J Bone Metab.  2013 Nov;20(2):83-88. 10.11005/jbm.2013.20.2.83.

Bone Morphogenetic Protein-2 Desensitizes MC3T3-E1 Osteoblastic Cells to Estrogen Through Transcriptional Downregulation of Estrogen Receptor 1

• Ishibashi

• KMID: 2286298
• DOI: http://doi.org/10.11005/jbm.2013.20.2.83

Abstract

BACKGROUND
Estrogens exert preferable effects on bone metabolism through two estrogen receptors (ERs), ER1 and ER2, which activate the transcription of a set of genes as ligand-dependent transcription factors. Thus, growth factors and hormones which modulate ER expression in the bone, if any, may possibly modulate the effect of estrogens on bone metabolism. However, research as to which of these molecules regulate the expression of ERs in osteoblasts has not been well documented.
METHODS
A reporter assay system developed in this study was used to explore molecules that modulate ER1 expression in MC3T3-E1 osteoblastic cells. Gene expression was analyzed by reverse transcription-polymerase chain reaction.
RESULTS
A pilot study using the reporter system revealed that bone morphogenetic protein (BMP)-2 negatively regulated ER1, but not ER2, expression in MC3T3-E1 cells. Consistently, estradiol-induced reporter activity via an estrogen responsive element was strongly suppressed in MC3T3-E1 cells pretreated with BMP-2.
CONCLUSIONS
BMP-2 desensitizes osteoblastic cells to estrogen through downregulation of ER1 expression.

Keyword

Bone morphogenetic proteins; Osteoblasts; Receptors estrogen

MeSH Terms

Bone Morphogenetic Proteins
Down-Regulation*
Estrogen Receptor alpha*
Estrogens*
Gene Expression
Intercellular Signaling Peptides and Proteins
Metabolism
Osteoblasts*
Pilot Projects
Receptors, Estrogen
Transcription Factors
Bone Morphogenetic Proteins
Estrogen Receptor alpha
Estrogens
Intercellular Signaling Peptides and Proteins
Receptors, Estrogen
Transcription Factors

Figure

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  Fig. 1 Construction of the reporter plasmids used in this study.

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  Fig. 2 Bone morphogenetic protein-2 downregulates a reporter activity driven by mouse estrogen receptor1 gene promoter in MC3T3-E1 cells. (A) MC3T3-E1-MERAluc1 cells (clone#1-4) were treated with 1alpha,25-dihydroxy-vitamin D3 (1,25-[OH]2D3; 10 nM), estradiol (E2; 100 nM), 4-hydroxy-tamoxifen (4-OH-Tam; 100 nM), phorbol-12-myristate-13-acetate (PMA; 10 nM), dibutyryl cyclic adenosine monophosphate (dbcAMP; 1 mM), recombinant mouse epidermal growth factor (rmEGF; 100 ng/mL) and recombinant human bone morphogenetic protein-2 (rhBMP-2; 250 ng/mL). After a 48-hour incubation, these cells were harvested and lysed then, luciferase activities in the lysates were measured. Luciferase activities in the vehicle-treated cells were set at 1. (B) MC3T3-E1 cells were transiently transfected with pMERAluc1 (0.5 µg) along with pSV-β-gal (0.3 µg) for normalization of transfection efficiency, incubated for 24 hours and exposed by rhBMP-2 (250 ng/mL) or vehicle. Forty-eight hours later, the cells were harvested and lysed in LCβ lysis buffer. Luciferase activities were measured as described in the Materials and Methods section. The results are indicated as relative values when the normalized luciferase activity of the vehicle-treated cells is set at 1. *P<0.05.

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  Fig. 3 Bone morphogenetic protein-2 downregulates expression of estrogen receptor (ER)1 mRNA, but not that of ER2, in MC3T3-E1 in a time-dependent manner. Total RNAs were isolated from MC3T3-E1 cells treated with bone morphogenetic protein (BMP)-2 or vehicle for the indicated periods, and estrogen receptor (ER)1 expression was examined by reverse transcription-polymerase chain reaction (RT-PCR) analysis. ER1 mRNA expression decreased from 6 to 24 hours after BMP-2 exposure (A). On the other hand, ER1 mRNA expression did not largely change over the period in the vehicle-treated MC3T3-E1 cells (B). Glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) expression was also analyzed as an internal control.

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  Fig. 4 Bone morphogenetic protein-2 suppresses estradiol (E2)-induced estrogen responsive element (ERE)-mediated reporter activation in MC3T3-E1 cells. MC3T3-E1 cells transfected with pERE-G-luc, a reporter plasmid harboring the firefly luciferase gene under the control of a β-globin gene promoter, were pretreated with recombinant human bone morphogenetic protein-2 (rhBMP-2; 250 ng/mL) for 48 hours. These cells were then treated with estradiol (E2; 100 nM) for 24 hours, and the induced luciferase activities were evaluated. pCMVsport-β-gal, a reporter plasmid designed to express a β-galactosidase gene constitutively, was simultaneously transfected for normalization. The normalized values of the luciferase activity for the non-treated MC3T3-E1 cells are set at 1. Data are presented as the mean±SD of the three experiments. **P<0.01.

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