RESUMO
BACKGROUND: Emerging studies suggest that enhanced glycolysis accompanies inflammatory responses. Virtually nothing is known about the relevance of glycolysis in patients with allergic asthma. OBJECTIVES: We sought to determine whether glycolysis is altered in patients with allergic asthma and to address its importance in the pathogenesis of allergic asthma. METHODS: We examined alterations in glycolysis in sputum samples from asthmatic patients and primary human nasal cells and used murine models of allergic asthma, as well as primary mouse tracheal epithelial cells, to evaluate the relevance of glycolysis. RESULTS: In a murine model of allergic asthma, glycolysis was induced in the lungs in an IL-1-dependent manner. Furthermore, administration of IL-1ß into the airways stimulated lactate production and expression of glycolytic enzymes, with notable expression of lactate dehydrogenase A occurring in the airway epithelium. Indeed, exposure of mouse tracheal epithelial cells to IL-1ß or IL-1α resulted in increased glycolytic flux, glucose use, expression of glycolysis genes, and lactate production. Enhanced glycolysis was required for IL-1ß- or IL-1α-mediated proinflammatory responses and the stimulatory effects of IL-1ß on house dust mite (HDM)-induced release of thymic stromal lymphopoietin and GM-CSF from tracheal epithelial cells. Inhibitor of κB kinase ε was downstream of HDM or IL-1ß and required for HDM-induced glycolysis and pathogenesis of allergic airways disease. Small interfering RNA ablation of lactate dehydrogenase A attenuated HDM-induced increases in lactate levels and attenuated HDM-induced disease. Primary nasal epithelial cells from asthmatic patients intrinsically produced more lactate compared with cells from healthy subjects. Lactate content was significantly higher in sputum supernatants from asthmatic patients, notably those with greater than 61% neutrophils. A positive correlation was observed between sputum lactate and IL-1ß levels, and lactate content correlated negatively with lung function. CONCLUSIONS: Collectively, these findings demonstrate that IL-1ß/inhibitory κB kinase ε signaling plays an important role in HDM-induced glycolysis and pathogenesis of allergic airways disease.
Assuntos
Asma/metabolismo , Hipersensibilidade/metabolismo , Interleucina-1beta/metabolismo , Pulmão/metabolismo , Nariz/patologia , Mucosa Respiratória/metabolismo , Escarro/metabolismo , Animais , Antígenos de Dermatophagoides/imunologia , Células Cultivadas , Estudos de Coortes , Modelos Animais de Doenças , Feminino , Glicólise , Humanos , Proteínas I-kappa B/metabolismo , Interleucina-1beta/genética , Ácido Láctico/metabolismo , Pulmão/patologia , Masculino , Camundongos , Pessoa de Meia-Idade , Neutrófilos/patologia , Proteínas Proto-Oncogênicas/metabolismo , Pyroglyphidae , RNA Interferente Pequeno/genética , Mucosa Respiratória/patologia , Transdução de SinaisRESUMO
NF-κB activation within the epithelium has been implicated in the pathogenesis of asthma, yet the exact role of epithelial NF-κB in allergen-induced inflammation and airway remodeling remains unclear. In the current study, we used an intranasal house dust mite (HDM) extract exposure regimen time course in BALB/c mice to evaluate inflammation, NF-κB activation, airway hyperresponsiveness (AHR), and airway remodeling. We used CC10-IκBαSR transgenic mice to evaluate the functional importance of epithelial NF-κB in response to HDM. After a single exposure of HDM, mRNA expression of proinflammatory mediators was significantly elevated in lung tissue of wild-type (WT) mice, in association with increases in nuclear RelA and RelB, components of the classical and alternative NF-κB pathway, respectively, in the bronchiolar epithelium. In contrast, CC10-IκBαSR mice displayed marked decreases in nuclear RelA and RelB and mRNA expression of proinflammatory mediators compared with WT mice. After 15 challenges with HDM, WT mice exhibited increases in inflammation, AHR, mucus metaplasia, and peribronchiolar fibrosis. CC10-IκBαSR transgenic mice displayed marked decreases in neutrophilic infiltration, tissue damping, and elastance parameters, in association will less peribronchiolar fibrosis and decreases in nuclear RelB in lung tissue. However, central airway resistance and mucus metaplasia remained elevated in CC10-IκBαSR transgenic mice, in association with the continued presence of lymphocytes, and partial decreases in eosinophils and IL-13. The current study demonstrates that following airway exposure with an asthma-relevant allergen, activation of classical and alternative NF-κB pathways occurs within the airway epithelium and may coordinately contribute to allergic inflammation, AHR, and fibrotic airway remodeling.
Assuntos
Antígenos de Dermatophagoides/toxicidade , Células Epiteliais/metabolismo , Regulação da Expressão Gênica/imunologia , Pulmão/imunologia , NF-kappa B/fisiologia , Pyroglyphidae/imunologia , Administração Intranasal , Remodelação das Vias Aéreas/imunologia , Animais , Antígenos de Dermatophagoides/administração & dosagem , Bronquíolos/patologia , Líquido da Lavagem Broncoalveolar/citologia , Linhagem Celular , Eosinófilos/imunologia , Epitélio/patologia , Fibrose , Humanos , Proteínas I-kappa B/genética , Mediadores da Inflamação/metabolismo , Interleucina-13/imunologia , Pulmão/efeitos dos fármacos , Pulmão/patologia , Linfócitos/imunologia , Macrófagos/imunologia , Metaplasia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Inibidor de NF-kappaB alfa , NF-kappa B/biossíntese , NF-kappa B/genética , Neutrófilos/imunologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Método Simples-Cego , Uteroglobina/genéticaRESUMO
RATIONALE: Obesity is a major risk factor for asthma; the reasons for this are poorly understood, although it is thought that inflammatory changes in adipose tissue in obesity could contribute to airway inflammation and airway reactivity in individuals who are obese. OBJECTIVES: To determine if inflammation in adipose tissue in obesity is related to late-onset asthma, and associated with increased markers of airway inflammation and reactivity. METHODS: We recruited a cohort of obese women with asthma and obese control women. We followed subjects with asthma for 12 months after bariatric surgery. We compared markers in adipose tissue and the airway from subjects with asthma and control subjects, and changes in subjects with asthma over time. MEASUREMENTS AND MAIN RESULTS: Subjects with asthma had increased macrophage infiltration of visceral adipose tissue (P < 0.01), with increased expression of leptin (P < 0.01) and decreased adiponectin (p < 0.001) when controlled for body mass index. Similar trends were observed in subcutaneous adipose tissue. Airway epithelial cells expressed receptors for leptin and adiponectin, and airway reactivity was significantly related to visceral fat leptin expression (rho = -0.8; P < 0.01). Bronchoalveolar lavage cytokines and cytokine production from alveolar macrophages were similar in subjects with asthma and control subjects at baseline, and tended to increase 12 months after surgery. CONCLUSIONS: Obesity is associated with increased markers of inflammation in serum and adipose tissue, and yet decreased airway inflammation in obese people with asthma; these patterns reverse with bariatric surgery. Leptin and other adipokines may be important mediators of airway disease in obesity through direct effects on the airway rather than by enhancing airway inflammation.
Assuntos
Asma/etiologia , Asma/patologia , Obesidade/complicações , Obesidade/patologia , Adipocinas/metabolismo , Tecido Adiposo , Adulto , Asma/metabolismo , Cirurgia Bariátrica , Biomarcadores/metabolismo , Índice de Massa Corporal , Estudos de Casos e Controles , Estudos de Coortes , Feminino , Humanos , Inflamação , Mediadores da Inflamação/metabolismo , Pessoa de Meia-Idade , Obesidade/metabolismo , Fatores de RiscoRESUMO
A number of Smoothened (SMO) pathway antagonists are currently undergoing clinical trials as anticancer agents. These drugs are proposed to attenuate tumor growth solely through inhibition of Hedgehog (HH), which is produced in tumor cells but acts on tumor stromal cells. The pivotal argument underlying this model is that the growth-inhibitory properties of SMO antagonists on HH-producing cancer cells are due to their off-target effects. Here, we show that the tumorigenic properties of such lung cancer cells depend on their intrinsic level of HH activity. Notably, reducing HH signaling in these tumor cells decreases HH target gene expression. Taken together, these results question the dogma that autocrine HH signaling plays no role in HH-dependent cancers, and does so without using SMO antagonists.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Proteínas Hedgehog/metabolismo , Neoplasias Pulmonares/metabolismo , Animais , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Humanos , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Nus , Transdução de Sinais , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Transplante Heterólogo , Proteína GLI1 em Dedos de ZincoRESUMO
Exposure to the environmental toxicant arsenic, through both contaminated water and food, contributes to significant health problems worldwide. In particular, arsenic exposure is thought to function as a carcinogen for lung, skin, and bladder cancer via mechanisms that remain largely unknown. More recently, the Hedgehog signaling pathway has also been implicated in the progression and maintenance of these same cancers. Based on these similarities, we tested the hypothesis that arsenic may act in part through activating Hedgehog signaling. Here, we show that arsenic is able to activate Hedgehog signaling in several primary and established tissue culture cells as well as in vivo. Arsenic activates Hedgehog signaling by decreasing the stability of the repressor form of GLI3, one of the transcription factors that ultimately regulate Hedgehog activity. We also show, using tumor samples from a cohort of bladder cancer patients, that high levels of arsenic exposure are associated with high levels of Hedgehog activity. Given the important role Hedgehog signaling plays in the maintenance and progression of a variety of tumors, including bladder cancer, these results suggest that arsenic exposure may in part promote cancer through the activation of Hedgehog signaling. Thus, we provide an important insight into the etiology of arsenic-induced human carcinogenesis, which may be relevant to millions of people exposed to high levels of arsenic worldwide.
Assuntos
Arsenitos/toxicidade , Exposição Ambiental/efeitos adversos , Substâncias Perigosas/toxicidade , Proteínas Hedgehog/metabolismo , Compostos de Sódio/toxicidade , Neoplasias da Bexiga Urinária/induzido quimicamente , Animais , Arsenitos/intoxicação , Bovinos , Células Endoteliais/efeitos dos fármacos , Substâncias Perigosas/intoxicação , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células NIH 3T3 , Transdução de Sinais/efeitos dos fármacos , Compostos de Sódio/intoxicaçãoRESUMO
The secreted protein Hedgehog (Hh) plays a critical instructional role during metazoan development. In Drosophila, Hh signaling is interpreted by a set of conserved, downstream effectors that differentially localize and interact to regulate the stability and activity of the transcription factor Cubitus interruptus. Two essential models that integrate genetic, cell biological, and biochemical information have been proposed to explain how these signaling components relate to one another within the cellular context. As the molar ratios of the signaling effectors required in each of these models are quite different, quantitating the cellular ratio of pathway components could distinguish these two models. Here, we address this important question using a set of purified protein standards to perform a quantitative analysis of Drosophila cell lysates for each downstream pathway component. We determine each component's steady-state concentration within a given cell, demonstrate the molar ratio of Hh signaling effectors differs more than two orders of magnitude and that this ratio is conserved in vivo. We find that the G-protein-coupled transmembrane protein Smoothened, an activating component, is present in limiting amounts, while a negative pathway regulator, Suppressor of Fused, is present in vast molar excess. Interestingly, despite large differences in the steady-state ratio, all downstream signaling components exist in an equimolar membrane-associated complex. We use these quantitative results to re-evaluate the current models of Hh signaling and now propose a novel model of signaling that accounts for the stoichiometric differences observed between various Hh pathway components.
Assuntos
Proteínas Hedgehog/metabolismo , Animais , Citosol/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Insetos , Cinesinas/metabolismo , Modelos Biológicos , Proteínas Serina-Treonina Quinases/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Recombinantes/química , Proteínas Repressoras/metabolismo , Transdução de Sinais , Frações Subcelulares/metabolismo , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Sonic hedgehog (SHH) plays an important instructional role in vertebrate development, as exemplified by the numerous developmental disorders that occur when the SHH pathway is disrupted. Mutations in the SHH gene are the most common cause of sporadic and inherited holoprosencephaly (HPE), a developmental disorder that is characterized by defective prosencephalon development. SHH HPE mutations provide a unique opportunity to better understand SHH biogenesis and signaling, and to decipher its role in the development of HPE. Here, we analyzed a panel of SHH HPE missense mutations that encode changes in the amino-terminal active domain of SHH. Our results show that SHH HPE mutations affect SHH biogenesis and signaling at multiple steps, which broadly results in low levels of protein expression, defective processing of SHH into its active form and protein with reduced activity. Additionally, we found that some inactive SHH proteins were able to modulate the activity of wt SHH in a dominant negative manner, both in vitro and in vivo. These findings show for the first time the susceptibility of SHH driven developmental processes to perturbations by low-activity forms of SHH. In conclusion, we demonstrate that SHH mutations found in HPE patients affect distinct steps of SHH biogenesis to attenuate SHH activity to different levels, and suggest that these variable levels of SHH activity might contribute to some of the phenotypic variation found in HPE patients.