Iron overload induces G1 phase arrest and autophagy in murine preosteoblast cells

This study aimed to investigate the cell cycle arrest and autophagy induced by iron overload in MC3T3-E1 cells. MC3T3-E1 cells were cultured in different concentrations of ferric ammonium citrate (FAC), and Perls' Prussian blue reaction was used to detect the iron levels of the cells. CCK-8 assays were used to detect the growth of MC3T3-E1. The level of reactive oxygen species (ROS) within cells was investigated with DCFH-DA. PI staining was used to analyze the cell cycle distribution of MC3T3-E1 cells. Finally, the expression levels of cell cycle related proteins, autophagy related proteins, AKT, p38 MAPK, Stat3, and their downstream proteins were detected with Western blot assays. The results showed that the iron levels of MC3T3-E1 cells increased with increasing concentrations of FAC. High levels of ferric ion inhibited proliferation of MC3T3-E1 cells and increased their ROS levels. Additionally, iron overload induced G1arrest in MC3T3-E1 cells and down-regulated the expression of Cyclin D₁ , Cyclin D₃ , CDK2, CDK4 and CDK6, but up-regulated p27 Kip1. In addition, the expression levels of Beclin-1 and LC3 II increased, but that of p62 decreased. Further experiments showed that the phosphorylation of AKT and its downstream proteins p-GSK-3β(Ser9) and p-mTOR (Ser2448) were decreased. The levels of p-p38 and p53 were up-regulated while those of cdc25A and p-ERK 1/2 were down-regulated. Phosphorylation of Stat3 and its downstream proteins was all decreased. These results show that iron overload generates ROS, blocks the PI3K/AKT and Jak/Stat3 signal pathways, and activates p38 MAPK, subsequently inducing G1 arrest and autophagy in MC3T3-E1 cells.