Functional Analysis of the Matricellular Protein SPARC with Novel Monoclonal Antibodies
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
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Summary |
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Key Words: SPARC collagen matricellular monoclonal antibody extracellular matrix
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Introduction |
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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 2001; Hohenester and Engel 2002
). 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)
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 IV and VIII, vitronectin, and thrombospondin-1 (Brekken and Sage 2001
). SPARC has therefore been proposed to modulate cellECM 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. 1997
) 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. 1987; Nomura et al. 1988
; Sage et al. 1989a
; Brekken and Sage 2001
). 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. 1987
; Sage et al. 1989a
). Lane et al. (1994)
showed SPARC in angiogenic microvasculature of developing brain and dermal wounds, and Iruela-Arispe et al. (1995)
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. 1995
). 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. 2001,2002
) and implanted biomaterials (Puolakkainen et al. 2003
), or tumor cells (Brekken et al. 2003
). 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 36 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. 2002
,2003
). 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 MAbSPARC complexes or are sensitive to conformational changes of SPARC induced by the MAbs themselves.
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Materials and Methods |
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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. 2000) 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 1015-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. 1997
), 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 ASepharose 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) 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 0.10.5 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 1970) 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
15%, collagen III
13%, laminin-1
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 1993) 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. 2000) 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. 2003). 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 GSepharose beads. 200 µl of anti-SPARC MAb tissue culture supernatant or 25 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 GSepharose beads and the precipitate was washed three times with 20% lysis buffer (Brekken et al. 2000). 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 (57 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 1991) (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 512) 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 90100% 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 SPARCMab solution was added to a 96-well tissue culture plate (Corning) in triplicate. BAECs (passages 57) were counted and were added to the SPARCMAb 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, 1994), 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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Results |
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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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Discussion |
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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), 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. 1991
; Maillard et al. 1992
). For example, MAbs ON2 (also known as Mab2) and IIIA3A8 were both characterized as binding to the central region of SPARC between amino acids 18146 (Villarreal et al. 1991
), whereas other anti-SPARC MAbs (Mab3, Mab6, Mab12) bind to distinct although undefined epitopes (Malaval et al. 1991
; Maillard et al. 1992
).
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. 1989).
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.
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Acknowledgments |
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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.
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Footnotes |
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2 Present address: Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL.
3 Present address: Departments of Surgery and Pharmacology and The Hamon Center for Therapeutic Oncology Research, UT-Southwestern Medical Center, Dallas, TX.
4 Present address: Department of Medicine, Gazes Cardiac Research Institute, Medical University of South Carolina, Charleston, SC.
5 Present address: Department of Immunology, Zymogenetics, Inc., Seattle, WA.
Received for publication August 26, 2003; accepted February 25, 2004
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