Cell culture and reagents
MC3T3-E1 mouse pre-osteoblast cell line, obtained from Shandong Provincial Key Laboratory of Oral Tissue Regeneration, were maintained with 5% CO2 at 37ºC in α-MEM (Hyclone, Logan, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; Hyclone, Logan, USA), 100 U/ml penicillin (Solarbo, Beijing, China) and 100 µg/ml streptomycin (Solarbo, Beijing, China). For osteogenic induction, the cells were cultured in α-MEM supplemented with 5% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, 10-8 mol/l dexamethason (Sigma, St, Louis, MO), 50 mg/l ascorbic acid (Sigma, St, Louis, MO) and 10 mmol/l β-glycerophosphate (Sigma, St, Louis, MO). The medium was switched every two days.
TNF-α was purchased from PeproTech（Rochy Hill, NJ, USA), and the concentration of 0.5 ng/ml was selected based on our preliminary concentration screening study. The neutralizing antibody against TNFR2, anti-mouse TNFR2/CD120b/TNFRSF1B neutralizing antibody (catalog# 50128-RN204), was from Sino Biological (Beijing, China). A normal rabbit IgG, which is an unconjugated rabbit polyclonal antibody and is not directed against any known antigen, was purchased from Cell Signaling Technology (catalog# 2729, Beverly, MA, USA) and used as a negative control. The inhibitor of EphB4 auto-phosphorylation, NVP-BHG712, was obtained from MedChemExpress (HY-13258). The ERK inhibitor U0126 were purchased from Cell Signaling Technology (Danvers, MA, USA).
Cell proliferation assay
Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan) was used to evaluate cell proliferation/survival. Briefly, MC3T3-E1 cells were seeded in 96-well plates at a density of 3×103 cells per well and cultured in regular culture medium for 24h. The medium was then switched to α-MEM supplemented with 0.1% FBS and 0.5ng/ml murine TNF-α (Peprotech Inc, Rocky Hill, NJ, USA). After 24h or 48h of TNF-α stimulation, 10 μl of CCK-8 solution was added to each well and the plates were incubated for 2.5h at 37°C. The optical density was measured at 450 nm using the SPECTROstar Nano Microplate Reader (BMG Labtech Inc, Ortenberg, Germany).
RNA isolation & reverse transcription-polymerase chain reaction (RT-PCR)
Our previous study has proved that low doses of TNF-α promotes expressions of osteogenesis-related markers at 7 and 14 days . Considering that the main aim of this study is to investigate the signaling pathways mediating the pro-osteogenic effect of low concentrations of TNF-α, we selected 24h or 48h after osteogenic induction as the observation time points in this study. To determine the mRNA levels, total RNA was extracted from the cells using Trizol® reagent (TaKaRa Biotech, Tokyo, Japan) according to the manufacturer’s instructions, and was reverse transcribed using Reverse Transcriptase (TaKaRa Biotech, Tokyo, Japan). RT-PCR was performed using SYBR® Primix Ex TaqTM (TaKaRa Biotech, Tokyo, Japan) with Roche 480 LightCycler System (Roche Diagnostics, Mannheim, Germany). Each sample was prepared in triplicate, and each experiment was repeated at least 3 times. GAPDH was used as a loading control. The sequences of the primers for amplification of mouse Tnfr2, Runx2, Bsp, Ephb4 and Gapdh were shown in Table 1. The relative gene expression levels were calculated using the 2-ΔΔCT method.
Western blot analysis
Total cell lysates were extracted from MC3T3-E1 cells by incubation with ice-cold RIPA (Solarbo, Beijing, China) containing 1% PMSF (Solarbo, Beijing, China) for 30min, and the protein concentrations were measured using a bicinchoninic acid (BCA) protein assay kit (Solarbo, Beijing, China). For immunoblot analysis, 20 µg of protein lysates per sample were denatured in 5×SDS-PAGE loading buffer (Beyotime, Shanghai, China) at 100℃ for 5min. The samples were then run on 10% SDS-PAGE gels (Beyotime, Shanghai, China), and transferred to polyvinylidene fluoride (PVDF) membranes (Invitrogen, Carlsbad, CA, UAS) for 1h at 100 V. The membranes were subsequently blocked with 5% defatted milk for 1h at room temperature and incubated with the primary antibodies overnight at 4ºC. The anti-mouse primary antibodies used in this study were listed as following: RUNX2 (1:1000, catalog no. 12556S; CST, Danvers, MA, USA), BSP (1:1000, catalog no. 5468S CST, Danvers, MA,USA), EphB4 (1:1000, catalog no. A00690; Boster, China), TNFR2 (1:1000, catalog no. ab19139; abcam, Danvers, MA, USA), p38 (1:1000,catalog no. ab170099; abcam, Danvers, MA, USA), p-p38 (1:1000,catalog no. ab195049; abcam, Danvers, MA, USA), JNK1+2+3 (1:1000,catalog no. ab179461; abcam, Danvers, MA, USA), p-JNK1+2+3 (1:5000,catalog no. ab124956; abcam, Danvers, MA, USA), ERK1/2 (1:10000,catalog no. ab184699; abcam, Danvers, MA, USA), and p-ERK1/2 (1:8000,catalog no. ab76299; abcam, Danvers, MA, USA). The membranes were then incubated with an HRP-linked goat anti-rabbit secondary antibody (1:5000, catalog no. 7074P2; CST, Danvers, MA, USA) for 1h at room temperature. For normalization, defatted milk-blocked membranes were incubated with an HRP-linked anti-mouse GAPDH primary antibody (1:20000, catalog no. HRP-60004; Proteintech, Wuhan, China) for 1h at room temperature. Protein bands were visualized using the Chemiluminescent HRP Substrate (Merck Millipore, Billerica, MA, USA). Quantification of the band intensity was carried out using the Image J Software (NIH, Bethesda, MD, USA).
ALP activity assay
After osteogenic induction for 7d or 14d, the cell lysates were extracted from the MC3T3-E1 cells using 1% Triton X-100 for 30min on ice. The cell lysates were centrifuged at 1.2×104 g for 5min at 4ºC, and the ALP activity was evaluated using an Alkaline Phosphatase Assay Kit according to the instructions of the manufacturer (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). ALP activity was calculated according to the concentration of the phenol in a standard well and adjusted according to the protein concentration of each sample.
For immunofluorescent staining, MC3T3-E1 cells were washed with cold PBS, fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100 in 1% bovine serum albumin for 30min, blocked with 5% bovine serum albumin for 1 hour at room temperature, and incubated overnight at 4˚C with polyclonal rabbit anti-TNFR2 antibody (1:100 dilution, 19272-1-AP, Proteintech, USA). Cells were then incubated with Alexa Fluor 594-conjugated goat anti-rabbit secondary antibody (1:100 dilution, SA00006-4, Proteintech, USA). The cell nuclei were stained with 4, 6-diamino-2-phenylindole (DAPI). Stained MC3T3-E1 cells were visualized under a fluorescent microscope (Olympus, BX51, Japan), and images were captured by a CCD camera (CoolSNAP-Pro cf., Media Cybernetics, USA). The fluorescence intensity was counted in selected merged microscopic images by Image-Pro Plus 6.0 software.
Lentivirus-mediated shRNA interference targeting TNFR2
siRNAs specifically targeting TNFR2 were designed and synthesized by Hanbio Biotechnology Co., Ltd. (Shanghai, China). An siRNA with no homology to any known mouse or human gene was also synthesized to serve as a negative control. Synthesized siRNAs were duplexed, ligated into the pHBLV-Zsgreen-PURO expression vector (Hanbio Biotechnology Co., Ltd., Shanghai, China), and confirmed by gene sequencing. The resulting lentiviral vectors were then transfected into human 293T cells with pSPAX2 and pMD2G using LipofiterTM Liposomal Transfection Reagent (Hanbio Biotechnology Co., Ltd., Shanghai, China). Forty-eight hours and 72 hours after the transfection, the supernatant was collected twice, and centrifuged at 2,000g for 10min to remove cell debris. The lentiviral particles were then concentrated by centrifugation at 82,700𝑔 for 2 hours and resuspended in opti-MEM. After the titration, the lentiviral particles were stored at -80°C.
For lentiviral infection, MC3T3-E1 cells were seeded onto six-well plates and cultured in the regular culture medium for 12h. Cells were then incubated with the corresponding lentiviral particles (MOI=50) in 1 ml of the regular culture medium supplemented with 5μg/ml polybrene. Four hours later, another 1 ml of the regular culture medium containing 5μg/ml polybrene was added to each well. The plates were incubated at 37°C for 16h before the medium was switched to the regular culture medium. Forty-eight hours after the lentiviral transduction, green fluorescence (from ZsGreen) was captured by a fluorescence microscope (Olympus IX81, Tokyo, Japan). Stably transduced cells were selected with 8μg/ml puromycin (Sigma, St, Louis, MO) and were maintained in culture medium containing 2μg/ml puromycin.
All data were expressed as the means ± standard error of the mean (SEM) from at least three replicates for each experiment. Differences between more than two experimental groups and negative control group were analyzed by one-way ANOVA followed by Tukey HSD comparison test. Variance between two groups was compared by two-way t-test with GraphPad Prism software (version 6, by MacKiev Software, Boston, MA, USA). p<0.05 was considered statistically significant.