Osteoblastic MC3T3-E1 cells were purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured in α-Minimum Essential Media (αMEM; Invitrogen Life Technologies, Carlsbad, CA, USA) with 10% fetal bovine serum (FBS; Invitrogen Life Technologies) at 37°C in 5% CO₂. Subsequent to reaching 85–95% confluence, the MC3T3-E1 cells were washed with 0.1% phosphate-buffered saline (PBS) and detached with 0.25% trypsin (dissolved in 0.1% PBS; Ameresco Inc., Framingham, MA, USA) with 0.025% EDTA and subcultured. To upregulate the HIF-1α, a murine HIF-1α coding sequence was amplified and cloned into a eukaryotic expression vector, pcDNA3.1 (+) (Invitrogen Life Technologies), and confirmed by sequencing. HIF-1α-pcDNA3.1 (+), or chloramphenicol acetyl transferase (CAT)-pcDNA3.1 (+) vectors were then transfected into MC3T3-E1 cells to upregulate the HIF-1α level or act as a control, respectively. The positive clone, MC3T3-E1 (HIF-1α), and MC3T3-E1 (Con) were selected in the presence of 800 µg/ml G418 and maintained in medium containing G418 (Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 400 µg/ml. To suppress HIF-1α expression, HIF-1α-specific small interfering (si)RNAs and siRNA control (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) were utilized at a concentration of 40 nM. Each siRNA was transfected into the MC3T3-E1 cells using Lipofectamine® 2000 (Invitrogen Life Technologies).

Total mRNA was extracted from the MC3T3-E1 or MC3T3-E1 (HIF-1α) cells with the RNeasy® Mini kit (Qiagen, Valencia, CA, USA), and an RNase inhibitor (Promega Corp., Madison, WI, USA) was then added. A SYBR® Green RT-qPCR kit (Takara, Tokyo, Japan) was used for the RT-qPCR analysis of HIF-1α mRNA, and tubulin was used as a reference gene. The ∆∆Ct method was used for relative quantification (17).

Whole MC3T3-E1 or MC3T3-E1 (HIF-1α) cells were collected and lyzed with a cell lysis reagent (Pierce, Rockford, IL, USA). Protein samples were then treated with a protease inhibitor cocktail kit (Roche Biochemicals, Basel, Switzerland) and quantified with a bicinchoninic acid assay kit (Thermo Fisher Scientific, Inc., Rockford, IL, USA). SDS-PAGE gel (8–12%) was used to separate the protein samples, which were then transferred to a polyvinylidene difluoride membrane. HIF-1α and tubulin protein levels were detected by immunoblot analysis using rabbit polyclonal antibodies against mouse HIF-1α (#ab82832) or tubulin (#ab18251; 1:500; Abcam, Cambridge, UK). Goat anti-rabbit immunoglobulin G conjugated to horseradish peroxidase (Pierce) and an enhanced chemiluminescence detection system (SuperSignal® West Femto; Pierce) were used for detection. The HIF-1α level was expressed as a percentage relative to tubulin expression.

MC3T3-E1 cells with overexpression of HIF-1α or CAT were seeded in 96-well plates. Upon reaching 85% confluence, the medium was substituted with αMEM containing 2% FBS. At different time-points post-normoxia or -hypoxia treatment, with or without siRNA transfection, the MTT assay (Invitrogen Life Technologies) was conducted according to the manufacturer's instructions. The optical density was then measured at 570 nm using a spectrophotometer.

MC3T3-E1 or MC3T3-E1 (HIF-1α) cells were seeded on six-well plates and treated with hypoxia for 24 or 48 h. The activity of caspase 3 was determined as previously described (18). Briefly, MC3T3-E1 or MC3T3-E1 (HIF-1α) cells were pelleted and resuspended in lysis buffer, prior to being incubated with Ac-DEVD-AMC fluorogenic peptide substrates (BD Pharmingen, San Diego, CA, USA) for caspase 3 for 30 to 60 min at 37°C. The yellow-green fluorescence of the reaction product was monitored on a spectrofluorometer by setting the excitation and emission wavelengths to 380 and 440 nm, respectively. The amount of yellow-green fluorescence was proportional to the amount of active caspase 3 present in the samples. The increase in caspase activity was expressed as a relative value to the control group.

MC3T3-E1 or MC3T3-E1 (HIF-1α) cells were seeded into Nunc™ LabTek™ II chamber slides (Nalge Nunc International Corp., Rochester, NY, USA) and subjected to hypoxia with or without siRNA transfection. The cells were then fixed, washed and stained with 1 µg/ml Hoechst 33528 (Invitrogen Life Technologies) using standard procedures (19). Apoptotic cells were screened and counted under a fluorescence microscope (Carl Zeiss, Oberkochen, Germany) using a 4,6-diamidino-2-phenylindole filter set.

SPSS 16.0 software (SPSS, Inc., Chicago, IL, USA) was used for statistical analyses. The Student's t-test was used to analyze the difference between two groups. Data are presented as the mean ± standard error of the mean, and P<0.05 was considered to indicate a statistically significant difference.

To explore the effect of hypoxia on the MC3T3-E1 cell line, the viability and HIF-1α protein levels of the cells were examined. In hypoxic and normoxic conditions, the viability of the cells was observed by MTT assay. The relative cell viability decreased significantly after 16 h in hypoxia, as compared with the cell viability in the normoxic condition (Fig. 1A). When the MC3T3-E1 cells were cultured in 1% O₂ conditions for 8 h or longer, the relative HIF-1α mRNA level became higher than that of cells cultured in 20% O₂ conditions, particularly when the cells were cultured for >16 h, as demonstrated by fluorescence qPCR (Fig. 1B). Western blot analysis was also conducted to analyze HIF-1α expression at the protein level, as shown in Fig. 1C. The HIF-1α expression in the MC3T3-E1 cells was significantly higher when the cells were cultured under hypoxic conditions for 16 and 24 h. These results suggest that the hypoxic condition reduces the viability of MC3T3-E1 cells and induces HIF-1α protein expression.

As stated previously, the viability of cells was decreased and the expression of HIF-1α was increased by the hypoxic condition. In order to elucidate the effect of HIF-1α expression on the viability decrease in the MC3T3-E1 cell line caused by hypoxia, the viability of cells with forced expression of HIF-1α was investigated using an MTT assay. As shown in Fig. 2A and B, significantly high levels of HIF-1α expression were confirmed in the HIF-1α-pcDNA3.1-transfected cells, as compared with the hypoxic and control groups. In addition, as shown in Fig. 2C, the MTT assay demonstrated that the viability of the MC3T3-E1 cells was increased by the forced expression of HIF-1α. These results showed that the effect of the forced HIF-1α expression was in contrast to the effect of hypoxia on the viability of MC3T3-E1 cells.

To detect the role of HIF-1α in the hypoxia-induced decrease in cell viability, MC3T3-E1 cells were cultured under hypoxic conditions and transfected with siRNA. The MTT assay and western blot analysis were then conducted to confirm the relative expression levels of HIF-1α to tubulin and determine cell viability. As shown in Fig. 3A, low levels of HIF-1α mRNA expression were found post-siRNA transfection. The western blotting results also demonstrated that the HIF-1α expression in the cells transfected with HIF-1α-siRNA was significantly lower than that in the siRNA-control group (Fig. 3B). The viability of the MC3T3-E1 cells post-siRNA transfection under hypoxic conditions was determined by MTT assay. Fig. 3C shows that the viability of the cells was reduced by HIF-1α-knockdown. These results suggest that HIF-1α-knockdown enhances the hypoxia-induced decrease in cell viability.

In order to explore the possible mechanism by which HIF-1α attenuates the hypoxia-induced decrease in MC3T3-E1 cell viability, the effects of HIF-1α on the hypoxia-induced osteoblast apoptosis and the activity of caspase 3 were investigated. The cells were transfected with pcDNA3.1 (+) (control) and HIF-1α-pcDNA3.1 (+) under hypoxic conditions. As shown in Fig. 4A, forced HIF-1α expression significantly suppressed the hypoxia-induced apoptosis after 24 and 48 h. By contrast, when the cells were transfected with siRNA (control) and two types of siRNA-HIF-1α during hypoxia, the levels of HIF-1α in the siRNA-HIF-1α-transfected cell groups were decreased and the percentage of cells undergoing apoptosis was increased significantly (Fig. 4B). In addition, the activity of caspase 3 was examined; as shown in Fig. 4C, the activity of caspase 3 was inhibited in the cells with forced HIF-1α expression under hypoxia after 24 and 48 h. By contrast, in the siRNA-HIF-1α-transfected osteoblasts, the hypoxia-induced caspase 3 activity was enhanced (Fig. 4D). These results show that HIF-1α inhibits hypoxia-induced osteoblast apoptosis.