Regulated shedding of transmembrane chemokines by the disintegrin and metalloproteinase 10 facilitates detachment of adherent leukocytes.
CX3CL1 (fractalkine) and CXCL16 are unique members of the chemokine family because they occur not only as soluble, but also as membrane-bound molecules. Expressed as type I transmembrane proteins, the ectodomain of both chemokines can be proteolytically cleaved from the cell surface, a process known as shedding. Our previous studies showed that the disintegrin and metalloproteinase 10 (ADAM10) mediates the largest proportion of constitutive CX3CL1 and CXCL16 shedding, but is not involved in the phorbolester-induced release of the soluble chemokines (inducible shedding). In this study, we introduce the calcium-ionophore ionomycin as a novel, very rapid, and efficient inducer of CX3CL1 and CXCL16 shedding. By transfection in COS-7 cells and ADAM10-deficient murine embryonic fibroblasts combined with the use of selective metalloproteinase inhibitors, we demonstrate that the inducible generation of soluble forms of these chemokines is dependent on ADAM10 activity. Analysis of the C-terminal cleavage fragments remaining in the cell membrane reveals multiple cleavage sites used by ADAM10, one of which is preferentially used upon stimulation with ionomycin. In adhesion studies with CX3CL1-expressing ECV-304 cells and cytokine-stimulated endothelial cells, we demonstrate that induced CX3CL1 shedding leads to the release of bound monocytic cell lines and PBMC from their cellular substrate. These data provide evidence for an inducible release mechanism via ADAM10 potentially important for leukocyte diapedesis.
Hundhausen
Christian
C
Institute of Biochemistry, Christian-Albrechts-University, Kiel, Germany.
Schulte
Alexander
A
Schulz
Beate
B
Andrzejewski
Michael G
MG
Schwarz
Nicole
N
von Hundelshausen
Philipp
P
Winter
Ulrike
U
Paliga
Krzysztof
K
Reiss
Karina
K
Saftig
Paul
P
Weber
Christian
C
Ludwig
Andreas
A
D051722:ADAM Proteins / Q000235:genetics / Q000378:metabolism*; D000072197:ADAM10 Protein; D053829:Amyloid Precursor Protein Secretases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D019556:COS Cells; D002448:Cell Adhesion; D002462:Cell Membrane / Q000378:metabolism; D054428:Chemokine CX3CL1; D000075743:Chemokine CXCL16; D020523:Chemokines, CX3C / Q000378:metabolism*; D019743:Chemokines, CXC / Q000378:metabolism*; D002522:Chlorocebus aethiops; D019483:Disintegrins / Q000378:metabolism*; D006801:Humans; D007962:Leukocytes / Q000276:immunology*; D053506:Matrix Metalloproteinase 10 / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D051116:Receptors, Scavenger / Q000378:metabolism*; D014162:Transfection
C515538:CX3CL1 protein, human; C423727:CXCL16 protein, human; D054428:Chemokine CX3CL1; D000075743:Chemokine CXCL16; D020523:Chemokines, CX3C; D019743:Chemokines, CXC; C515539:Cx3cl1 protein, mouse; D019483:Disintegrins; D008565:Membrane Proteins; D051116:Receptors, Scavenger; D053829:Amyloid Precursor Protein Secretases; D051722:ADAM Proteins; D053506:Matrix Metalloproteinase 10; D000072197:ADAM10 Protein; C491236:ADAM10 protein, human