Regulated Shedding of Transmembrane Chemokines by the Disintegrin and Metalloproteinase 10 Facilitates Detachment of Adherent Leukocytes

Abstract

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.

Site‐directed mutagenesis of the chemokine receptor CXCR6 suggests a novel paradigm for interactions with the ligand CXCL16

“…This also suggests that rather than simply acting to tether the chemokine to the cell membrane, the mucin stalk also infers a particular conformation upon the chemokine domain of CXCL16, making it subtly distinct from that of the soluble chemokine. Previous studies have shown that endogenous shedding of the membrane-bound forms of CXCL16 and the related chemokine CX3CL1 is carried out by the actions of the metalloproteinase ADAM10 [32,33]. Although the exact site of cleavage has not been defined at the primary sequence level, SDS-PAGE analysis suggests that cleavage is likely to take place at the membrane interface, shedding CXCL16 into the supernatant with a significant proportion of the mucin stalk still attached [33].…”

Glia in pathological pain: A role for fractalkine

“…As discussed, the extracellular domain of fractlakine can be proteolytically cleaved from the cell surface, a process known as shedding (Hundhausen et al, 2007), and occurs via cleavage of its mucin stalk. In vitro exposure of neuronal mixed cultures and/or brain slices to glutamate, TNF, or interferon-gamma is sufficient to induce fractalkine shedding (Chapman et al, 2000, Erichsen et al, 2003.…”

The TspanC8 Subgroup of Tetraspanins Interacts with A Disintegrin and Metalloprotease 10 (ADAM10) and Regulates Its Maturation and Cell Surface Expression

“…Indeed, transgenic overexpression of ADAM10 prevents amyloid plaque deposition in a mouse model of Alzheimer disease, suggesting that ADAM10 is a promising target for disease treatment in humans (6). ADAM10 targets in the vasculature include the platelet-activating collagen receptor glycoprotein VI (7,8) and endothelial proteins with roles in angiogenesis and inflammation, such as vascular endothelial growth factor receptor 2 (9), the junctional adhesion molecule VE-cadherin (10), and transmembrane chemokines CX3CL1 and CXCL16 (11). Despite the importance of ADAM10, the regulation of its trafficking, activation, and localization to targets is poorly understood.…”