Oestrogen receptor α regulates the odonto/osteogenic differentiation of stem cells from apical papilla via ERK and JNK MAPK pathways

Abstract

Objectives: Oestrogen receptor (ER) is a common nucleus receptor that is essential for the regulation of cell growth, proliferation and differentiation. This study was to examine whether ERα can affect the proliferation and odonto/osteogenic differentiation of stem cells from apical papilla (SCAPs).

Materials and methods: Stem cells from apical papillas were isolated, purified and then transfected with ERα lentiviruses. The proliferation capacity was investigated by cell counting kit-8 (CCK-8) assay and flow cytometry. The odonto/osteogenic differentiation ability was analysed by alkaline phosphatase (ALP) activity, alizarin red staining, western blot assay (WB) and real-time RT-PCR. MAPK pathway and its downstream transcriptional factors were explored by WB assay.

Results: As indicated by CCK-8 assay and flow cytometry, ERα had no significant effect on the proliferation of SCAPs. When ERα was overexpressed, the ALP activity and the formation of calcified nodules were significantly enhanced in SCAPs. Moreover, the odonto/osteogenic markers (DMP1/DMP1, DSPP/DSP, RUNX2/RUNX2, OCN/OCN) in SCAPs were significantly up-regulated at both mRNA and protein levels. On the contrary, the odonto/osteogenic differentiation ability of SCAPs was remarkably inhibited after suppression of ERα. Mechanistically, the protein levels of phosphorylated ERK and JNK significantly increased after ERα overexpression. Moreover, some downstream transcriptional factors of MAPK pathway were simultaneously activated by ERα overexpression.

Conclusions: Together, the data accumulated here indicated that ERα can enhance the odonto/osteogenic differentiation of SCAPs via ERK and JNK MAPK pathways.

Figure 1

Characterization of SCAPs and ERα expression in transfected SCAPs. A, Flow cytometry revealed that SCAPs were positive for STRO‐1, CD73, CD90 and CD105 and negative against CD34 and CD45. B, The ERα expression at day 3 in Scramble group, sh‐ERα group, GFP group and ERα group respectively. *P < .05 or **P < .01. C, The ERα expression at day 3 in Scramble group, sh‐ERα group, GFP group and ERα group respectively. *P < .05 or **P < .01. D, Greyscale analyses of (C) by Image‐Pro Plus 5.0 software

Figure 2

Effects of ERα on the proliferation of SCAPs. A, CCK‐8 assay for Scramble group, sh‐ERα group, GFP group and ERα group at day 0, 1, 3, 5, 7 and 9 respectively. B, Cell cycle phases in Scramble group, sh‐ERα group, GFP group and ERα group respectively

Figure 3

Effects of ERα on ALP and mineralization capacity of SCAPs. A, ALP activity of SCAPs in Scramble, sh‐ERα, GFP and ERα groups at day 5 respectively. *P < .05 or **P < .01. B, The formation of mineralized nodules in SCAPs in 4 groups at day 14. C, Mineralized nodules under the inverted microscope in 4 groups. Scale bars = 100 μm. D, Calcium contents in 4 groups by CPC assay. *P < .05

Figure 4

Effects of ERα on the odonto/osteogenic differentiation of SCAPs. A, Real‐time RT‐PCR analysis of DMP1, RUNX2, DSPP and OCN gene levels in Scramble and sh‐ERα groups at day 0, 3 and 7 respectively. *P < .05. B, Real‐time RT‐PCR analysis of DMP1, RUNX2, DSPP and OCN gene levels in GFP and ERα groups at day 0, 3 and 7 respectively. *P < .05 or **P < .01. C, The protein levels of OCN, DSP, RUNX2 and DMP1 in Scramble and sh‐ERα groups at day 0, 3 and 7 respectively. D, Grayscale analyses of (C). *P < .05 or **P < .01. E, The protein levels of OCN, DSP, RUNX2 and DMP1 in GFP and ERα groups at day 0, 3 and 7 respectively. F, Grayscale analyses of (E). *P < .05 or **P < .01

Figure 5

Effects of ERα on MAPK pathway and its downstream transcription factors of SCAPs. A, Protein levels of p38, p‐p38, ERK, p‐ERK, JNK and p‐JNK in the cytoplasm of transfected SCAPs in Scramble, sh‐ERα, GFP and ERα groups at 48 hours. β‐ACTIN served as an internal control. B, Quantitative analysis for the ratios of p‐p38/p38, p‐ERK/ERK and p‐JNK/JNK. *P < .05 or **P < .01. C, Protein levels of c‐Fos, p‐c‐Fos, c‐Jun, p‐c‐Jun, Elk1 and p‐Elk1 in the nuclei of transfected SCAPs in Scramble, sh‐ERα, GFP and ERα groups at 48 hours. H3 served as an internal control