RESUMO
Access to knowledge has never been easier in the internet age, and so it is important that students develop skills to discriminate undependable information from reliably investigated research. We have created an exercise that teaches good research practice by exploring the history, ethics, and design of clinical trials. Students apply their understanding of these principles through an assessed systematic review and meta-analysis (SRMA) exercise. Here, a clinically themed hypothesis is tested using a structured literature search in conjunction with an eligibility matrix to map study design, ethics, subject selection, randomization and blinding, methodological standards, study power, and other potential sources of interstudy heterogeneity. Data extracted from selected studies are used to produce a forest plot with an aggregated effect size, confidence range, and measure of interstudy heterogeneity. A funnel plot is then used in conjunction with the eligibility matrix to evaluate study bias tendency, and, in this way, students reflect on the factors that promote disparate conclusion-making among studies with a common research focus. This exercise produced a normally distributed grade-profile across three academic-year cohorts, and comparison of individual exercise grade with year-long aggregated average suggested students who performed less well on conventional assignments engaged successfully with the systematic nature of this assessment. Those opting to use this format for their final-year capstone project also performed above their grade point average from the preceding year. We suggest that SRMA offers a readily applied method for students to quantitatively explore how differences in experimental research practices influence study dependability.
Assuntos
Exercício Físico , Estudantes , HumanosRESUMO
The existence of a nutrient sensitive "autocatakinetic" regulator of embryonic tissue growth has been hypothesised since the early 20th century, beginning with pioneering work on the determinants of foetal size by the Australian physiologist, Thorburn Brailsford-Robertson. We now know that the mammalian target of rapamycin complexes (mTORC1 and 2) perform this essential function in all eukaryotic tissues by balancing nutrient and energy supply during the first stages of embryonic cleavage, the formation of embryonic stem cell layers and niches, the highly specified programmes of tissue growth during organogenesis and, at birth, paving the way for the first few breaths of life. This review provides a synopsis of the role of the mTOR complexes in each of these events, culminating in an analysis of lung branching morphogenesis as a way of demonstrating the central role mTOR in defining organ structural complexity. We conclude that the mTOR complexes satisfy the key requirements of a nutrient sensitive growth controller and can therefore be considered as Brailsford-Robertson's autocatakinetic centre that drives tissue growth programmes during foetal development.
Assuntos
Pulmão/embriologia , Complexos Multiproteicos/metabolismo , Organogênese/fisiologia , Serina-Treonina Quinases TOR/metabolismo , Gastrulação/fisiologia , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Alvo Mecanístico do Complexo 2 de Rapamicina , Transdução de SinaisRESUMO
Birt-Hogg-Dubé (BHD) syndrome is an autosomal dominant disorder where patients are predisposed to kidney cancer, lung and kidney cysts and benign skin tumors. BHD is caused by heterozygous mutations affecting folliculin (FLCN), a conserved protein that is considered a tumor suppressor. Previous research has uncovered multiple roles for FLCN in cellular physiology, yet it remains unclear how these translate to BHD lesions. Since BHD manifests hallmark characteristics of ciliopathies, we speculated that FLCN might also have a ciliary role. Our data indicate that FLCN localizes to motile and non-motile cilia, centrosomes and the mitotic spindle. Alteration of FLCN levels can cause changes to the onset of ciliogenesis, without abrogating it. In three-dimensional culture, abnormal expression of FLCN disrupts polarized growth of kidney cells and deregulates canonical Wnt signalling. Our findings further suggest that BHD-causing FLCN mutants may retain partial functionality. Thus, several BHD symptoms may be due to abnormal levels of FLCN rather than its complete loss and accordingly, we show expression of mutant FLCN in a BHD-associated renal carcinoma. We propose that BHD is a novel ciliopathy, its symptoms at least partly due to abnormal ciliogenesis and canonical Wnt signalling.
Assuntos
Síndrome de Birt-Hogg-Dubé/fisiopatologia , Cílios/fisiologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Sequência de Bases , Síndrome de Birt-Hogg-Dubé/genética , Linhagem Celular , Polaridade Celular , Proliferação de Células , Centrossomo/fisiologia , Cílios/patologia , Humanos , Rim/fisiologia , Microtúbulos/fisiologia , Dados de Sequência Molecular , Análise de Sequência de DNA , Via de Sinalização WntRESUMO
Sprouty2 (Spry2) acts as a central regulator of tubular growth and branch patterning in the developing mammalian lung by controlling both magnitude and duration of growth factor signalling. To determine if this protein coordinates airway and vascular growth factor signalling, we tested the hypothesis that Spry2 links the primary cue for airway outgrowth, fibroblast growth factor-10 (FGF-10), to genomic events underpinning the expression and release of vascular endothelial growth factor-A (VEGF-A). Using primary fetal distal lung epithelial cells (FDLE) from rat, and immortalised human bronchial epithelial cells (16HBE14o-), we identified a nuclear sub-population of Spry2 which interacted with regions of the rat and human VEGF-A promoter spanning the hypoxia response element (HRE) and adjacent 3' sites. In FDLE cultured at the PO2 of the fetal lung, FGF-10 relieved the Spry2 interaction at the HRE region by promoting clearance of a 39â¯kDa form and this was accompanied by histone-3 S10K14 phosphoacetylation, promoter de-methylation, hypoxia inducible factor-1α activation and VEGF-A expression. This repressive characteristic of nuclear Spry2 was relieved in 16HBE14o- by shRNA knockdown, and stable expression of mutants (C218A; C221A) that do not interact with the VEGF-A promoter HRE region. We conclude that nuclear Spry2 acts as a molecular link which co-ordinates airway and vascular growth of the cardiopulmonary system. This identifies Spry2 as a contributing determinant of design optimality in the mammalian lung.
Assuntos
Células Epiteliais/metabolismo , Feto , Pulmão , Proteínas do Tecido Nervoso/metabolismo , Transdução de Sinais/fisiologia , Animais , Feto/irrigação sanguínea , Feto/embriologia , Fator 10 de Crescimento de Fibroblastos/genética , Fator 10 de Crescimento de Fibroblastos/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Pulmão/irrigação sanguínea , Pulmão/embriologia , Pulmão/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/genética , Ratos , Ratos Sprague-Dawley , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
BACKGROUND: Precision medicine aims to combat the variability of the therapeutic response to a given medicine by delivering the right medicine to the right patient. However, the application of precision medicine is predicated on a prior quantitation of the variance of the reference range of normality. Airway pathophysiology provides a good example due to a very variable first line of defence against airborne assault. Humans differ in their susceptibility to inhaled pollutants and pathogens in part due to the magnitude of trans-epithelial resistance that determines the degree of epithelial penetration to the submucosal space. This initial 'set-point' may drive a sentinel event in airway disease pathogenesis. Epithelia differentiated in vitro from airway biopsies are commonly used to model trans-epithelial resistance but the 'reference range of normality' remains problematic. We investigated the range of electrophysiological characteristics of human airway epithelia grown at air-liquid interface in vitro from healthy volunteers focusing on the inter- and intra-subject variability both at baseline and after sequential exposure to drugs modulating ion transport. METHODOLOGY/PRINCIPAL FINDINGS: Brushed nasal airway epithelial cells were differentiated at air-liquid interface generating 137 pseudostratified ciliated epithelia from 18 donors. A positively-skewed baseline range exists for trans-epithelial resistance (Min/Max: 309/2963 Ω·cm2), trans-epithelial voltage (-62.3/-1.8 mV) and calculated equivalent current (-125.0/-3.2 µA/cm2; all non-normal, P<0.001). A minority of healthy humans manifest a dramatic amiloride sensitivity to voltage and trans-epithelial resistance that is further discriminated by prior modulation of cAMP-stimulated chloride transport. CONCLUSIONS/SIGNIFICANCE: Healthy epithelia show log-order differences in their ion transport characteristics, likely reflective of their initial set-points of basal trans-epithelial resistance and sodium transport. Our data may guide the choice of the background set point in subjects with airway diseases and frame the reference range for the future delivery of precision airway medicine.
Assuntos
Resistência das Vias Respiratórias/efeitos dos fármacos , Amilorida/farmacologia , Mucosa Nasal/fisiologia , Adolescente , Adulto , Impedância Elétrica , Fenômenos Eletrofisiológicos , Células Epiteliais/efeitos dos fármacos , Feminino , Humanos , Transporte de Íons/efeitos dos fármacos , Masculino , Mucosa Nasal/efeitos dos fármacos , Estatística como Assunto , Adulto JovemRESUMO
Serum and glucocorticoid-inducible kinase 1 (SGK1) is a protein kinase that contributes to the hormonal control of renal Na(+) retention by regulating the abundance of epithelial Na(+) channels (ENaC) at the apical surface of the principal cells of the cortical collecting duct (CCD). Although glucocorticoids and insulin stimulate Na(+) transport by activating SGK1, the responses follow different time courses suggesting that these hormones act by different mechanisms. We therefore explored the signaling pathways that allow dexamethasone and insulin to stimulate Na(+) transport in mouse CCD cells (mpkCCDcl4). Dexamethasone evoked a progressive augmentation of electrogenic Na(+) transport that became apparent after ~45 min latency and was associated with increases in SGK1 activity and abundance and with increased expression of SGK1 mRNA Although the catalytic activity of SGK1 is maintained by phosphatidylinositol-OH-3-kinase (PI3K), dexamethasone had no effect upon PI3K activity. Insulin also stimulated Na(+) transport but this response occurred with no discernible latency. Moreover, although insulin also activated SGK1, it had no effect upon SGK1 protein or mRNA abundance. Insulin did, however, evoke a clear increase in cellular PI3K activity. Our data are consistent with earlier work, which shows that glucocorticoids regulate Na(+) retention by inducing sgk1 gene expression, and also establish that this occurs independently of increased PI3K activity. Insulin, on the other hand, stimulates Na(+) transport via a mechanism independent of sgk1 gene expression that involves PI3K activation. Although both hormones act via SGK1, our data show that they activate this kinase by distinct physiological mechanisms.
Assuntos
Dexametasona/farmacologia , Proteínas Imediatamente Precoces/metabolismo , Insulina/farmacologia , Córtex Renal/enzimologia , Túbulos Renais Coletores/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Córtex Renal/efeitos dos fármacos , Túbulos Renais Coletores/efeitos dos fármacos , CamundongosRESUMO
Throughout gestation, low oxygen tensions are a dominant feature of the fetal environment and so may be important in sustaining a normal pattern of lung morphogenesis until the moment of birth. As breathing begins, the equilibration of the lung lumen to postnatal PO2 evokes a series of physiologic and morphogenic maturation events that are partially reversible by hypoxia. In this review, we discuss the experimental evidence that fetal and perinatal oxygen tensions differently influence lung morphogenesis through oxygen- and redox-responsive signaling pathways and identify five loci at which this regulation may occur: (I) proliferation of undifferentiated lung mesenchyme as governed by hypoxia-regulated transcription factors (HIF-1alpha, C/EBPbeta); (II) transient production of reactive oxygen species (ROS) and nuclear oxidation of the perinatal lung epithelium; (III) nuclear transport and oxidation of thioredoxin in hand with the acute activation of nuclear factor- kappaB (NF-kappaB); (IV) ROS-evoked chronic rise in intracellular glutathione and thioredoxin redox buffering capacity; and (V) NF-kappaB-dependent increase in transepithelial Na+ transport and lung lumenal fluid clearance. Although not exhaustive, this analysis leads us to the conclusion that redox events that occur in the lung during gestation, parturition, and the early neonatal period may dramatically influence the expression of genes and physiological events that are crucial to the successful transition from fetal to postnatal lung maturation.
Assuntos
Células Epiteliais/citologia , Regulação da Expressão Gênica , Pulmão/crescimento & desenvolvimento , Pulmão/fisiologia , Oxirredução , Animais , Transporte Biológico , Núcleo Celular/metabolismo , Proliferação de Células , Glutationa/metabolismo , Humanos , Hipóxia , Imuno-Histoquímica , Lipopolissacarídeos/metabolismo , Pulmão/embriologia , Pulmão/metabolismo , Modelos Biológicos , NF-kappa B/metabolismo , Oxigênio/metabolismo , Placenta/metabolismo , Ratos , Espécies Reativas de Oxigênio , Fenômenos Fisiológicos Respiratórios , Sódio/metabolismo , Tiorredoxinas/metabolismoRESUMO
The regulation of cytokine gene transcription and biosynthesis involves the reduction-oxidation (redox)-sensitive nuclear factor-kappaB (NF-kappaB), whose activation is mediated by an upstream kinase that regulates the phosphorylation of inhibitory-kappaB (IkappaB). It was hypothesized that lipopolysaccharide (LPS)-induced biosynthesis of interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha in vitro is regulated by redox equilibrium. In alveolar epithelial cells, we investigated the role of L-buthionine-(S,R)-sulfoximine (BSO), an irreversible inhibitor of gamma-glutamylcysteine synthetase, the rate-limiting enzyme in GSH biosynthesis, 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU), which inhibits glutathione oxidized disulfide reductase, pyrrolidine dithiocarbamate (PDTC), an antioxidant/prooxidant thiuram, and N-acetyl-L-cysteine (NAC), an antioxidant and GSH precursor, in regulating LPS-induced cytokine biosynthesis and IkappaB-alpha/NF-kappaB signaling. BSO blockaded the phosphorylation of IkappaB-alpha, reduced its degradation, and inhibited NF-kappaB activation, besides augmenting LPS-mediated biosynthesis of cytokines. BCNU up-regulated LPS-induced release of cytokines, an effect associated with partial phosphorylation/degradation of IkappaB-alpha and inhibition of the DNA binding activity. PDTC, which partially affected LPS-induced IkappaB-alpha phosphorylation/degradation, otherwise blockading NF-kappaB activation, reduced LPS-dependent up-regulation of cytokine release. Pretreatment with BSO did not abolish the NAC-dependent reduction of LPS-induced cytokine release, despite the fact that NAC marginally amplified IkappaB-alpha phosphorylation/degradation and suppressed NF-kappaB activation. These results indicate that cytokines are redox-sensitive mediators and that the IkappaB-alpha/NF-kappaB pathway is redox-sensitive and differentially implicated in mediating redox-dependent regulation of LPS-induced release of proinflammatory cytokines.
Assuntos
Citocinas/biossíntese , Proteínas I-kappa B , Lipopolissacarídeos/metabolismo , Oxirredução , Alvéolos Pulmonares/citologia , Animais , Antineoplásicos Alquilantes/farmacologia , Carmustina/farmacologia , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Glutationa/metabolismo , Inibidor de NF-kappaB alfa , NF-kappa B/metabolismo , Fosforilação , Ratos , Ratos Sprague-Dawley , Transdução de SinaisRESUMO
Oxygen-sensing pathways have been extensively explored in the context of homeostatic responses to hypoxic episodes; however, little is known of their involvement in the morphogenesis of respiratory structures (mitochondria, placenta, lung) during development in utero. This review identifies four essential loci where oxygen signalling pathways may cue the development of respiratory structures as: (i). mitochondrial biogenesis coupled with muted oxidative function dependent on the hypoxia-sustained production of NO; (ii). the generation of oxygen gradients which drive trophoblast differentiation and the formation of the chorionic gas exchange interface of the placenta; (iii). the proliferation and epithelial/endothelial differentiation of mesenchyme during the initiation of lung morphogenesis; and (iv). the regulation of epithelial fluid secretion/absorption in the lung. The identification of these oxygen-regulated developmental stages clarifies the close association between oxygen availability, reactive oxygen species and the morphogenesis of gas exchange structures and bears with it the implication that these pathways set the scope for aerobic metabolic performance throughout life.
Assuntos
Oxigênio/metabolismo , Animais , Adesão Celular , Diferenciação Celular , Divisão Celular , Endotélio/citologia , Células Epiteliais/citologia , Humanos , Hipóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia , Ligantes , Pulmão/metabolismo , Potenciais da Membrana , Mitocôndrias/metabolismo , Modelos Biológicos , NADPH Oxidases/metabolismo , Óxido Nítrico/química , Óxido Nítrico/metabolismo , Placenta/metabolismo , Ratos , Espécies Reativas de Oxigênio , Transdução de Sinais , Fatores de Transcrição/metabolismo , Veias Umbilicais/metabolismoRESUMO
Hochachka's "Hypoxia Defense Strategies" identify oxygen signalling, metabolic arrest, channel arrest and coordinated suppression of ATP turnover rates as key factors that determine the ability of organisms to survive exposure to chronic hypoxia. In this review, I assess the developmental role played by these phenomena in the morphogenesis of the gas exchange tissues that define the pathway for oxygen transport to cytochrome c oxidase. Key areas of regulation lie in: (I) the suppression of fetal mitochondrial oxidative function in hand with mitochondrial biogenesis (metabolic arrest), (II) the role of hypoxia-driven oxygen signalling pathways in directing the scope of non-differentiated stem cell proliferation in placenta and lung development and (III) the regulation of epithelial fluid secretion/absorption in the lung through the oxygen-dependent modulation of Na+ conductance pathways. The identification of developmental roles for Hochachka's "Hypoxia Defense Strategies" in directing the morphogenesis of gas exchange structures bears with it the implication that these strategies are fundamental to establishing the scope for aerobic metabolic performance throughout life.
Assuntos
Hipóxia Celular/fisiologia , Pulmão/fisiologia , Oxigênio/fisiologia , Troca Gasosa Pulmonar/fisiologia , Transdução de Sinais , Trifosfato de Adenosina/metabolismo , Animais , Sobrevivência Celular , Regulação da Expressão Gênica , Canais Iônicos/fisiologia , Placenta/metabolismoRESUMO
BACKGROUND/AIMS: Chemokine signaling from airway epithelium regulates macrophage recruitment to the lung in inflammatory diseases such as asthma. This study investigates the mechanism by which the α-melanocyte stimulating hormone-derived tripeptide, KPV, and the agonist of the dominant melanocortin receptor in airway epithelium (MC3R), γ-melanocyte stimulating hormone (γ-MSH), suppress inflammation in immortalised human bronchial airway epithelium. METHODS: TNFα and rhino syncitial virus (RSV)-evoked nuclear factor-κB (NFκB) signaling was measured in immortalised human bronchial epithelial cells (16HBE14o-) in response to KPV and γMSH. Cellular and systemic inflammatory signaling was measured by NFκB reporter gene and chemokine (IL8, eotaxin) secretion, respectively. RESULTS: KPV and γMSH evoked a dose-dependent inhibition of NFκB, matrix metalloproteinase-9 activity, IL8 and eotaxin secretion. The KPV effect was associated with its nuclear import, IκBα stabilisation and suppressed nuclear translocation of YFP-tagged p65RelA. Competition assays revealed an interaction between KPV and the Imp-α3 binding site on p65RelA which may involve blockade of the importin-α armadillo domain 7 and 8. In contrast, the γMSH anti-inflammatory effect required MC3R whose apical expression occurred in epithelium distributed along the length of the respiratory tree in vivo. CONCLUSION: KPV and γMSH respectively suppress NFκB signalling in airway epithelium by: i) inhibition of p65RelA nuclear import and, ii) epithelial MC3R activation. Melanocortin peptides therefore provide a robust mechanism for targeting airway inflammation in lung disease.
RESUMO
BACKGROUND AND PURPOSE: Glucocorticoids appear to control Na⺠absorption in pulmonary epithelial cells via a mechanism dependent upon serum and glucocorticoid-inducible kinase 1 (SGK1), a kinase that allows control over the surface abundance of epithelial Na⺠channel subunits (α-, ß- and γ-ENaC). However, not all data support this model and the present study re-evaluates this hypothesis in order to clarify the mechanism that allows glucocorticoids to control ENaC activity. EXPERIMENTAL APPROACH: Electrophysiological studies explored the effects of agents that suppress SGK1 activity upon glucocorticoid-induced ENaC activity in H441 human airway epithelial cells, whilst analyses of extracted proteins explored the associated changes to the activities of endogenous protein kinase substrates and the overall/surface expression of ENaC subunits. KEY RESULTS: Although dexamethasone-induced (24 h) ENaC activity was dependent upon SGK1, prolonged exposure to this glucocorticoid did not cause sustained activation of this kinase and neither did it induce a coordinated increase in the surface abundance of α-, ß- and γ-ENaC. Brief (3 h) exposure to dexamethasone, on the other hand, did not evoke Na⺠current but did activate SGK1 and cause SGK1-dependent increases in the surface abundance of α-, ß- and γ-ENaC. CONCLUSIONS AND IMPLICATIONS: Although glucocorticoids activated SGK1 and increased the surface abundance of α-, ß- and γ-ENaC, these responses were transient and could not account for the sustained activation of ENaC. The maintenance of ENaC activity did, however, depend upon SGK1 and this protein kinase must therefore play an important but permissive role in glucocorticoid-induced ENaC activation.
Assuntos
Canais Epiteliais de Sódio/metabolismo , Proteínas Imediatamente Precoces/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Mucosa Respiratória/metabolismo , Linhagem Celular , Dexametasona/farmacologia , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Glucocorticoides/farmacologia , Humanos , Proteínas Imediatamente Precoces/antagonistas & inibidores , Potenciais da Membrana/efeitos dos fármacos , Fosfatidilinositol 3-Quinase/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Subunidades Proteicas/metabolismo , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/enzimologia , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
Tuberous sclerosis complex (TSC) is a genetic condition characterized by the growth of benign tumours in multiple organs, including the brain and kidneys, alongside intellectual disability and seizures. Identification of a causative mutation in TSC1 or TSC2 is important for accurate genetic counselling in affected families, but it is not always clear from genetic data whether a sequence variant is pathogenic or not. In vitro functional analysis could provide support for determining whether an unclassified TSC1 or TSC2 variant is disease-causing. We have performed a detailed functional analysis of four patient-derived TSC2 mutations, E92V, R505Q, H597R and L1624P. One mutant, E92V, functioned similarly to wild-type TSC2, whereas H597R and L1624P had abnormal function in all assays, consistent with available clinical and segregation information. One TSC2 mutation, R505Q, was identified in a patient with intellectual disability, seizures and autistic spectrum disorder but who did not fulfil the diagnostic criteria for TSC. The R505Q mutation was also found in two relatives, one with mild learning difficulties and one without apparent phenotypic abnormality. R505Q TSC2 exhibited partially disrupted function in our assays. These data highlight the difficulties of assessing pathogenicity of a mutation and suggest that multiple lines of evidence, both genetic and functional, are required to assess the pathogenicity of some mutations.
Assuntos
Mutação/genética , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Adolescente , Animais , Células Cultivadas , Criança , Pré-Escolar , Doenças Genéticas Inatas/genética , Doenças Genéticas Inatas/fisiopatologia , Genótipo , Células HEK293 , Humanos , Lactente , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Complexos Multiproteicos , Fenótipo , Fosforilação/genética , Multimerização Proteica/genética , Estabilidade Proteica , Proteínas/metabolismo , Serina-Treonina Quinases TOR , Proteína 1 do Complexo Esclerose Tuberosa , Proteína 2 do Complexo Esclerose TuberosaRESUMO
Tumors that form as a result of heightened mammalian target of rapamycin (mTOR) signaling are highly vascularized. This process of angiogenesis is regulated through hypoxia-inducible factor (HIF)-mediated transcription of angiogenic factors. It is recognized that inhibition of mTOR with rapamycin can diminish the process of angiogenesis. Our work shows that activation of mTOR by Ras homologue enriched in brain (Rheb) overexpression potently enhances the activity of HIF1alpha and vascular endothelial growth factor (VEGF)-A secretion during hypoxia, which is reversed with rapamycin. Mutants of Rheb, which do not bind guanine nucleotide (D60K, D60V, N119I, and D122N) and are unable to activate mTOR, inhibit the activity of HIF when overexpressed. We show that regulatory associated protein of mTOR (Raptor) interacts with HIF1alpha and requires an mTOR signaling (TOS) motif located in the N terminus of HIF1alpha. Furthermore, a mutant of HIF1alpha lacking this TOS motif dominantly impaired HIF activity during hypoxia and was unable to bind to the co-activator CBP/p300. Rapamycin treatments do not affect the stability of HIF1alpha and modulate HIF activity via a Von Hippel-Lindau (VHL)-independent mechanism. We demonstrate that the high levels of HIF activity in cells devoid of TSC2 can be reversed by treatments with rapamycin or the readdition of TSC2. Our work explains why human cancers with aberrant mTOR signaling are prone to angiogenesis and suggests that inhibition of mTOR with rapamycin might be a suitable therapeutic strategy.
Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias/metabolismo , Neovascularização Patológica/metabolismo , Proteínas Quinases/metabolismo , Transdução de Sinais , Motivos de Aminoácidos/genética , Animais , Antibióticos Antineoplásicos/farmacologia , Hipóxia Celular/efeitos dos fármacos , Hipóxia Celular/genética , Linhagem Celular Tumoral , Nucleotídeos de Guanina/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Camundongos , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Mutação de Sentido Incorreto , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/genética , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Proteínas Quinases/genética , Estrutura Terciária de Proteína/efeitos dos fármacos , Estrutura Terciária de Proteína/genética , Proteína Enriquecida em Homólogo de Ras do Encéfalo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Sirolimo/farmacologia , Serina-Treonina Quinases TOR , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Fatores de Transcrição de p300-CBP/genética , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
It has been previously reported that amiloride suppresses inflammatory cytokine biosynthesis. However, the molecular mechanism involved has yet to be ascertained. Therefore, the immunoregulatory potential mediated by amiloride and the underlying signaling transduction pathway was investigated. Exposure of alveolar epithelial cells to amiloride or its analog, 5-(N,N-hexamethylene)-amiloride (HMA), reduced, in a dose-dependent manner, lipopolysaccharide (LPS)-induced secretion of interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha. This inhibitory effect was associated with the augmentation of a counter antiinflammatory response, mediated by IL-6 and IL-10. Analysis of the mechanism implicated revealed the involvement of an inhibitory kappaB (IkappaB-alpha)/nuclear factor kappaB (NF- kappaB)-sensitive pathway. Amiloride and HMA suppressed the phosphorylation of IkappaB-alpha mediated by LPS, thereby allowing its cytosolic accumulation. Furthermore, both inhibitors interfered with the nuclear translocation of selective NF-kappaB subunits, an effect associated with blockading the DNA-binding activity of NF-kappaB. Recombinant IL-10 blockaded LPS-induced biosynthesis of IL-1beta and TNF-alpha and reduced NF-kappaB activation. Immunoneutralization of endogenous IL-10 reversed the inhibitory effect of amiloride on proinflammatory cytokines and restored the DNA-binding activity of NF-kappaB. These results indicate that amiloride acts as a novel dual immunoregulator in the alveolar epithelium: it downregulates an inflammatory signal and at the same time upregulates an antiinflammatory response. This biphasic effect is IL-10 sensitive and is associated with the selective targeting of the IkappaB-alpha/NF-kappaB signaling transduction pathway.
Assuntos
Amilorida/análogos & derivados , Amilorida/farmacologia , Citocinas/biossíntese , Proteínas de Ligação a DNA/biossíntese , Diuréticos/farmacologia , Células Epiteliais/metabolismo , Proteínas I-kappa B , Lipopolissacarídeos/farmacologia , NF-kappa B/biossíntese , Transporte Ativo do Núcleo Celular , Animais , Antineoplásicos/farmacologia , Células Cultivadas , Relação Dose-Resposta a Droga , Regulação para Baixo , Ensaio de Imunoadsorção Enzimática , Células Epiteliais/imunologia , Regulação da Expressão Gênica , Interleucina-1/biossíntese , Interleucina-10/biossíntese , Interleucina-6/biossíntese , Pulmão/citologia , Pulmão/embriologia , Modelos Biológicos , Inibidor de NF-kappaB alfa , Fosforilação , Regiões Promotoras Genéticas , Ratos , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Fatores de Tempo , Fator de Necrose Tumoral alfa/biossíntese , Regulação para CimaRESUMO
Chorioamnionitis is associated with increased risks of perinatal respiratory failure; however, components of the inflammatory acute-phase response are known to actively promote lung maturation. To manipulate this relationship, we examined the effect of the thymic immunomodulator thymulin on fetal lung mesenchyme-epithelial differentiation during exposure to Escherichia coli lipopolysaccharide (LPS). Gestation day 14 fetal rat lung explants were cultured for 96 h at fetal (23 mmHg) or ambient (142 mmHg) Po(2). Airway surface complexity (ASC, perimeter/ radical area(2)) was greater at fetal vs. ambient Po(2); however, exposure to 0.1-50 microg/ml LPS significantly raised ASC at 2 microg/ml in ambient Po(2) explants. LPS (50 microg/ml) depressed ASC in both conditions to untreated ambient Po(2) control values without changes in necrosis or apoptosis. To manipulate LPS-evoked TNF-alpha and IL-6 release, we exposed explants and A549 cells to combinations of 50 microg/ml LPS, 10 microM ZnCl(2), and 0.1-1,000 ng/ml thymulin at either Po(2). Thymulin+Zn(2+) suppressed and potentiated LPS-evoked TNF-alpha and IL-6 release, yielding an IC(50(TNF-alpha)) of 0.5 +/- 0.01 ng/ml and EC(50(IL-6)) of 1.4 +/- 0.3 ng/ml in A549 cells. This was accompanied by activation of the p38 MAPKMAPKAP-K2 pathway with sustained expression of TNF-alpha and IL-6 transcripts at ambient Po(2). LPS+thymulin+Zn(2+)-treated explants showed proliferation of CCAAT-enhancer binding protein-beta (C/EBPbeta) and fibroblast growth factor-9 immunoreactive mesenchyme, which was abolished by IL-6 antisense oligonucleotides. The posttranscriptional suppression of immunogenic TNF-alpha synthesis coupled with raised IL-6 and C/EBPbeta-dependent mesenchyme proliferation suggests a role for bioactive thymulin in regulating regenerative repair in the fetal lung.
Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/imunologia , Interleucina-6/imunologia , Pulmão/imunologia , Fator Tímico Circulante/farmacologia , Fator de Necrose Tumoral alfa/imunologia , Adenocarcinoma , Adjuvantes Imunológicos/farmacologia , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Diferenciação Celular , Divisão Celular , Linhagem Celular Tumoral , Feto , Fator 10 de Crescimento de Fibroblastos , Fator 9 de Crescimento de Fibroblastos , Fatores de Crescimento de Fibroblastos/metabolismo , Humanos , Hiperplasia , Interleucina-6/genética , Interleucina-6/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Lipopolissacarídeos/farmacologia , Pulmão/citologia , Pulmão/embriologia , Mesoderma/citologia , Oxigênio/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/imunologia , Fator de Necrose Tumoral alfa/genética , Zinco/farmacologiaRESUMO
In an attempt to elaborate in vitro on a therapeutic strategy that counteracts an inflammatory signal, we previously reported a novel immunopharmacological potential of glutathione, an antioxidant thiol, in regulating inflammatory cytokines. In the present study, we investigated the hypothesis that selective regulation of phosphodiesterases (PDEs), a family of enzymes that controls intracellular cAMP/cGMP degradation, differentially regulates proinflammatory cytokines. Selective PDE1 inhibition (8-methoxymethyl-3-isobutyl-1-methylxanthine) blockaded lipopolysaccharide-endotoxin (LPS)-mediated biosynthesis of interleukin (IL)-6, but this pathway had no inhibitory effect on tumor necrosis factor-alpha (TNF-alpha). Furthermore, inhibition of PDE3 (amrinone) abolished the effect of LPS on IL-6, but attenuated TNF-alpha production. Reversible competitive inhibition of PDE4 (rolipram) exhibited a potent inhibitory effect on IL-6 and a dual, biphasic (excitatory/inhibitory) effect on TNF-alpha secretion. Blockading PDE5 (4-[[3',4'-(methylenedioxy)benzyl] amino]-6-methoxyquinazoline) showed a high potency in reducing IL-6 production, but in a manner similar to the inhibition of PDE4, exhibited a biphasic effect on TNF-alpha biosynthesis. Simultaneous inhibition of PDE5, 6, and 9 (zaprinast), purported to specifically elevate intracellular cGMP, reduced, in a dose-independent manner, IL-6 and TNF-alpha biosynthesis. Finally, nonselective inhibition of PDE by pentoxifylline suppressed LPS-mediated secretion of IL-6 and TNF-alpha. The involvement of specific PDE isoenzymes in differentially regulating LPS-mediated inflammatory cytokine biosynthesis indicates a novel approach to unravel the potential therapeutic targets that these isozymes constitute during the progression of inflammation within the respiratory epithelium.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Células Epiteliais/metabolismo , Interleucina-6/biossíntese , Lipopolissacarídeos/farmacologia , Inibidores de Fosfodiesterase/farmacologia , Alvéolos Pulmonares/metabolismo , Fator de Necrose Tumoral alfa/biossíntese , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Células Epiteliais/efeitos dos fármacos , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Alvéolos Pulmonares/citologia , Alvéolos Pulmonares/efeitos dos fármacos , Ratos , Sistema Respiratório/metabolismo , Sistema Respiratório/patologiaRESUMO
In this report we investigated the immunopharmacological role of selective and nonselective phosphodiesterase (PDE) inhibition in regulating the inhibitory-kappaB (IkappaB-alpha)/nuclear factor-kappaB (NF-kappaB) signaling transduction pathway. In fetal alveolar type II epithelial cells, PDE blockade at the level of the diverging cAMP/cGMP pathways differentially regulated the phosphorylation and degradation of IkappaB-alpha, the major cytosolic inhibitor of NF-kappaB. Whereas selective inhibition of PDEs 1, 3, and 4, by the action of 8-methoxymethyl-3-isobutyl-1-methylxanthine, amrinone, and rolipram, respectively, exhibited a tendency to augment the translocation of NF-kappaB(1) (p50), RelA (p65), RelB (p68), and c-Rel (p75), selective blockade of PDE 5, 6, and 9, by the action of 4-[[3',4'-(methylenedioxy)benzyl]amino]-6-methoxyquinazoline and zaprinast, attenuated lipopolysaccharide-endotoxin (LPS)-mediated NF-kappaB translocation. Pentoxifylline, a nonspecific PDE inhibitor, reversed the excitatory effect of LPS on NF-kappaB subunit nuclear localization, in a dose-dependent manner. Furthermore, analysis of NF-kappaB activation under the same conditions revealed a biphasic effect mediated by LPS. PDEs 1, 3, and 4 inhibition was associated with up-regulating NF-kappaB transcriptional activity. In contrast, blockading the activity of PDEs 5, 6, and 9 negatively attenuated LPS-mediated NF-kappaB activation, similar to the effect of 3,7-dihydro-3,7-dimethyl-1-(5-oxohexyl)-1H-purine-2,6-dione (pentoxifylline). These results indicate that selective and nonselective interference with the control of the dynamic equilibrium of cyclic nucleotides via PDE isoenzyme regulation represents an immunoregulatory mechanism that requires the differential, biphasic targeting of the IkappaB-alpha/NF-kappaB pathway.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Células Epiteliais/efeitos dos fármacos , NF-kappa B/antagonistas & inibidores , Inibidores de Fosfodiesterase/farmacologia , Alvéolos Pulmonares/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Actinas/metabolismo , Animais , Núcleo Celular/efeitos dos fármacos , Células Cultivadas , Citosol/metabolismo , DNA/genética , DNA/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Inflamação/tratamento farmacológico , Inflamação/patologia , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Lipopolissacarídeos/farmacologia , NF-kappa B/genética , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Fosforilação , Alvéolos Pulmonares/citologia , Ratos , Transdução de Sinais/genética , Translocação Genética/efeitos dos fármacosRESUMO
In fetal pneumocytes, increasing P(O(2)) can raise apical Na(+) conductance (G(Na(+))) and increase the abundance of epithelial Na(+) channel subunit (alpha-, beta-, and gamma-ENaC) mRNA, suggesting that the rise in G(Na(+)), which may be important to the perinatal maturation of the lung, reflects O(2)-evoked ENaC gene expression. However, we now show that physiologically relevant increases in P(O(2)) do not affect alpha-, beta-, and gamma-ENaC mRNA abundance in pneumocytes maintained (approximately 48 h) in hormone-free medium or in medium supplemented with dexamethasone and tri-iodothyronine, although the response does persist in cells maintained in medium containing a complex mixture of hormones/growth factors. However, parallel electrometric studies revealed clear increases in G(Na(+)) under all tested conditions and so it is now clear that O(2)-evoked increases in G(Na(+)) can occur without corresponding increases in ENaC mRNA abundance. It is therefore unlikely that this rise in G(Na(+)) is secondary to O(2)-evoked ENaC gene expression.