The disintegrin-like metalloproteinase ADAM10 is involved in constitutive cleavage of CX3CL1 (fractalkine) and regulates CX3CL1-mediated cell-cell adhesion.
The CX3C chemokine fractalkine (CX3CL1) exists as a membrane-expressed protein promoting cell-cell adhesion and as a soluble molecule inducing chemotaxis. Transmembrane CX3CL1 is converted into its soluble form by defined proteolytic cleavage (shedding), which can be enhanced by stimulation with phorbol-12-myristate-13-acetate (PMA). PMA-induced CX3CL1 shedding has been shown to involve the tumor necrosis factor-alpha-converting enzyme (TACE), whereas the constitutive cleavage in unstimulated cells remains elusive. Here we demonstrate a role of the closely related disintegrin-like metalloproteinase 10 (ADAM10) in the constitutive CX3CL1 cleavage. The hydroxamate GW280264X, capable of blocking TACE as well as ADAM10, proved to be an effective inhibitor of the constitutive and the PMA-inducible CX3CL1 cleavage in CX3CL1-expressing ECV-304 cells (CX3CL1-ECV-304), whereas GI254023X, preferentially blocking ADAM10 but not TACE, reduced the constitutive cleavage only. Overexpression of ADAM10 in COS-7 cells enhanced constitutive cleavage of CX3CL1 and, more importantly, in murine fibroblasts deficient of ADAM10 constitutive CX3CL1 cleavage was markedly reduced. Thus, ADAM10 contributes to the constitutive shedding of CX3CL1 in unstimulated cells. Addressing the functional role of CX3CL1 shedding for the adhesion of monocytic cells via membrane-expressed CX3CL1, we found that THP-1 cells adhere to CX3CL1-ECV-304 cells but detach in the course of vigorous washing. Inhibition of ADAM10-mediated CX3CL1 shedding not only increased adhesive properties of CX3CL1-ECV-304 cells but also prevented de-adhesion of bound THP-1 cells. Our data demonstrate that ADAM10 is involved in the constitutive cleavage of CX3CL1 and thereby may regulate the recruitment of monocytic cells to CX3CL1-expressing cell layers.
Hundhausen
Christian
C
Institute for Biochemistry, Christian-Albrechts-University, Kiel, Germany.
Misztela
Dominika
D
Berkhout
Theo A
TA
Broadway
Neil
N
Saftig
Paul
P
Reiss
Karina
K
Hartmann
Dieter
D
Fahrenholz
Falk
F
Postina
Rolf
R
Matthews
Vance
V
Kallen
Karl-Josef
KJ
Rose-John
Stefan
S
Ludwig
Andreas
A
D051722:ADAM Proteins; D000072198:ADAM17 Protein; D053829:Amyloid Precursor Protein Secretases; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors / Q000378:metabolism; D019556:COS Cells; D002448:Cell Adhesion / Q000502:physiology*; D002460:Cell Line; D054428:Chemokine CX3CL1; D020523:Chemokines, CX3C / Q000378:metabolism / Q000502:physiology*; D002522:Chlorocebus aethiops; D004151:Dipeptides / Q000737:chemistry / Q000494:pharmacology; D015536:Down-Regulation / Q000502:physiology; D010450:Endopeptidases / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D006877:Hydroxamic Acids / Q000737:chemistry / Q000494:pharmacology; D008565:Membrane Proteins / Q000378:metabolism / Q000502:physiology*; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors; D051379:Mice; D009000:Monocytes / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000031:analogs & derivatives / Q000494:pharmacology; D014407:Tumor Cells, Cultured
C555398:3-(formylhydroxyamino)-2-(3-phenyl-1-propyl)butanoic acid (2,2-dimethyl-1-methylcarbamoyl-1-propyl)amide; C515538:CX3CL1 protein, human; D054428:Chemokine CX3CL1; D020523:Chemokines, CX3C; C515539:Cx3cl1 protein, mouse; D004151:Dipeptides; D004791:Enzyme Inhibitors; D006877:Hydroxamic Acids; D008565:Membrane Proteins; C016658:phorbolol myristate acetate; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human; C509282:Bace1 protein, mouse; D051722:ADAM Proteins; D008666:Metalloendopeptidases; D000072198:ADAM17 Protein; C000606462:ADAM17 protein, human; C000606463:Adam17 protein, mouse; D013755:Tetradecanoylphorbol Acetate