To investigate the role of p53 in hypoxia induced inhibition of osteogenic differentiation of periodontal ligament cells (PDLCs).

PDLCs were cultured under hypoxia (1% O₂) or normoxia (20% O₂) for 48 h. Western blot was used to detect the expression of p53 and HIF-1α at 12, 24, and 48 h. After transfection with siRNAs targeting HIF-1α (Si-HIF1α), HIF-1α and p53 expression was furthered detected. After transfection with siRNAs targeting p53 (Si-p53), p53 and HIF-1α expression, changes of alkaline phosphatase (ALP) activity, and mRNA expression of osteogenic markers ALP, collagen-I (COL1), and runt related transcription factor 2 (RUNX2) were detected to evaluate osteogenic differentiation of PDLCs under hypoxia. The data were statistically analyzed with SPSS 13.0 software package.

Compared with the value (0.309±0.052) under normoixa, the relative expression of HIF-1α to GAPDH protein in PDLCs under hypoxia for 12, 24 and 48 h was significantly increased to 0.801±0.049, 0.881±0.037, and 0.936±0.039, respectively (t=6.901, 9.041, and 9.704; P=0.002, 0.0008, and 0.0006). The relative expression of p53 to GAPDH protein in PDLCs was significantly increased from 0.233±0.035 under normoixa to 0.463±0.036, 0.612±0.040, and 0.858±0.034 under hypoxia for 12, 24, and 48 h, respectively (t=4.595, 7.140, and 12.84; P=0.010, 0.002, and 0.0002). After PDLCs were transfected with Si-HIF1α and further cultured under hypoxia, HIF-1α expression in the HIF1α-Si1 and HIF1α-Si2 groups was significantly decreased by 64.57% and 59.94% at the protein level, and by 66.67% and 63.67% at the mRNA level compared with the NC-Si group, respectively (t_protein=9.326 and 6.985, P_protein=0.0007 and 0.002; t_RNA=5.319 and 5.015, P_RNA=0.006 and 0.008); p53 expression in the HIF1α-Si1 and HIF1α-Si2 groups was decreased by 36.47% and 38.41% at the protein level, and by 33.43% and 30.67% at the mRNA level, respectively (t_protein=4.645 and 4.135, P_protein=0.011 and 0.029; t_RNA=4.373 and 3.912, P_RNA=0.012 and 0.017). After PDLCs were transfected with Si-p53, p53 expression in the p53-Si1 and p53-Si2 groups was significantly decreased by 56.41% and 51.24% compared with the NC-Si control group (t=8.194 and 6.621, P=0.0012 and 0.0027). However, no significant changes in HIF1α expression were observed in the p53-Si1 and p53-Si2 groups compared with the NC-Si group (t=1.167 and1.391, P=0.308 and 0.237). After PDLCs were transfected with Si-p53 and further cultured under hypoxia for 48 h, ALP activity in the p53-Si1 and Si2 p53-groups was significantly increased by 2.05-fold and 2.17-fold compared with the NC-Si control group (t=4.889 and 4.346, P=0.008 and 0.012); ALP mRNA in the p53-Si1 and p53-Si2 groups was significantly increased by 2.14-fold and 2.05-fold than that of the NC-Si control group (t=5.423 and 4.078, P=0.006 and 0.015); COL1 mRNA was significantly increased by 2.86-fold and 3.03-fold (t=7.56 and 6.89, P=0.002 and 0.002); and RUNX2 mRNA was significantly increased by 3.41-fold and 3.71-fold (t=8.15 and 12.21, P=0.001 and 0.0003).

Hypoxia increases HIF-1α and p53 expression, and hypoxia inhibits osteogenic differentiation of PDLCs via upregulation of p53.