Dexamethasone and 1,25-dihydroxyvitamin D3 reduce oxidative stress-related DNA damage in differentiating osteoblasts

Abstract

The process of osteoblast differentiation is regulated by several factors, including RUNX2. Recent reports suggest an involvement of RUNX2 in DNA damage response (DDR), which is important due to association of differentiation with oxidative stress. In the present work we explore the influence of two RUNX2 modifiers, dexamethasone (DEX) and 1,25-dihydroxyvitamin D3 (1,25-D3), in DDR in differentiating MC3T3-E1 preosteoblasts challenged by oxidative stress. The process of differentiation was associated with reactive oxygen species (ROS) production and tert-butyl hydroperoxide (TBH) reduced the rate of differentiation. The activity of alkaline phosphatase (ALP), a marker of the process of osteoblasts differentiation, increased in a time-dependent manner and TBH further increased this activity. This may indicate that additional oxidative stress, induced by TBH, may accelerate the differentiation process. The cells displayed changes in the sensitivity to TBH in the course of differentiation. DEX increased ALP activity, but 1,25-D3 had no effect on it. These results suggest that DEX might stimulate the process of preosteoblasts differentiation. Finally, we observed a protective effect of DEX and 1,25-D3 against DNA damage induced by TBH, except the day 24 of differentiation, when DEX increased the extent of TBH-induced DNA damage. We conclude that oxidative stress is associated with osteoblasts differentiation and induce DDR, which may be modulated by RUNX2-modifiers, DEX and 1,25-D3.

Figure 1

Phase-contrast micrographs of mouse MC3T3-E1 preosteoblasts treated with dexamethasone (DEX) or 1,25-dihydroxyvitamin D3 (1,25-D3) at indicated concentrations on the day 0 and 3, 6, and 24 days after induction of their differentiation as compared with untreated control cells. Images were taken at 100× magnification with Olympus CKX41 microscope (Olympus, Tokyo, Japan) equipped with Olympus DP20 digital camera.

Figure 2

Mean intracellular reactive oxygen species (ROS) levels in differentiating mouse MC3T3-E1 preosteoblasts treated 10 min with 50 μM tert-butyl hydroperoxide at 37 °C (black bars) expressed by fluorescence of 2',7'-dichlorofluorescein (DCF) oxidatively converted from dichlorodihydrofluorescein diacetate as compared with untreated cells (empty bars). Error bars denote SEM, n = 5 for each measurement; * p < 0.05, *** p < 0.001 as compared with controls.

Figure 3

Mean activity of alkaline phosphatase (ALP) in differentiating mouse MC3T3-E1 preosteoblasts treated 10 min with 50 μM tert-butyl hydroperoxide at 37 °C (black bars) expressed as change in absorbance at 405 nm as compared with untreated cells (empty bars). Error bars denote SEM, n = 5 for each measurement.

Figure 4

DNA damage expressed as mean percentage of DNA in the alkaline version comet tail of differentiating mouse MC3T3-E1 preosteoblasts treated 10 min with 50 μM tert-butyl hydroperoxide at 37 °C (black bars) as compared with untreated cells (empty bars). Error bars denote SEM, n = 100 in each treatment; p < 0.001 as compared with unexposed sample each day.

Figure 5

Mean activity of alkaline phosphatase (ALP) in differentiating mouse MC3T3-E1 preosteoblasts with 1 μM dexamethasone (upper panel, black bars) or 10 nM 1,25-dihydroxyvitamin D3 (lower panel, black bars) in growth medium expressed as change in absorbance at 405 nm as compared with untreated cells (empty bars). Error bars denote SEM, n = 5 for each measurement.

Figure 6

DNA damage induced by tert-butyl hydroperoxide (TBH) and expressed as mean percentage of DNA in tail of comets in alkaline version of the comet assay in differentiating mouse MC3T3-E1 preosteoblasts. The cells differentiate with 1 μM dexamethasone (DEX, upper panel, black bars) or 10 nM 1,25-dihydroxyvitamin D3 (1,25-D3, lower panel, black bars) in growth medium and on the indicated days they were incubated 10 min at 37 °C with 50 μM TBH and DNA damage was measured by comet assay. Error bars denote SEM, n = 100 in each treatment; * p < 0.05, ** p < 0.01, *** p < 0.001 as compared with controls containing neither DEX, nor 1,25-D3.