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Cathepsin S Promotes Human Preadipocyte Differentiation: Possible Involvement of Fibronectin Degradation

Cathepsin S Promotes Human Preadipocyte Differentiation: Possible Involvement of Fibronectin... We previously showed that the cysteine protease cathepsin S (CTSS), known to degrade several components of the extracellular matrix (ECM), is produced by human adipose cells and increased in obesity. Because ECM remodeling is a key process associated with adipogenesis, this prompted us to assess the potential role of CTSS to promote preadipocyte differentiation. Kinetic studies in primary human preadipocytes revealed a modest increase in CTSS gene expression and secretion at the end of differentiation. CTSS activity was maximal in preadipocyte culture medium but decreased thereafter, fitting with increased release of the CTSS endogenous inhibitor, cystatin C, during differentiation. Inhibition of CTSS activity by an exogenous-specific inhibitor added along the differentiation, resulted in a 2-fold reduction of lipid content and expression of adipocyte markers in differentiated cells. Conversely, the treatment of preadipocytes with human recombinant CTSS increased adipogenesis. Moreover, CTSS supplementation in preadipocyte media markedly reduced the fibronectin network, a key preadipocyte-ECM component, the decrease of which is required for adipogenesis. Using immunohistochemistry on serial sections of adipose tissue of obese subjects, we showed that adipose cells staining positive for CTSS are mainly located in the vicinity of fibrosis regions containing fibronectin. Herein we propose that CTSS may promote human adipogenesis, at least in part, by degrading fibronectin in the early steps of differentiation. Taken together, these results indicate that CTSS released locally by preadipocytes promotes adipogenesis, suggesting a possible contribution of this protease to fat mass expansion in obesity. GROWTH OF ADIPOSE tissue mass involves both hypertrophia and hyperplasia of the adipocytes (1). These processes result respectively from the increase of lipid accumulation in the adipocytes and the formation of new adipocytes from precursors cells, the preadipocytes. Most of the knowledge regarding molecular and morphological changes during preadipocyte differentiation originates from studies on murine cell lines. Preadipocytes differentiate into mature adipocytes when treated with a well-characterized inducing cocktail (2). The sequence of events that leads to the expression of adipocyte-specific genes involves the activation of several transcriptional factors, notably the peroxisome-proliferator-activated receptor (PPAR)-γ, CCAAT/enhancer-binding proteins (C/EBP)-α, and sterol regulatory element binding protein 1c (SREBP1c) (3). PPARγ and C/EBPα control the expression of several adipocyte genes such as fatty acid binding protein (aP2) and fatty acid transporter (CD36). SREBP1c increases the expression of many lipogenic genes including the fatty acid synthase (FAS) (4). Fat mass growth is also associated with an important extracellular matrix (ECM) remodeling. ECM is composed of structural and adhesion proteins interacting with cell surface receptors, the integrins, to initiate cellular events like proliferation, differentiation, and apoptosis. During preadipocyte differentiation, the change of cell shape from fibroblastic morphology to rounded adipocyte is accompanied by major variations in expression and structure of several ECM components (5). In cell lines, the decrease of fibronectin, a key component of ECM, is required to promote adipogenesis (6, 7). We previously identified a novel biomarker of adiposity, the cathepsin S (CTSS) (8). The CTSS is a potent cysteine protease that has the ability to degrade several ECM proteins (9). CTSS is the only member of cysteine protease family that can retain proteolytic activity at neutral pH (10). The activity of CTSS is regulated by the endogenous inhibitor, cystatin C (11). We demonstrated that CTSS protein is expressed and secreted by the human adipose tissue and is up-regulated in obesity (8). In morbidly obese subjects, CTSS protein decreased in both adipocytes and the circulation after a significant fat mass loss induced by gastric surgery (12). We hypothesized that increased expression and secretion of CTSS by adipocytes of obese subjects might affect adipogenesis by the degradation of certain ECM elements. To determine the CTSS role in adipogenesis, we herein studied the consequences of inhibition or supplementation of CTSS in culture media of human preadipocytes. More specifically, we explored the possibility that a potential effect of CTSS on adipogenesis may be mediated through fibronectin degradation. Our results showed that CTSS facilitates adipogenesis, especially in the early steps of the differentiation, and that its effect is associated with fibronectin degradation. Materials and Methods Cell culture and protocol design Human sc adipose tissues were obtained from young women undergoing plastic surgery. This study was approved by the Ethics Committees of Hôtel-Dieu (Paris). Human preadipocytes were isolated and cultured according to the method originally described by Hauner (13) with minor modifications. Briefly, minced adipose tissue was digested in DMEM containing 2% albumin and 1 mg/ml collagenase for 1 h at 37 C. The digested material was filtered through a double-layered cotton mesh. The isolated cells were centrifuged at 250 × g for 10 min. The resulting pellet was washed with PBS and was next resuspended in erythrocyte lysis buffer [154 mm NH4Cl, 5.7 mm K2HPO4, and 0.1 mm EDTA (pH 7.0)] at 250 × g for 10 min. Finally, the pellet was washed and resuspended in DMEM and 10% fetal bovine serum. The cells were grown in DMEM and 10% fetal bovine serum and were used at passage 2 for the differentiation. Preadipocytes at d 0 were then cultured in differentiation medium containing 50 nm insulin, 100 nm dexamethasone, 0.25 mm 3-isobutyl-1-methylxanthine, and 100 nm rosiglitazone for the first 3 d. Next, medium was replaced by a culture medium containing 50 nm insulin, 100 nm dexamethasone, and 100 nm rosiglitazone and changed every 2 d until accumulation of lipid droplets (d 10–15). Lipid accumulation was assessed by staining paraformaldehyde-fixed cells with the Oil red O as described (14). In some experiments, 2 nm of a synthetic CTSS inhibitor (Z-Phe-Leu-COCHO; Calbiochem, La Jolla, CA) (inhibitory constant for CTSS: 0.185 nm] (15) or 50 nm of human recombinant CTSS (Calbiochem) were added to the medium at various time points, as indicated in the result section. CTSS synthetic inhibitor was dissolved in dimethylsulfide, and control cells were treated with dimethylsulfide alone (0.1%). In each culture conditions, cell viability was verified using a lactate dehydrogenase-cytotoxicity assay kit (BioVision, Mountain View, CA). In some experiments, cystatin C neutralizing antibody (R&D Systems, Minneapolis, MN) (inhibitory constant for cystatin C: 7.5 μg/ml) was used at concentration of 20 μg/ml. Determination of CTSS and cystatin C secretions in preadipocyte culture media CTSS and cystatin C protein levels were determined in the media of preadipocytes incubated during 48 h at different times of differentiation by using CTSS and cystatin C ELISA kits (Krka Laboratory, Inc., Novo mesto, Czech Republic; Biovendor Laboratory Medicine, Inc., Modrice, Czech Republic). Determination of CTSS enzymatic activity in preadipocyte culture media CTSS enzymatic activity was assessed in 48-h conditioned media of preadipocytes at different times of differentiation by using CTSS activity assay kit (BioVision Research Products). This kit is a fluorescence-based assay that uses the preferred CTSS substrate (Val-Val-Arg) labeled with amino-4-trifluoremethyl coumarin. The released amino-4-trifluoremethyl coumarin cleaved by CTSS is quantified using a fluorescence plate reader. Proteolysis of fibronectin by human CTSS To evaluate the ability of CTSS to degrade fibronectin in vitro, recombinant human CTSS (Calbiochem) (50 nm) was incubated with 0.4 mg/ml of human purified fibronectin (Sigma-Aldrich, Lyon, France) for 3 h. All reactions occurred at 37 C in DMEM in the presence or not of the CTSS inhibitor (Calbiochem) at either 20 nm or 20 μm. Fibronectin incubation without CTSS was also performed as controls. All samples were resolved by electrophoresis through a 4–20% Tris-glycine gel and stained with Coomassie Brilliant Blue (Invitrogen, Cergy Pontoise, France). RNA quantification by real-time PCR RNA extraction was performed using the RNeasy RNA minikit (QIAGEN, Courtaboeuf, France). Total RNAs (1 μg) were reversed transcribed using random hexamers and SuperScript II reverse transcriptase (Invitrogen). Real-time PCR was performed on GeneAmp 7000 sequence detection system (Applied Biosystems, Foster City, CA) as previously described (16). We used 18S rRNA (rRNA control Taqman assay kit; Applied Biosystems) as normalization control for gene expression. Western blot analysis Protein extracts of preadipocytes and adipocytes were prepared in cell lysing buffer containing 1× PBS, 0.1% sodium dodecyl sulfate, 1% IGEPAL CA-630 (Sigma-Aldrich), 0.5% sodium desoxycholate, and protease inhibitors. Proteins extracts (15 μg) were then resolved on 4–20% Tris-glycine gel. After protein transfer, the membranes were subsequently stained with ponceau red to verify loading of equal protein amounts. After blocking by Tris-buffered saline containing 0.2% Tween 20 (TBS-T) and 3% albumin for 2 h, filters were incubated overnight with the primary antibody diluted in TBS-T/3% albumin. Fibronectin antibody (diluted 1:1000) was bought from BD Bioscience (BD Transduction Laboratories, San Jose, CA). Integrin-β1 antibody (diluted 1:1000) was bought from Santa Cruz Biotechnology (Santa Cruz, CA). After washing, membranes were incubated with the secondary antibody coupled to peroxidase (diluted in PBS-Tween 20/3% BSA) for 1 h. Membranes were extensively washed and incubated with the enhanced chemiluminescence detection solution (Amersham Biosciences, Buckinghamshire, UK) and immediately exposed to x-ray films. Immunohistochemistry in human adipose tissue Surgical biopsies of sc white adipose tissue of 13 obese women (mean ± sem: body mass index 50.34 ± 1.84 kg/m2, age 37 ± 3.06 yr) were fixed in 4% paraformaldehyde, dehydrated, paraffin embedded, and then sectioned (thin sections, 5 μm thick). CTSS protein was detected with an antihuman CTSS polyclonal antibody (goat antihuman; Santa Cruz Biotechnology). Fibronectin protein was detected with a monoclonal antibody (BD Transduction Laboratories, San Jose, CA). Dewaxed, rehydrated sections were processed through the following incubation steps: 1) antigen unmasking by incubating tissue sections with 0.1% trypsin in PBS, as described (16); 2) hydrogen peroxide 3% in water for 20 min at room temperature (RT) to block endogenous peroxidases; 3) TBS-T/casein 0.02M solution (TBS-TC) for 15 min at RT; 4) incubation with polyclonal antihuman CTSS antibody or monoclonal antihuman fibronectin antibody diluted to 1:100 in TBS-TC overnight at 4 C; 5) swine multilink biotinylated immunoglobulins (Dako Cytomation, Trappes, France) diluted 1:200 in TBS-TC for 20 min at RT; 6) standard streptavidin-biotin-peroxidase complex method was applied using a commercially available kit (ABCYS GMR4–61; Biospa, Milan, Italy); and 7) visualization of staining using diaminobenzidine and counterstaining with Mayer’s hematoxylin. Specificity tests were performed by omission of primary antibodies from staining and use of preimmune serum instead of the first antiserum. Processed slide images were acquired by a microscope-camera system (Leica, Rueil-Malmaison, France). Immunocytofluorescence Primary cultures of preadipocytes were grown on glass coverslips in 24-well dishes. Cells were rinsed with PBS and fixed for 15 min in 4% paraformaldehyde. Cell were washed with PBS/0.15 m glycine and then permeabilized in BSA 3%/PBS/0.1% Tween 20 for 5 min. After blocking in BSA 3%/PBS for 30 min, primary antibody (antifibronectin; BD Bioscience) diluted 1:200 in PBS/3% BSA was incubated for 2 h. Cells were then washed and incubated for 2 h with the corresponding fluorescein isothiocyanate-conjugated secondary antibody Cy 2 (Amersham Biosciences) diluted in PBS/3% BSA. Nuclei were stained with 4′-6 diamidino-2-phenyl indole-2HCl. Negative controls were performed by omitting primary antibody. Finally, cells stained were mounted and examined by a BX 41 fluorescence microscope (Olympus, Rungis, France). Statistics The values given are means ± sem. The significance of difference between the experimental group and control was assessed by Student’s t test. The difference was considered significant when the P < 0.05. Results Expression and secretion profiles of CTSS and cystatin C during adipogenesis We examined the kinetic of CTSS and cystatin C gene expressions as well as other usual markers of adipogenesis at three times of differentiation. After preadipocytes were hormonally stimulated at d 0, the gene expression of the adipogenic transcriptional factors, C/EBPα, PPARγ, and SREBP1c increased by 6-, 5-, and 12-fold, respectively, as well as their target genes, leptin, aP2, and FAS that increased by 300-, 200-, and 30-fold, respectively, at d 15 of differentiation (Fig. 1, A and B). As shown Fig. 1C, the expression of CTSS and cystatin C genes increased, respectively, by 2- and 13-fold at d 15 of adipogenesis. Fig. 1 Open in new tabDownload slide Evolution of adipocyte markers and CTSS/cystatin C mRNAs during human preadipocyte differentiation. Total RNA was extracted from preadipocytes at d 0 (onset of the differentiation), 5, and 15, and mRNA expression of adipocyte markers was evaluated by quantitative real-time PCR. A, Kinetics of mRNA expressions of the master adipogenic transcriptional factors, PPARγ, C/EBPα, and SREBP1c. B, Evolution of mRNA of adipocyte markers, leptin, aP2, and FAS. C, Evolution of gene expressions of CTSS and cystatin C. Results are expressed as fold increase, compared with d 0. Values are means ± sem of three independent experiments performed in triplicates. *, P < 0.05 vs. d 0. Fig. 1 Open in new tabDownload slide Evolution of adipocyte markers and CTSS/cystatin C mRNAs during human preadipocyte differentiation. Total RNA was extracted from preadipocytes at d 0 (onset of the differentiation), 5, and 15, and mRNA expression of adipocyte markers was evaluated by quantitative real-time PCR. A, Kinetics of mRNA expressions of the master adipogenic transcriptional factors, PPARγ, C/EBPα, and SREBP1c. B, Evolution of mRNA of adipocyte markers, leptin, aP2, and FAS. C, Evolution of gene expressions of CTSS and cystatin C. Results are expressed as fold increase, compared with d 0. Values are means ± sem of three independent experiments performed in triplicates. *, P < 0.05 vs. d 0. To evaluate the concomitant protein secretions of CTSS and cystatin C, we measured their respective proteins in 48-h conditioned media from preadipocytes during differentiation at d 0, 5, and 15. CTSS and cystatin C protein secretions increased, respectively, by 2- and 9-fold at d 15 of adipogenesis (Fig. 2A). CTSS enzymatic activity was measured on 48-h conditioned media of preadipocytes and at d 5 and 15 of differentiation. As shown in Fig. 2B, CTSS activity was maximal in the media of undifferentiated preadipocytes and strongly decreased afterward. To precisely evaluate the time period that CTSS decreases during the first days of differentiation, we conducted another set of experiments in which we measured CTSS activity in preadipocyte media at d 0, 1, 2, 3, and 7 of differentiation. As shown in Fig. 2C, CTSS decreased mainly from d 1 of differentiation. Furthermore, the inhibition of cystatin C activity by adding its neutralizing antibody in adipocyte media during 24 h significantly increases CTSS activity (Fig. 2D). This observation argues for an inhibitory effect of cystatin C on CTSS activity in cultured human preadipocytes. Thus, the coordinated regulation of CTSS and cystatin C release by adipocytes ensures a high proteolytic activity specifically during the early steps of differentiation. Fig. 2 Open in new tabDownload slide Secretion patterns of CTSS and cystatin C during adipogenesis. A, Kinetic of CTSS and cystatin C protein secretions measured in 48-h conditioned media by using ELISA tests. These results represent one experiment performed in triplicate, which is representative of two independent experiments. B, CTSS activity measured in 48-h conditioned media during adipogenesis in three independent experiments performed in triplicates. RFU, Relative fluorescence unit. C, CTSS activity measured at more time points during the first days of differentiation realized in two independent experiments performed in triplicates. D, Day 10 differentiated adipocytes were incubated in adipogenic medium containing nonimmune goat antibody or anticystatin C. CTSS activity was measured in medium after 24 h of incubation. These results represent one experiment performed in triplicate, which is representative of two independent experiments. Results are expressed as fold increase or decrease, compared with d 0. *, P < 0.05 vs. d 0. Fig. 2 Open in new tabDownload slide Secretion patterns of CTSS and cystatin C during adipogenesis. A, Kinetic of CTSS and cystatin C protein secretions measured in 48-h conditioned media by using ELISA tests. These results represent one experiment performed in triplicate, which is representative of two independent experiments. B, CTSS activity measured in 48-h conditioned media during adipogenesis in three independent experiments performed in triplicates. RFU, Relative fluorescence unit. C, CTSS activity measured at more time points during the first days of differentiation realized in two independent experiments performed in triplicates. D, Day 10 differentiated adipocytes were incubated in adipogenic medium containing nonimmune goat antibody or anticystatin C. CTSS activity was measured in medium after 24 h of incubation. These results represent one experiment performed in triplicate, which is representative of two independent experiments. Results are expressed as fold increase or decrease, compared with d 0. *, P < 0.05 vs. d 0. CTSS effect on preadipocyte differentiation In an attempt to evaluate the importance of CTSS in the process of human preadipocyte differentiation, we chose to modulate CTSS activity in preadipose cells by using a synthetic inhibitor or through supplementation with human recombinant CTSS. After each treatment, the level of differentiation at d 10 was assessed by oil red staining and adipocyte markers gene expressions. The CTSS inhibitor (2 nm) was added at d 0 until the end of the culture period. As shown in Fig. 3, this treatment reduced lipid accumulation and gene expression of adipocyte markers including PPARγ, aP2, CD36, and SREBP1c in 10 d differentiated cells, indicating that inhibition of CTSS reduces adipogenesis. Fig. 3 Open in new tabDownload slide Effect of CTSS inhibition on adipogenesis. Preadipocyte were differentiated in the presence of adipogenic medium containing CTSS inhibitor (2 nm) or vehicle added from d 0 until d 10 of differentiation. A, Light microscopy of oil red O staining of adipocyte differentiated in the presence of synthetic CTSS inhibitor or vehicle. Lipid accumulation was evaluated by measuring oil red O staining. B, Effect of CTSS inhibitor on gene expression of adipocyte genes, PPARγ, aP2, CD36, and SREBP1c measured at d 10 of the differentiation. Results are expressed as fold decrease, compared with control. Values are means ± sem of three independent experiments performed in triplicates. *, P < 0.05 vs. control. Fig. 3 Open in new tabDownload slide Effect of CTSS inhibition on adipogenesis. Preadipocyte were differentiated in the presence of adipogenic medium containing CTSS inhibitor (2 nm) or vehicle added from d 0 until d 10 of differentiation. A, Light microscopy of oil red O staining of adipocyte differentiated in the presence of synthetic CTSS inhibitor or vehicle. Lipid accumulation was evaluated by measuring oil red O staining. B, Effect of CTSS inhibitor on gene expression of adipocyte genes, PPARγ, aP2, CD36, and SREBP1c measured at d 10 of the differentiation. Results are expressed as fold decrease, compared with control. Values are means ± sem of three independent experiments performed in triplicates. *, P < 0.05 vs. control. Conversely, to increase CTSS activity, human recombinant CTSS protein was used at a concentration of 50 nm previously shown to degrade efficiently certain components of the ECM in vitro (9). The preadipocytes were treated for 3 h either at d 0, before switching to the differentiation medium, or d 5. At d 10, a significant 2-fold increase in oil red staining and adipocyte markers (PPARγ, aP2, CD36, SREBP1c, and FAS) was observed specifically in the cells treated with CTSS at d 0 (Fig. 4A). By contrast, the levels of gene expression for adipocyte markers were not significantly altered when CTSS was added at d 5 (Fig. 4B). Thus, submitting preadipocytes to high CTSS activity before the induction of differentiation facilitates adipogenesis, in mirror with the reducing effect of CTSS inhibition. Fig. 4 Open in new tabDownload slide Effect of recombinant human CTSS protein on adipogenesis. CTSS (50 nm) or vehicle was added at d 0 or 5 of the differentiation during 3 h followed by the addition of adipogenic medium without recombinant protein until d 10 of the differentiation. A, Light microscopy of oil red O staining of adipocyte treated with CTSS recombinant protein or vehicle at d 0. Lipid accumulation was evaluated by measuring oil red O staining. B, Effect of CTSS supplementation at d 0 (CTSS D0) or 5 (CTSS D5) on gene expression of adipocyte genes, PPARγ, aP2, CD36, FAS, and SREBP1c measured at d 10 of the differentiation. Results are expressed as fold increase, compared with control. Values are means ± sem of three independent experiments performed in triplicates. *, P < 0.05 vs. control. Fig. 4 Open in new tabDownload slide Effect of recombinant human CTSS protein on adipogenesis. CTSS (50 nm) or vehicle was added at d 0 or 5 of the differentiation during 3 h followed by the addition of adipogenic medium without recombinant protein until d 10 of the differentiation. A, Light microscopy of oil red O staining of adipocyte treated with CTSS recombinant protein or vehicle at d 0. Lipid accumulation was evaluated by measuring oil red O staining. B, Effect of CTSS supplementation at d 0 (CTSS D0) or 5 (CTSS D5) on gene expression of adipocyte genes, PPARγ, aP2, CD36, FAS, and SREBP1c measured at d 10 of the differentiation. Results are expressed as fold increase, compared with control. Values are means ± sem of three independent experiments performed in triplicates. *, P < 0.05 vs. control. CTSS degrades fibronectin network It is known the expression of fibronectin, a key component of preadipocyte ECM, decreases during adipogenesis in cell lines. Here we showed that fibronectin protein expression is reduced during adipogenesis in our model of primary culture of human preadipocytes (Fig. 5A). Moreover, immunocytofluorescence experiments revealed that the structure of the fibronectin network was dramatically modified in differentiated adipose cells, compared with preadipocytes (Fig. 5B). Fig. 5 Open in new tabDownload slide Fibronectin expression during human adipogenesis. A, Western blotting of fibronectin in preadipocyte (Prea) and adipocyte (Adip) extracts. B, Phase-contrast pictures showed cell morphology at the stage of preadipocytes (d 0) and adipocytes (d 10). The pictures showed immunocytofluorescence of fibronectin network at the preadipocyte and adipocyte stages. Fig. 5 Open in new tabDownload slide Fibronectin expression during human adipogenesis. A, Western blotting of fibronectin in preadipocyte (Prea) and adipocyte (Adip) extracts. B, Phase-contrast pictures showed cell morphology at the stage of preadipocytes (d 0) and adipocytes (d 10). The pictures showed immunocytofluorescence of fibronectin network at the preadipocyte and adipocyte stages. Given the importance of the fibronectin remodeling on adipogenesis, we tested the capacity of CTSS to cleave fibronectin in vitro. Purified human recombinant CTSS at 50 nm was incubated with human fibronectin protein for 3 h, with or without the synthetic CTSS inhibitor, and the reaction products were analyzed by gel electrophoresis. As shown in Fig. 6A, CTSS efficiently degrades fibronectin in 3 h, a process markedly reduced in presence of the synthetic CTSS inhibitor. We further tested whether the supplementation of medium with CTSS increases degradation of fibronectin synthesized by preadipocytes. For this purpose, we added 50 nm of recombinant CTSS during 3 h on preadipocytes and then performed immunoblotting against fibronectin on both total cell extracts and conditioned media. As shown in Fig. 6B, CTSS cleaves fibronectin into several fragments that were followed in the medium, whereas integrin-β1, which represents the subunit of the main receptor of fibronectin is preserved. We verified by immunocytofluorescence the effect of CTSS on the fibronectin network in preadipocytes. This network is strongly altered when CTSS was added in the medium, as shown in Fig. 6C. Fig. 6 Open in new tabDownload slide Proteolysis of fibronectin by recombinant human CTSS. A, Fibronectin (FN) (0.4 mg/ml) was incubated at 37 C with 50 nm of human recombinant CTSS diluted in DMEM at neutral pH for 3 h in the absence (−) or presence of synthetic CTSS inhibitor at 20 nm or 20 μm. Control (Cont) represents fibronectin without adding CTSS. The products were analyzed as described in Materials and Methods. B, Western blotting against fibronectin and integrin-β1 in whole-cell extracts and preadipocyte (Prea) conditioned medium in the absence (Cont) or presence of CTSS recombinant protein (50 nm) during 3 h. The results are representative of two independent experiments. C, Immunocytofluorescence was performed with an antifibronectin antibody on preadipocytes treated with CTSS as above. Fig. 6 Open in new tabDownload slide Proteolysis of fibronectin by recombinant human CTSS. A, Fibronectin (FN) (0.4 mg/ml) was incubated at 37 C with 50 nm of human recombinant CTSS diluted in DMEM at neutral pH for 3 h in the absence (−) or presence of synthetic CTSS inhibitor at 20 nm or 20 μm. Control (Cont) represents fibronectin without adding CTSS. The products were analyzed as described in Materials and Methods. B, Western blotting against fibronectin and integrin-β1 in whole-cell extracts and preadipocyte (Prea) conditioned medium in the absence (Cont) or presence of CTSS recombinant protein (50 nm) during 3 h. The results are representative of two independent experiments. C, Immunocytofluorescence was performed with an antifibronectin antibody on preadipocytes treated with CTSS as above. Immunolocalization of CTSS and fibronectin in sc white adipose tissue of obese subjects We previously demonstrated that the number of CTSS-positive adipocytes is higher in sc white adipose tissue of obese compared with nonobese subjects (12). We then reconsidered the localization of CTSS in adipocytes of obese adipose tissue in relationship with the fibronectin staining. We observed that CTSS immunostaining was heterogeneous in mature adipocytes of white adipose tissue. Some adipocytes tested completely negative, whereas others stained strongly positive to the CTSS antibody (Fig. 7A). Besides, fully mature adipocytes tested negative for fibronectin (Fig. 7B). Interestingly, a strong positivity for fibronectin was observed in some extracellular fibrosis areas (Fig. 7B). ECM fibers were not completely positive for fibronectin, suggesting the presence of others ECM components such as collagens and proteoglycans. In serial sections of white adipose tissue, we observed that CTSS-positive adipocytes were mainly localized very close to fibrosis areas rich in fibronectin (Fig. 7, C and D). This feature may suggest a possible local role for adipose-derived CTSS protein in degradation of certain ECM components. Fig. 7 Open in new tabDownload slide Immunohistochemical detection of CTSS and fibronectin proteins in human sc white adipose tissue of obese subject. The CTSS immunopositivity was heterogeneous (A, ×100). Some adipocytes tested negative (A, asterisks) and others strongly positive (A, arrows). The strong positive staining was frequently observed in adipocytes close to fibrosis areas (F), as shown in A (×100) and C (×60, arrows). Fibronectin tested negative in fully mature adipocytes, and a strong positivity was observed in fibrosis areas (B, ×20, arrows). Serial sections stained, respectively, for CTSS (C, ×40) and fibronectin (D, ×40) show CTSS positivity (arrows) in adipocytes close to fibrosis areas rich in fibronectin (arrows). Fully mature adipocytes tested negative for fibronectin (×60, asterisks). Fig. 7 Open in new tabDownload slide Immunohistochemical detection of CTSS and fibronectin proteins in human sc white adipose tissue of obese subject. The CTSS immunopositivity was heterogeneous (A, ×100). Some adipocytes tested negative (A, asterisks) and others strongly positive (A, arrows). The strong positive staining was frequently observed in adipocytes close to fibrosis areas (F), as shown in A (×100) and C (×60, arrows). Fibronectin tested negative in fully mature adipocytes, and a strong positivity was observed in fibrosis areas (B, ×20, arrows). Serial sections stained, respectively, for CTSS (C, ×40) and fibronectin (D, ×40) show CTSS positivity (arrows) in adipocytes close to fibrosis areas rich in fibronectin (arrows). Fully mature adipocytes tested negative for fibronectin (×60, asterisks). Discussion The present study provides the first evidence that CTSS released by human preadipocytes intervenes in the regulation of adipogenesis. The role of CTSS in human preadipocytes was highlighted by the finding that the inhibition of its activity decreased adipogenesis, whereas supplementation of culture media with CTSS significantly promoted adipogenesis. To study the effect of CTSS on adipogenesis, we used primary cultures of human preadipocytes. Murine cell models, most notably the 3T3-L1 cell line, have been the basis for the majority of adipogenesis studies. However, there is a growing concern that adipogenesis differs between human and murine systems. For example, the resistin was first identified in the 3T3-L1 cells in which its expression increased along the differentiation, whereas in human preadipocytes its expression decreased along adipogenesis (17, 18). These discrepancies argue for the importance of human preadipocyte cell models use in exploratory research, particularly in adipogenesis. Analysis of CTSS expression and secretion during human preadipocyte differentiation revealed a modest increase of CTSS during differentiation as compared with the other usual markers of adipogenesis. Because a balance between cathepsins and their endogenous inhibitor, cystatin C, determines their enzymatic activity, we concomitantly examined cystatin C secretion profiles during adipogenesis. Our results showed that the secretion of cystatin C increased more importantly than CTSS during preadipocyte differentiation. Consistent with these findings, CTSS activity strongly decreased during differentiation, suggesting a critical role of CTSS in the early steps of adipogenesis. Besides, the addition of CTSS recombinant protein on preadipocytes led to increase adipogenic markers, whereas adding CTSS when adipogenesis is engaged did not affect differentiation. These results, together with the specific pattern of CTSS/cystatin C secretions and the decrease of CTSS activity during adipogenesis, suggests an early role of CTSS in facilitating preadipocyte differentiation. Future investigations are necessary to determine the molecular mechanisms by which CTSS and cystatin C expressions are regulated during adipogenesis. We further explored the possibility that CTSS may promote adipogenesis via its suspected role in ECM remodeling. Proteases such as metalloproteinases and plasmin that degrade certain components of ECM were suggested to regulate adipogenesis (19–22). Other unknown proteinases, such as family of cysteine proteases, may also contribute in adipose ECM remodeling and thereby the regulation of adipogenesis. The role of CTSS in such process might be argued based on a network of concordant indirect and direct arguments produced by previous studies and our work. The expression of CTSS is dramatically increased in obesity. We previously showed that CTSS protein expression significantly augments in adipose tissue of morbidly obese subjects, whereas cystatin C protein expression is similar in obese and lean subjects (8) (data not shown). Thus, the increase of CTSS in adipose tissue could lead to local degradation of some ECM proteins, promoting the development of fat mass. Indeed, CTSS degrades several ECM proteins in vitro such as elastin, laminin, and types I and IV collagens (9, 23). The involvement of CTSS in the excessive ECM degradation has been demonstrated in others tissues such as in the vascular wall in which CTSS is involved in the progression of atherosclerosis (24). The role of CTSS in the degradation of ECM components has never been explored in human adipose tissue. We here demonstrated a role of CTSS in the degradation of fibronectin, which appears as a key element regulating adipogenesis. Cell cultures on fibronectin matrices inhibited human preadipocyte adipogenesis (25). We confirmed this result with our preadipocytes (data not shown). TGFβ, a strong inhibitor of adipocyte differentiation, stimulates the synthesis of fibronectin by preadipocytes (26, 27). Fibronectin down-regulates gene expression of lipogenic proteins and thereby interfere with morphological changes necessary for new gene expression (28). In agreement with these results obtained in cell lines, we here showed that there was an important change of fibronectin network and a decreased of its expression during human preadipocyte differentiation. Besides, it was previously showed that proteolytic degradation of fibronectin yields specific fragments that activate adipogenesis (29). We presently demonstrated that CTSS degrades preadipocyte synthesized fibronectin. This effect is associated with an increase of adipogenic markers such as PPARγ and SREBP1c as well as lipogenic target genes CD36, aP2, and FAS. This result suggests a critical role of CTSS in the early steps of adipogenesis in which fibroblastic preadipocytes change morphology and fibronectin starts to decrease. Further studies are required to assess the impact of CTSS on fibronectin proteolysis in adipose tissue. Interestingly, immunohistochemistry study of human adipose tissue revealed that fibronectin is not expressed in fully differentiated adipocytes as it was previously reported, supporting the idea that fibronectin synthesis decreased during adipocyte development (30). Nevertheless, we observed a strong staining of fibronectin in fibrosis regions. In serial sections of adipose tissue, we showed that adipose cells with strong CTSS staining are preferentially localized close to fibrosis regions containing fibronectin. These fibrosis regions are increased in adipose tissue of obese subjects than non obese ones (data not shown). These results may support the idea of possible role of CTSS in ECM degradation, in particular fibronectin. Further studies are necessary to unravel adipose ECM remodeling and implication of proteinases in this process during fat mass development. In conclusion, our results showed that CTSS promotes the early steps of human preadipocyte differentiation. This effect may be mediated through the degradation of fibronectin. Our results open further avenues of research to elucidate the physiological functions of CTSS in human adipose tissue. Future studies are necessary for determining the physiological role of CTSS on adipose tissue biology in vivo. Acknowledgments We thank Dr. Pierre Niccolo and Dr. Philippe Sellam for adipose tissue availability and Franck Viguier (Cytogenetic Department) for fluorescence microscopy availability. We also thank Martine Pinçon-Raymond (Institut National de la Santé et de la Recherche Médicale, Unité 505) for helpful discussions concerning extracellular matrix and Michele Guerre-Millo for critical reading of the manuscript. This work was supported by the French National Agency of Research (OBCAT no. ANR-05-PCOD-026-01), the Contrat de Recherche Clinique ALFEDIAM and the BQR (Bonus Quality Research) of Paris 6 University. S.T. and R.C. received a grant from Institut National de la Santé et de la Recherche Médicale/Conseil Regional Ile de France (2003–2005), and S.T. received support from the Fondation pour la Recherche Médicale (2006). 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Eur J Histochem 42 : 183 – 188 Google Scholar PubMed WorldCat Copyright © 2006 by The Endocrine Society http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Endocrinology Oxford University Press

Cathepsin S Promotes Human Preadipocyte Differentiation: Possible Involvement of Fibronectin Degradation

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Oxford University Press
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Copyright © 2006 by The Endocrine Society
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0013-7227
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1945-7170
DOI
10.1210/en.2006-0386
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Abstract

We previously showed that the cysteine protease cathepsin S (CTSS), known to degrade several components of the extracellular matrix (ECM), is produced by human adipose cells and increased in obesity. Because ECM remodeling is a key process associated with adipogenesis, this prompted us to assess the potential role of CTSS to promote preadipocyte differentiation. Kinetic studies in primary human preadipocytes revealed a modest increase in CTSS gene expression and secretion at the end of differentiation. CTSS activity was maximal in preadipocyte culture medium but decreased thereafter, fitting with increased release of the CTSS endogenous inhibitor, cystatin C, during differentiation. Inhibition of CTSS activity by an exogenous-specific inhibitor added along the differentiation, resulted in a 2-fold reduction of lipid content and expression of adipocyte markers in differentiated cells. Conversely, the treatment of preadipocytes with human recombinant CTSS increased adipogenesis. Moreover, CTSS supplementation in preadipocyte media markedly reduced the fibronectin network, a key preadipocyte-ECM component, the decrease of which is required for adipogenesis. Using immunohistochemistry on serial sections of adipose tissue of obese subjects, we showed that adipose cells staining positive for CTSS are mainly located in the vicinity of fibrosis regions containing fibronectin. Herein we propose that CTSS may promote human adipogenesis, at least in part, by degrading fibronectin in the early steps of differentiation. Taken together, these results indicate that CTSS released locally by preadipocytes promotes adipogenesis, suggesting a possible contribution of this protease to fat mass expansion in obesity. GROWTH OF ADIPOSE tissue mass involves both hypertrophia and hyperplasia of the adipocytes (1). These processes result respectively from the increase of lipid accumulation in the adipocytes and the formation of new adipocytes from precursors cells, the preadipocytes. Most of the knowledge regarding molecular and morphological changes during preadipocyte differentiation originates from studies on murine cell lines. Preadipocytes differentiate into mature adipocytes when treated with a well-characterized inducing cocktail (2). The sequence of events that leads to the expression of adipocyte-specific genes involves the activation of several transcriptional factors, notably the peroxisome-proliferator-activated receptor (PPAR)-γ, CCAAT/enhancer-binding proteins (C/EBP)-α, and sterol regulatory element binding protein 1c (SREBP1c) (3). PPARγ and C/EBPα control the expression of several adipocyte genes such as fatty acid binding protein (aP2) and fatty acid transporter (CD36). SREBP1c increases the expression of many lipogenic genes including the fatty acid synthase (FAS) (4). Fat mass growth is also associated with an important extracellular matrix (ECM) remodeling. ECM is composed of structural and adhesion proteins interacting with cell surface receptors, the integrins, to initiate cellular events like proliferation, differentiation, and apoptosis. During preadipocyte differentiation, the change of cell shape from fibroblastic morphology to rounded adipocyte is accompanied by major variations in expression and structure of several ECM components (5). In cell lines, the decrease of fibronectin, a key component of ECM, is required to promote adipogenesis (6, 7). We previously identified a novel biomarker of adiposity, the cathepsin S (CTSS) (8). The CTSS is a potent cysteine protease that has the ability to degrade several ECM proteins (9). CTSS is the only member of cysteine protease family that can retain proteolytic activity at neutral pH (10). The activity of CTSS is regulated by the endogenous inhibitor, cystatin C (11). We demonstrated that CTSS protein is expressed and secreted by the human adipose tissue and is up-regulated in obesity (8). In morbidly obese subjects, CTSS protein decreased in both adipocytes and the circulation after a significant fat mass loss induced by gastric surgery (12). We hypothesized that increased expression and secretion of CTSS by adipocytes of obese subjects might affect adipogenesis by the degradation of certain ECM elements. To determine the CTSS role in adipogenesis, we herein studied the consequences of inhibition or supplementation of CTSS in culture media of human preadipocytes. More specifically, we explored the possibility that a potential effect of CTSS on adipogenesis may be mediated through fibronectin degradation. Our results showed that CTSS facilitates adipogenesis, especially in the early steps of the differentiation, and that its effect is associated with fibronectin degradation. Materials and Methods Cell culture and protocol design Human sc adipose tissues were obtained from young women undergoing plastic surgery. This study was approved by the Ethics Committees of Hôtel-Dieu (Paris). Human preadipocytes were isolated and cultured according to the method originally described by Hauner (13) with minor modifications. Briefly, minced adipose tissue was digested in DMEM containing 2% albumin and 1 mg/ml collagenase for 1 h at 37 C. The digested material was filtered through a double-layered cotton mesh. The isolated cells were centrifuged at 250 × g for 10 min. The resulting pellet was washed with PBS and was next resuspended in erythrocyte lysis buffer [154 mm NH4Cl, 5.7 mm K2HPO4, and 0.1 mm EDTA (pH 7.0)] at 250 × g for 10 min. Finally, the pellet was washed and resuspended in DMEM and 10% fetal bovine serum. The cells were grown in DMEM and 10% fetal bovine serum and were used at passage 2 for the differentiation. Preadipocytes at d 0 were then cultured in differentiation medium containing 50 nm insulin, 100 nm dexamethasone, 0.25 mm 3-isobutyl-1-methylxanthine, and 100 nm rosiglitazone for the first 3 d. Next, medium was replaced by a culture medium containing 50 nm insulin, 100 nm dexamethasone, and 100 nm rosiglitazone and changed every 2 d until accumulation of lipid droplets (d 10–15). Lipid accumulation was assessed by staining paraformaldehyde-fixed cells with the Oil red O as described (14). In some experiments, 2 nm of a synthetic CTSS inhibitor (Z-Phe-Leu-COCHO; Calbiochem, La Jolla, CA) (inhibitory constant for CTSS: 0.185 nm] (15) or 50 nm of human recombinant CTSS (Calbiochem) were added to the medium at various time points, as indicated in the result section. CTSS synthetic inhibitor was dissolved in dimethylsulfide, and control cells were treated with dimethylsulfide alone (0.1%). In each culture conditions, cell viability was verified using a lactate dehydrogenase-cytotoxicity assay kit (BioVision, Mountain View, CA). In some experiments, cystatin C neutralizing antibody (R&D Systems, Minneapolis, MN) (inhibitory constant for cystatin C: 7.5 μg/ml) was used at concentration of 20 μg/ml. Determination of CTSS and cystatin C secretions in preadipocyte culture media CTSS and cystatin C protein levels were determined in the media of preadipocytes incubated during 48 h at different times of differentiation by using CTSS and cystatin C ELISA kits (Krka Laboratory, Inc., Novo mesto, Czech Republic; Biovendor Laboratory Medicine, Inc., Modrice, Czech Republic). Determination of CTSS enzymatic activity in preadipocyte culture media CTSS enzymatic activity was assessed in 48-h conditioned media of preadipocytes at different times of differentiation by using CTSS activity assay kit (BioVision Research Products). This kit is a fluorescence-based assay that uses the preferred CTSS substrate (Val-Val-Arg) labeled with amino-4-trifluoremethyl coumarin. The released amino-4-trifluoremethyl coumarin cleaved by CTSS is quantified using a fluorescence plate reader. Proteolysis of fibronectin by human CTSS To evaluate the ability of CTSS to degrade fibronectin in vitro, recombinant human CTSS (Calbiochem) (50 nm) was incubated with 0.4 mg/ml of human purified fibronectin (Sigma-Aldrich, Lyon, France) for 3 h. All reactions occurred at 37 C in DMEM in the presence or not of the CTSS inhibitor (Calbiochem) at either 20 nm or 20 μm. Fibronectin incubation without CTSS was also performed as controls. All samples were resolved by electrophoresis through a 4–20% Tris-glycine gel and stained with Coomassie Brilliant Blue (Invitrogen, Cergy Pontoise, France). RNA quantification by real-time PCR RNA extraction was performed using the RNeasy RNA minikit (QIAGEN, Courtaboeuf, France). Total RNAs (1 μg) were reversed transcribed using random hexamers and SuperScript II reverse transcriptase (Invitrogen). Real-time PCR was performed on GeneAmp 7000 sequence detection system (Applied Biosystems, Foster City, CA) as previously described (16). We used 18S rRNA (rRNA control Taqman assay kit; Applied Biosystems) as normalization control for gene expression. Western blot analysis Protein extracts of preadipocytes and adipocytes were prepared in cell lysing buffer containing 1× PBS, 0.1% sodium dodecyl sulfate, 1% IGEPAL CA-630 (Sigma-Aldrich), 0.5% sodium desoxycholate, and protease inhibitors. Proteins extracts (15 μg) were then resolved on 4–20% Tris-glycine gel. After protein transfer, the membranes were subsequently stained with ponceau red to verify loading of equal protein amounts. After blocking by Tris-buffered saline containing 0.2% Tween 20 (TBS-T) and 3% albumin for 2 h, filters were incubated overnight with the primary antibody diluted in TBS-T/3% albumin. Fibronectin antibody (diluted 1:1000) was bought from BD Bioscience (BD Transduction Laboratories, San Jose, CA). Integrin-β1 antibody (diluted 1:1000) was bought from Santa Cruz Biotechnology (Santa Cruz, CA). After washing, membranes were incubated with the secondary antibody coupled to peroxidase (diluted in PBS-Tween 20/3% BSA) for 1 h. Membranes were extensively washed and incubated with the enhanced chemiluminescence detection solution (Amersham Biosciences, Buckinghamshire, UK) and immediately exposed to x-ray films. Immunohistochemistry in human adipose tissue Surgical biopsies of sc white adipose tissue of 13 obese women (mean ± sem: body mass index 50.34 ± 1.84 kg/m2, age 37 ± 3.06 yr) were fixed in 4% paraformaldehyde, dehydrated, paraffin embedded, and then sectioned (thin sections, 5 μm thick). CTSS protein was detected with an antihuman CTSS polyclonal antibody (goat antihuman; Santa Cruz Biotechnology). Fibronectin protein was detected with a monoclonal antibody (BD Transduction Laboratories, San Jose, CA). Dewaxed, rehydrated sections were processed through the following incubation steps: 1) antigen unmasking by incubating tissue sections with 0.1% trypsin in PBS, as described (16); 2) hydrogen peroxide 3% in water for 20 min at room temperature (RT) to block endogenous peroxidases; 3) TBS-T/casein 0.02M solution (TBS-TC) for 15 min at RT; 4) incubation with polyclonal antihuman CTSS antibody or monoclonal antihuman fibronectin antibody diluted to 1:100 in TBS-TC overnight at 4 C; 5) swine multilink biotinylated immunoglobulins (Dako Cytomation, Trappes, France) diluted 1:200 in TBS-TC for 20 min at RT; 6) standard streptavidin-biotin-peroxidase complex method was applied using a commercially available kit (ABCYS GMR4–61; Biospa, Milan, Italy); and 7) visualization of staining using diaminobenzidine and counterstaining with Mayer’s hematoxylin. Specificity tests were performed by omission of primary antibodies from staining and use of preimmune serum instead of the first antiserum. Processed slide images were acquired by a microscope-camera system (Leica, Rueil-Malmaison, France). Immunocytofluorescence Primary cultures of preadipocytes were grown on glass coverslips in 24-well dishes. Cells were rinsed with PBS and fixed for 15 min in 4% paraformaldehyde. Cell were washed with PBS/0.15 m glycine and then permeabilized in BSA 3%/PBS/0.1% Tween 20 for 5 min. After blocking in BSA 3%/PBS for 30 min, primary antibody (antifibronectin; BD Bioscience) diluted 1:200 in PBS/3% BSA was incubated for 2 h. Cells were then washed and incubated for 2 h with the corresponding fluorescein isothiocyanate-conjugated secondary antibody Cy 2 (Amersham Biosciences) diluted in PBS/3% BSA. Nuclei were stained with 4′-6 diamidino-2-phenyl indole-2HCl. Negative controls were performed by omitting primary antibody. Finally, cells stained were mounted and examined by a BX 41 fluorescence microscope (Olympus, Rungis, France). Statistics The values given are means ± sem. The significance of difference between the experimental group and control was assessed by Student’s t test. The difference was considered significant when the P < 0.05. Results Expression and secretion profiles of CTSS and cystatin C during adipogenesis We examined the kinetic of CTSS and cystatin C gene expressions as well as other usual markers of adipogenesis at three times of differentiation. After preadipocytes were hormonally stimulated at d 0, the gene expression of the adipogenic transcriptional factors, C/EBPα, PPARγ, and SREBP1c increased by 6-, 5-, and 12-fold, respectively, as well as their target genes, leptin, aP2, and FAS that increased by 300-, 200-, and 30-fold, respectively, at d 15 of differentiation (Fig. 1, A and B). As shown Fig. 1C, the expression of CTSS and cystatin C genes increased, respectively, by 2- and 13-fold at d 15 of adipogenesis. Fig. 1 Open in new tabDownload slide Evolution of adipocyte markers and CTSS/cystatin C mRNAs during human preadipocyte differentiation. Total RNA was extracted from preadipocytes at d 0 (onset of the differentiation), 5, and 15, and mRNA expression of adipocyte markers was evaluated by quantitative real-time PCR. A, Kinetics of mRNA expressions of the master adipogenic transcriptional factors, PPARγ, C/EBPα, and SREBP1c. B, Evolution of mRNA of adipocyte markers, leptin, aP2, and FAS. C, Evolution of gene expressions of CTSS and cystatin C. Results are expressed as fold increase, compared with d 0. Values are means ± sem of three independent experiments performed in triplicates. *, P < 0.05 vs. d 0. Fig. 1 Open in new tabDownload slide Evolution of adipocyte markers and CTSS/cystatin C mRNAs during human preadipocyte differentiation. Total RNA was extracted from preadipocytes at d 0 (onset of the differentiation), 5, and 15, and mRNA expression of adipocyte markers was evaluated by quantitative real-time PCR. A, Kinetics of mRNA expressions of the master adipogenic transcriptional factors, PPARγ, C/EBPα, and SREBP1c. B, Evolution of mRNA of adipocyte markers, leptin, aP2, and FAS. C, Evolution of gene expressions of CTSS and cystatin C. Results are expressed as fold increase, compared with d 0. Values are means ± sem of three independent experiments performed in triplicates. *, P < 0.05 vs. d 0. To evaluate the concomitant protein secretions of CTSS and cystatin C, we measured their respective proteins in 48-h conditioned media from preadipocytes during differentiation at d 0, 5, and 15. CTSS and cystatin C protein secretions increased, respectively, by 2- and 9-fold at d 15 of adipogenesis (Fig. 2A). CTSS enzymatic activity was measured on 48-h conditioned media of preadipocytes and at d 5 and 15 of differentiation. As shown in Fig. 2B, CTSS activity was maximal in the media of undifferentiated preadipocytes and strongly decreased afterward. To precisely evaluate the time period that CTSS decreases during the first days of differentiation, we conducted another set of experiments in which we measured CTSS activity in preadipocyte media at d 0, 1, 2, 3, and 7 of differentiation. As shown in Fig. 2C, CTSS decreased mainly from d 1 of differentiation. Furthermore, the inhibition of cystatin C activity by adding its neutralizing antibody in adipocyte media during 24 h significantly increases CTSS activity (Fig. 2D). This observation argues for an inhibitory effect of cystatin C on CTSS activity in cultured human preadipocytes. Thus, the coordinated regulation of CTSS and cystatin C release by adipocytes ensures a high proteolytic activity specifically during the early steps of differentiation. Fig. 2 Open in new tabDownload slide Secretion patterns of CTSS and cystatin C during adipogenesis. A, Kinetic of CTSS and cystatin C protein secretions measured in 48-h conditioned media by using ELISA tests. These results represent one experiment performed in triplicate, which is representative of two independent experiments. B, CTSS activity measured in 48-h conditioned media during adipogenesis in three independent experiments performed in triplicates. RFU, Relative fluorescence unit. C, CTSS activity measured at more time points during the first days of differentiation realized in two independent experiments performed in triplicates. D, Day 10 differentiated adipocytes were incubated in adipogenic medium containing nonimmune goat antibody or anticystatin C. CTSS activity was measured in medium after 24 h of incubation. These results represent one experiment performed in triplicate, which is representative of two independent experiments. Results are expressed as fold increase or decrease, compared with d 0. *, P < 0.05 vs. d 0. Fig. 2 Open in new tabDownload slide Secretion patterns of CTSS and cystatin C during adipogenesis. A, Kinetic of CTSS and cystatin C protein secretions measured in 48-h conditioned media by using ELISA tests. These results represent one experiment performed in triplicate, which is representative of two independent experiments. B, CTSS activity measured in 48-h conditioned media during adipogenesis in three independent experiments performed in triplicates. RFU, Relative fluorescence unit. C, CTSS activity measured at more time points during the first days of differentiation realized in two independent experiments performed in triplicates. D, Day 10 differentiated adipocytes were incubated in adipogenic medium containing nonimmune goat antibody or anticystatin C. CTSS activity was measured in medium after 24 h of incubation. These results represent one experiment performed in triplicate, which is representative of two independent experiments. Results are expressed as fold increase or decrease, compared with d 0. *, P < 0.05 vs. d 0. CTSS effect on preadipocyte differentiation In an attempt to evaluate the importance of CTSS in the process of human preadipocyte differentiation, we chose to modulate CTSS activity in preadipose cells by using a synthetic inhibitor or through supplementation with human recombinant CTSS. After each treatment, the level of differentiation at d 10 was assessed by oil red staining and adipocyte markers gene expressions. The CTSS inhibitor (2 nm) was added at d 0 until the end of the culture period. As shown in Fig. 3, this treatment reduced lipid accumulation and gene expression of adipocyte markers including PPARγ, aP2, CD36, and SREBP1c in 10 d differentiated cells, indicating that inhibition of CTSS reduces adipogenesis. Fig. 3 Open in new tabDownload slide Effect of CTSS inhibition on adipogenesis. Preadipocyte were differentiated in the presence of adipogenic medium containing CTSS inhibitor (2 nm) or vehicle added from d 0 until d 10 of differentiation. A, Light microscopy of oil red O staining of adipocyte differentiated in the presence of synthetic CTSS inhibitor or vehicle. Lipid accumulation was evaluated by measuring oil red O staining. B, Effect of CTSS inhibitor on gene expression of adipocyte genes, PPARγ, aP2, CD36, and SREBP1c measured at d 10 of the differentiation. Results are expressed as fold decrease, compared with control. Values are means ± sem of three independent experiments performed in triplicates. *, P < 0.05 vs. control. Fig. 3 Open in new tabDownload slide Effect of CTSS inhibition on adipogenesis. Preadipocyte were differentiated in the presence of adipogenic medium containing CTSS inhibitor (2 nm) or vehicle added from d 0 until d 10 of differentiation. A, Light microscopy of oil red O staining of adipocyte differentiated in the presence of synthetic CTSS inhibitor or vehicle. Lipid accumulation was evaluated by measuring oil red O staining. B, Effect of CTSS inhibitor on gene expression of adipocyte genes, PPARγ, aP2, CD36, and SREBP1c measured at d 10 of the differentiation. Results are expressed as fold decrease, compared with control. Values are means ± sem of three independent experiments performed in triplicates. *, P < 0.05 vs. control. Conversely, to increase CTSS activity, human recombinant CTSS protein was used at a concentration of 50 nm previously shown to degrade efficiently certain components of the ECM in vitro (9). The preadipocytes were treated for 3 h either at d 0, before switching to the differentiation medium, or d 5. At d 10, a significant 2-fold increase in oil red staining and adipocyte markers (PPARγ, aP2, CD36, SREBP1c, and FAS) was observed specifically in the cells treated with CTSS at d 0 (Fig. 4A). By contrast, the levels of gene expression for adipocyte markers were not significantly altered when CTSS was added at d 5 (Fig. 4B). Thus, submitting preadipocytes to high CTSS activity before the induction of differentiation facilitates adipogenesis, in mirror with the reducing effect of CTSS inhibition. Fig. 4 Open in new tabDownload slide Effect of recombinant human CTSS protein on adipogenesis. CTSS (50 nm) or vehicle was added at d 0 or 5 of the differentiation during 3 h followed by the addition of adipogenic medium without recombinant protein until d 10 of the differentiation. A, Light microscopy of oil red O staining of adipocyte treated with CTSS recombinant protein or vehicle at d 0. Lipid accumulation was evaluated by measuring oil red O staining. B, Effect of CTSS supplementation at d 0 (CTSS D0) or 5 (CTSS D5) on gene expression of adipocyte genes, PPARγ, aP2, CD36, FAS, and SREBP1c measured at d 10 of the differentiation. Results are expressed as fold increase, compared with control. Values are means ± sem of three independent experiments performed in triplicates. *, P < 0.05 vs. control. Fig. 4 Open in new tabDownload slide Effect of recombinant human CTSS protein on adipogenesis. CTSS (50 nm) or vehicle was added at d 0 or 5 of the differentiation during 3 h followed by the addition of adipogenic medium without recombinant protein until d 10 of the differentiation. A, Light microscopy of oil red O staining of adipocyte treated with CTSS recombinant protein or vehicle at d 0. Lipid accumulation was evaluated by measuring oil red O staining. B, Effect of CTSS supplementation at d 0 (CTSS D0) or 5 (CTSS D5) on gene expression of adipocyte genes, PPARγ, aP2, CD36, FAS, and SREBP1c measured at d 10 of the differentiation. Results are expressed as fold increase, compared with control. Values are means ± sem of three independent experiments performed in triplicates. *, P < 0.05 vs. control. CTSS degrades fibronectin network It is known the expression of fibronectin, a key component of preadipocyte ECM, decreases during adipogenesis in cell lines. Here we showed that fibronectin protein expression is reduced during adipogenesis in our model of primary culture of human preadipocytes (Fig. 5A). Moreover, immunocytofluorescence experiments revealed that the structure of the fibronectin network was dramatically modified in differentiated adipose cells, compared with preadipocytes (Fig. 5B). Fig. 5 Open in new tabDownload slide Fibronectin expression during human adipogenesis. A, Western blotting of fibronectin in preadipocyte (Prea) and adipocyte (Adip) extracts. B, Phase-contrast pictures showed cell morphology at the stage of preadipocytes (d 0) and adipocytes (d 10). The pictures showed immunocytofluorescence of fibronectin network at the preadipocyte and adipocyte stages. Fig. 5 Open in new tabDownload slide Fibronectin expression during human adipogenesis. A, Western blotting of fibronectin in preadipocyte (Prea) and adipocyte (Adip) extracts. B, Phase-contrast pictures showed cell morphology at the stage of preadipocytes (d 0) and adipocytes (d 10). The pictures showed immunocytofluorescence of fibronectin network at the preadipocyte and adipocyte stages. Given the importance of the fibronectin remodeling on adipogenesis, we tested the capacity of CTSS to cleave fibronectin in vitro. Purified human recombinant CTSS at 50 nm was incubated with human fibronectin protein for 3 h, with or without the synthetic CTSS inhibitor, and the reaction products were analyzed by gel electrophoresis. As shown in Fig. 6A, CTSS efficiently degrades fibronectin in 3 h, a process markedly reduced in presence of the synthetic CTSS inhibitor. We further tested whether the supplementation of medium with CTSS increases degradation of fibronectin synthesized by preadipocytes. For this purpose, we added 50 nm of recombinant CTSS during 3 h on preadipocytes and then performed immunoblotting against fibronectin on both total cell extracts and conditioned media. As shown in Fig. 6B, CTSS cleaves fibronectin into several fragments that were followed in the medium, whereas integrin-β1, which represents the subunit of the main receptor of fibronectin is preserved. We verified by immunocytofluorescence the effect of CTSS on the fibronectin network in preadipocytes. This network is strongly altered when CTSS was added in the medium, as shown in Fig. 6C. Fig. 6 Open in new tabDownload slide Proteolysis of fibronectin by recombinant human CTSS. A, Fibronectin (FN) (0.4 mg/ml) was incubated at 37 C with 50 nm of human recombinant CTSS diluted in DMEM at neutral pH for 3 h in the absence (−) or presence of synthetic CTSS inhibitor at 20 nm or 20 μm. Control (Cont) represents fibronectin without adding CTSS. The products were analyzed as described in Materials and Methods. B, Western blotting against fibronectin and integrin-β1 in whole-cell extracts and preadipocyte (Prea) conditioned medium in the absence (Cont) or presence of CTSS recombinant protein (50 nm) during 3 h. The results are representative of two independent experiments. C, Immunocytofluorescence was performed with an antifibronectin antibody on preadipocytes treated with CTSS as above. Fig. 6 Open in new tabDownload slide Proteolysis of fibronectin by recombinant human CTSS. A, Fibronectin (FN) (0.4 mg/ml) was incubated at 37 C with 50 nm of human recombinant CTSS diluted in DMEM at neutral pH for 3 h in the absence (−) or presence of synthetic CTSS inhibitor at 20 nm or 20 μm. Control (Cont) represents fibronectin without adding CTSS. The products were analyzed as described in Materials and Methods. B, Western blotting against fibronectin and integrin-β1 in whole-cell extracts and preadipocyte (Prea) conditioned medium in the absence (Cont) or presence of CTSS recombinant protein (50 nm) during 3 h. The results are representative of two independent experiments. C, Immunocytofluorescence was performed with an antifibronectin antibody on preadipocytes treated with CTSS as above. Immunolocalization of CTSS and fibronectin in sc white adipose tissue of obese subjects We previously demonstrated that the number of CTSS-positive adipocytes is higher in sc white adipose tissue of obese compared with nonobese subjects (12). We then reconsidered the localization of CTSS in adipocytes of obese adipose tissue in relationship with the fibronectin staining. We observed that CTSS immunostaining was heterogeneous in mature adipocytes of white adipose tissue. Some adipocytes tested completely negative, whereas others stained strongly positive to the CTSS antibody (Fig. 7A). Besides, fully mature adipocytes tested negative for fibronectin (Fig. 7B). Interestingly, a strong positivity for fibronectin was observed in some extracellular fibrosis areas (Fig. 7B). ECM fibers were not completely positive for fibronectin, suggesting the presence of others ECM components such as collagens and proteoglycans. In serial sections of white adipose tissue, we observed that CTSS-positive adipocytes were mainly localized very close to fibrosis areas rich in fibronectin (Fig. 7, C and D). This feature may suggest a possible local role for adipose-derived CTSS protein in degradation of certain ECM components. Fig. 7 Open in new tabDownload slide Immunohistochemical detection of CTSS and fibronectin proteins in human sc white adipose tissue of obese subject. The CTSS immunopositivity was heterogeneous (A, ×100). Some adipocytes tested negative (A, asterisks) and others strongly positive (A, arrows). The strong positive staining was frequently observed in adipocytes close to fibrosis areas (F), as shown in A (×100) and C (×60, arrows). Fibronectin tested negative in fully mature adipocytes, and a strong positivity was observed in fibrosis areas (B, ×20, arrows). Serial sections stained, respectively, for CTSS (C, ×40) and fibronectin (D, ×40) show CTSS positivity (arrows) in adipocytes close to fibrosis areas rich in fibronectin (arrows). Fully mature adipocytes tested negative for fibronectin (×60, asterisks). Fig. 7 Open in new tabDownload slide Immunohistochemical detection of CTSS and fibronectin proteins in human sc white adipose tissue of obese subject. The CTSS immunopositivity was heterogeneous (A, ×100). Some adipocytes tested negative (A, asterisks) and others strongly positive (A, arrows). The strong positive staining was frequently observed in adipocytes close to fibrosis areas (F), as shown in A (×100) and C (×60, arrows). Fibronectin tested negative in fully mature adipocytes, and a strong positivity was observed in fibrosis areas (B, ×20, arrows). Serial sections stained, respectively, for CTSS (C, ×40) and fibronectin (D, ×40) show CTSS positivity (arrows) in adipocytes close to fibrosis areas rich in fibronectin (arrows). Fully mature adipocytes tested negative for fibronectin (×60, asterisks). Discussion The present study provides the first evidence that CTSS released by human preadipocytes intervenes in the regulation of adipogenesis. The role of CTSS in human preadipocytes was highlighted by the finding that the inhibition of its activity decreased adipogenesis, whereas supplementation of culture media with CTSS significantly promoted adipogenesis. To study the effect of CTSS on adipogenesis, we used primary cultures of human preadipocytes. Murine cell models, most notably the 3T3-L1 cell line, have been the basis for the majority of adipogenesis studies. However, there is a growing concern that adipogenesis differs between human and murine systems. For example, the resistin was first identified in the 3T3-L1 cells in which its expression increased along the differentiation, whereas in human preadipocytes its expression decreased along adipogenesis (17, 18). These discrepancies argue for the importance of human preadipocyte cell models use in exploratory research, particularly in adipogenesis. Analysis of CTSS expression and secretion during human preadipocyte differentiation revealed a modest increase of CTSS during differentiation as compared with the other usual markers of adipogenesis. Because a balance between cathepsins and their endogenous inhibitor, cystatin C, determines their enzymatic activity, we concomitantly examined cystatin C secretion profiles during adipogenesis. Our results showed that the secretion of cystatin C increased more importantly than CTSS during preadipocyte differentiation. Consistent with these findings, CTSS activity strongly decreased during differentiation, suggesting a critical role of CTSS in the early steps of adipogenesis. Besides, the addition of CTSS recombinant protein on preadipocytes led to increase adipogenic markers, whereas adding CTSS when adipogenesis is engaged did not affect differentiation. These results, together with the specific pattern of CTSS/cystatin C secretions and the decrease of CTSS activity during adipogenesis, suggests an early role of CTSS in facilitating preadipocyte differentiation. Future investigations are necessary to determine the molecular mechanisms by which CTSS and cystatin C expressions are regulated during adipogenesis. We further explored the possibility that CTSS may promote adipogenesis via its suspected role in ECM remodeling. Proteases such as metalloproteinases and plasmin that degrade certain components of ECM were suggested to regulate adipogenesis (19–22). Other unknown proteinases, such as family of cysteine proteases, may also contribute in adipose ECM remodeling and thereby the regulation of adipogenesis. The role of CTSS in such process might be argued based on a network of concordant indirect and direct arguments produced by previous studies and our work. The expression of CTSS is dramatically increased in obesity. We previously showed that CTSS protein expression significantly augments in adipose tissue of morbidly obese subjects, whereas cystatin C protein expression is similar in obese and lean subjects (8) (data not shown). Thus, the increase of CTSS in adipose tissue could lead to local degradation of some ECM proteins, promoting the development of fat mass. Indeed, CTSS degrades several ECM proteins in vitro such as elastin, laminin, and types I and IV collagens (9, 23). The involvement of CTSS in the excessive ECM degradation has been demonstrated in others tissues such as in the vascular wall in which CTSS is involved in the progression of atherosclerosis (24). The role of CTSS in the degradation of ECM components has never been explored in human adipose tissue. We here demonstrated a role of CTSS in the degradation of fibronectin, which appears as a key element regulating adipogenesis. Cell cultures on fibronectin matrices inhibited human preadipocyte adipogenesis (25). We confirmed this result with our preadipocytes (data not shown). TGFβ, a strong inhibitor of adipocyte differentiation, stimulates the synthesis of fibronectin by preadipocytes (26, 27). Fibronectin down-regulates gene expression of lipogenic proteins and thereby interfere with morphological changes necessary for new gene expression (28). In agreement with these results obtained in cell lines, we here showed that there was an important change of fibronectin network and a decreased of its expression during human preadipocyte differentiation. Besides, it was previously showed that proteolytic degradation of fibronectin yields specific fragments that activate adipogenesis (29). We presently demonstrated that CTSS degrades preadipocyte synthesized fibronectin. This effect is associated with an increase of adipogenic markers such as PPARγ and SREBP1c as well as lipogenic target genes CD36, aP2, and FAS. This result suggests a critical role of CTSS in the early steps of adipogenesis in which fibroblastic preadipocytes change morphology and fibronectin starts to decrease. Further studies are required to assess the impact of CTSS on fibronectin proteolysis in adipose tissue. Interestingly, immunohistochemistry study of human adipose tissue revealed that fibronectin is not expressed in fully differentiated adipocytes as it was previously reported, supporting the idea that fibronectin synthesis decreased during adipocyte development (30). Nevertheless, we observed a strong staining of fibronectin in fibrosis regions. In serial sections of adipose tissue, we showed that adipose cells with strong CTSS staining are preferentially localized close to fibrosis regions containing fibronectin. These fibrosis regions are increased in adipose tissue of obese subjects than non obese ones (data not shown). These results may support the idea of possible role of CTSS in ECM degradation, in particular fibronectin. Further studies are necessary to unravel adipose ECM remodeling and implication of proteinases in this process during fat mass development. In conclusion, our results showed that CTSS promotes the early steps of human preadipocyte differentiation. This effect may be mediated through the degradation of fibronectin. Our results open further avenues of research to elucidate the physiological functions of CTSS in human adipose tissue. Future studies are necessary for determining the physiological role of CTSS on adipose tissue biology in vivo. Acknowledgments We thank Dr. Pierre Niccolo and Dr. Philippe Sellam for adipose tissue availability and Franck Viguier (Cytogenetic Department) for fluorescence microscopy availability. We also thank Martine Pinçon-Raymond (Institut National de la Santé et de la Recherche Médicale, Unité 505) for helpful discussions concerning extracellular matrix and Michele Guerre-Millo for critical reading of the manuscript. This work was supported by the French National Agency of Research (OBCAT no. ANR-05-PCOD-026-01), the Contrat de Recherche Clinique ALFEDIAM and the BQR (Bonus Quality Research) of Paris 6 University. S.T. and R.C. received a grant from Institut National de la Santé et de la Recherche Médicale/Conseil Regional Ile de France (2003–2005), and S.T. received support from the Fondation pour la Recherche Médicale (2006). 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Eur J Histochem 42 : 183 – 188 Google Scholar PubMed WorldCat Copyright © 2006 by The Endocrine Society

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Endocrinology – Oxford University Press

Published: Oct 1, 2006

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