Acetylcholine-induced activation of M3 muscarinic receptors stimulates robust matrix metalloproteinase gene expression in human colon cancer cells

Abstract

Previously, we showed that ACh-induced proliferation of human colon cancer cells is mediated by transactivation of epidermal growth factor (EGF) receptors (EGFRs). In the present study, we elucidate the molecular mechanism underlying this action. ACh-induced proliferation of H508 colon cancer cells, which express exclusively M3 muscarinic receptors (M3Rs), was attenuated by anti-EGFR ligand binding domain antibody, a broad-spectrum matrix metalloproteinase (MMP) inhibitor, anti-MMP7 antibody, a diphtheria toxin analog that blocks release of an EGFR ligand [heparin-binding EGF-like growth factor (HBEGF)], and anti-HBEGF antibody. Conditioned media from ACh-treated H508 cells induced proliferation of SNU-C4 colon cancer cells that express EGFR but not M3R. These actions were attenuated by an EGFR inhibitor and by anti-EGFR and anti-HBEGF antibodies. In H508, but not SNU-C4, colon cancer cells, ACh caused a striking dose- and time-dependent increase in levels of MMP7 mRNA and MMP7 protein. Similarly, ACh induced robust MMP1 and MMP10 gene transcription. ACh-induced MMP1, MMP7, and MMP10 gene transcription was attenuated by atropine, anti-EGFR antibody, and chemical inhibitors of EGFR and ERK activation. In contrast, inhibitors of phosphatidylinositol 3-kinase and NF-kappaB activation did not alter MMP gene transcription. Collectively, these findings indicate that MMP7-catalyzed release of HBEGF mediates ACh-induced transactivation of EGFR and consequent proliferation of colon cancer cells. ACh-induced activation of EGFR and downstream ERK signaling also regulates transcriptional activation of MMP7, thereby identifying a novel feed-forward mechanism for neoplastic cell proliferation.

Fig. 1.

Effects of various agents on proliferation of H508 human colon cancer cells. A: dose-response of ACh-induced proliferation of H508 cells. H508 cells were incubated for 5 days at 37°C with 10–1,000 μM ACh. B: effect of heparin-binding epidermal growth factor-like growth factor (HBEGF) antibody, CRM197, and matrix metalloproteinase (MMP) inhibitor (GM6001) on cell proliferation. H508 cells were incubated with the indicated reagents alone or in combination for 5 days at 37°C. C and D: neutralizing antibody to MMP7 blocks ACh- and recombinant MMP7 (rMMP7)-stimulated cell proliferation. H508 cells were incubated with ACh alone, rMMP7 alone, or the indicated combined reagents for 5 days at 37°C. Cell proliferation was determined using the CellTiter 96 AQ_ueous One solution assay. Values are means ± SE of ≥3 separate experiments. *P < 0.05 vs. ACh-stimulated cells (Student's t-test).

Fig. 2.

Effects of conditioned media from H508 cells (A) and addition of HBEGF to untreated H508 media (B) on proliferation of SNU-C4 human colon cancer cells. A: SNU-C4 cells were incubated with ACh or conditioned media from H508 cells and inhibitors for 5 days at 37°C. B: SNU-C4 cells were incubated with reagents alone in serum-free media or in combination in untreated H508 media. Values are means ± SE of ≥3 separate experiments. *P < 0.05 vs. ACh-stimulated cells. **P < 0.05 vs. cells stimulated by H508 supernatant alone. ***P < 0.05 vs. cells stimulated by HBEGF alone in untreated H508 media.

Fig. 3.

H508 cells express multiple epidermal growth factor (EGF) receptor (EGFR) ligands. A: relative mRNA levels of 3 EGFR ligands were measured by quantitative real-time PCR (Q-PCR). B: ACh-induced H508 cell proliferation is not affected by blocking amphiregulin (AR) with a neutralizing antibody. H508 cells were incubated with ACh alone, recombinant AR (rAR) alone, AR antibody alone, or combined reagents for 5 days at 37°C. Cell proliferation was determined using the CellTiter 96 AQ_ueous One solution assay. TGFα, transforming growth factor-α. Values are means ± SE of 3 separate experiments. *P < 0.05 vs. AR-stimulated cell proliferation.

Fig. 4.

ACh enhances H508 cell colony formation. A: representative photographs of colonies formed in soft agar assay in the presence and absence of ACh (300 μM). Dark spots represent H508 cell colonies. B: ACh-induced colony formation is attenuated by atropine and by anti-MMP7 and anti-EGFR antibodies. Values are means ± SE of 3 separate experiments. *P < 0.05 vs. ACh-stimulated colonies.

Fig. 5.

ACh stimulates time- and dose-dependent MMP7 mRNA and protein expression. A: ACh induces time-dependent upregulation of MMP7 transcription. Levels of MMP7 mRNA were determined by Q-PCR. B: ACh causes dose-dependent increases in MMP7 gene transcription. Q-PCR was performed using cDNAs that were synthesized from total RNAs prepared from H508 cells that were stimulated by different concentrations of ACh for 4 h. Fold increase was determined by comparison with controls. C: MMP7 mRNA decay was measured by Q-PCR after addition of actinomycin D (1 μg/ml) in the presence or absence of 100 μM ACh. Values are expressed as percentage of mRNA remaining compared with value at time 0 (i.e., before addition of ACh). *P < 0.05 vs. ACh-stimulated cells. D: ACh induces MMP7 protein expression determined by ELISA. In H508 cell extracts, at the indicated times after addition of 100 μM ACh, increases in MMP7 protein above basal levels were determined by ELISA. E: ACh (100 μM) induces upregulation of MMP7 transcription in H508 cells but not in SNU-C4 cells. HBEGF (10 ng/ml) upregulates MMP7 gene transcription in H508 and SNU-C4 colon cancer cells. Q-PCR was performed using cDNAs that were synthesized from total RNAs prepared from cells that were stimulated by test agents for 4 h. Values are means ± SE of ≥3 separate experiments.

Fig. 6.

Spectrum and mechanism of ACh-induced MMP gene transcription. A: actions of inhibitors of key signaling molecules on ACh-induced upregulation of MMP7 gene transcription. H508 cells were incubated with 100 μM ACh for 4 h in the absence or presence of the indicated concentrations of atropine, EGFR antibody, EGFR inhibitors (PD168393 and AG1478), MEK inhibitors (PD98059 and U0126), phosphatidylinositol 3′-kinase (PI3K) inhibitor (LY294002), and NF-κB inhibitor (SN50). MMP7 mRNA was measured by Q-PCR. Values are means ± SE of ≥3 separate experiments. B: actions of ACh on expression of MMP genes in H508 colon cancer cells. MMP mRNA was measured by Q-PCR using primers shown in Table 1. C: actions of inhibitors of key signaling molecules on ACh-induced upregulation of MMP1, MMP7, and MMP10 gene transcription. Inhibitors and concentrations are the same as those used in Fig. 5A. ↓, Attenuation; −, no effect.

Fig. 7.

Model depicting mechanisms underlying ACh-induced activation of MMP7, EGFR, ERK signaling, and upregulation of MMP1, MMP7, and MMP10 gene transcription. Interaction of ACh with M₃ muscarinic receptor (M₃R) results in activation of MMP7. MMP7 catalyzes release of HBEGF from pro-HBEGF. HBEGF is an EGFR ligand. Activation of EGFR results in post-receptor ERK signaling, which stimulates MMP1, MMP7, and MMP10 gene transcription. Agents that block this cascade include a muscarinic receptor inverse agonist (atropine), inhibitors of MMP7 activation (GM6001 and anti-MMP7), an inhibitor of pro-HBEGF cleavage (CRM197), inhibitors of EGFR activation (AG1478, PD168393, anti-HBEGF, and anti-EGFR antibodies), and inhibitors of MEK (U0126 and PD98059). As indicated by the large curved arrow, our findings provide evidence that we have identified a novel feed-forward mechanism regulated by muscarinic signaling whereby increased MMP7 gene transcription can replenish activated MMP7.