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 2

Rb–Raf-1 interaction is induced by nicotine in cultured cells and is elevated in tumor samples.

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Rb–Raf-1 interaction is induced by nicotine in cultured cells and is ele...
(A) Nicotine induces the binding of Raf-1 to Rb in HAEC and A549 cells as seen by IP/Western blotting. Rb–Raf-1 interaction was also observed in other NSCLC cell lines like H441, H226, and H23 (B) as well as in SAECs, NHBEs, and HMEC-Ls (C). (D) Nicotine-induced Rb–Raf-1 binding is mediated by α7-nAChRs in A549 cells. Quiescent A549 cells were stimulated with 1 μM nicotine in the presence or absence of 1 μM of the indicated antagonists; IP/Western blot shows the inhibition of the Rb–Raf-1 interaction by α7-nAChRs inhibitors α-bungarotoxin and MAA. (E) Penetratin–Raf-1 (Pen–Raf-1) peptide conjugate abrogated nicotine-induced S-phase entry in A549 cells (black bars) and HAECs (gray bars) whereas a scrambled peptide conjugate, Pen-Rafscram, had no effect. (F) The penetratin–Raf-1 conjugate abolished Rb–Raf-1 binding in nicotine-stimulated A549 cells and HAECs, whereas the scrambled peptide conjugate (Pen–Rafscram) did not; Rb–Raf-1 interaction was measured by IP/Western blot. (G) NSCLC tumors (N) contained more Rb–Raf-1 complexes than adjacent normal tissue (T). Rb–Raf-1 interaction was assessed by IP/Western blot on nuclear extracts. (H) ChIP assays on human NSCLC tumor samples show that more Raf-1 was present on cdc6 and cdc25A promoters in tumor samples compared with adjacent normal lung tissues.