Journal of Histochemistry and Cytochemistry, Vol. 47, 1495-1506, December 1999, Copyright © 1999, The Histochemical Society, Inc.
SPARC, a Matricellular Glycoprotein with Important Biological Functions
Qi Yana and
E. Helene Sagea
a Department of Vascular Biology, Hope Heart Institute, Seattle, Washington
Correspondence to:
E. Helene Sage, Dept. of Vascular Biology, Hope Heart Institute, 528 18th Ave., Seattle, WA 98122.
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Summary |
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SPARC (secreted protein, acidic and rich in cysteine) is a unique matricellular glycoprotein that is expressed by many different types of cells and is associated with development, remodeling, cell turnover, and tissue repair. Its principal functions in vitro are counteradhesion and antiproliferation, which proceed via different signaling pathways. SPARC consists of three domains, each of which has independent activity and unique properties. The extracellular calcium binding module and the follistatin-like module have been recently crystallized. Specific interactions between SPARC and growth factors, extracellular matrix proteins, and cell surface proteins contribute to the diverse activities described for SPARC in vivo and in vitro. The location of SPARC in the nuclear matrix of certain proliferating cells, but only in the cytosol of postmitotic neurons, indicates potential functions of SPARC as a nuclear protein, which might be involved in the regulation of cell cycle progression and mitosis. High levels of SPARC have been found in adult eye, and SPARC-null mice exhibit cataracts at 12 months of age. This animal model provides an excellent opportunity to confirm and explore some of the properties of SPARC, to investigate cataractogenesis, and to study SPARC-related family proteins, e.g., SC1/hevin, a counteradhesive matricellular protein that might functionally compensate for SPARC in certain tissues. (J Histochem Cytochem 47:14951505, 1999)
Key Words:
SPARC, extracellular matrix, proliferation, counteradhesion, cataract, nuclear protein, SC1
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Introduction |
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SPARC (SECRETED PROTEIN, ACIDIC AND RICH IN CYSTEINE), also termed osteonectin as a major noncollagenous protein of bone matrix (Termine et al. 1981
) or BM-40 as a component of the matrix of a basement membrane tumor (Mann et al. 1987
), is a calcium binding matricellular glycoprotein secreted by many different types of cells. Matricellular proteins comprise a nonhomologous group of extracellular regulatory macromolecules that mediate cellmatrix interactions but may not contribute significantly to extracellular matrix structure (Bornstein 1995
). The matricellular class of secreted glycoproteins includes SPARC, thrombospondins 1 and 2, tenascins C and X, and osteopontin. Although structurally unrelated, they appear to perform related functions, e.g., they exhibit counteradhesive effects that lead to cell rounding and changes in cell shape that result in the disruption of cellmatrix interactions. Events that are characterized by changes in cell shape and motility often require expression of these proteins, e.g., tissue renewal, tissue remodeling, and embryonic development. Therefore, the matricellular proteins are different from traditional extracellular matrix proteins such as fibronectin, laminin, fibrillar collagens, and vitronectin, all of which are adhesive proteins and contribute to the structural stability of the extracellular matrix.
SPARC is a prototype of the matricellular proteins. As the product of a single gene, the sequence of SPARC has been highly conserved among species, with the mammalian (Termine et al. 1981
; Mason et al. 1986
), amphibian (Damjanovski et al. 1992
), and avian proteins (Bassuk et al. 1993
) showing more than 70% amino acid identity. SPARC from the nematode C. elegans has an identity to human SPARC of 38% (Schwarzbauer and Spencer 1993
). The expression pattern of this protein has provided clues to its potential functions. SPARC is expressed at high levels in bone tissue, is distributed widely in many other tissues and cell types (Maillard et al. 1992
), and is associated generally with remodeling tissues, e.g., tissues undergoing morphogenesis, mineralization, angiogenesis, and pathological responses to injury and tumorigenesis. Experiments in vitro provided evidence that SPARC (a) disrupts cell adhesion (Murphy-Ullrich et al. 1995
), (b) promotes changes in cell shape (Sage et al. 1989b
; Lane and Sage 1990
), (c) inhibits the cell cycle (Funk and Sage 1991
, Funk and Sage 1993
), (d) regulates cell differentiation (Bassuk et al. 1999
), (e) inactivates cellular responses to certain growth factors (Hasselaar and Sage 1992
; Raines et al. 1992
; Kupprion et al. 1998), and (f) regulates extracellular matrix and matrix metalloprotease production (Hasselaar et al. 1991
; Tremble et al. 1993
). SPARC has demonstrated activities in angiogenesis (reviewed in Sage and Vernon 1995
, Sage 1996
), tumorigenesis (reviewed in Jendraschak and Sage 1996
; Ledda et al. 1997
; Sage 1997
), cataractogenesis (Gilmour et al. 1998
; Norose et al. 1998
; Bassuk et al. 1999
), and wound healing (reviewed in Reed and Sage 1996
).
The expression of SPARC in adult tissues is usually more limited (Lane and Sage 1994
), e.g., SPARC has been identified in tumors (Schulz et al. 1988
; Porter et al. 1995
) and in tissues involved in repair and turnover. In contrast, the high levels of SPARC expressed in the adult eye relative to the immature eye (Yan et al. 1998
) indicate that this protein might play an important role in the maintenance of ocular physiological functions. Interestingly, SPARC-null mice of three genetic backgrounds all exhibit a predominant phenotype: opacity of the lens, or cataract (Gilmour et al. 1998
; Norose et al. 1998
). The SPARC-null experimental model therefore provides an excellent opportunity to understand the biology of SPARC and to confirm and explore some of the properties of this unique protein. This review focuses primarily on some new information about SPARC and also summarizes some important features of this protein.
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Structure and Properties of SPARC |
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SPARC is encoded by a single gene in all species studied to date (Mason et al. 1986
; Schwarzbauer and Spencer 1993
). Although SPARC (after cleavage of the signal sequence) is a 32-kD protein (Mason et al. 1986
), the secreted form migrates at 43 kD on SDS-PAGE, in part due to the addition of carbohydrate (Sage et al. 1984
). The human protein consists of 286 residues divided into three distinct domains (Figure 1). The N-terminal domain (residues 152 after a 17-amino-acid signal sequence) is an acidic region rich in Asp and Glu. Domain I binds several calcium ions with low affinity (Maurer et al. 1992
) and interacts with hydroxyapatite (Romberg et al. 1985
). It has therefore been implicated in the mineralization of cartilage and bone. This N-terminal domain contains the major immunological epitopes of SPARC (Stenner et al. 1984
; Malaval et al. 1991
). It also exhibits the most divergent sequence among the family of SPARC-like proteins (discussed below). Therefore, antibodies against SPARC have not been found to crossreact with or recognize SPARC-like proteins (Yan et al. 1998
; Gooden et al. 1999
).

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Figure 1.
Modular structure of human SPARC and the location and functions of synthetic peptides. Three domains and their residue numbers are shown. Boxes designate synthetic peptides of murine/human SPARC and their functions on cultured cells. PAI, plasminogen activator inhibitor; FN, fibronectin; TSP, thrombospondin; C, cysteine; MMPs, matrix metalloproteases. Reprinted with permission from Nature Medicine 3:144146, 1997.
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One feature of Domain I is the two adjacent N-terminal Glu3 and Glu4, which can act as amine acceptor sites in transglutaminasec-catalyzed crosslinking modification (Hohenadl et al. 1995
). Transglutaminases catalyze a Ca2+-dependent transfer reaction between the
-carboxamide group of a peptide-bound glutamine residue and various primary amines.
-Glutamyl-
-lysine crosslinks are formed in or between proteins by reaction with the
-amino group of lysine residues (Aeschlimann and Paulsson 1994
). SPARC is a predominant glutaminyl substrate (amine acceptor) in the chondrocyte matrix and is co-expressed with transglutaminasec in maturing cartilage (Aeschlimann et al. 1993
). The formation of SPARC oligomers or complexes by transglutaminase has been identified in cartilage and is believed to stabilize the extracellular matrix of this tissue (Aeschlimann et al. 1995
). SPARC is widespread in connective tissue and is also found in basement membrane (Mann et al. 1987
; Maillard et al. 1992
), where it presumably binds collagen IV. Whether SPARC is crosslinked to components in other matrices is presently not known. In addition to transglutaminase-catalyzed crosslinking, disulfide crosslinks between SPARC and another Cys-rich protein have also been reported (Zhou et al. 1998
). This tissue-specific posttranslational modification contributes to the diverse biological functions proposed for SPARC.
The second domain is a Cys-rich, follistatin-like (FS) domain (residues 53137), in which all the Cys residues are disulfide-bonded, and with an N-linked complex carbohydrate at Asn99. The FS domain is homologous to follistatin and Kazal-type protease inhibitors (Patthy 1991
), but neither inhibition of activin A-mediated mesoderm induction nor protease inhibitory function has been identified for SPARC (Maurer et al. 1992
). It also contains two copper binding sites (Vernon and Sage 1989
), one of which, the sequence KGHK (residues 119122), stimulates cell proliferation and angiogenesis (Funk and Sage 1993
; Lane et al. 1994
). Glycosylation of SPARC at Asn99 of the FS domain is another example of a posttranslational modification that alters the function of the protein. A difference in glycosylation between bone and platelet SPARC has been claimed to account for their structural and functional heterogeneity (Kelm and Mann 1991
). Bone SPARC has a high mannose-type oligosaccharide, whereas platelet SPARC has a complex-type oligosaccharide. Differences in their molecular weight are apparent by SDS-PAGE, and each form of SPARC exhibits a different collagen binding specificity (Kelm and Mann 1991
). Whether different glycosylation patterns exist for SPARC derived from other cells or tissues needs to be examined. For example, SPARC from bovine aortic endothelial cells binds albumin with high affinity (Sage et al. 1984
; Sage 1986
), whereas SPARC from mouse parietal yolk sac cells and fetal calf ligament fibroblasts binds collagens III and V preferentially. It is therefore possible that differences in the interaction of SPARC with other proteins could be due to carbohydrate.
Domain III (EC domain) (the extracellular calcium binding domain, residues 138286) is largely
-helical and contains a canonical pair of EF-hands, with high-affinity calcium binding sites. Based on the sequence, four putative domains were originally distinguished in SPARC (Engel et al. 1987
). Domain III was predominantly
-helical and contained a binding site for collagen IV (Mayer et al. 1991
). The C-terminal Domain IV contains the EF-hand motif with a high-affinity calcium binding site (Pottgiesser et al. 1994
) that also interacts with endothelial cells (Murphy-Ullrich et al. 1995
; Sage et al. 1995
). Further study indicated that domains III and IV were not independent but represent one domain (hence termed the EC domain) binding both calcium and collagens (Pottgiesser et al. 1994
). The structure of the FS and EC domains has been elucidated by X-ray crystallography (Hohenester et al. 1996
, Hohenester et al. 1997
). The EF-hand pair interacts tightly with an amphiphilic N-terminal helix and has been defined as a novel calcium binding module (Hohenester et al. 1996
). This domain also has a binding epitope of moderate affinity for collagen Types I and IV (Sasaki et al. 1998
). An endogenous protease cleavage site is located at a LeuLeu bond in position 197/198 in the
-helical region (Mann et al. 1987
; Mayer et al. 1991
). Extraction of SPARC from the mouse EngelbrethHolmSwarm tumor in the presence of 2-mercaptoethanol resulted in two disulfide-linked fragments (Mann et al. 1987
). Peptide bond 197/198 can be cleaved by endogenous proteolysis when SPARC is extracted from tissues. Analysis by SDS-PAGE under reducing conditions results in several peptides that can be detected by anti-SPARC IgG (unpublished data).
To map functional domains of SPARC, our laboratory has tested synthetic peptides that represent different regions of the protein. We have defined their activities on cultured cells and on the chick chorioallantoic membrane in vivo (Lane and Sage 1990
, Lane and Sage 1994
; Iruela-Arispe et al. 1995
), as summarized in Figure 1. These studies have proved useful in our understanding of the various functions of SPARC.
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Interaction with Other Proteins |
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SPARC is a high-affinity calcium binding protein. Both the function and structure of SPARC are modulated by Ca2+ ions. Binding of calcium causes an increase in the
-helicity and hence a change in conformation that reduces the susceptibility of the EC domain to proteinases and alters its affinity for collagen (Engel et al. 1987
; Maurer et al. 1992
; Pottgiesser et al. 1994
). The EF-hand pair in the EC domain of SPARC represents a novel protein module, with calcium as a key factor (Hohenester et al. 1996
). SPARC also binds Cu2+ and Fe2+ (Vernon and Sage 1989
). SPARC residues 113130 and KGHK (119122) (Lane et al. 1994
) were shown to bind Cu2+ with high affinity (Table 1). Peptides containing these sequences resulting from proteolysis of SPARC could regulate angiogenesis in vitro (Lane et al. 1994
) and in vivo (Iruela-Arispe et al. 1995
).
Binding to cytokines is another major characteristic of SPARC. SPARC was shown to bind the PDGF (platelet-derived growth factor) dimers AB and BB, but not AA. This specific interaction of SPARC with the B-chain prevented binding of PDGF to its receptors on fibroblasts (Raines et al. 1992
). The affinity of SPARC for this important growth factor could regulate the availability of PDGF dimers and thus affect the biological activity of PDGF. Vascular endothelial growth factor (VEGF) has low but significant sequence homology with PDGF (20% similarity in amino acid sequence). A recent study showed that SPARC also binds to VEGF and that this interaction interfered with the binding of VEGF to human microvascular endothelial cells. In addition, it reduced the association of VEGF with its cell surface receptor Flt-1. SPARC therefore inhibits endothelial cell proliferation induced by VEGF (Kupprion et al. 1999
). This activity was found to reside in peptides from the FS domain and in the EF-hand sequence of the EC domain (Kupprion et al. 1999
). Our laboratory had also shown that SPARC diminished the response of bovine endothelial cells to basic fibroblast growth factor (bFGF) with respect to migration and proliferation (Hasselaar and Sage 1992
). Recently, Motamed et al. 1999
have shown that SPARC inhibits the phosphorylation of FGF receptor 1 in myoblasts. The demonstration that SPARC interacts with these growth factors and inhibits cell proliferation induced by VEGF, bFGF, and PDGF indicates the importance of SPARC in angiogenesis.
Another important and widely-distributed growth factor, TGF-ß, which is associated with the rapid remodeling of connective tissues and has been shown to regulate the expression of extracellular matrix proteins (Kingsley 1994
; Reed et al. 1994
), was demonstrated to augment SPARC mRNA levels via a nuclear posttranscriptional mechanism in human fibroblasts (Wrana et al. 1991
). Recent data indicate that SPARC can magnify TGF-ß1 expression (mRNA and protein) in cultured mouse mesangial cells (Francki et al. in press
). SPARC-null mesangial cells also showed significantly decreased synthesis of TGF-ß1 mRNA, and addition of SPARC to SPARC-null cells in culture restored the expression of TGF-ß1 to levels typical of wild-type cells (Francki et al. in press
). Given the coincidence of TGF-ß and SPARC in embryonic development and wound healing (Sage et al. 1989a
; Pelton et al. 1991
; Reed et al. 1994
), the modulation of SPARC by TGF-ß and that of TGF-ß by SPARC are likely to be significant. Further experiments are needed to (a) address the mechanism(s) of the induction, (b) investigate whether there is a specific interaction between SPARC and TGF-ß, (c) test whether the induction also applies to other types of cells, and (d) confirm the findings in vivo.
The third group of molecules that interact with SPARC is the extracellular matrix proteins. SPARC has been shown to bind to collagen Type I (Termine et al. 1981
; Iruela-Arispe et al. 1996
), Types II and III (Sage et al. 1989b
), Type IV (Mayer et al. 1991
; Maurer et al. 1995
), Type V (Sage et al. 1989b
), and Type VIII (Sage et al. 1989b
). Specific interaction between SPARC and certain of the collagens could result in the remodeling of extracellular matrix. The binding of SPARC to collagen Types I and IV has received considerable attention. For example, in Mov-13 mice, which harbor an embryonic lethal mutation in the
1(I) collagen gene, the lack of collagen Type I significantly impairs the deposition of SPARC in the extracellular matrix (Iruela-Arispe et al. 1996
). Likewise, cells from SPARC-null mice produce significantly diminished levels of collagen I in comparison to cells from wild-type mice (Francki et al. in press
). Although a functional relationship between SPARC and collagen I is suggested by these studies, further work is needed to elucidate how SPARC regulates the production of collagen I and how their interaction might modulate counteradhesion and/or cell invasion (Iruela-Arispe et al. 1996
). SPARC protein isolated from tumor basement membrane, as well as recombinant human SPARC, was shown to bind to basement membrane collagen IV in the presence of calcium (Mayer et al. 1991
; Nischt et al. 1991
). Because collagen IV is a predominant component of basement membrane that forms a sheet-like scaffold, aberrations of SPARC could lead to an abnormal basal lamina. For example, SPARC-null mice were found to have ruptured lens capsules (Gilmour et al. 1998
). SPARC was also localized to most basement membranes along the body wall and sex muscles of C. elegans. A reduction of SPARC in C. elegans resulted in a lack of gut granules, small size, sterility, and larval lethality (Fitzgerald and Schwarzbauer 1998
).
Another extracellular matrix protein, the adhesive glycoprotein vitronectin, is deposited at different extracellular matrix sites, particularly in the vessel wall, the skin, and in association with various cancers (Dahlback et al. 1986
). It was recently shown that vitronectin was co-localized with SPARC in vivo and interacted via the heparin binding region with the C-terminal EF-hand of SPARC (Rosenblatt et al. 1997
). These two proteins have opposing effects on cell adhesion and might therefore account in part for the differential morphoregulatory processes required of cells in remodeling tissues.
Finally, SPARC has been shown to bind to the surface of endothelial cells (Table 1). Scatchard analysis indicated 4.2 x 107 sites/cell and a KI of 2.4 nM. Iodinated proteins from plasma membranes were affinity-chromatographed on a C-terminal SPARC peptide (residues 254273); several proteins with apparent masses ranging from 153 to 100 kD (unreduced) or from 153 to 122 kD (reduced) were eluted with the soluble peptide (Yost and Sage 1993
). These proteins represent candidates for a SPARC receptor(s) that mediates the biological activity of this protein on endothelial cells. Characterization of cell surface receptors for SPARC is critical to an understanding of the specific pathways mediated by this protein.
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Antiproliferation and Counteradhesion |
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Antiproliferation and counteradhesion are two major functions of SPARC defined in vitro, each of which proceeds via a different signaling pathway (Motamed et al. 1999
). Earlier studies indicated that SPARC is a potent cell cycle inhibitor that arrests cells in mid-G1, independently of discernible changes in cell shape (Funk and Sage 1991
, Funk and Sage 1993
; Sage et al. 1995
). SPARC also inhibits the proliferation of endothelial, smooth muscle, mesangial, and fibroblastic cells, stimulated in vitro with VEGF, PDGF, bFGF, and fetal bovine serum (FBS) (Hasselaar and Sage 1992
; Raines et al. 1992
; Kupprion et al. 1999
; Bradshaw et al. 1999
). Furthermore, this antiproliferative property of SPARC has been verified on cells derived from SPARC-null mice. Fibroblasts, mesangial cells, and smooth muscle cells isolated from SPARC-null mice exhibited a higher rate of proliferation relative to their wild-type counterparts. Null cells were also more sensitive to the inhibition of cell cycle progression induced by recombinant SPARC (Bradshaw et al. 1999
). We have attempted to understand the mechanism(s) of the cell cycle inhibition mediated by SPARC through several approaches. Although SPARC exhibits specific binding to bovine endothelial cells (Yost and Sage 1993
), neither a SPARC receptor nor a transmembrane SPARC binding protein has been defined. By the use of inhibitors of major signaling proteins, one can begin to identify mediators through which SPARC exerts its antiproliferative and counteradhesive functions. The general protein tyrosine kinase (PTK) inhibitors, such as herbimycin A and genistein, protected against the counteradhesive effect of SPARC on bovine aortic endothelial cells, but the antiproliferative effect was not susceptible to these inhibitors (Motamed and Sage 1998
). The study concluded that the counteradhesive effect of SPARC on endothelial cells is mediated through a tyrosine phosphorylation-dependent pathway, whereas its antiproliferative function is partially dependent on signal transduction via a G-protein-coupled receptor (Motamed and Sage 1998
). In addition, the increased proliferation rate of SPARC-null cells has been shown to be associated with an elevated level of the cell cycle regulatory protein cyclin A (Bradshaw et al. 1999
). If cell cycle progression is inhibited by SPARC, it is likely that SPARC functions in cell differentiation. In fact, SPARC has exhibited prominent expression during the terminal differentiation of cultured human keratinocytes (Ford et al. 1993
) and in terminally differentiated retinal ganglion cells in vivo (Yan et al. 1998
). Moreover, SPARC is believed to regulate terminal differentiation of lens epithelial cells, because disruption of the SPARC locus in mice resulted in the abnormal differentiation of lens fibers (Bassuk et al. 1999
).
SPARC is well known for its counteradhesive function, achieved in part by the dissolution of focal adhesion complexes and reorganization of actin stress fibers. Extensive studies in vitro have documented these findings and have defined the sequences in SPARC that are responsible for counteradhesion (Lane and Sage 1994
). The anti-spreading and focal adhesion disassembly in bovine endothelial cells mediated by SPARC or a C-terminal peptide containing the calcium binding EF-hand are believed to involve the tyrosine phosphorylation of focal adhesion-associated proteins, e.g., paxillin (Motamed and Sage 1998
; Young et al. 1998
). Cell adhesion and subsequent spreading on a compatible substrate are essential requirements for proper growth and survival of cells. Disruption of the extracellular matrix could lead to cell rounding and apoptosis (Chen et al. 1997
). Conversely, changes in cell shape are temporarily necessary for cells undergoing migration and proliferation. Counteradhesive proteins might also regulate cell survival by their control of cell shape. For example, thrombospondin 1 and its Type I repeat peptides induced apoptosis of endothelial cells (Guo et al. 1997
). In contrast, SPARC has not been found to induce apoptosis in bovine retinal microvascular endothelial cells (unpublished data), although it induces endothelial cell rounding (Lane et al. 1994
).
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SPARC in the Nucleus |
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SPARC is a secreted glycoprotein. Cultured cells often exhibit a typical staining pattern for SPARC, i.e., a granular, perinuclear location that outlines the Golgi apparatus. The culture medium of subconfluent cells also contains abundant SPARC protein, consistent with the concept that SPARC is secreted. However, recent work in our laboratory has challenged this view and has raised questions concerning potential functions of SPARC as a nuclear or cytoplasmic protein (Gooden et al. 1999
). The novel observation in this study is that SPARC was detected in the nuclear matrix of cells by immunocytochemistry, both in vivo (chick embryonic Day E2) and in vitro (adult bovine aortic endothelial cells pretreated to remove soluble proteins and chromatin; embryonic chicken cells, E2 and E10). Moreover, SPARC was expressed at high levels in the cytoplasm of M-phase cells at metaphase and anaphase. These findings were confirmed by the use of several anti-SPARC antibodies and by immunoblotting of isolated nuclei and cytoplasmic fractions. Furthermore, it was shown that SPARC was taken up and translocated to the nuclei of cultured embryonic chicken cells (Gooden et al. 1999
). We have analyzed SPARC expression in E12 chick embryo retina by immunocytochemistry in vivo (Figure 2). Our results are consistent with the report by Gooden et al. 1999
: (a) SPARC is a component of the nuclear matrix during interphase; (b) expression of intracellular SPARC is correlated with specific stages of the cell cycle, e.g., strong immunoreactivity was detected during metaphase (Figure 2B and Figure 2C, arrows) and anaphase (Figure 2B, black arrowhead); and cells of late telophase were devoid of SPARC immunoreactivity (Figure 2B, white arrowheads; Figure 2C, arrowhead); and (c) ganglion cell neurons, which are postmitotic, showed cytoplasmic staining but no nuclear labeling (Figure 2D, arrows), similar to that described for adult bovine retinal ganglion cell neurons (Yan et al. 1998
). Control experiments have consistently shown that the recognition of SPARC by anti-SPARC IgG is a specific reaction (Figure 2A). It is possible, however, that a novel protein containing a crossreactive epitope, or an alternatively-spliced form of SPARC, might result in a similar staining pattern. However, the protein recognized by the anti-SPARC antibodies was approxmately 43 kD, and no other bands were detected in the nuclear fraction (Gooden et al. 1999
).

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Figure 2.
SPARC in E12 chick embryo retina. The eyeball was fixed in methyl Carnoy's solution (60% methanol, 30% chloroform, and 10% glacial acetic acid) for 24 hr at 4C, embedded in paraffin, and sectioned at 6 µm. (A) Control for primary antibody, mouse IgG1 at 100 nM. (BD) Monoclonal anti-SPARC IgG1 (100 nM; Haematological Technologies, Essex Junction, VT). Immunoreactivity was visualized with a mouse IgGavidinbiotinylated peroxidase complex developed with diaminobenzidine and hydrogen peroxide. Sections were counterstained with hematoxylin. A small proportion of the chick embryo retinal cells were dividing, and different stages of M-phase cells can be seen at the outer ventricular border (the pigment epithelium is off the bottom of the photos, AC). Retinal cells in the control section (A, includes two metaphase cells, arrows) showed no immunoreactivity. In the anti-SPARC IgG-treated sections (BD), SPARC protein was detected in the nuclei of metaphase (B,C, arrows), anaphase cells (B, black arrowhead), and some retinal cells at unidentified stages (C). However, SPARC protein was not detected in the cells of late telophase or at the end of cytokinesis (B, white arrowheads; C, arrowhead). Staining with anti-SPARC IgG was apparent in ganglion neuron cytosol (D, arrows) but their nuclei were devoid of reactivity. Bar = 10 µm.
Figure 3.
Expression of SPARC protein in adult bovine lens epithelium. Control sections exposed to mouse IgG1 (A,a). Sections exposed to monoclonal anti-SPARC IgG1 (B,b and C,c). Immunohistochemistry was performed with avidinbiotinperoxidase and sections were counterstained with hematoxylin (AC), Texas Red-conjugated goat anti-mouse IgG (ac). The lens epithelial nuclei were counterstained for 30 min with Hoechst 33258 fluorochrome (blue, c) (0.1 µg/ml in distilled water; Flow Laboratories, McLean, VA). SPARC is prominent in the cytosol of lens epithelial cells, but not in the nuclei (B,c, arrows). C is an en face section of lens epithelium. Note the predominance of SPARC protein (nuclei are obscured by cytoplasmic staining). The basement membrane showed no immunoreactivity with anti-SPARC IgG1. BM, basement membrane. Bar = 10 µm.
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The association of SPARC with the nucleus in active, proliferating embryonic cells, but not in cells that are mature (Yan et al. 1998
) or postmitotic (Figure 2D), indicates a potential role of SPARC in the regulation of mitosis during development. Further studies will address a number of questions. What is the role of intracellular SPARC? What is the mechanism responsible for the nuclear translocation of SPARC? What are the molecular characteristics of nuclear SPARC? Several secreted proteins have recently been reported to function not only via cell surface receptor-mediated intracellular signaling but via nuclear translocation and/or direct association with nuclear substrates (Henderson 1997
). Understanding why SPARC is present in the nuclear matrix will contribute greatly to our appreciation of the biological functions of this protein.
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SPARC and Lens |
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A SPARC-null animal model was necessary to confirm observations in vitro, because it eliminates any background contributed by endogenous SPARC. Two laboratories generated colonies of SPARC-null mice in different strains. Targeted disruption of the SPARC locus in mouse embryonic stem cells was achieved by the insertion of neomycin-resistance genes into exon 4 of the SPARC gene derived from 129/Sv mice (Norose et al. 1998
). Cells carrying the disrupted SPARC gene were injected into C57Bl/6J blastocysts to generate chimeras. Another SPARC-null colony was produced by Gilmour et al. 1998
. A targeted replacement of exon 6 in the SPARC allele deleted half of the follistatin module and resulted in premature termination of transcription. Chimeras derived from targeted embryonic stem cells (129/SvEv x MF1) transmitted the disrupted SPARC allele through the germline and produced neither SPARC mRNA nor protein (Gilmour et al. 1998
). SPARC-null mice are generally indistinguishable from their wild-type counterparts in viability, fertility, body size, and weight. However, some of the SPARC-null mice have curled or kinked tails (A. Bradshaw, personal communication), develop osteopenia (Delaney et al. 1998), and show accelerated healing of dermal wounds (Reed et al. unpublished data). Most prominent is the development of lenticular opacity or cataract with 100% penetrance in SPARC-null mice, regardless of the genetic background. Lens opacity was observed in the posterior subcapsular cortex of the SPARC-null mice by slit-lamp ophthalmoscopy as early as 12 months after birth (Norose et al. 1998
; Bassuk et al. 1999
). This opacity increases gradually and persistently as the mice grow; by 68 months, all of the SPARC-null mice, independent of sex, displayed mature cataract. The report by Gilmour et al. 1998
claimed severe cataract formation and rupture of the lens capsule around 6 months of age. The timing, in fact, is consistent with the observations of Norose et al. 1998
and Bassuk et al. 1999
, who used slit-lamp ophthamoscopy to detect early posterior opacity, which is invisible to the human eye at this stage. This predominant phenotype is very interesting, partly because it was unexpected. In fact, the lens is considered to be an organ extremely sensitive to any changes in the surrounding environment; it responds to pathological stimulation by loss of its transparency, an easily detected phenotype. In addition, lens epithelium is advantageous for study because it contains a single cell type (the lens epithelial cell) that can undergo both mitosis and terminal differentiation to form the lens fiber cell, which constitutes the body of the lens throughout life (Wride 1996
). The epithelial cell has no contact with any other cell type, and it presumably receives nutrients and growth/differentiation signals from the aqueous humor (anterior) and vitreous humor (posterior) via the lens capsule. Therefore, the SPARC-null mouse is a fortuitous model in which to study both cataractogenesis and the biological functions of SPARC.
Why does the lack of SPARC in the mouse lead to cataract? Although the mechanism of cataractogenesis is far from clear, lens epithelial cell proliferation, differentiation, and migration are critical processes in the normal development and maintenance of lens transparency (Zelenka et al. 1997
). In addition, extracellular matrix proteins comprise the lens capsule and mediate its appropriate physiological permeability as well as its structure. Because SPARC is involved in the regulation of cell proliferation, migration, shape, adhesion, and matrix production, the lack of SPARC might well contribute to cataract formation. Moreover, SPARC might be concerned with other aspects related to cataractogenesis, e.g., the regulation of gap junction formation or dissolution (Gong et al. 1997
). Because the lens consists of only one cell type, gene compensation by other cells is less likely in lens in comparison to other types of tissues, e.g., skin. SPARC-like proteins such as SC1 might be expressed to a lesser degree in the lens or vitreous of SPARC-null mice. Alternatively, SPARC-like proteins might not rescue the altered functions in the lens. These hypotheses are under investigation in our laboratory.
SPARC appears to be an essential gene for lens transparency in mice; does the same conclusion apply to human lens? The importance of SPARC to the human lens is not known. Although the high level of homology between the mouse and human SPARC genes indicates that the cataract found in SPARC-null mice could be manifested in humans, more study is needed. We started to explore this question by characterization of the expression of SPARC protein in human and bovine eye (unpublished observations). Lens epithelial cells have been shown to synthesize and secrete SPARC (Sawhney 1995
; Bassuk et al. 1999
), an observation we have recently confirmed in bovine lens (Figure 3). Adult bovine lens epithelial cells contain abundant SPARC in the cytosol (Figure 3C and Figure 3c), but nuclei were devoid of staining (Figure 3B and Figure 3c, arrows). Figure 3c, an en face section of lens epithelium, shows prominent expression of SPARC protein in the cells. The basement membrane showed no immunoreactivity with anti-SPARC IgG by immunocytochemical staining. However, the basement membrane is actually a virtual repository for SPARC, as revealed by immunoblotting (unpublished observations) and radioimmunoprecipitation (Maillard et al. 1992
). That SPARC is not detectable in the lens basement membrane by immunocytochemical methods (Figure 3; and Bassuk et al. 1999
) might be explained as follows: (a) the three-dimensional structure of SPARC in the lens basement membrane might preclude its recognition by anti-SPARC IgGs; (b) SPARC might be masked by other molecule(s); or (c) binding to collagen IV, a major constituent of lens basement membrane, might have altered or masked the epitope. Interestingly, we have found that the most abundant sources of SPARC for the human and bovine lens are the aqueous and vitreous humors (unpublished observations). This study clarifies that SPARC is produced in the adult human eye and suggests that SPARC might be important in ocular physiology. What would be the role of SPARC in human cataract? A recent study reported that SPARC mRNA was augmented in epithelia dissected from human age-related cataractous lenses (Kantorow et al. 1998
). Whether the increased levels of SPARC are the cause or the result of senile cataract is unknown. We do know that deficiency of SPARC results in cataract in mice. Likewise, too much of this protein is likely to disturb the normal chemical balance of the lens and contribute to the disease as well.
 |
SPARC Family-related Proteins |
---|
Four proteins have been described to date in which the FS and EC domains (including the EF-hand-related calcium binding site) are well-conserved (Figure 4). These proteins are the rat brain protein SC1 (Johnston et al. 1990
; McKinnon et al. 1996
) and its human homologue hevin, isolated from high endothelial venules (Girard and Springer 1995
), the quail retina protein QR1 (Guermah et al. 1991
), the TGF-ß-induced protein TSC-36/glioma-secreted follistatin-related protein FRP (Shibanuma et al. 1993
; Zwijsen et al. 1994
), and the human testicular proteoglycan testican (Alliel et al. 1993
) (Figure 4). Within this group, a follistatin-like module is followed by the EC domain (Figure 4), each of which exhibits similarity among the SPARC family of proteins. However, the acidic Domain I exhibits considerably less identity within this group. With respect to functional similarities, hevin has been shown to inhibit the attachment and spreading of human endothelial cells, because hevin-treated cells displayed a rounded shape and did not form focal adhesions (Girard and Springer 1996
). Moreover, both QR1 and TSC-36 are associated with cell cycle inhibition (Shibanuma et al. 1993
; Casado et al. 1996
).

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Figure 4.
SPARC and related proteins. Three prominent modules are shown. The N-terminal domains I of each protein exhibit considerably reduced identity, although all are acidic. A follistatin-like, 10-Cys-containing module (FS) is followed by the extracellular calcium-binding domain (EC). TY designates a thyroglobulin-like domain in testican. Triangles represent characterized modules in testican and TSC-36/FRP. Modified from Maurer et al. 1995.
|
|
Among the SPARC-related proteins, SC1 exhibits the highest similarity to SPARC, with an identity between mouse SPARC and mouse SC1 of 70% at the amino acid level (Soderling et al. 1997
). Comparison of the distribution of SC1 and SPARC mRNA in mouse tissues showed noncoincidental expression in some organs but coincidental expression in adrenal gland and brain (Soderling et al. 1997
). It has also been demonstrated that SPARC and SC1 are expressed at high levels in the normal adult central nervous system (Mendis and Brown 1994
; Mendis et al. 1995
, Mendis et al. 1996
; McKinnon and Margolskee 1996
). The overlapping expression of SC1 and SPARC in certain tissues raised the possibility that they might compensate functionally, at least in part, for each other.
QR1 is a retina-specific gene encoding an extracellular protein that regulates retinal differentiation (Casado et al. 1996
). Quail QR1 and mouse SPARC share 61% identity in their final 200 residues. The expression of SPARC, SC1, and QR1 in the retina and brain indicates that they are likely to mediate functions in the central nervous system and in the maintenance of ocular functions (Yan et al. 1998
), although their roles are not defined at this time.
SPARC- and/or SC1-null mice could be the best models to address the question of functional compensation of the SPARC family-related proteins. The cataract exhibited in SPARC-null mice is an exciting opportunity for us to test whether SC1 could correct this phenotype in vivo. It is possible that SC1 might not be available to the lens. Therefore, exogenous SC1 delivered to the vitreous and/or aqueous humor might prevent cataract in SPARC-null mice if SC1 is a functional equivalent of SPARC.
SPARC has emerged as an intriguing protein with a unique set of functions. Its counteradhesive and antiproliferative properties have been verified in various types of cells in vitro. The SPARC-null animal models have revealed the necessity and importance of this protein to normal physiology. Future studies will address how SPARC mediates counteradhesion and antiproliferation, the specific interactions between SPARC and other molecules, why the lack of SPARC causes cataract, the role of SPARC in the nucleus, and whether SC1/hevin, a counteradhesive protein structurally related to SPARC, is a functional equivalent of SPARC. We are only just beginning to appreciate the breadth of function conferred by the matricellular group of proteins.
 |
Footnotes |
---|
Presented at the 50th Meeting of the Histochemical Society, April 1617, 1999, Bethesda, Maryland. 
 |
Acknowledgments |
---|
Supported by National Science Foundation grant EEC-9814404, National Institutes of Health grant GM-40711, a pilot study award from the Nathan Shock Center for Aging Research at the University of Washington, and by a grant from the J. Hartford Foundation to QY.
We thank Drs John Clark and Anita Hendrickson for advice and comments.
Received for publication June 1, 1999; accepted June 1, 1999.
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