C2C12 cells (American Type Culture Collection, Manassas, VA, USA) were cultured in Dulbecco's modified Eagle's medium (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich; Merck Millipore), 100 U/ml penicillin and 100 mg/ml streptomycin. MC3T3-E1 cells were cultured in α-minimum essential medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin. C2C12 is a mouse myoblast cell line capable of osteoblastic differentiation, which has been identified as a type of mesenchymal stem cell (26,27). MC3T3-E1 is an osteoblast precursor cell line. In order to induce osteoblast differentiation, medium was added with 50 mg/ml ascorbic acid, 10 mM sodium β-glycerophosphate, 1 µM dexamethasone and 50 ng/ml bone morphogenetic protein 2 (all obtained from Sigma-Aldrich; Merck Millipore). To block the activation of the ERK1/2 signaling pathway, 20 µM U0126 (Sigma-Aldrich; Merck Millipore) was added to the medium.

STC2 cDNA was cloned into a plenti6 vector (Invitrogen; Thermo Fisher Scientific, Inc.). STC2 short hairpin RNA (shRNA) plasmid was purchased from GeneChem Co., Ltd. (Shanghai, China). Cells were transfected using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol for 6 h at 37°C.

Cells were plated in 96 or 24-well plates at a density of 0.5×10⁴ cells/well and cultured in differentiation medium for 0, 3 or 6 days. The medium was changed every 3 days. ALP activity and staining was completed with 1-Step NBT/BCIP substrate solution (Thermo Fisher Scientific, Inc.) as previously described (28). For mineralization detection, cells were fixed in 70% ethanol for 10 min after 7–14 days induction to differentiation, then stained with Alizarin red solution (2%, pH 4.2) for 15 min at room temperature, then washed with deionized water for removal of nonspecific staining.

Total mRNA was extracted using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.). cDNA was prepared using PrimeScript RT-PCR kit (Takara Bio, Inc., Otsu, Japan) and was subsequently used for the RT-qPCR analysis performed using FastStart Universal SYBRGreen Master (Roche Diagnostics GmbH, Mannheim, Germany). Primers used were as described by Wang et al (29). The thermocycling conditions were as follows: First cycle for 10 min at 95°C, followed by and 40 cycles for 15 sec at 95°C and 1 min at 60°C. The primer sequences were as follows: Forward 5′-CAATAAGGTAGTGAACAGAC-3′, and reverse, 5′-CTTCAAGCCATACTGGTCT-3′ for osteocalcin (OCN); forward, 5′-CCTGGTAAAGATGGTGCC-3′ and reverse, 5′-CACCAGGTTCACCTTTCGCACC-3′ for collagen type I α 1 chain (Col1α1); forward, 5′-GAATGCACTACCCAGCCAC-3′ and reverse, 5′-TGGCAGGTACGTGTGGTAG-3′ for runt-related transcription factor 2 (Runx2); forward, 5′-GTCAAGAGTCTTAGCCAAACTC-3′ and reverse, 5′-AAATGATGTGAGGCCAGATGG-3′ for osterix (OSX); and forward, 5′-CATGGCCTTCCGTGTTCCTA-3′ and reverse, 5′-CCTGCTTCACCACCTTCTTGAT-3′ for GAPDH. Results were quantified using the 2^−∆∆Cq method (30).

Cells were harvested and treated with lysis buffer [50 mM Tris-HCl (pH 6.8), 100 mM dithiothreitol, 2% SDS, 10% glycerol, and 1 mM phenylmethylsulfonyl fluoride]. Cell lysates were centrifuged at 12,000 × g for 15 min at 4°C, and cell debris was then discarded. Proteins were quantified by bicinchoninic acid assay and 40 µg samples were electrophoresed on 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes. Following blocking with milk, membranes were incubated with primary antibodies against STC2 (1:500; Santa Cruz Biotechnology, Dallas, TX, USA; cat. no. sc-14350), phosphorylated (p)-ERK1/2 (1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA; cat. no. 9102) and β-actin (1:3,000; Beyotime Institute of Biotechnology, Haimen, China; cat. no. AF0003) overnight at 4°C, followed by incubation with horseradish peroxidase (HRP)-labeled anti-mouse (cat. no. 7076) or anti-rabbit (cat. no. 7074) IgG secondary antibodies (1:2,000; Cell Signaling Technology, Inc.) for 1 h at room temperature. The proteins were visualized using an enhanced chemiluminescent substrate for detection of HRP (Beyotime Institute of Biotechnology).

Data are presented as the mean ± standard deviation of three independent experiments. Statistical analysis was performed using unpaired Student's t test with Microsoft Excel software (Microsoft Corporation, Redmond, WA, USA). P<0.05 was considered to indicate a statistically significant difference.

STC2 was expressed in the C2C12 and MC3T3-E1 cell lines (Fig. 1A) and STC2 protein expression levels were increased during differentiation of MC3T3-E1 cells to osteoblasts (Fig. 1B).

To investigate the function of STC2 in osteoblast differentiation, STC2 expression was silenced with a specific shRNA in MC3T3-E1 cells (Fig. 1C). ALP activity is an early marker for osteoblast differentiation; therefore, it was used in the present study to determine whether cells were differentiating. It was revealed that the ALP activity was significantly lower in the shSTC2 group of MC3T3-E1 cells compared with the scramble group (P<0.05; Fig. 1D and E). Following the induction of osteoblast differentiation, the mineralized nodules detected by Alizarin red staining on day 14 were markedly reduced in the shSTC2 group of MC3T3-E1 cells when compared with the control cells (Fig. 1F).

The mRNA expression levels of various osteoblast-associated genes were quantified in order to determine the molecular mechanisms underlying the effect STC2 may have on osteoblast differentiation. It was determined that mRNA expression levels of Runx2 (Fig. 2A), Col1α1 (Fig. 2B), OSX (Fig. 2C) and OCN (Fig. 2D) were significantly reduced in the shSTC2 group of the MC3T3-E1 cells compared with the scramble group at 3 and 6 days of induction (P<0.01).

Overexpression of STC2 facilitates osteoblast differentiation and mineralization. To confirm the function of STC2 in osteoblast differentiation, STC2 cDNA was transfected into MC3T3-E1 cells (Fig. 3A) and the cells were treated with differentiation medium. The ALP activity was significantly higher in the STC2 overexpressing cells compared with the control cells (P<0.05; Fig. 3B and C) and an increased number of mineralized nodules were observed in the STC2 overexpressing cells compared with the control cells on day 12 following the induction (Fig. 3D). The mRNA expression levels of osteoblast-specific genes, including Runx2 (Fig. 4A), Col1α1 (Fig. 4B), OSX (Fig. 4C), and OCN (Fig. 4D) were significantly increased at 3 and 6 days of induction, with the exception of OCN at 3 days as no significant difference between the expression levels of the different groups was identified.

STC2 regulates ERK phosphorylation. To determine how STC2 promoted the differentiation of MC3T3-E1 cells, the present study investigated the MAPK/ERK signaling pathway using western blotting. It was determined that the protein expression level of p-ERK was reduced in the shSTC2 group cells (Fig. 5A). However, in the MC2T3-E1 cell line transfected with a STC2 overexpressing plasmid, p-ERK expression was increased compared with the control cells (Fig. 5B). These findings suggest that the regulation of ERK phosphorylation by STC2 may be essential to osteoblast differentiation.

In order to validate the aforementioned findings, the cells were treated with U0126, an effective and selective inhibitor of MAPK kinase (a kinase upstream of ERK1/2) to block the activation of the ERK1/2 signaling pathway and the expression levels of osteoblast-specific genes were determined (Fig. 6). It was demonstrated that treatment of MC3T3-E1 cells with 20 µM U0126 reduced the upregulation of Runx2 (Fig. 6A), OSX (Fig. 6C), and OCN (Fig. 6D) in the 3 and 6 day groups compared with the controls. However, this was not observed for Col1α1 expression in cells that were stimulated by overexpression of STC2 at 6 days.