Bone marrow aspirates were obtained from iliac crest biopsies of two healthy donors (male, 26 years old, left calcaneus fracture and female, 32 years old, right plateau fracture, required open reduction and internal fixation, bone graft) during routine surgical procedures. The bone marrow was obtained during the bone graft procedures. The present study was approved by the Ethical Committee of the Second Affiliated Hospital of Nanchang University (Nanchang, China) and all the patients provided informed consent. Mononucleated cells were isolated from the bone marrow by density gradient centrifugation at 1,000 × g for 15 min using human lymphocyte separation medium (density 1.077±0.0001 g/ml; Beijing Solarbio Science & Technology, Co., Ltd., Beijing, China). The cells were subsequently seeded and grown in Low Glucose Dulbecco’s modified Eagle’s medium (DMEM; Hyclone Laboratories, Inc., Logan, UT, USA), supplemented with 15% fetal bovine serum (FBS; Hyclone Laboratories, Inc.), at 37°C in a humidified atmosphere containing 5% CO₂/95% air. BMSCs were observed under a phase contrast microscope (IX71; Olympus Corp., Guangzhou, China).

To induce adipogenesis, the BMSCs were seeded at a density of 1×10⁴ cell/cm² in 24-well plates (Corning Incorporated, Corning, NY, USA). Following a 24 h culture, the cells were treated with adipogenic medium, consisting of growth medium supplemented with StemPro^® Adipogenesis Differentiation kit (Gibco Life Technologies, Carlsbad, CA, USA). After 14 days, the adipogenesis was visualized by light microscopy (IX71; Olympus Corp.), with or without staining by Oil Red O (Bogu Biotechnology Co., Ltd, Shanghai, China), as described previously (10,11). Subsequently, the BMSCs were treated with 0, 0.1, 1, 10 or 100 μM CGA (#C3878; Sigma-Aldrich, St. Louis, MO, USA) for 15 min, and the expression levels of markers, including Erk1/2, PPARγ and CEBPα, were assessed by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blotting. Furthermore, the expression levels of the signaling pathways, which regulate cell proliferation, including PI3K/Akt and cyclin D1, were determined by RT-qPCR and western blotting in the BMSCs treated with 0.1, 1, 10 or 100 μM CGA (treated group) or 0 μM CGA (control group) for 48 h.

The cells were washed twice with phosphate-buffered saline (PBS; Hyclone; Thermo Fisher Scientific, Rockford, IL, USA) and then lysed on ice with radioimmunoprecipitation buffer (50 mM Tris, 150 mM NaCl, 1.0% NP-40, 0.5% sodium deoxycholate and 0.1% SDS; pH 7.4) supplemented with 10 μl/ml protease inhibitor cocktail (cat. no. 11836170001) and PhosStop (one tablet per 10 ml; cat no. #4906845001; Roche, Nutley, NJ, USA). The protein content was quantified using a Bicinchoninic Acid Protein Assay kit (Pierce Biotechnology, Inc., Rockford, IL, USA), according to the manufacturer’s instructions. For western blotting, 30–50 μg of the protein samples were separated by 10% SDS-PAGE [H₂O, 30% acrylamide, 1.5MTris-HCl (pH 8.8), 10% SDS, 10% ammonium persulfate and tetramethylethylenediamine] and transferred onto polyvinylidene difluoride membranes (Ruiqi Biotechnology Co., Ltd, Shanghai, China). The membranes were then incubated for 8–12 h at 4–8°C with the following primary antibodies, purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA): Rabbit polyclonal phosphorylated (p)-SHP-2 (Tyr580; cat. no. #5431), rabbit monoclonal p-p44/42 MAPK (T202/Y204; D13.14.4E; cat. no. 4370S), rabbit monoclonal p-Akt (ser473; cat. no. 4060S), mouse monoclonal cyclin D1 (cat. no. 2926), and Santa Cruz Biotechnology, Inc.: Rabbit polyclonal SH-PTP2 (cat. no. sc-280), rabbit polyclonal Akt1/2/3 (H-136; cat. no. sc-8312) and mouse monoclonal β-Actin (C4; cat. no. sc-47778). Primary antibodies (1:1,000) and horseradish-peroxidase-conjugated secondary antibodies (1:1,000; HRP-labeled Goat Anti-Rabbit IgG(H+L), A0208 and HRP-labeled Goat Anti-Mouse IgG(H+L), A0216, Beyotime, Nanjing, China) were diluted with Tris-buffered saline-0.1% Tween^® 20 (100 mmol/l Tris HCl, pH 7.5; 150 mmol/l NaCl), containing 5% bovine serum albumin (#A7906; Sigma-Aldrich). Membranes were incubated with secondary antibodies for 1–2 h at room temperature (12). The immunoreactive signals were then visualized using the Enhanced Chemiluminescence Plus system (Zhongshan Biotechnology Co., Ltd, Beijing, China).

An ERK inhibitor (10 μM PD98059; #9900; Cell Signaling Technology, Inc.) and PI3K inhibitor (0.1 μM Ly294002; #S1105; Selleck, Shanghai, China) were added to the cells (1×10⁵ cells/ml) at 37°C, 2 h prior to the addition of CGA. siRNA specific to Shp2, forward 5′-GAAUAUGGCGUCAUGCGUGTT-3′ and reverse 5′-CACGCAUGACGCCAUAUUCTT-3′ (13) and scrambled siRNA, forward 5′-AGUUAUAAGGCGGUCGUGCTT-3′ and reverse 5′-GCACGACCGCCUUAUAACUTT-3′ were synthesized by Invitrogen Life Technologies (Carlsbad, CA, USA). The siRNAs were transfected into the BMSCs using Lipofectamine^® 2000 reagent (Invitrogen Life Technologies) (14) for 6 h, and the cells were recovered in DMEM supplemented with 10% FBS. The cells were then harvested and reseeded at 1×10⁵ cells/ml in 96-well plates for subsequent bromodeoxyuridine (BrdU) and RT-qPCR assays.

The BrdU assay was performed using a Cell Proliferation kit (cat. no. #2750; Merck Millipore, Darmstadt, Germany), according to the manufacturer’s instructions. The incorporation of BrdU was detected by horseradish-peroxidase substrate (#S131125; Tiandz, Inc., Beijing, China) and the color intensity was measured at 450 nm (ELx800; Bio-Tek Instruments, Inc., Winooski, VT, USA).

RT-qPCR was performed, as described in our previous study (5). The PCR conditions used were as follows: 95°C for 3 min, followed by 40 cycles of 95°C for 20 sec, 52°C for 30 sec and 60°C for 30 sec. The results were analyzed using the cycle threshold (Ct) relative quantification method (^ΔΔCt), using the iQ5 Optical System Software version 2.0 (Bio-Rad Laborotories, Inc., Hercules, CA, USA). The Ct values were obtained using ABI 7300 software (Applied Biosysystems Life Technologies, Foster City, CA, USA). The fold change of the relative expression of mRNA was determined using the 2^−ΔΔCt method (15).

The BMSC suspensions were washed twice with PBS. For direct assays, 5×10⁴ cells were incubated with 1 mg/ml (1/1,000 dilution) fluorescein isothiocyanate-conjugated CD29, CD90 and CD105, or phycoerythrin-conjugated CD34, CD45 and CD166 (#ab81289, #ab10925 and #ab93758; Abcam, Shanghai, China) in the dark for 15 min, and were analyzed by flow cytometric analysis using a fluorescence-activated cell sorting (FACS)Calibur flow cytometer (BD Biosciences, San Jose, CA, USA) with the use of CellQuest software (TreeStar.FlowJo.v5.7; BD Biosciences, San Jose, CA, USA).

All the data are expressed as the mean ± standard deviation. One-way analysis of variance, followed by Student’s t-test were used to determine statistically significant differences between the experimental and control groups. Statistical analyses were conducted using SPSS 13.0 (SPSS Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference (5).

Following 2–3 days of culture, the non-adherent cells were removed by replacing the low glucose DMEM, and the attached nucleated cells were visualized. These cells displayed an elongated fibroblast-like morphology under phase-contrast microscopy (Fig. 1A). Furthermore, the immunophenotypes of the expanded clonal BMSCs were analyzed by FACS. The BMSCs expressed the CD29, CD90, CD105 and CD166 markers, but not the CD34 or CD45 markers (Fig. 1B). This phenotype remained unchanged for >20 passages, consistent with the results observed in a previous study by our group (5).

The effects of CGA on the proliferation of BMSCs was investigated using a BrdU assay. Cell proliferation was significantly enhanced following treatment with CGA at doses between 0.1 and 10 μM, compared with the control group and the group treated with 100 μM CGA (Fig. 2A). Cell death was observed following treated of the cells with 100 μM CGA. To determine the mechanism involved in the proliferation of BMSCs, the effects of CGA on the growth of BMSCs, cultured in phenol red-free medium containing 1% FBS, were investigated, at 48 h. Alterations in the phosphorylation of Akt and the expression of cyclin D1 in response to stimulation with CGA, were determined. The phosphorylation of Akt and the protein expression levels of cyclin D1 were increased in response to treatment with CGA at concentrations of 0.1, 1 and 10 μM (Fig. 2B); however, the phosphorylation of Erk was not enhanced by treatment with CGA at these concentrations (data not shown). In addition, the inhibition of PI3K/Akt, by the specific inhibitor, LY294002, reduced the proliferation of cells and deceased the expression levels of cyclin D1 (Fig. 2C and D).

Shp2 is a major signal cytoplasmic tyrosine phosphatase and contains two SH2 domains upstream of PI3K/Akt/cyclin D1, which have been implicated in mitogenic responses (16). Therefore, the present study investigated whether Shp2 was also a regulator of CGA-induced BMSC proliferation. The expression levels of Shp2 were suppressed in the BMSCs, which were transfected with siRNA. The proliferation of BMSCs was inhibited and activation of the PI3K/Akt/cyclin D1 pathways was induced by the combination of siRNA and Shp2 (10 nM), compared with the mock-transfected cells (Fig. 3).

The activation or inhibition of the PI3K/Akt and Erk signaling pathways has previously been observed to regulate the adipogenic differentiation of pre-adipocyte cell lines (17,18). However, the role of CGA in the adipogenesis of BMSCs and the underlying mechanisms remain to be elucidated. The present study assessed the effect of Shp2 inhibition on the Erk and Akt signaling pathways on BMSC adipogenic differentiation, in the absence or presence of CGA. The phosphorylation of Erk was significantly enhanced following treatment with CGA, at a dose between 0.1 and 10 μM, compared with the control group at 15 min (Fig. 4A); however, this effect was inhibited by treatment with the NSC-87877 Shp2 PTPase activity inhibitor (Fig. 4B).

Notably, treatment with NSC87887 and PD98059 had little effect on the adipogenesis of BMSCs in differentiation medium, however, increases in the expression levels of PPARγ and CEBPα were detected (Fig. 5A). The addition of LY294002 to the differentiation medium decreased the number of lipid-containing adipocytes, and significantly inhibited the expression levels of PPARγ and CEBPα (Fig. 5A). The activation of CGA and inhibition of Akt resulted in reduced adipogenic differentiation of the BMSCs (Fig. 5B). These results suggested that other downstream pathways, rather than Akt, were involved. As shown in Fig. 5, NSC87887 and PD98059 inhibited the inhibition of adipogenic differentiation of BMSCs in the presence of CGA. Therefore, treatment with CGA inhibited the adipogenic differentiation of BMSCs and the expression of PPARγ and CEBPα by enhancing the phosphorylation of Erk, but not Akt.