Tumor Necrosis Factor- (cid:1) -converting Enzyme Mediates the Inducible Cleavage of Fractalkine*

Fractalkine (FK, CX3CL1) is a novel multidomain protein expressed on the surface of endothelial cells. As a full-length transmembrane protein, FK binds cells expressing CX3CR1, its cognate receptor, with high affin-ity. Proteolytic cleavage of FK releases a soluble form that is a potent chemoattractant for monocytes, T cells, and natural killer cells. Activation of protein kinase C dramatically increases the rate of this cleavage. Regulation of FK cleavage is critical for maintaining the balance between the immobilized and soluble forms, but the protease responsible has not been identified. Here we report that tumor necrosis factor- (cid:1) -converting enzyme (TACE) is primarily responsible for the inducible cleavage of FK. After transfection into host cells, the proteolytic cleavage of FK was blocked by TACE-spe-cific inhibitors and was not detected in cells genetically altered to remove TACE activity. In contrast, the constitutive cleavage of FK was not mediated by TACE and proceeded normally in TACE-null fibroblasts. We con-clude that TACE is primarily responsible for the inducible cleavage of FK. These studies identify a potentially important link between local generation of potent cyto-kines and control of the balance between the cell adhesion and chemotactic properties of FK. Fractalkine structurally

Fractalkine (FK) 1 is a structurally unusual protein in which a chemokine-like domain is located atop a mucin stalk connected to a transmembrane domain (1). FK is expressed on the surface of endothelial cells (2), neurons (3), and epithelial cells (4), and we and others have shown that full-length FK can efficiently capture cells expressing CX3CR1, its cognate receptor. A soluble form of FK has also been described and is thought to be produced by cleavage at a di-arginine sequence (1,3) next to the transmembrane domain. Incubation of cells with phorbol 12-myristate 13-acetate (PMA; Ref. 5) or interleukin-1␤ (6) up-regulates cleavage and releases soluble FK, which is a potent chemoattractant for monocytes (1), natural killer cells (7), and T cells (1).
A number of functionally diverse proteins exist both in full-length, membrane-bound forms and in soluble forms generated by post-translational cleavage. These include tumor necrosis factor-␣ (TNF-␣), which is involved in inflammatory responses (8); L-selectin, a cell adhesion molecule (9,10); ␤-amyloid precursor protein, which has been implicated in Alzheimer's disease (11); and angiotensin-converting enzyme, which is involved in blood pressure regulation (12). An appropriate balance between the full-length and soluble forms of these proteins appears to be critical for homeostasis. For example, a failure of the proper cleavage of the ␤-amyloid precursor protein by secretase leads to formation of the amyloid-␤ peptide, found in the plaques of Alzheimer's patients (12). The mechanism for regulating the cleavage of FK is unknown and may have important implications for understanding how cells are attracted to and captured by and eventually migrate through epithelial cells, including the vascular endothelium. Here we report that the protease responsible for the inducible cleavage of FK is TNF-␣-converting enzyme (TACE; Ref. 8). FK is also cleaved constitutively by a non-TACE protease, and neither the inducible nor the constitutive cleavage requires the di-arginine sequence. These studies provide the first evidence that TACE, a well-characterized protease critical for the production of the potent cytokine TNF-␣, is involved in the generation of functional chemokines.
Recombinant Cell Lines-ECV cells expressing full-length FK (7) were a generous gift from Drs. Daniel Dairaghi and Thomas J. Schall (ChemoCentryx, San Carlos, CA) and were maintained in minimal essential medium and Earle's balanced salt solution supplemented with 10% fetal calf serum and antibiotics. ECV cells were also transfected with full-length FK in which the FLAG epitope (GSDYKDDDDK) was inserted between the chemokine domain and the mucin stalk or with a mutated form of FK in which the arginines at positions 347 and 348 were changed to alanines. All cDNA constructs were made in pcDNA3 (Invitrogen, Carlsbad, CA), and all transfections were done with Lipo-fectAMINE (Life Technologies), following the manufacturer's instructions. Chinese hamster ovary (CHO)-M1 (13) cells, which do not process TACE to an active form, were a generous gift from Dr. Joan Massague (Memorial Sloan-Kettering Hospital, New York, NY). These cells express a neomycin resistance gene, and transfectants were produced by co-transfecting full-length FK and a zeocin resistance gene, pZeoSV2. These cells were maintained in minimal essential medium and Earle's balanced salt solution supplemented with 10% fetal calf serum and penicillin and streptomycin, Geneticin (800 g/ml), and zeocin (400 g/ml; Invitrogen). Stably transfected cell lines were selected by flow * This study was supported by National Institutes of Health Grants HL 63894 and HL 52773 (to I. F. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. § Present Address: Berlex Biosciences, 15049 San Pablo Ave., Richmond, CA 94804.
FK Cleavage Assay-Transfected cell lines were seeded at ϳ2.5 ϫ 10 6 cells/100-mm plate 1 day before the experiment. One hour before the experiment, the medium was changed to serum-free minimal essential medium and Earle's balanced salt solution; 20 min before the addition of PMA, metalloprotease inhibitors (or control vehicle) were added. PMA (1 M dissolved in Me 2 SO) was added to the cells for 30 min. The conditioned medium was collected, spun for 30 min at 30,000 ϫ g to remove cellular debris, and concentrated with a Centricon column (Cen-triPlus YM-10). One-third of the concentrated conditioned medium was mixed with 4 ϫ sample buffer (0.004% bromphenol blue, 120 mM Tris-HCl, pH 6.8, 20% glycerol, 6% SDS, and 10% 2-mercaptoethanol) and added to each lane of an SDS 10% polyacrylamide gel. In experiments involving transfected fibroblasts, it was necessary to deglycosylate the proteins before detection by Western blotting. This was done using a glycoprotein deglycosylation kit (Calbiochem), following the manufacturer's instructions. After transfer of proteins to nitrocellulose membranes, the blots were probed with the biotinylated anti-human FK antibody (0.1 g/ml; R & D Systems), followed by streptavidin-horseradish peroxidase (1:1000 dilution; Amersham Pharmacia Biotech). Blots were developed with the ECL system (Amersham Pharmacia Biotech), and band intensities were quantitated on a Molecular Imager FX instrument (Bio-Rad, Hercules, CA). In some experiments, cell lysates were examined for FK content. After the conditioned medium was collected, the cells were lysed by adding immunoprecipitation buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 10 mM Na pyrophosphate, and 1% Triton X-100) supplemented with protease inhibitors (1 M leupeptin, 1 mM phenylmethylsulfonyl fluoride, 2.1 g/ml aprotinin, and 1 g/ml pepstatin). The cell lysates were precleared by incubation with 100 l of anti-rabbit IgG-agarose (Sigma) that had been blocked by preincubation with 2% (w/v) bovine serum albumin in immunoprecipitation buffer for 2 h at 4°C. The samples were spun for 10 min in a microfuge, and FK was immunoprecipitated by incubation with rabbit anti-human FK antibody (6 g/ml; Torrey Pines) overnight at 4°C. Anti-rabbit IgG-agarose (100 l, preblocked with 2% (w/v) bovine serum albumin) was used to precipitate the FK-antibody complex. Samples were resuspended in SDS sample buffer, incubated for 10 min without heating, and electrophoresed as described above.

RESULTS
To study the cleavage of FK, we used cell lines that stably expressed either wild-type or mutated forms of full-length FK. Each was expressed at the cell surface at similar levels, as determined by flow cytometry (data not shown). Soluble FK was detected in the conditioned medium of transfected epithe- lial cells, and its concentration was markedly increased by the addition of PMA for 30 min (Fig. 1A). Inclusion of EDTA in the medium reduced cleavage, indicating that the protease activity depends on divalent cations. The di-arginine sequence in FK located in juxtoposition to the transmembrane domain has been implicated as the cleavage site. To examine the importance of this sequence in the proteolysis of FK, we mutated these two arginines to alanines. This mutation had no effect on the PMA-induced cleavage (Fig. 1B).
Metalloproteases are divalent cation-dependent enzymes and are known to cleave membrane proteins. We therefore asked whether cleavage of FK was mediated by a metalloprotease. The metalloprotease inhibitor 1,10-phenanthroline (14) partially blocked FK cleavage, and the effect was dose-dependent ( Fig. 2A); additional inhibition was not seen at higher concentrations (data not shown). Similar results were obtained with GM6001 (15), a metalloprotease inhibitor of broad specificity (Fig. 2B). TAPI-2 is a hyroxamate-based inhibitor of matrix metalloproteases that has high activity against TACE, the protease that cleaves TNF-␣ (13,16). TAPI-2 blocked PMAinduced FK cleavage effectively and in a dose-dependent manner (Fig. 3). These data suggested that the enzyme that cleaved FK is very similar or possibly identical to TACE.
We next examined FK cleavage in CHO-M1 cells, a cell line in which known substrates of TACE are not shed (13,17). After transfection with full-length FK, we noted higher levels of soluble FK in the conditioned medium of wild-type CHO cells than in the medium of transfected ECV304 cells (Figs. 1-3), suggesting a higher rate of constitutive cleavage (Fig. 4A). Addition of PMA induced further cleavage of FK in both wildtype CHO and CHO-M1 cells but at a markedly lower level in the CHO-M1 cells (Fig. 4A). Addition of TAPI-2 did not affect the amount of soluble FK in the medium, confirming the absence of TACE activity in the CHO-M1 cells (data not shown). Western blots of the cell lysates confirmed that FK was expressed at comparable levels in the CHO wild-type and CHO-M1 cell lines (Fig. 4B).
To determine whether TACE was responsible for the constitutive cleavage, we followed the accumulation of soluble FK in the conditioned medium of unactivated transfected CHO and CHO-M1 cells (Fig. 5). The rate of accumulation of soluble FK was similar in these two cell lines, particularly at the later time points. These data suggest that, unlike the PMA-induced cleavage of FK, TACE is not primarily responsible for the constitu-tive cleavage. Consistent with this result, TAPI-2 had little or no effect on the rate of constitutive cleavage of FK (data not shown).
To further test the hypothesis that TACE was the protease responsible for the induced cleavage of FK, we transfected TACE Ϫ/Ϫ fibroblasts with FK. Similar surface expression of FK was obtained in stably transfected TACE Ϫ/Ϫ and TACE ϩ/ϩ fibroblasts (Fig. 6). Western blots of cell lysates also indicated very similar levels of FK expression in the wild-type and TACE Ϫ/Ϫ fibroblasts (data not shown). Addition of PMA re- sulted in robust cleavage of FK in the wild-type but not in the TACE Ϫ/Ϫ fibroblasts (Fig. 6, B and C). DISCUSSION Emerging in vivo data suggest an important role for FK in transplant rejection (18,19) and susceptibility to coronary artery disease (20), but it is not clear whether this is attributable to full-length FK acting as an adhesion molecule, to soluble FK acting as a chemoattractant, or to both. Because regulation of the cleavage event that generates soluble FK is likely to have profound effects on the biological functions of FK, we sought to identify the responsible protease(s). The major finding in this paper is that TACE, a well-characterized protease that generates biologically active TNF-␣, accounts for virtually all of the inducible cleavage of FK. In contrast, the constitutive cleavage of FK was not mediated by TACE and was not blocked by typical metalloprotease inhibitors. The di-arginine sequence of fractalkine at the plasma membrane interface was not required for either the inducible or the constitutive cleavage.
Several lines of evidence support the claim that TACE is responsible for the inducible cleavage of FK. First, cleavage was blocked by chelation of divalent cations with EDTA. Second, three structurally diverse metalloprotease inhibitors blocked cleavage. Third, there was little inducible cleavage detected when FK was expressed in CHO cells that had been genetically modified to prevent the generation of proteolytically active TACE. Fourth, little or no inducible cleavage of FK was evident in fibroblasts derived from TACE Ϫ/Ϫ mice. Taken together, these data provide strong evidence that virtually all of the inducible cleavage of FK is mediated by TACE. Interestingly, the constitutive cleavage of FK seen in both the ECV and CHO cells was also present in the TACE-null CHO-M1 and TACE Ϫ/Ϫ fibroblasts. These data indicate that TACE is not responsible for the constitutive cleave of FK.
Several full-length membrane proteins are cleaved to generate soluble forms that retain biological activity. These include such structurally and functionally unrelated proteins as angiotensin-converting enzyme (12), amyloid precursor protein (11), the receptor for interleukin-6, (13), and TNF-␣ (8). In many cases, cleavage occurs in a stalk region near the plasma membrane and is often up-regulated by activators of protein kinase C (12). The proteinases that mediate this cleavage have been termed sheddases or secretases, and many are zinc-dependent metalloproteinases (12). TACE is a member of a family of proteins containing a disintegrin and metalloprotease domain (ADAMS proteins). There are Ͼ30 ADAMS proteins, and they are involved in functions as diverse as neurogenesis, myotube formation, and proteolytic cleavage of cell surface proteins (for a recent review of the ADAMS proteins, see Ref. 21). To date, TACE is the only ADAMS protein that has been cloned and characterized in vivo. In addition to TNF-␣, TACE cleaves L-selectin (9), transforming growth factor-␣ (9), ␤-amyloid precursor protein (11), and the interleukin-6 receptor (22) but not angiotensin-converting enzyme (23). The physiological importance of TACE is underscored by the fact that TACE-null mice are not viable (9). The current study adds FK to the list of proteins that are post-translationally modified by TACE.
Like other members of the ADAMS family of metalloproteases, TACE is a multidomain, type I transmembrane protein consisting of a zinc-dependent catalytic domain, a disintegrin domain that mediates the binding to integrins, a transmembrane domain, and a cytoplasmic tail. As noted above, TACE cleaves a number of membrane-bound proteins, but amino acid sequences at the cleavage sites of these proteins do not resemble the TNF-␣ cleavage site, which under physiological conditions is between alanine 76 and valine 77 (12). Analysis of the cleavage site of TACE substrates does not reveal the presence of a well-defined consensus sequence but instead suggests that the length of the stalk between the cleavage site and the plasma membrane is important (12). One interpretation of these data is that the binding of TACE to its substrate is the critical event and that, once bound, the protease can cleave at a number of different amino acid sequences. Consistent with this hypothesis, deletion of at least 12 amino acids was necessary to completely block shedding of TNF-␣ (24). The di-arginine sequence near the plasma membrane has been implicated as the cleavage site that generates soluble FK (1, 3), but the evidence for this has not been direct. In this study, we found that mutation of these two arginines to alanines had no effect on either the inducible or the constitutive cleavage of FK. These data suggest that extensive mutatgenesis of the FK stalk just amino-terminal to the plasma membrane may be required to create a noncleavable form of FK.
Recently, Matloubian et al. (25) described CXCL16, a second member of the CX3C family of chemokines. CXCL16 is expressed on dendritic cells, and binds to naive CD8 T cells, natural killer T cells, and a subset of CD4 T cells (25). CXCL16 exists in both membrane-bound and soluble forms, and it is reasonable to speculate that soluble CXCL16 might attract T cells to the dendritic cells. This chemokine may therefore serve to facilitate interactions between dendritic cells and T cells. FIG. 6. Reduction in PMA-induced FK cleavage in TACE-null fibroblasts. Fibroblasts from TACE wild-type (TACE ϩ/ϩ ) and TACE-deficient (TACE Ϫ/Ϫ ) mice were transfected with FK, and the PMA-inducible cleavage was determined. A, FK was expressed at comparable levels in wild-type (FK/TACE ϩ/ϩ ) and TACE-deficient (FK/TACE Ϫ/Ϫ ) fibroblasts. B, soluble FK in the conditioned medium was measured after incubation with PMA. Untransfected fibroblasts (TACE ϩ/ϩ ) are shown as a negative control for the Western blot. The broadness of the FK bands is attributable to heavy glycosylation by the fibroblast cell line. Data shown are representative of three experiments. DMSO, Me 2 SO.
The protease responsible for CXCL16 cleavage has not been characterized, but the findings presented here make TACE a likely candidate. Whether mice expressing a noncleaveable form of CXCL16 would have an immune deficit remains to be determined.
In summary, we have found that TACE, the ADAMS family metalloprotease that cleaves TNF-␣, is primarily responsible for the inducible cleavage of FK. The relative contributions of the full-length and soluble forms of FK in mediating cell-cell interactions are not known, but the identification of TACE as the cleavage enzyme should aid in this effort, as well as facilitate creation of a mouse with a noncleavable form of FK. This novel role for TACE identifies a potentially important link between local cytokine production and the cleavage of FK in activated cells.
Note-While this paper was in the review process, Garton et al. (26) reported very similar results.