SPARC (osteonectin/BM-40)

SPARC (Secreted ProteinAcidic and Rich in Cysteine) is a prototype of a family of biologically active glycoproteins that bind to cells and to extracellular matrix (ECM) components. It is expressed spatially and temporally during embryogenesis, tissue remodeling and repair. SPARC is a modular protein (34 kDa) comprised of three structural domains, one or more of which are implicated in the regulation of cell adhesion, proliferation, matrix synthesis/turnover. Rapid proteolysis of SPARC by extracellular proteases accounts for its transient detection in the extracellular environment. The proposed roles of SPARC in the development of cataracts and the regulation of angiogenesis during wound healing and tumor growth account for the recent attention it has received from the biomedical community.     

Calcium-dependent binding of basement membrane protein BM-40 (osteonectin, SPARC) to basement membrane collagen type IV

Basement membrane protein BM-40, prepared from the mouse Engelbreth-Holm-Swarm tumor, was used in native, denatured and proteolytically processed form for binding to various extracellular matrix proteins. BM-40 and its derivatives were also characterized by CD spectroscopy, calcium binding and epitope analysis. Of several basement membrane proteins tested only collagen IV showed a distinct and calcium-dependent binding of BM-40 in an immobilized ligand assay. This interaction was specific as shown by a low activity of other collagen types (I, III, V, VI) in direct binding and competition assays. The binding was reduced or abolished by metal-ion-chelating or chaotropic agents, high salt and reduction of disulfide bonds in BM-40. Fragment studies indicated that domains III (alpha-helix) and/or IV (EF hand) of BM-40 possess the binding site(s) for collagen IV, while the N-terminal domains I and II provide the major antigenic determinants. A major BM-40-binding site on collagen IV was dependent on a triple-helical conformation and could be localized to a pepsin fragment from the central portion of the triple-helical domain, in agreement with electron microscopic visualization of BM-40--collagen-IV complexes.     

Cloning and complete amino acid sequences of human and murine basement membrane protein BM-40 (SPARC, osteonectin)

Amino acid sequences of 285 and 286 residues, respectively, were deduced for mouse and human BM-40 from cDNA clones isolated from expression libraries. The sequences showed 92% identity and were also essentially identical to those of bone osteonectin and of the parietal endoderm protein SPARC. About 60% of the mouse BM-40 sequence was confirmed by Edman degradation. Two of the seven disulfide bonds were localized which apparently separate two distinct domains of mouse BM-40.     

The matricellular protein SPARC/osteonectin as a newly identified factor up-regulated in obesity

Alterations in the expression level of genes may contribute to the development and pathophysiology of obesity. To find genes differentially expressed in adipose tissue during obesity, we performed suppression subtractive hybridization on epididymal fat mRNA from goldthioglucose (GTG) obese mice and from their lean littermates. We identified the secreted protein acidic and rich in cysteine (SPARC), a protein that mediates cell-matrix interactions and plays a role in modulation of cell adhesion, differentiation, and angiogenesis. SPARC mRNA expression in adipose tissue was markedly increased (between 3- and 6-fold) in three different models of obesity, i.e. GTG mice, ob/ob mice, and AKR mice, after 6 weeks of a high fat diet. Immunoblotting of adipocyte extracts revealed a similar increase in protein level. Using a SPARC-specific ELISA, we demonstrated that SPARC is secreted by isolated adipocytes. We found that insulin administration to mice increased SPARC mRNA in the adipose tissue. Food deprivation had no effect on SPARC expression, but after high fat refeeding SPARC mRNA levels were significantly increased. Our results reveal both hormonal and nutritional regulation of SPARC expression in the adipocyte, and importantly, its alteration in obesity. Finally, we show that purified SPARC increased mRNA levels of plasminogen activator inhibitor 1 (PAI-1) in cultured rat adipose tissue suggesting that elevated adipocyte expression of SPARC might contribute to the abnormal expression of PAI-1 observed in obesity. We propose that SPARC is a newly identified autocrine/paracrine factor that could affect key functions in adipose tissue physiology and pathology.     

Structural variability of BM-40/SPARC/osteonectin glycosylation: implications for collagen affinity

We performed a detailed investigation of N-glycan structures on BM-40 purified from different sources including human bone, human platelets, mouse Engelbreth-Holm-Swarm (EHS) tumor, and human BM-40 recombinantly expressed in 293 and osteosarcoma cells. These preparations were digested with endoglycosidases and N-glycans were further characterized by sequential exoglycosidase digestion and high-performance liquid chromatography (HPLC) analyses. Bone BM-40 carries high-mannose structures as well as biantennary complex type N-glycans, whereas the protein from platelets and 293 cells has exclusively bi- and triantennary complex type structures. BM-40 derived from the EHS tumor carries biantennary complex type and additional hybrid structures. Using the osteosarcoma-derived MHH-ES1 cell line we successfully expressed a recombinant BM-40 that bears at least in part the bone-specific high-mannose N-glycosylation in addition to complex type and hybrid structures. Using chromatography on Concanavalin-A Sepharose, we further purified a fraction enriched in high-mannose structures. This array of differentially glycosylated BM-40 proteins was assayed by surface plasmon resonance measurements to investigate the binding to collagen I. BM-40 carrying high-mannose structures binds collagen I with higher affinity, suggesting that differentially glycosylated forms may have different functional roles in vivo.     

Association of SPARC (osteonectin, BM-40) with extracellular and intracellular components of the ciliated surface ectoderm of Xenopus embryos

SPARC (Secreted Protein, Acidic, Rich in Cysteine) was detected by immunohistochemistry in the sensorial layer of the bilayered embryonic epidermis of Xenopus laevis during neurulation, when a subset of the sensorial cells are selected to differentiate into ciliated cell precursors. After the ciliated cells had intercalated into the outer layer and had undergone ciliogenesis, intense SPARC immunostaining was associated with the cilia and remained associated with the cilia throughout their persistence on the epidermis. Circumferential SPARC immunostaining was also detected at the interface between surface epithelial cells. Animal cap explants indicated that the embryonic activation of SPARC expression in the dorsal ectoderm does not require signaling from factors secreted by the underlying mesoderm. Immunoelectron microscopy revealed that SPARC is intimately associated with the 9 + 2 microtubule arrays of cilia. Our data indicate that SPARC plays a role in the development and function of the surface ciliated epidermis of Xenopus embryos. We propose that the counter-adhesive activity of SPARC facilitates the intercalation of ciliary cell precursors to the surface epithelial layer, where its Ca(2+)-binding abilities promote cell-cell adhesion. Based on its association with ciliary microtubule arrays, we also propose that intracellular SPARC may play a role in regulating ciliary beat frequency and polarity.     

Hevin/SC1, a matricellular glycoprotein and potential tumor-suppressor of the SPARC/BM-40/Osteonectin family

Hevin is an extracellular matrix-associated, secreted glycoprotein belonging to the secreted protein acidic and rich in cysteine (SPARC) family of matricellular proteins. It contains three conserved structural domains that are implicated in the regulation of cell adhesion, migration, and proliferation. Hevin is expressed during embryogenesis and tissue remodeling and is especially prominent in brain and vasculature. Its down-regulation in a number of cancers and the possibility of its functional compensation by SPARC has led to recent interest in hevin as a tumor suppressor and regulator of angiogenesis.     

Solubilization of protein BM-40 from a basement membrane tumor with chelating agents and evidence for its identity with osteonectin and SPARC

Up to 80% of the calcium-binding protein BM-40 could be extracted from a tumor basement membrane with a physiological buffer containing 10 mM EDTA. About half of its amino acid sequence was determined by Edman degradation demonstrating identity with cDNA deduced sequences of bone osteonectin and SPARC.     

Secreted protein acidic and rich in cysteine (SPARC/osteonectin/BM-40) binds to fibrinogen fragments D and E, but not to native fibrinogen.

Secreted protein acidic and rich in cysteine (SPARC/osteonectin/BM-40) is a matricellular protein that functions in wound healing. Fibrinogen is a plasma protein involved in many aspects of wound healing, such as inflammation, fibrosis and thrombosis. In this study, the binding of SPARC to both native and plasmin-cleaved fibrinogen under physiological conditions was examined by the use of a surface plasmon resonance (SPR) biosensor. We show that SPARC binds to plasmin-cleaved fibrinogen, but not to native fibrinogen. SPARC binds to both fibrinogen fragments D and E fg D and fg E with similar dissociation constants (8.67 x 10(-8) M for Fg D and 1.61 x 10(-7) M for Fg E). Results from endothelial cell proliferation assays show that the binding of SPARC to Fg E suppressed the inhibition of proliferation by SPARC, whereas the binding of SPARC to Fg D did not influence the activity of SPARC on the cell cycle. The interaction of SPARC with fibrinogen fragments D and E, which are produced as a result of proteolytic activation of fibrinolysis, reveals potential storage sites in provisional extracellular matrix for SPARC during the wound healing process and indicates a regulatory role of SPARC in fibrinolysis and angiogenesis.     

Immunochemical and tissue analysis of protease generated neoepitopes of BM-40 (osteonectin, SPARC) which are correlated to a higher affinity binding to collagens.

Proteolytic cleavage at single sites in the extracellular calcium-binding module of BM-40/SPARC/osteonectin either by an unknown endogenous protease (L197-L198) or several matrix metalloproteinases (E196-L197) was previously shown to enhance collagen binding activity 10-fold. Polyclonal rabbit antibodies were now obtained against synthetic peptide antigens containing either an N-terminal L197 or L198 and characterized by radioimmunoassay, ELISA, immunoblots and immunohistology. These neoepitope-specific antibodies reacted with proteolytically processed but not with uncleaved mouse and human BM-40. The cross-reaction between the two different neoepitopes was < 1%, indicating the immunodominant role of the N-terminal residues. Analysis of a basement membrane producing mouse tumor demonstrated extensive cleavage at the L198 site, which correlated with a calcium-dependent binding to the matrix. A variable degree of this cleavage was also detected in BM-40 obtained from adult mouse bone and several other tissues. Negligible or much lower levels of conversion were detected at the MMP-specific L197 site, however. Immunogold staining of mouse heart and a basement membrane-producing mouse tumor showed a distinct extracellular labeling for BM-40 and the L198 neoepitope but only a very weak reaction for the L197 neoepitope. This strongly indicates that these neoepitopes are generated in vivo and emphasizes a specific biological role for the proteolytic activation of BM-40.     

High-affinity and low-affinity calcium binding and stability of the multidomain extracellular 40-kDa basement membrane glycoprotein (BM-40/SPARC/osteonectin).

Reversible binding of calcium ions to a single high-affinity binding site in the 40-kDa basement membrane protein (BM-40) caused a 33% increase of alpha-helicity, an about 60% change in intrinsic fluorescence and a dramatic increase of the rate of cleavage by alpha-chymotrypsin. All these effects exhibited identical dependencies on calcium concentration from which a dissociation constant Kd = 0.6 microM was determined. Calcium release was accompanied by an increase of the frictional ratio in solution but not by denaturation which occurred at about equal guanidine.HCl concentration for both calcium-saturated and calcium-depleted protein (midpoint 1.5 M). The cleavage sites for alpha-chymotrypsin are located in or near to the EF-hand domain IV of calcium-depleted BM-40 (also known as SPARC, i.e. secreted protein acidic and rich in cysteine, and osteonectin). These and other data indicate that binding occurs in the EF-hand domain from which a large conformational change is transmitted. Low-affinity calcium-binding sites in the N-terminal glutamic-acid-rich domain I of BM-40 were identified by human leukocyte elastase which was found to cleave very specifically in the middle of this domain. From the increase of cleavage rate with increasing calcium concentration a Kd greater than or equal to 10 mM was estimated. It is suggested that variations of calcium levels in the extracellular space in this range may regulate functions of BM-40 such as collagen binding and that high-affinity binding is important for stabilization, folding and secretion during biosynthesis.     

Chaperone-like activity revealed in the matricellular protein SPARC

SPARC (Secreted Protein, Acidic and Rich in Cysteine) is a matricellular glycoprotein that modulates cell proliferation, adhesion, migration, and extracellular matrix (ECM) production. In this report chaperone-like activity of SPARC was identified in a thermal aggregation assay in vitro. Ultraviolet circular dichroism (UVCD) spectroscopy determined that SPARC was stable at temperatures up to 50 degrees C. Unfolding and aggregation of the chaperone target protein, alcohol dehydrogenase (ADH), were initiated at 50 degrees C. SPARC inhibited the thermal aggregation of ADH in a concentration-dependent manner, with maximal inhibition at a 1:4 molar ratio of SPARC:ADH. Synergy between the chaperone-like activities of SPARC and alphaB-crystallin, a small heat shock protein and molecular chaperone in the lens, was observed in SPARC-alphaB-crystallin double -/- mice.     

Impaired wound healing in mice lacking the basement membrane protein nidogen 1

Nidogens 1 and 2 are ubiquitous basement membrane (BM) components, whose interactions in particular with laminin, collagen IV and perlecan have been considered important for BM formation. Genetic deletion of either NID gene does not reveal BM alterations suggesting compensatory roles for nidogens 1 and 2. However, neurological deficits in nidogen 1 null mice, not seen in the absence of nidogen 2, also suggest isoform specific functions. To test this further, skin wound healing which requires BM reformation was studied in adult nidogen 1 deficient mice. Although re-epithelialization was not altered, the newly formed epidermis showed marked hyperproliferation and a delay in differentiation at day 10 post injury. Distinct to control wounds, there was also considerable α-smooth muscle actin staining in the dermis of nidogen 1 deficient wounds at this time point. Further, laminin deposition and distribution of the β1 and β4 integrin chains were also significantly altered whereas the deposition of other BM components, including nidogen 2, was unchanged. Surprisingly, these differences between control and mutant wounds at day 10 post wounding did not affect the ultrastructural appearance of the dermo-epidermal BM suggesting a non-structural role for nidogen 1 in wound repair.     

SPARC, a matricellular protein: at the crossroads of cell-matrix.

SPARC is a multifunctional glycoprotein that belongs to the matricellular group of proteins. It modulates cellular interaction with the extracellular matrix (ECM) by its binding to structural matrix proteins, such as collagen and vitronectin, and by its abrogation of focal adhesions, features contributing to a counteradhesive effect on cells. SPARC inhibits cellular proliferation by an arrest of cells in the G1 phase of the cell cycle. It also regulates the activity of growth factors, such as platelet-derived growth factor (PDGF), fibroblast growth factor (FGF)-2, and vascular endothelial growth factor (VEGF). The expression of SPARC in adult animals is limited largely to remodeling tissue, such as bone, gut mucosa, and healing wounds, and it is prominent in tumors and in disorders associated with fibrosis. The crystal structure of two of the three domains of the protein has revealed a novel follistatin-like module and an extracellular calcium-binding (EC) module containing two EF-hand motifs. The follistatin-like module and the EC module are shared by at least four other proteins that comprise a family of SPARC-related genes. Targeted disruption of the SPARC locus in mice has shown that SPARC is important for lens transparency, as SPARC-null mice develop cataracts shortly after birth. SPARC is a prototypical matricellular protein that functions to regulate cell-matrix interactions and thereby influences many important physiological and pathological processes.     

SPARC, a matricellular protein: at the crossroads of cell-matrix communication.

SPARC is a multifunctional glycoprotein that belongs to the matricellular group of proteins. It modulates cellular interaction with the extracellular matrix (ECM) by its binding to structural matrix proteins, such as collagen and vitronectin, and by its abrogation of focal adhesions, features contributing to a counteradhesive effect on cells. SPARC inhibits cellular proliferation by an arrest of cells in the G1 phase of the cell cycle. It also regulates the activity of growth factors, such as platelet-derived growth factor (PDGF), fibroblast growth factor (FGF)-2, and vascular endothelial growth factor (VEGF). The expression of SPARC in adult animals is limited largely to remodeling tissue, such as bone, gut mucosa, and healing wounds, and it is prominent in tumors and in disorders associated with fibrosis. The crystal structure of two of the three domains of the protein has revealed a novel follistatin-like module and an extracellular calcium-binding (EC) module containing two EF-hand motifs. The follistatin-like module and the EC module are shared by at least four other proteins that comprise a family of SPARC-related genes. Targeted disruption of the SPARC locus in mice has shown that SPARC is important for lens transparency, as SPARC-null mice develop cataracts shortly after birth. SPARC is a prototypical matricellular protein that functions to regulate cell-matrix interactions and thereby influences many important physiological and pathological processes.     

Impaired bone fracture healing in matrix metalloproteinase-13 deficient mice

Vascular and cellular invasion into the cartilage is a critical step in the fracture healing. Matrix metalloproteinase-13 (MMP-13) is a member of the zinc-dependent endopeptidase family and plays an important role in remodeling of extracellular matrix. Therefore we investigated the possible involvement of MMP-13 in a murine model of stabilized bone fracture healing. Repair of the fracture in MMP-13 deficient (MMP-13(-/-)) mice was significantly delayed and characterized by a retarded cartilage resorption in the fracture callus. Immunohistochemistry indicated severe defects in vascular penetration and chondroclast recruitment to the fracture callus in MMP-13(-/-) mice. Consistent with the observations, the chondrocyte pellets cultured from the MMP13(-/-) mice exhibited diminished angiogenic activities when the pellets were co-cultured with endothelial cells. These results suggest that MMP-13 is crucial to the process of angiogenesis during healing of fracture, especially in the cartilage resorption process.     

Calcium binding domains and calcium-induced conformational transition of SPARC/BM-40/osteonectin, an extracellular glycoprotein expressed in mineralized and nonmineralized tissues

SPARC, BM-40, and osteonectin are identical or very closely related extracellular proteins of apparent Mr 43,000 (Mr 33,000 predicted from sequence). They were originally isolated from parietal endoderm cells, basement membrane producing tumors, and bone, respectively, but are rather widely distributed in various tissues. In view of the calcium binding activity reported for osteonectin, we analyzed the SPARC sequence and found two putative calcium binding domains. One is an N-terminal acidic region with clusters of glutamic acid residues. This region, although neither gamma-carboxylated nor homologous, resembles the gamma-carboxyglutamic acid (Gla) domain of vitamin K dependent proteins of the blood clotting system in charge density, size of negatively charged clusters, and linkage to the rest of the molecule by a cysteine-rich domain. The other region is an EF-hand calcium binding domain located near the C-terminus. A disulfide bond between the E and F helix is predicted from modeling the EF-hand structure with the known coordinates of intestinal calcium binding protein. The disulfide bridge apparently serves to stabilize the isolated calcium loop in the extracellular protein. As observed for cytoplasmic EF-hand-containing proteins and for Gla domain containing proteins, a major conformational transition is induced in BM-40 upon binding of several Ca2+ ions. This is accompanied by a 35% increase in alpha-helicity. A pronounced sigmoidicity of the dependence of the circular dichroism signal at 220 nm on calcium concentration indicates that the process is cooperative. In view of its properties, abundance, and wide distribution, it is proposed that SPARC/BM-40/osteonectin has a rather general regulatory function in calcium-dependent processes of the extracellular matrix.     

Impaired skin wound healing in peroxisome proliferator-activated receptor (PPAR)alpha and PPARbeta mutant mice

We show here that the alpha, beta, and gamma isotypes of peroxisome proliferator-activated receptor (PPAR) are expressed in the mouse epidermis during fetal development and that they disappear progressively from the interfollicular epithelium after birth. Interestingly, PPARalpha and beta expression is reactivated in the adult epidermis after various stimuli, resulting in keratinocyte proliferation and differentiation such as tetradecanoylphorbol acetate topical application, hair plucking, or skin wound healing. Using PPARalpha, beta, and gamma mutant mice, we demonstrate that PPARalpha and beta are important for the rapid epithelialization of a skin wound and that each of them plays a specific role in this process. PPARalpha is mainly involved in the early inflammation phase of the healing, whereas PPARbeta is implicated in the control of keratinocyte proliferation. In addition and very interestingly, PPARbeta mutant primary keratinocytes show impaired adhesion and migration properties. Thus, the findings presented here reveal unpredicted roles for PPARalpha and beta in adult mouse epidermal repair.     

A prototypic matricellular protein in the tumor microenvironment--where there's SPARC, there's fire

Within the tumor microenvironment is a dynamic exchange between cancer cells and their surrounding stroma. This complex biologic system requires carefully designed models to understand the role of its stromal components in carcinogenesis, tumor progression, invasion, and metastasis. Secreted protein acidic and rich in cysteine (SPARC) is a prototypic matricellular protein at the center of this exchange. Two decades of basic science research combined with recent whole genome analyses indicate that SPARC is an important player in vertebrate evolution, normal development, and maintenance of normal tissue homeostasis. Therefore, SPARC might also play an important role in the tumor microenvironment. Clinical evidence indicates that SPARC expression correlates with tumor progression, but tightly controlled animal models have shown that the role of SPARC in tumor progression is dependent on tissue and tumor cell type. In this Prospectus, we review the current understanding of SPARC in the tumor microenvironment and discuss current and future investigations of SPARC and tumor-stromal interactions that require careful consideration of growth factors, cytokines, proteinases, and angiotropic factors that might influence SPARC activity and tumor progression.     

Crystal structure and mapping by site-directed mutagenesis of the collagen-binding epitope of an activated form of BM-40/SPARC/osteonectin.

The extracellular calcium-binding domain (positions 138-286) of the matrix protein BM-40 possesses a binding epitope of moderate affinity for several collagen types. This epitope was predicted to reside in helix alphaA and to be partially masked by helix alphaC. Here we show that deletion of helix alphaC produces a 10-fold increase in collagen affinity similar to that seen after proteolytic cleavage of this helix. The predicted removal of the steric constraint was clearly demonstrated by the crystal structure of the mutant at 2.8 A resolution. This constitutively activated mutant was used to map the collagen-binding site following alanine mutagenesis at 13 positions. Five residues were crucial for binding, R149 and N156 in helix alphaA, and L242, M245 and E246 in a loop region connecting the two EF hands of BM-40. These residues are spatially close and form a flat ring of 15 A diameter which matches the diameter of a triple-helical collagen domain. The mutations showed similar effects on binding to collagens I and IV, indicating nearly identical binding sites on both collagens. Selected mutations in the non-activated mutant DeltaI also reduced collagen binding, consistent with the same location of the epitope but in a more cryptic form in intact BM-40.