Abstract
This study shows that the high affinity alpha-chain of the interleukin (IL)-15 receptor exists not only in membrane-anchored but also in soluble form. Soluble IL-15Ralpha (sIL-15Ralpha) can be detected in mouse sera and cell-conditioned media by enzyme-linked immunosorbent assay and by immunoprecipitation and Western blotting. This protein has a molecular mass of about 30 kDa because of the presence of a single N-glycosylation site, which is reduced to 26 kDa after N-glycosidase treatment. Transmembrane IL-15Ralpha is constitutively converted into its soluble form by proteolytic cleavage that involves tumor necrosis factor-alpha-converting enzyme (TACE), and this process is further enhanced by phorbol 12-myristate 13-acetate (PMA) stimulation. The hydroxamate GW280264X, which is capable of blocking TACE and the closely related disintegrin-like metalloproteinase 10 (ADAM10), effectively inhibited both spontaneous and PMA-inducible cleavage of IL-15Ralpha, whereas GI254023X, which preferentially blocks ADAM10, was ineffective. Overexpression of TACE but not ADAM10 in COS-7 cells enhanced the constitutive and PMA-inducible cleavage of IL-15Ralpha. Moreover, murine fibroblasts deficient in TACE but not ADAM10 expression exhibited a significant reduction in the spontaneous and inducible IL-15Ralpha shedding, whereas a reconstitution of TACE in these cells restored the release of sIL-15Ralpha, thereby suggesting that TACE-mediated proteolysis may represent a major mechanism for sIL-15Ralpha generation in mice. The existence of natural sIL-15Ralpha offers novel insights into the complex biology of IL-15 and envisages a new level for therapeutic intervention.
Highlights
This study shows that the high affinity ␣-chain of the demonstrating a widespread existence of soluble receptors and interleukin (IL)-15 receptor exists in mem- membrane-anchored ligands
The A hydroxamate GW280264X, which is capable of blockHDR9, 2011 ing tumor necrosis factor-␣-converting enzyme (TACE) and the closely related disintegrin-like metalloproteinase 10 (ADAM10), effectively inhibited both spontaneous and phorbol 12-myristate 13-acetate (PMA)-inducible cleavage of IL-15R␣, whereas GI254023X, which preferentially blocks
To analyze a glycosylation pattern of sIL-15R␣, IL-15R␣ was precipitated from L929-conditioned medium usding and soluble receptor generation, we investigated the involvement of these two proteases
Summary
Nol blue), boiled for 5 min, and analyzed on 10% SDS-PAGE. The resolved proteins were transferred onto nitrocellulose (Bio-Rad). Antibodies, Inhibitors, Recombinant Proteins, and Vec- were blocked for 1 h in phosphate-buffered saline containing 0.05%. IgG2b fusion proteins (FPs) were produced as described previously (35). HDR9, 2011 Recombinant sIL-15R␣ was produced as described previously (32). The purity of recombinant sIL-15R␣ was analyzed by SDS-PAGE and ru Western blotting using specific anti-IL-15R␣ antibodies. F A broad-spectrum hydroxamic acid-based MP inhibitor batimaincubations with primary and secondary antibodies and washing with phosphate-buffered saline/Tween 20, visualization of specific proteins was carried out by an enhanced chemiluminescence (ECL) method using ECL Western blotting detection reagents (Amersham Biosciences) according to the manufacturer’s instructions. 15R␣ expression was evaluated by incubation of cells with IL-15-IgG2b FP as described previously (31), and analyzed by flow cytometry using. Cells were stimulated with PMA (200 ng/ml) in fetal calf serum-free medium. Cells were seeded at 5 ϫ 105/well in 6-well
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