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

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 microM, 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.

Fig. 1.

Growth-promoting effect of m3 mAChR in a colon cancer cell line T84. Quiescent T84 cells were incubated with carbachol (1–100 μM) for 48 h, and an MTT assay was carried out as material and method. Data are expressed as fold induction over control and are mean SE (n=5)

Fig. 2A–C.

Involvement of ERK in m3 mAChR-mediated cell growth. A Effect of carbachol on ERK activity. T84 cells were stimulated with increasing concentrations of carbachol (1–100 μM) for 5 min, and ERK activity was determined by immunoblotting with anti-phospho-ERK antibody. Data was reproduced in two other separate experiments; B Effect of the MEK inhibitor PD98059 on carbachol-stimulated ERK activity. T84 cells were pretreated with either vehicle (0.1% DMSO) or PD98059 (0.1–10 μM) for 20 min, followed by stimulation with 10 µM carbachol for 5 min. ERK assay was examined; C Effect of PD98059 on carbachol-stimulated T84 cell growth. T84 cells were stimulated with 10 µM carbachol for 48 h in the presence of either vehicle (0.1% DMSO) or 10 µM PD98059, and an MTT assay was performed. Data are expressed as fold induction over control and are mean SE (n=5)

Fig. 3A,B.

Transactivation of EGFR by mAChR; requirement of EGFR tyrosine kinase and functionally active in signal transduction. A Effect of the EGFR tyrosine kinase inhibitor AG1478 on EGFR transactivation stimulated by carbachol. T84 cells were pretreated with either vehicle (0.1% DMSO) or 10 μM AG1478 for 15 min, followed by stimulation with 10 µM carbachol for 5 min. EGFR in T84 lysates was immunoprecipitated, and its phosphorylation was studied by immunoblotting using an anti-phosphotyrosine antibody. Data was a representative of three independent experiments; B Effect of carbachol on association of GRB2 and Shc with EGFR. GRB2 in T84 lysates stimulated with 10 μM carbachol was immunoprecipitated and subjected to immunoblotting using an anti-phosphotyrosine antibody. Identical results were obtained in two other separate experiments

Fig. 4A,B.

Effect of the transmembrane metalloproteinase inhibitor, GM6001, and the EGFR neutralizing antibody LA-1 on EGFR transactivation. A T84 cells were preincubated with either vehicle (0.1% DMSO) or 50 μM of GM6001 for 18 h and subsequently stimulated with 10 µM carbachol. Tyrosine phosphorylation of EGFR was examined; B T84 cells were preincubated with either 10 mg/ml mouse Ig G or the same concentration of LA-1 for 20 min and stimulated with carbachol. Then transactivation was examined. These data were reproduced in two additional experiments

Fig. 5.

Effect of AG1478 or LA-1 on carbachol-stimulated T84 cell growth. The T84 cells were pretreated with 10 μM AG1478 or 10 mg/ml LA-1 for 20 min, followed by stimulation with 10 µM carbachol for 48 h. In each experiment, vehicle (0.1% DMSO) alone or mouse Ig G was included as the control, respectively. MTT assay was performed as above. Data are expressed as fold induction over control and are mean SE (n=5)