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

Abstract

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 alpha7 nAChR, expressed by oral keratinocytes (KCs). This study was designed to gain mechanistic insight into alpha7-mediated morbidity of tobacco products in the oral cavity. We investigated the signaling pathways downstream of alpha7 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 alpha7 antagonist alpha-bungarotoxin (alphaBTX) or transfected with anti-alpha7 small interfering RNA (siRNA-alpha7). The use of pathway inhibitors revealed that signaling through the Ras/Raf-1/MEK1/ERK steps mediated alpha7-dependent up-regulation of STAT-3. Targeted mutation of the alpha7 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 alphaBTX or siRNA-alpha7. Thus, nuclear transactivation of STAT-3 in KCs exposed to tobacco products is mediated via intracellular signaling downstream from alpha7, 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 alpha7 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.

Receptor-mediated tobacco toxicity: Alterations of the NF-κB expression and activity downstream of α7 nicotinic receptor in oral keratinocytes

“…The intracellular signaling downstream of α7 leading to upregulation of NF-κB proceeded through the Ras/Raf-1/MEK1/ERK steps. Previous studies identified α7 is one of the major acetylcholine-gated ion channels on the KC cell membrane that mediates acute effects of tobacco products on oral KCs (Arredondo et al, 2006b), whereas the effects of longterm exposures (lasting 5 days) are predominantly mediated by the α3-made nAChR channels, such as α3β2 . Interestingly, NF-κB is targeted by both the α7-and the α3-coupled pathways.…”

“…KCs were transfected with siRNAs and MEK1 kinase mutants following a standard protocol . The monolayers grown to ∼80% confluence in 6-well plates were incubated for 24 h in KGM pre-exposed to ETS in the chambers of a sidestream smoke exposure system, as detailed elsewhere (Arredondo et al, 2006b), or containing an equivalent concentration of nicotine (10 μM), and used in the biochemical assays described below.…”

“…The real-time PCR, the in-cell western assay (LI-COR Lincoln, NE) and EMSA were performed as described in detail elsewhere (Arredondo et al, 2006b). For real-time PCR, the primers of the gene encoding human NF-κB were designed with the assistance of the Primer Express software version 2.0 computer program (Applied Biosystems, Foster City, CA), and the service Assays-on-Design provided by Applied Biosystems.…”

“…Targeted mutation of the α7 gene prevented ERK1/2 activation by nicotine. Using the electrophoretic mobility shift assay (EMSA), we have also demonstrated that an increased protein-binding activity of STAT-3 caused by ETS/nicotine was mediated by JAK-2 (Arredondo et al, 2006b). …”

“…The α7 subunit is first expressed on the cell surfaces of oral keratinocytes (KCs) comprising the lower third portion of the gingival epithelium, and becomes abundant at the terminal stage of cell development in the gingival epithelium (Nguyen et al, 2000). In a recent study, we found that the α7 inhibitor α-bungarotoxin (αBtx) and transfection with the small interfering RNA against α7 (siRNA-α7) can block effects of environmental tobacco smoke (ETS) and pure nicotine on human KCs (Arredondo et al, 2006b;Chernyavsky et al, 2005). The signaling through the Ras/Raf-1/MEK1/ERK steps mediated, in the most part, the α7-dependent effects.…”