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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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 1

Nicotine induces Rb inactivation and S-phase entry in lung cells.

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Nicotine induces Rb inactivation and S-phase entry in lung cells. (A) Ni...
(A) Nicotine induces S-phase entry. Quiescent H23, H441, H226, and A549 cells were treated with 1 μM nicotine for 18 hours in the presence or absence of a panel of nAChR subunit inhibitors. hex, hexamethonium bromide; α-BT, α-bungarotoxin. (B) A similar experiment was performed using normal lung cells, namely NHBEs, SAECs, and HMEC-Ls. (C) Transfection of α7-nAChR siRNA ablated the mitogenic activity of nicotine on A549 cells, whereas a nontargeting control siRNA had no effect. (D) Western blotting analysis showed that α7-nAChR siRNA ablated the expression of α7-nAChR in A549 cells, which was quantitated using densitometric analysis. A nontargeting siRNA sequence was used as a control for all experiments. (E) An in vitro assay using full-length Rb as a substrate to measure cyclin D–associated kinase activity in quiescent and nicotine-stimulated A549 cells. The lower panel shows a Coomassie Blue staining of the gel indicating comparable amounts of substrate in all lanes. (F) A similar assay as in E, showing cyclin E–associated kinase activity using histone H1 as a substrate. (G) Nicotine induces the dissociation of E2F1 from Rb in A549 cells, with concomitant phosphorylation of Rb. WB, Western blot. (H) Nicotine caused increased recruitment of E2F1 on the E2F-responsive cdc6 promoter and cdc25A accompanied by concomitant dissociation of Rb. PCR for the c-Fos promoter was taken as the negative control.