Cigarette smoke extract (CSE) promotes acetylcholine (ACh) mediated inflammation and oxidative stress by PEBP1/Raf-mediated MEK and ERK pathway in human bronchial epithelial cells

Abstract

Chronic cigarette smoking can activate inflammation and oxidative/nitrosative stress often increasing expression of non neuronal cholinergic system in epithelial cells. PEBP1 binds to Raf, inhibiting Raf-mediated MEK and ERK pathway regulating the mechanism of oxidative stress and inflammation. We aimed to investigate whether the long term exposure to CSE (0 to 20% for 7 days) promotes inflammation and oxidative/nitrosative stress by autocrine ACh production and via PEBP1 Raf-mediated MEK and ERK pathway activation in bronchial epithelial cell line (16HBE). We evaluated the ACh binding and Ros production by flowcytometry, Choline Acetyltransferase (ChAT), NOX4, PEBP1and ERk1/2 phosphorylation by western blot, the IL-8 release by ELISA, in 16HBE CSE treated. The effect of Tiotropium (Spiriva®), Olodaterol and Hemicholinium (HCh) a potent choline uptake blocker was tested. We showed increased levels of ChAT, autocrine ACh binding, pPEBP1, pERK1/2, Ros, NOX4 and IL-8 in CSE treated 16HBE compared to untreated cells. HCh, reducing levels of autocrine ACh synthesis and binding, downregulated PEBP1 and ERK1/2 phosphorylation as well as Ros, Nox4 and IL-8 production in CSE treated cells. Tiotropium (Spiriva®) or Olodaterol are able to reduce the levels of Ros, NoX4, IL-8 and ACh binding. Cigarette smoke, promoting the synthesis of autocrine ACh, generates the detachment of PEBP1 to Raf, increasing Raf-mediated MEK and ERK pathway activation and the related inflammatory and oxidative mediators in epithelium. Anticholinergic and long-acting β2-agonist drugs are able to control these mechanisms.