Regulation of human cbfa1 gene transcription in osteoblasts by selective estrogen receptor modulators (SERMs).
Cbfa1, a transcription factor critical for bone formation, plays a key role in osteoblast recruitment and differentiation. We have cloned and characterized a 3.0 KB 5'-flanking region of the human cbfa1 gene isolated from a P1 human genomic library. DNA sequencing revealed several known canonical nuclear transcription factor binding sites, including two AP1 and six OSE2 binding sites in the proximal promoter region. Although no estrogen (E2)-response element (ERE) binding sites were identified, E2 has been shown to regulate gene activity via AP1 promoter sites. We examined the effect of selective estrogen receptor modulators (SERMs) on human cbfa1 gene promoter activity using cell-based luciferase reporter transcriptional assays. Three characterized SERMs, tamoxifen, raloxifene, and ICI 178,180, all upregulated cbfa1-luciferase (cbfa1Luc) gene activity 5- to 10-fold in a dose-dependent manner. This effect was mediated by both ER alpha and ER beta. Mutational analysis demonstrated that the minimal promoter region for basal of SERM-activated transcription was mapped to adjacent AP1-like and OSE2 binding sites within -93 and +7 of the transcription start condon. Further, electrophoretic mobility shift assays (EMSAs) demonstrate that ICI 178,180 increased binding of AP1 and OSE2 site by ER alpha and cbfa1, respectively. These studies suggest that SERMs can modulate bone-specific cbfa1 gene expression in a human osteosarcoma cell line.
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Harvard-Thorndike and Charles A. Dana Research Laboratories, Division of Bone and Mineral Metabolism, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02115, USA.
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Alexander
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D001483:Base Sequence; D002874:Chromosome Mapping; D050659:Core Binding Factor Alpha 1 Subunit; D004305:Dose-Response Relationship, Drug; D017930:Genes, Reporter; D006801:Humans; D008156:Luciferases / Q000235:genetics; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins; D010006:Osteoblasts / Q000187:drug effects* / Q000502:physiology; D012516:Osteosarcoma; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D020845:Selective Estrogen Receptor Modulators / Q000494:pharmacology*; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects*; D014407:Tumor Cells, Cultured
D050659:Core Binding Factor Alpha 1 Subunit; D009363:Neoplasm Proteins; D020845:Selective Estrogen Receptor Modulators; D014157:Transcription Factors; D008156:Luciferases