The disintegrin-like metalloproteinase ADAM10 is involved in constitutive cleavage of CX3CL1 (fractalkine) and regulates CX3CL1-mediated cell-cell adhesion

Abstract

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-␣-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 un- IntroductionLeukocyte recruitment to inflammatory sites involves a sequence of adhesive events that are mediated by different classes of adhesion molecules expressed on the endothelium and the leukocytes. 1 Whereas adhesion molecules of the selectin family usually contribute to the rolling of leukocytes under flow, members of the integrin family are involved in establishing a stable shear-resistant cell adhesion. Chemokines are thought to play a role in modulating cell adhesion by inducing shedding of L-selectin and by increasing functional integrins on the leukocyte surface. Thus, besides acting as chemoattractants in the tissue, chemokines can promote the transition from an early to a late adhesion type in the course of leukocyte recruitment.Within the chemokine family a transmembrane molecule termed CX3C chemokine ligand 1 (CX3CL1), or fractalkine, has been identified that by itself induces adhesion. 2 CX3CL1 is encoded as a 95-kDa multidomain molecule consisting of a chemokine domain linked to a transmembrane domain via a mucin-rich stalk. The chemokine is expressed on endothelial cells, 2 epithelial cells, 3,4 smooth muscle cells, 5,6 dendritic cells, 7,8 neurons, 9,10 and macrophages. 11 In vitro, CX3CL1 induces cell adhesion by interaction with its receptor CX3CR1 expressed on monocytes, T cells, mast cells, and natural killer cells. 2,[12][13][14] This adhesion does not require signaling of the receptor, is resistant to physiologic shear flow, and is independent of extracellular calcium. 2,15,16 Besides its activity as an adhesion molecule, CX3CL1 can be cleaved from the cell membrane to produce a soluble 80-kDa molecule that induces chemotaxis of CX3CR1-expressing leukocytes. 2 In vivo, upregulation of CX3CL1 has been found in atherosclerotic blood vessels, 6,11 rejected transplants, 1...

ADAM10 regulates FasL cell surface expression and modulates FasL-induced cytotoxicity and activation-induced cell death

“…We also used two recently described hydroxamate-based compounds that differ in their capacity to block the activities of the two disintegrin-like MPs ADAM17 and ADAM10. 23 Whereas GW280264X potently blocks both enzymes, GI254023X possesses comparable inhibitory potency for ADAM10 only and blocks ADAM17 with more than 100-fold reduced potency. The preferential ADAM10 inhibitor GI254023X blocked the constitutive release of sFasL into the medium with a similar potency as the mixed ADAM10/ ADAM17 inhibitor GW280264X (Figure 1b).…”

“…The hydroxamate based inhibitors GW280264X and GI254023X were described elsewhere. 23 The cloning of mADAM10 in pcDNA3.1 (Invitrogen, Karlsruhe, Germany) was reported previously. 24 ADAM17 was kindly provided by Carl Blobel (NY, USA).…”

The Fas ligand intracellular domain is released by ADAM10 and SPPL2a cleavage in T-cells

“…However, as the ADAM family members ADAM10 and ADAM17 have been implicated in TNFa processing, 21 we examined a potential role for both proteases in FasL shedding and production of APL in 293 cells. In the first experiment, we used the metalloproteinase inhibitor GI254023X, which preferentially blocks ADAM10, 22 on stable 293 transfectants overexpressing hFasL, and analyzed FasL processing by immunoblotting. Figure 2a demonstrates that incubation of the cells with the ADAM inhibitor increases the amount of full-length FasL (upper panel, G247 antibody), whereas production of the 17-kDa APL fragment declines (lower panel, M2 antibody).…”

“…Members of the ADAM protease family are involved in the proteolysis of various substrates, such as Notch, EGF ligands and others ( 34 and references therein). By using different inhibitors specific for the disintegrin-like metalloproteinase ADAM10 22,23 and by using a genetic siRNA knockdown approach, we have shown that ADAM10 is one of the proteases responsible for ectodomain shedding of overexpressed FasL in 293 cells and of endogenous FasL in Tcells. Blockage of ADAM10 (but not of ADAM17) activity results in an accumulation of full-length FasL and a decrease in the FasL ADAM10 cleavage product APL.…”

“…Phytohemagglutinin (PHA), the SPP protease inhibitor (Z-LL) 2 -ketone and clastolactacystin (for simplicity, referred to as lactacystin) were purchased from Calbiochem, and IL-2 from Roche Diagnostics (Mannheim, Germany). The ADAM10 inhibitor GI254023X ((2R,3S)-3-(formyl-hydroxyamino)-2-(3-phenyl-1-propyl) butanoic acid ((1S)-2,2-dimethyl-1-methylcarbamoyl-1-propyl) amide) has been described elsewhere, 22 the MMP and TACE inhibitor TAPI-2 (N-(R)-(2-(Hydroxyaminocarbonyl)methyl)-4-methylpentanoyl-L-t-butyl-alanyl-L-alanine, 2-aminoethyl amid) were purchased from Calbiochem. The small molecule protease inhibitors INCB-3619, INCB-3420, INCB-8765 and INCB-3420 were obtained from P. Scherle (Incyte Corporation, Wilmington, Delaware, USA) and have been described elsewhere.…”