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Originally published online as doi:10.1189/jlb.0403141 on July 15, 2003

Published online before print July 15, 2003
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(Journal of Leukocyte Biology. 2003;74:389-394.)
© 2003 by Society for Leukocyte Biology

ADAM-17-independent shedding of L-selectin

Bruce Walcheck1, Shelia R. Alexander, Catherine A. St. Hill and Erik Matala

The Center for Immunology and the Departments of Veterinary PathoBiology and Laboratory Medicine and Pathology, University of Minnesota Academic Health Center, University of Minnesota, St. Paul

1Correspondence: University of Minnesota, 295j AS/VM Bldg., 1988 Fitch Ave., St. Paul, MN 55108. E-mail: walch003{at}umn.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
L-selectin is expressed by leukocytes and facilitates their adhesion under flow along the walls of blood vessels. As do a variety of membrane proteins, L-selectin undergoes ectodomain shedding. Using approaches that monitor full-length L-selectin in short-term assays, it has been determined that L-selectin shedding is defective in tumor necrosis factor {alpha}-converting enzyme (ADAM-17)-deficient cells. In this study, we examined the steady-state levels of L-selectin on ADAM-17-deficient cells using a monoclonal antibody to the cytoplasmic region of L-selectin, which allows for the detection of total L-selectin (full-length and the membrane-associated cleavage fragment). We demonstrate that ADAM-17-deficient cells generate a 6-kDa transmembrane fragment of L-selectin. Although inducible L-selectin shedding by phorbol 12-myristate 13-acetate stimulation was not observed by these cells in short-term assays, basal turnover did occur, resulting in the production of soluble L-selectin, as determined by enzyme-linked immunosorbent assay. L-selectin turnover was greatly increased upon ADAM-17 reconstitution. Truncating the juxtamembrane region of L-selectin blocked ADAM-17-independent shedding as did a hydroxymate metalloprotease inhibitor. Together, these findings demonstrate that a metalloprotease activity separate from ADAM-17 can use the cleavage domain of L-selectin. We speculate that separate proteolytic mechanisms of L-selectin shedding may regulate distinct antiadhesive mechanisms, such as inducible shedding for the rapid dissociation of cell–cell interactions and constitutive shedding for the homeostatic maintenance of high serum levels of soluble L-selectin, a potential adhesion buffer.

Key Words: inflammation • adhesion • endoproteolysis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
L-selectin is a type-1 membrane protein that is composed of extracellular domains structurally similar to a C-type lectin, epidermal growth factor, and a complement binding regulatory protein [1 ]. The role of L-selectin in initiating the accumulation of free-flowing leukocytes along the vascular wall by adhering to ligands presented by the endothelium and attached leukocytes is well established [1 2 3 4 5 ]. This process directs lymphocytes to secondary lymphoid organs and leukocytes to various inflammatory sites [1 ]. Recently, it has been reported that L-selectin may also be important for human pregnancy, as it is expressed by trophoblasts and involved in blastocyst implantation on the uterine wall [6 ].

L-selectin can be released as a soluble fragment by endoproteolysis [7 8 9 10 ], similar to a number of structurally and functionally diverse membrane proteins that undergo ectodomain shedding upon proteolysis within a membrane-proximal stalk region [11 , 12 ]. The maintenance of high levels of soluble L-selectin in normal serum of humans and mice (1.6–1.9 µg/ml) indicates that L-selectin shedding is a physiological process [13 14 15 16 ]. Cell activation by various means (e.g., phorbol esters, chemoattractants, cytokines, and stress) results in very rapid L-selectin turnover [7 , 17 , 18 ]. In fact, neutrophils can shed essentially all of their L-selectin molecules in a matter of a few minutes [7 , 17 ]. L-selectin also undergoes a slower basal turnover, which has been described to occur by primary cells and various cell lines expressing recombinant L-selectin [9 , 10 , 19 , 20 ].

L-selectin shedding generates a 6-kDa membrane-associated cleavage fragment that consists of the cytoplasmic region, transmembrane domain, and the first 11 amino acids of the extracellular juxtamembrane region [8 , 10 ]. The proteolytic processing of L-selectin within its cleavage domain is relaxed in sequence specificity but requires a certain physical length and secondary structure [10 , 20 , 21 ]. Early studies demonstrated that the protease activity involved in inducible L-selectin shedding functioned in cis and could be abrogated by hydroxymate metalloprotease inhibitors [22 23 24 25 ]. More recently, Peschon et al. [26 ] generated mice lacking functional tumor necrosis factor {alpha} (TNF-{alpha})-converting enzyme, also referred to as a disintegrin and metalloprotease (ADAM-17), and demonstrated that ADAM-17-deficient cells were defective in inducible L-selectin shedding. The proteolytic process of basal L-selectin shedding, however, may not be as exclusive. Studies on the structural requirements of L-selectin for basal and inducible shedding indicate that these events may be mediated by separate proteases [10 , 20 ]. For instance, a proline substitution at the P1, P2', or P3' position of the L-selectin cleavage site has been shown to block inducible but not basal L-selectin shedding, whereas an alanine substitution at the P1 position enhanced basal but not inducible L-selectin shedding [10 , 20 ]. In addition, the epidermal growth factor domain of L-selectin has been shown to be critical for inducible but not basal turnover [10 ]. However, because of the ubiquitous expression and predominant role of ADAM-17 in ectodomain shedding, it has been difficult to directly establish whether other proteolytic mechanisms may be involved in L-selectin turnover at the cellular level.

In this report, we use ADAM-17-deficient cells to further investigate L-selectin shedding. This system allows for the examination of ADAM-17-independent cleavage mechanisms, as well as for ADAM-17 reconstitution and the expression of L-selectin constructs to study structural relationships. Evidence is provided here that L-selectin undergoes ADAM-17-independent ectodomain shedding proximal to the membrane by a metalloprotease mechanism. An additional L-selectin cleavage mechanism may serve as a novel therapeutic target to regulate L-selectin adhesion events.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies
The DREG-200 monoclonal antibody (mAb) directed against the ectodomain of L-selectin and the CA21 mAb directed against the cytoplasmic region of L-selectin have been described previously [8 , 9 ]. The EL112 mAb, specific to the ectodomain of E-selectin, was purchased from LigoCyte Pharmaceuticals (Bozeman, MT) and used as an isotype-matched negative control mAb [immunoglobulin G1 (IgG1)]. Phycoerythrin-conjugated, F(ab)'2 goat anti-mouse IgG secondary antibody was purchased from Jackson Immunoresearch (West Grove, PA). Horseradish peroxidase-conjugated, goat anti-mouse IgG was purchased from Pierce (Rockford, IL). Biotinylation of mAb was performed using N-hydroxysuccinimide bound via a disulfide bond to biotin (Pierce), as per the manufacturer’s instructions.

Cells and transduction
The 300.19 mouse pre-B cell line [27 28 29 ] and the ADAM-17-deficient mouse fibroblast cell line (kindly provided by Dr. Jacques Peschon, Amgen Inc., Seattle, WA) [30 ] have been described previously. For cell activation, the phorbol ester phorbol 12-myristate 13-acetate (PMA; Sigma Chemical Co., St. Louis, MO) was used at 100 ng/ml. This compound was dissolved in 100% ethanol and diluted more than 1000-fold for all whole cell assays. Human L-selectin and mouse ADAM-17 were stably expressed in these cells by transduction. The human L-selectin constructs used in this report have been described previously [9 ]. L-selectin cDNAs were ligated into the pDON-AI retroviral plasmid (Takara, Shiga, Japan). Mouse ADAM-17 cDNA (kindly provided by Dr. Roy Black, Amgen Inc.) was ligated in a murine stem cell virus (MSCV)-based bicistronic retroviral vector coexpressing enhanced green fluorescent protein (EGFP; MigR1, kindly provided by Dr. Warren Pear, University of Pennsylvania, Philadelphia). Retrovirus generation and transduction were performed as described previously [9 ]. ADAM-17 expression was estimated based on EGFP fluorescence. L-selectin expression was determined by various immunoassays as described below.

Immunoassays
Flow cytometry, immunoprecipitation, immunoblotting, and enzyme-linked immunosorbent assay (ELISA) were performed as described previously [9 , 17 , 31 ]. Antibody-labeled cells were analyzed (10,000 cells/sample) or sorted by flow cytometry on a FACSCalibur instrument (Becton Dickinson Immunocytometry Systems, San Jose, CA). ELISA was performed by chemiluminescence and analyzed on a Fluoroskan Ascent FL luminometric plate reader (LabSystems, Helsinki, Finland). The relative concentration of soluble L-selectin in conditioned tissue-culture media was calculated by comparing the chemiluminescent intensity to a standard curve of titrated, soluble L-selectin of known concentration (R&D Systems, Minneapolis, MN), as per the manufacturer’s instructions.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
L-selectin undergoes ADAM-17-dependent and -independent shedding
A genetic deficiency in ADAM-17 by mice is lethal [26 ], thus cells derived from fetal tissues have been important for identifying substrates of ADAM-17. For our study, we used a well-validated fibroblastic cell line derived from ADAM-17-deficient mice [30 ]. Such cells have been used to investigate the shedding of various molecules [32 ], including the leukocyte determinants proTNF-{alpha}, TNF receptor (TNFR)I, TNFRII, interleukin (IL)-6R, and TNF-related activation-induced cytokine (TRANCE) [26 , 30 , 33 , 34 ].

The cells used here were confirmed to lack ADAM-17 by a polymerase chain reaction screen on genomic DNA using primers specific to the mouse ADAM-17 metalloprotease domain (data not shown). The ADAM-17-deficient cells also did not express detectable levels of endogenous L-selectin; therefore, the cells were transduced with recombinant human L-selectin, resulting in expression levels equivalent to leukocytes. It has been demonstrated that L-selectin does not undergo rapid shedding by ADAM-17-deficient thymocytes upon protein kinase C (PKC) activation with a phorbol ester [26 ]. Similarly, we found that ADAM-17-deficient cells activated with PMA demonstrated little-to-no down-regulation of L-selectin expression (Fig. 1A ). In contrast to the ADAM-17-deficient cells, PMA activation of leukocytes [9 , 17 ] and transduced 300.19 cells (a mouse pre-B cell line) resulted in a considerable down-regulation of L-selectin expression (Fig. 1C) . Upon restoring ADAM-17 expression, we found that L-selectin expression was down-regulated in a spontaneous manner, which was not significantly increased by PMA stimulation, as determined by flow cytometry (Fig. 1B) and ELISA (data not shown). We as well as others have shown that truncation of the juxtamembrane cleavage region of L-selectin abrogates L-selectin shedding [9 , 10 , 20 , 21 ]. As anticipated, such a mutation also prevented the shedding of L-selectin by the ADAM-17-reconstituted cells (Fig. 1B) . In contrast to the ADAM-17-deficient cells, 300.19 cells when transduced with ADAM-17 did not demonstrate a striking down-regulation in L-selectin expression (Fig. 1C) , indicating that an overexpression of ADAM-17 alone may not have accounted for the spontaneous shedding of L-selectin.



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  		Figure 1. The reconstitution of ADAM-17-deficient cells results in a considerable down-regulation of L-selectin expression. (A) Effects of PKC stimulation on L-selectin expression by ADAM-17-deficient cells. ADAM-17-deficient cells expressing wild-type L-selectin were treated with PMA to induce cell activation or carrier alone (Resting), as indicated. Cells were stained with DREG-200 to detect L-selectin or an appropriate isotype-negative control mAb (shaded graph; mAb control for activated cells is shown). Cell-staining levels were examined by flow cytometry, and 10,000 cells were examined per sample. (B) Effects of ADAM-17 reconstitution on L-selectin expression. Equivalent numbers of ADAM-17-deficient cells expressing wild-type L-selectin were untreated (WT L-selectin), transduced with a MSCV-based bicistronic retroviral vector (MigR1) that coexpresses ADAM-17 and EGFP (WT L-selectin, ADAM-17), or transduced to express ADAM-17 and also stimulated with PMA (WT L-selectin, ADAM-17, PMA). ADAM-17-deficient cells expressing wild-type L-selectin were also transduced with an empty MigR1 vector (WT L-selectin, MigR1). Finally, ADAM-17-deficient cells expressing cleavage-resistant L-selectin were transduced with ADAM-17 (CR L-selectin, ADAM-17). After transduction (48–72 h), the cells were stained with DREG-200. (C) Effects of ADAM-17 overexpression on L-selectin expression. The mouse pre-B cell line 300.19 expressing wild-type L-selectin was untreated (WT L-selectin), activated with PMA (WT L-selectin, PMA), or transduced with ADAM-17 (WT L-selectin, ADAM-17). The cells were then stained with DREG-200. (B and C) All cells were dual-analyzed by flow cytometry for their expression of L-selectin (y-axis) and EGFP (x-axis) as indicated. Nonspecific antibody labeling was determined using the appropriate isotype-negative control mAb (above and data not shown). Both axes represent Log10 fluorescence. PMA activation was performed similarly for all experiments (100 ng/ml; 1x106 cells; 37°C for 30 min). Representative data from multiple repetitions are shown.

 
An examination of L-selectin shedding by ADAM-17-deficient cells has previously been performed only by flow cytometric analysis, which assessed the down-regulation of full-length L-selectin molecules in short-term assays (<=30 min; see above and ref. [26 ]). In this study, we examined the steady-state levels of L-selectin on ADAM-17-deficient cells by immunoprecipitation and immunoblotting using a mAb to the cytoplasmic region of L-selectin. This approach allows for the detection of total L-selectin (i.e., full-length and the membrane-associated cleavage fragment) [8 , 9 , 23 ]. It is interesting that a full-length species (75 kDa) and a 6-kDa species of L-selectin were detected (Fig. 2A ). It is known that membrane-proximal cleavage of L-selectin results in a 6-kDa transmembrane fragment [8 ], which suggests that L-selectin underwent proteolysis by the ADAM-17-deficient cells within its cleavage region. We investigated whether the cleavage-resistant L-selectin mutant was susceptible to shedding by the ADAM-17-deficient cells. Equal numbers of ADAM-17-deficient cells expressing similar levels of wild-type or cleavage-resistant L-selectin (Fig. 2B) were detergent-lysed, and the L-selectin was immunoprecipitated with an anticytoplasmic region mAb. Immunoblot analysis revealed that the ADAM-17-deficient cells expressing cleavage-resistant L-selectin did not produce a transmembrane fragment (Fig. 2A) . In addition, these cells did not generate soluble L-selectin, as determined by ELISA (data not shown).



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  		Figure 2. L-selectin undergoes proteolysis within its cleavage domain by ADAM-17-deficient cells. (A) Steady-state L-selectin expression by ADAM-17-deficient cells. Equal numbers of ADAM-17-deficient cells expressing wild-type (WT) or cleavage-resistant (CR) L-selectin were detergent-lysed and immunoprecipitated with the mAb CA21, specific to the cytoplasmic region of L-selectin. Immunoprecipitated L-selectin was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. The resolved proteins were transferred to nitrocellulose and immunoblotted with CA21. The upper bands indicate full-length L-selectin, and the lower band indicates a 6-kDa membrane-associated fragment of L-selectin, as indicated. (B) Wild-type and CR L-selectin expression by the ADAM-17-deficient cells. ADAM-17-deficient cells transduced with wild-type or CR L-selectin cDNA were stained for L-selectin and sorted by flow cytometry to obtain cell populations that expressed similar levels of L-selectin on their cell surface. Immediately before detergent-lysing the transductants for immunoprecipitation, both transductants were stained with DREG-200 to determine the relative expression levels of L-selectin by flow cytometry, as described in Figure 1 .

 
To further examine L-selectin turnover by the ADAM-17-deficient cells, we used an ELISA to monitor the generation of soluble L-selectin. This approach has been used previously to quantitate soluble L-selectin levels in serum and media supernatants [9 , 14 , 15 ]. As shown in Figure 3A , the production of soluble L-selectin by ADAM-17-deficient cells was examined at time points up to 6 h and also compared with ADAM-17-reconstituted cells. Consistent with the production of the transmembrane fragment of L-selectin, the ADAM-17-deficient cells were found to generate soluble L-selectin.



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  		Figure 3. ADAM-17-deficient cells mediate L-selectin shedding by a metalloprotease activity. (A) ADAM-17-deficient and -reconstituted cells generate soluble L-selectin as determined by ELISA. ADAM-17-deficient and -reconstituted cells expressing L-selectin (Fig. 1B , top two panels) were each plated at 2.5 x 105 cells/well, media supernatant was collected at the indicated time points (data from the ADAM-17-reconstituted cells are shown only at the 2-h time point), and the levels of soluble L-selectin were determined. The y-axis represents ng/ml soluble L-selectin. (B) Effects of a hydroxymate metalloprotease inhibitor on L-selectin shedding by ADAM-17-deficient cells. Equal numbers of mock (pDON-AI empty vector) and L-selectin-transduced, ADAM-17-deficient cells were plated (2.5x105 cells/well). The ADAM-17-deficient cells were then treated with the hydroxymate metalloprotease inhibitor KD-IX-73-4 (50 µg/ml) or carrier alone (dimethyl sulfoxide). Media supernatant was then collected at 4 h from all samples and compared for levels of soluble L-selectin by ELISA. The y-axis represents the luminescence intensity of soluble L-selectin. For all assays described above, each transductant was plated in triplicate wells, and the supernatant from each well was analyzed in duplicate. Each bar represents the mean ± SD. Representative data from multiple repetitions are shown.

 
ADAM-17-independent, L-selectin shedding occurs by a metalloprotease
The proteolysis of L-selectin by the ADAM-17-deficient cells appears to occur in the receptor’s membrane-proximal stalk region, which is characteristic of metalloprotease sheddases. Prior to the cloning and characterization of ADAM-17, the involvement of a metalloprotease in inducible L-selectin shedding was determined by using hydroxymate metalloprotease inhibitors [22 23 24 25 ]. We examined whether a metalloprotease might be involved in ADAM-17-independent, L-selectin shedding by using the hydroxamic acid-based peptide inhibitor KD-IX-73-4. Equal numbers of ADAM-17-deficient cells were treated with KD-IX-73-4 or carrier alone, and ELISA then determined the levels of soluble L-selectin in the media supernatant. As shown in Figure 3B , KD-IX-73-4 at 50 µg/ml, a concentration that we have previously shown to abrogate shedding and not other cell functions [22 , 23 ], reduced L-selectin shedding by 81.1 ± 7.1% SD.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Consistent with previous findings [26 ], ADAM-17 can mediate L-selectin shedding, as shown here upon the reconstitution of ADAM-17-deficient cells. We found that the reconstitution of ADAM-17 expression resulted in high-level L-selectin turnover (Fig. 1B) . This spontaneous shedding may be the result of ADAM-17 induction or overexpression. However, overexpression of ADAM-17 by the 300.19 hematopoietic cell line did not result in such vigorous L-selectin shedding. The ADAM-17-deficient cells used here have been transduced with Ras [30 ], which is known to signal through the mitogen-activated protein kinase (MAPK) cascades [35 ]. We have found in initial studies that inhibition of the extracellular-regulated kinase (Erk) MAPK pathway by the pharmacological inhibitor U0126, diminished the shedding of L-selectin from ADAM-17-reconstituted cells by ~60% (data not shown). It is interesting that others have shown that inducible L-selectin shedding is blocked by inhibition of the Erk and p38 MAPK pathways [18 , 36 ].

We also demonstrate in this study that L-selectin shedding can take place in the absence of ADAM-17. This proteolytic process appears to occur within the cleavage region of L-selectin, as indicated by the production of a 6-kDa transmembrane fragment and that truncating this region blocked shedding. In addition, the production of soluble L-selectin by the ADAM-17-deficient cells was greatly reduced by a hydroxymate metalloprotease inhibitor. These data provide the first direct evidence that L-selectin can undergo ectodomain shedding by an ADAM-17-independent, metalloprotease process.

Similar to L-selectin, various other ADAM-17 substrates can undergo shedding in ADAM-17-deficient cells, such as fractalkine, amyloid precursor protein (APP), proTNF-{alpha}, TRANCE, proTNF-{alpha}, IL-6R, and tyrosine kinase receptor A [30 , 32 33 34 , 37 38 39 ]. The additional L-selectin sheddase appears to be a metalloprotease with the same juxtamembrane spatial requirements as ADAM-17. The ADAM family currently consists of >30 members (www.uta.fi/%7Eloiika/ADAMs/HADAMs.htm), of which five can apparently function as sheddases (ADAM-9, ADAM-10, ADAM-12, ADAM-17, and ADAM-19; refs. [40 41 42 43 44 45 46 47 ]). In addition, matrix metalloprotease-7 can also mediate the cleavage of proTNF-{alpha} and FasL [48 , 49 ]. ADAM-10 is the most similar to ADAM-17 in terms of protein sequence and the structural properties of their catalytic domains [50 ], and certain substrates can be cleaved by both ADAMs, including APP, cellular prion protein, IL-6R, and proTNF-{alpha} [34 , 41 , 44 , 51 , 52 ].

Many of the membrane proteins that undergo ectodomain shedding do so in a basal and inducible manner [11 , 12 ]. For proteins such as IL-6R and cellular prion protein, basal and inducible shedding have been shown to involve separate proteolytic mechanisms [34 , 44 ]. This may also be the case for L-selectin as indicated by findings that basal and inducible shedding require distinct structural features of L-selectin [10 , 20 ]. It is possible that ADAM-17 may play a predominant role in inducible L-selectin shedding, whereas the proteolytic process of steady-state L-selectin turnover may not be as exclusive. A limitation of our studies is that an ADAM-17-deficient fibroblast cell line was examined. However, L-selectin expression has been shown to occur by hematopoietic and nonhematopoietic primary cells [1 , 6 ], and L-selectin shedding has been well validated in hematopoietic and nonhematopoietic cell lines [20 , 21 ]. Of importance is that ADAM-17-independent, L-selectin shedding may primarily account for the serum levels of soluble L-selectin. This is based on recent findings that the serum levels of soluble L-selectin from chimeric mice with ADAM-17-deficient bone marrow were equivalent to that of control animals [53 ].

In summary, ectodomain shedding provides particular growth factors and cytokines with a juxtacrine and paracrine activity. Similarly, L-selectin shedding may have a dual role, and this may require separate proteolytic mechanisms. For instance, rapid L-selectin shedding appears to have an immediate influence on leukocyte accumulation along the vascular wall [23 , 54 ], whereas constitutive shedding may allow for homeostatic regulation of the high serum levels of soluble L-selectin [13 14 15 ]. The soluble form of L-selectin is functional and can influence leukocyte-endothelium interactions [14 , 16 ] and thus may serve as an adhesion buffer to diminish inflammation. Indeed, a reduced level of serum L-selectin has been shown to correlate with susceptibility to inflammatory disease, such as acute respiratory distress syndrome [55 ].


   ACKNOWLEDGEMENTS
 
This work was supported in part by funds from the National Institutes of Health (1R01 HL61613) and the C. M. Iverson Charitable Trust/American Cancer Society (RPG0005201CSM). We thank Drs. Peschon, Black, and Pear for providing reagents, Polly Mattila for technical assistance, and Lisa Adwan for assistance with the manuscript.

Received April 7, 2003; accepted June 12, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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