Tiron Has Negative Effects on Osteogenic Differentiation via Mitochondrial Dysfunction in Human Periosteum-Derived Cells

Abstract

Tiron is a potent antioxidant that counters the pathological effects of reactive oxygen species (ROS) production due to oxidative stress in various cell types. We examined the effects of tiron on mitochondrial function and osteoblastic differentiation in human periosteum-derived cells (hPDCs). Tiron increased mitochondrial activity and decreased senescence-associated β-galactosidase activity in hPDCs; however, it had a detrimental effect on osteoblastic differentiation by reducing alkaline phosphatase (ALP) activity and alizarin red-positive mineralization, regardless of H₂O₂ treatment. Osteoblast-differentiating hPDCs displayed increased ROS production compared with non-differentiating hPDCs, and treatment with tiron reduced ROS production in the differentiating cells. Antioxidants decreased the rates of oxygen consumption and ATP production, which are increased in hPDCs during osteoblastic differentiation. In addition, treatment with tiron reduced the levels of most mitochondrial proteins, which are increased in hPDCs during culture in osteogenic induction medium. These results suggest that tiron exerts negative effects on the osteoblastic differentiation of hPDCs by causing mitochondrial dysfunction.

Keywords: mitochondria; osteoblastic differentiation; periosteum-derived cells; tiron.

Figure 1

Biological activity of tiron in hPDCs. (A) The viability of hPDCs was unchanged after culture with 1 mM tiron compared to that of control cells. (B) Treatment with 1 mM tiron significantly increased the MMP in hPDCs, regardless of the passage. (C) Treatment with 1 mM tiron clearly decreased H₂O₂-induced β-galactosidase activity in hPDCs. (*** p < 0.001).

Figure 2

Effects of tiron on osteogenic phenotypes of hPDCs. Tiron reduced osteoblastic differentiation of hPDCs by reducing (A,C) ALP activity and (B,D) alizarin red-positive mineralization. CTL: DMEM; OM: osteogenic induction media. (*** p < 0.001).

Figure 3

Effects of tiron on osteogenic phenotypes of hPDCs treated with H₂O₂. (A,B) Treatment with 1 mM tiron reduced ALP expression and activity in H₂O₂-treated hPDCs. (C,D) Alizarin red-positive mineralization and calcium content were clearly reduced in H₂O₂-treated hPDCs. CTL; DMEM., OM; osteogenic induction media. (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 4

Effects of tiron on ROS production in osteoblast-differentiating hPDCs. (A) ROS production was significantly increased in hPDCs after 7 days of culture in osteogenic induction medium. (B) MMP was significantly increased in hPDCs in a time-dependent manner in osteogenic induction medium. (C) In flow cytometry analysis, antioxidants reduced ROS levels in hPDCs. The 50% value (percentage) was the control. (D,E) Effects of antioxidants on mitochondrial activity in osteoblast-differentiating hPDCs. Antioxidants significantly reduced the oxygen consumption rate (OCR) (D) and the rate of ATP production (E), which were both clearly increased during osteoblastic differentiation. Exceptions were treatment with 0.1 mM NAC that did not have a significant effect on OCR and treatments with 0.1 mM tiron, 0.1 mM NAC, and 0.01 mM Mito-TEMPO that did not have significant effects on the rate of ATP production. Control: DMEM; OM: osteogenic-induction medium; T: tiron; N: NAC; MT: Mito-TEMPO. (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 5

Effects of tiron on the level of mitochondrial proteins in osteoblast-differentiating hPDCs. (A,B) The levels of mitochondrial proteins were clearly increased in hPDCs cultured in osteogenic-induction medium compared with those in DMEM. Tiron markedly reversed the increases in mitochondrial protein levels induced by osteogenic induction medium, except for HSP 60 and VDAC. CTL: DMEM; OM: osteogenic induction medium; T: tiron. (* p < 0.05, ** p < 0.01, *** p < 0.001).