Copyright ©The Histochemical Society, Inc.

Functional Analysis of the Matricellular Protein SPARC with Novel Monoclonal Antibodies

Mariya T. Sweetwyne1,2,, Rolf A. Brekken1,3,, Gail Workman, Amy D. Bradshaw4, Juliet Carbon, Anthony W. Siadak5, Carrie Murri and E. Helene Sage

Department of Vascular Biology, The Hope Heart Institute, Seattle, Washington

Correspondence to: Dr. E. Helene Sage, Hope Heart Program, Benaroya Research Institute at Virginia Mason, 1201 9th Avenue, Seattle, WA 98101-2795. E-mail: hsageBRI{at}hopeheart.org


    Summary
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
SPARC (osteonectin, BM-40) is a matricellular glycoprotein that is expressed in many embryogenic and adult tissues undergoing remodeling or repair. SPARC modulates cellular interaction with the extracellular matrix (ECM), inhibits cell adhesion and proliferation, and regulates growth factor activity. To explore further the function and activity of this protein in tissue homeostasis, we have developed several monoclonal antibodies (MAbs) that recognize distinct epitopes on SPARC. The MAbs bind to SPARC with high affinity and identify SPARC by ELISA, Western blotting, immunoprecipitation, immunocytochemistry, and/or immunohistochemistry. The MAbs were also characterized in functional assays for potential alteration of SPARC activity. SPARC binds to collagen I and laminin-1 through an epitope defined by MAb 293; this epitope is not involved in the binding of SPARC to collagen III. The other MAbs did not interfere with the binding of SPARC to collagen I or III or laminin-1. Inhibition of the anti-adhesive effect of SPARC on endothelial cells by MAb 236 was also observed. Functional analysis of SPARC in the presence of these novel MAbs now confirms that the activities ascribed to this matricellular protein can be assigned to discrete subdomains. (J Histochem Cytochem 52:723–733, 2004)

Key Words: SPARC • collagen • matricellular • monoclonal antibody • extracellular matrix


    Introduction
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
SPARC (OSTEONECTIN, BM-40) is a prototypical matricellular protein that regulates cell function in the context of the ECM but does not serve a structural role. Other members of the matricellular class of proteins include thrombospondin 1 and 2, tenascin-C, osteopontin, and the CCN [cyr61, CTGF (connective tissue growth factor), Nov] family of proteins (Bornstein and Sage 2002Go). These proteins, although structurally diverse, have a common functional theme, i.e., mediation of cell–ECM interactions.

SPARC is a 32-kD protein that contains three modules: a C-terminal extracellular (EC) module with two Ca2+-binding EF hands is preceded by a follistatin-like module and an N-terminal acidic module (Brekken and Sage 2001Go; Hohenester and Engel 2002Go). De-adhesion and anti-proliferation are two major functions identified for SPARC in vitro. Although a cell surface receptor specific for SPARC has not been identified conclusively, Yost and Sage (1993)Go demonstrated that SPARC binds to the surface of bovine aortic endothelial cells (BAECs). In addition, SPARC binds to protein constituents of the ECM, including collagen types I–V and VIII, vitronectin, and thrombospondin-1 (Brekken and Sage 2001Go). SPARC has therefore been proposed to modulate cell–ECM interactions and to influence cell behavior during ECM remodeling. Cleavage of SPARC in the EC module by certain matrix metalloproteinases (MMPs) also increases the affinity of SPARC for collagens I, IV, and V (Sasaki et al. 1997Go) and might direct the activity of SPARC to regions of the ECM that are being remodeled.

Studies of the expression of SPARC in lower vertebrates and invertebrates have shown that SPARC is spatially and temporally regulated during development (Holland et al. 1987Go; Nomura et al. 1988Go; Sage et al. 1989aGo; Brekken and Sage 2001Go). SPARC can be detected in mouse embryos at day 9 in the heart primordia, somites, and extraembryonic membranes. Later in development SPARC is found in bone, epithelia such as those associated with the gut and skin, and blood vessels. In the adult, the expression of SPARC appears to be limited largely to renewing or remodeling tissues, such as gut or bone, in addition to tissues undergoing repair (Holland et al. 1987Go; Sage et al. 1989aGo). Lane et al. (1994)Go showed SPARC in angiogenic microvasculature of developing brain and dermal wounds, and Iruela-Arispe et al. (1995)Go demonstrated that SPARC was expressed by endothelial cells in capillaries of the chick chorioallantoic membrane that were associated with maximal growth. Moderate immunoreactivity for SPARC was also identified in steroidogenic cells, chrondocytes, placental trophoblasts, vascular smooth muscle cells, and endothelial cells in normal adult human tissue (Porter et al. 1995Go). High levels of SPARC were again noted in fibroblasts and endothelial cells involved in tissue repair and in malignant tumors.

SPARC-null mice have defects in collagen fibril formation that affect their responses to insults, such as dermal wounding (Bradshaw et al. 2001Go,2002Go) and implanted biomaterials (Puolakkainen et al. 2003Go), or tumor cells (Brekken et al. 2003Go). These results indicate that SPARC is associated with changes in the formation and deposition of ECM. In addition, SPARC-null mice develop cataracts within the first 3–6 months after birth, a consequence in part of structural alterations in the lens capsule basement membrane that lead to increased permeability and osmotic changes within the lens (Yan et al. 2002Go,2003Go). Studies with SPARC-null mice support the hypothesis that the function of SPARC is contextual and that regulated expression of SPARC is important for development and host response to changes in tissue homeostasis.

By the use of novel MAbs specific for SPARC, we now show that SPARC interacts with different ECM proteins via distinct epitopes. One MAb blocked SPARC from binding directly to certain ECM proteins, whereas another inhibited SPARC-mediated rounding of BAECs. Our data indicate that the effects of SPARC on endothelial cells are mediated through cell surface components that are either more efficiently ligated in the presence of MAb–SPARC complexes or are sensitive to conformational changes of SPARC induced by the MAbs themselves.


    Materials and Methods
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
Generation and Screening of Hybridomas
Recombinant human SPARC (rhuSPARC) was produced and purified as described (Bradshaw et al. 2000Go) and was used as an immunogen. Male SPARC-null mice (C57Bl/6 x129SVJ) were immunized IP with 50 µg of rhuSPARC in Ribi R-700 adjuvant (Corixa; Hamilton, MT). After two boosts with the same mixture on days 21 and 42 and a final IP boost without adjuvant on day 63, spleens were collected under sterile conditions on day 66. Splenocytes were fused with P3-X63-Ag8.653 mouse myeloma cells (American Type Culture Collection; Rockville, MD) (Kearney et al. 1979Go) according to established procedures (Lane 1985Go). The resulting cell mixture was plated into multiple 96-well tissue culture plates in medium consisting of Iscove's modified Dulbecco's medium (Gibco; Grand Island, NY) supplemented with 10% (v/v) Fetal Clone I serum (HyClone Laboratories; Logan, UT), 10% (v/v) BM Condimed H1 (Roche Applied Science; Indianapolis, IN), 2 mM L-glutamine (Gibco), 100 U/ml penicillin G and 100 µg/ml streptomycin sulfate (Gibco), and HAT (0.1 mM hypoxanthine, 0.4 mM aminopterin, 0.016 mM thymidine; Gibco). The plates were fed on days 4 and 7 after fusion by aspiration and replacement with fresh medium of approximately three-fourths of the medium content in each well.

Two days after the last feeding, supernatants from the fusion plates were screened by indirect ELISA. Immulon II plates (Thermo Labsystems; Franklin, MA) were coated with 0.5 µg/ml, 55 µl/well of rhuSPARC (Bradshaw et al. 2000Go) diluted in 10 mM PBS (Gibco) overnight at 4C. Unbound antigen was removed, wells were washed twice with PBS containing 0.05% Tween-20 (PBST), and were blocked for 1 hr at room temperature with PBST containing 1% (w/v) bovine serum albumin (BSA) (200 µl/well; Sigma, St Louis, MO). After removal of the blocking solution, the wells were washed twice with PBST and culture supernatants from the fusion plates were replica-plated onto the ELISA plates at 55 µl/well. The plates were incubated for 1 hr at RT, after which the supernatants were removed and the wells were washed four times with PBST. For detection of bound mouse antibody, peroxidase-conjugated Fc-specific goat anti-mouse IgG reagent (Southern Biotechnology Associates; Birmingham, AL) was diluted 1:5000 in Iscove's modified Dulbecco's medium containing 2% Fetal Clone I serum and was added to the wells at 55 µl/well. After a 1-hr incubation at RT, the wells were washed five times with PBST. 3,3',5,5'-Tetramethylbenzidine (TMB) substrate in substrate buffer (Genetic Systems; Redmond, WA) was prepared according to the manufacturer's instructions and was added to the wells (55 µl/well). After a 10–15-min incubation at RT, color development in the wells was terminated by the addition of 1 N H2SO4, and the optical density of the wells was read on an ELISA plate reader (Molecular Devices; Sunnyvale, CA) at 450 nm. All supernatants that were positive in this assay were subsequently evaluated in a second indirect ELISA using recombinant murine SC1, a homologue of SPARC (Soderling et al. 1997Go), as a negative control protein to ensure that the positive supernatants in the initial assay contained antibody that was specific for rhuSPARC.

Hybridomas in wells with anti-rhuSPARC specificity were cloned at least twice by culturing the cells with a limiting dilution technique in Costar 96-well half-area plates (Corning; Acton, MA) and by assessing specificity of antibody produced by the clones as described above. MAbs produced by each of the final clones were evaluated for IgG subclass and light chain composition with the Mouse Monoclonal Antibody Isotyping kit (Roche Applied Science).

Antibody Purification
IgG antibodies were purified from tissue culture supernatants by chromatography on protein A–Sepharose with the Pierce ImmunoPure Binding/Elution buffering system (Pierce; Rockford, IL). IgGs were evaluated for purity by SDS-PAGE and staining with Coomassie Brilliant Blue R and for reactivity with SPARC by indirect ELISA. The hybridoma AON-1 produced by Bolander et al. (1989)Go was obtained from the Developmental Studies Hybridoma Bank (University of Iowa, Department of Biological Sciences; Iowa City, IA) and was further subcloned in our laboratory. The derived AON-1 clone was grown in Iscove's medium (Gibco) with 10% fetal calf serum (FCS), and the IgG was purified from tissue culture supernatant by protein A chromatography as described above.

ELISA
Recombinant human SPARC was biotinylated by incubation of biotinamidocaproate NHS-ester (Sigma) with rhuSPARC at a 20:1 molar ratio in Hank's balanced salt solution (HBSS) (Gibco). The solution was rotated gently at RT for 1.5 hr, after which the reaction was stopped with Tris-HCl/glycine (pH 8) at a final concentration of 10 mM. Biotinylated rhuSPARC was separated from excess biotin on a PD-10 column (Amersham; Piscataway, NJ). For the capture ELISA, microtiter plates coated with protein G (Pierce,) or purified IgG (100 ng/well) were blocked with 5% casein acid hydrolysate (Sigma) in PBST and were incubated with biotinylated rhuSPARC at various concentrations. Peroxidase-conjugated NeutrAvidin (NA-Hrp) (Pierce) was added as a second layer and reactive wells were developed with the peroxidase substrate TMB (BioFx Laboratories; Owings Mills, MD). Reactions were stopped after 15 min with 1 M H3PO4 and were read spectrophotometrically at 450 nm (Molecular Devices).

Competition ELISA experiments were performed with MAbs labeled with biotin (biotinamidocaproate NHS ester; Sigma). Microtiter plates were coated with SPARC and were blocked as above. Approximately 0.1–0.5 µg/ml of labeled MAb was incubated with either buffer alone, an antibody of irrelevant specificity, or the other unlabeled anti-SPARC MAbs at an equimolar to 100-fold molar excess. The binding of the labeled MAb was assessed by addition of NA-Hrp and was developed with TMB as above. The assay was done in triplicate at least twice for each combination of labeled and unlabeled MAbs.

We employed ELISA to assess binding of SPARC to collagen type I, collagen type III (Chemicon International; Temecula, CA), or laminin-1 (BD Biosciences; Bedford, MA). To determine the coating efficiency, we coated each ECM protein (20 nM) onto 96-well assay plates (Nalge Nunc; Rochester, NY), and the percentage of the total protein remaining in solution vs protein bound to the assay plate was compared by SDS-PAGE. Briefly, 1.0 nmol of each protein was added in 150 µl of sensitizing buffer (15 mM Na2CO3 + 35 mM NaHCO3, pH 9.6) and allowed to incubate for 90 min at 37C. Supernatant was removed from the wells and bound protein was eluted in SDS-PAGE buffer containing 100 mM dithiothreitol (DTT) (Laemmli 1970Go) at 90C for 10 min. Eluant and supernatant, along with a standard titration of each protein, were separated by SDS-PAGE (7.5% gels). Ratios of eluant and supernatant gave the following coating efficiencies at 90 min: collagen I {approx}15%, collagen III {approx}13%, laminin-1 {approx}22%. Wells of microtiter plates were coated with equimolar amounts of the indicated ECM protein, after which biotinylated rhuSPARC, in the presence or absence of different concentrations of the anti-SPARC MAbs, was added to the wells. Binding of SPARC was assessed with NA-Hrp as described above.

Immunoblotting
Four µg of purified rhuSPARC or mouse parietal yolk sac carcinoma (PYS) SPARC (Funk and Sage 1993Go) was resolved by SDS-PAGE on 12% gels under reducing (100 mM DTT added to protein samples) and non-reducing conditions. Proteins were transferred to Immun-Blot PVDF membranes (Bio-Rad; Hercules, CA) and were blocked for 1 hr at RT with AquaBlock (East Coast Biologics; North Berwick, ME). The membranes were incubated with anti-SPARC MAbs at 2 µg/ml in PBST + 10% AquaBlock for 1 hr at RT, followed by washes with PBST. The membranes were developed after incubation with peroxidase-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories; West Grove, PA) by SuperSignal West Pico chemiluminescence substrate (Pierce) according to the manufacturer's instructions. Equal transfer of the proteins was confirmed by staining of the transferred gels with Coomassie Brilliant Blue R.

Recombinant huSPARC (10 µg) (Bradshaw et al. 2000Go) was digested with TPCK-treated trypsin (USB; Cleveland, OH) at a 1:100 enzyme to substrate molar ratio, at 37C for 30 min. Samples were immediately resolved by SDS-PAGE on 16% Tricine gels (Invitrogen; Carlsbad, CA) under reducing conditions. Western blots were performed as described above.

Recombinant huSPARC was also digested with the active form of MMP-3 (R&D Systems; Mineapolis, MN) at a 1:100 enzyme to substrate ratio (by weight) (Sage et al. 2003Go). Pro-MMP-3 was activated via incubation with 1 mM aminophenylmercuric acetate (APMA) at 37C for 1 hr. 10 µg of rhuSPARC was incubated with activated MMP-3 at 37C for 16 hr. Samples were immediately reduced and resolved on 16% Tricine gels by SDS-PAGE. Western blots were performed as described above.

Immunoprecipitation
200 µg of tissue lysate from mouse testis or 500 µl of conditioned medium (CM) from either SPARC-infected or control Spodoptera frugiperda (Sf9) cultures or PYS cells was pre-cleared with protein G–Sepharose beads. 200 µl of anti-SPARC MAb tissue culture supernatant or 2–5 µg of purified IgG was mixed with the pre-cleared lysate or CM for 2 hr at 4C. The IgG was precipitated by the addition of protein G–Sepharose beads and the precipitate was washed three times with 20% lysis buffer (Brekken et al. 2000Go). Proteins were resolved after disulfide bond reduction by SDS-PAGE on a 12% polyacrylamide gel. The presence of SPARC in the precipitate was determined by Western blotting with a goat polyclonal anti-SPARC antibody (0.2 µg/ml) (R&D Systems).

Immunohistochemistry and Immunocytochemistry
Formalin- and methyl Carnoy's-fixed tissues embedded in paraffin were sectioned by the Histopathology Laboratory (University of Washington; Seattle, WA). Methyl Carnoy's-fixed sections were deparaffinized under standard conditions. If necessary, endogenous peroxidases were blocked in methanol with 1.0% H2O2 for 30 min. Some of the sections were then treated with Autozyme (10 µl enzyme concentrate/1 ml buffer for 6 min at RT) (BioMeda; Foster City, CA). The sections were incubated with primary antibody for 1 hr, washed with PBST, incubated for 1 hr with the appropriate peroxidase-labeled secondary antibody (Jackson ImmunoResearch Laboratories), developed with Stable DAB (ResGen; Huntsville, AL), counterstained with hematoxylin, and coverslipped in Permount (Fisher; Fair Lawn, NJ). Sections of frozen tissue (5–7 µm) were air-dried, fixed in fresh acetone for 5 min, rehydrated in PBST, and blocked in PBST containing 20% AquaBlock for 30 min. The slides were incubated with primary antibodies and were developed as described above.

We screened MAbs AON-1, 175, 236, 255, 293, and 303 for reactivity with BAECs (Funk and Sage 1991Go) (passage 10) plated onto glass coverslips and grown to 70% confluence in Dulbecco's modified Eagle's medium (DMEM) (Gibco) with 10% FCS. Cells were washed once with PBS, incubated at 37C for 30 min in serum-free DMEM (SFM), fixed with 3% formaldehyde/SFM for 20 min at RT, and rinsed with PBS. Cells were blocked with 5% normal goat serum (NGS) in PBST for 20 min at RT and the anti-SPARC MAbs were added at concentrations of 2.5, 5, or 10 µg/ml for 1 hr at RT. Polyclonal goat anti-SPARC IgG (R&D Systems) was used as a positive control at concentrations of 5 and 10 µg/ml. Negative controls were normal mouse serum and the secondary antibody alone. Reactivity was detected by incubation of the coverslips with the appropriate fluorescein isothiocyanate (FITC)-conjugated secondary antibody (Jackson ImmunoResearch Laboratories). The coverslips were rinsed, after which the nuclei were stained with 20 µg/ml Hoechst 33258 dye in PBS (Molecular Probes; Eugene, Oregon) for 3 min at RT. The coverslips were then washed in PBS and were mounted on slides with Vectashield mounting medium (Vector Laboratories; Burlingame, CA).

Adhesion Assays
BAECs (passages 5–12) were plated in DMEM containing 5% FCS (Gibco) onto 96-well tissue culture plates at a concentration of 104 cells/well (Falcon; BD Biosciences, Bedford, MA) and were grown to 90–100% confluence. For determination of the optimal concentration of rhuSPARC for analysis of the MAbs, rhuSPARC was added to the cells in a titration from 0.5 to 40 µg/ml and the cells were monitored visually. The de-adhesion associated with SPARC was concentration-dependent and was evident over the titration range. De-adhesion was observed within 6 hr and persisted for up to 24 hr. To evaluate the effect of the anti-SPARC MAbs on SPARC-induced de-adhesion, we preincubated 5 µg/ml of rhuSPARC in DMEM containing 5% FCS with a 10-fold molar excess of anti-SPARC MAb and added the solution to BAECs. De-adhesion of cells was scored visually after 24 hr as rounding in <25% of the plated cells (–), >25% of cells (+), >50% of cells (++), >75% of cells (+++), and >90% of cells (++++).

For cell-rounding assays, rhuSPARC was preincubated with a 10-fold molar excess of MAb in serum-free DMEM overnight at 4C on a rocker platform. The SPARC–Mab solution was added to a 96-well tissue culture plate (Corning) in triplicate. BAECs (passages 5–7) were counted and were added to the SPARC–MAb solution such that the final concentration of cells was 7.5 x 104 cells/ml and that of rhuSPARC 10 µg/ml. Cells were photographed at 30, 60, and 90 min after plating. At 90 min, quantification was performed according to a Rounding Index (Lane and Sage, 1994Go), in which attached cells were scored as spread, partially spread, or round. Each condition was graphed as a percentage of the Rounding Index of the control cells receiving IgG + SPARC.


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 Materials and Methods
 Results
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 Literature Cited
 
Characterization of MAbs
Table 1 summarizes information on the class/subclass of the different anti-SPARC antibodies, as well as the relative reactivity of the MAbs by ELISA, Western blotting, immunohistochemistry, and immunoprecipitation. Differences in the reactivity of the MAbs by indirect and capture ELISA and their reactivity with mouse SPARC were noted. Figure 1 shows the reactivity of the MAbs in a representative indirect ELISA.


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Table 1

Summary of properties of monoclonal antibodies specific for SPARCa

 


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Figure 1

Indirect ELISA showing reactivity of anti-SPARC MAbs with rhuSPARC bound to wells of an ELISA plate. The indicated MAb was titrated across the plate in triplicate and was detected with a peroxidase-conjugated goat anti-mouse IgG. Secondary antibody was detected by the peroxidase substrate TMB. Mean absorbance and standard deviation at 450 nm are shown. Key at upper left indicates MAb by clone number.

 
The MAbs represent at least six distinct epitope groups on SPARC, as determined by competitive ELISA (Table 2) and by the characterization summarized in Table 1. In the competition ELISA, the binding of biotinylated MAb 175 to SPARC was competed by unlabeled MAbs 175, 236, and 303, and the reciprocal binding by labeled 236 and 303 was competed by unlabeled 175. Interestingly, MAbs 236 and 303 did not block one another in the competition assays and had distinct reactivity profiles, as shown in Table 1. MAb 236 did not bind to mouse SPARC (PYS column; Table 1) nor did it immunoprecipitate SPARC, whereas 303 reacted with mouse SPARC and immunoprecipitated SPARC. MAb 175, although competed by 236 and 303, did not recognize SPARC by Western blotting analysis, an indication that the epitope for 175 overlaps that of 236 and 303 but is not identical. A similar situation was evident with respect to MAbs 255, 303, and AON-1, the last a commercially available anti-SPARC MAb. AON-1 crossblocked the binding of 255 and 303 to SPARC, and 255 and 303 crossblocked the binding of AON-1 to SPARC, but 255 and 303 did not crossblock each other. MAbs 255 and 303 have similar characteristics; however, 255 had a higher affinity for SPARC by ELISA (Table 1). MAb 293 represents a distinct epitope and was not competed by the other MAbs that were evaluated in the competition assay. Although AON-1 was purchased as a hybridoma line, it is important to note that we subcloned a line and purified AON-1 from the subclone.


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Table 2

Competitive ELISA of anti-SPARC monoclonal antibodies

 
Western blotting analysis revealed that 4/7 MAbs reacted with SPARC under both non-reducing and reducing conditions. Figure 2 shows the reactivity of three of the MAbs (236, 255, and 303), as well as AON-1, with mouse (PYS) and rhuSPARC. The MAbs reacted more consistently and efficiently with reduced SPARC than with the disulfide-bonded form. MAb 236 did not react with mouse SPARC by ELISA (Table 1) nor with non-reduced mouse SPARC by Western blotting. However, MAb 236 did react with mouse SPARC after reduction of disulfide bonds. MAb 255 reacted with mouse SPARC by ELISA (Table 1) but also showed differential binding to mouse SPARC under reducing and non-reducing conditions.



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Figure 2

Western blotting analysis of the anti-SPARC MAbs. Mouse SPARC (Lanes 1, 3) or rhuSPARC (Lanes 2, 4) was separated by SDS-PAGE under reducing (+DTT) and non-reducing (–DTT) conditions and was transferred to PVDF membranes, which were incubated with indicated MAbs (AON-1, 236, 255, 303) followed by peroxidase-conjugated goat anti-mouse IgG secondary antibodies. Reactivity was visualized by incubation with chemiluminescent substrate and exposure to film. Arrows indicate SPARC under reducing ({approx}43 kD) and non-reducing ({approx}37 kD) conditions. Molecular weight standards in kD are indicated at left.

 
The MAbs were also screened for immunoprecipitation of SPARC from Sf9 (recombinant human) and PYS cell-conditioned medium and tissue lysates (Table 1). MAbs 303, 293, 255, and 175 immunoprecipitated SPARC from mouse testis, as did a control goat polyclonal anti-SPARC antibody (Figure 3) . MAbs 236 and 275, both IgG1K, immunoprecipitated SPARC weakly from mouse testis, although 275 precipitated rhuSPARC more efficiently (Table 1). The differences in the intensity of the SPARC band might be due in part to a difference in the amount of precipitating MAb used in the assay, because hybridoma tissue culture supernatant was used instead of purified MAb. Figure 3 also shows the VH band from the precipitating IgG. The secondary antibody used in the assay might have a stronger crossreaction to mouse IgG1 VH than other mouse IgG isotypes. Given that MAbs 236, 275, and 303 are all IgG1, the reactivity might also represent the relative amount of precipitating IgG.



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Figure 3

Immunoprecipitation of SPARC from testis by anti-SPARC MAbs. 200 µg of total protein from mouse testis tissue lysate was incubated with the indicated MAb or a control affinity-purified goat anti-SPARC antibody (Gt {alpha} SP). The antibodies were precipitated with protein G-conjugated Sepharose beads. Proteins were resolved by SDS-PAGE under reducing conditions, transferred to a PVDF membrane, and probed for SPARC with goat anti-SPARC IgG. SPARC ({approx}43 kD) and the IgG heavy chain (VH) are indicated. PYS CM, conditioned medium from mouse PYS cells. Molecular weight standards in kD are indicated at right.

 
To identify the domains of SPARC recognized by several of the MAbs, we digested SPARC with trypsin and MMP-3 and performed immunoblotting of the cleavage fragments with MAbs 236, 303, and 255. Both 236 and 255 bound to a fragment of 22 kD in the trypsin digest, whereas 303 failed to recognize any of the SPARC digestion products (Figure 4) . The approximate 22-kD peptide is an N-terminal fragment representing approximately half of the sequence of SPARC, i.e., part of the follistatin and E-C domains (amino acids 53–138) (Sage et al. 1989bGo). Therefore, MAb 303 is likely to recognize the extreme C-terminal region of SPARC, which is included in the EC domain (amino acids 138–286), contains the second Ca2+-binding loop, and binds to cells (Brekken and Sage 2001Go). Similar results were obtained with digests of SPARC incubated with MMP-3: MAb 303 did not recognize any of the larger primary cleavage products, whereas MAb 255 was highly reactive with a band of 24 kD, probably representing a slightly larger version of the peptide termed J2 (data not shown) (Sage et al. 2003Go). However, MAb 236 reacted with two minor cleavage products of 30 kD and 35 kD (data not shown). These data indicate that the three MAbs shown in Figure 4 each recognize distinct epitopes on SPARC.



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Figure 4

Anti-SPARC MAbs recognize different regions within the SPARC sequence. 10 µg rhuSPARC was digested with trypsin in PBS at a 1:100 enzyme to substrate molar ratio at 37C for 30 min. Samples were immediately reduced, resolved by SDS-PAGE, electrotransferred to PVDF membranes, and incubated with MAbs 236, 303, and 255 (replicate blots with MAbs as indicated on figure). Lanes 1 and 5, undigested SPARC; Lanes 2 and 6, SPARC digested with trypsin; Lane 3, trypsin; Lane 4, PBS. Trypsin- and PBS-only lanes for MAb 255 blot showed no specific bands.

 
Tissues examined for IHC reactivity with the MAbs included skin, testis, retina, and kidney, as well as frozen sections of human tumor xenografts grown in SCID mice (Table 1). Paraffin-embedded sections of human skin showed a striking difference in reactivity between AON-1 and two of our MAbs (Figure 5) . MAbs 236 and 293 displayed prominent immunoreactivity with SPARC along the dermal/epidermal junction below the stratum spinosum and stratum basale. However, AON-1 reacted principally in the stratum spinosum and stratum granulosum (above the stratum spinosum; Figure 5). MAbs 236 and 293 also stained cells within the upper papillary dermis. Nerve bundles and vasculature in the dermis were evident in the presence of AON-1, 236, and 293. In testis, MAbs 255 and 236 recognized SPARC in the seminiferous tubules, whereas AON-1 exhibited limited reactivity in these areas. Staining of Sertoli cells, and occasionally Leydig cells, was also noted (Figure 6 , arrows).



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Figure 5

IHC reactivity of anti-SPARC MAbs on human skin. Formalin-fixed, paraffin-embedded serial sections of human skin were deparaffinized and incubated with either purifed MAb or hybridoma supernatant (identified on each panel), followed by peroxidase-conjugated goat anti-mouse IgG. Reactivity was developed with the peroxidase substrate DAB and the slides were counterstained with hematoxylin. DEJ, dermal/epidermal junction; S, stratum spinosum; blue arrow, blood vessel; red arrow, nerve bundle. Control, incubation with PBS and secondary antibody. Magnification x400.

 


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Figure 6

IHC reactivity of anti-SPARC MAbs on human testis. Formalin-fixed, paraffin-embedded serial sections were deparaffinized and incubated with MAbs (identified on each panel) as described in the legend to Figure 5. L, Leydig cells; S, Sertoli cell; ST, seminiferous tubule; TM, testicular matrix. Control, incubation with PBS and secondary antibody. Magnification x200.

 
Several of the MAbs were reactive with BAECs grown on coverslips. Each MAb displayed a consistent pattern of cytoplasmic staining, with areas of intense focal reactivity around or near the nucleus (data not shown).

Functional Analysis of SPARC in the Presence of Anti-SPARC MAbs
Biotinylated SPARC was used to assess the binding of SPARC to equimolar amounts of collagen I, collagen III, and laminin-1 by ELISA (Figure 7) . All reagents were first evaluated for their binding to SPARC and/or ECM proteins after biotinylation. Unlabeled SPARC competed with biotinylated SPARC for binding to each protein. The MAbs had differential effects on the binding of SPARC to the ECM proteins. AON-1 inhibited the binding of SPARC to collagen types I and III and to laminin-1 by 75%, 50%, and 80%, respectively. However, MAb 293 was the only other MAb tested that had a significant inhibitory effect in this assay. MAb 293 inhibited the binding of SPARC to collagen I and laminin-1 by 50% but it did not interfere with the binding of SPARC to collagen III (Figure 7). Neither BSA nor irrelevant, anti-mouse MAbs, used as negative controls, inhibited the binding of biotinylated SPARC to ECM proteins (data not shown).



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Figure 7

Effects of anti-SPARC MAbs on the interaction of SPARC with ECM components by ELISA. Equimolar amounts of (A) collagen type I, (B) collagen type III, and (C) laminin-1 were coated onto the wells of an ELISA plate and the level of biotinylated SPARC bound in the presence of indicated MAbs was determined by development with peroxidase-conjugated NeutrAvidin. Controls included competition with unlabeled SPARC and addition of peroxidase-conjugated NeutrAvidin only. Bars denote the binding of SPARC to ECM proteins in the presence of the respective MAbs. In the absence of MAb, binding by biotinylated SPARC was defined as 100%. Error bars were calculated by error propagation of at least nine wells (n=9–12) for each MAb. *, p values <0.5.

 
SPARC is a de-adhesive protein that induces rounding of BAECs (Sage et al. 1989bGo) in a concentration- and time-dependent manner. The effects of the MAbs on the de-adhesive activity of SPARC are summarized in Table 3. MAb 175 augmented the de-adhesive effect of SPARC on confluent BAEC monolayers, whereas MAbs 293 and AON-1 significantly reduced SPARC-induced de-adhesion (Table 3). We also examined the effect of the MAbs in the presence or absence of SPARC on the adhesion of BAECs to tissue culture plastic (Figure 8) . In this experiment, BAECs were plated in the presence of SPARC and/or the MAbs and were monitored visually for adherence at 30, 60, and 90 min after plating. At 90 min, the degree of cell rounding/spreading was quantified by a Rounding Index (Figure 8). Two of the MAbs exhibited effects in this assay: 255 augmented the activity of SPARC on cell rounding, whereas 236 was inhibitory (Figure 8). These data indicate that MAb 236 is function-blocking with respect to the rounding (inhibition of spreading) activity of SPARC on BAECs.


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Table 3

Effects of anti-SPARC MAbs on SPARC-induced de-adhesion of BAECsa

 


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Figure 8

Effects of anti-SPARC MAb on the inhibition of adhesion of BAECs by SPARC. BAECs were plated in the presence of medium only (no Ab), mouse IgG isotype control (IgG), or the indicated MAbs at 0.3 µM in the presence (red bars) or absence of SPARC (blue bars) at 0.03 µM (1.0 µg/ml). Cells were quantified by a Rounding Index after 90 min. Rounding Indices were plotted as a percentage of the Rounding Index of Control (IgG + SP). Error bars represent SEM (n=6) for each condition. *, p value <0.5.

 

    Discussion
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
We have produced and characterized several novel MAbs specific for the matricellular protein SPARC. The MAbs have proven to be useful for analyzing the interaction of SPARC with ECM proteins and with cells in vitro, and for studying the distribution of SPARC in tissues. A summary of these reagents and their properties is shown in Table 1.

SPARC modulates cell response to changes in the extracellular environment by influencing the expression and activity of growth factors, ECM-modifying enzymes, and structural ECM proteins, such as collagen I. That SPARC associates with collagen and other ECM proteins is established, and studies in SPARC-null mice demonstrate that this interaction is critical for an appropriate host response to injury. Previous work in which SPARC-null mice were challenged with dermal wounds, porous and non-porous biomaterials, and tumor cells demonstrates that mice lacking SPARC have significant differences from wild-type counterparts in their response to such insults. The MAbs characterized here will aid significantly in our understanding of how SPARC regulates the response of host tissue to changes in the extracellular environment.

SPARC interacts with several proteins that reside in the ECM. However, identification of subdomains of SPARC that bind to different ECM proteins has remained largely elusive. We now provide evidence that SPARC interacts with at least three components of the ECM (collagen types I and III, and laminin-1) through at least two distinct epitopes. The binding of SPARC to collagen I and laminin-1 was inhibited by MAb 293, but 293 did not block the binding of SPARC to collagen III. Interestingly, AON-1 blocked SPARC from binding to collagen types I and III as well as to laminin-1. These results are consistent with those of Bolander et al. (1989)Go, who first described AON-1 and suggested that the epitope for AON-1 was obscured by intermolecular interactions between SPARC and ECM components. However, to our knowledge the epitope on SPARC bound by AON-1 has not been defined. Other anti-SPARC/osteonectin MAbs have been described and their binding to SPARC mapped to various regions of the protein (Villarreal et al. 1991Go; Maillard et al. 1992Go). For example, MAbs ON2 (also known as Mab2) and IIIA3A8 were both characterized as binding to the central region of SPARC between amino acids 18–146 (Villarreal et al. 1991Go), whereas other anti-SPARC MAbs (Mab3, Mab6, Mab12) bind to distinct although undefined epitopes (Malaval et al. 1991Go; Maillard et al. 1992Go).

The function of SPARC is measured in vitro most consistently through its effect on cell adhesion. We found two MAbs that enhance (255) or inhibit (236) the rounding effect of SPARC on BAEC (Figure 8). In addition, MAb 175 mimicked the de-adhesive activity of SPARC (Table 3). Although MAb 175 reacts with mouse SPARC and immunoprecipitates native SPARC, it does not react with denatured SPARC by immunoblotting or IHC analysis. The nature of the interactions between MAbs and SPARC in the context of cell adhesion is at this point not clear.

MAb 293 and AON-1 both inhibited the effect of SPARC in the de-adhesion assay using confluent BAECs (Table 3). However, neither was found to inhibit the effect of SPARC on cell rounding when the cells were plated in the presence of SPARC and MAb (data not shown). This difference might be related to the effects of 293 and AON-1 on the blocking of SPARC from binding to ECM proteins, as shown in Figure 7. Therefore, the effect of the MAbs might indeed differ in the context of different substrates. Immunohistochemical analysis shows that AON-1 is restricted to a reaction with SPARC that is largely intracellular, and Figure 7 shows that AON-1 blocks SPARC from interaction with ECM proteins. These results indicate that the epitope on SPARC recognized by AON-1 is not accessible when SPARC is bound to ECM proteins, which is consistent with the initial characterization of this MAb (Bolander et al. 1989Go).

SPARC-null mice do not show a substantially impaired phenotype (in captivity). Therefore, it is likely that SPARC acts as a subtle modulator of cell adhesion, proliferation, and ECM deposition. This characteristic makes SPARC an ideal protein for studying the complexities of ECM modification and assembly without altering the experimental system (in vitro or in vivo) to an unrealistic extent. That only a 10-fold excess of some of our MAbs was sufficient to block (or enhance) SPARC-mediated cell rounding should allow the testing of the various effects of these reagents in vivo. We anticipate that, through the use of these MAbs, we will be able to map the regions of SPARC that interact with a variety of resident ECM components and further our understanding of how SPARC influences tissue homeostasis.


    Acknowledgments
 
Supported in part by grants from the National Institutes of Health (F32 HL10352 to R.A.B., K01 AR002220 to A.D.B., and R01 GM40711 and R01 HL59574 to E.H.S., and R01 GM40711-16, a Minorities in Research Supplement, to M.T.S.), by a grant from The Gilbertson Foundation to The Hope Heart Institute, and by NSF-Engineering Research Center program grant #EEC-9529161 from the National Science Foundation to the University of Washington.

We acknowledge members of the Sage and Wight laboratories. We would also like to thank Drs T. Barker and R. Vernon for stimulating discussions and Eileen Neligan for assistance with the manuscript.


    Footnotes
 
1 These authors contributed equally to this work. Back

2 Present address: Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL. Back

3 Present address: Departments of Surgery and Pharmacology and The Hamon Center for Therapeutic Oncology Research, UT-Southwestern Medical Center, Dallas, TX. Back

4 Present address: Department of Medicine, Gazes Cardiac Research Institute, Medical University of South Carolina, Charleston, SC. Back

5 Present address: Department of Immunology, Zymogenetics, Inc., Seattle, WA. Back

Received for publication August 26, 2003; accepted February 25, 2004


    Literature Cited
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 

Bolander ME, Robey PG, Fisher LW, Conn KM, Prabhakar BS, Termine JD (1989) Monoclonal antibodies against osteonectin show conservation of epitopes across species. Calcif Tissue Int 45:74–80[Medline]

Bornstein P, Sage EH (2002) Matricellular proteins: extracellular modulators of cell function. Curr Opin Cell Biol 14:608–616[CrossRef][Medline]

Bradshaw AD, Bassuk JA, Francki A, Sage EH (2000) Expression and purification of recombinant human SPARC produced by baculovirus. Mol Cell Biol Res Commun 3:345–351[CrossRef][Medline]

Bradshaw AD, Reed MJ, Carbon JG, Pinney E, Brekken RA, Sage EH (2001) Increased fibrovascular invasion of subcutaneous polyvinyl alcohol sponges in SPARC-null mice. Wound Repair Regen 9:522–530[CrossRef][Medline]

Bradshaw AD, Reed MJ, Sage EH (2002) SPARC-null mice exhibit accelerated cutaneous wound closure. J Histochem Cytochem 50:1–10[Abstract/Free Full Text]

Brekken RA, Overholser JP, Stastny VA, Waltenberger J, Minna JD, Thorpe PE (2000) Selective inhibition of vascular endothelial growth factor (VEGF) receptor 2 (KDR/Flk-1) activity by a monoclonal anti-VEGF antibody blocks tumor growth in mice. Cancer Res 60:5117–5124[Abstract/Free Full Text]

Brekken RA, Puolakkainen P, Graves DC, Workman G, Lubkin SR, Sage EH (2003) Enhanced growth of tumors in SPARC null mice is associated with changes in the ECM. J Clin Invest 111:487–495[Abstract/Free Full Text]

Brekken RA, Sage EH (2001) SPARC, a matricellular protein: at the crossroads of cell-matrix communication. Matrix Biol 19:816–827[Medline]

Funk SE, Sage EH (1991) The Ca2(+)-binding glycoprotein SPARC modulates cell cycle progression in bovine aortic endothelial cells. Proc Natl Acad Sci USA 88:2648–2652[Abstract]

Funk SE, Sage EH (1993) Differential effects of SPARC and cationic SPARC peptides on DNA synthesis by endothelial cells and fibroblasts. J Cell Physiol 154:53–63[Medline]

Hohenester E, Engel J (2002) Domain structure and organisation in extracellular matrix proteins. Matrix Biol 21:115–128[CrossRef][Medline]

Holland PW, Harper SJ, McVey JH, Hogan BL (1987) In vivo expression of mRNA for the Ca++-binding protein SPARC (osteonectin) revealed by in situ hybridization. J Cell Biol 105:473–482[Abstract]

Iruela-Arispe ML, Lane TF, Redmond D, Reilly M, Bolender RP, Kavanagh TJ, Sage EH (1995) Expression of SPARC during development of the chicken chorioallantoic membrane: evidence for regulated proteolysis in vivo. Mol Biol Cell 6:327–343[Abstract]

Kearney JF, Radbruch A, Liesegang B, Rajewsky K (1979) A new mouse myeloma cell line that has lost immunoglobulin expression but permits the construction of antibody-secreting hybrid cell lines. J Immunol 123:1548–1550[Abstract]

Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685[Medline]

Lane RD (1985) A short-duration polyethylene glycol fusion technique for increasing production of monoclonal antibody-secreting hybridomas. J Immunol Methods 81:223–228[CrossRef][Medline]

Lane TF, Iruela-Arispe ML, Johnson RS, Sage EH (1994) SPARC is a source of copper-binding peptides that stimulate angiogenesis. J Cell Biol 125:929–943[Abstract]

Lane TF, Sage EH (1994) The biology of SPARC, a protein that modulates cell-matrix interactions. FASEB J 8:163–173[Abstract/Free Full Text]

Maillard C, Malaval L, Delmas PD (1992) Immunological screening of SPARC/osteonectin in nonmineralized tissues. Bone 13:257–264[Medline]

Malaval L, Darbouret B, Preaudat C, Jolu JP, Delmas PD (1991) Intertissular variations in osteonectin: a monoclonal antibody directed to bone osteonectin shows reduced affinity for platelet osteonectin. J Bone Miner Res 6:315–323[Medline]

Nomura S, Wills AJ, Edwards DR, Heath JK, Hogan BL (1988) Developmental expression of 2ar (osteopontin) and SPARC (osteonectin) RNA as revealed by in situ hybridization. J Cell Biol 106:441–450[Abstract]

Porter PL, Sage EH, Lane TF, Funk SE, Gown AM (1995) Distribution of SPARC in normal and neoplastic human tissue. J Histochem Cytochem 43:791–800[Abstract/Free Full Text]

Puolakkainen P, Bradshaw AD, Kyriakides TR, Reed M, Brekken R, Wight T, Bornstein P (2003) Compromised production of extracellular matrix in mice lacking secreted protein, acidic and rich in cysteine (SPARC) leads to a reduced foreign body reaction to implanted biomaterials. Am J Pathol 162:627–635[Abstract/Free Full Text]

Sage H, Vernon RB, Decker J, Funk S, Iruela-Arispe ML (1989a) Distribution of the calcium-binding protein SPARC in tissues of embryonic and adult mice. J Histochem Cytochem 37:819–829[Abstract]

Sage H, Vernon RB, Funk SE, Everitt EA, Angello J (1989b) SPARC, a secreted protein associated with cellular proliferation, inhibits cell spreading in vitro and exhibits Ca+2-dependent binding to the extracellular matrix. J Cell Biol 109:341–356[Abstract]

Sage EH, Reed M, Funk SE, Truong T, Steadele M, Puolakkainen P, Maurice DH (2003) Cleavage of the matricellular protein SPARC by matrix metalloproteinase 3 produces polypeptides that influence angiogenesis. J Biol Chem 278:37849–37857[Abstract/Free Full Text]

Sasaki T, Gohring W, Mann K, Maurer P, Hohenester E, Knauper V, Murphy G (1997) Limited cleavage of extracellular matrix protein BM-40 by matrix metalloproteinases increases its affinity for collagens. J Biol Chem 272:9237–9243[Abstract/Free Full Text]

Soderling JA, Reed MJ, Corsa A, Sage EH (1997) Cloning and expression of murine SC1, a gene product homologous to SPARC. J Histochem Cytochem 45:823–835[Abstract/Free Full Text]

Villarreal XC, Malaval L, Mann KG, Delmas P, Long GL (1991) Epitope mapping of two monoclonal antibodies to the central portion of human osteonectin. Calcif Tissue Int 48:138–141[Medline]

Yan Q, Blake D, Clark JI, Sage EH (2003) Expression of the matricellular protein SPARC in murine lens. SPARC is necessary for the structural integrity of the capsular basement membrane. J Histochem Cytochem 51:503–511[Abstract/Free Full Text]

Yan Q, Clark JI, Wight TN, Sage EH (2002) Alterations in the lens capsule contribute to cataractogenesis in SPARC-null mice. J Cell Sci 115:2747–2756[Abstract/Free Full Text]

Yost JC, Sage EH (1993) Specific interaction of SPARC with endothelial cells is mediated through a carboxyl-terminal sequence containing a calcium-binding EF hand. J Biol Chem 268:25790–25796[Abstract/Free Full Text]