhanced phagocytic clearance of eosinophils by macrophages. Finally, we show that R-roscovitine induces apoptosis in murine peripheral blood and spleen-derived eosinophils; despite this, R-roscovitine did not modulate the tissue and lumen eosinophilia characteristic of the ovalbumin mouse model of airway eosinophilia. CONCLUSION AND CLINICAL RELEVANCE: These data demonstrate that R-roscovitine is capable of inducing rapid apoptosis and secondary necrosis in eosinophils but does not affect the onset or improve the resolution of eosinophilic airway inflammation in vivo.
Assuntos
Apoptose/efeitos dos fármacos , Quinases Ciclina-Dependentes/antagonistas & inibidores , Eosinófilos/citologia , Eosinófilos/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Purinas/farmacologia , Animais , Apoptose/imunologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/imunologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Eosinófilos/imunologia , Feminino , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina , Roscovitina , Fatores de TempoRESUMO
Previously, we used cDNA expression profiling to identify genes associated with glucocorticoid (Gc) sensitivity. We now identify which of these directly influence Gc action. Interferon-inducible protein 16 (IFI16), bone morphogenetic protein receptor type II (BMPRII), and regulator of G-protein signaling 14 (RGS14) increased Gc transactivation, whereas sialyltransferase 4B (SIAT4B) had a negative effect. Amyloid beta (A4) precursor-protein binding, family B, member 1 (APBB1/Fe65) and neural cell expressed developmentally down-regulated 9 (NEDD9) were without effect. Only IFI16 potentiated Gc repression of NF-kappaB. In addition, IFI16 affected basal expression, and Gc induction of endogenous target genes. IFI16 did not affect glucocorticoid receptor (GR) expression, ligand-dependent repression of GR expression, or the ligand-dependent induction of GR phosphorylation on Ser-211 or Ser-203. Coimmunoprecipitation revealed an interaction, suggesting that IFI16 modulation of GR function is mediated by protein crosstalk. Transfection analysis with GR mutants showed that the ligand-binding domain of GR binds IFI16 and is the target domain for IFI16 regulation. Analysis of human lung sections identified colocalization of GR and IFI16, suggesting a physiologically relevant interaction. We demonstrate that IFI16 is a novel modulator of GR function and show the importance of analyzing variation in Gc sensitivity in humans, using appropriate technology, to drive discovery.
Assuntos
Glucocorticoides/farmacologia , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Receptores de Glucocorticoides/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Western Blotting , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/genética , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Células Cultivadas , Biologia Computacional , Imunofluorescência , Humanos , Immunoblotting , Técnicas Imunoenzimáticas , Imunoprecipitação , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , NF-kappa B/genética , NF-kappa B/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/genética , Fosforilação , Regiões Promotoras Genéticas , Proteínas RGS/genética , Proteínas RGS/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Glucocorticoides/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ativação TranscricionalRESUMO
In addition to the core circadian oscillator, located within the suprachiasmatic nucleus, numerous peripheral tissues possess self-sustaining circadian timers. In vivo these are entrained and temporally synchronized by signals conveyed from the core oscillator. In the present study, we examine circadian timing in the lung, determine the cellular localization of core clock proteins in both mouse and human lung tissue, and establish the effects of glucocorticoids (widely used in the treatment of asthma) on the pulmonary clock. Using organotypic lung slices prepared from transgenic mPER2::Luc mice, luciferase levels, which report PER2 expression, were measured over a number of days. We demonstrate a robust circadian rhythm in the mouse lung that is responsive to glucocorticoids. Immunohistochemical techniques were used to localize specific expression of core clock proteins, and the glucocorticoid receptor, to the epithelial cells lining the bronchioles in both mouse and human lung. In the mouse, these were established to be Clara cells. Murine Clara cells retained circadian rhythmicity when grown as a pure population in culture. Furthermore, selective ablation of Clara cells resulted in the loss of circadian rhythm in lung slices, demonstrating the importance of this cell type in maintaining overall pulmonary circadian rhythmicity. In summary, we demonstrate that Clara cells are critical for maintaining coherent circadian oscillations in lung tissue. Their coexpression of the glucocorticoid receptor and core clock components establishes them as a likely interface between humoral suprachiasmatic nucleus output and circadian lung physiology.
Assuntos
Bronquíolos/fisiologia , Ritmo Circadiano/fisiologia , Células Epiteliais/fisiologia , Pulmão/fisiologia , Animais , Bronquíolos/citologia , Bronquíolos/efeitos dos fármacos , Bronquíolos/fisiopatologia , Técnicas de Cultura de Células , Proteínas de Ciclo Celular/metabolismo , Células Epiteliais/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Luciferases/genética , Pulmão/efeitos dos fármacos , Pulmão/fisiopatologia , Neoplasias Pulmonares/cirurgia , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Naftalenos/farmacologia , Proteínas Nucleares/metabolismo , Proteínas Circadianas Period , Fatores de Transcrição/metabolismoRESUMO
Any model of apoptosis must explain the mechanism of action of the Bcl-2 family of proteins. This has proved to be unusually difficult. This review concentrates on some of the newly isolated members of this growing family and attempts to provide an insight into the complexity of interactions through which the Bcl-2 proteins modulate apoptosis.
Assuntos
Apoptose/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Diferenciação Celular , Humanos , Proteína X Associada a bcl-2RESUMO
Irradiated leukocytes or mononuclear leukocytes, from 16 out of 30 patients with Hodgkin's disease and from one patient with the Sézary syndrome, stimulated in culture subnormal (3H)thymidine incorporation by allogeneic lymphocytes from normal individuals. This abnormality was not demonstrated in any of 30 other patients with non-Hodgkin's lymphomas. Subnormal mixed leukocyte culture reaction activation was caused by suppression of the mixed leukocyte reaction by patients' cells. Inhibition of the reaction by patient mononuclear leukocytes was corrected when adherent cells were removed or when protein synthesis was inhibited with cycloheximide. The inhibitory cells were probably lymphocytes since selective removal of phagocytic cells did not remove the inhibition by other patient mononuclear leukocytes. The presence in culture of as few as 2,500 granulocytes per mm3 also reduced responses when target cells were from patients with Hodgkin's disease. Patient cells no longer suppressed the mixed leukocyte reaction after patients entered clinical remission which suggests that suppression is a reversible, disease-related abnormality. Thus, the immune deficiency with advance Hodgkin's disease caused by ly lymphocyte depletion may be compounded by a relative excess of suppressor lymphocytes. The overall immunodeficiency may be further compounded by suppression of immune response by granulocytes at even physiologic concentrations.
Assuntos
Doença de Hodgkin/imunologia , Leucócitos/imunologia , Dermatite Esfoliativa/imunologia , Doença de Hodgkin/sangue , Humanos , Técnicas In Vitro , Leucemia Linfoide/imunologia , Teste de Cultura Mista de Linfócitos , Linfoma não Hodgkin/imunologia , Timidina , TrítioRESUMO
To evaluate whether immunological enhancement plays a role in adaptation to renal allografts, we studied sera from transplant recipients to determine whether those which suppressed mixed leukocyte culture (MLC) responses in vitro contained alloantibodies reactive with donor cells. Sera from five of nine renal transplant recipients consistently and specifically suppressed autologous autologous MLC responses to donor cells without impairing the blastogenic responses to third-party leukocytes, soluble antigens, or nonspecific mitogenic agents. In three of the five cases the suppressive activity of the serum was striking; in two cases the effect was less marked but still readily demonstrable in studies designed to evaluate the dose of serum which provided optimal suppression of MLC responses. Serum from one of the recipients nonspecifically suppressed blastogenic activity both to donor cells and other stimuli. No alloantibody reactive with donor leukocytes was found in any of the sera which exhibited suppressive activity in MLC, whereas in one patient, serum which contained antibody reactive with donor cells did not suppress MLC response to that donor. These findings suggest that, if the serum factors which suppress MLC responses in vitro are enhancing antibodies, they are not detectable even with very sensitive techniques either because they are present in very low concentrations, belong to immunoglobulin classes other than IgA, IgG, or IgM, or are complexed with donor antigen in such a way that their ability to react with fresh donor cells in vitro is blocked.
Assuntos
Isoanticorpos/análise , Transplante de Rim , Imunologia de Transplantes , Testes Imunológicos de Citotoxicidade , Imunofluorescência , Humanos , Teste de Cultura Mista de Linfócitos , Linfócitos/metabolismo , Timidina/metabolismo , Transplante Homólogo , TrítioRESUMO
DR3 (Ws1, Apo3, LARD, TRAMP, TNFSFR12) is a member of the death domain-containing tumor necrosis factor receptor (TNFR) superfamily, members of which mediate a variety of developmental events including the regulation of cell proliferation, differentiation, and apoptosis. We have investigated the in vivo role(s) of DR3 by generating mice congenitally deficient in the expression of the DR3 gene. We show that negative selection and anti-CD3-induced apoptosis are significantly impaired in DR3-null mice. In contrast, both superantigen-induced negative selection and positive selection are normal. The pre-T-cell receptor-mediated checkpoint, which is dependent on TNFR signaling, is also unaffected in DR3-deficient mice. These data reveal a nonredundant in vivo role for this TNF receptor family member in the removal of self-reactive T cells in the thymus.
Assuntos
Receptores do Fator de Necrose Tumoral/imunologia , Linfócitos T/imunologia , Animais , Apoptose/genética , Apoptose/imunologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Regulação da Expressão Gênica/imunologia , Humanos , Camundongos , Camundongos Knockout , Receptores do Fator de Necrose Tumoral/genética , Membro 25 de Receptores de Fatores de Necrose Tumoral , Recombinação Genética , Linfócitos T/citologiaRESUMO
OBJECTIVES: This study investigated concerns that have been raised about past and future health effects caused by high power transmissions of high frequency (7-30 MHz) radio waves from military antenna systems at Akrotiri, Cyprus. METHODS: A cross-sectional study of three villages (two exposed, one unexposed) collected longitudinal and short-term radiofrequency measurements. Health data were collected using questionnaires containing information on demographic factors, specific illnesses, general health (SF-36 well-being questionnaire), reproductive history, childhood illnesses, risk perception and mortality. Analysis was with SPSS v11.5 using cross tabulations of non-parametric data and tests for significance. Key health outcomes were subjected to logistic regression analysis. RESULTS: Field strengths within the two "exposed" villages were a maximum of 0.30 (Volts/Vm(-1) metre) from the 17.6 MHz military transmissions and up to 1.4 Vm(-1) from unspecified sources, mainly cell phone frequencies. The corresponding readings in the control village were <0.01 Vm(-1). Compared with the control village there were highly significant differences in the reporting of migraine (OR 2.7, p<0.001), headache (OR 3.7, p<0.001), and dizziness (OR 2.7, p<0.001). Residents of the exposed villages showed greater negative views of their health in all eight domains of the SF-36. There were also higher levels of perceived risk, particularly to noise and electromagnetic "pollution". All three villages reported higher values of risk perception than a UK population. There was no evidence of birth abnormalities or differences in gynaecological or obstetric history. Numbers of cancers were too small to show differences. CONCLUSION: It was clear that even this close (1-3 km) to powerful transmissions, the dominant sources of radiofrequency fields were cell phone and national broadcast systems. There was no excess of cancer, birth defects or obstetric problems. There was heightened risk perception and a considerable excess of migraine, headache and dizziness, which appears to share a gradient with radiofrequency exposure. The authors report this association but suggest this is unlikely to be an effect of radiofrequency and more likely to be antenna visibility or aircraft noise.
Assuntos
Exposição Ambiental/efeitos adversos , Nível de Saúde , Ondas de Rádio/efeitos adversos , Adulto , Causas de Morte , Criança , Estudos Transversais , Chipre/epidemiologia , Transtorno Depressivo/epidemiologia , Transtorno Depressivo/etiologia , Tontura/epidemiologia , Tontura/etiologia , Saúde Ambiental/estatística & dados numéricos , Feminino , Transtornos da Cefaleia/epidemiologia , Transtornos da Cefaleia/etiologia , Humanos , Masculino , Análise de Regressão , Fatores de Risco , Inquéritos e QuestionáriosRESUMO
BACKGROUND: In the United Kingdom, tuberculosis (TB) predominantly affects the most deprived populations, yet the extent to which deprivation affects TB care outcomes is unknown. METHODS: Since 2011, the North West TB Cohort Audit collaboration has undertaken quarterly reviews of outcomes against consensus-defined care standard indicators for all individuals notified with TB. We investigated associations between adverse TB care outcomes and Index of Multiple Deprivation (IMD) 2010 scores measured at lower super output area of residence using logistic regression models. RESULTS: Of 1831 individuals notified with TB between 2011 and 2014, 62% (1131/1831) came from the most deprived national quintile areas. In single variable analysis, greater deprivation was significantly associated with increased likelihood of the completion of a standardised risk assessment (OR 2.99, 95%CI 5.27-19.65) and offer of a human immunodeficiency virus test (OR 1.72, 95%CI 1.10-2.62). In multivariable analysis, there were no significant associations. CONCLUSIONS: TB patients in the most deprived areas had similar care indicators across a range of standards to those of individuals living in the more affluent areas, suggesting that the delivery of TB care in the North West of England is equitable. The extent to which the cohort review process contributes to, and sustains, this standard of care deserves further study.
Assuntos
Tuberculose/epidemiologia , Tuberculose/terapia , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Etnicidade , Feminino , Humanos , Lactente , Recém-Nascido , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Fatores de Risco , Fatores Socioeconômicos , Resultado do Tratamento , Reino Unido/epidemiologia , Adulto JovemRESUMO
Colorectal cancer is a major cause of cancer deaths in Western countries, but epidemiological data suggest that dietary modification might reduce these by as much as 90%. Cyclo-oxygenase 2 (COX2), an inducible isoform of prostaglandin H synthase, which mediates prostaglandin synthesis during inflammation, and which is selectively overexpressed in colon tumours, is thought to play an important role in colon carcinogenesis. Curcumin, a constituent of turmeric, possesses potent anti-inflammatory activity and prevents colon cancer in animal models. However, its mechanism of action is not fully understood. We found that in human colon epithelial cells, curcumin inhibits COX2 induction by the colon tumour promoters, tumour necrosis factor alpha or fecapentaene-12. Induction of COX2 by inflammatory cytokines or hypoxia-induced oxidative stress can be mediated by nuclear factor kappa B (NF-kappaB). Since curcumin inhibits NF-kappaB activation, we examined whether its chemopreventive activity is related to modulation of the signalling pathway which regulates the stability of the NF-kappaB-sequestering protein, IkappaB. Recently components of this pathway, NF-kappaB-inducing kinase and IkappaB kinases, IKKalpha and beta, which phosphorylate IkappaB to release NF-kappaB, have been characterised. Curcumin prevents phosphorylation of IkappaB by inhibiting the activity of the IKKs. This property, together with a long history of consumption without adverse health effects, makes curcumin an important candidate for consideration in colon cancer prevention.
Assuntos
Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Curcumina/farmacologia , Inibidores Enzimáticos/farmacologia , Isoenzimas/metabolismo , NF-kappa B/efeitos dos fármacos , Prostaglandina-Endoperóxido Sintases/metabolismo , Antineoplásicos/farmacologia , Ácidos Cafeicos/farmacologia , Ciclo-Oxigenase 2 , Relação Dose-Resposta a Droga , Humanos , Quinase I-kappa B , Proteínas I-kappa B/efeitos dos fármacos , Proteínas I-kappa B/metabolismo , Isoenzimas/efeitos dos fármacos , Proteínas de Membrana , NF-kappa B/genética , NF-kappa B/metabolismo , Álcool Feniletílico/análogos & derivados , Álcool Feniletílico/farmacologia , Polienos/farmacologia , Prostaglandina-Endoperóxido Sintases/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Acetato de Tetradecanoilforbol/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Quinase Induzida por NF-kappaBRESUMO
Under axenic growth all 14 syngens of Paramecium aurelia have 4 types of esterases. The three major types (A, B and cathodal C) vary independently in electrophoretic mobility among the syngens. Using these three esterases, stocks can be keyed to a syngen, except for the groupings 1-3-5 and 7-13. Using 5 other enzymes only syngens 1 and 5 cannot be distinguished. Most syngens differ from each other in 6 out of the 8 enzymes. An axenically-grown stock of Paramecium multimicronucleatum collected in Costa Rica has the same types of esterases as P. aurelia. Two of the types (A and C) are similar in mobility to those found in syngens 7 and 13, but its B esterase differs in mobility from all the known syngens of P. aurelia.
RESUMO
The unfolded protein response (UPR) occurs following the accumulation of unfolded proteins in the endoplasmic reticulum (ER) and orchestrates an intricate balance between its prosurvival and apoptotic arms to restore cellular homeostasis and integrity. However, in certain neurodegenerative diseases, the apoptotic arm of the UPR is enhanced, resulting in excessive neuronal cell death and disease progression, both of which can be overcome by modulating the UPR. Here, we describe a novel crosstalk between glucocorticoid receptor signaling and the apoptotic arm of the UPR, thus highlighting the potential of glucocorticoid therapy in treating neurodegenerative diseases. Several glucocorticoids, but not mineralocorticoids, selectively antagonize ER stress-induced apoptosis in a manner that is downstream of and/or independent of the conventional UPR pathways. Using GRT10, a novel selective pharmacological modulator of glucocorticoid signaling, we describe the importance of the transrepression arm of the glucocorticoid signaling pathway in protection against ER stress-induced apoptosis. Furthermore, we also observe the protective effects of glucocorticoids in vivo in a Drosophila model of Huntington's disease (HD), wherein treatment with different glucocorticoids diminished rhabdomere loss and conferred neuroprotection. Finally, we find that growth differentiation factor 15 has an important role downstream of glucocorticoid signaling in antagonizing ER stress-induced apoptosis in cells, as well as in preventing HD-mediated neurodegeneration in flies. Thus, our studies demonstrate that this novel crosstalk has the potential to be effectively exploited in alleviating several neurodegenerative disorders.
Assuntos
Proteínas do Tecido Nervoso/metabolismo , Doenças Neurodegenerativas/metabolismo , Receptores de Glucocorticoides/metabolismo , Animais , Apoptose/genética , Apoptose/fisiologia , Drosophila , Estresse do Retículo Endoplasmático/genética , Estresse do Retículo Endoplasmático/fisiologia , Células HeLa , Humanos , Proteínas do Tecido Nervoso/genética , Doenças Neurodegenerativas/genética , Receptores de Glucocorticoides/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Resposta a Proteínas não Dobradas/genética , Resposta a Proteínas não Dobradas/fisiologiaRESUMO
We have examined the effect of changes in the concentration of extracellular calcium on parathyroid hormone mRNA in both short-term (hours) and long-term (days) cultures of bovine parathyroid tissue. Using a 32P-labeled PreProPTH cDNA probe, PTH mRNA was measured by gel blot hybridization of total RNA from tissue slices incubated for 4 h in low (0.5 mM) or high (5 mM) calcium concentrations and also by dot blot hybridization of cytoplasmic RNA extracted from aggregates of partially dispersed cells cultured up to 72 h in low (0.4 mM), normal (1 mM), or high (3 mM) calcium concentrations. PTH mRNA was unchanged over 4 h while high calcium had suppressed PTH secretion. However PTH mRNA did respond during long-term culture. By 24 h in high calcium there was a 50% suppression which was maintained for a further 48 h. PTH mRNA in normal calcium remained unchanged over 72 h while in low calcium it had increased slightly by 48 h. In contrast to the effect seen in cultured bovine parathyroid cells, PTH mRNA in human parathyroid adenoma cells cultured for 48 h in high calcium was decreased by only 10%.
Assuntos
Cálcio/farmacologia , Glândulas Paratireoides/efeitos dos fármacos , Hormônio Paratireóideo/genética , RNA Mensageiro/genética , Adenoma/genética , Animais , Bovinos , Técnicas de Cultura , Citoplasma , Eletroforese em Gel de Ágar , Hibridização de Ácido Nucleico , Hormônio Paratireóideo/metabolismo , Neoplasias das Paratireoides/genética , RNA/isolamento & purificação , RadioimunoensaioRESUMO
The aim of this study was to investigate the mechanism by which translation of parathyroid hormone (PTH) mRNA is regulated with regard to the subcellular distribution of PTH mRNA and RNA:protein interactions. Sucrose density ultracentrifugation of RNA from bovine parathyroid cells indicated that there was no evidence for a pool of nonribosomal PTH mRNA, and the extracellular calcium concentration had no effect on polysome size. UV cross-linking studies revealed two proteins in parathyroid cell cytosol which bound specifically to the 5'-untranslated region (UTR) of PTH mRNA with molecular masses of 66 and 68 kD while proteins with apparent molecular masses of 48 and 70 kD bound to the 3'-UTR. In vitro translation assays indicated that parathyroid cell cytosol contains factors that inhibit translation of PTH mRNA. Fractionation of cytosol revealed that this effect was associated with proteins within the molecular mass range 30-90 kD. To determine which sequences in PTH mRNA mediate translational regulation, RNA was synthesized from luciferase gene constructs containing the 5'- and/or 3'-UTR of PTH mRNA, and translated in vitro. Addition of parathyroid cell cytosol reduced the translation of RNA containing the 5'- and 3'-UTR of PTH mRNA by 44 +/- 7% but had no effect on the translation of RNA containing only the luciferase coding region. Translation of RNA containing only the 5'-UTR of PTH mRNA was unchanged; however, cytosol reduced the translation of RNA containing the 3'-UTR by 31 +/- 9%. These data demonstrate a role for RNA:protein interactions in the regulation of PTH synthesis and that translational control is mediated primarily through interactions with the 3'-UTR of PTH mRNA.
Assuntos
Regulação da Expressão Gênica , Glândulas Paratireoides/metabolismo , Hormônio Paratireóideo/genética , Animais , Northern Blotting , Cálcio/farmacologia , Bovinos , Células Cultivadas , Citosol/fisiologia , Glândulas Paratireoides/citologia , Polirribossomos/efeitos dos fármacos , Ligação Proteica/fisiologia , Biossíntese de Proteínas/efeitos dos fármacos , Biossíntese de Proteínas/fisiologia , Processamento de Proteína Pós-Traducional , RNA Mensageiro/análise , RNA Mensageiro/metabolismoRESUMO
We reported previously that genetic polymorphisms of the alpha 2-adrenergic receptor are associated with hyperinsulinemia, diabetes mellitus, and hypertension in blacks. The evolutionary driving force for maintaining such deleterious mutations in the black population is unknown. Recognizing that vascular alpha 2-adrenergic receptors mediate cold-induced vasoconstriction and that temperature maintenance is a primary thrust of cellular metabolism, we postulated that vascular alpha 2-adrenergic receptors contribute significantly to metabolic heat generation in homeotherms such as humans. Using aerobic lactate production as an indicator of thermogenesis, we measured metabolic heat production in HT29 cells that expressed the gene encoding human vascular alpha 2-adrenergic receptors. Epinephrine, an alpha 2-adrenergic receptor agonist, increased net lactate efflux from 226 +/- 20 to 280 +/- 20 nmol/min (mean +/- SE) (P = .06). Clonidine, a more specific alpha 2-adrenergic agonist, increased lactate efflux from 110 +/- 6 to 156 +/- 8 nmol/min (P < .01). Similarly, in the presence of physiological concentrations of glucose (5.5 mmol/L), insulin increased lactate production from 123 +/- 6 to 175 +/- 10 nmol/min (P < .01). Because differences in aerobic glycolysis may also explain the heat intolerance and abnormal fuel homeostasis found in genetically hypertensive rats, we also measured lactate production in cultured vascular smooth muscle cells isolated from stroke-prone spontaneously hypertensive rats (SHRSP) and normotensive control Wistar-Kyoto rats (WKY). Vascular smooth muscle cells from SHRSP had significantly greater lactate efflux compared with cells from normotensive WKY (296 +/- 4 versus 172 +/- 2 nmol/min, P < .001). These differences were not due to abnormalities in glucose uptake, as lactate efflux was greater in SHRSP cells compared with WKY cells when dextrose was replaced with equimolar concentrations of fructose (230 +/- 6 versus 138 +/- 2 nmol/min, P < .001). alpha 2-Adrenergic agonists increase lactate efflux in HT29 cells, and abnormalities in vascular smooth muscle lactate metabolism in genetically hypertensive rats is independent of altered glucose uptake. These data provide support for our hypothesis that balanced polymorphisms of the alpha 2-adrenergic receptor could offer protection against cold stress by increasing the thermogenic response associated with aerobic lactate production.
Assuntos
Agonistas alfa-Adrenérgicos/farmacologia , Lactatos/metabolismo , Animais , Regulação da Temperatura Corporal , Células Cultivadas , Transtornos Cerebrovasculares/genética , Clonidina/farmacologia , Epinefrina/farmacologia , Predisposição Genética para Doença , Humanos , Insulina/farmacologia , Ácido Láctico , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Ratos , Ratos Endogâmicos SHR/genética , Ratos Endogâmicos WKYRESUMO
We studied the effect of yohimbine, a drug that inhibits presynaptic alpha 2-adrenergic receptors and increases the neuronal release of norepinephrine from the central and sympathetic nervous systems, on tolerance to cardiovascular stress in 10 untrained, healthy subjects. Using radioligand binding of tritiated yohimbine to platelets, these subjects were found to have a normal complement of alpha 2-adrenergic receptors (174 +/- 18 [+/- SEM] receptors/platelet) with normal Kd (1.93 +/- 0.17 nmol/l). Lower body negative pressure was used to test responses to cardiovascular stress in the subjects after they received either placebo or 20 mg yohimbine. Graded lower body negative pressure from 0 to -40 mm Hg significantly decreased systolic blood pressure from 116 +/- 3.7 to 106 +/- 5.8 mm Hg, increased heart rate from 54 +/- 3 to 68 +/- 7 beats/min, decreased forearm blood flow from 1.8 +/- 0.21 to 1.36 +/- 0.25 ml/100 ml/min, and increased forearm vascular resistance from 55.76 +/- 12.1 to 77.26 +/- 15.8 mm Hg/ml/min. Yohimbine increased the blood pressure at rest and during lower body negative pressure, but these changes were not significantly different from values recorded from the individuals when they were given placebo. Compared with placebo, however, yohimbine significantly increased forearm blood flow at rest (1.80 +/- 0.21 vs. 2.66 +/- 0.31 ml/100 ml/min, p less than 0.05) and during -40 mm Hg of lower body negative pressure (1.36 +/- 0.25 vs. 1.91 +/- 0.28 ml/100 ml/min, p less than 0.05). We also found that yohimbine significantly increased the plasma insulin concentration in these fasted subjects (9.4 +/- 2.4 vs. 14.5 +/- 1.4 ng/ml, p less than 0.05) without inducing hypoglycemia.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Antagonistas Adrenérgicos alfa/farmacologia , Sistema Cardiovascular/fisiopatologia , Gravitação , Hipotensão Ortostática/fisiopatologia , Ioimbina/farmacologia , Adulto , Pressão Sanguínea/efeitos dos fármacos , Feminino , Antebraço/irrigação sanguínea , Humanos , Pressão Negativa da Região Corporal Inferior , Masculino , Pessoa de Meia-Idade , Norepinefrina/sangue , Receptores Adrenérgicos alfa/metabolismo , Fluxo Sanguíneo Regional , DescansoRESUMO
Hereditary vitamin D resistant rickets has been associated with a number of mutations within the DNA and ligand binding domains of vitamin D receptors (VDR). The aim of our study was to identify and characterize the causative mutations in three kindreds with this condition. Resistance of 1,25(OH)2D3 was confirmed in cultured skin fibroblasts in which there was no induction of 24-hydroxylase activity; binding of 1,25(OH)2D3 to VDR was undetectable in patients 1 and 2, but normal in patients 3 and 4. The coding region of the VDR gene was sequenced to seek mutations. A mutation in the VDR gene of patient 1 resulted in a STOP codon, patient 2 showed a 56 bp deletion leading to frameshift and premature termination of VDR; a point mutation of A to C lying within the hormone-binding domain was shown for patients 3 and 4, who were siblings. Transactivation studies confirmed that these were functional mutations. Gel shift assays using nuclear extract from patient 3 demonstrated that the mutation that altered a conserved amino acid (glutamine-259) known to be involved in heterodimerization with other nuclear receptors affected protein: protein interactions.