The expression and pharmacological characterization of nicotinic acetylcholine receptor subunits in HBE16 airway epithelial cells.

This study characterizes the expression and the biological effects of the nicotinic acetylcholine receptor (nAChR) on human airway epithelial cells. Cultured HBE16 airway epithelial cells were incubated with either nicotine or cigarette smoke extract (CSE). The nAChR gene and protein expression in cells were detected by reverse transcriptase-polymerase chain reaction (RT-PCR), real-time PCR, and western blot. The protein expression of the nAChR subunits, α1, α5, and α7, were evaluated by immunohistochemistry. Cells were subsequently transfected with α1-, α5-, and α7-specific siRNAs, and the effects of nicotine on the production of the pro-inflammatory factors, TNF-α, IL-8, and IL-6 in transfected cells were analyzed using an enzyme-linked immunosorbent assay and real-time PCR. We detected α1, α5, α7, and β2 subunits in untreated HBE16 cells, and their expression was elevated after nicotinic incubation. Importantly, the most significant increase in expression was observed in the α5 and α7 subunits. However, CSE did not cause a significant enhancement in the expression of these genes and proteins. Cells pretreated with nicotine prior to lipopolysaccharide (LPS) stimulation exhibited a lower production of TNF-α, IL-8, and IL-6 compared to LPS-treated (only) cells. Cells that were transfected with α7 siRNA and subsequently incubated with nicotine and LPS, exhibited a higher expression of TNF-α, IL-8, and IL-6 compared with non-transfected cells or α1 and α5 siRNA-transfected cells. In α1- and α5-siRNA-transfected cells, the expression of TNF-α, IL-8, and IL-6 showed no significant difference compared with non-transfected cells. Therefore, we concluded that α1, α5, α7, and β2 nAChR subunits are highly expressed in human bronchial epithelial cells (HBE16) after nicotinic incubation and that the α7 subunit is involved in the nicotine-induced inhibitory effect on the production of inflammatory factors. Moreover, α1, α5, and β2 subunits did not play an important role in this process.

Li Q ，Zhou X ，Kolosov VP ，Perelman JM ... -  《-》

Nicotine reduces TNF-α expression through a α7 nAChR/MyD88/NF-ĸB pathway in HBE16 airway epithelial cells.

To explore the signaling mechanism associated with the inhibitory effect of nicotine on tumor necrosis factor (TNF)- α expression in human airway epithelial cells. HBE16 airway epithelial cells were cultured and incubated with either nicotine or cigarette smoke extract (CE). Cells were then transfected with α1, α5, or α7 nicotinic acetylcholine receptor (nAChR)-specific small interfering RNAs (siRNAs). The effects of nicotine on the production of proinflammatory factors TNF-α, in transfected cells were analyzed. Furthermore, we assayed the expression levels of myeloid differentiation primary response gene 88 (MyD88) protein, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65 protein, NF-κB activity and NF-κB inhibitor alpha (I-κBα) expression in cells after treatment with nicotine or α7 nAChR inhibitor, α -bungarotoxin (α-BTX). The production of TNF-α was lower in cells pretreated with nicotine before lipopolysaccharide (LPS) stimulation, compared with LPS-only-treated cells. In contrast, in α7 siRNA-transfected cells incubated with nicotine and LPS, TNF-α expression was higher than that in non-transfected cells or in α1 or α5 siRNA-transfected cells. Addition of MyD88 siRNA or the NF-κB inhibitor pyridine-2,6-dithiocarboxylic acid (PDTC) also reduced TNF-α expression. Furthermore, we found that nicotine decreased MyD88 protein, NF-κB p65 protein, NF-κB activity and phospho-I-κBα expression induced by CE or LPS. The inhibitor α-BTX could reverse these effects. Nicotine reduces TNF-α expression in HBE16 airway epithelial cells, mainly through an α7 nAChR/MyD88/NF-κB pathway.

Li Q ，Zhou XD ，Kolosov VP ，Perelman JM ... -  《-》

Nicotine suppresses inflammatory factors in HBE16 airway epithelial cells after exposure to cigarette smoke extract and lipopolysaccharide.

Cigarette smoke is a major cause of chronic inflammatory pulmonary disease, leading to inflammation, mucin (MUC) production, tissue damage, and remodeling. It is also well known that the major addictive component of cigarette smoke is nicotine. This study focused on the role of nicotine in the development of inflammatory pulmonary disease induced by cigarette smoke. HBE16 human airway epithelial cells were treated with serial dilutions of cigarette smoke chloroform extract (CE), lipopolysaccharide (LPS), and nicotine. The release of MUC5AC, tumor necrosis factor (TNF)-α, interleukin (IL)-8, and IL-6 protein were assayed by enzyme-linked immunosorbent assay. The MUC5AC protein also was observed by immunofluorescence. The expression of MUC5AC, TNF-α, IL-8, and IL-6 mRNA were detected by real-time polymerase chain reaction. We found that the mRNA of the proinflammatory mediators TNF-α, IL-8, and IL-6, as well as MUC5AC was highly expressed after CE and LPS stimulation. Nicotine did not cause an excessive expression of TNF-α, IL-8, and IL-6, nor did it affect protein production from the MUC5AC gene. Nicotine not only failed to stimulate production of TNF-α, IL-8, and IL-6, but its presence was shown to suppress the activation resulting from exposure to CE and LPS (P < 0.05). Preincubation with nicotine also would reduce the level of MUC5AC protein in culture supernatants of CE- and LPS-treated cells. However, mRNA expression of MUC5AC showed no significant change in nicotine-treated cells when compared with normal control cells. This distinctive pattern implies that nicotine may have potential to suppress airway inflammation and maintain the mucus over retention in airway secretory cells to some extent, thus forming a balance between mucus hyperproduction and hypersecretion in airways exposed to smoking and LPS.

Li Q ，Zhou X ，Kolosov VP ，Perelman JM ... -  《-》

Nicotine activates cell-signaling pathways through muscle-type and neuronal nicotinic acetylcholine receptors in non-small cell lung cancer cells.

Carlisle DL ，Liu X ，Hopkins TM ，Swick MC ，Dhir R ，Siegfried JM ... -  《PULMONARY PHARMACOLOGY & THERAPEUTICS》

Nicotine up-regulates IL-8 expression in human gingival epithelial cells following stimulation with IL-1β or P. gingivalis lipopolysaccharide via nicotinic acetylcholine receptor signalling.

Cigarette smoking is an important risk factor for periodontal disease. The aim of this study is to evaluate the effect of nicotine, a major component of cigarette smoke, on interleukin-8 (IL-8) production and cellular signalling via nicotinic acetylcholine receptors (nAChRs) in human gingival epithelial cells (HGECs). Messenger RNA (mRNA) expression of nAChR subunits in three different HGEC lines (epi 4, Tfx and E6E7) was assessed using reverse transcription-polymerase chain reaction (RT-PCR). HGECs were stimulated by 1×10(-3)M nicotine in the presence or absence of IL-1β or Porphyromonas gingivalis lipopolysaccharide (LPS). IL-8 production was then examined using real-time PCR and enzyme-linked immunosorbent assay. Nicotine-mediated signalling in the epi 4 cell line was also evaluated by Western blotting. HGECs expressed several nAChR subunits. Nicotine increased the secretion of IL-8 from HGECs that were cultured in the presence of IL-1β or P. gingivalis LPS and also induced the phosphorylation of extracellular signal-regulated kinase (ERK) in epi 4. Pretreatment with non-selective nAChR antagonist or intracellular calcium chelator reduced the nicotine-induced phosphorylation of ERK. Furthermore, nicotine-induced IL-8 secretion was decreased by pretreatment with non-selective nAChR antagonist, ERK1/2 inhibitor or intracellular calcium chelator. These findings indicate that nicotine increases IL-8 production in gingival epithelial cells via ERK phosphorylation following Ca(2+) signalling after nAChR activation.

Kashiwagi Y ，Yanagita M ，Kojima Y ，Shimabukuro Y ，Murakami S ... -  《-》