Functional analysis of the I.3, I.6, pII and I.4 promoters of CYP19 (aromatase) gene in human osteoblasts and their role in vitamin D and dexamethasone stimulation.
OBJECTIVE
Current evidence suggests that extragonadal estrogens play an important role in bone metabolism. Estrogen biosynthesis is catalyzed by P450aromatase, encoded by the CYP19 gene. The aims of this paper were to study CYP19 gene expression in human osteoblasts under several hormone and cytokine treatments and to define promoter regions involved in this regulation.


METHODS
CYP19 transcript levels were measured from primary human osteoblasts and MG-63 cells by real-time PCR in basal conditions, and in response to seven different hormones and cytokines. Four promoters of CYP19 gene were cloned upstream of the luciferase gene and transfected into MG-63 cells. The effect of vitamin D and dexamethasone in these promoter activities was evaluated.


RESULTS
Vitamin D and dexamethasone were potent stimulators of CYP19 transcription, while testosterone and 17beta-estradiol stimulated moderately. Promoter pII proved the most potent in driving transient luciferase expression. Promoter I.4 displayed moderate activity, while promoters I.3 and I.6 were weak. A region upstream of exon I.3, including exon I.6, was identified as containing repressor elements of promoter pII. Promoter I.3 activity was modulated by repressors located within exon I.3, while an enhancer of promoter I.4 was detected within exon I.4. In the absence of fetal calf serum, dexamethasone stimulation was observed on promoters I.3 and I.4, while vitamin D stimulation acted only on promoter I.3.


CONCLUSIONS
Four regulatory regions of promoters pII, I.3 and I.4 are relevant to CYP19 expression in human osteoblasts. Vitamin D and dexamethasone modulate transcription through these regions.
Enjuanes
Anna
A
URFOA-IMIM, Hospital del Mar, Universitat Autònoma de Barcelona, E-08003 Barcelona, Spain. aenjuanes@clinic.ub.es
Garcia-Giralt
Natalia
N
Supervía
August
A
Nogués
Xavier
X
Ruiz-Gaspà
Silvia
S
Bustamante
Mariona
M
Mellibovsky
Leonardo
L
Grinberg
Daniel
D
Balcells
Susana
S
Díez-Pérez
Adolfo
A
D000368:Aged; D001141:Aromatase / Q000096:biosynthesis / Q000235:genetics*; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D003907:Dexamethasone / Q000494:pharmacology*; D004958:Estradiol / Q000494:pharmacology; D005260:Female; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D006801:Humans; D007375:Interleukin-1 / Q000494:pharmacology; D020738:Leptin / Q000494:pharmacology; D008297:Male; D008875:Middle Aged; D010006:Osteoblasts / Q000201:enzymology*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D013739:Testosterone / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D014807:Vitamin D / Q000494:pharmacology*
D007375:Interleukin-1; D020738:Leptin; D014409:Tumor Necrosis Factor-alpha; D014807:Vitamin D; D013739:Testosterone; D004958:Estradiol; D003907:Dexamethasone; D001141:Aromatase