Inflammatory Bowel Diseases

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

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*

Supplemental Author Material
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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.

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

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