Gastroenterology

Gastroenterology

Volume 123, Issue 6, December 2002, Pages 1962-1971
Gastroenterology

Basic–Alimentary Tract
Helicobacter pylori CagA protein activates serum response element–driven transcription independently of tyrosine phosphorylation,☆☆

https://doi.org/10.1053/gast.2002.37044Get rights and content

Abstract

Background & Aims: Infection with Helicobacter pylori possessing the cag pathogenicity island (PAI) is associated with severe gastritis and gastric cancer. CagA protein, one of the products of cag PAI, is translocated into epithelial cells, where cytoskeletal rearrangements occur as a result of CagA tyrosine (Tyr) phosphorylation. Here we identify a new role for CagA protein as an activator of host cell signaling. Methods: We transfected CagA into epithelial cells and analyzed its effect on transcription by reporter assays. The mechanism of reporter activation was assessed by electrophoretic mobility shift assays (EMSA) and immunoblots. Responsible regions of CagA for reporter activation were determined by truncation and mutagenesis of cagA gene. Results: In HeLa cells, expression of CagA increased serum response element (SRE)-driven and serum response factor (SRF)-driven transcription by 40-fold and 3.3-fold, respectively, but did not affect nuclear factor κB– or AP-1–driven transcription. CagA-mediated SRE activation was also observed in gastric cell lines. Immunoblotting and EMSA revealed that transfection of CagA enhanced phosphorylation of and DNA binding by Elk1. Furthermore, involvement of Ras and MEK in CagA-mediated Elk1 phosphorylation was observed. SRE activation was dependent on several regions within the C-terminal portion of CagA (CagA873-1002), and independent of Tyr phosphorylation. Conclusions: The C-terminal portion of CagA enhances SRE-driven transcription by activating an upstream signaling cascade without requiring CagA Tyr phosphorylation. This result suggests that translocated CagA regulates 2 distinct cellular responses: phosphorylation-dependent cytoskeletal rearrangement and phosphorylation-independent transcriptional activation.

GASTROENTEROLOGY 2002;123:1962-1971

Section snippets

Cell line

HeLa cells, a human cervical carcinoma cell line, were obtained from the Riken Cell Bank (Tsukuba, Japan). Cells were grown at 37°C in 5% CO2 in Dulbecco's modified Eagle medium supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, and 10% fetal bovine serum. Three gastric carcinoma cell lines—AGS, TMK1, and MKN45 cells—were obtained and grown as previously described.19, 22, 30

Plasmids

The full-length cagA gene and its various fragments were amplified from H. pylori strain 26695 by polymerase

Helicobacter CagA protein activates serum response element–driven transcription in epithelial cells

To investigate the roles of translocated CagA protein in epithelial cells, we first transfected HeLa cells with the expression vector pTX-CagA, which contains the H. pylori ATCC26695 cagA gene. As shown by immunoblotting with an anti-CagA antibody, CagA was expressed in a dose-dependent manner (Figure 1A).

. Expression of CagA protein and analysis of CagA-induced reporter gene activation. (A) HeLa cells were transfected with the indicated amount of CagA expression vector pTX-CagA for 24 hours.

Discussion

Several studies have revealed that the type I strain of H. pylori transports CagA protein into host epithelial cells via the bacterial PAI secretion system, and that translocated CagA protein is phosphorylated on a Tyr residue.38, 39, 40, 41 In these reports, the function of CagA protein is linked to cellular cytoskeletal rearrangements through Tyr phosphorylation. In this study we have demonstrated a new role for translocated CagA as a regulator of cytoplasmic signaling and gene transcription.

Acknowledgements

The authors thank Mitsuko Tsubouchi for her excellent technical assistance.

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    Address requests for reprints to: Yoshihiro Hirata, M.D., Department of Gastroenterology, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. e-mail: [email protected]; fax: (81) 3-3814-0021.

    ☆☆

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

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