The expression of Akt/mTOR in VSMC calcification induced by high phosphate and its regulation of Cbfα1

Abstract

Objective: To observe the expression of protein kinase B (Akt) / mammalian target of rapamycin (mTOR) induced by high phosphorus in rat vascular smooth muscle cells (VSMC) calcification model, and its modulation on the expression of core binding factor alpha 1 (Cbfα1). Methods: Rat VSMC cells were cultured in vitro, and then divided into two groups: normal phosphorus group (Pi 1.3 mmol/L) and high phosphorus group (Pi 2.6 mmol/L). At day 7, calcium deposition was detected by Alizarin stain. The mRNA levels of Cbfα1 and osteopontin (OPN) were determined by real-time PCR. The protein expressions of p-Akt (ser473), p-mTOR (S2448), Cbfα1 and OPN were quantified by Western blot. Then, VSMC cultured with high phosphorus were treated with Akt inhibitor (Wortmannin) and mTOR inhibitor (Rapamycin) with different concentrations. After 24 h, the mRNA levels of Cbfα1 and OPN were determined and after 48 h, the protein expressions of p-Akt, p-mTOR, Cbfα1 and OPN were quantified. Also, at day 7, calcium deposition was also visualized by Alizarin stain. Results: After 7 days, compared with normal phosphorus group, calcium deposition was more obvious in high phosphorus group. The mRNA expressions of Cbfα1 and OPN increased significantly and the protein expressions of p-Akt, p-mTOR, Cbfα1 and OPN up-regulated significantly in high phosphorus group (all P<0.05). After treated with Wortmannin or Rapamycin for 24 h, compared with high phosphorus group, the mRNA expressions of Cbfα1 and OPN decreased significantly in high phosphorus + Wortmannin (30, 50 and 100 nmol/L) groups (all P<0.05) and high phosphorus + Rapamycin (1, 10 and 100 ng/ml) groups (all P<0.05). After treated with Wortmannin or Rapamycin for 48 h, compared with high phosphorus group, the protein expressions of p-Akt, Cbfα1 and OPN down-regulated significantly in high phosphorus + Wortmannin (30, 50 and 100 nmol/L) groups (all P<0.05). It showed a dose-dependent down-regulation of p-mTOR, Cbfα1 and OPN in high phosphorus + Rapamycin (1, 10, 100 ng/ml) groups (all P<0.05). After 7 days, compared with high phosphorus group, calcium deposition decresased significantly in high phosphorus + Wortmannin and high phosphorus + Rapamycin groups. Conclusions: High phosphorus can induce VSMC calcification. Akt and mTOR are involved in VSMC calcification induced by high phosphorus through the activation of Cbfα1.