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 2013
*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: email@example.com).
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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