Introduction

Secreted protein acidic and rich in cysteine (SPARC),¹ also known as osteonectin and BM-40, is a secreted multifunctional glycoprotein. SPARC belongs to the class of matricellular proteins that also includes thrombospondin 1 and 2, osteopontin, and tenascins C and X. SPARC is an extracellular matrix (ECM)-associated protein that does not seem to contribute to the structural integrity of the ECM. SPARC modulates cell shape, adhesion, and migration by disrupting cell- ECM interactions, in part, through its binding to several resident proteins of the ECM, such as fibrillar collagens (type I, III, and V), collagen type IV, and vitronectin (1). In addition to its counteradhesive function, SPARC modulates angiogenesis, stimulates the production of matrix metalloproteinases (2, 3) and ECM proteins, and influences the activity of cytokines and growth factors such as platelet-derived growth factor and vascular endothelial growth factor (4, 5). Furthermore, SPARC has been described as an inhibitor of cell cycle progression from G1 to S phase (4, 6).

SPARC is highly expressed during embryonic development and in tissues undergoing wound repair or active remodeling (7). Pathological conditions, such as cancer metastasis, arthritis, diabetes, and kidney disease, are characterized by elevated expression of SPARC (7). Mice with a homozygous-null mutation in the SPARC gene develop normally but show a progressive early-onset cataractogenesis with disruption of the structural integrity of the lens capsule (8, 9, 10). The SPARC-null mutation also results in accelerated wound healing (11), decreased bone formation leading to osteopenia (12, 13), and enhanced tumor progression (14).

In previous work, we proposed a novel role for SPARC during the development of adipose tissue associated with massive obesity. Obesity is a progressive disorder that is associated with an excessive expansion of adipose tissue. Both adipocyte hypertrophy and hyperplasia contribute to the growth of adipose tissue mass in obesity. Adipose tissue hyperplasia is a process that involves preadipocyte recruitment, followed by proliferation and adipocyte differentiation. We previously showed that SPARC is produced by adipocytes and that its expression is up-regulated in obese ob/ob mice and during high-fat (HF) diet- and drug-induced obesity (15). These three models of obesity are known to be associated mainly with hypertrophy of fat cells. Taken together, our observations suggest that SPARC participates in the homeostasis of adipose tissue. However, Bradshaw et al. (16) have shown that SPARC-null mice display increased subcutaneous fat deposition and that fat tissue expansion is associated with an increase in adipocyte size and number. This observation indicated that SPARC might be involved in the control of adipose tissue expansion. However, this adipose tissue accumulation is not accompanied by an increase in body weight; hence, the authors suggested that sparc^{- /-} mice might be resistant to obesity when given an HF diet. This study and ours provide only a glimpse into the complex role of SPARC in adipose tissue physiology and pathogenesis. For example, little is known about the regulation of SPARC expression during the early stages of adipocyte maturation and adipose tissue development. In this report, we show that SPARC overexpression is associated with adipocyte hyperplasia in an obese transgenic mouse model characterized by a large increase in fat cell number during adipose tissue development. In addition, we find that SPARC is expressed in human adipose tissue and that it is mainly produced by the adipocyte fraction. Consistent with these findings, we observe that SPARC is increased during adipose conversion of both human and murine preadipocytes. As a whole, our data point to the idea that SPARC is involved in cell–matrix interactions during adipose tissue hyperplasia and adipogenic differentiation.

Research Methods and Procedures

Total RNA Extraction and Northern Blotting

Total RNA from white adipose tissue or cells was isolated using the TRIzol reagent according to the manufacturer's instructions (Invitrogen, Carlsbad, CA). Northern blotting was conducted as previously described (15). Briefly, 10 g of denatured RNA was resolved by electrophoresis on 1.2% (wt/vol) agarose gels and transferred to positively charged nylon membranes (Millipore Corp., Billerica, MA). Blots were hybridized overnight at 42 °C with specific ³²P-labeled SPARC cDNA probes. SPARC cDNA was cloned by reverse transcriptase-polymerase chain reaction (PCR) from adipose tissue using the SuperScript one-step reverse transcriptase-PCR system kit (Life Technologies, Rockville, MD) and was labeled with [ -³²P] deoxycytidine triphosphate using the Rediprime II kit (Amersham Biosciences, Piscataway, NJ). Membranes were exposed to phosphor screen for 4 to 24 hours, and the signals were scanned with a STORM 840 PhosphorImager and quantified using the ImageQuant 5.0 software (Molecular Dynamics, Amersham Pharmacia Biotech, Piscataway, NJ). Blots were stripped and rehybridized with an 18S oligonucleotide probe as an indicator of RNA integrity and loading.

Real-time PCR

After treatment with DNase I (DNA-free; Ambion, Houston, TX), 1 g of total RNA was reverse transcribed using random priming, oligo (dT), and a reverse transcription system (Promega, Madison, WI), according to the manufacturer's instructions. Quantitative PCR was performed by monitoring in real-time the increase in fluorescence of the SYBR Green dye on an ABI PRISM 7000 Sequence Detector System (Applied Biosystems, Foster City, CA) as previously described (17). Gene-specific primers (Invitrogen, Carlsbad, CA) were designed using Primer Express software from Applied Biosystems (Table 1). Mouse 36B4 or ribosomal 18S primers were used for normalization.

Table 1. - Primers used for amplification of PCR products.

Full table

Cell Culture

Visceral adipose tissue was obtained during esthetic surgery under general anesthesia from non-obese subjects. The protocol was approved by the hospital's Committee on Ethics of the University Hospital Center of Toulouse (France), and individual informed consent was obtained. Cells were prepared and cultured as described below under "Adipocyte Differentiation." 3T3-L1 preadipocytes were obtained from American Type Culture Collection (Manassas, VA) and were cultured as described under "Adipocyte Differentiation." Human umbilical vein endothelial cells (HUVECs) were obtained from Clonetics (Cambrex Bio-Sciences, Emerainville, France). HUVECs were grown in EBM-2 (Cambrex, Walkersville, MD) supplemented with endothelial cell medium (Cambrex Bio-Sciences, Emerainville, France). HUVECs were tested positive for von Willebrand factor immunostaining.

Western Blotting

Protein extracts were obtained by lysing adipose tissue, HUVECs, or 3T3-L1 cells at different steps of differentiation in Laemmli buffer [ 3% w/v sodium dodecyl sulfate (SDS), 70 mM Tris-HCl, pH 7.0, and 11% (v/v) glycerol] . Protein concentration was measured using the bicinchoninic acid technique (BCA protein assay kit; Pierce Biotechnology, Rockford, IL), and 20 g of protein lysates was separated by SDS-polyacrylamide gel electrophoresis and analyzed by immunoblotting using the 303 monoclonal antibody against SPARC (18). Immunoreactive proteins were detected by enhanced chemiluminescence (Amersham Biosciences). Membranes were stripped and blotted with a polyclonal antibody directed to mitogen-activated protein kinase, Erk1/2 (Upstate Biotechnology, Waltham, MA), as a loading control. The blots were scanned and quantified using Scion Image software.

Transgenic Mice

Perigonadal adipose tissues were obtained from transgenic mice [ murine 3 adrenergic receptor (AR)^{- /-} , human 3 AR+/- , 2A human AR^+/- ] or from control mice (murine 3 AR^{- /-} , human 3 AR^+/- ). Mice were characterized as described previously (19). Transgenic mice expressed human 2A and 3 ARs on a murine 3 AR-null background. Just after weaning, mice were fed with a low-fat (LF) diet or an HF diet for 17 weeks (19). This study was conducted with permission from the local animal ethics board of the Research Institute of Toulouse (France).

Adipocyte Differentiation

Human preadipocytes were isolated as previously described (20). Adipose tissue samples were carefully dissected. All visible fibrous material and blood vessels were discarded. The remaining adipose tissue was finely minced and digested for 45 minutes in phosphate-buffered saline containing 1.5 mg/mL collagenase and 20 mg/mL bovine serum albumin, with gentle shaking at 37 °C. After centrifugation at 200g, the stromal vascular fraction was resuspended and incubated in an erythrocyte-lysing buffer (155 mM NH₄Cl, 5.7 mM K₂HPO₄, 0.1 mM EDTA) for 10 minutes to remove contaminating erythrocytes. The suspension was filtered through a 100-m screen. After additional washing and centrifugation, stromal cells were resuspended in Dulbecco's modified Eagle's medium (DMEM)/Ham's F-12 medium supplemented with 10% (v/v) fetal bovine serum (FBS) and were seeded at 60,000 cells/cm². After a 16-hour incubation period, the cells were changed into a medium consisting of DMEM/Ham's F-12 supplemented with 33 M biotin, 17 M pantothenate, 10 g/mL human transferrin, and 50 g/mL gentamicin in the presence of 66 nM insulin, 1 nM triiodothyronine, 100 nM cortisol, and, for the first 3 days, 1 g/mL ciglitazone. Medium was changed every 2 days. Adipogenesis was monitored by expression of an adipocyte-specific gene, adipocyte fatty acid-binding protein (aP2).

3T3-L1 preadipocytes (American Type Culture Collection) were grown in DMEM supplemented with 10% (v/v) calf serum, 50 U/mL penicillin, and 50 g/mL streptomycin until confluency. After 2 days (Day 0), differentiation was induced by changing the medium to DMEM containing 10% (v/v) FBS plus 0.25 mM 3-isobutyl-1-methylxanthine (Sigma, St. Louis, MO), 1 M dexamethasone (Sigma), 2 M insulin, and 0.01 M troglitazone (Sigma), a peroxisome proliferator-activated receptor (PPAR) activator. Three days later (Day 3), medium was replaced by DMEM supplemented with 10% (v/v) FBS and insulin. Cells were fed every 2 days. After 7 days of differentiation (Day 7), adipogenesis was analyzed by expression of an adipocyte-specific gene (PPAR) and cytologically by lipid accumulation.

Statistical Analysis

Data are represented as means standard error of the mean (SEM). Group means were compared by factorial ANOVA. Differences were considered statistically significant at p < 0.05.

Results

SPARC Protein Expression Is Elevated in White Adipose Tissue of Obese Transgenic Mice

We have previously described high levels of SPARC in adipose tissue of obese mice exhibiting adipocyte hypertrophy (15). Subsequently, we examined its regulation in adipose tissue from a mouse model of diet-induced obesity characterized by a large rise in fat cell number rather than an increase in the size of adipocytes. These transgenic mice express the 3 human AR and the 2 human AR on a murine 3 AR-null background (19). Animals were fed either an LF or an HF diet. After 17 weeks of diet, perigonadal fat tissue was removed and weighed. As shown in Figure 1A, no significant difference in fat pad weight was observed between LF-fed control (m3 AR^{- /-} ; h3 AR^+/- ) and transgenic (m3 AR^{- /-} ; h3 AR^+/- , h2 AR^+/- ) mice or between LF- and HF-fed control mice. However, fat pads from HF-fed transgenic mice were 2.5-fold higher than those of mice from other groups. As previously described (19), one of the major consequences of the development of obesity in these transgenic mice is a large increase in fat cell number. Expression of two molecular species (42 and 40 kDa) corresponding to SPARC is unchanged in adipose tissue from LF- and HF-fed control mice, indicating that, in this model, SPARC expression is not regulated by food lipids (Figure 1B and 1C). In contrast, HF-fed transgenic mice showed markedly elevated SPARC protein levels in white adipose tissue. However, we also noticed a slight decrease in SPARC protein levels in adipose tissue of LF-fed transgenic mice in comparison with LF-fed control mice. These findings show that, in obese mice, SPARC expression is associated with fat hyperplasia and support the idea that SPARC may be involved in fat cell recruitment during adipose tissue development.

Figure 1.

SPARC protein is elevated in white adipose tissue of obese transgenic mice. Controls (m3AR^{- /-} , h3AR^+/- ) and transgenic (m3AR^{- /-} , h3AR^+/- , h2AR^+/- ) mice were fed either an LF diet or an HF diet. After 17 weeks, perigonadal fat pads were removed, weighed, and processed for protein analysis. (A) Perigonadal fat pad weight of LF- or HF-fed control and transgenic mice. (B) Analysis of SPARC protein by Western blotting. Erk1/2 was used as a control for loading. (C) Autoradiograms were analyzed using Scion Image program. The intensity of the signals was normalized against the relative level of Erk1/2 expression in the corresponding sample. Values are the mean SEM of four samples. Asterisks represent statistically significant differences (ANOVA): * 0.01 < p < 0.05; ** 0.001 < p < 0.01; *** p < 0.001.

Full figure and legend (52K)

SPARC Is Produced by Human Adipocytes

To determine whether SPARC is expressed in human adipose tissue, SPARC mRNA was analyzed by Northern blot. Figure 2A shows that, similar to adipose tissue in rodents, both transcripts encoding SPARC were detected in human adipose tissue (15). Using a monoclonal antibody to human SPARC, we found that human white adipose tissue contains SPARC protein. SPARC in HUVECs is shown as a control (Figure 2B). Within the fat pad, adipocytes are surrounded by a stromal-vascular fraction that contains fat cell precursors and a large series of other cell types, such as fibroblasts, macrophages, endothelial cells, and smooth muscle cells. After separation of mature fat cells from non-fat cells by collagenase digestion and adipocyte flotation, SPARC mRNA levels in the two fractions were analyzed by real-time PCR. SPARC mRNA was detected mainly in the adipocyte fraction (Figure 2C). However, we observed low levels of SPARC mRNA in stromal cells. We confirmed that adequate separation had been achieved by determining aP2 expression. However, we cannot totally exclude that SPARC expression in stromal fraction could be caused by contamination. These results indicate that the main source of SPARC mRNA in human adipose tissue is the fat cells themselves.

Figure 2.

SPARC is produced by human adipocytes. (A) Ten micrograms of total RNA isolated from two different human visceral adipose tissue samples was analyzed for SPARC mRNA by Northern blot. SPARC transcripts are indicated by arrows. (B) Thirty micrograms of protein extracted from human visceral adipose tissue or HUVECs was separated by SDS-polyacrylamide gel electrophoresis. Immunoblot analysis was performed with a monoclonal antibody to SPARC, and immunoreactive species were visualized by enhanced chemiluminescence. (C) Adipocyte and stromal-vascular fractions from human adipose tissue were separated by collagenase digestion followed by centrifugation. SPARC and aP2 mRNA levels were analyzed in these two fractions by real-time quantitative PCR. mRNA expression data were normalized to 18S rRNA level in the corresponding sample. Values are the mean standard deviation of three independent experiments. Asterisks represent statistically significant differences (ANOVA): * 0.01 < p < 0.05; ** 0.001 < p < 0.01; *** p < 0.001.

Full figure and legend (60K)

SPARC Expression Increases During Adipocyte Differentiation

We next studied the expression of SPARC during the differentiation of human primary preadipocytes. After digestion of human adipose tissue by collagenase treatment, the stromal-vascular fraction was resuspended in DMEM/F-12 medium supplemented with 10% (v/v) FBS. After a 16-hour incubation period, the differentiation process was initiated by addition of an adipogenic mixture, and the medium was changed every 2 days. Using real-time PCR assays, we analyzed SPARC mRNA expression every 5 days over a 15-day interval. SPARC transcripts increased by 3-fold throughout the differentiation process (Figure 3A). A similar time-course was observed for aP2, a marker of fully differentiated adipocytes.

Figure 3.

Expression of SPARC is modulated during adipose conversion. (A) Stromal cells from human adipose tissue were maintained in culture and were induced to differentiate in vitro as described in Research Methods and Procedures. SPARC mRNA and aP2 mRNA were analyzed by real-time PCR throughout this process. mRNA expression data were normalized to 18S rRNA level in the corresponding sample. Values are the mean SEM of three independent experiments conducted with three different subjects. (B) SPARC and PPAR mRNA levels during 3T3-L1 adipocyte differentiation were determined by real-time quantitative PCR. mRNA expression data were normalized to 36B4 mRNA levels in the corresponding sample. Values are the mean SE of three independent experiments. (C) Analysis of SPARC protein during 3T3-L1 cell differentiation by Western blot. The same membrane was blotted with an antibody to Erk1/2 as a control for equal loading of samples. (D) Quantification of SPARC expression during 3T3-L1 cell differentiation. The intensity of the signals was calculated from Scion Image program and normalized against the relative level of Erk1/2 expression in the corresponding sample. Asterisks represent statistically significant differences (ANOVA): * 0.01 < p < 0.05; ** 0.001 < p < 0.01; *** p < 0.001.

Full figure and legend (53K)

We next determined the profile of SPARC expression during the differentiation of the murine 3T3-L1 cell line. Confluent preadipocytes (Day 0) were induced to differentiate by incubation with adipogenic medium for 3 days. The differentiation process was followed by incubation of the cells in medium supplemented with insulin alone (Day 3). Real-time PCR assays at various stages of differentiation (Figure 3B) showed that SPARC mRNA was strongly induced in confluent preadipocytes (Day 0 vs. Day - 2) and that addition of the adipogenic mixture provoked a decrease in SPARC levels. However, we observed a 2-fold increase in SPARC levels at the end of the differentiation process. Expression of PPAR mRNA as a mature adipocyte marker showed a similar enhancement at Day 5, as pictured in Figure 3B (right). The expression pattern of SPARC during adipose conversion was confirmed by Western blot. We observed that the levels of SPARC protein correlated with those of SPARC transcript (Figure 3Cand 3D). To summarize, our results indicate that SPARC expression is regulated during murine and human adipogenesis and that SPARC is characteristic of committed and fully differentiated cells.

Discussion

Our study provides evidence that SPARC is expressed in the human adipocyte. We show that both SPARC transcript and protein are present in human adipose tissue and that the main source of SPARC production is the adipocyte compared with stromal cells, in which endothelial cells express SPARC protein (Figure 2B). There seems to be a difference in the production of SPARC between rodents and humans, because we have previously shown in mice that SPARC was synthesized in both adipocytes and stromal cells (15). SPARC is increased during adipose conversion of human primary and murine 3T3-L1 preadipocytes. Using real-time PCR assays, we found that the SPARC transcript was increased 3-fold in human primary preadipocyte cultures as a consequence of adipocyte differentiation. During 3T3-L1 preadipocyte differentiation, SPARC transcript and protein levels showed a similar biphasic pattern. SPARC is expressed in the preadipocyte, and its expression is increased to an important extent when the cells reach confluency. One day after the induction of differentiation, SPARC returned to basal levels and was maintained throughout the time that the adipogenic mixture remained in the growth medium. Finally, at the end of the differentiation period, SPARC increased (Day 7). The reason for this biphasic SPARC expression during 3T3-L1 differentiation is not known. Furthermore, at variance with our data, Takahashi et al. (21) found that SPARC mRNA levels are increased throughout the differentiation process. These differences in expression pattern could be related to the different sensitivity of experimental approaches (Northern blot vs. real-time quantitative PCR in our experiments). Our result agrees with that obtained for expression of thrombospondin 1, a gene belonging to the same family. The expression of thrombospondin 1 decreased at Day 2 after induction of differentiation and, subsequently, increased at Day 6, indicating that some members of the matricellular protein group need to be down-regulated during early phases of the differentiation process (22). This early reduction of SPARC expression appears to be important. In fact, inhibition of 3T3-L1 adipocyte differentiation by overexpression of Wnt was found to prevent the decrease in SPARC mRNA (23). This observation suggests that the differentiation process, rather than the various agents present in the adipogenic mixture, induces the reduction of SPARC. It is possible that enhanced production of SPARC serves as a signal for the cellular arrest that is crucial for adipogenesis (24). After induction, growth-arrested 3T3-L1 preadipocytes re-enter the cell cycle and undergo one or two rounds of cell division (25, 26). It has been reported that SPARC has the capacity to block the cell cycle of smooth muscle cells and mesangial cells by preventing progression through S phase (4, 6). We suggest that high levels of SPARC block the preadipocyte cell cycle at Day 0 and that the decrease in SPARC at Day 1 might permit preadipocytes to re-enter the cell cycle.

In an attempt to understand the action of SPARC in adipose tissue development, we examined SPARC in adipose tissue from a transgenic mouse model with HF diet-induced obesity. Because of the role of catecholamines in adipose tissue (27), we generated transgenic mice (m3 AR^{- /-} ; h3 AR^+/- , h2 AR^+/- ) exhibiting a "human-like" pattern of AR expression in adipocytes. HF-fed transgenic mice, which express 2 AR (m3 AR^{- /-} ; h3 AR^+/- , h2 AR^+/- ), develop obesity, whereas HF-fed control mice (m3 AR^{- /-} ; h3 AR^+/- ) do not. The obese transgenic mice had no metabolic disturbances, no elevated blood glucose, and no modification of insulin and free fatty acid levels at the end of the HF diet (19). In this model, we observed that SPARC protein was up-regulated in adipose tissue from obese transgenic mice. As previously described, the major consequence of the development of obesity in these transgenic mice is a large increase in fat cell number without fat cell hypertrophy (19). We have previously described high levels of SPARC in adipose tissue of other obese models including gold thioglucose-treated and ob/ob mice, which exhibit adipocyte hypertrophy and high plasma insulin levels (15). We suggest that SPARC might play a role in both hypertrophy and hyperplasia phenomena. Our present studies show that SPARC expression is independent of food lipids and obesity-associated disorders and might be involved in the control of adipose tissue growth. Recently, Bradshaw et al. (16) reported that SPARC-null mice exhibit increased adiposity associated with an increase in both adipocyte size and number. The authors suggested that the known effects of SPARC on the regulation of cell shape and production of ECM could limit the accumulation of adipose tissue. Clearly, SPARC plays a complex role in adipose tissue development, which is not surprising given the wide impact of SPARC on major cell responses including adhesion and proliferation, expression of ECM proteins and proteinases, and growth factor functions. Furthermore, SPARC-null mice display decreased bone remodeling leading to profound osteopenia (12). Osteoblastic cells from these mice are more highly disposed to differentiate in adipocytes than those of wild-type mice (13). These data may explain why sparc^{- /-} mice did not show a modification in body weight and suggest that SPARC could act on differentiation of precursor cells to direct the cells in an osteoblastic or an adipogenic pathway.

Finally, the low level of SPARC protein in adipose tissue of lean transgenic mice compared with lean control mice indicates that catecholamines might control SPARC gene expression in adipose tissue. This possibility is currently being studied.

In conclusion, we showed that SPARC is expressed in human adipose tissue, with higher levels in the human adipocyte compared with the stromal-vascular cells, and that production of SPARC is modulated during adipose conversion. In addition, our data show a potential role for SPARC in the control of adipose tissue hyperplasia and fat pad development. The future challenge is to decipher the mechanisms by which SPARC affects these integrated biological responses.

Notes

¹ Nonstandard abbreviations: SPARC, secreted protein acidic and rich in cysteine; ECM, extracellular matrix; HF, high-fat; PCR, polymerase chain reaction; HUVEC, human umbilical vein endothelial cell; SDS, sodium dodecyl sulfate; AR, adrenergic receptor; LF, low-fat; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; aP2, adipocyte fatty acid-binding protein; PPAR, peroxisome proliferator-activated receptor SEM, standard error of the mean

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Acknowledgments

We thank Drs. Stephane Rocchi and Anne Johnston for discussion and critical reading of our manuscript and Drs. Amy Bradshaw and David Graves for invaluable help and critical review of this manuscript. This work was supported, in part, by the Institut National de la Santé et de la Recherche Médicale, LIPHA-MERCK (Lyon, France), and by NIH Grant ROI-GM 40711, and by the European Community's FP6 EUGENE 2 (LSHM-CT-2004-512013). C.C. was a recipient of a fellowship from l'Association pour la Recherche sur le Cancer.