Transcriptional regulation of a BMP-6 promoter by estrogen receptor alpha.
The effects of 17beta-estradiol (E2) and ICI 182,780 (ICI) on activity of a BMP-6 promoter were compared in osteoblast-like and breast cancer cells transiently transfected with ERalpha. E2 but not ICI stimulated BMP-6 reporter activity in breast cancer cells, whereas the opposite was observed in osteoblast-like cells, associated with lack of AF-2 dependence of the response, and absent intranuclear localization of ERalpha, suggesting the involvement of a distinct ERalpha-dependent response mechanism in osteoblasts.


BACKGROUND
Previous studies suggest that the tissue-selective effect of antiestrogens on bone reflects the ability of these compounds to target certain osteoblast regulatory genes. To explore this hypothesis, we examined whether antiestrogens preferentially stimulate the bone morphogenetic protein 6 (BMP-6) promoter in bone cells, and if so, whether this activity is associated with a distinct estrogen receptor (ER)alpha-dependent response mechanism to that in other cell types.


METHODS
We compared the effects of 17beta-estradiol (E2) and ICI 182,780 (ICI) on activity of a 4.3-kb BMP-6 reporter construct in osteoblast-like cells (human MG63 and SaOS-2 cells and rat ROS 17/2.8 cells), human MCF-7 and T47-D breast cancer cell lines, and HepG2 hepatoma cells, after transient transfection with ERalpha, ERbeta, and mutant ER constructs.


RESULTS
E2, but not ICI, stimulated BMP-6 reporter activity by approximately 100% in MCF-7, T47-D cells, and HepG2 cells when transfected with ERalpha. In contrast, in ERalpha-transfected osteoblast-like cells, an increase in reporter activity of approximately 75% was observed after treatment with ICI but not E2. The response of MG63 cells to ICI and MCF-7 cells to E2 both required ERalpha as opposed to ERbeta and the ERalpha activation function (AF)-1 activation domain. However, whereas the AF-2 domain was also required for E2 to stimulate reporter activity in MCF-7 cells, the response to ICI in MG63 cells was AF-2 independent. In further studies where we compared the intracellular distribution of ERalpha associated with these responses, E2-dependent stimulation of the BMP-6 reporter in MCF-7 cells was associated with intranuclear localization of ERalpha, whereas extranuclear localization was seen in rat osteosarcoma cells (ROS) cells treated with ICI.


CONCLUSIONS
Antiestrogens selectively stimulate BMP-6 reporter activity in osteoblast-like cells through a distinct ERalpha-dependent mechanism characterized by independence of the AF-2 domain and extranuclear localization of ERalpha.
Ong
Delia B
DB
Academic Rheumatology, University of Bristol, Bristol, United Kingdom.
Colley
Shane M
SM
Norman
Michael R
MR
Kitazawa
Sohei
S
Tobias
Jonathan H
JH
D000818:Animals; D055418:Bone Morphogenetic Protein 6; D019485:Bone Morphogenetic Proteins / Q000096:biosynthesis / Q000235:genetics*; D045744:Cell Line, Tumor; D004958:Estradiol / Q000031:analogs & derivatives* / Q000502:physiology; D047628:Estrogen Receptor alpha / Q000502:physiology*; D005260:Female; D000077267:Fulvestrant; D006801:Humans; D010012:Osteogenesis; D011401:Promoter Regions, Genetic; D051381:Rats; D015398:Signal Transduction; D014158:Transcription, Genetic; D015854:Up-Regulation
C525816:BMP6 protein, human; C525818:Bmp6 protein, rat; D055418:Bone Morphogenetic Protein 6; D019485:Bone Morphogenetic Proteins; D047628:Estrogen Receptor alpha; D000077267:Fulvestrant; D004958:Estradiol