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Originally published In Press as doi:10.1074/jbc.M302946200 on July 16, 2003

J. Biol. Chem., Vol. 278, Issue 39, 37849-37857, September 26, 2003
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Cleavage of the Matricellular Protein SPARC by Matrix Metalloproteinase 3 Produces Polypeptides That Influence Angiogenesis*

E. Helene Sage {ddagger} §, May Reed ¶, Sarah E. Funk {ddagger}, Thao Truong ¶, Melissa Steadele {ddagger}, Pauli Puolakkainen {ddagger} ||, Donald H. Maurice ** and James A. Bassuk {ddagger}{ddagger}

From the {ddagger}Department of Vascular Biology, The Hope Heart Institute, Seattle, Washington 98104, the Departments of Medicine and {ddagger}{ddagger}Urology, University of Washington, Seattle, Washington 98195, and the **Department of Pharmacology and Toxicology, Queen's University, Kingston, Ontario K7L 3N6, Canada

Received for publication, March 21, 2003 , and in revised form, June 19, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
SPARC, a matricellular protein that affects cellular adhesion and proliferation, is produced in remodeling tissue and in pathologies involving fibrosis and angiogenesis. In this study we have asked whether peptides generated from cleavage of SPARC in the extracellular milieu can regulate angiogenesis. Matrix metalloproteinase (MMP)-3, but not MMP-1 or 9, showed significant activity toward SPARC. Limited digestion of recombinant human (rhu)SPARC with purified catalytic domain of rhuMMP-3 produced three major fragments, which were sequenced after purification by HPLC. Three synthetic peptides (Z-1, Z-2, and Z-3) representing motifs from each fragment were tested in distinct assays of angiogenesis. Peptide Z-1 (3.9 kDa, containing a Cu2+-binding sequence KHGK) exhibited a biphasic effect on [3H]thymidine incorporation by cultured endothelial cells and stimulated vascular growth in the chick chorioallantoic membrane (CAM). In contrast, peptides Z-2 (6.1 kDa, containing Ca2+-binding EF hand-1) and Z-3 (2.2 kDa, containing neither Cu2+-binding motifs nor EF hands), inhibited cell proliferation in a concentration-dependent manner and exhibited no effects on vessel growth in the CAM. Reciprocal results were obtained in a migration assay in native collagen gels: peptide Z-1 was ineffective over a range of concentrations, whereas Z-2 or Z-3 stimulated cell migration. Therefore, proteolysis of SPARC by MMP-3 produced peptides that regulate endothelial cell proliferation and/or migration in vitro in a mutually exclusive manner. One of these peptides containing KHGK also demonstrated a concentration-dependent effect on angiogenesis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The matricellular proteins are a functional class of secreted proteins that mediate interactions between cells and the extracellular matrix (ECM)1 (1). Members of this group include SPARC and its homolog hevin/SC1, thrombospondin 1 and 2, tenascin C and X, osteopontin, CYR61, and potentially some of the cell surface-associated proteoglycans (2). Rather than serving primarily structural roles, these proteins act as extracellular modulators of signaling events that occur, for example, between cell-surface receptors and the ECM (1). In fact, regulation of cell adhesion, migration, proliferation, survival, and/or differentiation has been established for each of the matricellular proteins identified to date. These fundamental effects of the matricellular group have implicated most if not all of them in the morphogenesis of tissues and/or their restructuring as a result of injury or repair. Notably, SPARC, thrombospondin 1, and thrombospondin 2 have been shown to be prominent in angiogenesis (1, 3–5).

Several functions mediated by SPARC are relevant to vascular biology and angiogenesis: (a) SPARC inhibits the spreading of endothelial cells and fibroblasts; moreover, it disrupts focal adhesions, alters the distribution of actin, and enhances the permeability of endothelial monolayers (reviewed in Ref. 6); (b) SPARC regulates cell proliferation by its block of the cell cycle in mid-G1 and its interactions with platelet-derived growth factor and vascular endothelial growth factor (VEGF), which abrogate their binding to cognate receptors (7–10); (c) SPARC is released by platelets, macrophages, fibroblasts, and capillary endothelial cells at sites of renal vascular injury, invasive carcinoma, fibrosis, and wound repair (5, 6, 11); (d) SPARC regulates the production of fibronectin, laminin, plasminogen activator inhibitor-1, and certain matrix metalloproteinases (MMP) (Refs. 12 and 13; reviewed in Ref. 6), as well as the activation of MT-MMP1 (14), in vitro. The production of SPARC by nascent vessels, its stimulation of MMPs, its interaction with VEGF, and its regulation of proteins associated with cell-ECM interactions, support a role for SPARC in the growth and/or regression of blood vessels.

An interesting corollary to the involvement of matricellular proteins in biological processes such as angiogenesis is the release of bioactive peptides from these domain-rich proteins by endogenous tissue proteolysis (15). In the case of SPARC, synthetic peptides representing motifs in each of the three domains have been used to map many of the functions described above (16, 17). Interestingly, two relatively short sequences that bind Cu2+ with high affinity affected endothelial cell proliferation, and one of them, KGHK, stimulated angiogenesis in vivo (18). Moreover, KGHK/Cu2+ was shown to be released from native SPARC by plasmin (17), and peptide(s) containing this sequence were identified as natural cleavage products in the chick chorioallantoic membrane (CAM) model of angiogenesis (18).

In the present study we have asked whether certain members of the MMP family with established activities in angiogenesis could cleave SPARC into fragments with sequence motifs that could regulate one or more aspects of endothelial cell function, e.g. in a CAM assay to quantify vessel growth, the radial invasion of matrix by aggregated cells (RIMAC) assay, which measures migration of cells in native collagen gels, and an assay of DNA synthesis in vitro. We have shown that MMP-3 (stromelysin 1) cleaves SPARC into three major fragments, the sequencing of which allowed us to synthesize peptides with motifs previously identified as modulators of angiogenesis, as well as some additional ones, in an extended sequence context. The penultimate Cu2+-binding sequence was found to be an effective stimulator of the endothelial cell cycle and of angiogenesis in vivo. In contrast, a peptide comprising the Ca2+-binding EF hand-1 inhibited endothelial cell proliferation but stimulated migration in collagen gels. A third peptide, lacking both Cu2+- and Ca2+-binding motifs, also stimulated migration but was substantially less effective as a cell-cycle inhibitor. Thus, we have shown that MMP-3 releases peptides from SPARC that contain sequence motifs with mutually exclusive effects, within a given range of concentration, on endothelial cell migration and proliferation. Additionally, a peptide containing the Cu2+-binding sequence KHGK exhibited a concentration-dependent effect on angiogenesis in vivo.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Recombinant Proteins—Recombinant human (rhu) SPARC with a C-terminal hexahistidine tag (SPARC-His) was prepared as described (19, 20), with the following modifications. An 8 M urea extract of bacterial paste derived from 25 liters of a fed-batch fermentation (21) was applied to a 40-cm3 column of nickel-nitriloacetic acid resin (Ni-NTA, Qiagen, Valencia, CA). Endogenous bacterial proteins were removed by washing with pH 6.0 buffer. rhuSPARC-His that was immobilized onto the Ni-NTA resin was renatured at pH 8.5 by a reverse gradient from 8 to 0 M urea in 0.1 M NaH2PO4, 0.05 M Tris-HCl, 0.5 M NaCl, 5 mM oxidized glutathione, 1 mM reduced glutathione, 0.5% octylthioglucoside. rhuSPARC-His was eluted at pH 4.5, dialyzed against 0.1 N acetic acid, and lyophilized.

The catalytic domain of human proMMP-3, lacking the N-terminal 34 amino acids and the C-terminal hemopexin-like domain ({Delta}248–460), was expressed in Escherichia coli, refolded from inclusion bodies, purified as described previously (22), and provided as a gift from Dr. Hideaki Nagase (Imperial College, London, UK).

Cleavage of rhuSPARC-His with Active Form MMP-3 ({Delta}248–460)—1–5 mg rhuSPARC-His was digested with active form MMP-3 in 0.05 M Tris-HCl (pH 7.5), 0.15 M NaCl, 0.01 M CaCl2 for 16 h at 37 °C. Optimal digestion conditions were obtained at a 1:100 (enzyme:substrate) ratio by weight. Digestion was terminated by the addition of EDTA to 0.01 M. Following the addition of 3 volumes of 0.1% trifluoroacetic acid, the digestion mixture was injected onto a Vydac C4 reverse-phase HPLC column that had been repeatedly conditioned by the process described below. Development of the column was achieved at 1 ml/min by linear gradients from 0.1% trifluoroacetic acid (A) to n-propanol/0.1% trifluoroacetic acid (B), according to the following schedule: 0% B, 10 min; 0–15% B, 15 min; 15–60% B, 100 min; 60–100% B, 10 min; 100–0% B, 20 min. Fractions were dried by vacuum centrifugation and were stored at –80 °C.

Amino Acid Sequencing of rhuSPARC-His Fragments—Cleavage products of rhuSPARC-His were fractionated on 16.5% polyacrylamide electrophoretic gels that contained 0.1% SDS and were transferred to a polyvinyldenefluoride membrane in 10 mM morpholinosulfonic acid (pH 11.0) that contained 20% methanol by volume, for 30 min at 0.3 A. Transferred peptides were visualized by staining with Coomassie Brilliant Blue R-250, excised from the membrane, and subjected to N-terminal Edman degradation and sequencing on an Applied Biosystems 477A protein sequencer.

Synthetic Peptides—Peptides were synthesized by Zymogenetics, Inc. (Seattle, WA) and were purified by reverse-phase HPLC. Concentration and purity were determined by amino acid analysis. In all cases, peptides exhibited >90% purity, with no contaminating (unexpected) residues present. The sequences, molecular weights, and pIs of these peptides, designated Z-1, Z-2, and Z-3, are shown in Table I.


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  		TABLE I
Synthetic peptides representing MMP-3 cleavage products of human SPARC

 

Proliferation and Migration Assays—BAE cells were isolated and subcultured as previously described (7). Cell proliferation was measured by the incorporation of [3H]thymidine (10 mCi/ml, PerkinElmer Life Sciences, Boston, MA) according to (7). Confluent cells were released by incubation in trypsin, and were resuspended in Dulbecco's modified Eagle's medium (DMEM) containing 2% fetal calf serum at 5000 cells/ml. The cells were plated in triplicate (0.5 ml/well, 2 cm2), and peptides were added immediately at concentrations ranging from 0.05–1.0 mM. After 18 h, 2 µCi/ml [3H]thymidine was added to each well for an additional 2 h. After removal of the media, the cell layers were rinsed twice with ice-cold 5% trichloroacetic acid and were solubilized by shaking in 0.5 ml of 0.4 M NaOH. Incorporated cpm were measured by scintillation counting. Each experiment was performed a minimum of three times.

Endothelial migration and cord morphogenesis were assessed by the RIMAC assay (23). Aggregates of BAE or porcine aortic endothelial (PAE) cells (a gift of J. Waltenberger, Uppsala, Sweden) were placed in native type I collagen gels (0.5 mg/ml; BD Biosciences, Bedford, MA), in the presence or absence of fibroblast growth factor (FGF)-2 (20 ng/ml, Scios, Mountain View, CA). DMEM was used as control buffer and as diluent for the peptides. Assays were conducted in quadruplicate, and experiments were performed a minimum of three times.

Angiogenesis Assay—Quail CAM assays were performed and quantified as described in Parsons-Wingerter et al. (24). Peptides Z-1, Z-2, and Z-3, each at concentrations of 0.1 and 1.0 mM in a total volume of 500 µl of PBS, were applied to CAMs cultured in 6-well tissue culture plates. Control CAMs received an equal volume of PBS alone. After 24 h, images were photographed digitally at 10x, and arterial vessel growth was quantified by calculation of the fractal dimension (Df) of skeletonized binary images (24).

Digestion with MMPs—Purified rhuSPARC (20 µg) was iodinated with IodobeadsTM (Pierce, Rockford, IL) as previously described (10). 125I-SPARC (100 µl) was concentrated at 5,000 x g for 5 min in a Centricon-30 tube (Amicon, Beverly, MA). A volume of retentate equivalent to 3–6 x 105 cpm (~100 ng) was incubated with huMMP-1 (30 ng/µl, 5–10 µl), rhuMMP-3 (0.4 µg/ml, 0.5–5 µl), or huMMP-9 (10 ng/µl, 5–10 µl) in buffer A (50 mM Tris-HCl, 150 mM NaCl, 1 mM CaCl2, pH 7.5), in a total volume of 50 µl. All MMPs were a gift from Dr. H. Nagase. Samples were incubated 16–18 h at 37 °C, after which they were diluted 1:1 (by volume) in SDS-PAGE sample buffer (25) containing 50 mM dithiothreitol and were boiled for 5 min. The resulting digests were resolved on 16.5% polyacrylamide tricine gels (BioRad, Hercules, CA), and proteins/peptides were visualized by autoradiography. rhuTIMP-1 (5 µl, 0.5 µg/µl) (a gift from Dr. H. Nagase) was added to some reaction mixtures prior to incubation (16–18 h) at 37 °C. Some digests of SPARC, after resolution by SDS-PAGE, were electroblotted onto nitrocellulose membranes and were subsequently exposed to guinea pig anti-SPARC peptide 2.3 IgG (5 µg/ml). For production of this antibody, 8 copies of murine (m) SPARC peptide 2.3 (Thr113–Glu129) were synthesized on a branched Lys multiple antigen peptide core (Biosynthesis, Inc., Lewisville, TX). The resulting macromolecule, which has a high molar ratio of peptide to core molecule, is highly antigenic and requires no carrier protein to elicit an antibody response (26). The antiserum was highly specific for peptide 2.3 and did not react with SPARC by ELISA. After exposure to 125I-protein A (PerkinElmer Life Sciences/Dupont, Burbank, CA), the blots were exposed to x-ray film at –70 °C.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Published studies from several laboratories have identified a number of different functions of SPARC, many of which are related to de-adhesion and anti-proliferation of cultured cells (4–6). The use of synthetic peptides representing domain-specific sequences of SPARC has confirmed some of the functional properties attributed to SPARC, and has identified smaller regions or subdomains and their sequences to which certain activities of SPARC could be assigned. However, most of the synthetic peptides were chosen arbitrarily, e.g. to isolate a Ca2+ binding region or a sequence with a purportedly stabilized secondary structure, and prior to the crystallographic studies performed on domains II and III (16, 27). Having identified peptides of SPARC with specific activities on several types of cells in vitro, we questioned whether one or more of these sequences were present in vivo, and whether natural proteolysis of SPARC could occur as a result of tissue injury. In this study we have therefore asked the following questions: (a) Are there peptides generated from the cleavage of SPARC by certain MMPs that influence the process of angiogenesis, and (b) are the previously identified short sequence motifs of SPARC active in the context of larger peptides?

Cleavage of SPARC by MMP-3—In Fig. 1A is shown an incubation of 125I-SPARC individually with three different MMPs (1, 3, and 9) that are relevant to angiogenesis. MMP-3, but not MMP-1 or 9, exhibited reproducible catalytic activity toward SPARC. The cleavage of SPARC by MMP-3 was concentration-dependent (Fig. 1B, lanes 2–5) and was inhibited by TIMP-1 (Fig. 1B, lane 6).



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  		FIG. 1.
Cleavage of SPARC by MMP-3, but not by MMP-1 or MMP-9. A, iodinated rhuSPARC (~100 ng) was incubated 16 h at 37 °C in buffer alone (lane 1) and with 150 ng of MMP-1 (lane 2), 400 ng of MMP-3 (lane 3), or 50 ng of MMP-9 (lane 4). Digests were treated with 50 mM dithiothreitol and were resolved by SDS-PAGE on 16.5% gels; cleavage products were visualized by autoradiography. Lanes are derived from the same autoradiogram. B, iodinated rhuSPARC was incubated 16 h at 37 °C in buffer alone (lane 1), and with 0.2 µg (lane 2), 0.4 µg(lane 3), 1.2 µg(lane 4), and 2 µg(lane 5) of rhuMMP-3, respectively. Lane 6, rhuSPARC incubated with 0.4 µg MMP-3 and 2.5 µg rhuTIMP-1. Similar levels of inhibition were noted with 1 µg of TIMP-1. Arrowheads denote bands of approx. Mr 33,000 and 14,000.

 

Electroblotting of an MMP-3 digest of SPARC onto a nitrocellulose filter, followed by exposure to an antibody recognizing hSPARC peptide 2.3 (Thr114–Gly130), revealed a band of approximate Mr 14,000 (not shown). Interestingly, peptide 2.3 contains the sequence KKGHK, which has a high affinity for Cu2+ and is angiogenic in vivo and in vitro (17, 18, 28). Incubation of peptide 2.3 with MMP-3 resulted in multiple and extensive cleavages, predominantly at Lys123, Leu124, and His125–Leu126, as detected by tandem mass spectrometry (not shown). These data indicate that this sequence is protected within the native structure of SPARC, a conclusion borne out by crystallographic data (27) and cell proliferation assays (29). In summary, MMP-3, but not MMP-1 or 9, displayed consistent proteolytic activity toward SPARC. One of the fragments released from SPARC was of approximate Mr 14,000 and contained a Cu2+-binding sequence with reported angiogenic activity.

For the purpose of sequencing major cleavage fragments of SPARC produced by MMP-3, rhuSPARC was subjected to digestion under conditions that we had previously determined as optimal for this study: an enzyme-to-substrate ratio of 1:100 (by weight) for 16 h at 37 °C, in a buffer containing 10 mM Ca2+ for maintenance of native structure. Peptides were fractionated by HPLC on a C4 column, as shown in Fig. 2A, and peaks J1 and J2+J3 were further resolved by SDS-PAGE (Fig. 2, inset), prior to electroblotting and Edman degradation. The peptide sequences obtained from this procedure are shown in Fig. 2B, and their size distributions are summarized in Table II. Peptide J1 (Mr 9,300) covered the N-terminal-third of the protein (Gln2–Pro87), whereas peptide J2 (Mr 17,700) extended from Ile88 to Pro237, with the remainder of the protein (49 amino acids) cleaved into smaller fragments. Importantly, EF hand-2 (Asp257–Glu268) is absent from peptide J2, although J2 comprises much of the C-terminal EC domain of SPARC. In addition, it contains the Cu2+-binding sequence KKGHK (Lys119–Lys123) and EF hand-1 (Asp165–Lys176). Sequencing of peptide J3 (Mr 10,600, Met150–Pro237) revealed it to be derived from J2 (Fig. 2, A and B), which also contains EF hand-1 but lacks KKGHK. The net result of the limited digestion of rhuSPARC by MMP-3 is: (a) liberation of fragment J1 (Table II and Fig. 2A), (b) production of fragment J2-J3 (Table II and Fig. 2B), and (c) subsequent release of fragment J3 from J2-J3 (Table II and Fig. 2B).



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  		FIG. 2.
Purification and sequences of SPARC peptides produced by limited proteolysis by MMP-3. Purified rhuSPARC-His was digested with active form MMP-3 ({Delta}248–460) at a 1:100 enzyme to substrate ratio (by weight) for 16 h at 37 °C. A, peptides were fractionated on a C4 column by reverse-phase HPLC by development with a linear gradient as described under "Experimental Procedures." Peaks containing peptides J1 and J2+J3 are identified. Inset, Coomassie Blue-stained polyvinyldenefluoride membrane from 16.5% Tris-Tricine polyacrylamide electrophoretic gels containing 0.1% SDS that show J1 (lane 1, faint band at 9 kDa), J2 (lane 2, 18 kDa; lane 5 from separate HPLC fractionation), and J3 (lane 3, 11 kDa). In left margin are relative size markers in kDa. B, sequence analysis of peptide J2 (upper panel) and J3 (lower panel). Cleavage sites are indicated in bold type. EF hands-1 and -2 are underlined. Note that bacterial rhuSPARC lacks one amino acid (the N-terminal Ala) and is therefore shown as a sequence of 285 residues, rather than the 286 residues that comprise human SPARC.

 

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  		TABLE II
Size distribution of MMP-3-generated rhuSPARC-His fragments

 

Fig. 3 is a model of SPARC depicting the major fragments produced by the proteolytic cleavage of SPARC by MMP-3. Two domains of SPARC, as defined by Hohenester et al. (27), are shown in blue (follistatin [FS] domain II) and in green (extracellular Ca2+-binding [EC] domain III). The N-terminal domain I (approximately the initial 50 amino acids) is not shown. Peptide J2 (shown in yellow in Fig. 3B) encompasses portions of both domains II and III, whereas peptide J3 (shown in yellow in Fig. 3C) is limited to domain III. Both, however, contain EF hand-1 (identified by Ca2+ as orange spheres) (Fig. 3, B and C).



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  		FIG. 3.
Major fragments produced by proteolytic cleavage of SPARC by MMP-3. Shown are molecular models derived from A chain coordinates of Protein Data Bank Accession 1BMO [PDB] (27). A, domain I (the structure of which is unsolved) is linked to the follistatin domain II (FS, blue), which is linked to the extracellular calcium-binding domain III (EC, green). His6-COOH designates the covalent linkage of a histidine hexamer to the C terminus of the recombinant protein (19). Calcium ions are designated by orange spheres. Incubation of rhuSPARC-His with active MMP-3 ({Delta}248–460) results in cleavage between Pro87–Ile88 and Pro237–Leu238 to yield the Mr 17,700 fragment J2 (yellow structure in panel B). Further proteolysis of J2 by cleavage between Arg149–Met150 creates the Mr 10,600 fragment J3 (yellow structure in panel C). Sequences are shown in Fig. 2B. NAG, carbohydrate post-translational modification that is present in mammalian SPARC.

 

SPARC Peptide Z-1 Exhibits a Biphasic Effect on Angiogenesis—From the data shown in Figs. 2 and 3, we identified novel sequence motifs, or previously described motifs within extended sequences, as candidates for the production of synthetic peptides, termed Z-1, Z-2, and Z-3, as described in Table I. These peptides provided several advantages for assays of angiogenesis in vivo and in vitro: (a) a high degree of purity with no detectable endotoxin, (b) a molecular size and solubility properties advantageous for concentration-dependent assay in the CAM and RIMAC settings, and (c) copious amounts of standardized preparation that allowed for multiple experiments.

HPLC-purified peptides Z-1–Z-3 correspond to subfragments of 3 major MMP-3 cleavage products of rhuSPARC (Tables I and II). Z-1 exhibited a stimulatory effect in the quail CAM assay (Fig. 4), with an increase in vessel density at a concentration of 0.1 mM, and an inhibitory effect (return to basal state) at 1.0 mM (Fig. 4, B and C, respectively). Peptide Z-1 contains the Cu2+ binding sequence KHGK (Table I and Fig. 2B), for which a nearly identical effect on BAE cell proliferation was previously reported (7, 29). In contrast, peptides Z-2 and Z-3 displayed minimal effects on vascular density in the CAM that were found not to be statistically significant (data not shown).



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  		FIG. 4.
SPARC peptide Z-1 inhibits angiogenesis in the CAM. Quail CAMs (n = 5 per sample) were incubated with PBS (A) or SPARC peptide Z-1, at concentrations of 0.1 mM (B), and 1.0 mM (C) in PBS, for 48 h, as described under "Experimental Procedures." After fixation and image capture, patterns representing the arterial tree were binarized and skeletonized, prior to quantification of vascular growth by fractal dimension (D). Scale bars in A–C = 1 mm.

 

Biphasic and Inhibitory Effects of SPARC Peptides on Endothelial Cell Proliferation—Standard [3H]thymidine incorporation assays were performed with BAE cells in the presence of different concentrations of peptides Z-1, Z-2, and Z-3, as shown in Fig. 5. Peptide Z-1 exhibited a biphasic effect on thymidine incorporation, with a 4-fold stimulation at a concentration of 0.2 mM (Fig. 5A). This result parallels the effect seen with peptide Z-1 in the CAM assay (Fig. 4). In contrast, both peptides Z-2 and Z-3 demonstrated only inhibitory activities in the BAE cell proliferation assay (Fig. 5B). Peptide Z-2 exhibited an ED50 at slightly less than 0.2 mM, whereas peptide Z-3 was considerably less effective in this assay, with an ED50 {approx} 0.5 mM (Fig. 5B). Since a 20-mer peptide containing a partial EF hand-1 sequence has previously shown minimal activity in proliferation assays, it is likely that the extended sequence of peptide Z-2, a 50-mer, has conferred additional stability on this nondisulfide-bonded Ca2+-binding sequence. Peptide Z-3, which lacks both EF hands, showed diminished activity in the thymidine incorporation assay, relative to Z-1 and Z-2 (Fig. 5B).



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  		FIG. 5.
Biphasic and inhibitory effects of SPARC peptides on endothelial cell proliferation. BAE cells were incubated with a range of concentrations of SPARC peptides Z-1, Z-2, and Z-3 for 24 h. DNA synthesis was measured by incorporation of [3H]thymidine into cellular DNA. Results are reported as percent of control incorporation by cells treated with buffer alone, and are the means of triplicate values + S.E., from one of three representative experiments. A, peptide Z-1 (filled squares); B, peptides Z-2 (open squares), and Z-3 (filled triangles).

 

SPARC Peptides Z-2 and Z-3 Enhance Endothelial Cell Migration through Native Collagen Gels—Aggregates of PAE cells were embedded in collagen gels, with different concentrations of peptides Z-1, Z-2, and Z-3 (singly or in combination), FGF-2, or buffer alone, and analyzed for distance of radial migration, as described for the RIMAC assay (23). In contrast to their null and inhibitory effects in the CAM and thymidine incorporation assays (Figs. 4 and 5, respectively), peptides Z-2 and Z-3 stimulated PAE cell migration in the RIMAC assay, whereas peptide Z-1, which exhibited biphasic effects in the CAM and proliferation assays, was ineffective (Fig. 6). At concentrations of 0.2 mM, peptides Z-2 and Z-3 stimulated PAE cell migration to essentially the same extent as seen with FGF-2 at 20 ng/ml (Fig. 6). None of the peptides was able to block the stimulatory effect of FGF-2 over a range of concentrations, and combinations of Z-2 or Z-3 (0.05–0.4 mM) with FGF-2 (20 ng/ml) were not synergistic (data not shown). Similar results were obtained with BAE cells (not shown).



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  		FIG. 6.
Effects of SPARC peptides on endothelial cell migration through native collagen gels. Aggregates of PAE cells were embedded within native collagen I gels, as described for the RIMAC assay under "Experimental Procedures." Quantification of cell migration was performed on triplicate cultures incubated with DMEM alone, peptides Z-1, Z-2, or Z-3 at a concentration of 0.2 mM in DMEM, or 20 ng/ml FGF-2. Values are plotted as percent of radial migration exhibited by cells in DMEM alone and represent means + S.E.

 

A summary of the activities of peptides Z-1, Z-2, and Z-3, and their location within intact SPARC and within the peptides (J1, J2, and J3) derived from the proteolysis of SPARC by MMP-3, are shown in Fig. 7.



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  		FIG. 7.
Summary of locations, characteristics, and activities of SPARC peptides. A, diagram of huSPARC showing domains I-III, with two basic Cu2+-binding sequences depicted in II and two Ca2+-binding EF hands (1 and 2) in III. Peptides J1-J3, major products produced by cleavage of rhuSPARC with MMP-3, are shown in black. Synthetic peptides Z-1 to Z-3 are shown in gray. Amino acid residues are numbered. B, activities of SPARC peptides Z-1, Z-2, and Z-3 in vivo and in vitro.

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study we have shown that the matricellular protein SPARC can be cleaved in a reproducible, concentration-dependent manner by MMP-3, a reactivity that was inhibited by TIMP-1. Limited digestion of rhuSPARC with MMP-3 produced three major peptides (J1–J3) containing sequence motifs that influenced endothelial cell behavior and angiogenesis: Cu2+-binding KHGK, and Ca2+-binding EF hand-1. To facilitate study of these motifs on cultured endothelial cells and on CAMs, we synthesized shorter versions of J2 and J3 (termed Z-2 and Z-3) and tested them over a range of concentrations in assays of endothelial cell proliferation and migration, as well as angiogenesis. Peptide Z-1, containing the motif KHGK/Cu2+, exhibited a biphasic effect on [3H]thymidine incorporation by endothelial cells in vitro and on angiogenesis, but did not affect the migration of endothelial cells through native collagen gels. In contrast, peptide Z-2, containing EF hand-1, and Z-3, lacking both Cu2+- and Ca2+-binding motifs, exhibited inhibitory effects on proliferation but stimulated migration to levels seen with 20 ng/ml FGF-2. Peptides Z-2 and Z-3 were inactive in the CAM assay of developmental angiogenesis. Therefore, discrete motifs within major MMP-3-derived peptides of SPARC influenced endothelial cell proliferation, migration, and angiogenesis in a mutually exclusive manner.

Proteolytic fragments of several components of the ECM, including collagen types XVIII (endostatin), IV (tumstatin, canstatin, arresten), XV (restin), and VIII (vastatin), as well as the laminin {gamma}1 chain, have been shown to regulate angiogenesis (15, 30, 31). In addition, fragments from molecules as diverse as calreticulin (vasostatin) (32), high molecular weight kininogen (kininostatin) (33), platelet factor 4, plasminogen (angiostatin), and prolactin have demonstrated activity on endothelial cells in vitro and on angiogenesis in vivo (reviewed in Ref. 15). Several of the matricellular proteins, which are characteristically expressed in areas of vascular remodeling and tissue repair, give rise to peptides that exhibit angiostimulatory or angioinhibitory activities in vitro and in vivo, e.g. osteopontin, thrombospondin 1, and SPARC (1, 15). One of the more remarkable examples is the plasmin-mediated release of the proangiogenic Cu2+-binding peptide KGHK from SPARC, which stimulated endothelial cell proliferation and cord formation in vitro and vessel growth in the CAM (17, 18). It is important to note that some of the fragment(s) exhibit activities that differ from those of the parent protein, e.g. KGHK versus SPARC.

Limited digestion of SPARC with MMP-3 produced initially a cleavage at Pro87–Ile88, giving rise to peptides J1 and J2, as well as proteolysis of several peptide bonds at the C terminus of SPARC (beginning at Pro237–Leu238) (Fig. 2). These latter small peptides were not recovered and comprise ~17% of the total sequence. Thus EF hand-2 (Asp257–Glu268), previously characterized as peptide 4.2 (mThr254–Gly273) that mimics most of the functions of SPARC (e.g. de-adhesion, cell cycle inhibition, dissolution of focal contacts) (34, 35), was not a part of this study. Further limited proteolysis of SPARC by MMP-3 resulted in the identification of a scissile bond in J2 at Arg149-Met150, and gave rise to peptide J3 containing EF hand-1 and the same C-terminal amino acid (Pro 237) as J2 (Fig. 2B). EF hand-1 (Asp165–Lys176) binds Ca2+ but differs from the canonical EF hand-2 (see below). Previously studied as synthetic peptide 3.2 (mLys154–Lys173), this sequence exhibited essentially no effect on focal adhesion dissolution (35). In the context of a larger synthetic peptide (termed Z-2 in this study, Mr 6,100), the EF hand-1 motif was also weakly inhibitory with respect to thymidine incorporation but stimulated endothelial cell migration in native collagen gels (Figs. 5 and 6). Although the molecular basis for this effect is not known, enhancement of cell migration within collagen is a newly identified activity for this sequence motif in SPARC.

Peptide J1 (Glu2–Pro87) (Fig. 7) comprises all of SPARC domain I, an acidic, low affinity Ca2+-binding region thought to interact with mineralized components of bone. Domain I (amino acids 1–52) is poorly conserved within the SPARC family. Because several studies describing functions of this region have been published (reviewed in Ref. 6), we focused on the C-terminal sequence of J1 extending into the FS domain (II) and reexamined the Cu2+-binding motif KHGK, in the context of the synthetic peptide Z-1 (Mr 3,900) (Fig. 7). The sequence KHGK was previously studied as a 20-mer peptide termed 2.1, and as a 10-mer, 2.1a (29). Both exhibited a biphasic effect on the incorporation of [3H]thymidine by BAE cells, with stimulation between 0.025 mM and 0.2 mM and inhibition at concentrations in excess of 0.4 mM (29). In the present study, using the extended sequence Z-1, we also found a biphasic effect on thymidine incorporation by BAE cells (Fig. 5), which was ~2-fold at 0.05 mM and maximal at 0.2 mM, as well as a stimulation of vascular growth in the CAM (at 0.1 mM), followed by a return to baseline at 1.0 mM (Fig. 4). Of interest was the lack of effect of a range of concentrations of peptide Z-1 on endothelial cell migration in collagen gels (Fig. 6). Thus, the effects of the high-affinity Cu2+- and Ca2+-binding motifs of SPARC on endothelial cell adhesion and proliferation appear to be mutually exclusive.

Previous experience with the use of peptides in vitro has demonstrated the necessity of testing a broad range of concentrations under a variety of culture conditions, for confirmation of biological activity (29, 36). In earlier studies we had shown a stimulatory effect of SPARC peptide 2.1 on DNA synthesis on three different types of endothelial cells, over a concentration range of 0.025–0.2 mM, as well as an inhibitory effect at concentrations in excess of 0.4 mM (29). Moreover, a maximal effect on the dissolution of focal adhesions in BAE cells was observed at 0.2 mM peptide 2.1 (35). Our present study included an extended sequence of the 10-mer 2.1a (containing the active residues KHGK) that was denoted peptide Z-1 (Table I). Interestingly, Z-1 exhibited similar concentration-dependent effects on BAE cell proliferation, data indicating that KHGK-Cu2+ is active within extended flanking regions of amino acids within the FS domain of SPARC (Fig. 7). KHGK (as peptide 2.1) has been shown either to mimic or to synergize with the EF hand-2 sequence of SPARC in many of the activities observed in vitro: binding to collagen and endothelial cells, focal adhesion dissolution, antiproliferation, and regulation of several proteins associated with G1 -> S progression (10, 34, 35, 37). These similar or synergistic effects between the two sequences are consistent with their location in the three-dimensional structure of SPARC (27, 38), a factor that could influence the effective concentrations of peptide required for bioactivity in different assays. The concentrations of peptides Z-1 to Z-3 used in this study are similar to those used recently on vascular cells for fragments of two proteins: the laminin {gamma}1 chain (a 12-mer at 0.1 mM) (39) and the type III Ca2+-binding repeat of thrombospondin 1 containing RGD (0.1 mM) (40), but are considerably less effective than a 17-mer derived from kininostatin, which inhibited endothelial cell migration to vitronectin at an IC50 of 0.2 µM (33). The study by Colman et al. (33) also identified a second sequence within kininostatin (a 16-mer) that potently inhibited proliferation but not migration, and was also inhibitory in the CAM assay, results consistent with those reported here for peptide Z-1 (Fig. 7).

The MMPs are a large family (~24 members) of structurally and functionally related zinc endopeptidases that exert decisive roles in the turnover of ECM in the context of tissue morphogenesis and remodeling (41). Several novel functions have recently been described for MMP-3 that underscore the importance of the identification of its substrates in various pathologies. For example, transgenic mice overexpressing MMP-3 in mammary tissue exhibited spontaneous development of malignant tumors that were reversed by coexpression of TIMP-1 (42). In apolipoprotein E-deficient mice (a model for atherosclerosis) that were also null for MMP-3, there was an increase in plaque size but a decrease in aneurysm formation, in comparison with MMP-3+/+ mice (43). Further evidence in vivo for the consequences of impaired degradation of ECM/fibrillar collagen by MMPs can be found in a murine model of adipose tissue development, in which inhibition of MMP activity was associated with impaired adipose accumulation that was attributed to the formation of an excessive collagen-enriched ECM (44). Since SPARC-null mice exhibit both attenuated levels of collagen and increased amounts of adipose tissue (45, 46), a condition collectively mimicking the results of Lijnen et al. (44), it becomes interesting to speculate that the absence of one or more MMPs (e.g. MMP-3) might produce a phenotype in mice similar to the lack of an MMP-3 substrate, or a regulator of an ECM substrate, e.g. SPARC. Another matricellular protein, osteopontin, has recently been identified as a substrate of MMP-3 and MMP-7 (47). In analogy to SPARC, fragments of osteopontin were identified in tumor cell lines and in remodeling tissues, and cleavage of osteopontin by MMP-3 and -7 enhanced its stimulatory effects on cell adhesion and migration (47).

Under the reaction conditions that we used, we did not detect cleavage of native mSPARC or refolded, rhuSPARC by MMP-1 or -9, consistent with our previous work (16, 48) but different from reports claiming proteolysis of mSPARC (termed BM-40 in these studies) by MMP-13 (murine homolog of human MMP-1) (49) and of rhuSPARC by MMP-2, 3, 7, and 9 (50). These discrepancies could be accounted for essentially by two factors: differences in protein conformation resulting from its source and purification, and differences in the conditions of proteolysis. As both our group (19, 20) and Sasaki et al. (50) have used rhuSPARC that, despite differences in source and purification procedures, showed activity and/or physical chemical characteristics consonant with a correctly folded structure, we favor the latter possibility, i.e. the enzyme to substrate ratios and diminished incubation times. Whereas Sasaki et al. (50) report minimal cleavage of SPARC by MMPs (including MMP-3) at a 1:100 enzyme to substrate ratio (by weight) after 24 h, peptides J1–3 in our study were generated reproducibly at this ratio, within 16 h. Importantly, cleavage of SPARC by one or more of the MMPs used in studies from the Timpl laboratory has revealed an enhanced (~10-fold) Ca2+-dependent binding to both fibrillar and basement membrane collagens (49, 50). Moreover, antibodies specific for the cleaved neoepitope at Leu198 revealed the presence of this fragment of SPARC in tissues (51). Further work will be necessary to clarify the ultimate specificity of MMP-3 for native SPARC, and the biological consequences thereof.

We have identified novel cleavage products of SPARC produced by MMP-3, including a sequence containing EF hand-1, a variant Ca2+-binding loop thought to function as a structural element in the native protein (38). This region (contained within peptides J2 and J3) was stimulatory for cell migration but inactive in both proliferation and CAM assays. Effects of SPARC on various stages of angiogenesis have been documented (reviewed in Refs. 1 and 6). Typically associated with growing/regressing capillaries rather than larger, established vessels (18), SPARC was identified as a highly and differentially expressed pan endothelial marker in both normal and tumor-associated endothelium (52). It is now generally accepted that angiogenesis on the part of the host is required for the growth and metastasis of most solid tumors, and many of the matricellular proteins have demonstrated roles in the regulation of neoplastic progression via their effects on neovessel formation (reviewed in Ref. 1). It is still not clear whether SPARC or specific fragments of SPARC are normally involved in the regulation of angiogenesis. Moreover, the presence of inflammatory cells, cytokines, specific proteinases, and their inhibitors, as well as a requirement for ECM, serves to complicate the interpretation of different injury models. It is undisputed, however, that MMPs are important contributors to disease progression and/or resolution vis-à-vis their effects on the activation of other proteases, ECM degradation, and growth factor release or activation (30, 41, 53). Conversely, SPARC can regulate the activation as well as the production and release of certain of the MMPs, including MMP-3 (12, 13). Solving the regulatory loops in a tissue or model system, and the contribution of each variable to resolution of the biological process, remains a challenging but realistic endeavor.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants GM40711, HL59475, and P50 DK47659. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| Present address: Dept. of Gastroenterological & General Surgery, Helsinki University Central Hospital, Helsinki FIN-00290, Finland. Back

§ To whom correspondence should be addressed: Dept. of Vascular Biology, The Hope Heart Institute, 1124 Columbia St., Suite 723, Seattle, WA 98104-2046. Tel.: 206-903-2026; Fax: 206-903-2044; E-mail: hsage{at}hopeheart.org.

1 The abbreviations used are: ECM, extracellular matrix; BAE, bovine aortic endothelial; CAM, chick choriallantoic membrane; DMEM, Dulbecco's modified Eagle's medium; EC, extracellular Ca2+ binding; FGF, fibroblast growth factor; FS, follistatin; MMP, matrix metalloproteinase; PAE, porcine aortic endothelial; rhu, recombinant human; RIMAC, radial invasion of matrix by aggregated cells; TIMP, tissue inhibitor of metalloproteinase; VEGF, vascular endothelial growth factor; PBS, phosphate-buffered saline; HPLC, high performance liquid chromatography. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Earl Davie and Brad McMullen for sequencing work, Dr. Richard Johnson (Amgen, Inc.) for his collaboration during the initial phase of the project, Dr. Robert Vernon for assistance with the RIMAC assay, and Zymogenetics, Inc., for synthesis of peptides. Eileen Neligan is gratefully acknowledged for her assistance with the article.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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