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Growth-promoting effect of muscarinic acetylcholine receptors in colon cancer cells

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Abstract

Purpose

G-protein-coupled receptors are known to mediate cell growth via divergent signaling pathways. It has been reported that colon cancer cells express muscarinic acetylcholine receptor (mAChR) although their functional role is largely unknown. The aim of this study is to elucidate possible mechanisms responsible for the growth-promoting effect of mAChRs in colon cancer cells by using colon cancer cell line T84.

Methods

Carbachol, a stable mAChR agonist, dose-dependently induced cell growth with a maximal effect observed at 100 μM, equipotent with 1 nM EGF. 4-DAMP, a specific antagonist of subtype 3 mAChR, inhibited the stimulatory effect by carbachol, suggesting that the growth-promoting effect was receptor-mediated. Carbachol also dose-dependently stimulated extracellular signal-regulated protein kinase (ERK) activation. This effect was inhibited by PD98059, an inhibitor of extracellular signal-regulated protein kinase kinase, which also blocked carbachol activation of cell proliferation, indicating that the p21Ras-ERK pathway is an important signaling cascade in the mitogenic effect. To investigate how mAChR activated the p21Ras-ERK pathway, transactivation of epidermal growth factor receptor (EGFR) was examined.

Results

Carbachol induced tyrosine phosphorylation of EGFR, which was abolished by an EGFR tyrosine kinase inhibitor AG1478. Transactivation by carbachol was also abrogated by a metalloproteinases (MMPs) inhibitor GM6001 or an EGFR-blocking antibody (LA-1), suggesting that binding of EGFR ligand(s) produced by MMPs may initiate transactivation in a manner dependent on EGFR tyrosine kinase. The tyrosine-phosphorylated EGFR was immunoprecipitated together with GRB2 and tyrosine-phosphorylated Shc, indicating that transactivated EGFR is able to generate downstream signals. AG 1478 and LA-1 inhibited carbachol stimulation of cell growth.

Conclusions

Taken together, our results indicate that the growth-promoting effect of subtype 3 mAChR in colon cancer cells may depend on transactivated EGFR-ERK pathways. EGFR not only receives external stimuli but also serves as a scaffold for downstream signaling molecules.

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Fig. 1.
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Abbreviations

ERK:

Extracellular signal-regulated protein kinase

MAPK:

Mitogen-activated protein kinase

EGF:

Epidermal growth factor

GPCR:

G-protein-coupled receptor

mAChR:

Muscarinic acetylcholine receptor

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Authors and Affiliations

  1. Second Department of Internal Medicine, School of Medicine, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, 142-8666, Tokyo, Japan

    J.-I. Ukegawa, Y. Takeuchi, S. Kusayanagi & K. Mitamura

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  1. J.-I. Ukegawa
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  2. Y. Takeuchi
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  3. S. Kusayanagi
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  4. K. Mitamura
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Correspondence to Y. Takeuchi.

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Ukegawa, JI., Takeuchi, Y., Kusayanagi, S. et al. Growth-promoting effect of muscarinic acetylcholine receptors in colon cancer cells. J Cancer Res Clin Oncol 129, 272–278 (2003). https://doi.org/10.1007/s00432-003-0433-y

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  • DOI: https://doi.org/10.1007/s00432-003-0433-y

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