Nicotine induces cell proliferation by β-arrestin–mediated activation of Src and Rb–Raf-1 pathways
Piyali Dasgupta, … , Eric Haura, Srikumar Chellappan
Piyali Dasgupta, … , Eric Haura, Srikumar Chellappan
Published August 1, 2006
Citation Information: J Clin Invest. 2006;116(8):2208-2217. https://doi.org/10.1172/JCI28164.
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Research Article Oncology

Nicotine induces cell proliferation by β-arrestin–mediated activation of Src and Rb–Raf-1 pathways

Abstract

Recent studies have shown that nicotine, a component of cigarette smoke, can stimulate the proliferation of non-neuronal cells. While nicotine is not carcinogenic by itself, it has been shown to induce cell proliferation and angiogenesis. Here we find that mitogenic effects of nicotine in non–small cell lung cancers (NSCLCs) are analogous to those of growth factors and involve activation of Src, induction of Rb–Raf-1 interaction, and phosphorylation of Rb. Analysis of human NSCLC tumors show enhanced levels of Rb–Raf-1 complexes compared with adjacent normal tissue. The mitogenic effects of nicotine were mediated via the α7-nAChR subunit and resulted in enhanced recruitment of E2F1 and Raf-1 on proliferative promoters in NSCLC cell lines and human lung tumors. Nicotine stimulation of NSCLC cells caused dissociation of Rb from these promoters. Proliferative signaling via nicotinic acetylcholine receptors (nAChRs) required the scaffolding protein β-arrestin; ablation of β-arrestin or disruption of the Rb–Raf-1 interaction blocked nicotine-induced proliferation of NSCLCs. Additionally, suppression of β-arrestin also blocked activation of Src, suppressed levels of phosphorylated ERK, and abrogated Rb–Raf-1 binding in response to nicotine. It appears that nicotine induces cell proliferation by β-arrestin–mediated activation of the Src and Rb–Raf-1 pathways.

Authors

Piyali Dasgupta, Shipra Rastogi, Smitha Pillai, Dalia Ordonez-Ercan, Mark Morris, Eric Haura, Srikumar Chellappan

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Figure 4

β-arrestin–1 mediates Src activation upon nAChR signaling.

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β-arrestin–1 mediates Src activation upon nAChR signaling. (A) Transfect...
(A) Transfection of β-arrestin–1 siRNA inhibited nicotine-induced, but not serum-induced, S-phase entry in A549 cells. A nontargeting siRNA sequence was used as a control for all experiments. (B) Western blot analysis showing the suppression of β-arrestin in A549 cells upon transfection of β-arrestin–1 siRNA. Ablation of β-arrestin–1 significantly reduced nicotine-induced phosphorylated Src and phosphorylated ERK levels, whereas levels of total Src and ERK remained constant. Western blotting for actin served as the loading control. (C) Densitometric analysis was performed to quantitate the results obtained by Western blotting. (D) Nicotine induced the binding of Src to β-arrestin in A549 cells. Transfection of β-arrestin–1 siRNA abrogated the binding of c-Src to β-arrestin, whereas a nontargeting siRNA sequence had no effect. (E) Nicotine-induced stimulation of quiescent A549 cells for 15 minutes led to the binding of β-arrestin–1 to Src and to β-nAChR. (F) The binding of β-arrestin to nAChRs seen in E was confirmed by IP using β-nAChR antibody and Western blotting for β-arrestin. (G) Transfection of β-arrestin–1 siRNA inhibited Rb–Raf-1 binding in nicotine-stimulated A549 lysates, whereas a non-targeting siRNA sequence had no effect. No effect of β-arrestin–1 siRNA was seen on serum-induced Rb–Raf-1 binding in A549 cells.