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

ABSTRACT: Nicotine modulates multiple inflammatory responses in the lung through the nicotinic acetylcholine receptor subtype alpha7 (?7). Previously we reported that ?7 modulates both the hematopoietic and epithelium responses in the lung to the bacterial inflammogen, lipopolysaccharide (LPS). Here we apply immunohistochemistry, flow cytometry and RNA-Seq analysis of isolated distal lung epithelium to further define ?7-expression and function in this tissue. Mouse lines were used that co-express a bicistronic tau-green fluorescent protein (tGFP) as a reporter of ?7 (?7G) expression and that harbor an ?7 with a specific point mutation (?7E260A:G) that selectively uncouples it from cell calcium-signaling mechanisms. The tGFP reporter reveals strong cell-specific ?7-expression by alveolar macrophages (AM), Club cells and ATII cells. Ciliated cells do not express detectible tGFP, but their numbers decrease by one-third in the ?7E260A:G lung compared to controls. Transcriptional comparisons (RNA-Seq) between ?7G and ?7E260A:G enriched lung epithelium 24 hours after challenge with either intra-nasal (i.n.) saline or LPS reveals a robust ?7-genotype impact on both the stasis and inflammatory response of this tissue. Overall the ?7E260A:G lung epithelium exhibits reduced inflammatory cytokine/chemokine expression to i.n. LPS. Transcripts specific to Club cells (e.g., CC10, secretoglobins and Muc5b) or to ATII cells (e.g., surfactant proteins) were constitutively decreased in in the ?7E260A:G lung, but they were strongly induced in response to i.n. LPS. Protein analysis applying immunohistochemistry and ELISA also revealed ?7-associated differences suggested by RNA-Seq including altered mucin protein 5b (Muc5b) accumulation in the ?7E260A:G bronchia, that in some cases appeared to form airway plugs, and a substantial increase in extracellular matrix deposits around ?7E260A:G airway bronchia linings that was not seen in controls. Our results show that ?7 is an important modulator of normal gene expression stasis and the response to an inhaled inflammogen in the distal lung epithelium. Further, when normal ?7 signaling is disrupted, changes in lung gene expression resemble those associated with long-term lung pathologies seen in humans who use inhaled nicotine products.

SUBMITTER: Gahring LC

PROVIDER: S-EPMC5383308 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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

Gahring Lorise C LC Myers Elizabeth J EJ Dunn Diane M DM Weiss Robert B RB Rogers Scott W SW

PloS one 20170406 4

Nicotine modulates multiple inflammatory responses in the lung through the nicotinic acetylcholine receptor subtype alpha7 (α7). Previously we reported that α7 modulates both the hematopoietic and epithelium responses in the lung to the bacterial inflammogen, lipopolysaccharide (LPS). Here we apply immunohistochemistry, flow cytometry and RNA-Seq analysis of isolated distal lung epithelium to further define α7-expression and function in this tissue. Mouse lines were used that co-express a bicist ...[more]

PMID: 28384302

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Project description:We and others have shown that one of the mechanisms of growth regulation of small cell lung cancer cell lines and cultured pulmonary neuroendocrine cells is by the binding of agonists to the alpha7 neuronal nicotinic acetylcholine receptor. In addition, we have shown that the nicotine-derived carcinogenic nitrosamine, 4(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is a high affinity agonist for the alpha7 nicotinic acetylcholine receptor. In the present study, our goal was to determine the extent of alpha7 mRNA and protein expression in the human lung.Experiments were done using reverse transcription polymerase chain reaction (RT-PCR), a nuclease protection assay and western blotting using membrane proteins.We detected mRNA for the neuronal nicotinic acetylcholine receptor alpha7 receptor in seven small cell lung cancer (SCLC) cell lines, in two pulmonary adenocarcinoma cell lines, in cultured normal human small airway epithelial cells (SAEC), one carcinoid cell line, three squamous cell lines and tissue samples from nine patients with various types of lung cancer. A nuclease protection assay showed prominent levels of alpha7 in the NCI-H82 SCLC cell line while alpha7 was not detected in SAEC, suggesting that alpha7 mRNA levels may be higher in SCLC compared to normal cells. Using a specific antibody to the alpha7 nicotinic receptor, protein expression of alpha7 was determined. All SCLC cell lines except NCI-H187 expressed protein for the alpha7 receptor. In the non-SCLC cells and normal cells that express the alpha7 nAChR mRNA, only in SAEC, A549 and NCI-H226 was expression of the alpha7 nicotinic receptor protein shown. When NCI-H69 SCLC cell line was exposed to 100 pm NNK, protein expression of the alpha7 receptor was increased at 60 and 150 min.Expression of mRNA for the neuronal nicotinic acetylcholine receptor alpha7 seems to be ubiquitously expressed in all human lung cancer cell lines tested (except for NCI-H441) as well as normal lung cells. The alpha7 nicotinic receptor protein is expressed in fewer cell lines, and the tobacco carcinogen NNK increases alpha7 nicotinic receptor protein levels.

Project description:Tobacco is a known cause of oral disease but the mechanism remains elusive. Nicotine (Nic) is a likely culprit of pathobiological effects because it displaces the local cytotransmitter acetylcholine from the nicotinic receptors (nAChRs) expressed by oral keratinocytes (KCs). To gain a mechanistic insight into tobacco-induced morbidity in the oral cavity, we studied effects of exposures to environmental tobacco smoke (ETS) versus equivalent concentration of pure Nic on human and murine KCs. Both ETS and Nic up-regulated expression of cell cycle and apoptosis regulators, differentiation marker filaggrin, and signal transduction factors at both the mRNA and protein levels. These changes could be abolished in cultured human oral KCs transfected with anti-alpha3 small interfering RNA or treated with the alpha3beta2-preferring antagonist alpha-conotoxin MII. Functional inactivation of alpha3-mediated signaling in alpha3-/- mutant KCs prevented most of the ETS/Nic-dependent changes in gene expression. To determine relevance of the in vitro findings to the in vivo situation, we studied gene expression in oral mucosa of neonatal alpha3+/+ and alpha3-/- littermates delivered by heterozygous mice soon after their exposures to ETS or equivalent concentration of pure Nic in drinking water. In addition to reverse transcriptase-polymerase chain reaction and Western blot, the ETS/Nic-dependent alterations in gene expression were also detected by semiquantitative immunofluorescence assay directly in KCs comprising murine oral mucosa. Only wild-type mice consistently developed significant (P < 0.05) changes in the gene expression. These results identified alpha3beta2 nAChR as a major receptor mediating effects of tobacco products on KC gene expression. Real-time polymerase chain reaction demonstrated that in all three model systems the common genes targeted by alpha3beta2-mediated ETS/Nic toxicity were p21, Bcl-2, NF-kappaB, and STAT-1. The expression of the nAChR subunits alpha5 and beta2 and the muscarinic receptor subtypes M(2) and M(3) was also altered. This novel mechanism offers innovative solutions to ameliorate the tobacco-related cell damage and intercede in disease pathways, and may shed light on general mechanisms regulating and driving tobacco-related morbidity in human cells.

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

Publications

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

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