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S100B Suppresses the Differentiation of C3H/10T1/2 Murine Embryonic Mesenchymal Cells Into Osteoblasts

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S100B Suppresses the Differentiation of C3H/10T1/2 Murine Embryonic Mesenchymal Cells Into Osteoblasts 3878 MOLECULAR MEDICINE REPORTS 14: 3878-3886, 2016 S100B suppresses the differentiation of C3H/10T1/2 murine embryonic mesenchymal cells into osteoblasts DONG LI1, KAIHUA LI1, GANG CHEN1, JIANLONG XIA1, TING YANG1, PING CAI1, CHEN YAO1, YONGJIANG YANG2, SHICHANG YAN2, RIHUA ZHANG3 and HUI CHEN2 1Department of Orthopedics, Jiangsu Province Hospital of TCM, Affiliated Hospital of Nanjing University of TCM, Nanjing, Jiangsu 210029; 2Department of Orthopedics, Benq Hospital, Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu 210019; 3The Laboratory Animal Center, Jiangsu Province Hospital, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu 210029; P.R. China Received August 10, 2015; Accepted August 9, 2016 DOI: 10.3892/mmr.2016.5697 Abstract. S100 calcium-binding protein B (S100B) is c-Jun N-terminal kinase (JNK) phosphorylation. The results expressed and released by adipocytes, and is positively corre- suggest that S100B inhibits osteogenesis, however stimulates lated with body mass index, however, the direct effects of adipogenesis. The ERK pathway is involved in the regulation S100B on adipocytes remain unclear. Bone marrow-derived of osteogenesis, whereas the JNK pathway is involved in the mesenchymal stem cells have the capacity to differentiate regulation of adipogenesis. into osteoblasts and adipocytes, which is important for bone metabolism. The current study aimed to determine the effect of Introduction S100B on adipogenesis and osteogenesis. The mouse embryo cell line C3H/10T1/2 was used to build cell models with varying Osteoporosis and obesity are two of the most common chronic levels of S100B protein expression. Western blot analysis was conditions and pose major health threats worldwide, with both performed to assess the expression of various marker proteins. showing increasing prevalence rates, however, the associa- Oil red O staining and alizarin red S staining were used to tion between osteoporosis and obesity is complex. The bone detect adipogenesis and osteogenesis, respectively. S100B marrow is the only place in mammalian tissues where bone overexpression was associated with a significant increase in and fat lie adjacent to each other, in osteoporosis, adipogenesis oil red O staining and a significant reduction in alizarin red S is increased at the expense of osteogenesis from common staining. Runt-related transcription factor-2 and bone morpho- osteoporotic bone marrow cells (1,2). Bone marrow-derived genetic protein 2 expression levels were significantly increased mesenchymal stem cells (BM-MSCs) have the capacity to in the S100B underexpression group, however not in the S100B differentiate into osteoblasts and adipocytes, and osteopo- overexpression group. By contrast, the expression levels of rosis is partially attributable to the alteration of the balance the adipogenesis markers peroxisome proliferator-activated of BM-MSC differentiation into osteoblasts and adipocytes. receptor γ and CCAAT-enhancer-binding protein α was signif- BM-MSC differentiation is regulated by hormones, cytokines, icantly increased in the S100B overexpression group, however and genes. The differentiation of BM-MSCs into adipocytes is not in the S100B underexpression group. Osteogenesis stimu- accompanied by a marked increase in the expression of adipo- lation increased extracellular signal-regulated kinase (ERK) cyte markers, including peroxisome proliferator-activated phosphorylation, and adipogenesis stimulation increased receptor γ (PPARγ) and CCAAT-enhancer-binding protein α (C/EBPα). Similarly, the differentiation of BM-MSCs into osteoblasts is regulated by bone morphogenetic proteins (BMPs) and runt-related transcription factor-2 (RUNX2) (3-6). Correspondence to: Dr Hui Chen, Department of Orthopedics, A deeper understanding of the differentiation of BM-MSCs Benq Hospital, Affiliated Hospital of Nanjing Medical University, into osteoblasts or adipocytes will provide insight into the 71 Hexi Ave, Nanjing, Jiangsu 210019, P.R. China pathophysiology and treatment of osteoporosis. E-mail: [email protected] S100 calcium-binding protein B (S100B), an important Dr Rihua Zhang, The Laboratory Animal Center, Jiangsu Province member of the S100 family, is ubiquitously expressed in Hospital, The First Affiliated Hospital of Nanjing Medical human tissue, including fat tissues, and is associated with a University, 140 Hanzhong Road, Nanjing, Jiangsu 210029, P.R. China variety of human diseases such as neurodegenerative disor- E-mail: [email protected] ders (7), malignant melanoma (7), trauma with or without brain injury (8-10) and obesity (11). Serum S100B levels are positively Key words: S100B, osteogenesis, adipogenesis, extracellular correlated with body mass index (11), and S100B expression signal-regulated kinase, c-Jun N-terminal kinase is increased by diet-induced obesity (12). Adipocytes express and secrete S100B protein, which may act as an adipokine by modulating the immune response and metabolism. However, LI et al: S100B AND BM-MSC DIFFERENTIATION 3879 the direct effect of S100B on osteoporosis and obesity remains Fisher Scientific, Inc.), penicillin (100 U/ml) and streptomycin to be investigated (13). Therefore, the current study aimed to sulfate (100 µg/ml; cat. no. 15070063; Life Technologies; determine the effect of S100B on MSC differentiation into Thermo Fisher Scientific, Inc.), and maintained at 37˚C in a adipocytes and osteoblasts. humid incubator containing 5% CO2. In the present study, an in vitro model of osteogenesis and The expression constructs were transfected into adipogenesis was established using the mouse embryo cell C3H/10T1/2 cells using the X-tremeGENE HP DNA Transfec- line C3H/10T1/2 (ATCC number, CCL-226) in a monolayer tion Reagent (cat. no.; 06366236001; Roche Diagnostics, Basel, and high-density cultures. The current study presents novel Switzerland). After 48 h, the cells were cultured in a selective evidence concerning the effect of S100B on cell differentia- medium containing 300 µg/ml G418 or 3 mg/ml blasticidin tion, including adipogenic and osteogenic differentiation. (cat. nos., N6386 and 11033102, respectively; Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) for 1 week, and resis- Materials and methods tant colonies, which indicated successful transfectants, were selected. Construction of expression plasmids. C57B/6 mice were purchased from Nanjing Qingzilan Technologies Co., Osteoblast differentiation. Osteogenic differen- Ltd. (Nanjing, China). Animals were housed at 23±1˚C in tiation was induced as described previously (14). Confluent a 12/12-h light/dark cycle and a humidity of 45±5%, and C3H/10T1/2 cells (the day on which confluence was allowed free access to a normal chow diet and water. The reached was considered day 0) were incubated for 12 days study was approved by the ethics committee of the Animal in an osteogenic induction medium consisting of MEM Care Facility of Nanjing Medical University, Nanjing, China. containing 10% FBS, 0.1 mM dexamethasone (cat. no. D4902; Total mRNA was isolated using TRIzol® reagent Sigma-Aldrich; Merck Millipore, Darmstadt, Germany), (cat. no. 15596‑026; Invitrogen; Thermo Fisher Scientific, Inc., 10 mM β-glycerophosphate (cat. no. G9422; Sigma-Aldrich; Waltham, MA, USA). This mRNA was reverse-transcribed into Merck Millipore) and 50 mM ascorbic acid. The induction complementary (c)DNA via reverse transcription-polymerase medium was changed every 2 days. The presence and extent of chain reaction (RT-PCR) using the ThermoScript™ RT-PCR bone matrix mineralization was evaluated using alizarin red S System for First-Strand cDNA Synthesis kit; cat. no. 11146024, staining. Thermo Fisher Scientific, Inc.). Subsequently, this cDNA was used as the template DNA. PCR (cat. no. 4464268; the Adipocyte differentiation. Adipocyte differentiation was Platinum Multiplex PCR Master Mix, 2X; Thermo Fisher induced as described previously (14). C3H/10T1/2 cells were Scientific, Inc.) was performed to clone S100B cDNA using seeded on plates, and allowed to grow for 2 days to reach appropriate primers. confluence (considered day 0). Cell differentiation was induced Plasmids overexpressing S100B, termed pcDNA3.1(+) by culturing the cells in MEM containing 10% FBS, 0.5 mM A-S100B, were constructed. The coding sequences of mouse 3-isobutyl-1-methylxanthine (cat. no. I7018; Sigma-Aldrich; S100B were amplified using RT‑PCR and mRNAs isolated Merck Millipore), 1 µg/ml porcine insulin (cat. no. I0320000; from the white adipose tissue of mice using TRIzol® reagent Sigma-Aldrich; Merck Millipore) and 1 mM dexamethasone. (cat. no. 15596-026; Invitrogen; Thermo Fisher Scientific, Following 48 h of incubation, the medium was replaced with Inc.). The primer sequences (including the sites of restriction MEM containing 10% FBS and 1 µg/ml insulin. On day 4, the enzymes) were as follows: Forward, 5'-CGT GAA TTC ATG medium was replaced with fresh medium (MEM containing TCC GAG CTG GGA AG-3' and reverse, 5'-GCT GTC GAC 10% FBS), and the incubation was continued for 12 days. Lipid GGG TCA CTC ATG TTC AAA GAA GT-3'. The PCR products droplets were evaluated using oil red O staining. were subcloned into the pcDNA3.1(+)A expression vector and then confirmed by sequencing. Alkaline phosphatase staining. The differentiation of Subsequently, miRNA-S100B expression plasmids were C3H/10T1/2 cells into osteoblasts. After 4 days, alkaline phos- constructed. Three distinct domains within the coding region phatase (ALP) staining was performed
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