Conditional deletion of epithelial IKKβ impairs alveolar formation through apoptosis and decreased VEGF expression during early mouse lung morphogenesis

<p>Abstract</p> <p>Background</p> <p>Alveolar septation marks the beginning of the transition from the saccular to alveolar stage of lung development. Inflammation can disrupt this process and permanently impair alveolar formation resulting in alveolar hypoplasia as seen in bronchopulmonary dysplasia in preterm newborns. NF-κB is a transcription factor central to multiple inflammatory and developmental pathways including dorsal-ventral patterning in fruit flies; limb, mammary and submandibular gland development in mice; and branching morphogenesis in chick lungs. We have previously shown that epithelial overexpression of NF-κB accelerates lung maturity using transgenic mice. The purpose of this study was to test our hypothesis that targeted deletion of NF-κB signaling in lung epithelium would impair alveolar formation.</p> <p>Methods</p> <p>We generated double transgenic mice with lung epithelium-specific deletion of IKKβ, a known activating kinase upstream of NF-κB, using a cre-<it>loxP </it>transgenic recombination strategy. Lungs of resulting progeny were analyzed at embryonic and early postnatal stages to determine specific effects on lung histology, and mRNA and protein expression of relevant lung morphoreulatory genes. Lastly, results measuring expression of the angiogenic factor, VEGF, were confirmed <it>in vitro </it>using a siRNA-knockdown strategy in cultured mouse lung epithelial cells.</p> <p>Results</p> <p>Our results showed that IKKβ deletion in the lung epithelium transiently decreased alveolar type I and type II cells and myofibroblasts and delayed alveolar formation. These effects were mediated through increased alveolar type II cell apoptosis and decreased epithelial VEGF expression.</p> <p>Conclusions</p> <p>These results suggest that epithelial NF-κB plays a critical role in early alveolar development possibly through regulation of VEGF.</p

Inhibition of NFκB abrogates induction of NGF in primary murine lung fibroblasts treated with nicotine.

<p>Primary murine lung fibroblasts from C57BL/6J mice were serum starved overnight, treated with nicotine (50 µg/ml), and caffeic acid phenyl ester (CAPE, 10 and 20 µM). Cells and media were collected after 72 hours. NGF ELISA was performed on the media. NGF levels were normalized to total protein concentration of the cells in each condition. n = 3–4 in 3 separate experiments, *p<0.05 compared to control, error bars represent ±SEM.</p

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

<div><p>Rationale</p><p>Airway 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.</p><p>Methods</p><p>Primary lung fibroblasts isolated from wild type and α7 nicotinic acetylcholine receptor (α7 nAChR) deficient mice were treated with nicotine (50 µg/ml) <i>in vitro</i> 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.</p><p>Results</p><p>NGF levels were elevated in the bronchoalveolar lavage fluid of nicotine-exposed mice, current smokers, and asthmatic children. Nicotine increased NGF secretion in lung fibroblasts <i>in vitro</i> 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.</p><p>Conclusion</p><p>Nicotine 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.</p></div

Nicotine-exposed fibroblasts stimulate increased contractile protein expression in airway smooth muscle cells.

<p>Using a transwell system, primary murine lung fibroblasts were cultured on an upper chamber transwell membrane and primary murine ASM cells were cultured on the lower plastic chamber. Both chambers were cultured in serum free medium. The upper chamber containing fibroblasts was treated with of nicotine (50 µg/ml) for 72 hours. Primary murine ASM cells were harvested for protein isolation. Phosphorylated (p-MLC) and total myosin light chain (total MLC) expression was measured by using immunoblot analysis with densitometry. ASM cells cultured in proximity to nicotine treated fibroblasts (<b>N</b>) have significantly increased higher p-MLC/total MLC ratio compared to controls (<b>C</b>). n = 6, *p<0.05, error bars represent ±SEM.</p

Nicotine increases NGF levels in bronchoalveolar lavage fluid.

<p><b>A.</b> BAL fluid was collected from C57BL/6J and Chrna7^−/− mice administered nicotine (100 µg/ml) in the drinking water <i>ad libitum</i> for 8 weeks. Using ELISA with a standard curve, NGF levels were measured. Wild type mice exposed to nicotine had significantly increased NGF levels in BAL fluid when compared to untreated mice, whereas Chrna7^−/− mice exposed to nicotine did not. n = 4–8 mice, *p<0.05 compared to wild type exposed to nicotine, error bars represent ±SEM. <b>B.</b> BAL fluid was collected from adult non-smokers and smokers in a veteran medical clinic for NGF ELISA. Line represents mean NGF level. <b>C.</b> BAL fluid was collected from children with severe asthma and healthy adult controls. BAL fluid from all asthmatic children had detectable levels of NGF, but the healthy adult controls had minimal to no detectable levels. Line represents mean NGF level, *p<0.05.</p

Nicotine stimulates NFκB/p65 pathway in primary murine lung fibroblasts.

<p>Primary murine lung fibroblasts from C57BL/6J and Chrna7^−/− mice were serum starved overnight, treated with nicotine (50 µg/ml) and harvested after 24 hours. <b>A–C.</b> Cytoplasmic (<b>A, C</b>) and nuclear (<b>B, C</b>) fractions were isolated for Western blot analysis. Bar graphs show densitometry data as fold change compared to wild type/untreated cells. Densitometry was normalized to endogenous controls histone and β-tubulin for nuclear and cytoplasmic extracts, respectively. n = 4–5 separate experiments. *p<0.05 compared to untreated control. <b>D–F.</b> Chromatin immunoprecipitation assay was performed using antibodies to IgG, histone, and p65 for the immunoprecipitation as previously described in Materials and Methods. Stimulation with TNF-α was used as a positive control. qRT-PCR analysis using primers for putative NFκB binding sites in the NGF promoter was performed. Results are reported as % input (<b>D</b>) and fold increase binding (<b>E</b>) at NFkB binding site. Fibroblasts treated with nicotine demonstrate an increase in NFκB binding compared to untreated cells (<b>F</b>). Figure is representative of 3 separate experiments. <b>G.</b> NFκB dependent transcriptional activity was determined using an NFκB luciferase reporter assay as previously described in Materials and Methods. Primary lung fibroblasts from wild type and Chrna7^−/− animals were transfected with an NFκB luciferase reporter with renilla as control for transfection efficiency. Lung fibroblasts treated with nicotine (50 µg/ml) showed a time-dependent increase in NFκB transcriptional activity at 72 hours. n = 6 separate experiments, *p<0.05 compared to WT control, #p<0.05 compared to Chrna7^−/−, error bars represent ±SEM. <b>H.</b> Primary murine lung fibroblasts were transfected with p65 siRNA, and then treated with nicotine (50 µg/ml) after serum starvation. By densitometric analysis of western blot, approximately 60% knockdown of p65 expression was achieved 24 hours after transfection. n = 3, *p<0.01, error bars represent ±SEM. NGF levels were measured by ELISA in the media after 48 hours of treatment; the cell pellet was collected for normalization to total protein. siRNA knockdown of p65 abrogates NGF secretion into the media by nicotine. n = 4 separate experiments, *p<0.05, error bars represent ±SEM. <b>I.</b> Schematic summary of the above results showing increased p65 nuclear translocation, increased p65 binding to the NGF promoter, and increased NFκB transcriptional activity.</p

α7 nAChR is required for nicotine-stimulated NGF secretion in primary murine lung fibroblasts.

<p><b>A.</b> Primary murine lung fibroblasts from C57BL/6J mice were serum starved overnight and treated with nicotine (50 µg/ml) or 1% cigarette smoke extract (CSE) for 72 hours. Cells and media were then collected. NGF levels were measured by ELISA in the media and normalized to total protein concentration of the cells in each condition. Nicotine and CSE treatment induced NGF secretion into the media. n = 3–4 in 3 separate experiments, *p<0.05 compared to control, **p<0.05 compared to nicotine, error bars represent ±SEM. <b>B.</b> Primary murine lung fibroblasts from C57BL/6J and Chrna7^−/− mice were serum starved overnight, treated with nicotine (5 and 50 µg/ml) or 10 ng/ml IL-1β (used as a positive control), and then cells and media were collected after 72 hours. NGF levels were measured by ELISA in the media and normalized to total protein concentration of the cells in each condition. n = 3–4 in 3 separate experiments, *p<0.05 compared to wild type control, error bars represent ±SEM.</p