SPARC Inhibits Adipogenesis by Its Enhancement of β-Catenin Signaling
2008; Elsevier BV; Volume: 284; Issue: 2 Linguagem: Inglês
10.1074/jbc.m808285200
ISSN1083-351X
Autores Tópico(s)Curcumin's Biomedical Applications
ResumoSPARC (secreted protein acidic and rich in cysteine) modulates interactions between cells and extracellular matrix and is enriched in white adipose tissue. We have reported that SPARC-null mice accumulate significantly more fat than wild-type mice and maintain relatively high levels of serum leptin. We now show that SPARC inhibits adipogenesis in vitro. Specifically, recombinant SPARC inhibited (a) adipocyte differentiation of stromal-vascular cells isolated from murine white adipose tissue and (b) the expression of adipogenic transcription factors and adipocyte-specific genes. SPARC induced the accumulation and nuclear translocation of β-catenin and subsequently enhanced the interaction of β-catenin and T cell/lymphoid enhancer factor 1. The activity of integrin-linked kinase was required for the effect of SPARC on β-catenin accumulation as well as extracellular matrix remodeling. During adipogenesis, fusiform preadipocytes change into sphere-shaped adipocytes and convert the extracellular matrix from a fibronectin-rich stroma to a laminin-rich basal lamina. SPARC retarded the morphological changes exhibited by preadipocytes during differentiation. In the presence of SPARC, the deposition of fibronectin was enhanced, and that of laminin was inhibited; in parallel, the expression of α5 integrin was enhanced, and that of α6 integrin was inhibited. Lithium chloride, which enhances the accumulation of β-catenin, also inhibited the expression of α6 integrin. These findings demonstrate a role for SPARC in adipocyte morphogenesis and in signaling processes leading to terminal differentiation. SPARC (secreted protein acidic and rich in cysteine) modulates interactions between cells and extracellular matrix and is enriched in white adipose tissue. We have reported that SPARC-null mice accumulate significantly more fat than wild-type mice and maintain relatively high levels of serum leptin. We now show that SPARC inhibits adipogenesis in vitro. Specifically, recombinant SPARC inhibited (a) adipocyte differentiation of stromal-vascular cells isolated from murine white adipose tissue and (b) the expression of adipogenic transcription factors and adipocyte-specific genes. SPARC induced the accumulation and nuclear translocation of β-catenin and subsequently enhanced the interaction of β-catenin and T cell/lymphoid enhancer factor 1. The activity of integrin-linked kinase was required for the effect of SPARC on β-catenin accumulation as well as extracellular matrix remodeling. During adipogenesis, fusiform preadipocytes change into sphere-shaped adipocytes and convert the extracellular matrix from a fibronectin-rich stroma to a laminin-rich basal lamina. SPARC retarded the morphological changes exhibited by preadipocytes during differentiation. In the presence of SPARC, the deposition of fibronectin was enhanced, and that of laminin was inhibited; in parallel, the expression of α5 integrin was enhanced, and that of α6 integrin was inhibited. Lithium chloride, which enhances the accumulation of β-catenin, also inhibited the expression of α6 integrin. These findings demonstrate a role for SPARC in adipocyte morphogenesis and in signaling processes leading to terminal differentiation. Obesity is a major public health problem in the United States because of its high prevalence and causal relationship to many medical complications, including diabetes, high blood pressure, high blood cholesterol, heart disease, cancer, gallbladder disease, liver disease, arthritis, pulmonary complications, sleep disorders, and premature death. Obesity is characterized by excessive accumulation of white adipose tissue (WAT, 3The abbreviations used are: WAT, white adipose tissue; ECM, extracellular matrix; GM, growth medium; WT, wild type; C/EBPα, CAAT/enhancer-binding protein α; PPARγ, peroxisome proliferator-activated receptor γ; TCF/LEF, T-cell factor/lymphoid-enhancing factor; GSK3β, glycogen synthase kinase 3β; ILK, integrin-linked kinase; LN, laminin; FN, fibronectin; IBMX, 3-isobutyl-1-methylxanthine; GPDH, glycerol-3-phosphate dehydrogenase; ERK1/2, extracellular signal-regulated kinase 1/2; BSA, bovine serum albumin; RUNX2, runt-related transcription factor 2; DMEM, Dulbecco's modified Eagle's medium; SVC, stromal-vascular cell; RT, reverse transcription. fat). The cellular composition of WAT includes primarily adipocytes and preadipocytes as well as endothelial cells and macrophages. Obesity is the result of both over-proliferation (number) and overgrowth (size) of adipocytes. Adipocytes are not only the storage depots of energy but also the source of various cytokines and hormones. These so-called adipokines, e.g. tumor necrosis factor-α, leptin, adiponectin, and resistin, target the central nervous system and peripheral tissues (fat, liver, and muscle) to modulate energy metabolism (1Gregoire F.M. Smas C.M. Sul H.S. Physiol. Rev. 1998; 78: 783-809Crossref PubMed Scopus (1862) Google Scholar, 2Rosen E.D. Spiegelman B.M. Annu. Rev. Cell Dev. Biol. 2000; 16: 145-171Crossref PubMed Scopus (1053) Google Scholar). SPARC (secreted protein acidic and rich in cysteine) belongs to the family of matricellular proteins, which generally do not contribute to the structure of extracellular matrix (ECM) but regulate its interaction with cells. SPARC is typically anti-adhesive in vitro and regulates angiogenesis and collagen production/fibrillogenesis in vivo. It is also a major participant in wound healing, tumor progression, and inflammation (3Brekken R.A. Sage E.H. Matrix Biol. 2001; 19: 816-827Crossref PubMed Scopus (56) Google Scholar). Recent findings have attracted new interest in SPARC and its proposed role(s) in adipose tissue formation. SPARC-null mice exhibit significantly more fat accumulation than wild-type (WT) mice (4Bradshaw A.D. Graves D.C. Motamed K. Sage E.H. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6045-6050Crossref PubMed Scopus (181) Google Scholar); consistent with this observation, SPARC-null bone marrow cells showed an increased tendency to differentiate into adipocytes rather than osteoblasts (5Delany A.M. Kalajzic I. Bradshaw A.D. Sage E.H. Canalis E. Endocrinology. 2003; 144: 2588-2596Crossref PubMed Scopus (136) Google Scholar). Expression of SPARC in fat is enhanced in various murine obesity models that include diet-induced obesity, gold thioglucose treatment, and the ob/ob strain (6Tartare-Deckert S. Chavey C. Monthouel M.N. Gautier N. Van Obberghen E. J. Biol. Chem. 2001; 276: 22231-22237Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). In a clinical study, the plasma concentration of SPARC was correlated positively with body mass index (7Takahashi M. Nagaretani H. Funahashi T. Nishizawa H. Maeda N. Kishida K. Kuriyama H. Shimomura I. Maeda K. Hotta K. Ouchi N. Kihara S. Nakamura T. Yamashita S. Matsuzawa Y. Obes. Res. 2001; 9: 388-393Crossref PubMed Scopus (47) Google Scholar). These data imply that SPARC is involved in the regulation of adipocyte differentiation and adipose tissue turnover. Adipocytes are derived from mesenchymal stem cells, which first differentiate into preadipocytes and, subsequently, adipocytes, a process termed adipogenesis. Extensive studies have probed into mechanisms by which transcription factors and exogenous hormones regulate adipogenesis in cultured 3T3-L1/F442 cells. CAAT/enhancer-binding protein α (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ) are the key factors required for adipogenesis in addition to signaling mediated by insulin/insulin-like growth factor-1 and nuclear receptors (1Gregoire F.M. Smas C.M. Sul H.S. Physiol. Rev. 1998; 78: 783-809Crossref PubMed Scopus (1862) Google Scholar, 2Rosen E.D. Spiegelman B.M. Annu. Rev. Cell Dev. Biol. 2000; 16: 145-171Crossref PubMed Scopus (1053) Google Scholar). The Wnt/β-catenin pathway has been shown to inhibit adipogenesis and enhance osteoblastogenesis (1Gregoire F.M. Smas C.M. Sul H.S. Physiol. Rev. 1998; 78: 783-809Crossref PubMed Scopus (1862) Google Scholar, 8Nuttall M.E. Gimble J.M. Curr. Opin. Pharmacol. 2004; 4: 290-294Crossref PubMed Scopus (322) Google Scholar, 9Farmer S.R. Int. J. Obes. (Lond). 2005; 29: 13-16Crossref PubMed Scopus (341) Google Scholar). Activation of this pathway is sufficient to inhibit the differentiation and apoptosis of preadipocytes through an inhibition of C/EBPα and PPARγ (9Farmer S.R. Int. J. Obes. (Lond). 2005; 29: 13-16Crossref PubMed Scopus (341) Google Scholar, 10Ross S.E. Hemati N. Longo K.A. Bennett C.N. Lucas P.C. Erickson R.L. MacDougald O.A. Science. 2000; 289: 950-953Crossref PubMed Scopus (1531) Google Scholar). Wnt proteins bind to frizzled (Fz) receptors and low density lipoprotein receptor-related protein coreceptors to activate several signaling pathways. Importantly, the inhibition of glycogen synthase kinase 3β (GSK3β) via Wnt results in the stabilization of β-catenin in the cytoplasm as opposed to its proteasomal degradation. After translocation to the nucleus, β-catenin binds to and coactivates transcription factors that include members of the T-cell factor/lymphoid-enhancing factor (TCF/LEF) family. Moreover, constitutively activated Fz1 increases the stability of β-catenin, inhibits apoptosis, inhibits adipogenesis, and induces osteoblastogenesis (11Kennell J.A. MacDougald O.A. J. Biol. Chem. 2005; 280: 24004-24010Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). We have recently reported that SPARC regulates the activity of integrin-linked kinase (ILK) in lung fibroblasts (12Barker T.H. Baneyx G. Cardo-Vila M. Workman G.A. Weaver M. Menon P.M. Dedhar S. Rempel S.A. Arap W. Pasqualini R. Vogel V. Sage E.H. J. Biol. Chem. 2005; 280: 36483-36493Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). Another group also demonstrated that ILK activity mediates oncogenic effects of SPARC in glioma cells (13Shi Q. Bao S. Song L. Wu Q. Bigner D.D. Hjelmeland A.B. Rich J.N. Oncogene. 2007; 26: 4084-4094Crossref PubMed Scopus (123) Google Scholar). ILK also regulates the β-catenin pathway through its phosphorylation of GSK3β, an inhibition resulting in the stabilization of β-catenin (14Oloumi A. McPhee T. Dedhar S. Biochim. Biophys. Acta. 2004; 1691: 1-15Crossref PubMed Scopus (159) Google Scholar, 15Persad S. Troussard A.A. McPhee T.R. Mulholland D.J. Dedhar S. J. Cell Biol. 2001; 153: 1161-1174Crossref PubMed Scopus (207) Google Scholar). Further accumulation of free β-catenin in the cytoplasm is a consequence of the inhibition of E-cadherin production by ILK (14Oloumi A. McPhee T. Dedhar S. Biochim. Biophys. Acta. 2004; 1691: 1-15Crossref PubMed Scopus (159) Google Scholar, 16Tan C. Costello P. Sanghera J. Dominguez D. Baulida J. de Herreros A.G. Dedhar S. Oncogene. 2001; 20: 133-140Crossref PubMed Scopus (232) Google Scholar). SPARC represses expression of E-cadherin and promotes tumorigenesis in melanoma cells (17Robert G. Gaggioli C. Bailet O. Chavey C. Abbe P. Aberdam E. Sabatie E. Cano A. Garcia de Herreros A. Ballotti R. Tartare-Deckert S. Cancer Res. 2006; 66: 7516-7523Crossref PubMed Scopus (138) Google Scholar). Therefore, we hypothesized that SPARC could inhibit adipogenesis through ILK-β-catenin-mediated signaling. Herein we have established that SPARC inhibits adipogenesis and enhances osteoblastogenesis. SPARC not only retarded morphological changes in preadipocytes but also inhibited the expression of most adipocyte transcription factors and other adipocyte-specific genes. Significantly, SPARC inhibited the degradation of β-catenin and enhanced its nuclear translocation. ILK was required for the effect of SPARC on β-catenin accumulation. Consistent with its effects in other tissues (18Francki A. Bradshaw A.D. Bassuk J.A. Howe C.C. Couser W.G. Sage E.H. J. Biol. Chem. 1999; 274: 32145-32152Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 19Gruber H.E. Sage E.H. Norton H.J. Funk S. Ingram J. Hanley Jr., E.N. J. Histochem. Cytochem. 2005; 53: 1131-1138Crossref PubMed Scopus (66) Google Scholar, 20Bradshaw A.D. Puolakkainen P. Dasgupta J. Davidson J.M. Wight T.N. Helene Sage E. J. Investig. Dermatol. 2003; 120: 949-955Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar), SPARC also regulated the production of ECM proteins and integrins, in an ILK-dependent manner. These data identify a novel pathway by which SPARC inhibits adipogenesis. Mice—A colony of WT and SPARC-null mice has been described (21Norose K. Lo W.K. Clark J.I. Sage E.H. Howe C.C. Exp. Eye Res. 2000; 71: 295-307Crossref PubMed Scopus (45) Google Scholar). C57BL/6 WT and SPARC-null mice are maintained in a specific pathogen-free facility. All animal experiments conformed to NIH guidelines and were approved by the Institutional Animal Care and Use Committee. These WT and SPARC-null mice have a mixed genetic background (129SV × C57BL/6) with four to ten backcrosses into the C57BL/6 background. Reagents and Antibodies—Recombinant human SPARC was produced and purified by our laboratory as described (22Bradshaw A.D. Bassuk J.A. Francki A. Sage E.H. Mol. Cell Biol. Res. Commun. 2000; 3: 345-351Crossref PubMed Scopus (32) Google Scholar). The preparations of SPARC used for these studies contained 0.001-0.005 ng of endotoxin/μg protein as determined by the Limulus amebocyte lysate assay (Cape Cod, Inc., E. Falmouth, MA). The purity of SPARC was greater than 90%, and its activity was verified in an assay of cell proliferation (23Bradshaw A.D. Francki A. Motamed K. Howe C. Sage E.H. Mol. Biol. Cell. 1999; 10: 1569-1579Crossref PubMed Scopus (88) Google Scholar). Recombinant murine hevin protein was produced as described (24Sweetwyne M.T. Brekken R.A. Workman G. Bradshaw A.D. Carbon J. Siadak A.W. Murri C. Sage E.H. J. Histochem. Cytochem. 2004; 52: 723-733Crossref PubMed Scopus (38) Google Scholar). All chemicals were reagent grade (Sigma). Goat anti-mouse SPARC antibody was purchased fromR&D Systems (Minneapolis, MN); rabbit-anti-β-actin antibody was from Abcam, Inc. (Cambridge, MA); anti-ILK antibody was from Upstate Biotechnology, Inc. (Lake Placid, NY); rabbit anti-phospho-extracellular signal-regulated kinase 1/2 (ERK1/2; Thr-202/Tyr-204) and anti-ERK1/2 antibodies were from Cell Signaling Technology (Danvers, MA); anti-histones, rabbit anti-α5-integrin, and rat anti-α6-integrin antibodies were from Chemicon International (Temecula, CA); anti-mouse laminin (LN)-1, monoclonal anti-cellular fibronectin (FN), and monoclonal anti-vinculin antibodies were from Sigma; mouse anti-β-catenin antibody and a blocking antibody against α6 integrin (GoH3) were from BD Bioscience. Secondary antibody conjugates were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Alexa Fluor 488 phalloidin and Hoechst 33258 dye were purchased from Molecular Probes (Eugene, OR). Aquablock was from EastCoast Bio, Inc. (North Berwick, ME). Cell culture reagents, including Dulbecco's modified Eagle's medium (DMEM), DMEM/F-12, trypsin/EDTA, antibiotics, and fetal bovine serum were purchased from Invitrogen. Isolation and Differentiation of Preadipocytes from Adipose Tissue—Stromal-vascular cells (SVCs) from mouse adipose tissue were prepared as described (25Zhao L. Gregoire F. Sul H.S. J. Biol. Chem. 2000; 275: 16845-16850Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). The subcutaneous inguinal fat deposits from 3∼4-week-old male mice were dissected under sterile conditions, and the lymph nodes were carefully removed. The SVCs were obtained from the minced fat tissues by collagenase (Invitrogen) digestion (2 mg/ml at 37 °C for 60 min in DMEM, with orbital agitation). The resulting cell suspension was homogenized by pipetting with a 10-ml serological plastic pipette, passed through a 100-mm nylon filter, and centrifuged at 400 × g for 10 min. The floating (top) layer of mature adipocytes was removed, and the pellets were resuspended. Erythrocytes were removed by exposure to hypotonic buffer for 1 min. The cells were plated at a density of 3 × 104 cells/cm2 in DMEM/F12 supplemented with 10% fetal bovine serum, penicillin G, and streptomycin sulfate. When the cells reached confluence, differentiation was initiated by the addition of 0.1 mm dexamethasone, 0.25 mm 3-isobutyl-1-methylxanthine (IBMX), and 17 nm insulin in DMEM/F-12 containing 10% fetal bovine serum. After 48 h, the differentiation medium was replaced by DMEM/F-12 containing 10% fetal bovine serum and insulin only. Oil-Red-O Staining—Cells were fixed in 10% formalin for 10 min. After two washes in water, cells were stained with 0.5% Oil-Red-O in isopropanol:H2O (3:2, v:v) for 1 h. After washes with water to remove unbound dye, cells were photographed under a Leica inverted microscope equipped with a digital camera. Glycerol-3-phosphate Dehydrogenase (GPDH) Activity Assay—Cells were lysed in 50 mm Tris-HCl (pH 8.0) containing 1 mm 2-mercaptoethanol and 1 mm EDTA. Next, cell lysates were sonicated and centrifuged to clear debris. The supernatant was incubated with 0.12 mm β-NADH and 0.2 mm dihydroxyacetone phosphate in 100 mm triethanolamine/HCl buffer (pH 7.5), and the reaction was monitored by absorbance at 340 nm. Semiquantitative RT-PCR—Total RNA from (pre)adipocytes at day 0, 2, 4, 6, or 7 of adipogenesis was isolated with RNeasy spin columns (Qiagen). cDNA was synthesized with superscript II reverse transcriptase (Invitrogen). PCR was performed with TaqDNA polymerase (Invitrogen) with the following primers: PPARγ2 (forward, 5′-ggagattctcctgttgacccag-3′; reverse, 5′-ggcactcaatggccatgag-3′), C/EBPα (forward, 5′-cagttccagatcgcgcact-3′; reverse, 5′-ctagagatccagcgacccga-3′), C/EBPβ (forward, 5′-cgactacggttacgtgagcct-3′; reverse, 5′-cgacagctgctccaccttcttc-3′), C/EBPδ (forward, 5′-cgcagacagtggtgagctt-3′; reverse, 5′-tcctgtcgctcgcaggt-3′), lipoprotein lipase (forward, 5′-gcaagcaacacaaccaggc-3′; reverse, 5′-cctgggttagccaccgttt-3′), leptin (forward, 5′-atgtgctggagacccctgtg-3′; reverse, 5′-tcagcattcagggctaacatcc-3′), osteocalcin (forward, 5′-gcagacaccatgaggacca-3′; reverse, 5′-tggagctgctgtgacatcc-3′), runt-related transcription factor 2 (RUNX2, forward 5′-cccagccacctttacctaca-3′; reverse, 5′-tatggagtgctgctggtctg-3′), 36B4 (forward, 5′-ccagaggcaccattgaaattctg-3′; reverse, 5′-cgaagagaccgaatcccatatc-3′), LNα1 chain (forward, 5′-gatgccattggcctagagattg-3′; reverse, 5′-ggatgggaatgggagctga-3′), LNα4 chain (forward, catgggatcctattggcctg-3′; reverse, 5′-cacatagccgccttctgtgg-3′), LNγ1 chain (forward, 5′-acctggaccgtctgattgacc-3′; reverse, 5′-agctgcctcagcataccgtt-3′), α5 integrin (forward, 5′-gcgactggaatcctcaaga-3′; reverse, 5′-gctgcagactacggctctct-3′), and α6 integrin (forward, 5′-cttgagggaaacaccgtca-3′; reverse, 5′-cacaactcaagaaagaaacgg-3′). Immunoblotting—Cells cultured in media were lysed in radiolabeled immunoprecipitation assay buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 0.25% sodium deoxycholate, 0.1% Nonidet P-40, and a mixture of protease inhibitors), cleared by centrifugation, and analyzed by SDS-PAGE followed by detection with a specific antibody as detailed in the figure legends. Immunostaining—For immunohistochemistry, WAT was fixed with methyl Carnoy's solution for 24 h and subsequently embedded in paraffin. Five-μm-thick sections were deparaffinized and incubated with control goat IgG or goat anti-mouse SPARC IgG. Bound primary antibodies were detected with horseradish peroxidase-conjugated donkey anti-goat IgG, and histochemical reactions were performed with 3, 3′-diaminobenzidine (Vector Laboratories, Burlingame, CA) as the substrate. Sections were counterstained with hematoxylin. For immunocytochemistry, preadipocytes were plated on coverslips. At certain stages of differentiation, cells were fixed with 10% formalin for 10 min followed by permeabilization with 20% Aquablock and 0.1% Triton X-100. Specific primary antibodies were used as indicated, with fluorescein isothiocyanate- or rhodamine-conjugated secondary antibodies. Sections and cells were examined with a Leica DMR microscope, and images were captured digitally with an RT-Spot camera (Diagnostic Instruments, Sterling Heights, MI). Alkaline Phosphatase Staining—After 2 washes with phosphate-buffered saline, cells were fixed in methanol/acetone (1:1) for 10 min at -20 °C. The cells were subsequently washed twice with water and stained with 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium alkaline phosphatase substrate buffer (Sigma) for 45 min at 37 °C. Cells were photographed under a Leica inverted microscope equipped with a digital camera. ILK Activity Assay—200-250 μg of cell lysate was incubated with 5 μg of rabbit polyclonal anti-ILK antibody (Cell Signaling Technology) or IgG isotype control, and protein A-Sepharose beads (Amersham Biosciences). Complexes were washed with high salt lysis buffer and kinase reaction buffer, and kinase assays were performed with 5 μg of myelin basic protein as a substrate. Kinase activity was measured by immunoblotting with a specific antibody against phosphorylated myelin basic protein (Upstate Biotechnology). Subcellular Fractionation—Cells were separated into cytosolic and nuclear fractions (NUCLEI EZ Prep Nuclei Isolation kit, Sigma). The cells were scraped into Nuclei EZ lysis buffer and were pelleted by centrifugation after two washes with ice-cold serum-free DMEM. Cell pellets were washed again with Nuclei EZ lysis buffer. The nuclei were subsequently pelleted by centrifugation at 4 °C. Nuclear fractions were solubilized with cold Nuclei EZ storage buffer. Proteins comprising the different fractions were analyzed by SDS-PAGE under reducing conditions and, subsequently, by immunoblotting. Transfection of Small Interfering RNAs (siRNAs)—Equimolar amounts of nonspecific siRNAs or siRNAs targeting murine ILK or SPARC (Qiagen) were incubated with Hiperfect Transfection Reagent (Qiagen) according to the manufacturer's instructions. The oligo mixtures were added to preadipocyte cultures 18 h after plating the cells. Thirty hours later the media were replaced with standard differentiation media as described above. Luciferase Reporter Gene Assay—5 × 105 cells were transfected with 3 μg of Super 8× TOPflash (From Dr. Randall T. Moon, University of Washington, Seattle, WA) and 20 ng of pRenilla-TK (Promega, Madison, WI) by electroporation. Super 8× TOPflash contains eight TCF/LEF transcription factor binding sites upstream of the luciferase gene. Forty-eight hours later, cells were lysed in passive lysis buffer (Promega). Luciferase/Renilla assays were performed with the Dual-luciferase reporter assay kit (Promega). The average ratio of luciferase activity (relative light units) to Renilla activity was calculated. Experiments were repeated more than three times in triplicates. ECM Protein Deposition—For quantification of ECM proteins, cells were plated into 6-well tissue culture plates and were induced to differentiate as described above. The cells in each well were removed by repeated washing with cold Mg2+/Ca2+-free phosphate-buffered saline. Detached cells were counted and subsequently lysed with a lysis buffer (1% Nonidet P-40, 0.1% SDS, 10 mm Tris-HCl, pH 7.5, and 5 mm EDTA) containing a complete protease inhibitor mixture (Roche Applied Science). Proteins deposited on the plates were collected by scraping with a policeman into radiolabeled immunoprecipitation assay buffer. Protein concentrations were determined by a bicinchoninic acid (BCA) assay (Pierce). Proteins were resolved by SDS-PAGE and subjected to immunoblotting with antibodies against LN-1 or cellular FN. SPARC Inhibits Adipogenesis and Enhances Osteoblastogenesis—SPARC is expressed extensively in WAT and in most adipocytes (Fig. 1A) as previously reported (6Tartare-Deckert S. Chavey C. Monthouel M.N. Gautier N. Van Obberghen E. J. Biol. Chem. 2001; 276: 22231-22237Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 7Takahashi M. Nagaretani H. Funahashi T. Nishizawa H. Maeda N. Kishida K. Kuriyama H. Shimomura I. Maeda K. Hotta K. Ouchi N. Kihara S. Nakamura T. Yamashita S. Matsuzawa Y. Obes. Res. 2001; 9: 388-393Crossref PubMed Scopus (47) Google Scholar). Given the high levels of SPARC in WAT and the data linking SPARC with obesity (4Bradshaw A.D. Graves D.C. Motamed K. Sage E.H. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6045-6050Crossref PubMed Scopus (181) Google Scholar, 6Tartare-Deckert S. Chavey C. Monthouel M.N. Gautier N. Van Obberghen E. J. Biol. Chem. 2001; 276: 22231-22237Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 7Takahashi M. Nagaretani H. Funahashi T. Nishizawa H. Maeda N. Kishida K. Kuriyama H. Shimomura I. Maeda K. Hotta K. Ouchi N. Kihara S. Nakamura T. Yamashita S. Matsuzawa Y. Obes. Res. 2001; 9: 388-393Crossref PubMed Scopus (47) Google Scholar), we asked whether SPARC controlled one (or more) of the processes contributing to adipogenesis. We separated SVCs from murine WAT and established an in vitro adipogenesis model as described (25Zhao L. Gregoire F. Sul H.S. J. Biol. Chem. 2000; 275: 16845-16850Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). SVCs are composed mainly of preadipocytes, as verified by immunostaining (data not shown); contamination by either macrophages or endothelial cells is less than 5%, according to staining for CD31 and F4/80, respectively. At least 50% of cells differentiate into adipocytes by day 7 after induction of adipogenesis, although the actual differentiation capacity of cells varied between preparations by ∼10%. More than 90% of WT SVCs expressed SPARC. SPARC protein was distributed in a diffuse pattern in cells maintained in growth medium (GM), whereas the location of SPARC appeared more concentrated in the endoplasmic reticulum/Golgi-like compartment surrounding nuclei at day 2 of adipogenesis (Fig. 1B and supplemental Fig. 1). During the differentiation of preadipocytes, levels of SPARC protein were augmented at two different stages: the first, between 6 and 24 h, and the second, at days 3-4 of adipogenesis (Fig. 1C). The concentration of SPARC in conditioned media on day 1 of adipogenesis is ∼200 ng/ml. Levels of β-actin are diminished during differentiation due to the major cytoskeletal remodeling required for this process (26Spiegelman B.M. Ginty C.A. Cell. 1983; 35: 657-666Abstract Full Text PDF PubMed Scopus (341) Google Scholar). ERK1/2 is phosphorylated and activated only at an early stage of differentiation when mitotic clonal expansion occurs (Fig. 1C and supplemental Fig. 2) (27Tang Q.Q. Otto T.C. Lane M.D. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 44-49Crossref PubMed Scopus (644) Google Scholar). On day 7 of differentiation, cultured SPARC-null cells were fixed and stained with Oil-Red-O or were extracted for GPDH activity assays. Purified SPARC protein was added simultaneously to the culture with the differentiation medium and was replenished every 2 days. SPARC protein did not change the cellular apoptotic index, according to an acridine orange assay (data not shown). As shown in Fig. 2, A and B, 2 μg/ml SPARC significantly inhibited adipogenesis in these primary cultures. Both hevin (a SPARC homolog) and SPARC proteins are purified from insect cells. Hevin, as well as bovine serum albumin (BSA), were therefore used as controls, neither of which showed effects on adipogenesis, and SPARC protein after partial denaturation exhibited significantly less activity (Fig. 2B). The inhibitory effect of SPARC was concentration-dependent (Fig. 2C). The endogenous level of SPARC (200 ng/ml) should exert certain inhibitory effects on adipogenesis; consequently, anti-SPARC IgG rescued adipogenesis in the presence of SPARC and blocked SPARC activity by 80% (Fig. 2D). Preadipocytes and mesenchymal stem cells differentiate into osteoblasts under long term culture. SPARC significantly enhanced osteoblast formation 21 days after the induction of differentiation (supplemental Fig. 3). These data support previous reports that SPARC inhibits adipogenesis and enhances osteoblastogenesis (4Bradshaw A.D. Graves D.C. Motamed K. Sage E.H. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6045-6050Crossref PubMed Scopus (181) Google Scholar, 5Delany A.M. Kalajzic I. Bradshaw A.D. Sage E.H. Canalis E. Endocrinology. 2003; 144: 2588-2596Crossref PubMed Scopus (136) Google Scholar, 28Delany A.M. Amling M. Priemel M. Howe C. Baron R. Canalis E. J. Clin. Investig. 2000; 105: 915-923Crossref PubMed Scopus (235) Google Scholar). To confirm the effect of SPARC on adipocyte differentiation, we compared the differentiation in vitro of preadipocytes from WT and SPARC-null mice. Preadipocytes were isolated from epididymal WAT of each genotype and were induced to differentiate into adipocytes. After 7 days, more adipocytes and more fat accumulation were observed in cultures of SPARC-null preadipocytes compared with those of WT preadipocytes (Fig. 3, A-C). We also tested the capacity of these preadipocytes to differentiate into osteoblasts. On day 28, cells were stained for the osteoblast marker, alkaline phosphatase. There were fewer stained cells in SPARC-null preadipocytes (Fig. 3, D and E). These data further demonstrate that SPARC regulates mesenchymal stem cell lineage commitment. SPARC Inhibits Adipocyte Gene Expression and Enhances Osteoblast Gene Expression—Adipogenesis requires the sequential expression of adipogenesis transcription factors and adipocyte genes. We next examined the levels of specific mRNAs in the presence or absence of SPARC. By RT-PCR (Fig. 4A), the transcription factors C/EBPα, C/EBPβ, and PPARγ2 were all significantly decreased by SPARC, whereas C/EBPδ was unchanged. Adipocyte genes such as leptin and lipoprotein lipase were also substantially decreased by SPARC. Conversely, genes required for osteoblast differentiation were enhanced by SPARC (Fig. 4B). RUNX2 is one of the key transcription factors for osteoblast formation (29Lian J.B. Stein G.S. Curr. Pharm. Des. 2003;
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