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...
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-γ and TNF-α, 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-γ and TNF-α 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.
Fractalkine/CX3C-chemokine ligand 1 is expressed as a membrane-spanning adhesion molecule that can be cleaved from the cell surface to produce a soluble chemoattractant. Within the vasculature, fractalkine is known to be generated by endothelial cells, but to date there are no reports describing its expression by smooth muscle cells (SMC). In this study we demonstrate that IFN-γ and TNF-α, but not IL-1β, cooperate synergistically to induce fractalkine mRNA and protein expression in cultured aortic SMC. We also report the release of functional, soluble fractalkine from the membranes of stimulated SMC. This release is inhibited by the zinc metalloproteinase inhibitor batimastat, resulting in the accumulation of membrane-associated fractalkine on the SMC surface. Therefore, an SMC-derived metalloproteinase activity is involved in fractalkine shedding. While soluble fractalkine present in SMC-conditioned medium is capable of inducing calcium transients in cells expressing the fractalkine receptor (CX3CR1), blocking experiments using neutralizing Abs reveal that it can be inactivated without affecting the chemotactic activity of SMC-conditioned media on monocytes. However, membrane-bound fractalkine plays a major role in promoting adhesion of monocytic cells to activated SMC. This fractalkine-mediated adhesion is further enhanced in the presence of batimastat, indicating that shedding of fractalkine from the cell surface down-regulates the adhesive properties of SMC. Hence, during vascular inflammation, the synergistic induction of fractalkine by IFN-γ and TNF-α together with its metalloproteinase-mediated cleavage may finely control the recruitment of monocytes to SMC within the blood vessel wall.
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.
Here we describe the characterization of a novel human CC chemokine, tentatively named monocyte chemotactic protein (MCP-4). This chemokine was detected by random sequencing of expressed sequence tags in cDNA libraries. The full-length cDNA revealed an open reading frame for a 98-amino acid residue protein, and a sequence alignment with known CC chemokines showed high levels of similarity (59 -62%) with MCP-1, MCP-3, and eotaxin. MCP-4 cDNA was cloned into Drosophila S2 cells, and the mature protein (residues 24 -98) was purified from the conditioned medium. Recombinant MCP-4 induced a potent chemotactic response (EC 50 Chemokines are structurally and functionally related 8 -10-kDa polypeptides, involved in the recruitment of white blood cells into areas of inflammation and their subsequent activation (1, 2). In addition, some chemokines are able to regulate the proliferative potential of hematopoietic progenitor cells, endothelial cells, and certain types of transformed cells (3, 4). Based on whether the first two cysteine moieties are separated by one amino acid moiety or are adjacent, chemokines belong to the ␣-chemokine or CXC chemokine family (e.g. interleukin-8) or the -chemokine or CC chemokine family (e.g. RANTES 1 and MCP-1). CXC chemokines preferentially attract and affect neutrophils, while CC chemokines chemoattract and affect eosinophils, monocytes, and T cells with relative potencies that differ between the different members of this family.Chemokines express their biological responses through interaction with chemokine receptors (5). Two human CXC chemokine receptors (the interleukin-8A and interleukin-8B (6, 7)) and six human CC chemokine receptors (the MIP-1␣/RANTES receptor (CC-CKR-1) (8, 9), the MCP-1A and -B receptors (CC-CKR-2A and -B) (10, 11), the eotaxin/RANTES receptor (CC-CKR-3) (12-14), the promiscuous receptor on basophils CC-CKR-4 (15), and a new MIP-1␣/MIP-1/RANTES receptor (CC-CKR-5) (16)) have to date been cloned.Here we describe the identification, cloning, and in vitro expression of a novel human CC chemokine, tentatively named MCP-4. This chemokine shows a high level of amino acid sequence homology (62%) with human MCP-1. Following expression of MCP-4 in Drosophila S2 cells, an N terminus-processed protein (amino acid residues 24 -98) was purified from the conditioned medium, and alignment with the sequences of other mature forms of members of the CC chemokine family suggests that this polypeptide represents mature MCP-4. MCP-4 (residues 24 -98) binds and induces functional responses in freshly isolated human monocytes but not in polymorphonuclear leukocytes. We also show that binding of MCP-4 (residues 24 -98) to monocytes is at least in part due to the CC-CKR-2B receptor, a conclusion supported by data obtained in recombinant CHO cells expressing this receptor. Using immunohistochemical methods, we also show that MCP-4 is expressed in human atherosclerotic carotid and coronary arteries and that the expression is associated with vascular endothelial cells and macrophages.
ATP citrate (pro-S)-lyase (EC 4.1.3.8), a cytosolic enzyme that generates acetyl-CoA for cholesterol and fatty acid synthesis de novo, is a potential target for hypolipidaemic intervention. Here we describe the biological effects of the inhibition of ATP citrate-lyase on lipid metabolism in Hep G2 cells, and plasma lipids in rats and dogs, by using SB-204990, the cell-penetrant gamma-lactone prodrug of the potent ATP citrate-lyase inhibitor SB-201076 (Ki=1 microM). Consistent with an important role of ATP citrate-lyase in the supply of acetyl-CoA units for lipid synthesis de novo, SB-204990 inhibited cholesterol synthesis and fatty acid synthesis in Hep G2 cells (dose-related inhibition of up to 91% and 82% respectively) and rats (76% and 39% respectively). SB-204990, when administered orally to rats, was absorbed into the systemic circulation; pharmacologically relevant concentrations of SB-201076 were recovered in the liver. When administered in the diet (0.05-0. 25%, w/w) for 1 week, SB-204990 caused a dose-related decrease in plasma cholesterol (by up to 46%) and triglyceride levels (by up to 80%) in rats. This hypolipidaemic effect could be explained, at least in part, by a decrease (up to 48%) in hepatic very-low-density lipoprotein (VLDL) production as measured by the accumulation of VLDL in plasma after injection of Triton WR-1339. SB-204990 (25 mg/kg per day) also decreased plasma cholesterol levels (by up to 23%) and triglyceride levels (by up to 38%) in the dog, preferentially decreasing low-density lipoprotein compared with high-density lipoprotein cholesterol levels. Overall these results are consistent with the concept that ATP citrate-lyase is an important enzyme in controlling substrate supply for lipid synthesis de novo and a potential enzyme target for hypolipidaemic intervention.
(-)-Hydroxycitrate, a potent inhibitor of ATP citrate-lyase, was tested in Hep G2 cells for effects on cholesterol homoeostasis. After 2.5 h and 18 h incubations with (-)-hydroxycitrate at concentrations of 0.5 mM or higher, incorporation of [1,5-14C]citrate into fatty acids and cholesterol was strongly inhibited. This most likely reflects an effective inhibition of ATP citrate-lyase. Cholesterol biosynthesis was decreased to 27% of the control value as measured by incorporations from 3H2O, indicating a decreased flux of carbon units through the cholesterol-synthetic pathway. After 18 h preincubation with 2 mM-(-)-hydroxycitrate, the cellular low-density-lipoprotein (LDL) receptor activity was increased by 50%, as determined by the receptor-mediated association and degradation. Measurements of receptor-mediated binding versus LDL concentration suggests that this increase was due to an increase in the numbers of LDL receptors. Simultaneously, enzyme levels of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase as determined by activity measurements increased 30-fold. Our results suggest that the increases in HMG-CoA reductase and the LDL receptor are initiated by the decreased flux of carbon units in the cholesterol-synthetic pathway, owing to inhibition of ATP citratelyase. A similar induction of HMG-CoA reductase and LDL receptor was also found after preincubations of cells with 0.3 microM-mevinolin, suggesting that the underlying mechanism for this induction is identical for both drugs.
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