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Originally published In Press as doi:10.1074/jbc.M106434200 on August 8, 2001

J. Biol. Chem., Vol. 276, Issue 41, 37993-38001, October 12, 2001
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Tumor Necrosis Factor-alpha -converting Enzyme (ADAM17) Mediates the Cleavage and Shedding of Fractalkine (CX3CL1)*

Kyle J. GartonDagger §, Peter J. GoughDagger §, Carl P. Blobel, Gillian Murphy||, David R. Greaves**, Peter J. DempseyDagger Dagger , and Elaine W. RainesDagger §§

From the Dagger  Department of Pathology, University of Washington, Harborview Medical Center, Seattle, Washington 98104,  Memorial Sloan-Kettering Cancer Center, New York, New York 10021, || School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom, ** Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom, and Dagger Dagger  Pacific Northwest Research Institute, Seattle, Washington 98122

Received for publication, July 10, 2001, and in revised form, July 27, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Fractalkine (CX3CL1) is an unusual member of the chemokine family that is synthesized with its chemokine domain at the end of a mucin-rich, transmembrane stalk. This membrane-bound localization allows fractalkine to function as an adhesion molecule for cells bearing its receptor, CX3CR1. In addition, fractalkine can be proteolytically released from the cell surface, generating a soluble molecule that functions as a chemoattractant similar to the other members of the chemokine family. In this study, we have examined the mechanisms that regulate the conversion between these two functionally distinct forms of fractalkine. We demonstrate that under normal conditions fractalkine is synthesized as an intracellular precursor that is rapidly transported to the cell surface where it becomes a target for metalloproteinase-dependent cleavage that causes the release of a fragment containing the majority of the fractalkine extracellular domain. We show that the cleavage of fractalkine can be markedly enhanced by stimulating cells with phorbol 12-myristate 13-acetate (PMA), and we identify tumor necrosis factor-alpha converting enzyme (TACE; ADAM17) as the protease responsible for this PMA-induced fractalkine release. In addition, we provide data showing that TACE-mediated fractalkine cleavage occurs at a site distinct from the dibasic juxtamembrane motif that had been suggested previously based on protein sequence homologies. The identification of TACE as a major protease responsible for the conversion of fractalkine from a membrane-bound adhesion molecule to a soluble chemoattractant will provide new information for understanding the physiological function of this chemokine.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Chemoattractant cytokines, or chemokines, regulate mammalian inflammatory responses at multiple levels including the induction of leukocyte chemotaxis and activation of integrin-mediated adhesion (1, 2). To date more than 40 chemokines have been identified and classified, based on the spacing of N-terminal cysteine residues, into four groups: C, CC, CXC, and CX3C (3). Fractalkine (CX3CL1; FKN)1 is a unique member of the chemokine family; it is the sole CX3C chemokine cloned to date, and its N-terminal CX3C chemokine domain is attached to a glycosylated mucin-like transmembrane stalk (4, 5). This membrane-anchored localization of FKN has led to the suggestion that it functions as a cell adhesion molecule for circulating inflammatory cells (6-8). Data supporting this hypothesis have come from numerous in vitro experiments showing that immobilized FKN, either on glass substrata or monolayers of transfected cells, can support the capture and adhesion of leukocytes (7-9). These adhesive functions of FKN appear to be mediated by a single G-protein-coupled chemokine receptor, CX3CRI, expressed on monocytes, T-cells, natural killer cells, neurons, and microglia (8, 10). In addition to functioning as an adhesion molecule, FKN can be released from the cell surface by an uncharacterized protease to generate a soluble molecule that has chemotactic activity for cells bearing the CX3CR1 receptor (4, 5, 9). The dichotomous properties of these two forms of FKN suggest that the protease responsible for this cleavage event will play a key role in regulating FKN function in vivo.

Several recent findings are consistent with the hypothesis that FKN plays an important role in inflammatory responses in vivo. Under normal conditions FKN is predominantly expressed by epithelial cells, but increased expression of FKN can be detected in lesions of atherosclerosis, psoriatic plaques, and in human kidneys with glomerulonephritis (11-14). Immunoneutralization of the FKN receptor CX3CRI attenuates cardiac transplant rejection and leads to a marked inhibition of leukocyte recruitment and subsequent crescentric glomerulonephritis in rodent models of acute renal injury (15, 16). Furthermore, polymorphisms in the FKN receptor have been identified as a genetic risk factor for human coronary artery disease, an inflammatory disease of the vessel wall (17, 18). Together these observations suggest that FKN processing at the cell surface may play an important role in regulating FKN function in inflammation.

Evidence is beginning to emerge suggesting that proteolytic cleavage of cell surface proteins, or ectodomain shedding, is an important mechanism whereby cells can regulate the repertoire of proteins expressed on their surface (19). Several types of membrane proteins undergo ectodomain shedding, including cytokines (tumor necrosis factor-alpha (TNF-alpha )), cytokine receptors (IL-6 receptor (IL-6R), TNF-receptor I and II (TNF-RI and TNF-RII), macrophage colony-stimulating factor receptor I), growth factors (transforming growth factor-alpha (TGF-alpha ), heparin bound-epidermal growth factor), and adhesion molecules (L-selectin) (20-25). Cell surface proteolysis appears to be mediated largely by members of the metzincin superfamily of zinc-dependent proteases that include the matrix metalloproteinases (MMPs) and ADAMs (for A Disintegrin And Metalloproteinase) (26, 27). ADAMs are type I transmembrane proteins that contain a disintegrin-like and metalloproteinase-like domain. In addition to ectodomain shedding, ADAMs play an important role in epithelial and neural development, fertilization, myoblast fusion, and cell-cell interactions. The most well characterized ADAM to date is the TNF-alpha -converting enzyme (TACE; ADAM17), which has been implicated in the shedding of a number of diverse cell surface proteins including TNF-alpha , TNFRs I and II, TGF-alpha , L-selectin, and M-CSFR1.

In this study, we have examined the cellular processing of FKN and the mechanisms that lead to the release of the soluble ectodomain. We show that FKN can be cleaved from the cell surface by two distinct enzyme activities: a "constitutive" sheddase that causes the release of FKN from unstimulated cells, and an "inducible" sheddase that we identify as TACE. Furthermore, we provide evidence that the FKN cleavage occurs at a site distinct from the previously predicted dibasic motif in the juxtamembrane region. The implications of these findings for the regulation of FKN function in vivo are also discussed.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Culture and Reagents-- Cultured human umbilical vein endothelial cells (HUVECs) were isolated and grown as described previously (28). NIH3T3 cells were maintained in DMEM + 10% fetal calf serum (FCS). ADAM9- and ADAM17-deficient cells lines were generated by crossing ADAM 9 -/-2 and TACE Delta Zn/Delta Zn (24) mice with transgenic mice expressing an interferon-gamma (IFN-gamma )-inducible temperature-sensitive SV40 large T-antigen allele as described3 (29). Isolated ADAM-deficient cell lines were maintained at 32 °C in DMEM + 10% FCS + 5 units/ml IFN-gamma . For experiments, cells were plated in the absence of IFN-gamma and grown at 37 °C in DMEM + 10% FCS for 36 h to arrest immortalization by the SV40 large T-antigen. The following antibodies were used: polyclonal rabbit anti-hemagglutinin epitope tag (HA) (Zymed Laboratories Inc.), goat polyclonal anti-fractalkine (R & D Systems), PE-conjugated anti-CD8 (PharMingen), and PE- and peroxidase-conjugated anti-goat and anti-rabbit IgG (Jackson ImmunoResearch). Phorbol-12 myristate 13-acetate (PMA) and all other chemicals not specified were from Sigma. GM6001 was purchased from Elastin Products Co.

Generation of Expression Constructs and Retroviral Infection-- All constructs were generated using standard molecular biology techniques. A full-length murine TACE cDNA and cDNA encoding a TACE (Glu right-arrow Ala) active site mutant were kindly provided by Roy Black and Jacques Peschon (Immunex). HA epitope-tagged FKN was generated by PCR using primers containing a 5' BamHI site and a 3' HA-epitope tag and a NotI site for subcloning into retroviral vectors. The FKN juxtamembrane mutants Delta 1-Delta 5 were generated by overlap PCR using oligonucleotides encoding an uncleavable CD4 sequence (30). The sequences of all PCR-generated constructs were confirmed by sequencing. Primer sequences used for cloning are available upon request. All retroviral expression plasmids were constructed using the pBM-IRES-EGFP and pBM-IRES-CD8 retroviral vectors generously provided by G. Nolan (Stanford University). High titer retrovirus was prepared as described previously (31). For infection, 4 × 105 NIH3T3, HUVEC, or ADAM-deficient cells were plated into 25-cm2 tissue culture flasks 24 h prior to infection by incubation with 5 ml of virus stock for 12 h in the presence of 4 µg/ml Polybrene. After infection, retroviral supernatant was replaced with fresh medium, and cells were cultured for at least 48 h before use in subsequent experiments. Infection efficiencies were determined by analyzing expression of the EGFP or CD8 retroviral reporter genes by flow cytometry.

Fractalkine Shedding Assays-- Cells expressing human FKN were plated at a density of 6 × 105 cells per 60-mm dish in complete growth medium 36 h before stimulation. Cells were pretreated for 15 min by addition of GM6001 (50 µM final) or dimethyl sulfoxide (Me2SO) vehicle control directly to the culture medium. Cells were washed with serum-free medium and stimulated with 2.5 ml of serum-free medium with or without PMA (100 ng/ml), GM6001 (50 µM or the indicted concentration), or Me2SO control followed by incubation at 37 °C for 45 min or the indicated time. Following stimulation, supernatants were removed, cells washed once with cold phosphate-buffered saline (PBS), and subsequently lysed with 1 ml of RIPA buffer (PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 2 µg/ml aprotinin, 2 µg/ml leupeptin, 1 µg/ml pepstatin, 100 µg/ml phenylmethylsulfonyl fluoride, and 0.5 mM iodoacetamide). Resulting cell supernatants and lysates were cleared by centrifugation at 15,000 × g and stored at -80 °C until analysis. The amount of FKN in cellular lysates and supernatants was determined by ELISA using a goat polyclonal anti-fractalkine antibody (R & D Systems) according to the manufacturer's instructions. Levels of soluble FKN present in conditioned medium, cellular FKN present in detergent extracts, and the ratio of soluble/cellular FKN were determined for triplicate dishes and reported as the average ± S.D.

Protein Analysis, Immunoprecipitation, and Metabolic Labeling-- Cells expressing HA epitope-tagged human FKN were plated and stimulated as described above. Post-stimulation, cells were washed twice with PBS and lysed in 350 µl of RIPA buffer for 30 min on ice. Cell lysates were cleared by centrifugation at 15,000 × g for 15 min, and protein concentrations were determined using the BCA protein assay (Pierce). Lysates were separated by SDS-PAGE under reducing conditions, transferred to Immobilon polyvinylidene difluoride membranes (Millipore), and subsequently immunoblotted with specific antibodies, prior to visualization by enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech). For metabolic labeling, cells were washed twice with PBS and then incubated with DMEM lacking cysteine and methionine, supplemented with 10% dialyzed calf serum for 1 h. Cells were subsequently labeled for 30 min with 500 µCi/ml Translabel [35S]Met, [35S]Cys (ICN) and chased for the indicated times in DMEM + 10% FCS. Cells were washed twice with PBS, lysed as above, and FKN immunoprecipitated by overnight incubation with 5 µg of anti-HA antibody (Zymed Laboratories Inc.) and 30 µl of a 50% slurry of protein A-agarose (Santa Cruz Biotechnology). Immunoprecipitates were separated by 12% SDS-PAGE under reducing conditions and visualized by autoradiography. For cell surface protein biotinylation, cells were washed in cold PBS and incubated where indicated with 1 mg/ml NHS-LC-biotin (Pierce) in PBS for 45 min on ice. Labeling reagent was quenched with 0.1 M glycine, and cells were lysed in RIPA buffer as above. Where indicated, cell extracts were incubated with 100 µl of a 50% slurry of agarose-streptavidin for 1 h at 4 °C followed by centrifugation at 2500 rpm to remove biotinylated proteins. Equal volumes of the resulting cell extracts were separated by SDS-PAGE and immunoblotted with an anti-HA antibody as above.

Flow Cytometry and Cell Sorting-- Cell surface levels of FKN were determined by staining cells before and after stimulation with PMA (100 ng/ml) with a goat polyclonal anti-fractalkine antibody (R & D Systems) followed by PE-conjugated secondary antibodies. After fixation, FKN expression was measured by flow cytometry using a FACScan (Becton Dickinson) flow cytometer and data analysis with CellQuest software. Relative FKN cell surface expression is reported as the average mean fluorescence intensity of duplicate samples ± S.D. For reconstitution experiments, TACE-deficient dermal fibroblasts (TACE-KO-DF) were co-infected as above with an IRES-CD8 retrovirus encoding FKN and an IRES-EGFP retrovirus encoding wild-type TACE, a catalytically inactive TACE (Glu right-arrow Ala) mutant, or an empty vector control. Cells were stained with a PE-conjugated anti-CD8 antibody (PharMingen), and 5 × 105 EGFP(+)CD8(+) cells were isolated by fluorescence-activated cell sorting (FACS) using a FACSVantage cell sorter (Becton Dickinson).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Maturation, Processing, and Release of Human Fractalkine-- FKN can exist as two functionally distinct forms: a cell-associated form and a soluble form that has been proposed to arise by processing of the membrane-bound protein. To study the relationship between these two forms, we expressed human FKN containing a cytoplasmic, C-terminal, HA-epitope tag in NIH3T3 cells (Fig. 1A). Western blot analysis of detergent cell extracts using antibodies recognizing either the FKN extracellular domain or a cytoplasmic HA epitope tag revealed numerous species of FKN protein (Fig. 1B). Both antibodies recognize a 100-kDa form that has been suggested previously to represent mature FKN protein (4). Similar Western blots performed after depletion of cell surface proteins through their biotinylation and incubation with agarose-streptavidin confirmed that this 100-kDa form was the only FKN species present at the cell surface (Fig. 1C). A band of ~20 kDa that migrates close to the gel front is detected by antibodies against the cytoplasmic tail HA epitope tag but not by antibodies against the FKN extracellular domain (Fig. 1B). Conditioned cell media contained a single ~85-kDa FKN-immunoreactive protein that could only be detected with antibodies against the extracellular domain of FKN (Fig. 1B). An identical pattern of FKN processing was found in primary human umbilical vein endothelial cells (HUVECs; data not shown).


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  		Fig. 1.   FKN maturation and processing. A, schematic representation of cytoplasmic, C-terminally HA epitope-tagged FKN used in this study. B, NIH3T3 cells expressing HA epitope-tagged human FKN (FKN) or uninfected cells (WT) were cultured for 2 h in serum-free DMEM. Conditioned media (Sup) and detergent cell extracts (Cell) were analyzed by Western blotting with antibodies against the FKN extracellular domain (R & D Systems) or cytoplasmic HA epitope tag (Zymed Laboratories Inc.). Mature (mat), soluble (sol), and FKN C-terminal fragments (CTFs) were detected as shown. C, cell surface proteins of 3T3-FKNHA cells were biotinylated with NHS-LC-biotin (Pierce) where indicated, lysed with RIPA buffer, and incubated in the presence or absence of agarose-streptavidin (AG-Strep) to remove biotinylated proteins. 20 µl of the resulting cleared extracts were separated by SDS-PAGE and immunoblotted with an anti-HA antibody. D, cells were labeled for 30 min with 500 µCi/ml [35S]Met, [35S]Cys (Translabel, ICN) and chased for the indicated times in DMEM + 10% FCS. Cells were lysed, and FKN was isolated by immunoprecipitation with an anti-HA antibody and analyzed by SDS-PAGE autoradiography. Detection of the FKN C-terminal fragment required longer exposure (2 days versus 16 h).

To characterize further the maturation and processing of human FKN, we performed pulse-chase analysis of NIH3T3 cells expressing human FKN. FKN is synthesized as a 50-75-kDa precursor that undergoes rapid maturation, presumably by glycosylation, to yield 100-kDa mature FKN (Fig. 1D). Disappearance of mature FKN is associated with appearance of a small molecular weight fragment that can be immunoprecipitated with an antibody against the C-terminal HA epitope tag (Fig. 1D). These data suggest that FKN is initially synthesized as an intracellular precursor that undergoes glycosylation and transport to the cell surface as a 100-kDa glycoprotein. 100-kDa FKN can then be released from the cell surface yielding a soluble 85-kDa fragment that likely contains the majority of the glycosylated ectodomain and an ~20-kDa transmembrane cytoplasmic tail fragment.

Increased FKN Release by Activation of a PMA-sensitive Metalloproteinase Activity-- It has been reported recently (32) that FKN release from activated neurons and TNF-alpha -stimulated endothelial cells can be mediated by metalloproteinase activity. We therefore sought to identify the enzymatic activity responsible for FKN release and to characterize additional factors that regulate the generation of soluble FKN. When expressed in NIH3T3 cells or primary HUVECs, FKN is constitutively released from cells under normal growth conditions as determined by ELISA (Fig. 2A). Constitutive FKN release could be efficiently inhibited by a broad spectrum zinc-dependent metalloproteinase inhibitor, GM6001 (Fig. 2, A and B), and led to a corresponding increase in the amount of cell-associated FKN and a decrease in the ratio of soluble/cellular FKN consistent with decreased FKN shedding (Fig. 2B).


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  		Fig. 2.   A, metalloproteinase activity mediates the cleavage and shedding of FKN. Generation of soluble FKN was determined by ELISA using NIH3T3 cells and HUVECs expressing FKN, cultured in the presence or absence of the metalloproteinase inhibitor, GM6001 (50 µM). Non-transduced cells showed no detectable FKN signal by ELISA (not shown). B, HUVECs expressing FKN were cultured for 45 min in RPMI with or without PMA (100 ng/ml) or GM6001 (50 µM) as indicated. FKN concentrations in conditioned media or detergent cell extracts were determined by ELISA. Data represent averaged values of duplicate dishes. C, NIH3T3 cells expressing HA epitope-tagged FKN were stimulated with PMA (100 ng/ml) in the presence or absence of GM6001 (50 µM). Detergent cell extracts were analyzed by Western blotting with antibodies against the cytoplasmic tail HA epitope tag and the FKN extracellular domain. Positions of mature FKN (FKN-Mat), immature FKN (FKN-pre), and the FKN C-terminal cleavage fragment (FKN-CTF) are shown (NS, nonspecific band detected with the anti-FKN antibody present in non-transduced cells). D, FKN cell surface levels were measured by flow cytometry in 3T3-FKNHA cells stimulated with PMA (100 ng/ml) in the presence or absence of GM6001 (50 µM). Control NIH3T3 cells infected with empty GFP retrovirus show background staining.

The release of a wide variety of proteins from the cell surface has been shown to be stimulated by phorbol esters, such as PMA, through activation of a metalloproteinase-dependent ectodomain sheddase machinery. PMA stimulation of HUVECs expressing FKN led to an increase in soluble FKN levels, a concomitant decrease in cell-associated FKN, leading to an increase in the soluble/cellular FKN ratio consistent with increased shedding (Fig. 2B). PMA-induced metalloproteinase-dependent FKN shedding was also observed in NIH3T3 cells (Fig. 3), and the effects of PMA in both cell types could be completely reversed by prior treatment with GM6001. FKN shedding was confirmed by Western blot analysis of detergent cell extracts. PMA stimulation markedly decreased levels of full-length FKN and coordinately increased generation of the C-terminal fragment, with GM6001 completely blocking the effects of PMA stimulation on FKN processing (Fig. 2C). As we had determined previously that mature 100-kDa FKN is predominantly localized to the cell surface, we used flow cytometry to confirm that PMA stimulation altered FKN cell surface levels (Fig. 2D). PMA stimulation led to a marked decrease in FKN cell surface levels that could be blocked by GM6001 (Fig. 2D). Together these results support a model in which full-length mature FKN at the cell surface is cleaved by a metalloproteinase to release the majority of the FKN ectodomain, leaving a cell-associated cytoplasmic tail fragment. Furthermore, FKN shedding can be markedly enhanced by PMA stimulation that appears to further activate metalloproteinase activity.


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  		Fig. 3.   Constitutive and PMA-inducible FKN shedding are mediated by distinct metalloproteinase activities. FKN shedding from 3T3-FKNHA (A) and HUVEC-FKNHA (B) cells was determined in the presence or absence of PMA (100 ng/ml) after pretreatment with increasing concentrations of GM6001. FKN release into the media was measured by ELISA and expressed as a percentage of FKN release in the absence of GM6001. N.D. represents FKN levels below the detection limit by ELISA.

Constitutive and PMA-inducible FKN Shedding Are Mediated by Distinct Metalloproteinase Activities-- To begin to characterize the enzyme(s) responsible for constitutive and PMA-stimulated FKN release, we examined the sensitivity of these activities to the broad spectrum metalloproteinase inhibitor, GM6001. GM6001 inhibited constitutive as well as PMA-induced FKN release in a dose-dependent manner in both 3T3 and human endothelial cells (Fig. 3). However, in both cell types, constitutive FKN release was more sensitive to inhibition by GM6001 than PMA-stimulated FKN release, suggesting that two distinct metalloproteinase activities may be responsible for constitutive versus PMA-stimulated FKN release. GM6001 is expected to inhibit both matrix metalloproteinases (MMPs) and ADAM members of the metzincin superfamily of zinc-dependent proteases.

Endogenous TACE Function Is Required for Stimulated but Not Constitutive FKN Shedding-- The marked stimulation of FKN shedding in response to PMA, in addition to the inhibition studies above, suggested that ADAM proteases, such as TACE (ADAM17), were good candidates for releasing FKN. We therefore decided to analyze FKN shedding in cells derived from mice genetically deficient for specific ADAM protease activities. For these studies we used immortalized dermal fibroblasts and stomach epithelial cells isolated from mice obtained by crossing ADAM-deficient mice with transgenic mice expressing an IFN-gamma -inducible and temperature-sensitive SV40 large T-antigen allele3 (29). By using this system, ectodomain shedding can be determined in conditionally immortalized, yet non-transformed cells. Dermal fibroblasts derived from wild-type (WT), or ADAM 9, and TACE-deficient mice and stomach epithelial cells from ADAM9- and TACE-deficient mice were infected with retrovirus encoding FKN with a C-terminal HA epitope tag. Constitutive and PMA-inducible FKN shedding was determined by ELISA. Dermal fibroblasts and stomach epithelial cells specifically lacking TACE function failed to shed FKN in response to PMA stimulation as determined by either an increased release of soluble FKN or an increase in the FKN soluble/cell ratio (Fig. 4A). However, neither TACE nor ADAM9 protease function were required for constitutive FKN shedding, as these cells shed FKN under basal conditions at levels indistinguishable from WT dermal fibroblasts (Fig. 4A).


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  		Fig. 4.   TACE but not ADAM9 is required for PMA-induced FKN cleavage and shedding. A, dermal fibroblasts (DF) and stomach epithelial cells (Stm) isolated from TACE Delta Zn/Delta Zn (TACE-KO) (21) and ADAM9 -/-2 mice (ADAM9-KO) crossed with transgenic mice overexpressing an INFgamma -inducible and temperature-sensitive SV40 large T-antigen allele were infected with an FKNHA-IRES-EGFP retrovirus. FKN shedding was determined after PMA (100 ng/ml) stimulation in the presence or absence of GM6001 (50 µM) by ELISA measurements of conditioned media and detergent cell extracts. FKN shedding is expressed as both soluble FKN (percentage of unstimulated) and as the FKN-soluble/cell ratio. B, FKN cleavage in dermal fibroblasts from A was analyzed in detergent cell extracts by Western blotting with an anti-HA epitope tag antibody. FKN-Mat and FKN-CTF represent full-length FKN and the FKN C-terminal cleavage fragment, respectively. C, TACE-KO-DF cells were co-infected with an FKN-IRES-CD8 retrovirus and retrovirus encoding either wild-type TACE (TACE), a protease-dead TACE (Glu right-arrow Ala) mutant (TACEMUT), or EGFP alone as a control, and CD8(+)EGFP(+) cells were isolated by FACS sorting. FKN shedding in these cells was analyzed after stimulation with PMA (100 ng/ml) in the presence or absence of GM6001 (50 µM) by ELISA as in A.

To determine if the absence of PMA-stimulated FKN shedding in TACE-deficient cells was due to a loss of FKN cleavage, we used Western blot analysis to compare FKN processing in WT, ADAM9-, and TACE-deficient fibroblasts. As compared with WT or ADAM9-deficient cells, TACE-deficient fibroblasts showed no decrease in the amount of full-length, mature FKN in response to PMA and no corresponding increase in the levels of the FKN C-terminal cleavage fragment (Fig. 4B).

To confirm that the loss of PMA-induced FKN shedding in TACE-deficient cells was specifically due to the absence of TACE function, we used retroviral infection to reconstitute TACE expression. TACE-deficient dermal fibroblasts were co-infected with an FKN-IRES-CD8 retrovirus and an IRES-EGFP retrovirus encoding either WT-TACE, a catalytically inactive TACE (Glu right-arrow Ala) mutant, or an empty vector control. CD8(+)EGFP(+) doubly infected cells were isolated by FACS sorting, and FKN shedding was determined by ELISA. Expression of WT-TACE specifically rescued the loss of PMA-inducible FKN shedding in TACE-deficient dermal fibroblasts (Fig. 4C). In addition, overexpression of WT-TACE increased basal FKN shedding suggesting that TACE is not efficiently regulated when overexpressed in this particular cell type. In contrast, overexpression of TACE in TACE-deficient stomach epithelial cells did not increase constitutive FKN shedding but efficiently rescued PMA-induced FKN shedding (data not shown). We also found that overexpression of a catalytically inactive TACE (Glu right-arrow Ala) mutant efficiently blocked PMA-induced FKN shedding in NIH3T3 cells without affecting constitutive shedding.4 Together these results show that TACE function is required for PMA-inducible FKN shedding but not for constitutive FKN shedding. Furthermore, the constitutive FKN sheddase is not ADAM9 as FKN shedding under basal conditions is maintained in cells genetically deficient in ADAM9 function.

FKN Juxtamembrane Sequences Regulate PMA-inducible Shedding and Cell Surface Levels-- A putative cleavage site within the FKN juxtamembrane region has previously been assigned based upon sequence similarity to a dibasic amino acid sequence in syndecan-1, which can also undergo PMA-induced ectodomain shedding (30). Syndecan-1 shedding can be abrogated by replacement of the first 15 amino acids of the juxtamembrane region, including the dibasic motif, with the corresponding amino acid sequence of human CD4, a protein that is not cleaved in response to PMA stimulation (30). A series of similar FKN mutants (Delta 1, Delta 2, Delta 3) were constructed such that three sequential stretches of 15 amino acids within the juxtamembrane region were replaced with the uncleavable CD4 sequence (Fig. 5A). Two additional mutants (Delta 4 and Delta 5) were constructed in which the first 30 and 45 amino acids of the FKN juxtamembrane region, respectively, were replaced with the uncleavable CD4 sequence (Fig. 5A). The resulting constructs were expressed in NIH3T3 cells, and the shedding of FKN in response to PMA stimulation was determined by ELISA. Replacement of the first 15 amino acids (Delta 1, amino acids 1-15) or the third 15 amino acids (Delta 3, amino acids 31-45) with the uncleavable CD4 sequence had no effect on PMA-induced FKN shedding (Fig. 5B). In contrast, replacement of the second 15 amino acids (Delta 2, amino acids 16-30) almost completely abrogated PMA-induced FKN shedding (Fig. 5B). Replacement of the first 45 amino acids with the CD4 sequence (Delta 5) similarly resulted in a loss of PMA-induced FKN shedding. However, an FKN mutant containing a deletion of the first 30 amino acids (Delta 4), which includes amino acids deleted in the uncleavable Delta 2 mutant, was able to undergo PMA-induced shedding suggesting that the Delta 2 sequences are not absolutely required for PMA-stimulated shedding.


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  		Fig. 5.   FKN juxtamembrane sequence requirements for PMA-inducible cleavage and shedding. A, schematic of FKN juxtamembrane domain mutants. FKN juxtamembrane domain mutants were generated by replacing the indicated amino acids with an uncleavable juxtamembrane sequence from CD4. The first amino acid of the FKN ectodomain is labeled 1. Mutants Delta 1, Delta 2, and Delta 3 replace amino acids 1-15, 16-30, and 31-45 with the uncleavable CD4 sequence, respectively. Mutants Delta 4 and Delta 5 replace amino acids 1-30 and 1-45 with the CD4 sequence, respectively. All constructs contained a C-terminal, cytoplasmic HA epitope tag. The position of the dibasic motif that has previously been predicted to be the site of FKN cleavage is indicated by an asterisk. B, NIH3T3 cells were infected with retrovirus encoding wild-type (WT) FKN or FKN juxtamembrane domain mutants Delta 1-Delta 5 shown in A. FKN shedding in response to PMA (100 ng/ml) was determined by ELISA and expressed as the FKN-soluble/cell ratio. C, FKN cleavage in detergent cell extracts from NIH3T3 cells expressing WT or Delta 1-Delta 5 FKN mutants as in B was determined by Western blotting with an anti-HA epitope tag antibody. D, FKN cell surface levels measured by flow cytometry in NIH3T3 cells expressing WT or Delta 1-Delta 5 FKN in the presence or absence of PMA (100 ng/ml). Representative comparison of FKN cell surface expression by flow cytometry in NIH3T3 cells expressing cleavable (FKN-WT) or uncleavable (FKN-Delta 5) FKN in the presence of PMA (100 ng/ml). Control uninfected cells show background staining.

The lack of shedding of FKN mutants Delta 2 and Delta 5 in response to PMA was also confirmed by immunoblotting and was correlated with a lack of metalloproteinase-mediated cleavage. FKN mutants Delta 2 and Delta 5 did not show the PMA-induced reduction in the amount of full-length FKN that was visible for wild-type FKN and the other FKN mutants (Fig. 5C). Additionally, cleavage of mature 100-kDa FKN (FKN-Mat) in WT, Delta 1, Delta 3, and Delta 4 FKN also led to increased levels of the expected FKN C-terminal cleavage fragment (not shown). We determined whether the absence of PMA-inducible FKN cleavage and shedding led to an increase in the levels of FKN present at the cell surface. In the presence of PMA, the uncleavable FKN mutants Delta 2 and Delta 5 showed significantly higher cell surface expression levels when compared with either wild-type or cleavable FKN mutants (Delta 1, Delta 3, Delta 4) (Fig. 5D). These findings suggest that unlike syndecan-1, PMA-induced FKN cleavage cannot be inhibited by replacement of the juxtamembrane dibasic amino acid sequence with an uncleavable CD4 sequence. Our data support a model in which cleavage of the FKN juxtamembrane sequence relies upon recognition of a structural motif by TACE rather than by recognition of a specific conserved cleavage site that can be deleted to abrogate PMA-induced shedding.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

It has been shown previously that FKN can exist as either a membrane-anchored cell surface protein or as a soluble chemokine (4). In this study, we show that soluble FKN can be generated by TACE-dependent cleavage and ectodomain shedding. After synthesis as an intracellular precursor, FKN undergoes rapid maturation, presumably by glycosylation, followed by transport to the cell surface where it becomes a substrate for metalloproteinase-dependent cleavage and shedding. We show that FKN shedding can be mediated by two distinct metalloproteinase activities as follows: a constitutive FKN sheddase, active under normal cell culture conditions, and an inducible FKN sheddase that is rapidly activated by phorbol esters such as PMA. PMA stimulation leads to the rapid cleavage and release of cell surface FKN resulting in a corresponding increase in soluble FKN.

Several lines of evidence presented here suggest that constitutive and PMA-inducible FKN shedding are mediated by different metalloproteinases. First, the constitutive FKN sheddase(s) is efficiently inhibited by low micromolar concentrations of the broad spectrum metalloproteinase inhibitor GM6001, whereas the PMA-inducible sheddase requires ~5-10-fold higher concentrations for efficient inhibition. More directly, cells derived from mice genetically deficient in specific ADAM proteases show that TACE, but not ADAM9, is required for PMA-induced but not constitutive FKN cleavage and shedding. Additionally, the constitutive FKN sheddase is a metalloproteinase but not ADAM9, as basal levels of FKN shedding are maintained in cells derived from ADAM9 knockout mice and are still sensitive to the broad spectrum metalloproteinase inhibitor, GM6001.

In addition to FKN, several other proteins known to be shed from the cell surface display both constitutive and PMA-inducible shedding when analyzed in vitro. At present, the relative importance of constitutive versus PMA-induced shedding observed in vitro to physiologically relevant shedding in vivo is poorly understood. However, several lines of evidence suggest that shedding that can be stimulated by PMA in vitro may mimic physiologically important shedding processes. In vitro, shedding of TGF-alpha can be mediated by TACE in response to stimulation by PMA but can also be induced by several diverse, physiologically relevant, stimuli including serum, epidermal growth factor, fibroblast growth factor, and platelet-derived growth factor (33). However, in vivo TACE appears to be the major protease responsible for the shedding of TGF-alpha , due to the similarity between mice genetically deficient in TACE function and TGF-alpha null animals (21, 24). Additionally, expression of TACE is up-regulated under a variety of inflammatory conditions in vivo, suggesting that TACE becomes activated during inflammatory responses where it can mediate the shedding of a diverse set of proteins including TNF-alpha and FKN (34, 35). Moreover, recent data suggest that "constitutive" ectodomain shedding observed in vitro may actually represent a form of induced shedding stimulated by the serum component lysophosphatidic acid (36). To begin to understand the role of TACE-mediated shedding in regulating FKN function in vivo, we are currently generating mice selectively deficient for TACE expression in endothelial cells.

It has been reported previously (4, 30) that FKN cleavage occurs at a conserved dibasic motif, close to the transmembrane domain, based solely on sequence similarity to syndecan-1, which is also cleaved and shed in response to PMA stimulation. However, unlike syndecan, replacement of the dibasic motif with an uncleavable CD4 sequence does not abrogate PMA-induced FKN shedding. Furthermore, PMA-induced syndecan shedding does not require TACE function (30). In addition, we have found that the FKN C-terminal cleavage fragment is glycosylated (data not shown) ruling out cleavage at the proposed dibasic motif that is membrane-proximal to any predicted glycosylation sites. Together these observations suggest that shedding of syndecan and FKN in response to PMA is mediated by different metalloproteinases through cleavage at distinct juxtamembrane sites.

Relatively little is known about the mechanism by which TACE cleaves such a diverse but select group of cell surface proteins. Several lines of evidence suggest that the structure of the juxtamembrane stalk region, rather than the presence or absence of a specific amino acid sequence, determines the susceptibility of a transmembrane protein to shedding. Mutational analysis of the cleavage sites for TNF-alpha , TNFRI, L-selectin, IL-6R, and the amyloid precursor protein have revealed relaxed sequence specificities surrounding the cleavage sites (37-41). For example, TNF-alpha shedding can only be abolished by deletion of at least 10 amino acid residues surrounding the cleavage site (41). Therefore, some sequence changes may maintain the juxtamembrane domain structural integrity and cleavability, whereas others disrupt this structure and potential access by the sheddase enzyme. For example, exchange of the TNF-alpha cleavage sequence for the IL6-R cleavage sequence of the same length results in a chimeric protein that is resistant to shedding (42) despite the fact that both TNF-alpha and IL6-R are substrates for TACE. These observations may help explain why deletion of the second 15 amino acids of the FKN juxtamembrane domain abrogate PMA-induced shedding in some contexts but not others (compare mutants FKN-Delta 2 and Delta 4, Fig. 5).

The in vivo significance of FKN cleavage and shedding is not yet known. Although it is premature to draw definitive conclusions, it is tempting to speculate that metalloproteinase-mediated cleavage of FKN from the endothelial cell surface may regulate FKN function at several levels. First, if FKN acts predominantly as an adhesion molecule for circulating leukocytes, then FKN cleavage may be necessary to modify the high affinity interactions between endothelial FKN and its receptor CXC3R1 during leukocyte recruitment. In support of this model, metalloproteinase-mediated L-selectin shedding plays an important role in regulating leukocyte rolling velocity and transmigration in vivo (43). A second possibility is that FKN targeted to the cell surface may solely act as a pro-form for soluble FKN that can be rapidly released by cleavage in response to the appropriate inflammatory stimuli. TACE has clearly been shown to mediate the cleavage of other precursor molecules including pro-TNFalpha and pro-TGFalpha , and as such TACE may be a key protease in the cell surface pro-protein convertase machinery. Finally, FKN may indeed be a dual function protein acting both as an adhesion molecule and a soluble chemokine. Irrespective of the true in vivo role of FKN, TACE-mediated cleavage is likely to act as an important regulator of FKN function in vivo.

    ACKNOWLEDGEMENTS

We are grateful to Roy Black and Jacques Peschon for supplying TACE reagents and for many helpful discussions. We also thank Garry Nolan for providing retroviral expression plasmids and the Department of Immunology Flow Cytometry Facility (University of Washington) for assistance with cell sorting.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants HL18645 (to E. W. R) and DK59778 (to P. J. D.), the Paul G. Allen Foundation for Medical Research (to K. J. G), the American Heart Association (to P. J. G), and the British Heart Foundation (to D. R. G).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Both authors contributed equally to this work.

§§ To whom correspondence should be addressed: Dept. of Pathology, Harborview Medical Center, 325 9th Ave., Box 359791, Seattle, WA 98104-2499. Tel.: 206-341-5410; Fax: 206-341-5416; E-mail: ewraines@u.washington.edu.

Published, JBC Papers in Press, August 8, 2001, DOI 10.1074/jbc.M106434200

2 C. P. Blobel, manuscript in preparation.

3 P. J. Dempsey, manuscript in preparation.

4 K. J. Garton, manuscript in preparation.

    ABBREVIATIONS

The abbreviations used are: FKN, fractalkine (CX3CL1); ADAM, a disintegrin and metalloproteinase; Me2SO, dimethyl sulfoxide; EGFP, enhanced green fluorescent protein; ELISA, enzyme-linked immunoabsorbance assay; FACS, fluorescence-activated cell sorting; HA, hemagglutinin epitope tag; HUVEC, human umbilical vein endothelial cell; IL-6R, interleukin-6 receptor; IFN-gamma , interferon-gamma ; MMP, matrix metalloproteinase; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; PMA, phorbol 12-myristate 13-acetate; TACE, tumor necrosis factor-alpha -converting enzyme (ADAM17); TGF-alpha , transforming growth factor-alpha ; TNF-alpha , tumor necrosis factor-alpha ; TNFRI, tumor necrosis factor receptor I; WT, wild type; PAGE, polyacrylamide gel electrophoresis; FCS, fetal calf serum; PE, phycoerythrin; DMEM, Dulbecco's modified Eagle's medium.

    REFERENCES
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ABSTRACT
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RESULTS
DISCUSSION
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