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J. Biol. Chem., Vol. 278, Issue 26, 23656-23665, June 27, 2003
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From the Department of Molecular, Cell, and Developmental Biology, Molecular Biology Institute and Jonsson Comprehensive Cancer Center, University of California, Los Angeles, California 90095
Received for publication, December 19, 2002 , and in revised form, April 21, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The vascular endothelial growth factor (VEGF)1 gene produces several splice variants critical for capillary morphogenesis and tumor angiogenesis (11–13). Haploinsufficiency of this gene is incompatible with development due to major vascular abnormalities, as demonstrated by inactivation of the gene through homologous recombination (14, 15). Isoforms of VEGF are secreted by diverse cell types including smooth muscle, fibroblasts, and epithelial cells. These proteins function by activation of two tyrosine kinase receptors, VEGFR1 and VEGFR2, as well as by binding to non-receptor tyrosine kinase coreceptors such as neuropilin 1 and 2 on endothelial cells (16, 17).
ADAMTS1 was the first member of a growing family of ADAMTS extracellular proteases characterized by the presence of disintegrin-like metalloprotease and a variable number of thrombospondin-like domains (18, 19). Once secreted, ADAMTS1 activation requires furin cleavage and removal of the pro-domain. The active protease can undergo a secondary processing event that separates the catalytic subunit from the thrombospondin (TSP) repeats (20, 21). These TSP motifs in TSP1 and TSP2 have been shown to block angiogenesis by several, and likely not independent, mechanisms (22–24).
We demonstrated previously that ADAMTS1 is able to suppress capillary growth using multiple in vivo and in vitro assays (18). Interestingly, the ability of ADAMTS1 to inhibit neovascularization in vivo was greater than that of endostatin and TSP1 at the same molar ratio. The findings are intriguing and appear in marked contrast to the current paradigm that MMPs are pro-migratory and pro-angiogenic (25–28). In an effort to determine the mechanism(s) responsible for the angiostatic properties of ADAMTS1, we investigated its effects on endothelial cell growth and found that ADAMTS1 was able to drastically decrease VEGFR2 phosphorylation by a mechanism that involved direct binding and sequestration of VEGF165. The interaction was also verified in vivo using xenograft assays engineered to express ADAMTS1. Our results demonstrate that ADAMTS1 binds to VEGF165, and that this impacts the bioavailability of VEGF with consequences to receptor phosphorylation, endothelial proliferation, and angiogenesis.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Generation of Monoclonal Antibodies against ADAMTS1—Balb/c mice were immunized by intraperitoneal injections of purified ADAMTS1 (10 µg) (21) and complete Freud coadjuvant (300 µl). Additional immunizations were done with incomplete Freud coadjuvant at days 15 and 33 (intraperitoneal injection) and intravenously at day 45. At day 48, spleenocytes were obtained from immunized mice and fused with SP2 myeloma cells at a 4:1 ratio following established techniques (29). Conditioned media from hybridoma cultures were screened by ELISA against purified ADAMTS1 coated to plastic. Further characterization of positive clones was performed by immunoprecipitation, Western blot analysis, and immunohistochemistry. From 101 positive wells, twelve hybridomas were cloned by repeated limiting dilution based on epitopes and specific properties. Table I summarizes the selected monoclonal antibodies (mAbs).
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Endothelial Cell Proliferation—Quiescent BAEC were trypsinized and plated onto 24-well plates in Dulbecco's modified Eagle's medium supplemented with VEGF165 (R&D Systems) (25 ng/ml), basic fibroblast growth factor (FGF-2) (2 ng/ml) (provided by Dr. Gera Neufeld) or a combination of both, in the presence or absence of recombinant ADAMTS1/METH-1 protein (5 µg/ml). A pulse of 1 µCi/well of [3H]thymidine (Amersham Biosciences) was applied over the last 8 h prior to harvesting. Cells were washed and fixed in 10% trichloroacetic acid. Incorporation of [3H]thymidine was determined by scintillation counting, as described previously (30).
Phosphorylation Assays—Subconfluent cells were incubated overnight in serum-free medium and subsequently preincubated for 5 min with 0.1 mM Na3VO4 to inhibit phosphatase activity. Cultures were then washed once and pretreated with 1.5 ml of conditioned media from 293T cells expressing ADAMTS1 or vector alone for 15 min at 37 °C. The concentration of ADAMTS1 in the conditioned media ranges between 5.85 µg/ml (66 nM) and 16.7 µg/ml (191 nM). These values were obtained by ELISA against a standard curve of purified ADAMTS1 (data not shown). Cells were stimulated for 6 min at 37 °C with specified concentration of VEGF165. The incubation was terminated by removal of the medium and washes with cold phosphate-buffered saline/0.2 mM Na3VO4. Cells were solubilized in lysis buffer (1% Triton X-100, 10 mM Tris-HCl, pH 7.6, 150 mM NaCl, 30 mM sodium pyrophosphate, 50 mM sodium fluoride, 2.1 mM sodium orthovanadate, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 2 µg/ml of aprotinin) at 4 °C for 15 min. Insoluble material was removed by centrifugation at 4 °C for 30 min at 14,000 x g. Equal amounts of cell lysate were separated by SDS-PAGE and transferred to nitrocellulose membranes. Phosphorylated proteins were detected by immunoblotting using antiphosphotyrosine antibodies (polyclonal or mAb PY20, BD Transduction Laboratories) followed by secondary antibodies coupled with horseradish peroxidase and visualized by chemiluminescence (ECL kit, Pierce). Protein-loading control was assessed by Western blot using anti-VEGFR2 (A-3, Santa Cruz Biotechnology) and or anti-enolase antibodies.
Immunoprecipitation—Cell lysates were precleared with 40 µl of protein G-agarose (Roche Applied Science) for 1 h at 4 °C. Beads were discarded by centrifugation, and the supernatant was incubated with 1 µg/ml of the antiphosphotyrosine antibody (PY20) overnight at 4 °C followed by addition of protein G-agarose for 1 h under continuous agitation. Immunoprecipitates were washed three times with lysis buffer and extracted in 2x SDS-PAGE sample buffer by boiling 5 min, fractionated by one-dimensional SDS-PAGE, and further analyzed by Western blot with antiphosphotyrosine antibodies.
ADAMTS1 was immunoprecipitated from tumor lysates (450 µg) as described above using the mAb 5C6D5. The presence of coimmunoprecipitated VEGF was assessed by Western blot using polyclonal anti-VEGF, 375 (generous gift from Don Senger, Beth Israel Deaconess Medical Center, Boston). Levels of ADAMTS1 were determined by Western analysis (mAb 5C6D5).
Radiolabeling of VEGF—VEGF165 was labeled with 125I-Na using iodogen as coupling agent. Briefly, VEGF165 (2 µg) was incubated in 200 µl of borate buffer (0.01 M Na2B4O7, 0.14 M NaCl, pH 8.2) with 0.3 mCi of 125I-Na (Amersham Biosciences) in IODO-GEN-precoated iodination tubes (Pierce) 5 min at room temperature. The reaction was stopped by transfer to a fresh tube containing 40 µl of 0.4 mg/ml tyrosine in borate buffer for 1 min at room temperature and adding 200 µl of 1 mg/ml IK in phosphate-buffered saline/1% BSA. 125I-VEGF165 was separated from free iodine using size exclusion chromatography on Sephadex-G25 columns (Amersham Biosciences). The specific radioactivity of the purified iodinated VEGF165 was 18,886 cpm/ng. Quality and integrity of the labeled VEGF was assessed by SDS-PAGE followed by autoradiography of the dried gel.
Binding Assays—Subconfluent cells were incubated at low
serum (2% serum for HAEC and 0.1% for PAE and PAE-VEGFR2 cells) for 5 h at 37
°C, rinsed in binding buffer (media containing 20 mM HEPES,
0.2% gelatin) and equilibrated at 4 °C for 15 min. Indicated
concentrations of 125I-VEGF165 were added to the wells
in a final volume of 350 µl of binding buffer in the presence or absence of
ADAMTS1. After 1 h of incubation at 4 °C, cells were washed four times in
binding buffer and solubilized in 500 µl of lysis buffer (2% Triton X-100,
10% glycerol, 1 mg/ml BSA). Bound protein was assessed on a 
-counter.
Nonspecific binding was calculated in the presence of 20-fold excess of cold
VEGF165. Specific binding was determined by subtracting nonspecific
binding from total binding.
Purification of ADAMTS1 and Evaluation of VEGF Levels—Recombinant ADAMTS1 protein was purified from conditioned media of stable 293T cells expressing ADAMTS1 by heparin and Zn2+-chelate affinity chromatography (21). Conditioned media from 293T cells transfected with the vector alone was used as control. Presence of VEGF in the ADAMTS1 fractions was assessed by Western blot (Chemicon). Levels of ADAMTS1 were tested using the mAb 5D4E11B5 after stripping of the same membrane.
Gel Permeation Chromatography—ADAMTS-VEGF complexes purified by heparin chromatography were concentrated to 1 ml and dialyzed into 10 mM HEPES, 150 mM NaCl, 25 mM 1-decanesulfonic acid sodium salt (DSS). Sample was loaded onto a Superdex 75 HR 10/30, previously equilibrated in 10 mM HEPES, 150 mM NaCl, and 0.3% Zwittergent 3–12. Chromatography was carried out at 4 °C with a flow rate of 0.3 ml/min (at 1100 psi). Fractions of 300 µl were collected and evaluated by Western blots.
Binding of VEGF to Immobilized ADAMTS1—ADAMTS1 was immunoprecipitated from conditioned media of stably transfected 293T cells using the mAb 5C6D5. Protein A beads were washed three times with phosphate-buffered saline, 1% Triton X-100, 1% BSA, 1 mM CaCl2,1mM MgCl2, and equilibrated in binding buffer (50 mM HEPES, 150 mM NaCl, 0.5% Nonidet P-40, 0.5% BSA, 1 mM CaCl2,1mM MgCl2). Soluble ligands: VEGF165 (50 ng) (R&D Systems) and/or heparin (50 ng) (Sigma), were added to the pellets in a final volume of 500 µl of binding buffer, and incubated for 30 min at 4 °C. The beads were subsequently washed three times with binding buffer. Proteins bound to the immunoconjugates were subjected to SDS-PAGE, and the presence of coimmunoprecipitated VEGF was analyzed by Western blots. Immunoprecipitated ADAMTS1 was tested reproving the same membrane with the mAb 5D4E11B5.
Cross-linking Experiments Using Purified Proteins—Purified ADAMTS1 (1.25 µg) was incubated with VEGF165 (25 ng) in the presence or absence of heparin (50 ng) in 75 µl of binding buffer (50 mM HEPES, 100 mM NaCl, pH 7.2) for 2 h at room temperature. Bound proteins were cross-linked with disuccinimydil suberate (1.5 mM) (Pierce) for 30 min at room temperature. The reaction was stopped by adding 20 mM Tris, 50 mM glycine, 2 mM EDTA (pH7.4) for 15 min at room temperature. The samples were processed by SDS-PAGE, and protein complexes, indicating protein-protein interaction, were visualized by Western blots.
Cross-linking Experiments Using Conditioned Media—Subconfluent 293T or T47D cells expressing full-length or different deletion constructs of ADAMTS1 or pCDNA (negative control) grown on 24-well plates were washed twice with serum-free Dulbecco's modified Eagle's medium and incubated in 300 µl of serum-free media for 18 h in the presence or absence of VEGF165 (150 ng) or VEGF121 (125 ng) (provided by Dr. Gera Neufeld) with or without heparin (1.5 µg). Conditioned media was collected, and the interacting proteins were cross-linked and analyzed as described above.
Xenograft Tumor Assays and Tumor Protein Extraction—Control and ADAMTS1 tumors were generated in 6-week-old male nude mice (Charles River Laboratories) by subcutaneous flank injection of both T47D control (empty vector) or ADAMTS1 (5 x 106 cells/injection). When tumors reached 1,500 mm3, mice were euthanized, and dissected tumors were diced, sieved, and solubilized in lysis buffer (1% Triton X-100, 10 mM Tris-HCl, pH 7.6, 150 mM NaCl, 30 mM sodium pyrophosphate, 50 mM sodium fluoride, 2.1 mM sodium orthovanadate, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 2 µg/ml aprotinin) at 4 °C for 1 h. Insoluble material was removed by centrifugation at 4 °C for 1 h at 14,000 x g.
Expression of the Carboxyl-terminal (CT) Region of ADAMTS1 in T47D Cells—The construct for expression of the CT region of ADAMTS1 corresponding to Pro549–Ser951 was obtained by PCR of the full-length cDNA. A KpnI site was introduced at the 5'-end by site-directed mutagenesis using the following forward oligo: 5'TTTTCATGGTACCTGGGGAATGTGGG-3'. The reverse oligo used was 5'-ACTGCATTCTGCCTTTGTGCAAAAGTC-3'. The resulting PCR product was cloned into the pSecTag2/Hygro B vector (Invitrogen) using KpnI/EcoRV. Stable transfectant clones were generated using this plasmid in T47D cells by calcium phosphate transfection and selected with hygromycin-B (300 µg/ml).
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The mitogenic action of all VEGF isoforms is mediated through binding to the 205-kDa receptor tyrosine kinase VEGFR2 (31). Therefore, we tested whether activation of VEGFR2 was affected by ADAMTS1. VEGFR2 phosphorylation was strongly inhibited by ADAMTS1 in several endothelial cell cultures including PAE-VEGFR2 (Fig. 1B), BAEC (Fig. 1C) and human aortic endothelial cells (data not shown).
Stimulation of BAEC with increasing concentrations of VEGF165 resulted in a dose-dependent phosphorylation of VEGFR2 and ADAMTS1 reduced the levels of phospho-VEGFR2 in all cases (Fig. 1D). Together these results imply a direct link between VEGF165 signaling and the anti-angiogenic effects mediated by ADAMTS1.
As a possible explanation for the ADAMTS1 inhibition of VEGFR2 phosphorylation, we tested whether binding of VEGF165 to the endothelial cell surface was affected by ADAMTS1. 125I-VEGF165 bound to HAEC (Fig. 2A) and PAEVEGFR2 cells (Fig. 2B) in a concentration-dependent manner; however it did not bind to VEGFR2-deficient cells (PAE) (Fig. 2B). It has been documented that saturation binding occurs at 10 ng/ml with VEGF165 (32). The specific binding of 125I VEGF165 (16 ng/ml) to HAEC (Fig. 2A) was 14.7%, while saturation binding in PAE-VEGFR2 was lower, 8.9% after incubation with 12 ng/ml of radioiodinated VEGF (Fig. 2B). Presence of purified ADAMTS1 reduced 125I-VEGF165 specific binding by 69% (Fig. 2C).
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VEGF Co-purifies with ADAMTS1—It has been reported that TSP repeats in thrombospondin 1 and in connective tissue growth factor bind to VEGF165 and modulate its activity on endothelial cells (33, 34). The CT of ADAMTS1 contains three domains that share significant homology to the TSP repeats of the TSP1 molecule (18). Therefore, we investigated the possibility of an interaction between ADAMTS1 and VEGF165 as a regulatory mechanism to explain the effect of ADAMTS1 on VEGFR2 phosphorylation and the binding to the endothelial cell surface. We found that indeed VEGF (Fig. 3A, lanes 1–3) was present in the samples containing ADAMTS1 (Fig. 3A, lanes 5–7) after purification from heparin affinity chromatography (Fig. 3A, lanes 1 and 5) and from chromatography on a Zn2+-chelate affinity column (Fig. 3A, lanes 2 and 6, 3 and 7). Although detection of VEGF in the first purification step was not surprising, since VEGF interacts avidly with heparin (34, 36); its presence after Zn2+ chromatography was unexpected since this growth factor does not bind to Zn2+. To ascertain the degree of purification of VEGF from these two chromatography procedures in the absence of ADAMTS1, conditioned media from parental cell lines transfected with vector alone was subjected to the same chromatography purification. As expected, VEGF was eluted from heparin column at 1 M (Fig. 3B), whereas no binding was detected on Zn2+-chelate affinity chromatography in the absence of ADAMTS1 (Fig. 3C). 293T cells have been shown previously to secrete VEGF; our results concur with those findings (37, 38). These results provide evidence that VEGF binds to ADAMTS1.
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The two molecules can be dissociated and purified from one another by gel filtration chromatography (Fig. 4A). When bound to ADAMTS1, VEGF165 was unable to phosphorylate VEGFR2 (Fig. 4B, sample A). However, VEGF165 regained its activity when dissociated from the protease (Fig. 4B, sample 13).
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ADAMTS1 Binds to VEGF165—The interaction of ADAMTS1 with VEGF165 was also evaluated by co-immunoprecipitation and cross-linking assays. Conditioned media was collected from cells that were stably transfected with either vector alone (pCDNA) or ADAMTS1 and immunoprecipitated with anti-ADAMTS1 antibodies. The immunocomplexes were subsequently incubated with VEGF165, and binding was evaluated by Western blots. VEGF165 was detected in the ADAMTS1 immunocomplexes and addition of exogenous heparin increased binding of ADAMTS1 to VEGF165 (Fig. 5A). No VEGF165 was bound to immunoprecipitated complexes when media conditioned from cells expressing vector alone was used (Fig. 5A).
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Cross-linking experiments were conducted with disuccinimydil suberate to evaluate physical proximity of ADAMTS1 and VEGF165. Heterodimeric complexes of about 250 and 130 kDa were detected with VEGF antibodies in the presence of ADAMTS1 (Fig. 5B). VEGF antibodies also recognized 130-kDa species in the ADAMTS1 preparations, indicating presence of VEGF in ADAMTS1 preparations as demonstrated previously (Fig. 3). We predict that the 130-kDa form corresponds to the sum of ADAMTS1 (87 kDa) and VEGF (43 kDa). Addition of heparin enhanced the formation of the high molecular weight complexes; however, the presence of heparin was not a requirement (Fig. 5B).
To ascertain whether ADAMTS1-VEGF165 complexes were formed in vivo, we incubated 293T cells transfected with ADAMTS1 or empty vector with purified VEGF165 and heparin. Evaluation of VEGF after cross-linking revealed a shifted band of about 130 kDa only in the ADAMTS1-conditioned media (Fig. 5C). Under these experimental conditions, formation of the complex ADAMTS1-VEGF165 required heparin. To ensure the presence of ADAMTS1 in the 130-kDa band, the same blot was re-probed with anti-ADAMTS1 antibodies (Fig. 5C, blot to the right). Indeed a 130-kDa band was found only in the presence of VEGF165 and overlapped with the band identified by the VEGF antisera.
Cross-linking experiments were also performed with VEGF121; however, no interaction was found (Fig. 5D). These results indicated that the carboxyl-terminal heparin-binding domain of VEGF participates in an interaction with ADAMTS1.
ADAMTS1 and VEGF Form Complexes in Vivo—The in
vitro data implied that one possible mechanism for ADAMTS1 activity is
binding and inactivation of VEGF165 function. Therefore, we
investigated whether ADAMTS1 and VEGF formed complexes in vivo. The
experiments were performed using xenograft tumor lysates from T47D mammary
carcinoma cells expressing either ADAMTS1 or vector alone (pCDNA). As
previously described, we found that biochemical evaluation of VEGF from
tissues/tumors reveals multiple bands immunoreactive with several independent
VEGF antibodies (Fig.
6A) (39,
40), representing different
VEGF forms (17,
41) as well as possible
complexes formed with other proteins
(42–45).
Comparison between control and ADAMTS1 expressing tumors revealed a novel VEGF
immunoreactive species of 
130 kDa present exclusively in the ADAMTS1
tumor lysates (Fig.
6A). The 130-kDa band co-migrated and overlapped with a
species recognized by ADAMTS1 antibodies
(Fig. 6A).
Furthermore, VEGF was detected in immunoprecipitates of ADAMTS1 from tumor
lysates, whereas no VEGF was detected in lysates from pCDNA tumor tissue
treated in an identical manner (Fig.
6B). These in vivo results corroborate the
in vitro finding that ADAMTS1 and VEGF form a stable complex.
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Defining Domains of ADAMTS1 Involved in
VEGF165 Binding—To determine the
domain(s) of ADAMTS1 that participate in the interaction with
VEGF165, we used several deletion constructs
(Fig. 7A). In these
experiments, only the full-length P87-ADAMTS1 was able to bind to
VEGF165 (Fig.
7B). Levels of each deletion protein were determined by
Western blot analysis in a parallel experiment
(Fig. 7C). VEGF
appears as a rather diffused and wide band. It is pertinent to the
interpretation of these experiments to clarify that the VEGF antibodies have a
higher titer than the ADAMTS1 antibodies, with a limit of detection of 10
pg (VEGF) versus 500 pg in the case of ADAMTS1 antibodies (data not
shown).
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VEGF165 Binds to the CT Region of
ADAMTS1—Previous results indicated that the interaction of
VEGF165 to ADAMTS1 requires the two last TSP repeats and at least
part of the spacer region. To test this possibility, we generated a deletion
construct consisting of the CT of ADAMTS1 from the 1st TSP repeat to the last
residue (including the spacer region) (Fig.
8A). Fig.
8B shows the relative expression levels of several
clones, which showed a truncated protein of 50 kDa. We found that the
protein bound avidly to the cell surface of expressing cells and was only
released when in the presence of heparin
(Fig. 8B). As
anticipated, the CT region of ADAMTS1 binds to VEGF165, resulting
in a 90-kDa cross-linked complex (Fig.
8C). Purification of significant levels of the CT region
for evaluation on angiogenesis has been hindered due to its avidity to bind
matrix proteins. However we have evaluated the function of this domain in
phosphorylation assays and found significant decrease of VEGFR2
phosphorylation on VEGF165-stimulated PAE-VEGFR2 in the presence of
the CT-truncated protein as well as the full-length of ADAMTS1
(Fig. 8D).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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VEGF165 is one of the most specific mediators of tumor angiogenesis (46–48). The importance of the growth factor and its receptor VEGFR2 to pathophysiological states has been exemplified by studies using dominant-negative VEGFR2 (48), VEGFR2 kinase inhibitors (49), and neutralizing VEGF and VEGFR2 antibodies (47, 50, 51). In all cases, suppression of VEGF signaling resulted in inhibition of angiogenesis and concomitant reduction of tumor burden. Moreover, overexpression of VEGF and VEGFR2 is associated with invasion and metastasis of human cancers (52, 53), further demonstrating the impact of this growth factor in tumor biology.
As a paracrine mediator, one of the rate-limiting steps in VEGF signaling relates to its ability to reach the target cell. The rate of diffusion of VEGF is greatly impacted by the presence of a heparin-binding domain in its carboxyl-terminal. Splice variants of VEGF that lack this domain, such as VEGF121, have been shown to interact poorly with the extracellular environment and have a greater diffusion rate (54). Thus, the balance between the selective anchorage of VEGF by extracellular matrix proteins and its programmed release by matrix metalloproteases regulates the level of growth factor bioavailability and function. Consequently, alterations in either matrix-binding proteins or secretion of proteases have been shown to directly impact VEGF function (23, 55).
Our results demonstrate that ADAMTS1 binds to VEGF165, as indicated by cross-linking and co-immunoprecipitation studies and that this binding prevents the association of this growth factor with its receptor, affecting VEGFR2 phosphorylation and endothelial cell proliferation. The interaction of ADAMTS1 with VEGF165 occurs under culture conditions and is maintained through purification procedures. VEGF165 was detected in purified samples of ADAMTS1 after Zn2+-chelation chromatography, but not in control samples subjected to the same purification scheme. ADAMTS1 protein purified in this manner was still quite effective in the suppression of angiogenesis, as previously demonstrated (18, 21). Our data indicate that VEGF is present in purified ADAMTS1 from 293T-conditioned media. The levels of VEGF are not equimolar to ADAMTS1, and addition of exogenous VEGF results in more binding and inactivation of the growth factor. Also, we were unable to detect VEGF using silver staining of purified ADAMTS1 preparations. Consequently, ADAMTS1 is likely more potent than previously anticipated (18). Evaluation of the ADAMTS1-VEGF165 complex in VEGFR2 phosphorylation assays indicated no activity, demonstrating that once bound, VEGF165 is unable to activate its receptor. Using gel-filtration chromatography under denaturing conditions we have been able to separate both proteins and determine that the resulting VEGF165 is active, demonstrating that the reversibility of this interaction has the potential to impact biological processes.
Interestingly, we found that ADAMTS1 did not bind to VEGF121, indicating that the interaction requires the heparinbinding domain. Kuno et al. (20, 56) have found that ADAMTS1 binds to the extracellular matrix through the spacer/cysteine-rich region. We also found that the association of ADAMTS1 with cell surfaces and matrix can be blocked/competed by heparin (21). The cross-linking results suggest that heparin might be acting as a bridge between ADAMTS1 and VEGF. The function of heparin as a chaperone has been previously reported in similar protein-protein interactions (4, 57). In vivo, however, the interaction of ADAMTS1 to VEGF is not dependent on exogenous addition of heparin indicating that is either not necessary or that another heparin-like molecule, such a heparan-sulfate proteoglycan i.e. syndecan, could be the physiological mediator of this interaction.
The results presented in this study argue that the CT region of ADAMTS1 is responsible, at least in part, for the anti-angiogenic properties displayed by this molecule. Interestingly, we have previously reported that this protein is processed extracellularly resulting in the release of the CT end (21); this processing does not affect the catalytic function of the protease. Thus, ADAMTS1 processing might release two fragments with distinct/independent functions in the extracellular milieu and add to the list of extracellular matrix molecules whose functional features are multiplied by processing events. The contribution of the catalytic domain to the angio-inhibitory properties of ADAMTS1 is the subject of continuing work and is likely to be additive to the vascular inhibitory properties displayed by the carboxyl-terminal domain that we described here.
At this point, it is unclear whether the CT region in ADAMTS1 binds and
modulate other growth factors. Thrombospondin1 has been shown to bind to
several growth factors contributing to the regulation of many biological
processes (22,
58–60).
Although the murine ADAMTS1 protein has the RFK tripeptide known to activate
TGF-
in TSP1, the human ADAMTS1 homolog does not show conservation of
this activating motif. However, based on extensive flanking sequence homology
it is likely ADAMTS1 binds to TGF-, a possibility that deserves
examination.
The present study has extended and provided clarification to the mechanism by which ADAMTS1 acts to suppress angiogenesis. However, the overall contribution of ADAMTS1 as a physiological endogenous inhibitor remains to be fully clarified. Our previous results have indicated that added exogenously, ADAMTS1 is capable of blocking neovascularization in vivo and suppressing endothelial cell proliferation in vitro more effectively than recognized inhibitors, such as TSP1 and endostatin. These findings have to be reconciled, however, with the fact that loss of function mutations of ADAMTS1 in mice results in pathological states not directly linked to the vascular compartment (61). Although an extensive evaluation of the null animal with focused attention in angiogenic states is currently missing, it may not be surprising that ADAMTS1 functions in a multiplicity of biological scenarios with either partially overlapping or entirely independent functions. This has been the case for other extracellular matrix proteins and proteases, including TSP1 and TSP2, both recognized physiological inhibitors of neovascularization (62, 63). Studies that aim to determine the mechanism of action of ADAMTS1 and/or the TSP repeats are likely to enhance our understanding of the biology of angiogenesis and provide a platform for the development of drugs for pharmacological intervention in situations of unwanted neovascular growth.
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FOOTNOTES |
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Recipient of a postdoctoral fellowship from the Department of Defense (DOD)
Congressionally Directed Medical Research Programs (CDMRP) (DOD Breast Cancer
Research Program, DAMD17-02-1-0329).
Recipient of a scholarship from the Giannini Family Foundation.
¶ To whom correspondence should be addressed: Molecular Biology Institute, 611 Charles Young Drive East, Los Angeles, CA 90095. Tel.: 310-794-5763; Fax: 310-794-5766; E-mail: arispe{at}mbi.ucla.edu.
1 The abbreviations used are: VEGF, vascular endothelial growth factor;
ADAMTS1, a disintegrin and metalloproteinase with thrombospondin motifs; BAEC,
bovine aortic endothelial cells; BSA, bovine serum albumin; CT,
carboxyl-terminal region of ADAMTS1; DSS, decanesulfonic acid sodium salt;
HAEC, human aortic endothelial cells; HPLC, high pressure liquid
chromatography; mAb, monoclonal antibody; METH1, methalloprotease and
thrombospondin; Met, metalloproteinase construct; Met-Dis, metalloproteinase
and disintegrin-truncated construct; MMP, matrix metalloproteinase; PAE,
porcine aortic endothelial cells; TGF-, transforming growth factor
; TSP, thrombospondin; VEGFR, VEGF receptor; FGF, fibroblast growth
factor; ELISA, enzyme-linked immunosorbent assay.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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