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Annexin A2 Regulates A Disintegrin and Metalloproteinase 17–mediated Ectodomain Shedding of Pro–Tumor Necrosis Factor-α in Monocytes and Colon Epithelial Cells

Tsukamoto, Hironobu MD*; Tanida, Satoshi MD, PhD*; Ozeki, Keiji MD*; Ebi, Masahide MD, PhD*; Mizoshita, Tsutomu MD, PhD*; Shimura, Takaya MD, PhD*; Mori, Yoshinori MD, PhD*; Kataoka, Hiromi MD, PhD*; Kamiya, Takeshi MD, PhD*; Fukuda, Shinji PhD; Higashiyama, Shigeki PhD; Joh, Takashi MD, PhD*

doi: 10.1097/MIB.0b013e318281f43a
Original Basic Science Article

Background: Understanding the mechanism of tumor necrosis factor (TNF)-α shedding is important because TNF-α triggers inflammatory bowel disease development. A disintegrin and metalloproteinase (ADAM) 17 is a key enzyme for the shedding of not only the type 1 membrane-anchored protein, amphiregulin, but also the type 2 protein, TNF-α. However, the detailed mechanism by which ADAM17 cleaves type 1 and 2 membrane-anchored proteins is unclear. Annexin (ANX) A2 is involved in ADAM17-mediated amphiregulin shedding. In this study, we examined whether ANX A2 is involved in TNF-α shedding.

Methods: We prepared U937, HT29, and HCT116 cells overexpressing alkaline phosphatase (AP)-tagged proTNF-α and depleted ADAM17 and ANX A2. We assessed TNF-α release and shedding by measuring the TNF-α release concentration and AP activities in conditioned media after interleukin-1β or 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulation by enzyme-linked immunosorbent assay and AP assay, respectively. A direct association of ANX A2 with ADAM17 was examined with immunoprecipitation and Western blotting.

Results: Enzyme-linked immunosorbent assay and AP assay showed interleukin-1β–induced TNF-α shedding in HCT116 and HT29 cells and TPA-induced TNF-α release in U937 cells. KB-R7785 and ADAM17 depletion significantly blocked TNF-α shedding by TPA. ANX A2 depletion significantly inhibited TNF-α shedding by interleukin-1β and TPA. In contrast, ANX A2 depletion did not abrogate ADAM17-mediated amphiregulin and heparin-binding epidermal growth factor-like growth factor shedding. ANX A2 was directly associated with ADAM17.

Conclusions: ANX A2 was closely associated with ADAM17 and played an important role in TNF-α shedding by TPA. Inhibition of ANX A2 might be a new therapeutic strategy for prevention of TNF-α shedding during inflammatory bowel disease inflammation.

Article first published online 9 April 2013Supplemental Digital Content is Available in the Text.

*Department of Gastroenterology and Metabolism, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan; and

Department of Medical Biochemistry, Ehime University School of Medicine, Ehime, Japan.

Reprints: Satoshi Tanida, MD, PhD, Department of Gastroenterology and Metabolism, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya, Japan 4678601 (e-mail:

Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's Web site (

Supported by Grant-in Aid for scientific research C (KAKENHI no.24590949) from Japan Society for the Promotion of Science.

The authors have no conflicts of interest to disclose.

Received November 09, 2012

Accepted November 15, 2012

© Crohn's & Colitis Foundation of America, Inc.
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