Effects of Estradiol on the Proliferation and Odontoblastic Differentiation of Human Dental Pulp Cells 3

Results

 

Estradiol enhanced proliferation of HDPCs

The effect of estradiol on HDPCs viability was evaluated by a MTS assay. Estradiol stimulated cell viability of HDPCs in a dose-dependent manner after 5 and 7 days of treatment compared with the untreated group (Fig. 1).

To identify the influence of estradiol on proliferation of HDPCs, the BrdU incorporation in HDPCs treated with or without estradiol was assessed. Estradiol increased BrdU positive cells compared with the control, although not dose dependently (Fig. 2A). Also, subsequent RT-PCR for proliferating cell nuclear antigen (PCNA) was increased in estradiol-treated HDPCs compared with the untreated group (Fig. 2B).

To know whether estrogen receptors (ER)-mediated proliferation was activated in estradiol-treated HDPCs, the mRNA levels of ER-α and ER-β in estradiol–treated HDPCs were determined by RT-PCR. Both of ER-α and ER-β were increased by estradiol treatment, showing a peak at 1 μM concentration for 7 days after stimulation with estradiol (Fig. 3).

 

Estradiol reduced HDPCs viability in differentiation inductive media

For the differentiation condition of HDPCs, cells were cultured in differentiation inductive medium containing basic α-MEM added with 100 μM ascorbic acid and 10 mM β-glycerophosphate. Viability of HDPCs cultured in supplementally estradiol of different concentration (0.1, 1 and 10 μM) with differentiation inductive medium was reduced compared with the untreated group (Fig. 4).

In addition, the cells were incubated in the absence or presence of 1 μM estradiol for 7 days. Cell morphology changed after estradiol treatment was observed by microscopy (x40). HDPCs cultured in differentiation inductive medium only was spindle-shaped, by the 1 μM estradiol treatment, HDPCs became spherical and developed expanding processes (Fig. 5).

Estradiol stimulated odontoblastic differentiation of HDPCs

To investigate potential of HDPCs for the odontoblast-like differentiation after estradiol treatment, mRNA expression of several differentiation markers such as ALP, BSP, DMP-1 and DSPP and ALP activity were assessed in estradiol-treated HDPCs. In HDPCs, exposure to estradiol (0.1, 1 or 10 μM) for 7 days increased mRNA expression of ALP, DMP-1, DSPP and BSP. ALP was expressed weak until at 1 μM of concentration, but showed clear increase at 10 μM of concentration. BSP and DMP-1 showed a peak at 1 μM of concentration, while at 10 μM was decreased. DSPP expressiom showed markedly increased at 0.1 μM of concentration, but subquent gradually attenuated (Fig. 6).

Furthermore, the protein expression of DSPP and DMP-1, a well known odontogenic gene in estradiol-treated HDPCs with differentiation inductive medium for 7 days were determined by Western blot. Estradiol upregulated DSPP protein expression in a dose-dependent manner and increased markdly DMP-1 level at 0.1 μM of concentration (Fig. 7). Also, ALP activity of HDPCs in differentiation inductive medium was increased significantly by the estradiol treatment in dose-dependent manner (Fig. 8). As another index of odontoblastic-like differentiation, the formation of calcified nodules were detected by alizarin red-S staining in HDPCs treated with 0.1, 1 or 10 μM estradiol for 14 days. Color stained by alizarin red-S stain was thicker with estradiol treatment of 0.1 μM or 1 μM (Fig. 9A). Fig. 9B showed the results of quantification of calcium deposition for mineralization in estradiol-treated HDPCs for 14 days. Compared with the untreated control, alizarin red-S staining significantly increased in the presence of estradiol at 0.1 μM or 1 μM of concentradion.

 

Estradiol stimulated ERK phosphorylation in HDPCs

To investigate whether estrogen receptors-mediated differentiation was activated in estradiol-treated HDPCs, the mRNA levels of ER-α and ER-β were determined by RT-PCR after the cells were cultured in differentiation inductive medium supplementally with 0.1, 1 or 10 μM estradiol for 7 days. mRNA expression of ER-β decreased at 0.1 μM estradiol  treatment, but ER-α expression did not significantly decrease by estradiol exposure (Fig. 10). This result suggested that the ER-β may be associated with estradiol–induced differentiation of HDPCs.

In order to investigate whether ERK pathway is involved in estradiol-induced differentiation of HDPCs. The activation of ERK in the estradiol-treated HDPCs was assessed by Western blotting. As shown in Fig.11, phosphorylated ERK increased after the exposure of the cells to esradiol at a concentration of 0.1 or 1 μM for 7 days, while decreased at 10 μM estradiol.

 

 

Fig. 1. Estradiol increased cell viability in HDPCs. The cells were incubated with different concentration of estradiol for indicated days (1, 3, 5 or 7 days). The cell viability was determined using a MTS assay. Estradiol stimulated cell viability in a dose-dependent manner at 5 and 7 days of treatment, compared with untreated group. The data are represented as the mean ± S.E.M of three independent experiments.

 

Fig. 2. Estradiol stimulated proliferation in HDPCs. Analysis of BrdU-positive cells of estradiol-treated HDPCs. (A) BrdU cell proliferation assay was performed in the cell lysate treated with 0.1, 1, and 10 μM estradiol for 7 days. Estradiol increased BrdU-positive cells more than 2.5-fold compared with untreated group in HDPCs. (B) PCNA mRNA expression in estradiol-treated cells with different concentrations (0.1, 1 and 10 μM) was detected by RT-PCR. Estradiol upregulated PCNA mRNA compared with untreated control in HDPCs. The data are represented as the mean ± S.E.M of three independent experiments.

 

 

Fig. 3. Expression of estrogen receptors in the estradiol-treated HDPCs. The cells were treated with 0.1, 1 and 10 μM estradiol for 7 days, and expression of ER-α and ER-β were determined by RT-PCR. Estradiol markedly upregulated mRNA expression of ER-α and ER-β in HDPCs compared with uncontrol. The data are represented as the mean ± S.E.M of three independent experiments.

 

 

Fig. 4. Estradiol decreased cell viability in HDPCs cultured with differentiation inductive medium. Cells were cultured in differentiation inductive medium supplementally estradiol with 0.1, 1 and 10 μM for indicating days (1, 3, 5 and 7) and then cell viability was determined using a MTS assay. Estradiol decreased cell viability of HDPCs compared with control. The data are represented as the mean ± S.E.M of three independent experiments.

 

Fig. 5. Morphologic change in estradiol-treated HDPCs cultured with differentiation inductive medium. The cells were incubated in the absence or presence of 1 μM estradiol for 7 days. Cell morphology was observed by microscopy (x40). Estradiol induced morphologic change like spindle-shape or more spherical shape and developed expending processes in HDPCs cultured differentiation inductive medium compared with untreated control cells.

 

Fig. 6. Gene expression of odontoblastic/osteoblastic differentiation markers in estradiol-treated HDPCs. The cells were cultured in differentiation inductive medium with 0.1, 1 and 10 μM estradiol for 7 days. Expression levels of ALP, DMP-1, DSPP and BSP mRNA as odntoblastic/osteoblastic differentiation markers were determined using RT-PCR analysis. Estradiol upregulated ALP, DMP-1, DSPP and BSP mRNA.

 

Fig. 7. Protein expression of DSPP and DMP-1 in estradiol-treated HDPCs. The protein level of DSPP and DMP-1 in estradiol-treated HDPCs for 7 days with differentiation inductive medium were determined by Western blot. In HDPCs, estradiol upregulated DSPP in a dose-dependent manner and also markedly increased DMP-1 protein expression compared with untreated control cells.

 

 

Fig. 8. Estradiol enhanced ALP activity in HDPCs. The cells were cultured in differentiation inductive medium with 0.1, 1 and 10 μM estradiol for 7 days and then ALP activity was measured in each cell lysates using ALP activity assay kit. Estradiol increased ALP activity of HDPCs in a dose dependent manner. The data are represented as the mean ± S.E.M of three independent experiments.

 

Fig. 9. Alizarin red S stain in estradiol-treated HDPCs. The cells were cultured in differentiation inductive medium supplementally with 0.1, 1 and 10 μM estradiol for 14 days and then stained using Alizarin red S. (A) Alizarin red S stained-HDPCs were increased in the presence of estradiol, indicating that estradiol accelerated mineral deposition in HDPCs cultured with differentiation inductive medium. (B) Alizarin red S stained-HDPCs were normalized with untreated cells. The data are represented as the mean ± S.E.M of three independent experiments.

 

 

Fig. 10. Expression of estradiol receptors (ERs) in estradiol-treated HDPCs with differentiation inductive medium. The cells were cultured in differentiation inductive medium supplementally with 0.1, 1 and 10 μM estradiol for 7 days and ER-α and ER-β mRNA level were determined by RT-PCR analysis. Estradiol decreased mRNA expression of ER-β in differentiation inductive medium. The data are represented as the mean ± S.E.M of three independent experiments.

 

 

Fig. 11. ERK activation in estradiol-treated HDPCs. The cells were treated with various concentrations of estradiol (0.1, 1 and 10 μM ) for 7 days in differentiation inductive medium. The protein extracts (50 μg) were used for Western blotting. Phosphorylated ERK increased in estradiol-treated HDPCs at 0.1 or 1 μM concentration, but not at 10 μM.