Involvement of membrane-type bile acid receptor M-BAR/TGR5 in bile acid-induced activation of epidermal growth factor receptor and mitogen-activated protein kinases in gastric carcinoma cells

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Abstract

Bile acids, which have been implicated in gastrointestinal-tract cell carcinogenesis, share properties with tumor promoters in that both affect signal transduction pathways responsible for cell proliferation and apoptosis. In the present study, we demonstrate that EGFR–ERK1/2 is activated following treatment of AGS human gastric carcinoma cells with bile acids. EGFR phosphoactivation is ligand-dependent, since treatment of cells with HB–EGF antisera or CM197 (a selective inhibitor of HB–EGF) markedly inhibits deoxycholate (DC)-promoted activation. Membrane-type bile acid receptor (M-BAR)/TGR5 is a recently identified G-protein-coupled receptor (GPCR). In AGS cells, siRNAs that target M-BAR suppress DC-induced phosphorylation of EGFR. Furthermore, introduction of siRNAs targeting ADAM17 transcripts resulted in suppression of DC-induced activation of EGFR and ERK1/2. These results suggest that in AGS cells, DC transactivates EGFR through M-BAR- and ADAM/HB–EGF-dependent mechanisms.

Section snippets

Experimental procedures

Materials. Antibodies against EGFR, phospho-specific antibodies against Tyr1068-phosphorylated EGFR, and phospho- and non-specific antibodies to ERK1/2 were purchased from Santa Cruz Biotechnology (Santa Cruz). EGFR-neutralizing antibody (Clone 225) was purchased from Lab Vision Corporation. Amphiregulin- and HB–EGF-neutralizing antibodies were purchased from R&D. TGFα-neutralizing antibody was purchased from BioVision. Antibodies against ADAM10 and ADAM17 were purchased from AnaSpec Inc.

DC-induced ERK1/2 activation in AGS cells

DC induces a rapid activation of ERK1/2 in many different cell systems including liver [4], biliary [5], and gastric cells [6]. Consistent with previous findings, treatment of AGS cells with DC-induced phosphorylation of ERK-1/2 MAPK within 5 min that was detectable for at least 2 h (Fig. 1a).

DC activation of EGFR in AGS cells is ligand-dependent

Previous studies have implicated EGFR activation in bile acid-mediated activation of ERK1/2 in hepatocytes and cholangiocytes [4], [5]. We tested whether EGFR was also involved in the DC-induced activation

Discussion

We report that EGFR–ERK1/2 pathways are activated in AGS cells treated with DC. Our results suggest that the novel GPC receptor M-BAR functions in the transactivation event, and that transactivation of EGFR, specifically, occurs via an ADAM/HB–EGF dependent pathway. Our results provide new insights that may contribute to the development of therapeutic tools to treat or prevent gastrointestinal neoplasm.

Data from many reports make it clear that the EGF receptor functions within signaling

Acknowledgment

We thank Ms. Y. Okayama for excellent technical assistance.

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    This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan to H.Y.

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