SATB1 regulates SPARC expression in K562 cell line through binding to a specific sequence in the third intron

Special AT-rich binding protein 1 (SATB1), a cell type-specific nuclear matrix attachment region (MAR) DNA-binding protein, tethers to a specific DNA sequence and regulates gene expression through chromatin remodeling and HDAC (histone deacetylase complex) recruitment. In this study, a SATB1 eukaryotic expression plasmid was transfected into the human erythroleukemia K562 cell line and individual clones that stably over-expressed the SATB1 protein were isolated. Microarray analysis revealed that hundreds of genes were either up- or down-regulated in the SATB1 over-expressing K562 cell lines. One of these was the extra-cellular matrix glycoprotein, SPARC (human secreted protein acidic and rich in cysteine). siRNA knock-down of SATB1 also reduced SPARC expression, which was consistent with elevated SPARC levels in the SATB1 over-expressing cell line. Bioinformatics software Mat-inspector showed that a 17bp DNA sequence in the third intron of SPARC possessed a high potential for SATB1 binding; a finding confirmed by Chromatin immunoprecipitation (ChIP) with anti-SATB1 antibody. Our results show for the first time that forced-expression of SATB1 in K562 cells triggers SPARC up-regulation by binding to a 17bp DNA sequence in the third intron.     

Novel function of alternatively activated macrophages: stabilin-1-mediated clearance of SPARC

The matricellular protein SPARC (secreted protein acidic and rich in cysteine) has been implicated in development, differentiation, response to injury, and tumor biology by virtue of its regulation of extracellular matrix production/assembly and its antiadhesive and antiproliferative effects on different cell types. Despite numerous biological activities described for SPARC, cell surface receptors for this protein have not been identified. By phage display and in vitro-binding assays, we now show that SPARC interacts with stabilin-1, a scavenger receptor expressed by tissue macrophages and sinusoidal endothelial cells. The interaction is mediated by the extracellular epidermal growth factor-like region of stabilin-1 containing the sequence FHGTAC. Using FACS analysis and confocal microscopy, we demonstrate that stabilin-1 internalizes and targets SPARC to an endosomal pathway in Chinese hamster ovary cells stably transfected with this receptor. In human macrophages, stabilin-1 expression is required for receptor-mediated endocytosis of SPARC. SPARC was efficiently endocytosed by alternatively activated macrophages stimulated by IL-4 and dexamethasone, but not solely by Th1 or Th2 cytokines. A time course of ligand exposure to alternatively activated macrophages revealed that stabilin-1-mediated endocytosis of SPARC was followed by its targeting for degradation, similar to the targeting of acetylated low density lipoprotein, another stabilin-1 ligand. We propose that alternatively activated macrophages coordinate extracellular matrix remodeling, angiogenesis, and tumor progression via stabilin-1-mediated endocytosis of SPARC and thereby regulate its extracellular concentration.     

Isolation of a cDNA encoding a putative SPARC from the brine shrimp, Artemia franciscana

SPARC (Secreted protein, acidic, rich in cysteine) is an extracellular matrix-associated and anti-adhesive glycoprotein extensively studied in vertebrates. Its presence among invertebrates has been reported in nematodes and flies. We cloned a cDNA containing a complete open reading frame for SPARC from the brine shrimp, Artemia franciscana. The amino acid sequence identity between the Artemia and the fly SPARCs was 55%, whereas that of the Artemia and the nematode proteins was 45%. Artemia and vertebrates exhibited a sequence identity of 30% in the predicted aa sequences. The SPARC consisted of four domains commonly found among reported SPARCs. The protein comprised 291 amino acids, having a signal peptide, a follistatin-like domain, one N-glycosylation site and one calcium-binding EF-hand motif. Fourteen cysteine residues conserved among all the secreted forms of SPARCs were present in the Artemia SPARC, and four extra cysteine residues were also found in it. The extra residues were conserved among SPARCs of the arthropods and the nematode. Phylogenetic analyses showed that the sequences of SPARCs were grouped into those of vertebrates and invertebrates. Though the structural organization of SPARC was conserved among all the species studied, SPARC within a group was highly conserved within that group, but divergent between the two. Northern blots revealed the presence of a 1.1 kb mRNA, which was faintly expressed in embryos and considerably detected in prenauplii and nauplii. The isolation of a SPARC cDNA from Artemia franciscana provides intriguing features of the divergent protein, SPARC.     

SPARC, a secreted protein associated with morphogenesis and tissue remodeling, induces expression of metalloproteinases in fibroblasts through a novel extracellular matrix-dependent pathway

SPARC (osteonectin/BM40) is a secreted protein that modifies the interaction of cells with extracellular matrix (ECM). When we added SPARC to cultured rabbit synovial fibroblasts and analyzed the secreted proteins, we observed an increase in the expression of three metalloproteinases--collagenase, stromelysin, and the 92-kD gelatinase-- that together can degrade both interstitial and basement membrane matrices. We further characterized the regulation of one of these metalloproteinases, collagenase, and showed that both collagenase mRNA and protein are upregulated in fibroblasts treated with SPARC. Experiments with synthetic SPARC peptides indicated that a region in the neutral alpha-helical domain III of the SPARC molecule, which previously had no described function, was involved in the regulation of collagenase expression by SPARC. A sequence in the carboxyl-terminal Ca(2+)-binding domain IV exhibited similar activity, but to a lesser extent. SPARC induced collagenase expression in cells plated on collagen types I, II, III, and V, and vitronectin, but not on collagen type IV. SPARC also increased collagenase expression in fibroblasts plated on ECM produced by smooth muscle cells, but not in fibroblasts plated on a basement membrane-like ECM from Engelbreth-Holm-Swarm sarcoma. Collagenase was induced within 4 h in cells treated with phorbol diesters or plated on fibronectin fragments, but was induced after 8 h in cells treated with SPARC. A number of proteins were transiently secreted by SPARC-treated cells within 6 h of treatment. Conditioned medium that was harvested from cultures 7 h after the addition of SPARC, and depleted of residual SPARC, induced collagenase expression in untreated fibroblasts; thus, part of the regulation of collagenase expression by SPARC appears to be indirect and proceeds through a secreted intermediate. Because the interactions of cells with ECM play an important role in regulation of cell behavior and tissue morphogenesis, these results suggest that molecules like SPARC are important in modulating tissue remodeling and cell-ECM interactions.     

Regulation of gene expression by SPARC during angiogenesis in vitro. Changes in fibronectin, thrombospondin-1, and plasminogen activator inhibitor-1.

Angiogenesis in vitro, the formation of capillary-like structures by cultured endothelial cells, is associated with changes in the expression of several extracellular matrix proteins. The expression of SPARC, a secreted collagen-binding glycoprotein, has been shown to increase significantly during this process. We now show that addition of purified SPARC protein, or an N-terminal synthetic peptide (SPARC4-23), to strains of bovine aortic endothelial cells undergoing angiogenesis in vitro resulted in a dose-dependent decrease in the synthesis of fibronectin and thrombospondin-1 and an increase in the synthesis of type 1-plasminogen activator inhibitor. SPARC decreased fibronectin mRNA by 75% over 48 h, an effect that was inhibited by anti-SPARC immunoglobulins. Levels of thrombospondin-1 mRNA were diminished by 80%. Over a similar time course, both mRNA and protein levels of type 1-plasminogen activator inhibitor (PAI-1) were enhanced by SPARC and the SPARC4-23 peptide. The effects were dose-dependent with concentrations of SPARC between 1 and 30 micrograms/ml. In contrast, no changes were observed in the levels of either type I collagen mRNA or secreted gelatinases. Half-maximal induction of PAI-1 mRNA or inhibition of fibronectin and thrombospondin mRNAs occurred with 2-5 micrograms/ml SPARC and approximately 0.05 mM SPARC4-23. Strains of endothelial cells that did not form cords and tubes in vitro had reduced or undetectable responses to SPARC under identical conditions. These results demonstrate that SPARC modulates the synthesis of a subset of secreted proteins and identify an N-terminal acidic sequence as a region of the protein that provides an active site. SPARC might therefore function, in part, to achieve an optimal ratio among different components of the extracellular matrix. This activity would be consistent with known effects of SPARC on cellular morphology and proliferation that might contribute to the regulation of angiogenesis in vivo.     

The matricellular protein SPARC is internalized in Sertoli, Leydig, and germ cells during testis differentiation

The gene encoding the matricellular protein secreted protein, acidic and rich in cysteine (SPARC) was identified in a screen for genes expressed sex-specifically during mouse gonad development, as being strongly upregulated in the male gonad from very early in testis development. We present here a detailed analysis of SPARC gene and protein expression during testis development, from 11.5 to 15.5 days post coitum (dpc). Section in situ hybridization analysis revealed that SPARC mRNA is expressed by the Sertoli cells in the testis cords and the fetal Leydig cells, found within the interstitial space between the testis cords. Immunodetection with anti-SPARC antibody showed that the protein was located inside the testis cords, within the cytoplasm of Sertoli and germ cells. In the interstitium, SPARC was present intracellularly within the Leydig cells. The internalization of SPARC in Sertoli, Leydig, and germ cells suggests that it plays an intracellular regulatory role in these cell types during fetal testis development.     

The calcium-binding protein SPARC is secreted by Leydig and Sertoli cells of the adult mouse testis

In mammals, polypeptides secreted by cells of the testis are believed to influence spermatogenesis and to affect the behavior of the resident somatic cell populations. The 43,000-MW, secreted, calcium-binding glycoprotein SPARC (Secreted Protein, Acidic and Rich in Cysteine) is synthesized by a number of embryonic, fetal, and adult somatic cells and is associated with areas of cellular differentiation, proliferation, and morphological reorganization. Here, we report on the expression of SPARC in the testes of adult mice. By immunohistochemistry, SPARC was observed in the cytoplasm of Leydig cells and of Sertoli cells bearing late-stage, elongate spermatids. Testicular mRNA, translated in vitro, yielded a polypeptide of approximately 42,000 MW that bound anti-SPARC antibodies. Northern blot analysis revealed 2.3 kilobase (kb) SPARC mRNA in the testis, a size comparable to that of SPARC mRNA in nongonadal cells. Western blot assays of proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed an immunoreactive polypeptide of 43,000 MW in purified mouse Sertoli cells and their culture supernatants. Similar assays of testis interstitial fluid revealed 43,000 MW and 30,000 MW immunoreactive polypeptides. By indirect immunofluorescence, purified mouse Leydig cells cultured 24-48 h expressed SPARC in cytoplasmic granules. Cultured Leydig cells incorporated [35S]methionine into a secreted polypeptide of 43,000 MW that was recognized by anti-SPARC antibodies. In metal binding assays, purified SPARC bound Ca2+, Fe2+ and Cu2+. The function of SPARC in testes may be to sequester or transport certain metallic cations. Our recent discovery that SPARC induces changes in shape of certain nongonadal cell types also suggests that this glycoprotein may influence the functions of both Leydig and Sertoli cells by affecting their morphology.     

Downregulation of SPARC Expression Inhibits the Invasion of Human Trophoblast Cells In Vitro

Successful pregnancy depends on the precise regulation of extravilloustrophoblast (EVT) invasion into the uterine decidua. SPARC (secreted protein acidic and rich in cysteine) is a matricellular glycoprotein that plays critical roles in the pathologies associated with obesity and diabetes, as well as tumorigenesis. The objective of this study was to investigate the role of SPARC in the process of trophoblast invasion which shares many similarities with tumor cell invasion. By Western blot, higher expression of SPARC was observed in mouse brain, ovary and uterus compared to other mouse tissues. Immunohistochemistry analysis revealed a spatio-temporal expression of SPARC in mouse uterus in the periimplantation period. At the implantation site of d8 pregnancy, SPARC mainly accumulated in the secondary decidua zone (SDZ), trophoblast cells and blastocyst. The expression of SPARC was also detected in human placental villi and trophoblast cell lines. In a Matrigel invasion assay, we found SPARC-specific RNA interference significantly reduced the invasion of human extravilloustrophoblast HTR8/SVneo cells. Microarray analysis revealed that SPARC depletion upregulated the expression of interleukin 11 (IL11), KISS1, insulin-like growth factor binding protein 4 (IGFBP4), collagen type I alpha 1 (COLIA1), matrix metallopeptidase 9 (MMP9), and downregulated the expression of the alpha polypeptide of chorionic gonadotropin (CGA), MMP1, gap junction protein alpha 1 (GJA1), et al. The gene array result was further validated by qRT-PCR and Western blot. The present data indicate that SPARC may play an important role in the regulation of normal placentation by promoting the invasion of trophoblast cells into the uterine decidua.     

Matricellular protein SPARC is translocated to the nuclei of immortalized murine lens epithelial cells

The matricellular glycoprotein, secreted protein acidic and rich in cysteine (SPARC), has complex biological activities and is important for lens epithelial cell function and regulation of cataract formation. To understand how SPARC influences lens epithelial cell activity and homeostasis, we have studied the subcellular distribution of SPARC in murine lens epithelial cells in vitro. We demonstrate that endogenous SPARC is located in the cytoplasm of either quiescent or dividing lens epithelial cells in culture. However, cytoplasmic SPARC was translocated into the nuclei of immortalized lens epithelial cells upon a significant reduction of intracellular SPARC in these cells. Recombinant human (rh) SPARC added to the culture media was quickly and efficiently internalized into the cytosol of SPARC-null lens epithelial cells. Moreover, cytoplasmic rhSPARC was also translocated into the nucleus after exogenous rhSPARC was removed from the culture media. The translocation of SPARC into the nucleus was therefore triggered by the reduction of SPARC protein normally available to the cells. A mouse SPARC-EGFP chimeric fusion protein (70 kDa) was expressed in lens epithelial cells and 293-EBNA cells, and was observed both in the cytoplasm and culture medium, but not in the nucleus. SPARC does not appear to have a strong nuclear localization sequence. Alternatively, SPARC might pass through the nuclear pore complex by passive diffusion. SPARC therefore functions not only as an extracellular protein but also potentially as an intracellular protein to influence cellular activities and homeostasis.     

SPARC antagonizes the effect of basic fibroblast growth factor on the migration of bovine aortic endothelial cells.

Migration of endothelial cells is requisite to wound repair and angiogenesis. Since the glycoprotein SPARC (secreted protein, acidic and rich in cysteine) is associated with remodeling, cellular migration, and angiogenesis in vitro, we questioned whether SPARC might influence the motility of endothelial cells. In this study we show that, in the absence of serum, exogenous SPARC inhibits the migration of bovine aortic endothelial cells induced by bFGF. Similar results were obtained from two different assays, in which cell migration was measured in a Boyden chamber and in monolayer culture after an experimental wound. Without bFGF, the migration of endothelial cells was unaffected by SPARC. The inhibitory effect of SPARC on cell motility was dose-dependent, required the presence of Ca2+, was mimicked by synthetic peptides from the N- and C-terminal Ca(2+)-binding domains of the protein, and was not seen in the presence of serum. Modulation of the activities of secreted and cell-associated proteases, including plasminogen activators and metalloproteinases, appeared not to be responsible for the effects that we observed on the motility of endothelial cells. Moreover, a molecular interaction between SPARC and bFGF was not detected, and SPARC did not interfere with the binding of bFGF to high-affinity receptors on endothelial cells. Finally, in culture medium that contained serum, SPARC inhibited the incorporation of [3H]-thymidine into newly synthesized DNA, both in the absence and presence of bFGF. However, DNA synthesis was not affected by SPARC when the cells were plated on gelatin or fibronectin in serum-free medium. We propose that the combined action of a serum factor and SPARC regulates both endothelial cell proliferation and migration and coordinates these events during morphogenetic processes such as wound repair and angiogenesis.     

Inhibition of endothelial cell proliferation by SPARC is mediated through a Ca2+-binding EF-hand sequence

SPARC (secreted protein, acidic and rich in cysteine, also known as osteonectin and BM-40) is a metal-binding glycoprotein secreted by a variety of cultured cells and characteristic of tissues undergoing morphogenesis, remodeling, and repair. Recently it has been shown that SPARC inhibits the progression of the endothelial cell cycle in mid-G₁, and that a synthetic peptide (amino acids 54–73 of secreted murine SPARC, peptide 2.1) from a cationic, disulfide-bonded region was in part responsible for the growth-suppressing activity [Funk and Sage (1991): Proc Natl Acad Sci USA 88:2648–2652]. Moreover, SPARC was shown to interact directly with bovine aortic endothelial (BAE) cells through a C-terminal EF-hand sequence comprising a high-affinity Ca²⁺-binding site of SPARC and represented by a synthetic peptide (amino acids 254–273) termed 4.2 [Yost and Sage (1993): J Biol Chem 268:25790–25796]. In this study we show that peptide 4.2 is a more potent inhibitor of DNA synthesis that acts cooperatively with peptide 2.1 to diminish the incorporation of [³H]-thymidine by both BAE and bovine capillary endothelial (BCE) cells. At concentrations of 0.019–0.26 mM peptide 4.2, thymidine incorporation by BAE cells was decreased incrementally, relative to control values, from approximately 100 to 10%. Although somewhat less responsive, BCE cells exhibited a dose-responsive decrement in thymidine incorporation, with a maximal inhibition of 55% at 0.39 mM. The inhibitory effect of peptide 4.2 was essentially independent of heparin and basic fibroblast growth factor and was blocked by anti-SPARC peptide 4.2 IgG, but not by antibodies specific for other domains of SPARC. To identify residues that were necessary for inhibition of DNA synthesis, we introduced single amino acid substitutions into synthetic peptide 4.2 and tested their activities and cell-surface binding characteristics on endothelial cells. Two peptides displayed null to diminished effects in the bioassays that were concentration-dependent: peptide 4.2 K, containing an Asp₂₅₈ → Lys substitution, and peptide 4.2 AA, in which the two disulfide-bonded Cys (positions 255 and 271) were changed to Ala residues. Peptide 4.2 K, which failed to fulfill the EF-hand consensus formula, exhibited an anomalous fluorescence emission spectrum, in comparison with the wild-type 4.2 sequence, that was indicative of a compromised affinity for Ca²⁺. Moreover, ablation of the disulfide bond in peptide 4.2 AA potentially destabilized the Ca²⁺-binding loop structure, as assessed by fluorescence spectroscopy, such that the peptide competed poorly for the binding of [¹²⁵I]-peptide 4.2 to BAE cells. We conclude both that Ca²⁺-coordinating Asp at position 258 and the conformation of peptide 4.2 are necessary for the inhibition of DNA synthesis by SPARC in cultured endothelial cells.     

SPARC synthesis in pre-implantation and early post-implantation mouse embryos

Summary SPARC (secreted protein acidic and rich in cysteine), also known as osteonectin and BM-40, is a secreted protein associated with a variety of embryonic and adult tissue and cell types, including placenta, parietal and visceral endoderm, certain epithelia (e.g. gut, skin, glandular epithelia), and regions of active chondrogenesis and osteogenesis. Although much is known concerning the tissue distribution of this protein, neither the time and location of its initial appearance nor its functions during embryogenesis have been clearly established. We identified the location of SPARC on two-dimensional protein gels. By using two-dimensional gel analysis of both pre- and post-implantation stage mouse embryos, we find that SPARC is initially synthesized between 3.5 and 4.5 days of embryogenesis. This is the earliest time during development at which synthesis of SPARC has been demonstrated. Inner cell masses isolated from 4.5 day blastocysts synthesize SPARC indicating that either primitive ectoderm, primitive endoderm, or both produce this protein. SPARC synthesis is also detectable in isolated trophoblast vesicles. Thus, SPARC is synthesized not only in placenta, parietal endoderm, and visceral endoderm, but in the precursors of these tissues as well. Examination of 7.5 day embryos reveals that SPARC is synthesized in isolated parietal yolk sac and in whole extraembryonic and embryonic regions. Relative to other proteins, synthesis of SPARC was most prevalent in the parietal yolk sac. The possible implications of SPARC synthesis as early as 4.5 days are discussed.     

Pre-eclampsia onset and SPARC: A possible involvement in placenta development

Pre-eclampsia (PE) is a multisystem disorder commonly diagnosed in the latter half of pregnancy and it is a leading cause of intrauterine fetal growth retardation (IUGR). The aim of this study was to investigate the localization and the role of SPARC, secreted protein acidic, and rich in cysteine, in PE and PE–IUGR placentas in comparison with normal placentas. SPARC was mainly expressed in the villous and extravillous cytotrophoblastic cells in first trimester, whereas in PE, PE–IUGR and at term placentas, SPARC immunostaining was visible in both cytotrophoblastic cells and syncytiotrophoblast. SPARC expression significantly decreased in normal placenta from first to third trimester and a further significant reduction was demonstrated in PE and PE–IUGR. The latter downregulation of SPARC depends on hypoxic condition as shown by in vitro models. In conclusion, SPARC can play a pivotal role in PE and PE–IUGR onset and it should be considered as a key molecule for future investigations in such pathologies.     

Proteomic analysis identified N-cadherin, clusterin, and HSP27 as mediators of SPARC (secreted protein, acidic and rich in cysteines) activity in melanoma cells

Secreted protein, acidic and rich in cysteines (SPARC) is a secreted protein associated with increased aggressiveness of different human cancer types. In order to identify downstream mediators of SPARC activity, we performed a 2-DE proteomic analysis of human melanoma cells following antisense-mediated downregulation of SPARC expression. We found 23/504 differential spots, 15 of which were identified by peptide fingerprinting analysis. Three of the differential proteins (N-cadherin (N-CAD), clusterin (CLU), and HSP27) were validated by immunoblotting, confirming decreased levels of N-CAD and CLU and increased amounts of HSP27 in conditioned media of cells with diminished SPARC expression. Furthermore, transient knock down of SPARC expression in melanoma cells following adenoviral-mediated transfer of antisense RNA confirmed these changes. We next developed two different RNAs against SPARC that were able to inhibit in vivo melanoma cell growth. Immunoblotting of the secreted fraction of RNAi-transfected melanoma cells confirmed that downregulation of SPARC expression promoted decreased levels of N-CAD and CLU and increased secretion of HSP27. Transient re-expression of SPARC in SPARC-downregulated cells reverted extracellular N-CAD, CLU, and HSP27 to levels similar to those in the control. These results constitute the first evidence that SPARC, N-CAD, CLU, and HSP27 converge in a unique molecular network in melanoma cells.     

SPARC is a source of copper-binding peptides that stimulate angiogenesis

SPARC is a transiently expressed extracellular matrix-binding protein that alters cell shape and regulates endothelial cell proliferation in vitro. In this study, we show that SPARC mRNA and protein are synthesized by endothelial cells during angiogenesis in vivo. SPARC and peptides derived from a cationic region of the protein (amino acids 113- 130) stimulated the formation of endothelial cords in vitro; moreover, these peptides stimulated angiogenesis in vivo. Mapping of the active domain demonstrated that the sequence KGHK was responsible for most of the angiogenic activity; substitution of the His residue decreased the effect. We found that proteolysis of SPARC provided a source of KGHK, GHK, and longer peptides that contained these sequences. Although the Cu(2+)-GHK complex had been identified as a mitogen/morphogen in normal human plasma, we found KGHK and longer peptides to be potent stimulators of angiogenesis. SPARC113-130 and KGHK were shown to bind Cu2+ with high affinity; however, previous incubation with Cu2+ was not required for the stimulatory activity. Since a peptide from a second cationic region of SPARC (SPARC54-73) also bound Cu2+ but had no effect on angiogenesis, the angiogenic activity appeared to be sequence specific and independent of bound Cu2+. Thus, specific degradation of SPARC, a matrix-associated protein expressed by endothelial cells during vascular remodeling, releases a bioactive peptide or peptides, containing the sequence (K)GHK, that could regulate angiogenesis in vivo.     

Novel Role for Matricellular Proteins in the Regulation of Islet β Cell Survival: THE EFFECT OF SPARC ON SURVIVAL,PROLIFERATION, AND SIGNALING*

Understanding the mechanisms regulating islet growth and survival is critical for developing novel approaches to increasing or sustaining β cell mass in both type 1 and type 2 diabetes patients. Secreted protein acidic and rich in cysteine (SPARC) is a matricellular protein that is important for the regulation of cell growth and adhesion. Increased SPARC can be detected in the serum of type 2 diabetes patients. The aim of this study was to investigate the role of SPARC in the regulation of β cell growth and survival. We show using immunohistochemistry that SPARC is expressed by stromal cells within islets and can be detected in primary mouse islets by Western blot. SPARC is secreted at high levels by pancreatic stellate cells and is regulated by metabolic parameters in these cells, but SPARC expression was not detectable in β cells. In islets, SPARC expression is highest in young mice, and is also elevated in the islets of non-obese diabetic (NOD) mice compared with controls. Purified SPARC inhibits growth factor-induced signaling in both INS-1 β cells and primary mouse islets, and inhibits IGF-1-induced proliferation of INS-1 β cells. Similarly, exogenous SPARC prevents IGF-1-induced survival of primary mouse islet cells. This study identifies the stromal-derived matricellular protein SPARC as a novel regulator of islet survival and β cell growth.     

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.