- Research article
- Open Access
Proteomic study of different culture medium serum volume fractions on RANKL-dependent RAW264.7 cells differentiating into osteoclasts
- Qi Xiong†1,
- Lihai Zhang†1,
- Lingli Xin3,
- Yanpan Gao2,
- Ye Peng1,
- Peifu Tang1Email author and
- Wei Ge2Email author
© Xiong et al.; licensee BioMed Central. 2015
Received: 30 January 2015
Accepted: 22 April 2015
Published: 2 May 2015
Cultivation of osteoclasts is a basic tool for investigating osteolytic bone diseases. Fetal bovine serum (FBS) is the standard supplement used for in vitro cell culture medium. Typically, the serum volume fraction used for osteoclast cultivation is 10%. In this study, we investigated the use of a low serum (1% FBS) model for culturing osteoclasts.
To confirm the validity of this model for use in osteoclast research, we compared the capacity for osteoclastogenesis and bone resorption of RANKL-induced RAW 264.7 cells cultured in medium supplemented with 10% FBS and 1% FBS. Osteoclasts were successfully generated in medium supplemented with 1% FBS, and exhibited prolonged longevity and similar bone resorbing ability to those generated in medium supplemented with 10% FBS, although the osteoclasts were smaller in size. Proteomics and bioinformatics analyses were performed to assess the suitability of osteoclasts formed in low serum-containing medium for use in research focusing on osteoclast differentiation and function. Our study demonstrated that a total of 100 proteins were differentially expressed in cells cultured in medium containing 1% FBS, of which 29 proteins were upregulated, and 71 proteins were downregulated. Bioinformatics analysis showed that the electron transport chain and oxidative phosphorylation pathways were downregulated obviously; however, the osteoclast signaling pathway was unaffected. The data have been deposited to the ProteomeXchange with identifier PXD001935.
Our study provides clear evidence of the validity of the low serum model for use in studying RANKL-dependent osteoclasts differentiation and bone resorption with the advantage of prolonged survival time.
Osteoclasts are bone resorbing cells that are formed by the fusion of monocyte/macrophage cells induced by receptor activator of nuclear factor B ligand (RANKL), which is a member of the tumor necrosis factor superfamily [1,2]. Arthritis, osteoporosis, bone metastasis and other bone degrading diseases partially result from the abnormal function or overactivation of osteoclasts [3-5]. Given the critical role that osteoclasts play in such bone diseases, many studies of osteoclasts have been conducted. The cultivation of cells in vitro is an indispensable tool in basic cell and molecular biology studies  and culturing osteoclasts in vitro is an essential basis for exploring bone metabolism and the mechanisms of bone diseases.
Due to the identification of the RANKL/RANK signaling pathway as a crucial requirement for osteoclast formation, mature osteoclasts can now be obtained in vitro. Previously, in vitro models of osteoclast differentiation were mainly based on primary cell cultures, such as bone marrow macrophages, splenocytes, and peripheral blood monocytes induced by M-CSF and RANKL, all of which are poorly suited to molecular studies because of their limited availability and failure to produce pure populations of osteoclasts [7,8]. More recently, the mouse macrophage cell line RAW 264.7, a RANK-expressing cell line, is increasingly being used as a cellular model of osteoclast formation and function [2,9]. To obtain mature osteoclasts, RAW 264.7 cells are cultured in medium supplemented with FBS, and stimulated with RANKL. Conventionally, the serum volume fraction used is 10%; however, the use of varying serum concentrations in the osteoclast culture system has been reported. In fact, Vincent et al. demonstrated that RAW 264.7 cells cultured in serum-deprived medium could differentiate into tartrate resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts, and their resorptive activity was significantly enhanced . Nevertheless, the molecular changes associated with the differentiation were not mentioned in this study. Thus, the effects of the culture medium serum volume fraction on osteoclast formation and bone resorbing ability are still poorly defined.
To address these issues, we evaluated RANKL-dependent osteoclast formation (unless otherwise specified, osteoclast formation refers to RANKL-dependent osteoclast formation in present study) in both the conventional (10% FBS) and low serum (1% FBS) culture systems. The bone resorptive activities of the osteoclasts were investigated and the protein changes of RAW 264.7 cells cultured under both sets of conditions were compared in proteomics analyses, which provide a global view of protein expression changes in cells. Herein, we determined that the low serum fraction model did not interfere with the osteoclast signaling pathways and both culture systems could be used to obtain osteoclasts. Thus, either culture system can be used for specific applications according to the duration and intervention factors of the experiments.
Osteoclastogenesis in medium supplemented with 10% FBS and 1% FBS
Comparison of size of osteoclasts and area of resorbing surface
Identification and classification of differentially expressed proteins
Bioinformatics analysis of differentially expressed proteins
Enriched KEGG pathways analysis
12858 11740 22335 66142 67264 14775 17709 22333 20463
C = 197;O = 9;E = 0.27;R = 33.89;rawP = 7.62e-12;adjP = 1.37e-10
12858 11740 22335 66142 67264 17709 22333 20463
C = 148;O = 8;E = 0.20;R = 40.10;rawP = 3.05e-11;adjP = 2.75e-10
12858 56012 18263 # 14085 # 18770 # 66142 13200 208715 # 67264 27425 20018 # 110196 # 17709
C = 1184;O = 13;E = 1.60;R = 8.14;rawP = 6.66e-09;adjP = 4.00e-08
67264 12858 27425 17709 66142 20463
C = 147;O = 6;E = 0.20;R = 30.28;rawP = 5.36e-08;adjP = 2.41e-07
Cardiac muscle contraction
12858 17709 66142 20463
C = 81;O = 4;E = 0.11;R = 36.63;rawP = 4.70e-06;adjP = 1.69e-05
67264 12858 17709 66142 20463
C = 188;O = 5;E = 0.25;R = 19.73;rawP = 5.98e-06;adjP = 1.79e-05
Fetal bovine serum (FBS) is a cocktail of numerous factors required for cell maintenance, and is utilized routinely to supplement medium for in vitro cultivation of numerous cell-types, including osteoclasts . Culture media are typically supplemented with 10% FBS [12,13]; however, this parameter does not generally mimic the in vivo microenvironment.
In the present study, osteoclast formation was investigated in medium supplemented with 1% FBS, which simulates physiological conditions more closely than 10% FBS. Here, we demonstrate the successful formation of osteoclasts with similar bone resorbing ability from RANKL-induced RAW264.7 cells cultured in media supplemented with either 10% FBS or 1% FBS. However, larger osteoclasts were formed more rapidly in medium supplemented with 10% FBS compared with those formed using the low serum model, while the longevity of the osteoclasts was less prolonged. Subsequent proteomics analysis of the molecular mechanisms underlying these differences revealed a total of 100 differentially expressed proteins involved in 12 biological processes. Of these, 29 proteins were upregulated and 71 were downregulated. However, no significant changes in the expression of proteins involved in osteoclastogenesis pathways were detected.
Osteoclasts are formed in the monocyte/macrophage lineage from hematopoietic progenitors. Osteoclastogenesis includes a number of steps comprised of survival, differentiation, fusion and activation . Extensive investigations have demonstrated that the RANKL-mediated signaling pathway and downstream transcription factors play essential roles in the regulation of osteoclastogenesis. RANKL performs crucial regulation of osteoclastogenesis mediated by binding to its receptor, RANK, leading to the expression of a variety of osteoclast genes including TRAP, cathepsin K, calcitonin receptor, αvβ3-integrin and MMP-9 [15,16]. During osteoclastogenesis, RANKL induces the recruitment of TNF receptor-associated cytoplasmic factor 6 (TRAF6), which subsequently stimulates downstream signaling pathways, including IκB kinase (IKK), nuclear factor κB (NF-κB), c-Jun N-terminal kinase (JNK), Akt, c-Src, p38, ERK, activator protein 1 (AP-1), and nuclear factor and activator of transcription (NFATc1) [17,18]. No significant changes in the expression of these proteins were detected in this study, and the bone resorption capacity of osteoclasts formed under the two sets of conditions was similar. Hence, our results indicate that low serum medium does not change the expression of osteoclast biomarkers. In accordance with the observations reported by Vincent et al. , the osteoclasts formed in low serum medium in our study were smaller than those formed in medium supplemented with 10% FBS. Taken together, these observation indicate that osteoclasts formed in both mediums are similar, and confirm the validity of their use in research focusing on osteoclast differentiation and function.
Notably, our results revealed a significant difference in the size of osteoclasts generated in the two types of media. Therefore, we performed proteomics analysis and bioinformatics analysis to elucidate the molecular mechanisms underlying this difference. Our results clearly demonstrate downregulation of the electron transport chain (ETC.) and oxidative phosphorylation pathways, both of which are essential for ATP synthesis, in osteoclasts formed under low serum conditions. Previous studies have indicated that osteoclast formation is an energy consuming processes [19,20]. An et al. suggested that metabolism and re-direction of energy flow plays a critical role in osteoclast formation . Therefore, we imply that energy restriction partially contributed to the small size of osteoclasts formed in medium supplemented with 1% FBS. Additionally, studies have shown that perturbations in some mitochondrial ETC. complexes increase lifespan [22-24]. Thus, it can be speculated that these reports explain our observation that osteoclasts formed in medium containing 1% FBS exhibited prolonged long-term survival, which might be beneficial for transgene studies of osteoclasts.
Previous studies have shown that increased mitochondrial ETC. activity enhances bone resorption by osteoclasts ; however, in our study, no differences in the bone resorption ability of osteoclasts formed under both sets of conditions were observed despite the downregulation in ETC. activity observed under low serum conditions. This may be due to the hypoxic conditions under which osteoclasts were cultured in the previous study. Knowles et al. reported that osteoclasts exposed to a constant hypoxic environment exhibited increased bone resorption ability in a HIF-1α-dependent manner . Furthermore, Muzylak et al. found that osteoclasts generated in hypoxia showed an eight-fold increase in size compared with those cultured in normoxia . Moreover, bone resorption requires osteoclast attachment to bone surfaces and the formation of the ruffled border by αvβ3 and small Rho family GTPases . H+ ions are then pumped through the ruffled border to demineralize inorganic materials, and cathepsin K is secreted to digest organic materials . No significant differences in the expression of these proteins were detected in the current study, indicating that low serum culture conditions do not affect the bone resorption capacity of osteoclasts. In addition, we found that OPN was slightly upregulated, which might compromise ETC. activity. It is worth noting that researchers studying osteoclast energy metabolism should avoid culturing osteoclasts in low serum medium due to the interference in the metabolic and ETC. pathways.
In summary, compared with the typical culture conditions of medium supplemented with 10% FBS, cultivation of under low serum conditions (1% FBS) is associated with changes in a number of biological pathways in RAW 264.7 cells and influences osteoclasts formation without affecting bone resorption ability. Moreover, osteoclasts formed in medium supplemented with 1% FBS exhibited extended longevity. Accordingly, low serum conditions are suitable for studies requiring prolonged osteoclast survival, while these conditions should be avoided for studies of osteoclast metabolism. Our study provides an alternative model for studying osteoclast differentiation and bone resorption.
Materials and methods
Cells and reagents
RAW 264.7 cells were obtained from the Chinese Academy of Medical Sciences (Beijing, China). α-MEM (11095–080) and FBS (26140079) were obtained from Life Technologies. Recombinant mouse RANKL (462-TEC-010) was purchased from R&D Systems. Acid Phosphatase Leukocyte Kits (387–1) were purchases from Sigma Aldrich. Urea (17-1319-01) was obtained from GE healthcare. Protease inhibitor cocktails were obtained from Roche. TMT sixplex Isobaric Label Reagent Set (90061) was purchased from Thermo Scientific. Trypsin/Lys-C Mix (V5072) was purchased from Promega. BCA Protein Assay Kits (23227) were obtained from Thermo Scientific. Osteo Assay Surface 24 Well Plate (3987) was purchased from Corning.
RAW264.7 cell cultivation and osteoclastogenesis
RAW 264.7 cells at a density of 1.5 × 105 cells/ml in 6-well plates were cultured in α-MEM supplemented with either 10% (v/v) or 1% (v/v) FBS at 37°C in a 5% CO2 incubator; both groups were prepared in triplicate. On the next day, RAW 264.7 cells were harvested and used for subsequent proteomics analysis; the triplicates of each group were pooled and prepared for TMT labeling. To generate mature multinucleated osteoclasts, 30 ng/ml RANKL was added to both culture systems in 24-well plates. Cells were cultured for 9 days; both conditions were prepared in triplicate. The culture medium was refreshed every other day.
TRAP staining was performed to confirm the formation of mature osteoclasts. Mature osteoclasts were defined as TRAP-positive cells containing three of more nuclei. TRAP staining was performed using the Acid Phosphatase Leukocyte kit according to the manufacturer’s instructions. The number of TRAP-positive multinucleated osteoclasts was counted using inverted microscopy. Furthermore, the mean size of TRAP-positive multinucleated osteoclasts was calculated with Adobe Photoshop CS3 software in three random visual fields for each group.
Osteoclast resorption pit assay
An Osteo Assay Surface 24 Well Plate was used to evaluate the bone resorptive activities of osteoclasts. On Day 9, the medium from the wells was aspirated and 100 μl of 10% bleach solution was added to each well and incubated for 5 minutes. Toluidine blue staining was then performed to improve the contrast for resorbed pit image analysis. NIH Image J software was used to assess the total area of the resorbed pits.
Sample preparation and TMT labeling
Sample preparation and labeling were performed as described previously . In brief, cells were washed with cold PBS three times followed by the addition of lysis buffer. Cell lysates were clarified by centrifugation at 16,000 g for 10 minutes at 4°C. Supernatants were obtained and protein concentrations were measured with the BCA Protein Assay Kit. Then, 100 μg of protein was incubated in 10 mM dithiothreitol at 50°C for 1 h. Thereafter, proteins were incubated with 25 mM indole acetic acid in the dark for 2 h. Proteins were then digested with trypsin/ Lys-C Mix at a protein/protease ratio of 25:1 and incubated overnight at 37°C. The TMT Isobaric Label Reagent Set was used to labeling proteins following the manufacturer’s manual protocol. The proteins extracted from cells cultured in medium with 10% FBS or 1% FBS were labeled with 0.8 mg TMT6-126 or TMT6-127, respectively. Equal amounts of the labeled protein digests from each group were combined for mass spectrometry (MS) analysis.
High-performance liquid chromatography (HPLC)
Fractionation of combined protein digests was conducted as described previously . Briefly, the combined TMT labeled samples were dissolved in 100 μl 0.1% FA and transferred to a MS tube for HPLC analysis (UltiMate 3000 UHPLC, Thermo Scientific) using an Xbridge BEH300 C18 column (4.6 × 250 mm2, 5 μm, 300 Å, Waters). Fifty fractions were collected into microtubes at 1.5 min intervals. The fractions were dried under vacuum and dissolved in 20 μl 0.1% FA for further LC-MS/MS analysis.
LC-MS/MS was performed using a Q Exactive mass spectrometer. Following separation using a 120 min gradient elution at a flow rate of 0.3 μl/min using UltiMate 3000 RSLCano System (Thermo Scientific), the protein digests were analyzed with a directly interfaced Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Scientific). A home-made fused silica capillary column (75 μm × 150 mm, Upchurch, Oak Harbor, WA, USA) packed with C18 resin (300 Å, 5 μm, Varian Lexington, MA, USA) was used as the analytical column. Xcalibur 2.1.2 software was used with the Q Exactive mass spectrometer in data-dependent acquisition mode. Ten data-dependent MS/MS scans at 27% normalized collision energy (HCD) were performed, after which, a single full-scan mass spectrum in Orbitrap (400–1,800 m/z, 60, 000 resolution) was conducted.
Osteoclast numbers in five random fields viewed under light microscopy were counted in both groups from Day 1 to Day 9. Furthermore, the mean size of mature osteoclasts and the total area of the resorbed pits were assessed with Adobe Photoshop CS3 and Image J software. Statistical differences between parametric data sets were assessed using Student’s t-test. A value of P < 0.05 was considered to indicate statistical significance.
LC-MS/MS data were analyzed using the Thermo Scientific Proteome Discoverer software suite 1.3 with the SEQUEST search engine and the mouse FASTA database from UniProt (released on July 9, 2014). At least 1 unique peptide per protein had to be identified to list the protein as a hit. In the SEQUEST search engine, full trypsin specificity was selected, two missed cleavages were allowed, carbamidomethylation (C) and TMT 6-plex (K and peptide N-terminal) were set as the static modification, oxidation (M) was set as the dynamic modification, precursor ion mass tolerances were set at 20 ppm for all MS data acquired using an Orbitrap mass analyzer, and the fragment ion mass tolerance was set as 20 mmu for all MS/MS spectra acquired. The ratio of proteins labeled with TMT6-127 and TMT6-126 was adjusted using the β-actin ratio value as the internal control. The thresholds for downregulation and upregulation were set at 0.6 and 1.7 respectively. In addition, the UniprotKB/Swiss-Prot accession numbers were converted into Entrez Gene ID for subsequent analysis using UniProt online ID Mapping. The PANTHER Classification System online was used to classify the proteins. The WebGestalt online toolkit was used to conduct enrichment analysis. Cytoscape 3.1.1 software was used to visualize the protein-protein interactions matching the online pathway database and the significance level was set at 0.0001. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium  via the PRIDE partner repository with the data identifier PXD001935.
Q. Xiong, Y. Peng, P-F Tang and L-H Zhang are supported by the National Natural Science Foundation of China (31370947). W. Ge and Y-P Gao are supported by the National Natural Science Foundation of China (81373150).
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