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PubMed2000

Induction of TGF-beta1 by the matricellular protein SPARC in a rat model of glomerulonephritis.

UNLABELLED: Induction of TGF-beta1 by the matricellular protein SPARC in a rat model of glomerulonephritis. BACKGROUND: SPARC has been implicated as a counteradhesive and antiproliferative protein associated with deposits of extracellular matrix in renal disease. METHOD: We have examined the effect of recombinant SPARC containing a C-terminal His tag (rSPARC) in an acute model of mesangial cell injury that is induced in the rat by an antibody against the Thy1 antigen on the mesangial cell membrane. The recombinant protein was administered 24 hours after the induction of nephritis and was infused through day 4. RESULTS: rSPARC was localized to the renal glomeruli of rats treated with anti-Thy1 antibody. Type I collagen and fibronectin, as well as transforming growth factor-beta1 (TGF-beta1), were increased at day 5 in rats treated with rSPARC (N = 4, P < 0.05 vs. delivery buffer), but only minimal effects were seen on mesangial cell and endothelial cell proliferation. In primary cultures of rat mesangial cells, infusion of rSPARC was associated with increases in TGF-beta1 mRNA and in total, secreted TGF-beta1 protein. CONCLUSIONS: rSPARC stimulates expression of TGF-beta1 both in vitro and in vivo. Given the closely regulated expression of SPARC, TGF-beta1, and type I collagen in several animal models of glomerulonephritis, we propose that SPARC could be one of the major mediators of the induction of TGF-beta1 in renal disease.

Animals↗

PubMed2002

Localization of SPARC in developing, mature, and chronically injured human allograft kidneys.

BACKGROUND: The matricellular protein SPARC (secreted protein acidic and rich in cysteine) is expressed during development, tissue remodeling and repair. It functions as an endogenous inhibitor of cell proliferation, regulates angiogenesis, regulates cell adhesion to extracellular matrix, binds cytokines such as platelet derived growth factor and stimulates transforming growth factor-beta (TGF-beta) production. This study describes the expression of SPARC during human renal development, in normal kidneys and during renal allograft rejection. METHODS: A total of 60 renal specimens, including normal areas from tumor nephrectomies (N = 24), fetal kidneys (N = 27) and explanted renal allografts (N = 9), were included in the study. SPARC protein was localized by immunohistochemistry using two different antibodies. On consecutive sections SPARC mRNA was detected by in situ hybridization. RESULTS: In the normal adult kidney SPARC protein was expressed by visceral and parietal epithelial cells, collecting duct epithelium (CD), urothelium, smooth muscle cells of muscular arteries and focally in interstitial cells. During renal development immature glomeruli demonstrated a polarized SPARC expression in visceral epithelial cells at their surface abutting the capillary basement membranes. In the fully differentiated glomeruli the expression pattern mirrored that of the adult kidney. Furthermore, SPARC was abundantly expressed by derivatives of the ureteric bud, and smooth muscle cells of arterial walls. During chronic allograft rejection SPARC is expressed in neointimal arterial smooth muscle cells, infiltrating inflammatory cells as well as by interstitial myofibroblasts in areas of interstitial fibrosis. SPARC mRNA synthesis detected by in situ hybridization mirrored these protein expression patterns. CONCLUSION: These studies co-localize SPARC to several sites of renal injury previously shown to be sites of PDGF B-chain expression and/or activity. We speculate that SPARC could function as an accessory molecule in chronic PDGF-mediated sclerosing interstitial and vascular injury. SPARC localization to glomerular epithelial cells corresponds to similar findings in rodents, and may reflect its role in cell adhesion and /or regulation of cell shape.

Adult↗

PubMed2003

DNA oligonucleotide microarray technology identifies fisp-12 among other potential fibrogenic genes following murine unilateral ureteral obstruction (UUO): modulation during epithelial-mesenchymal transition.

BACKGROUND: Tubulointerstitial inflammation and fibrosis are pathologic hallmarks of end-stage renal disease (ESRD). Here we have used DNA microarray technology to monitor the transcriptomic responses to murine unilateral ureteral obstruction (UUO) with a view to identifying molecular modulators of tubulointerstitial fibrosis. METHODS: Using Affymetrix Mu74Av2 microarrays, gene expression 4 and 10 days postobstruction was investigated relative to control contralateral kidneys. Candidate profibrogenic genes were further investigated in epithelial cells undergoing epithelial to mesenchymal transition (EMT) in vitro. RESULTS: mRNA levels for 1091 gene/EST sequences, of a total of 12,488 displayed on the microarray, were altered twofold or greater by days 4 and 10 postobstruction compared to contralateral control kidneys. Genes were categorised into functional groups, including modulators of cytoskeletal and extracellular matrix metabolism, cell growth, signalling, and transcription/translational events. Among the potentially profibrogenic genes, whose mRNA levels were increased after UUO, were fibroblast-inducible secreted protein (fisp-12), the murine homologue of connective tissue growth factor (CTGF), collagen XVIIIalpha1, secreted protein acidic and rich in cysteine (SPARC), and src-suppressed C-kinase substrate (SSeCKS). A sustained increase in fisp-12 mRNA level was observed during EMT induced by transforming growth factor-beta1 (TGF-beta1) and epidermal growth factor (EGF). CONCLUSION: Altered gene expression in murine UUO has been demonstrated. Increased expression of fisp-12, SPARC, and SSeCKS has been shown in response to TGF-beta1 treatment and during EMT, suggesting that these genes may offer potential therapeutic targets against tubulointerstitial fibrosis.

A Kinase Anchor Proteins↗

PubMedDate not supplied

Increased fibrovascular invasion of subcutaneous polyvinyl alcohol sponges in SPARC-null mice.

The expression of SPARC (secreted protein acidic and rich in cysteine/osteonectin/BM-40) is elevated in endothelial cells participating in angiogenesis in vitro and in vivo. SPARC acts on endothelial cells to elicit changes in cell shape and to inhibit cell cycle progression. In addition, SPARC binds to and diminishes the mitotic activity of vascular endothelial growth factor. To determine the effect(s) of SPARC on angiogenic responses in vivo, we implanted polyvinyl alcohol sponges subcutaneously into wild-type and SPARC-null mice. On days 12 and 20 following implantation, SPARC-null mice showed increased cellular invasion of the sponges in comparison to wild-type mice. Areas of the sponge with the highest cell density exhibited the highest numbers of vascular profiles in both wild-type and SPARC-null animals. The endothelial component of the vessels was substantiated by immunoreactivity with three different markers specific for endothelial cells. Although sponges from SPARC-null relative to wild-type mice were populated by significantly more cells and blood vessels, an increase in the ratio of vascular to nonvascular cells was not apparent. No differences in the percentage of proliferating cells within the sponge were detected between wild-type and SPARC-null sections. However, elevated levels of vascular endothelial growth factor were associated with sponges from SPARC-null versus wild-type mice. An increase in vascular endothelial growth factor production was also observed in SPARC-null primary dermal fibroblasts relative to those of wild-type cells. In conclusion, we have shown that the fibrovascular invasion of polyvinyl alcohol sponges is enhanced in mice lacking SPARC, and we propose that increased levels of vascular endothelial growth factor account, at least in part, for this response.

Animals↗

PubMed2001

Role of angiotensin II in tubulointerstitial injury.

The renin angiotensin system (RAS) has been implicated in tubulointerstitial injury in a range of clinical and experimental settings. Angiotensin II, the major effector molecule of the RAS, in addition to its effects on systemic blood pressure and intrarenal hemodynamics, also acts as a local hormone and growth factor to modulate renal function and pathology. There is increasing evidence for a pivotal role of this hormone in influencing renal tubular and interstitial function and structure including regulation of multiple cytokines and chemokines, promoting infiltration of monocytes/macrophages, promoting cellular proliferation, and inducing apoptosis. Pathologic actions of angiotensin II lead to tubulointerstitial fibrosis and inflammation via a range of cytokines and chemokines including transforming growth factor (TNF)-beta1, osteopontin, tumor necrosis factor (TNF)-alpha, secreted protein acidic and rich in cysteine (SPARC), and RANTES (regulated on activation normal T-cell expression and secreted). Blockade of production of angiotensin II by an angiotensin-converting enzyme (ACE) inhibitor or angiotensin II receptor antagonism with an angiotensin type 1 receptor antagonist has been shown to attenuate tubulointerstitial injury and reduce expression of cytokines and matrix proteins. The role of angiotensin II in tubulointerstitial fibrosis and inflammation is addressed in this article.

Angiotensin II↗

PubMed1998

Rat and mouse epiblasts differ in their capacity to generate extraembryonic endoderm.

In this study we have compared the in vitro differentiation potential of epiblast tissue from mouse and rat embryos. Epiblasts were isolated from egg cylinder stage embryos by microdissection and placed in culture. Rat cultures were distinguished by the copious production of parietal endoderm cells. Mouse epiblasts, in contrast, did not produce parietal endoderm. This difference in capacity to regenerate extraembryonic endoderm marks a surprising distinction in development of the pluripotential lineage between these two closely related rodents.

Animals↗

PubMed2004

Genetic basis for the evolution of vertebrate mineralized tissue.

Mineralized tissue is vital to many characteristic adaptive phenotypes in vertebrates. Three primary tissues, enamel (enameloid), dentin, and bone, are found in the body armor of ancient agnathans and mammalian teeth, suggesting that these two organs are homologous. Mammalian enamel forms on enamel-specific proteins such as amelogenin, whereas dentin and bone form on collagen and many acidic proteins, such as SPP1, coordinately regulate their mineralization. We previously reported that genes for three major enamel matrix proteins, five proteins necessary for dentin and bone formation, and milk caseins and salivary proteins arose from a single ancestor by tandem gene duplications and form the secretory calcium-binding phosphoprotein (SCPP) family. Gene structure and protein characteristics show that SCPP genes arose from the 5' region of ancestral sparcl1 (SPARC-like 1). Phylogenetic analysis on SPARC and SPARCL1 suggests that the SCPP genes arose after the divergence of cartilaginous fish and bony fish, implying that early vertebrate mineralization did not use SCPPs and that SPARC may be critical for initial mineralization. Consistent with this inference, we identified SPP1 in a teleost genome but failed to find any genes orthologous to mammalian enamel proteins. Based on these observations, we suggest a scenario for the evolution of vertebrate tissue mineralization, in which body armor initially formed on dermal collagen, which acted as a reinforcement of dermis. We also suggest that mammalian enamel is distinct from fish enameloid. Their similar nature as a hard structural overlay on exoskeleton and teeth is because of convergent evolution.

Animals↗

PubMed2003

SPARC-null mice exhibit increased adiposity without significant differences in overall body weight.

Secreted protein acidic and rich in cysteine/osteonectin/BM-40 (SPARC) is a matrix-associated protein that elicits changes in cell shape, inhibits cell-cycle progression, and influences the synthesis of extracellular matrix (ECM). The absence of SPARC in mice gives rise to aberrations in the structure and composition of the ECM that result in generation of cataracts, development of severe osteopenia, and accelerated closure of dermal wounds. In this report we show that SPARC-null mice have greater deposits of s.c. fat and larger epididymal fat pads in comparison with wild-type mice. Similar to earlier studies of SPARC-null dermis, we observed a reduction in collagen I in SPARC-null fat pads in comparison with wild-type. Although elevated levels of serum leptin were observed in SPARC-null mice, their overall body weights were not significantly different from those of wild-type counterparts. The diameters of adipocytes from SPARC-null versus wild-type epididymal fat pads were 252 +/- 61 and 161 +/- 33 microm (means +/- SD), respectively, and there was an increase in adipocyte number within SPARC-null fat pads in comparison with wild-type pads. Thus the absence of SPARC appears to result in an increase in the size of individual adipocytes as well as an increase in the number of adipocytes per fat pad. In fat pads isolated from wild-type mice, SPARC mRNA was associated with both the stromal/vascular and adipocyte fractions. We propose that SPARC limits the accumulation of adipose tissue in mice in part through its demonstrated effects on the regulation of cell shape and production of ECM.

Adipose Tissue↗

PubMed1991

Transcription of a quail gene expressed in embryonic retinal cells is shut off sharply at hatching.

The avian neuroretina (NR) is part of the central nervous system and is composed of photoreceptors, neuronal cells, and Müller (glial) cells. These cells are derived from proliferating neuroectodermal precursors that differentiate after terminal mitosis and become organized in cell strata. Genes that are specifically expressed at the various stages of retinal development are presently unknown. We have isolated a quail (Coturnix coturnix japonica) cDNA clone, named QR1, encoding a 676-amino acid protein whose carboxyl-terminal portion shows significant similarity to those of the extracellular glycoprotein osteonectin/SPARC/BM40 and of the recently described SC1 protein. The QR1 cDNA identifies a mRNA detected in NR but not in other embryonic tissues examined. The levels of this mRNA are markedly reduced when nondividing NR cells are induced to proliferate by the v-src oncogene. QR1 expression in NR is limited to the middle portion of the inner nuclear layer, a localization that essentially corresponds to that of Müller cells. Transcription of QR1 takes place only during the late phase of retinal development and is shut off sharply at hatching. Signals that regulate this unique pattern of expression appear to originate within the NR, since the QR1 mRNA is transcribed in cultured NR cells and is shut off also in vitro at a time coinciding with hatching.

Amino Acid Sequence↗

PubMed1991

The Ca2(+)-binding glycoprotein SPARC modulates cell cycle progression in bovine aortic endothelial cells.

SPARC (secreted protein, acidic and rich in cysteine) is an extracellular, Ca2(+)-binding protein associated with cellular populations undergoing migration, proliferation, and/or differentiation. Active preparations of SPARC bind to specific components of the extracellular matrix and cause mesenchymal cells to assume a rounded phenotype. In this study we show that SPARC modulates the progression of bovine aortic endothelial cells through the cell cycle. At a concentration of 20 micrograms/ml, SPARC inhibited the incorporation of [3H]thymidine into newly synthesized DNA by approximately 70%, as compared to control cultures within 24 hr after the release from G0 phase. The effect was dose-dependent and reached greater than 90% inhibition at 30 micrograms of SPARC per ml after 24 hr. A 20-residue synthetic peptide (termed 2.1) from a non-Ca2(+)-binding, disulfide-rich domain of SPARC also exhibited a dose-dependent inhibition of [3H]thymidine uptake in endothelial cells within 24 hr after release from G0 phase. An inhibition of 50% was seen with peptide 2.1 at a 0.4 mM concentration. Peptides from other regions of the SPARC protein did not produce this effect. Maximum inhibition of [3H]thymidine uptake by SPARC and peptide 2.1 occurred during the early-to-middle G1 phase of the endothelial-cell cycle. From 0-12 hr after release from G0 phase, cells exhibited delayed entry into S phase, which normally occurred at 24 +/- 2 hr. These results were further corroborated by flow cytometry. In the presence of SPARC at 20 micrograms/ml, 72% fewer cells were in S phase after a 24-hr period; a similar, but less marked, reduction was seen with peptide 2.1. Peptide 2.1 did not cause cell rounding, whereas peptide 1.1, a highly efficient inhibitor of endothelial-cell spreading, exhibited essentially no activity with respect to cell-cycle progression. It therefore appears that the transient, inhibitory effect of SPARC on the entry of endothelial cells into S phase does not depend on the overt changes in cell shape mediated through cytoskeletal rearrangement.

Amino Acid Sequence↗

PubMed1992

The extracellular glycoprotein SPARC interacts with platelet-derived growth factor (PDGF)-AB and -BB and inhibits the binding of PDGF to its receptors.

Interactions among growth factors, cells, and extracellular matrix are critical to the regulation of directed cell migration and proliferation associated with development, wound healing, and pathologic processes. Here we report the association of PDGF-AB and -BB, but not PDGF-AA, with the extracellular glycoprotein SPARC. Complexes of SPARC and 125I-labeled PDGF-BB or -AB were specifically immunoprecipitated by anti-SPARC immunoglobulins. 125I-PDGF-BB and -AB also bound specifically to SPARC that was immobilized on microtiter wells or bound to nitrocellulose after transfer from SDS/polyacrylamide gels. The binding of PDGF-BB to SPARC was pH-dependent; significant binding was detectable only above pH 6.6. The interaction of SPARC with specific dimeric forms of PDGF affected the activity of this mitogen. SPARC inhibited the binding of PDGF-BB and PDGF-AB, but not PDGF-AA, to human dermal fibroblasts in a dose-dependent manner. The expression of SPARC and PDGF was minimal in most normal adult tissues but was increased after injury. Enhanced expression of both PDGF-B chain and SPARC was seen in advanced lesions of atherosclerosis. We suggest that the coordinate expression of SPARC and PDGF-B-containing dimers following vascular injury may regulate the activity of specific dimeric forms of PDGF in vivo.

Animals↗

PubMed1994

SPARC (secreted protein acidic and rich in cysteine) regulates endothelial cell shape and barrier function.

SPARC (secreted protein acidic and rich in cysteine) can be selectively expressed by the endothelium in response to certain types of injury and induces rounding in adherent endothelial cells in vitro. To determine whether SPARC might influence endothelial permeability, we studied the effect of exogenous SPARC on the movement of 14C-labeled bovine serum albumin across postconfluent bovine pulmonary artery endothelial cells. SPARC increased (P < 0.02) transendothelial albumin flux in a dose-dependent manner at concentrations > or = 0.5 microgram/ml. At a fixed dose (15 micrograms/ml), exposure times > or = 1 h augmented (P < 0.005) albumin flux by 1.3- to 3.6-fold; this increase was blocked by anti-SPARC antibodies but not by inhibition of protein synthesis. Barrier dysfunction was not associated with loss of cell viability. Monolayers exposed to SPARC exhibited a rounded morphology and intercellular gaps. Prior stabilization of F-actin with phallicidin protected against the changes in barrier function (P = 0.0001) that were otherwise induced by SPARC. Bovine aortic and retinal microvascular endothelia also responded to SPARC. We propose that SPARC regulates endothelial barrier function through F-actin-dependent changes in cell shape, coincident with the appearance of intercellular gaps, that provide a paracellular pathway for extravasation of macromolecules.

Actins↗

PubMed1996

Isolation and characterization of a cell surface albumin-binding protein from vascular endothelial cells.

Albumin-binding proteins identified in vascular endothelial cells have been postulated to contribute to the transport of albumin via a process involving transcytosis. In the present study, we have purified and characterized a 57- to 60-kDa (gp60) putative albumin-binding protein from bovine pulmonary microvessel endothelial cells. The endothelial cell membranes were isolated from cultured cells by differential centrifugation and solubilized with sodium cholate and urea. The solubilized extract was concentrated after dialysis by ethanol precipitation and reextracted with Triton X-100, and the resulting extract was subjected to DEAE-cellulose column chromatography. Proteins eluted from this column were further separated using preparative sodium dodecyl sulfate/polyacrylamide gel electrophoresis and used for immunizing rabbits. Fluorescence-activated cell sorter analysis using the anti-gp60 antibodies demonstrated the expression of gp60 on the endothelial cell surface. Affinity-purified anti-gp60 antibodies inhibited approximately 90% of the specific binding of 125I-labeled albumin to bovine pulmonary microvessel endothelial cell surface. The anti-gp60 antibodies reacted with gp60 from bovine pulmonary artery, bovine pulmonary microvessel, human umbilical vein, and rat lung endothelial cell membranes. Bovine anti-gp60 antibodies also reacted with bovine secreted protein, acidic and rich in cysteine (SPARC). However, bovine SPARC NH2-terminal sequence (1-56 residues) antibodies did not react with gp60, indicating that the endothelial cell-surface-associated albumin-binding protein gp60 was different from the secreted albumin-binding protein SPARC. We conclude that the endothelial cell-surface-associated gp60 mediates the specific binding of native albumin to endothelial cells and thus may regulate the uptake of albumin and its transcytosis.

Animals↗

PubMed1999

Induction of tenascin-C in cardiac myocytes by mechanical deformation. Role of reactive oxygen species.

Mechanical overload may change cardiac structure through angiotensin II-dependent and angiotensin II-independent mechanisms. We investigated the effects of mechanical strain on the gene expression of tenascin-C, a prominent extracellular molecule in actively remodeling tissues, in neonatal rat cardiac myocytes. Mechanical strain induced tenascin-C mRNA (3.9 +/- 0.5-fold, p < 0.01, n = 13) and tenascin-C protein in an amplitude-dependent manner but did not induce secreted protein acidic and rich in cysteine nor fibronectin. RNase protection assay demonstrated that mechanical strain induced all three alternatively spliced isoforms of tenascin-C. An angiotensin II receptor type 1 antagonist inhibited mechanical induction of brain natriuretic peptide but not tenascin-C. Antioxidants such as N-acetyl-L-cysteine, catalase, and 1, 2-dihydroxy-benzene-3,5-disulfonate significantly inhibited induction of tenascin-C. Truncated tenascin-C promoter-reporter assays using dominant negative mutants of IkappaBalpha and IkappaB kinase beta and electrophoretic mobility shift assays indicated that mechanical strain increases tenascin-C gene transcription by activating nuclear factor-kappaB through reactive oxygen species. Our findings demonstrate that mechanical strain induces tenascin-C in cardiac myocytes through a nuclear factor-kappaB-dependent and angiotensin II-independent mechanism. These data also suggest that reactive oxygen species may participate in mechanically induced left ventricular remodeling.

1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium ↗

PubMed1999

SPARC regulates the expression of collagen type I and transforming growth factor-beta1 in mesangial cells.

The matricellular protein SPARC is expressed at high levels in cells that participate in tissue remodeling and is thought to regulate mesangial cell proliferation and extracellular matrix production in the kidney glomerulus in a rat model of glomerulonephritis (Pichler, R. H., Bassuk, J. A., Hugo, C., Reed, M. J., Eng, E., Gordon, K. L., Pippin, J., Alpers, C. E., Couser, W. G., Sage, E. H., and Johnson, R. J. (1997) Am. J. Pathol. 148, 1153-1167). A potential mechanism by which SPARC controls both cell cycle and matrix production has been attributed to its regulation of a pleiotropic growth factor. In this study we used primary mesangial cell cultures from wild-type mice and from mice with a targeted disruption of the SPARC gene. SPARC-null cells displayed diminished expression of collagen type I mRNA and protein, relative to wild-type cells, by the criteria of immunocytochemistry, immunoblotting, and the reverse transcription-polymerase chain reaction. The SPARC-null cells also showed significantly decreased steady-state levels of transforming growth factor-beta1 (TGF-beta1) mRNA and secreted TGF-beta1 protein. Addition of recombinant SPARC to SPARC-null cells restored the expression of collagen type I mRNA to 70% and TGF-beta1 mRNA to 100% of wild-type levels. We conclude that SPARC regulates the expression of collagen type I and TGF-beta1 in kidney mesangial cells. Since increased mitosis and matrix deposition by mesangial cells are characteristics of glomerulopathies, we propose that SPARC is one of the factors that maintains the balance between cell proliferation and matrix production in the glomerulus.

Animals↗

PubMed2000

Calcium affinity, cooperativity, and domain interactions of extracellular EF-hands present in BM-40.

The structure and function of cytosolic Ca(2+)-binding proteins containing EF-hands are well understood. Recently, the presence of EF-hands in an extracellular protein was for the first time proven by the structure determination of the EC domain of BM-40 (SPARC (for secreted protein acidic and rich in cysteine)/osteonectin) (Hohenester, E., Maurer, P., Hohenadl, C., Timpl, R., Jansonius, J. N., and Engel, J. (1996) Nat. Struct. Biol. 3, 67-73). The structure revealed a pair of EF-hands with two bound Ca(2+) ions. Two unusual features were noted that distinguish the extracellular EF-hands of BM-40 from their cytosolic counterparts. An insertion of one amino acid into the loop of the first EF-hand causes a variant Ca(2+) coordination, and a disulfide bond connects the helices of the second EF-hand. Here we show that the extracellular EF-hands in the BM-40 EC domain bind Ca(2+) cooperatively and with high affinity. The EC domain is thus in the Ca(2+)-saturated form in the extracellular matrix, and the EF-hands play a structural rather than a regulatory role. Deletion mutants demonstrate a strong interaction between the EC domain and the neighboring FS domain, which contributes about 10 kJ/mol to the free energy of binding and influences cooperativity. This interaction is mainly between the FS domain and the variant EF-hand 1. Certain mutations of Ca(2+)-coordinating residues changed affinity and cooperativity, but others inhibited folding and secretion of the EC domain in a mammalian cell line. This points to a function of EF-hands in extracellular proteins during biosynthesis and processing in the endoplasmic reticulum or Golgi apparatus.

Amino Acid Motifs↗

PubMed2001

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.

Adipose Tissue↗

PubMed2002

Characterization of SMOC-1, a novel modular calcium-binding protein in basement membranes.

We have isolated the novel gene SMOC-1 that encodes a secreted modular protein containing an EF-hand calcium-binding domain homologous to that in BM-40. It further consists of two thyroglobulin-like domains, a follistatin-like domain and a novel domain. Recombinant expression in human cells showed that SMOC-1 is a glycoprotein with a calcium-dependent conformation. Results from Northern blots, reverse transcriptase-PCR, and immunoblots revealed a widespread expression in many tissues. Immunofluorescence studies with an antiserum directed against recombinant human SMOC-1 demonstrated a basement membrane localization of the protein and additionally its presence in other extracellular matrices. Immunogold electron microscopy confirmed the localization of SMOC-1 within basement membranes in kidney and skeletal muscle as well as its expression in the zona pellucida surrounding the oocyte.

Amino Acid Sequence↗