Regulation of CYP19 gene expression in primary human osteoblasts: effects of vitamin D and other treatments

Abstract

Objective: Extragonadal estrogen biosynthesis is relevant for the regulation of bone metabolism. The aims of this paper were: (i) to examine CYP19 (aromatase) gene expression in primary cultures of osteoblasts under several hormone and cytokine treatments and (ii) to study the promoter usage of CYP19 in these cells. Methods: Primary cultures of osteoblasts were obtained from healthy donors. The effects of vitamin D and other factors on CYP19 expression were analysed by semiquantitative RT-PCR. Furthermore, CYP19 alternative promoter usage under the different treatments was characterized by RT-PCR. Results: CYP19 transcripts were detected in cultured human osteoblasts in serum-free conditions. Vitamin D, dexamethasone, 17b-estradiol and testosterone increased transcript levels of CYP19, whereas interleukin-1b or tumor necrosis factor a decreased them. Aromatase mRNA produced under treatment with vitamin D was transcribed from promoters I.4 and I.3, while stimulation with dexamethasone or dexamethasone plus vitamin D also involved promoter I.2. Testosterone activated promoters I.2 and I.4. Conclusions: Our results suggested that vitamin D, testosterone, estrogens and glucocorticoids regulate CYP19 gene expression in human primary osteoblasts and the main promoter used appears to be promoter I.4. Promoters pII and I.3 seem to be related to basal transcription and may mediate estrogen stimulation, while promoter I.2 seems to play a role in the effect of glucocorticoids. These findings indicate that vitamin D and several hormones regulate local estrogen synthesis in human osteoblasts mainly through usage of promoters I.4 and I.3.

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

“…Consistently with results obtained in our previous study using primary hOBs (22), luciferase assays showed that pII, I.3 and I.4 promoters were active in MG-63 cells. Here we report that the sequence containing promoter pII up to exon I.3 (2 221 to þ 1) displayed high activity in MG-63 cells.…”

“…Our results therefore underscore the importance of exon I.6 in the regulation of pII or I.3 promoter activity in osteoblasts. Regarding promoter I.4, an important activity was observed in MG-63 cells, consistent with our previous findings in hOBs and those of other groups (15,16,22). Additionally the presence of an enhancer element in exon I.4 (þ 71 to þ 328) was demonstrated.…”

“…The protocol used for primary hOB culture has been previously described (22) 26 mol/l (R&D Systems) and recombinant human leptin (10 100 and 1000 ng/ml) (R&D Systems).…”

A common polymorphism in the 5'-untranslated region of the aromatase gene influences bone mass and fracture risk

“…Exon I.4 is the preferential transcript in adipose tissue, which also transcribes PII, I.3 and I.6 in smaller amounts (3,10,18). The main promoter used in bone tissue seems to be I.4, but bone cells have also been shown to express exons PII, I.6, I.3, I.2 and I.f in some culture conditions (16,19,20). In fact, a phenomenon of promoter switching depending upon the regulatory factors present in the media, including hormones and cytokines, and cell differentiation inducers has been shown in many tissues.…”

“…Expression of exon I.2 was first reported in the placenta, but it is also expressed in other tissues, including bone. Indeed, Enjuanes et al (20) have recently reported that 1,25-dihydroxyvitamin D3 and glucocorticoids induce I.2 expression by The mechanism underlying the association between CYP19 alleles and bone mass is unclear. It could be directly related to differences in the transcription response to promoters.…”