Membrane-anchored CD40 is processed by the tumor necrosis factor-alpha-converting enzyme. Implications for CD40 signaling.
The soluble form of CD40 (sCD40), which co-exists with the membrane-anchored form (mCD40), is a natural antagonist of mCD40/CD154 interaction. However, the mechanism leading to the production of sCD40 has never been investigated. Here, we show that the engagement of mCD40 on the surface of B lymphocytes by anti-CD40 antibody led to enhanced sCD40 release associated with decreased amounts of mCD40. This sCD40 production was not affected by vesicular traffic inhibitors but was completely blocked by a broad-spectrum synthetic metalloproteinase (MP) inhibitor (GM6001) or a membrane-anchored MP-specific inhibitor (dec-RVKR-cmk). Recombinant MP disintegrin tumor necrosis factor-alpha converting enzyme (TACE) cleaved the purified CD40 ectodomain/Fc chimeric protein in vitro, giving rise to an sCD40 form similar to that shed from B cell cultures. Moreover, spontaneous production of sCD40 by mCD40-transfected human embryonic kidney cells (constitutively expressing TACE) was enhanced by the overexpression of TACE and abrogated by co-transfection with a dominant-negative TACE mutant. These results provide strong evidence that sCD40 production is an active process regulated by the engagement of mCD40 and its proteolytic cleavage by TACE or a related MP disintegrin. Given the antagonistic activity of sCD40 on the CD40/CD154 interaction, this shedding mechanism might represent an important negative feedback control of CD40 functions.
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CNRS UMR 5540, IFR 66, Université Bordeaux 2, 146 rue Léo Saignat, 33076 Bordeaux, France.
Pitard
Vincent
V
Itai
Toshimitsu
T
Nagata
Shigekazu
S
Moreau
Jean-François
JF
Déchanet-Merville
Julie
J
D051722:ADAM Proteins; D000072198:ADAM17 Protein; D001402:B-Lymphocytes / Q000378:metabolism; D015153:Blotting, Western; D019013:CD40 Antigens / Q000378:metabolism*; D023201:CD40 Ligand / Q000378:metabolism; D002460:Cell Line; D002461:Cell Line, Transformed; D002462:Cell Membrane / Q000378:metabolism*; D004151:Dipeptides / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D005799:Genes, Dominant; D006801:Humans; D008666:Metalloendopeptidases / Q000378:metabolism*; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection
D019013:CD40 Antigens; D004151:Dipeptides; D004791:Enzyme Inhibitors; C078131:N-(2(R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl)-L-tryptophan methylamide; D011993:Recombinant Fusion Proteins; D023201:CD40 Ligand; D051722:ADAM Proteins; D008666:Metalloendopeptidases; D000072198:ADAM17 Protein; C000606462:ADAM17 protein, human