Activation of bone morphogenetic protein-6 gene transcription in MCF-7 cells by estrogen.
BACKGROUND
Bone morphogenetic protein-6 (BMP-6) is closely correlated with tumor differentiation and skeletal metastasis. Estrogen is considered as a stimulant for the initiation and promotion of breast cancer. Previous studies demonstrated that 17beta-estadiol (E2) can selectively increase the expression of BMP-6. This experiment is designed to detect the molecular mechanism of estrogen activating BMP-6 gene transcription in human estrogen receptor positive (ER+) breast cancer cell line MCF-7.


METHODS
After the treatment of MCF-7 cells with E2 at different concentrations (10(-11) mol/L, 10(-9) mol/L, 10(-7) mol/L), the BMP-6 expression level was examined through real-time polymerase chain reaction. Through restriction enzyme digestion, human BMP-6 1.2 kb long promoter, BMP-6 0.7 kb long promoter was cloned into pGL-3 basic vector; after the treatment with 10(-7) mol/L E2, luciferase activities of the two promoters were detected. Site-directed mutagenesis was performed to obtain the mutant forms of estrogen response element half-site (1/2 ERE) element and Sp1 sites in the BMP-6 promoter, the activities of these mutant form promoters were detected following the methods mentioned above. Chromatin immunoprecipitation (ChIP) assay was also used to confirm the binding of estrogen receptor alpha (ERalpha) on BMP-6 promoter in the presence of E2.


RESULTS
E2 dose dependently increased BMP-6 mRNA expression in human ER+ breast cancer cell line MCF-7. At a dose of 10(-7) mol/L E2, human BMP-6 1.2 kb promoter activity was increased by 90% compared with the control group treated with ethanol (P < 0.05). Both the 1/2 ERE response element mutant form and the Sp1 site mutant form of the BMP-6 promoter abolished the activation of the BMP-6 promoter's response to E2. Through ChIP assay, the binding of ERalpha on 1/2 ERE response element in BMP-6 promoter was further validated.


CONCLUSIONS
Estrogen induces BMP-6 expression in human ER+ breast cancer cell line MCF-7 through its receptor ERalpha binding on 1/2 ERE element in the BMP-6 promoter.
Zhang
Ming
M
Laboratory of Molecular Medicines, Medical College, Nankai University, Tianjin 300071, China.
Yan
Ji-dong
JD
Zhang
Lei
L
Wang
Qing
Q
Lü
Shu-jun
SJ
Zhang
Jie
J
Zhu
Tian-hui
TH
D055418:Bone Morphogenetic Protein 6; D019485:Bone Morphogenetic Proteins / Q000235:genetics*; D001943:Breast Neoplasms / Q000235:genetics*; D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D004958:Estradiol / Q000494:pharmacology*; D047628:Estrogen Receptor alpha / Q000502:physiology; D005260:Female; D006801:Humans; D044162:Parathyroid Hormone-Related Protein / Q000378:metabolism; D011401:Promoter Regions, Genetic; D015533:Transcriptional Activation / Q000187:drug effects*
C525816:BMP6 protein, human; D055418:Bone Morphogenetic Protein 6; D019485:Bone Morphogenetic Proteins; D047628:Estrogen Receptor alpha; D044162:Parathyroid Hormone-Related Protein; D004958:Estradiol