Abstract
BackgroundIn response to tissue damage or inflammation, adenosine-5′-triphosphate (ATP) is released into the extracellular compartment and has been demonstrated to augment inflammation via purinergic P2 receptors (P2Rs). Recently, ATP has been shown to be increased in the airways of COPD patients. In the present study, we examined the possible involvement of extracellular ATP in airway mucus hypersecretion during viral-induced COPD exacerbations.MethodsThe involvement of extracellular ATP in the release of a major airway mucin, MUC5AC, and its signal pathway was examined after stimulation with polyinosine-polycytidylic acid [poly(I:C)], a synthetic analog of dsRNA to mimic viral infection, and rhinovirus (RV) infection in NCI-H292 cells and differentiated airway epithelial cells from COPD patients.ResultsTreatment with poly(I:C) significantly increased the amount of extracellular ATP and induced MUC5AC release in NCI-H292 cells. Pre-treatment with a pannexin channel inhibitor, carbenoxolone (CBX), reduced the amount of extracellular ATP and suppressed MUC5AC release from poly(I:C)-treated cells. Pre-treatment with the P2R antagonist suramin significantly reduced the expression and release of MUC5AC. The inhibitory effects of CBX and suramin on the release of ATP and/or MUC5AC were replicated with RV infection. Pre-treatment with suramin also significantly reduced the expression and amount of extracellular EGFR ligands and the phosphorylation of EGFR and ERK in poly(I:C)-treated cells. In addition, pre-treatment with a P2Y2 receptor siRNA significantly suppressed the poly(I:C)-potentiated EGFR ligands and MUC5AC release. After poly(I:C) stimulation, the expression of MUC5AC in the differentiated cells from COPD patients was significantly higher than those from healthy subjects and the values of MUC5AC expression were inversely related with forced expiratory volume in 1 s (FEV1) % predicted. The inhibitory effects of CBX and suramin on poly(I:C)-potentiated MUC5AC expression were confirmed in differentiated airway epithelium from COPD patients.ConclusionsThese results demonstrate that dsRNA induces the release of ATP via pannexin channel and that the extracellular ATP is involved in the expression and release of MUC5AC, mainly via P2Y2R, in an autocrine manner. Modulation of this pathway could be a therapeutic target for viral-induced mucus hypersecretion in COPD exacerbations.
Highlights
In response to tissue damage or inflammation, adenosine-5′-triphosphate (ATP) is released into the extracellular compartment and has been demonstrated to augment inflammation via purinergic P2 receptors (P2Rs)
We evaluated the following points, using NCI-H292 cells and differentiated primary human bronchial epithelial cells (HBECs) from normal subjects and Chronic obstructive pulmonary disease (COPD) patients: (a) whether pannexin channels are involved in ATP release, (b) whether the inhibition of P2Rs, especially P2X7R and P2Y2R, affects MUC5AC production and the epidermal growth factor receptor (EGFR)-extracellular signal-regulated kinase (ERK) signaling pathway after stimulation with a synthetic double-stranded RNA (dsRNA) analogue, polyinosinic-polycytidylic acid [poly(I:C)], as a TLR3 ligand to mimic viral infection, and rhinovirus infection
Involvement of extracellular ATP on MUC5AC release in poly(I:C)-treated NCI-H292 cells In our previous study, we confirmed that TLR3 was expressed in NCI-H292 cells, a human pulmonary mucoepidermoid carcinoma cell line, and a synthetic dsRNA analogue, poly(I:C), used as a TLR3 ligand to mimic viral infection, induced the expression and release of MUC5AC from the cells [22]
Summary
In response to tissue damage or inflammation, adenosine-5′-triphosphate (ATP) is released into the extracellular compartment and has been demonstrated to augment inflammation via purinergic P2 receptors (P2Rs). ATP has been shown to be increased in the airways of COPD patients. Once released into the extracellular compartment in response to tissue damage or inflammation, extracellular ATP has been shown to act as a potent inducer of inflammation [4, 5]. In the airway of COPD patients, an increased concentration of extracellular ATP has been demonstrated, especially during acute smoke exposure [13], suggesting that extracellular ATP might be involved in the pathophysiology of COPD and exacerbations
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