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Published ahead of print on June 15, 2007, doi:10.1165/rcmb.2006-0364OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 37, pp. 494-500, 2007
© 2007 American Thoracic Society
DOI: 10.1165/rcmb.2006-0364OC

Inhibition of Airway Smooth Muscle Adhesion and Migration by the Disintegrin Domain of ADAM-15

Dong Lu1, ShaoPing Xie3, Maria B. Sukkar3, Xinjie Lu1, Michael F. Scully2 and Kian Fan Chung3

1 Molecular Immunology Section, and 2 Proteomics Section, Thrombosis Research Institute; and 3 Experimental Studies Unit, Airways Disease Section, National Heart and Lung Institute, Imperial College London, London, United Kingdom

Correspondence and requests for reprints should be addressed to Michael F. Scully, Ph.D., Thrombosis Research Institute, Emmanuel Kaye Building, Manresea Road, London SW3 6LR, UK. E-mail: mscully{at}tri-london.ac.uk


   Abstract
Top
 Abstract
CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Disintegrin and metalloprotease proteins (ADAMs) are membrane-anchored glycoproteins involved in cell adhesion, cell fusion, protein ecto-domain shedding, and intracellular signaling. We examined whether the disintegrin domain of ADAM-15 (named ddADAM-15) containing an Asp-Gly-Asp (RGD) integrin-binding motif could interfere with airway smooth muscle cell (ASMC) adhesion and migration. Recombinant ddADAM-15 adhered to human ASMCs with saturation kinetics, and was beta1-integrin dependent. ddADAM-15 inhibited the binding of fibrinogen but not of fibronectin to ASMCs. ddADAM-15 also inhibited platelet-derived growth factor (PDGF)-induced ASMC migration, and this was reversed by an anti–beta1-integrin antibody. PDGF induced the activation of phosphoinositol-3-kinase (PI3K) and p38 mitogen-activated protein kinase (MAPK), and selective inhibitors of these kinases inhibited PDGF-induced ASMC migration. ddADAM-15 did not inhibit PDGF-induced activation of PI3K or p38, thereby excluding these kinase pathways as a mechanism by which ddADAM-15 inhibits ASMC migration. ADAM-15 mRNA and protein were expressed under basal conditions, and both gene and protein expression were inhibited by PDGF. In summary, ddADAM-15 inhibits ASMC adhesion and migration through the beta1-integrin, without modulating signaling pathways involved in ASMC migratory responses.

Key Words: ADAM-15 • airway smooth muscle • beta1-integrin • platelet-derived growth factor



   CLINICAL RELEVANCE
Top
 Abstract
 CLINICAL RELEVANCE
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
The disintegrin domain of ADAM-15 is shown to be capable of controlling the migration of smooth muscle cells by modulating intracellular signaling. This property is likely to engender specific roles for each ADAMs protein in healthy or diseased airways.

 
Disintegrin and metalloprotease proteins (ADAMs) or metalloprotease/disintegrin/cysteine-rich (MDC) proteins are a family of more than 30 membrane-anchored glycoproteins (1–4), whose functions include proteolysis, cell adhesion, cell fusion, protein ecto-domain shedding, and intracellular signaling (5–7). ADAMs are composed of a pro-domain, a metalloproteinase, a disintegrin domain, and a cysteine-rich region containing an epidermal growth factor (EGF) repeat, a transmembrane domain, and a cytoplasmic tail. The disintegrin domains of ADAMs represent potential binding sites for cell adhesion and signaling receptors of the integrin superfamily. The integrin binding motif is characteristically a tripeptide sequence that is exposed within a loop and, together with the N- and C-terminal flanking regions, determines the specificity and affinity of ADAMs toward distinct integrins (8–10). Integrins provide a physical connection between the internal and external cellular environment through their cytoplasmic regions, which interact with cytoskeletal proteins, and constitute an interface in which mechanical forces, cytoskeletal reorganization, and biochemical signals may converge (11–15). ADAM-15 is unique in containing the integrin binding motif Arg-Gly-Asp (RGD) within its disintegrin region (16–19).

The airway smooth muscle cell (ASMC) through its contractile properties and geometric disposition around the airways contributes to the control of airway caliber. However, the ASMC is more than a contractile cell, since it can synthesize inflammatory cytokines and chemokines and has a proliferative capacity (20, 21). ASMCs are remodeled in disease states such as asthma, where they are in an increased proliferative state (22) and are increased in size and in number (23, 24). ASMCs interact with extracellular matrix (ECM) proteins such as collagen through integrin attachment, and attachment through the {alpha}5beta1-integrin promotes ASMC survival (25). It has been proposed that increased airway smooth muscle mass may be the result of migration of ASMC precursors such as myofibroblasts into the muscle bundle (26). Migration of the ASMC is dependent on its interaction with the surrounding matrix.

The role of ADAMs in ASMC biology is not known. We chose to study ADAM-15 and hypothesized that ADAM-15, through its disintegrin properties, may interfere with ASMC adhesion and migration. We synthesized the disintegrin domain of ADAM-15 (ddADAM-15), and to examine the specific role of the unique RGD peptide sequence contained within the disintegrin domain, we synthesized ddADAM-15 mutants that contained integrin-binding motifs of other ADAM proteins. We then determined the effect of ddADAM-15 and ddADAM-15 mutants on ASMC adhesion to fibrinogen and fibronectin, and on platelet-derived growth factor (PDGF)-induced ASMC migration. We also determined whether ADAM-15 is expressed in ASMC.


   MATERIALS AND METHODS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Preparation of ddADAM-15 and Mutants
A cDNA fragment that encodes the disintegrin-like domain of ADAM-15 (Met420 to Glu510) was amplified by PCR with pcDNA3 as a template. The PCR fragment was digested by EcoR1and BamH1, then cloned into the restricted vector pGEX-3X (Amersham Biosciences, Piscataway, NJ) at the carboxyl terminus of the glutathione S-transferase (GST) gene and transformed in DH5{alpha} Escherichia coli strain. For the generation of ddADAM15 containing a disintegrin loop derived from ADAM-2, -12, -19, -28, a two-run PCR method was developed. The first-run PCR generated the sequence encoding the individual loop and the second-run downstream elongates the sequence encoding the C-terminal residues. The products were ethanol-precipitated to reduce the volume for the digestion with BamHI and EcoRI, then gene-cleaned again before ligation. After ligation, the construct was transformed in the same DH5{alpha} E. coli strain.

Protein Expression and Purification
ddADAM-15 was expressed in E. coli. The culture was inoculated with an overnight seed culture (1%, vol/vol) in 2x YT/ampicillin medium (100 µg/ml). Isopropyl beta-D-thiogalactoside (0.1 mM) was added for induction. GST-ddADAM-15 was purified from the supernatant of sonicated cells by affinity chromatography, using glutathione-sepharose 4B columns in the presence of trypsin inhibitor (50 mg/ml), pepstatin (10 mg/ml), aprotinin (10 ml stock), EDTA (0.5 M) and phenylmethanesulfonyl fluoride (100 mM in ethanol). The recombinant ddADAMs were released from GST-fusion proteins by digestion with factor Xa (1:300, wt/wt, factor Xa: fusion protein) at 4°C for 12 hours. The product was purified by reverse-phase HPLC (Waters 515) with a Vydac C18 analytical column.

ASMC Cultures
Human bronchial tissue was obtained from the lungs of patients undergoing surgical resection for carcinoma. All patients gave informed consent as to the use of the lung tissue and the project was approved by the Ethics Committees of St. Mary's and Royal Brompton Hospitals. ASMCs were dissected out and prepared using established methods (27). Briefly, bands of smooth muscle from bronchi were cut out from surrounding connective tissue and resuspended in 1 ml Dulbecco's modified Eagle's medium (DMEM) containing 1 mg/ml collagenase and kept in a humidified atmosphere of 5% CO2 for 30 to 60 minutes. The resulting cell suspension was centrifuged under 200 x g for 5 minutes, and pellets from the centrifugation were resuspended in DMEM media supplemented with 10% fetal calf serum. The cells were grown to confluence, passaged, and transferred to T175 tissue culture flasks. ASMC characteristics were identified by light microscopy with the "hill and valley" appearance and by positive immunostaining of smooth muscle (SM) {alpha}-actin, SM myosin heavy chain, SM-22, and calponin.

RNA Extraction and Real-Time PCR
Confluent ASMCs in 6-well plates were serum-deprived in DMEM containing 0.5% bovine serum albumin (BSA) for 24 hours. Cells were then treated with PDGF for a further 24 hours. Total cellular RNA was extracted from ASMCs using the Qiagen RNeasy mini Kit (Qiagen, Crawley, UK) according to the manufacturer's instructions. Single-stranded complementary DNA (cDNA) was synthesized from 0.5 µg of RNA by RT-PCR using AMV reverse transcriptase and random hexamers. Real-time PCR was performed by monitoring PCR product accumulation in real-time during the entire PCR cycling by fluorescence detection of SYBR Green dye (Rotor Gene 3000; Corbett Life Science, Sydney, Australia). The primer sequence pairs used were derived from the target cDNA sequence: ADAM-15 sense: 5'-CCGACGGGCCCTGGAGAAAG-3' and antisense: 5'-GCTGGGCATAGGAGGCACAAC-3'. Cycling conditions were as follows: Step1, 15 minutes at 94°C; Step2, 20 seconds at 94°C; Step3, 20 seconds at 67°C; and Step4, 20 seconds at 72°C. Steps 2 to 4 were repeated for 50 cycles. Data acquired were analyzed using the software supplied from the manufacture. Relative quantification of gene expression was calculated with GAPDH as control.

Western Blot Analysis for ADAM-15
ASM cells were lysed on ice to extract cell protein using lysis buffer (1% Igepal CA-630, 0.5% sodium deoxycholate, 0.1% SDS in PBS, pH 7.4) along with the following inhibitors: 1 mM PMSF, 5µg/ml aprotinin, 1 mM Na3VO4, and 5µg/ml leupeptin. Protein extract (15 µg per lane) was electrophoresed on 4–12% Criterion XT gels (Bio-Rad, Herts, UK) and then transferred to a blot membrane using an LKB semi-dry transfer unit (LKB, Stockholm, Sweden). ADAM-15 was then detected using purified polyclonal antibody (28) (kindly provided by John Martin, University of Wales College of Medicine, Cardiff, Wales, UK) raised against the disintegrin domain of ADAM-15. The antibody was immunoreactive against the specific disintegrin domain but not against the cytoplasmic or metalloproteinase domains of ADAM-15. The specific band was visualized using the enhanced chemiluminescence detection reagents and analysis system (Amersham Biosciences).

ASMC Adhesion Assay
96-well plates were coated overnight by incubation with solutions of fibronectin, fibrinogen, or ddADAM-15. Harvested ASMCs were resuspended in Tyrode's wash buffer. Cells were spun at 1,000 x g for 5 minutes and resuspended in Tyrode's wash buffer. Cells were then spun and resuspended in Tyrode's buffer containing 2 mM of MnCl2. The cells were then treated with or without anti-integrin antibodies (5 µg/ml) or with the appropriate isotype control for 30 minutes before adding to microtiter wells (100 µl of 1 x 106 cells) coated with ddADAM-15 acting as a ligand. The plates were incubated for 1 hour at 25°C and wells were washed with Tyrode's wash buffer. The number of adherent cells was determined by measurement of endogenous acid phosphatase at an absorbance of 450/650 nm.

ASMC Migration Assay
Migration assays were performed using Transwell 8-µm permeable membranes (Fisher Scientific, Loughborough, UK) coated with a 1% collagen solution (Cohesion, Eindhoven, The Netherlands). Confluent ASMCs were serum-deprived in DMEM containing 0.5% BSA for 24 hours before being used in migration assays. Cells were resuspended at a density of 1 x 106 cells/ml, and 100 µl of cell suspension was used. PDGF and/or ddADAM-15 were added to the lower chamber and cells were allowed to migrate over a 16-hour period. Cells on the upper face of the membrane were removed and migrated cells on the lower face were fixed in 100% methanol and stained with Diff-Quik solution (Dade Behring, Eschborn, Germany). Cells were counted under x40 magnification in five random fields.

Flow Cytometric Analysis of ASMC Surface Integrins
ASMCs were serum-deprived in DMEM containing 0.5% BSA for 24 hours before treatment with PDGF (0.8 nM) for a further 16 hours. This stimulation period was chosen as it was used in the cell migration assays. Cells were trypsinized, washed in 1% PBS, and incubated with the primary monoclonal anti-integrin antibodies (Chemicon, Hampshire, UK) or the appropriate isotype control (Beckman Coulter, Buckinghamshire, UK) (2 µg/105 cells, 30 min). After a wash with PBS, cells were incubated with the fluorescein isothiocyanate (FITC)-tagged secondary antibody (2 µg/105 cells, 30 min; Beckman Coulter). Further washes were performed with PBS and cells were then resuspended in binding buffer. Flow cytometric analysis was performed using a Beckmann Coulter FC 500 flow cytometer.

Measurement of p38 MAPK and PI3 Kinase Phosphorylation
Measurement of phosphorylated p38 and PI3K and total p38 and PI3K protein was performed using Fast Activated Cell Based ELISA (FACE) assays (Active Motif, Rixensart, Belgium) according to the manufacturers' instructions. ASMCs were grown to 90% confluence in 96-well plates and placed in fresh serum-free supplemented DMEM for 24 hours before treatment.

Data Analysis
Data are presented as mean ± SEM. Comparison between groups were performed using one-way ANOVA followed by the Newman-Keuls post test to determine statistical significance. A P value of < 0.05 was taken as statistically significant.


   RESULTS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Binding of ddADAMs to ASMCs
ddADAM-15 and ddADAM-15s containing different disintegrin-like loops derived from ddADAM-2, -12, -19, and -28 (referred to as dd2ADAM-15, dd12ADAM-15, dd19ADAM-15, and dd28ADAM-15) were initially tested for their ability to bind ASMCs. ddADAM-15 bound to ASMCs in a dose-dependent and saturable manner, with maximal effect at 2.8 µM. The binding ability of ddADAM-15 was weaker when compared with the glycoproteins fibrinogen (2.8 µM) and fibronectin (2.8 µM), but stronger when compared with ddADAM-15 containing any other disintegrin-like loop (Figure 1A). We then determined whether ddADAM-15 competes with fibrinogen or fibronectin for adherence to ASMC. ddADAM-15 inhibited fibrinogen binding to ASMCs in a dose-dependent manner. A maximal effect of 90% was observed at 1.5 µM ddADAM-15, a 5-fold higher concentration than that causing maximal adherence. ddADAM-15 did not inhibit fibronectin attachment to ASMCs. Thus, ddADAM-15 may have overlapping integrin-binding sequences to fibrinogen, but not to fibronectin (Figure 1B).


Figure 1
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  		Figure 1. (A) Adhesion of ddADAM-15 (open triangles); ddADAM-15 mutants containing different disintegrin-like loops derived from ddADAM-2, -12, -19, and -28, referred to as dd2 (solid circles), dd12 (open squares), dd19 (inverted open triangles), and dd28-ADAM-15 (open diamonds), respectively; and fibrinogen (solid squares) and fibronectin (solid triangles) to ASMC. (B) Effect of ddADAM-15 on adhesion of fibrinogen and fibronectin (2.8 µM each) to ASMC. Data are expressed as % of adhesion in control cells not treated with ddADAM-15. (C) Effect of anti-integrin antibodies on adhesion of ddADAM-15 (2.8 µM) to ASMC. Data are expressed as % of adhesion to ddADAM-15 in the absence of anti-integrin antibodies. **P < 0.001 compared with control cells treated with ddADAM-15 only. Data shown as mean ± SEM from three to four ASMC donors.

 
To establish which integrin(s) were mediating ASMC adherence to ddADAM-15, blocking anti-human monoclonal antibodies (mAbs) against the {alpha}4, {alpha}5, {alpha}6, {alpha}V, beta1, and beta3 subunits and the {alpha}2beta1 and {alpha}IIbbeta3 complexes were used (10 µg/ml each). The anti-beta1 antibody caused ~ 80% inhibition of ddADAM-15 binding to ASMCs. The other mAbs used had no significant effect, indicating that this adhesion was predominantly beta1 dependent (Figure 1C). The appropriate isotype control did not cause any significant effect.

ddADAM-15 Inhibits PDGF-Induced ASMC Migration
We investigated whether ddADAM-15 modulates ASMC migration induced by PDGF-BB. ddADAM-15 alone had no effect on baseline ASMC migration (data not shown). PDGF stimulated ASMC migration and displayed a classical chemokine bell-shaped dose response curve, with maximal migratory activity observed at 2.2 nM (Figure 2A). Submaximal concentration of PDGF (0.8 nM) was used in subsequent experiments. ddADAM-15 inhibited PDGF induced ASMC migration in a dose-dependent manner, causing maximal inhibition of ~ 70% at 4 µM (Figure 2B). A single amino-acid substitution of arginine (R64) to alanine (A64) in the RGD tri-peptide disintegrin domain abolished the inhibitory effect of ddADAM-15, indicating an absolute requirement of the RGD-tripeptide for this inhibitory activity (Figure 2C). An anti-beta1 antibody completely inhibited PDGF-induced ASMC migration, whereas an anti-{alpha}V antibody had a partial effect, with ~ 50% inhibition. Antibodies directed against {alpha}5 or beta3 integrins had no effect on PDGF-induced migration (Figure 2D). Flow-cytometric analysis of beta1, beta3, {alpha}v, and {alpha}5 expression on ASMC revealed predominant expression of the beta1-integrin. PDGF (0.8 nM) had no effect on the expression of these integrins (Figure 3).


Figure 2
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  		Figure 2. (A) Effect of PDGF on ASMC migration. Serum-deprived ASMC were allowed to migrate over 16 hours. Data represent mean ± SEM from three ASMC donors. (B) Effect of ddADAM-15 on PDGF induced ASMC migration. **P < 0.001 compared with cell migration in the presence of PDGF alone (control). (C) Effect of a mutated ddADAM-15 on PDGF-induced migration. The RGD motif of ddADAM-15 was mutated to AGD (A64-ADAM-15). *P < 0.05 and **P < 0.001 compared with cell migration in the presence of PDGF alone (control). (D) Effect of anti-integrin antibodies on PDGF induced migration. **P < 0.001 compared with cell migration in the presence of PDGF alone (control).

 

Figure 3
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  		Figure 3. Integrin expression on ASMC and modulation by PDGF. Cells were treated with or without PDGF (0.8 nM) for 16 hours and integrin expression was examined by flow cytometry. (A) Representative histograms of integrin expression on ASMC. Lightly dotted lines represent untreated cells stained with isotype control primary antibody and FITC-conjugated IgG secondary antibody. Light solid lines and dark solid lines represent untreated and PDGF-treated ASMC, respectively, stained with the indicated integrin primary antibodies and secondary FITC-conjugated IgG antibody. (B) Mean fluorescence intensities of integrin expression on ASMC in the presence and absence of PDGF. Data represent mean ± SEM from four ASMC donors.

 
PI3Kinase and MAP Kinases in ASMC Migration and Effect of dd-ADAM15
We used chemical inhibitors to determine the role of PI3 kinase, as well as the p38, extracellular signal–regulated kinase (ERK), and c-jun N-terminal (JNK) MAP kinases in PDGF-induced ASMC migration. The PI3 kinase inhibitor LY294002 (10 µM) and the p38 inhibitor SB203580 (5 µM) almost completely inhibted PDGF-induced migration, whereas inhibition of ERK with PD98029 (10 µM) or JNK with SP600125 (10 µM) had no effect (data not shown). Treatment with these inhibitors alone had no effect on ASMC migration (data not shown). Consistent with these findings, we demonstrated phosphorylation of both p38 and PI3 kinase 10 and 15 minutes, respectively, after stimulation with PDGF (0.8 nM) (data not shown). Interestingly, however, ddADAM-15 did not inhibit PDGF-induced phosphorylation of PI3 kinase or p38, nor did it further inhibit PI3 kinase or p38 phosphorylation activity in the presence of their respective inhibitors, indicating that ddADAM-15 did not interfere with these signaling activation pathways (Figure 4).


Figure 4
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  		Figure 4. Effect of ddADAM-15 on PDGF-induced PI3 kinase (A) and p38 (B) phosphorylation determined by ELISA. Cells were pretreated with the PI3 kinase inhibitor LY294002 (10 µM) or the p38 inhibitor SB203580 (5 µM) for 30 minutes before treatment with PDGF and/or ddADAM-15. **P < 0.001 compared with cells treated with PDGF only. Data are mean ± SEM from three ASMC donors.

 
Endogenous Expression of ADAM-15 in ASMC
We determined whether there was endogenous expression of ADAM-15 in unstimulated ASMC, and whether endogenously expressed ADAM-15 inhibited PDGF-induced ASMC migration. Using quantitative real-time PCR, ADAM-15 mRNA was detected under basal conditions. PDGF caused a dose-dependent inhibition of endogenous ADAM-15 mRNA, particularly at concentrations of 0.8 and 1.6 nM (Figure 5A). PDGF (1.6 nM) caused a time-dependent inhibition of ADAM-15 expression, maximal after 24 hours (Figure 5B). We also determined that TGF-beta (10 ng/ml), IL-1beta (10 ng/ml), IL-4 (10 ng/ml), and EGF (40 ng/ml) also inhibited ADAM-15 gene expression to an extent similar to that of PDGF (Figure 5C). Using Western blotting, we detected the presence of a 110-kD protein band, corresponding to the molecular weight of ADAM-15, thereby demonstrating constitutive expression of ADAM-15 protein in ASMC. PDGF caused a concentration-dependent inhibition of endogenous ADAM-15 protein expression (Figure 6).


Figure 5
Figure 5
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  		Figure 5. Endogenous ADAM-15 expression in ASMC and modulation by PDGF. (A) ADAM-15 mRNA expression under basal conditions or after treatment with PDGF for 24 hours was determined by real-time PCR and normalized to GAPDH expression. (B) Time-course of ADAM-15 expression in the presence of PDGF (1.6 nM). (C) Effect of TGF-beta (10 ng/ml), IL-1beta (10 ng/ml), IL-4 (10 ng/ml), PDGF (40 ng/ml), and EGF (40 ng/ml) on the expression of ADAM-15 at 24 hours. Data are mean ± SEM from three to four ASMC donors. *P < 0.05 compared with unstimulated cells (control).

 

Figure 6
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  		Figure 6. (A) Representative Western blot of ADAM-15 protein expression under basal conditions or following treatment with PDGF for 48 hours. (B) Mean densitometric analysis ± SEM of ADAM-15 protein expression determined in ASMC from three donors. *P < 0.05 and **P < 0.001 compared with unstimulated cells (control).

 

   DISCUSSION
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
We have shown that ddADAM-15 can bind to ASMCs and that this binding can be modulated by different putative disintegrin-like loops within the ddADAM-15 scaffold. This adhesion was predominantly mediated by the beta1-integrin. Hence, ddADAM-15 can serve as preferential beta1-integrin antagonist as shown by inhibition of ASMC adhesion toward immobilized fibrinogen. Furthermore, ddADAM-15 inhibited PDGF-induced cell migration, with the RGD-motif of ddADAM-15 playing a crucial role. Finally, although ASMC migration was blocked by selective kinase inhibitors of PI3K and p38 MAPK, the inhibitory role of ddADAM-15 in that respect was not accompanied by inhibition of these kinases.

The adhesive-promoting activity of ddADAM-15 is specifically due to the integrin-binding RGD sequence located in a putative disintegrin-like loop of ddADAM-15, as the replacement of the putative disintegrin-like loop with those of ADAM-2, -12, and -19 showed a dramatic decrease in binding ability. Although both fibrinogen and fibronectin can use beta1-integrin binding to ASMC, only fibrinogen but not fibronectin binding was blocked by ddADAM-15 in a dose-dependent manner. This preferential antagonism implied that ddADAM-15 and fibrinogen may share the same or a closely related region of the beta1-integrin–binding site; such a region may not be involved in fibronectin binding despite the location of an RGD sequence in the 10th type III repeat of fibronectin, which is the major binding site for beta1-integrin, for example as with {alpha}5beta1 (29, 30). The role of RGD in ddADAM-15 may be limited, as this is the only ADAM family protein containing this sequence. It cannot be ruled out that other regions beyond the disintegrin-like loop may also play a role in integrin-binding ability, since ddADAM-15, as well as ddADAM-12 (which lacks the RGD-motif), can interact with beta1-associated {alpha}9 integrin (31), and {alpha}5 integrin (19), in other cell types.

Integrins play an important role in cell adhesion to ECM (32). Fibronectin, collagen I and IV, and laminin are important for survival of ASMCs, and these survival signals are mediated through the fibronectin receptor or {alpha}5beta1 integrin (25). Enhancement by fibronectin of IL-1beta–stimulated eotaxin release was mediated through multiple beta1-integrins expressed on ASMCs including {alpha}2beta1, {alpha}5beta1, {alpha}vbeta1, and also {alpha}vbeta3 (33). Similarly enhancement of PGDF-BB dependent proliferation by either collagen or fibronectin required multiple integrins including {alpha}2beta1, {alpha}4beta1, and {alpha}5beta1 integrins (34).

An important effect of ddADAM-15 is its inhibition of PDGF-induced ASMC migration in a dose-dependent manner within the range of 1–10 µM. The substitution of R64 into A64 in ddADAM-15 (RGD->AGD) abolished its inhibitory activity on ASMC migration, implying a crucial role for the RGD sequence. Given that the beta1-integrin subunit or its associated complex can interact with many ADAM proteins including ADAM-2, -7, -9, -10, -12, -17, -28, and -33, and given an earlier report describing the involvement of beta1- and beta3-integrins in endothelial cell migration (35), we tested the involvement of beta1- or beta3-integrins in mediating ASMC migration. The present study shows that ASMC migration is mediated predominantly by beta1-integrin but not by either beta3- or {alpha}5-integrin subunits as demonstrated by the use of specific integrin–blocking antibodies.

We confirmed the dependence of PDGF-induced ASMC migration on the activation of p38 MAPK and PI3K, as previously demonstrated (36, 37). Our data indicate that endogenous ADAM-15 through its disintegrin domain interacting with beta1-integrins may inhibit PDGF-induced ASMC migration, but without inhibition of p38 MAPK or PI3K activation triggered by the binding of beta1-integrin on the surface of ASMC to the RGD sequence in the extracellular matrix, that results in actin remodeling and chemotaxis. However, blockade of adhesion by ddADAM-15 may prevent the migration of an activated ASMC. The most obvious role for integrins in cell motility involves their ability to interface with the extracellular matrix and thus allow cells to adhere to its substratum essential for chemotaxis. The extracellular matrix collagen can be a ligand of the beta1-integrin subunit or beta1-associated integrin complex. ddADAM-15 may induce integrin conformation after its occupancy, which may reduce the integrin's ability to influence signaling pathways, since ddADAM-15 did not affect p38 and PI3K activation. We demonstrate for the first time the expression of ADAM-15 in human ASMC, and the inhibitory effect of PDGF on gene and protein expression of endogenous ADAM-15. The inhibitory effect may reduce any potential disintegrin effect of ADAM-15 to a level with little biological effect.

Apart from ADAM-15, other ADAMs such as ADAM-33 have been implicated in the lung pathophysiology. ADAM-33 has been reported as a susceptibility gene for asthma (38) and may influence asthma phenotype with functions related to airway wall remodeling. ADAM-33 has been shown to be overexpressed in ASMC in subjects with asthma (39). ADAM-8, which has an unknown function, can be induced during allergen challenge, and is expressed in inflammatory cells and epithelium but not in ASMC in mice (40). We have shown that ddADAM-15 has the ability to inhibit integrin-mediated ASMC adhesion and serves as an inhibitor of ASMC migration. The RGD-motif in ADAM-15 plays a key role in the interaction of ddADAM-15 with beta1-integrin. It is likely that there is diverse expression of different ADAMs in the airways and lungs that could play different roles in airway diseases.


   Acknowledgments
 
The authors are grateful for Dr. Robyn Clutterbuck for helpful discussions. They thank Dr. John Martin and Professor Carl Blobel for kindly providing the ADAM-15 antibody and cDNA.


   Footnotes
 
This work was supported by the Thrombosis Research Trust/Garry Weston Foundation (D.L., X.L., M.F.S.) and by a grant from the UK Wellcome Trust (to S.P.X., M.B.S.).

Originally Published in Press as DOI: 10.1165/rcmb.2006-0364OC on June 15, 2007

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form September 26, 2006

Accepted in final form May 15, 2007


   References
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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