ABSTRACT
The novel CXC-chemokine ligand 16 (CXCL16) functions as transmembrane adhesion molecule on the surface of APCs and as a soluble chemoattractant for activated T cells. In this study, we elucidate the mechanism responsible for the conversion of the transmembrane molecule into a soluble chemokine and provide evidence for the expression and shedding of CXCL16 by fibroblasts and vascular cells. By transfection of human and murine CXCL16 in different cell lines, we show that soluble CXCL16 is constitutively generated by proteolytic cleavage of transmembrane CXCL16 resulting in reduced surface expression of the transmembrane molecule. Inhibition experiments with selective hydroxamate inhibitors against the disintegrin-like metalloproteinases a disintegrin and metalloproteinase domain (ADAM)10 and ADAM17 suggest that ADAM10, but not ADAM17, is involved in constitutive CXCL16 cleavage. In addition, the constitutive cleavage of transfected human CXCL16 was markedly reduced in embryonic fibroblasts generated from ADAM10-deficient mice. By induction of murine CXCL16 in ADAM10-deficient fibroblasts with IFN-gamma and TNF-alpha, we show that endogenous ADAM10 is indeed involved in the release of endogenous CXCL16. Finally, the shedding of endogenous CXCL16 could be reconstituted by retransfection of ADAM10-deficient cells with ADAM10. Analyzing the expression and release of CXCXL16 by cultured vascular cells, we found that IFN-gamma and TNF-alpha synergize to induce CXCL16 mRNA. The constitutive shedding of CXCL16 from the endothelial cell surface is blocked by inhibitors of ADAM10 and is independent of additional inhibition of ADAM17. Hence, during inflammation in the vasculature, ADAM10 may act as a CXCL16 sheddase and thereby finely control the expression and function of CXCL16 in the inflamed tissue.
Subject(s)
Chemokines, CXC/biosynthesis , Chemokines, CXC/metabolism , Disintegrins/metabolism , Interferon-gamma/physiology , Membrane Proteins/biosynthesis , Membrane Proteins/metabolism , Metalloendopeptidases/metabolism , Receptors, Immunologic/biosynthesis , Receptors, Immunologic/metabolism , Tumor Necrosis Factor-alpha/physiology , ADAM Proteins , ADAM10 Protein , Amyloid Precursor Protein Secretases , Animals , COS Cells , Cell Line, Tumor , Cell Membrane/immunology , Cell Membrane/metabolism , Cells, Cultured , Chemokine CXCL16 , Chemokine CXCL6 , Cytokines/pharmacology , Endopeptidases/metabolism , Endothelium, Vascular/cytology , Endothelium, Vascular/immunology , Endothelium, Vascular/metabolism , Humans , Hydrolysis , Membrane Proteins/deficiency , Membrane Proteins/genetics , Membrane Proteins/physiology , Metalloendopeptidases/deficiency , Metalloendopeptidases/genetics , Metalloendopeptidases/physiology , Mice , Mice, Knockout , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/immunology , Muscle, Smooth, Vascular/metabolism , Protein Precursors/biosynthesis , Protein Precursors/metabolism , Protein Structure, Tertiary , Receptors, Scavenger , Solubility , Tetradecanoylphorbol Acetate/pharmacologyABSTRACT
We describe here a classical molecular modeling exercise that was carried out to provide a basis for the design of novel antagonist ligands of the CCR2 receptor. Using a theoretical model of the CCR2 receptor, docking studies were carried out to define plausible binding modes for the various known antagonist ligands, including our own series of indole piperidine compounds. On the basis of these results, a number of site-directed mutations (SDM) were designed that were intended to verify the proposed docking models. From these it was clear that further refinements would be necessary in the model. This was aided by the publication of a crystal structure of bovine rhodopsin, and a new receptor model was built by homology to this structure. This latest model enabled us to define ligand-docking hypotheses that were in complete agreement with the results of the SDM experiments.
Subject(s)
Receptors, Chemokine/antagonists & inhibitors , Receptors, Chemokine/metabolism , Amino Acid Substitution , Animals , Binding Sites , Binding, Competitive , CHO Cells , Cattle , Cell Line , Chemotaxis/drug effects , Cricetinae , Humans , Indoles/chemistry , Indoles/metabolism , Indoles/pharmacology , Kinetics , Models, Molecular , Monocytes/drug effects , Monocytes/physiology , Mutagenesis, Site-Directed , Piperidines/chemistry , Piperidines/metabolism , Piperidines/pharmacology , Radioligand Assay , Receptors, CCR2 , Receptors, Chemokine/chemistry , Receptors, Chemokine/genetics , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Rhodopsin/chemistry , Rhodopsin/genetics , Structural Homology, Protein , TransfectionABSTRACT
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.
