Estrogen Receptor α Regulates Dlx3-Mediated Osteoblast Differentiation

Abstract

Estrogen receptor α (ER-α), which is involved in bone metabolism and breast cancer, has been shown to have transcriptional targets. Dlx3 is essential for the skeletal development and plays an important role in osteoblast differentiation. Various osteogenic stimulators and transcription factors can induce the protein expression of Dlx3. However, the regulatory function of ER-α in the Dlx3 mediated osteogenic process remains unknown. Therefore, we investigated the regulation of Dlx3 and found that ER-α is a positive regulator of Dlx3 transcription in BMP2-induced osteoblast differentiation. We also found that ER-α interacts with Dlx3 and increases its transcriptional activity and DNA binding affinity. Furthermore, we demonstrated that the regulation of Dlx3 activity by ER-α is independent of the ligand (estradiol) binding domain. These results indicate that Dlx3 is a novel target of ER-α, and that ER-α regulates the osteoblast differentiation through modulation of Dlx3 expression and/or interaction with Dlx3.

Keywords: Dlx3; estrogen receptor α; osteoblast differentiation.

Fig. 1.

ER-α induces Dlx3-enhanced osteoblast differentiation. (A) C2C12 cells were transfected with an empty vector or the indicated combinations of Dlx3 and ER-α, stimulated with BMP2, and then stained for ALP activity. (B) C2C12 cells were transfected with an empty vector or the indicated combinations of Dlx3 and ER-α, stimulated with BMP2, and then harvested for RT-PCR. GAPDH is the loading control. (C) C2C12 cells were transfected with pCMV-β-gal, ALP-Luc, or BSP-Luc reporter vector along with the indicated combinations of Dlx3 and ER-α. Luciferase activity was then measured after 36 h. Data are expressed as means ± SEM of at least three experiments. ^*p < 0.05 by Student’s t-test.

Fig. 2.

ER-α physically associates with and induces the transcriptional activity of Dlx3. HEK293 cells were transfected with Myc-tagged Dlx3 and HA-tagged ER-α (A) or with HA-tagged ER-α and GFP-tagged Dlx3 (B), and the cell lysates were subjected to immunoprecipitation using an anti-Myc antibody and Protein A-Sepharose beads. The immunoprecipitated proteins were then analyzed using immunoblotting. (C) HEK293 cells were transfected with pCMV-β-gal and a DRE-Luc reporter vector along with the indicated combinations of Dlx3 and ER-α. Luciferase activity was then measured after 36 h. Data are expressed as means ± SEM of at least three experiments. ^*p < 0.05 by Student’s t-test. (D and E) HEK293 cells were transfected with pCMV-β-gal, DRE-Luc (D), or ERE-Luc (E) reporter vectors along with the indicated combinations of Dlx3 and ER-α. Transfected cells were treated for 12 h with estradiol (E2) and luciferase activity was then measured. Data are expressed as means ± SEM of at least three experiments. NS, not significant by Student’s t-test.

Fig. 3.

ER-α-ΔLBD interacts with and affects Dlx3 in BMP2-stimulated osteoblast differentiation. (A) A schematic of the ER-α structure. FL, full length; AF-1, activation function 1; DBD, DNA binding domain; LBD, ligand binding domain; AF-2, activation function 2. HEK293 cells were transfected with the indicated combinations of plasmids and 48 h later cell lysates were harvested and analyzed using immunoprecipitation and immunoblotting. (B) HEK293 cells were transfected with pCMV-β-gal, and DRE-Luc reporter vector along with the indicated combinations of Dlx3, ER-α-FL or ER-α-ΔLBD. Luciferase activity was measured after 36 h. Data are expressed as mean ± SEM of at least three experiments. ^*p < 0.05 by Student t-test. (C) C2C12 cells were transfected with pCMV-β-gal, ALP-Luc reporter vector along with the indicated combinations of Dlx3, ER-α-FL or ER-α-ΔLBD. Luciferase activity was measured after 36 h. Data are expressed as mean ± SEM of at least three experiments. ^*p < 0.05 by Student’s t-test. (D) C2C12 cells were transfected with an empty vector or the indicated combinations of Dlx3 and ER-α-FL or ER-α-ΔLBD, stimulated with BMP2, and then stained for ALP activity.

Fig. 4.

ER-α increases DNA binding affinity of Dlx3 in a ligand-independent manner. (A, B) HEK293 cells were transfected with an empty vector or indicated combinations of Dlx3 and ER-α-FL (A) or ER-α-ΔLBD (B). The cell lysates were incubated with biotin-labeled oligonucleotide Dlx3-responsive elements, and the DNA-bound proteins were analyzed by immunoblotting using an anti-Dlx3 antibody.

Fig. 5.

The proposed model for regulation of Dlx3 by ER-α. Dlx3 binds to DRE site on osteogenic genes such as ALP, BSP, COL1A1 and subsequently increases their activities. ER-α interacts and consequently reinforces Dlx3-mediated regulation in a ligand independent manner.