Nicotine Stimulates Nerve Growth Factor in Lung Fibroblasts through an NFκB-Dependent Mechanism

Abstract

RationaleAirway hyperresponsiveness (AHR) is classically found in asthma, and persistent AHR is associated with poor asthma control. Although airway smooth muscle (ASM) cells play a critical pathophysiologic role in AHR, the paracrine contributions of surrounding cells such as fibroblasts to the contractile phenotype of ASM cells have not been examined fully. This study addresses the hypothesis that nicotine promotes a contractile ASM cell phenotype by stimulating fibroblasts to increase nerve growth factor (NGF) secretion into the environment.MethodsPrimary lung fibroblasts isolated from wild type and α7 nicotinic acetylcholine receptor (α7 nAChR) deficient mice were treated with nicotine (50 µg/ml) in vitro for 72 hours. NGF levels were measured in culture media and in bronchoalveolar lavage (BAL) fluid from asthmatic, smoking and non-smoking subjects by ELISA. The role of the NFκB pathway in nicotine-induced NGF expression was investigated by measuring NFκB nuclear translocation, transcriptional activity, chromatin immunoprecipitation assays, and si-p65 NFκB knockdown. The ability of nicotine to stimulate a fibroblast-mediated, contractile ASM cell phenotype was confirmed by examining expression of contractile proteins in ASM cells cultured with fibroblast-conditioned media or BAL fluid.ResultsNGF levels were elevated in the bronchoalveolar lavage fluid of nicotine-exposed mice, current smokers, and asthmatic children. Nicotine increased NGF secretion in lung fibroblasts in vitro in a dose-dependent manner and stimulated NFκB nuclear translocation, p65 binding to the NGF promoter, and NFκB transcriptional activity. These responses were attenuated in α7 nAChR deficient fibroblasts and in wild type fibroblasts following NFκB inhibition. Nicotine-treated, fibroblast-conditioned media increased expression of contractile proteins in ASM cells.ConclusionNicotine stimulates NGF release by lung fibroblasts through α7 nAChR and NFκB dependent pathways. These novel findings suggest that the nicotine-α7 nAChR-NFκB- NGF axis may provide novel therapeutic targets to attenuate tobacco smoke-induced AHR.

Nicotinic alpha 7 receptor expression and modulation of the lung epithelial response to lipopolysaccharide

“…However, extensive analysis of these cells in immunohistochemical sections did not suggest any obvious abnormalities such as a difference in their distribution or numbers. While others [77] have reported α7 expression in lung fibroblasts, we did not detect the tGFP α7 expression reporter in these cells (not shown). Our genetic approach to define α7 transcript expression provides heretofore unavailable precision and sensitivity towards identifying those cells that do, and do not, express α7.…”

“…This is a substantial advancement over methods that rely on antibodies of unclear sensitivity and reliability (e.g., see discussion in [2,29]). While there is no direct measure of α7 expression in fibroblasts, our studies do not necessarily disagree with conclusion of studies demonstrating the effects of nicotine on lung fibroblasts [77] and there are additional considerations. For example the possibility of functional compensation in the α7KO by other nicotinic receptors is always an ongoing issue since this mouse exhibits the complete lack of receptor expression, an issue that at least in part is addressed in the α7 E260A:G mouse.…”

Revisiting nicotine’s role in the ageing brain and cognitive impairment

“…Accumulating evidence shows that nicotine can itself exert neurotrophic effects and together with nAChRs may have a crucial role in the development and maturation of neurones (Ferrea and Winterer, 2009). As noted above, nicotine activates α 7 nAChRs and can increase NGF expression through NF-κB-dependent pathways Martínez-Rodríguez et al, 2003;Hernandez and Terry, 2005;Wongtrakool et al, 2014). Indeed, nicotine increases the nuclear translocation and transcriptional activity of NF-κB and enhances p65 attachment to the promoter region of the NGF gene which ultimately increases NGF expression (Wongtrakool et al, 2014).…”

“…As noted above, nicotine activates α 7 nAChRs and can increase NGF expression through NF-κB-dependent pathways Martínez-Rodríguez et al, 2003;Hernandez and Terry, 2005;Wongtrakool et al, 2014). Indeed, nicotine increases the nuclear translocation and transcriptional activity of NF-κB and enhances p65 attachment to the promoter region of the NGF gene which ultimately increases NGF expression (Wongtrakool et al, 2014). Additionally, nicotine increases expression of mRNA for tyrosine receptor kinase A mRNA expression which mediates effects of NGF in the neurones (Garrido et al, 2003).…”

Cell Culture Techniques: Corticosteroid Treatment in A549 Human Lung Epithelial Cell

“…They are used in a wide variety of research areas such as virology, cancer research, or immunology, and allow further studies of intracellular activity, intracellular flow, or cellular interactions [2]. In the study of respiratory diseases such as asthma, it is very common to find reports based on cell cultures of human bronchial epithelial cells [3], human T cells [4], or murine lung fibroblast [5], used to analyze the underlying mechanisms, as well as, the possible development of new therapeutic targets.…”