Receptor‐mediated tobacco toxicity: cooperation of the Ras/Raf‐1/MEK1/ERK and JAK‐2/STAT‐3 pathways downstream of a7 nicotinic receptor in oral keratinocytes (original) (raw)

The use of tobacco products is associated with an increased incidence of periodontal disease, poor response to periodontal therapy, and a high risk for developing head and neck cancer. Nicotine and tobacco-derived nitrosamines have been shown to exhibit their pathobiologic effects due in part to activation of the nicotinic acetylcholine (ACh) receptors (nAChRs), mainly ␣7 nAChR, expressed by oral keratinocytes (KCs). This study was designed to gain mechanistic insight into ␣7-mediated morbidity of tobacco products in the oral cavity. We investigated the signaling pathways downstream of ␣7 nAChR in monolayers of oral KCs exposed for 24 h to aged and diluted sidestream cigarette smoke (ADSS) or an equivalent concentration of pure nicotine. By both real-time polymerase chain reaction (PCR) and In-cell Western, the KCs stimulated with ADSS or nicotine showed multifold increases of STAT-3. These effects could be completely blocked or significantly (P<0.05) diminished if the cells were pretreated with the ␣7 antagonist ␣-bungarotoxin (␣BTX) or transfected with anti-␣7 small interfering RNA (siRNA-␣7). The use of pathway inhibitors revealed that signaling through the Ras/Raf-1/MEK1/ ERK steps mediated ␣7-dependent up-regulation of STAT-3. Targeted mutation of the ␣7 gene prevented ERK1/2 activation by nicotine. Using the gel mobility shift assay, we demonstrated that an increased protein binding activity of STAT-3 caused by ADSS or pure nicotine was mediated by janus-activated kinase (JAK)-2. Activation of JAK-2/STAT-3 pathway could be prevented by ␣BTX or siRNA-␣7. Thus, nuclear transactivation of STAT-3 in KCs exposed to tobacco products is mediated via intracellular signaling downstream from ␣7, which proceeds via two complementary pathways. The Ras/Raf-1/MEK1/ERK cascade culminates in up-regulated expression of the gene encoding STAT-3, whereas recruitment and activation of tyrosine kinase JAK-2 phosphorylates it. Elucidation of this novel mechanism of nicotine-dependent nuclear transactivation of STAT-3 identifies oral ␣7 nAChR as a promising molecular target to prevent, reverse, or retard tobacco-related periodontal disease and progression of head and neck cancer by receptor inhibitors.-Arredondo, J., Chernyavsky, A. I., Jolkovsky, D. L., Pinkerton, K. E., Grando, S. A. Receptor-mediated tobacco toxicity: cooperation of the Ras/Raf-1/MEK1/ ERK and JAK-2/STAT-3 pathways downstream of ␣7 nicotinic receptor in oral keratinocytes.