Ectodomain shedding of Fcalpha receptor is mediated by ADAM10 and ADAM17

Abstract

FcalphaR (CD89) plays important roles in immunoglobulin A (IgA)-mediated immune responses. Soluble forms of FcalphaR (sFcalphaR) are found in the culture supernatants of FcalphaR-expressing cells, in human serum and in the serum of FcalphaR transgenic mice, and have been suggested to be produced through a proteolytic process. However, little is known about the mechanism involved in the proteolytic release of sFcalphaR. In this study, we investigated the shedding mechanism of FcalphaR and determined the nature of the proteinase involved in FcalphaR shedding. In chemical inhibitor assays, shedding of FcalphaR was dramatically inhibited by EDTA, EGTA and a broad-spectrum metalloproteinase inhibitor, GM6001, suggesting that a metalloproteinase was responsible for FcalphaR shedding. Overexpression of dominant-negative mutants of ADAM (a disintegrin and metalloproteinase) 10 and ADAM17 markedly inhibited the production of sFcalphaR. Finally, knockdown of both endogenous ADAM10 and endogenous ADAM17 inhibited FcalphaR shedding, demonstrating that ADAM10 and ADAM17 were involved in the shedding of FcalphaR. The characterization of ADAM10 and ADAM17 as sFcalphaR-releasing enzymes provides a novel insight into the molecular mechanism of sFcalphaR production and will help in further elucidation of the physiological and pathological roles of sFcalphaR.

Figure 1

Phorbol 12-myristate 13-acetate (PMA)-stimulated shedding of FcαR from U937 cells. (a) Detection of soluble FcαR (sFcαR) in the supernatant of U937 cells. A total of 2 × 10⁵ U937 cells were cultured in the presence of PMA (10 ng/ml) or ionomycin (5 μm) for 24 hr, after which the concentration of sFcαR in the supernatants was determined using enzyme-linked immunosorbent assay (ELISA) analysis. The control was the equivalent concentration of solvent (dimethylsulphoxide) for PMA and ionomycin added to the culture medium. Data are the mean ± standard error of the mean (SEM) from three independent experiments. (b) Biochemical analysis of sFcαR from U937 cells. A total of 1 × 10⁷ U937 cells were stimulated with 10 ng/ml of PMA for 24 hr. FcαR in the cell lysate and sFcαR in the supernatant were purified using MIP8a-coupled beads and treated with N-glycosidase F. Proteins were separated by electrophoresis on 10% sodium dodecyl sulphate–polyacrylamide, transferred onto nitrocellulose membranes and detected using rabbit anti-FcαR polyclonal IgG. The result is representative of five independent experiments. PGNase F, N-glycosidase F.

Figure 2

FcαR shedding is inhibited by metalloproteinase inhibitors. (a) A total of 2 × 10⁵ U937 cells were cultured in the presence of phorbol 12-myristate 13-acetate (PMA) (10 ng/ml) and leupeptin (2 μg/ml), pepstatin (2 μg/ml) or aprotinin (2 μg/ml) for 24 hr, and then the concentration of soluble FcαR (sFcαR) in the supernatants was determined using enzyme-linked immunosorbent assay (ELISA) analyses. (b), (c) and (d) A total of 2 × 10⁵ U937 cells were cultured in the presence of PMA (10 ng/ml) and EDTA (b), EGTA (c) or GM6001(d), at the indicated concentrations, for 24 hr, and then the concentration of sFcαR in the supernatants was determined using ELISA. Data are the mean ± standard error of the mean (SEM) from three independent experiments. *P <0·05, **P <0·01, ***P <0·001. NS, not significant.

Figure 3

FcαR shedding from stable transfectants of Chinese Hamster ovary (CHO) cells and rat basophilic leukaemia (RBL) cells. (a) and (b) Flow cytometry analysis of FcαR stably expressed on CHO (a) and RBL (b) cells. CHO or RBL cells (filled histogram) and CHO-CD89 or RBL-CD89 cells (black line) were incubated with fluorescein isothiocyanate (FITC)-conjugated MIP8a-F(ab′)₂ for 60 min on ice, then washed and analysed using flow cytometry. (c) and (d) FcαR shedding from CHO and RBL cells stably expressing FcαR. A total of 2 × 10⁵ cells were cultured with or without phorbol 12-myristate 13-acetate (PMA) (10 ng/ml) for 24 hr, and the concentration of soluble FcαR (sFcαR) in the supernatants was determined using enzyme-linked immunosorbent assay (ELISA). (e) and (f) CHO-CD89 cells or RBL-CD89 cells were cultured in the presence of leupeptin (2 μg/ml), pepstatin (2 μg/ml), aprotinin (2 μg/ml) or GM6001 (20 μm) for 24 hr. The concentration of sFcαR in the supernatants was determined using enzyme-linked immunosorbent assay (ELISA) analyses. Data are the mean ± standard error of the mean (SEM) of three independent experiments. *P <0·01, **P <0·001. NS, not significant.

Figure 4

ADAM10 and ADAM17 are responsible for FcαR shedding from 293T cells. (a) 293T cells were co-transfected with plasmids coding for FcαR and ADAM8ΔMP, ADAM10ΔMP, ADAM10E385A or ADAM17ΔMP. Supernatants were collected between 24 and 48 hr after transfection for detection of soluble FcαR (sFcαR) using enzyme-linked immunosorbent assay (ELISA). (b) and (c) Quantitive real-time polymerase chain reaction (PCR) analysis of the messenger RNA (mRNA) level in 293T cells transduced with short hairpin RNA (shRNA) against human ADAM10 and ADAM17. (d) FcαR was transfected into ADAM10 and ADAM17 knockdown 293T cells, and supernatants were collected between 24 and 48 hr after transfection for detection of sFcαR using ELISA. Data are the mean ± standard error of the mean (SEM) of three independent experiments. *P <0·05, **P <0·01, ***P <0·001. NS, not significant.

Figure 5

Depletion of endogenous ADAM10 and ADAM17 by short hairpin RNA (shRNA) reduces FcαR shedding from U937 cells. (a) and (b) Quantitive real-time PCR analysis of the messenger RNA (mRNA) level in U937 cells transduced with shRNA against human ADAM10 and ADAM17. These data are the mean ± standard error of the mean (SEM) of three independent experiments. (c) ADAM10 or ADAM17 knockdown U937 cells, or scramble shRNA U937 cells, were stimulated with 10 ng/ml of phorbol 12-myristate 13-acetate (PMA) for 24 hr, and the supernatants were collected to detect soluble FcαR (sFcαR) via enzyme-linked immunosorbent assay (ELISA). Data are the mean ± standard error of the mean (SEM) of five independent experiments. *P <0·01. NS, not significant.