RESUMEN
The small airway epithelium (SAE), the first site of smoking-induced lung pathology, exhibits genome-wide changes in gene expression in response to cigarette smoking. Based on the increasing evidence that the epigenome can respond to external stimuli in a rapid manner, we assessed the SAE of smokers for genome-wide DNA methylation changes compared with nonsmokers, and whether changes in SAE DNA methylation were linked to the transcriptional output of these cells. Using genome-wide methylation analysis of SAE DNA of nonsmokers and smokers, the data identified 204 unique genes differentially methylated in SAE DNA of smokers compared with nonsmokers, with 67% of the regions with differential methylation occurring within 2 kb of the transcriptional start site. Among the genes with differential methylation were those related to metabolism, transcription, signal transduction and transport. For the differentially methylated genes, 35 exhibited a correlation with gene expression, 54% with an inverse correlation of DNA methylation with gene expression and 46% a direct correlation. These observations provide evidence that cigarette smoking alters the DNA methylation patterning of the SAE and that, for some genes, these changes are associated with the smoking-related changes in gene expression.
Asunto(s)
Epigénesis Genética , Epitelio/metabolismo , Mucosa Respiratoria/metabolismo , Fumar/genética , Adulto , Estudios de Casos y Controles , Metilación de ADN/efectos de los fármacos , Epitelio/efectos de los fármacos , Femenino , Perfilación de la Expresión Génica , Genoma Humano , Estudio de Asociación del Genoma Completo , Humanos , Masculino , Persona de Mediana Edad , Fenotipo , Mucosa Respiratoria/efectos de los fármacos , Fumar/efectos adversos , Fumar/metabolismo , Sitio de Iniciación de la Transcripción , Adulto JovenRESUMEN
In cystic fibrosis (CF), excessive furin activity plays a critical role in the activation of the epithelial sodium channel (ENaC), dysregulation of which contributes to airway dehydration, ineffective mucociliary clearance (MCC), and mucus obstruction. Here, we report a highly selective, cell-permeable furin inhibitor, BOS-318, that derives selectivity by eliciting the formation of a new, unexpected binding pocket independent of the active site catalytic triad. Using human ex vivo models, BOS-318 showed significant suppression of ENaC, which led to enhanced airway hydration and an â¼30-fold increase in MCC rate. Furin inhibition also protected ENaC from subsequent activation by neutrophil elastase, a soluble protease dominant in CF airways. Additional therapeutic benefits include protection against epithelial cell death induced by Pseudomonas aeruginosa exotoxin A. Our findings demonstrate the utility of selective furin inhibition as a mutation-agnostic approach that can correct features of CF airway pathophysiology in a manner expected to deliver therapeutic value.
Asunto(s)
Fibrosis Quística , Furina , Fibrosis Quística/tratamiento farmacológico , Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Canales Epiteliales de Sodio/genética , Canales Epiteliales de Sodio/metabolismo , Furina/antagonistas & inhibidores , Humanos , Depuración MucociliarRESUMEN
IL-13 is known to affect many processes that contribute to an asthmatic phenotype, including inflammation, fibrosis, and mucus production. Members of the aquaporin (AQP) family of transmembrane water channels are targets of regulation in models of lung injury and inflammation. Therefore, we examined AQP mRNA and protein expression in allergen and IL-13-induced mouse models of asthma. Lungs from ovalbumin sensitized and ovalbumin challenged (OVA/OVA) and IL-13 treated mice showed airway thickening, increased mucus production, and pulmonary eosinophilia. Pulmonary function tests showed a significant increase in methacholine-induced airway hyperreactivity in OVA/OVA and IL-13-treated mice as compared with controls. Quantitative PCR analysis revealed differential regulation of AQPs in these two models. AQP1 and AQP4 mRNA expression was downregulated in the OVA/OVA model, but not in the IL-13 model. AQP5 mRNA was reduced in both models, whereas AQP3 was upregulated only in the IL-13 model. Western analysis showed that diminished expression of an apically localized aquaporin, (AQP5), and concomitant upregulation of a basolateral aquaporin (AQP3 or AQP4) are characteristic features of both inducible asthma models. These results demonstrate that aquaporins are common targets of gene expression in both allergen and IL-13 induced mouse models of asthma.
Asunto(s)
Acuaporina 5/biosíntesis , Acuaporina 5/metabolismo , Asma/metabolismo , Regulación de la Expresión Génica , Interleucina-13/biosíntesis , Animales , Acuaporina 1/biosíntesis , Acuaporina 4/biosíntesis , Broncoconstrictores/farmacología , Modelos Animales de Enfermedad , Femenino , Inflamación , Pulmón/metabolismo , Cloruro de Metacolina/farmacología , Ratones , Ratones Endogámicos BALB C , Modelos BiológicosRESUMEN
BACKGROUND: The expression of RET in the developing enteric nervous system (ENS) suggests that RET may contribute to adult intestinal function. ENS cholinergic nerves play a critical role in the control of colonic function through the release of acetylcholine (ACh). In the current study, we hypothesized that a RET-mediated mechanism may regulate colonic ion transport and motility through modulation of cholinergic nerves. METHODS: The effect of RET inhibition on active ion transport was assessed electrophysiologically in rat colonic tissue mounted in Ussing chambers via measurements of short circuit current (Isc) upon electrical field stimulation (EFS) or pharmacologically with cholinergic agonists utilizing a gastrointestinal (GI)-restricted RET inhibitor. We assessed the effect of the RET inhibitor on propulsive motility via quantification of fecal pellet output (FPO) induced by the acetylcholinesterase inhibitor neostigmine. KEY RESULTS: We found that enteric ganglia co-expressed RET and choline acetyltransferase (ChAT) transcripts. In vitro, the RET kinase inhibitor GSK3179106 attenuated the mean increase in Isc induced by either EFS or carbachol but not bethanechol. In vivo, GSK3179106 significantly reduced the prokinetic effect of neostigmine. CONCLUSION AND INFERENCES: Our findings provide evidence that RET-mediated mechanisms regulate colonic function by maintaining cholinergic neuronal function and enabling ACh-evoked chloride secretion and motility. We suggest that modulating the cholinergic control of the colon via a RET inhibitor may represent a novel target for the treatment of intestinal disorders associated with increased secretion and accelerated GI transit such as irritable bowel syndrome with diarrhea (IBS-D).
Asunto(s)
Neuronas Colinérgicas/efectos de los fármacos , Colon/efectos de los fármacos , Motilidad Gastrointestinal/efectos de los fármacos , Mucosa Intestinal/efectos de los fármacos , Transporte Iónico/efectos de los fármacos , Proteínas Proto-Oncogénicas c-ret/antagonistas & inhibidores , Animales , Colina O-Acetiltransferasa/metabolismo , Agonistas Colinérgicos/farmacología , Neuronas Colinérgicas/metabolismo , Colon/metabolismo , Defecación/efectos de los fármacos , Sistema Nervioso Entérico/efectos de los fármacos , Sistema Nervioso Entérico/metabolismo , Tránsito Gastrointestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Masculino , Proteínas Proto-Oncogénicas c-ret/metabolismo , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacosRESUMEN
Regulation of Cl(-) channel conductance by Ins(3,4,5,6)P(4) provides receptor-dependent control over salt and fluid secretion, cell volume homeostasis, and electrical excitability of neurones and smooth muscle. Ignorance of how Ins(3,4,5,6)P(4) is synthesized has long hindered our understanding of this signaling pathway. We now show Ins(3,4,5,6)P(4) synthesis by Ins(1,3,4,5,6)P(5) 1-phosphatase activity by an enzyme previously characterized as an Ins(3,4,5,6)P(4) 1-kinase. Rationalization of these phenomena with a ligand binding model unveils Ins(1,3,4)P(3) as not simply an alternative kinase substrate, but also an activator of Ins(1,3,4,5,6)P(5) 1-phosphatase. Stable overexpression of the enzyme in epithelial monolayers verifies its physiological role in elevating Ins(3,4,5,6)P(4) levels and inhibiting secretion. It is exceptional for a single enzyme to catalyze two opposing signaling reactions (1-kinase/1-phosphatase) under physiological conditions. Reciprocal coordination of these opposing reactions offers an alternative to general doctrine that intracellular signals are regulated by integrating multiple, distinct phosphatases and kinases.
Asunto(s)
Fosfatos de Inositol/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Transducción de Señal , Calcio/metabolismo , Humanos , FosforilaciónRESUMEN
Tumor metastasis is a highly complex, dynamic, and inefficient process involving multiple steps, yet it accounts for more than 90% of cancer-related deaths. Although it has long been known that fibrotic signals enhance tumor progression and metastasis, the underlying molecular mechanisms are still unclear. Identifying events involved in creating environments that promote metastatic colonization and growth are critical for the development of effective cancer therapies. Here, we show a critical role for lysyl oxidase (LOX) in establishing a milieu within fibrosing tissues that is favorable to growth of metastastic tumor cells. We show that LOX-dependent collagen crosslinking is involved in creating a growth-permissive fibrotic microenvironment capable of supporting metastatic growth by enhancing tumor cell persistence and survival. We show that therapeutic targeting of LOX abrogates not only the extent to which fibrosis manifests, but also prevents fibrosis-enhanced metastatic colonization. Finally, we show that the LOX-mediated collagen crosslinking directly increases tumor cell proliferation, enhancing metastatic colonization and growth manifesting in vivo as increased metastasis. This is the first time that crosslinking of collagen I has been shown to enhance metastatic growth. These findings provide an important link between ECM homeostasis, fibrosis, and cancer with important clinical implications for both the treatment of fibrotic disease and cancer.