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A novel promoter controls Cyp19a1 gene expression in mouse adipose tissue.

Zhao H, Innes J, Brooks DC, Reierstad S, Yilmaz MB, Lin Z, Bulun SE - Reprod. Biol. Endocrinol. (2009)

Bottom Line: Subcutaneous and brown adipose tissue did not contain detectable Cyp19a1 mRNA.Dexamethasone significantly induced activity of this adipose-specific promoter region.These results expand the known 5'-regulatory region of the murine Cyp19a1 gene to 75 kb upstream of the translation start site.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Obstetrics and Gynecology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, USA. h-zhao@northwestern.edu

ABSTRACT

Background: Aromatase, the key enzyme in estrogen biosynthesis, is encoded by the Cyp19a1 gene. Thus far, 3 unique untranslated first exons associated with distinct promoters in the mouse Cyp19a1 gene have been described (brain, ovary, and testis-specific). It remains unknown whether aromatase is expressed in other mouse tissues via novel and tissue-specific promoters.

Methods: Real-time PCR was used to examine the aromatase expression levels in various C57BL/6 mouse tissues. 5'-rapid amplification of cDNA ends (5'-RACE) was used to determine the transcriptional start sites of Cyp19a1 transcripts. Promoter activity was measured using serial deletion mutants of DNA fused to the luciferase reporter gene. Primary mouse adipose fibroblasts were isolated and cultured from 16-week-old mouse gonadal fat pads.

Results: We systematically analyzed Cyp19a1 expression in a large number of mouse tissues, and demonstrated for the first time that aromatase was expressed in the male but not female gonadal fat pad. Subcutaneous and brown adipose tissue did not contain detectable Cyp19a1 mRNA. We used 5'-RACE to clone a novel gonadal fat-specific untranslated first exon, which is spliced onto a common junction 15 bp upstream of the translation start site. This adipose-specific first exon was mapped to approximately 75 kb upstream of the translation start site. Transfection of luciferase reporter gene plasmids containing the promoter region upstream of the adipose-specific first exon into murine 3T3-L1 adipose fibroblasts demonstrated significant basal promoter activity conferred primarily by the sequence located at -343/-1 bp. Dexamethasone significantly induced activity of this adipose-specific promoter region. Adipose-specific Cyp19a1 mRNA was expressed in primary mouse adipose fibroblasts and significantly induced by dexamethasone alone or serum plus dexamethasone.

Conclusion: Taken together, this research identified a novel, adipose-specific first exon of Cyp19a1 and its hormonally regulated promoter region in male murine gonadal fat. These results expand the known 5'-regulatory region of the murine Cyp19a1 gene to 75 kb upstream of the translation start site. Cyp19a1 expression in mouse adipose tissue may play an important role in reproductive biology and lipid metabolism.

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Dexamethasone or dexamethasone plus FBS induced adipose-specific Cyp19a1 mRNA expression in primary MAF. Following overnight serum starvation, cells were incubated in serum-free DMEM/F-12 medium or DMEM/F-12 supplemented with 10% FBS in the absence or presence of 250 nM dexamethasone for 24 hours. Adipose-specific, ovarian-specific or total C yp19a1 mRNA levels were analyzed by real-time RT-PCR. *p < 0.05 for vs. vehicle treatment. #p < 0.05 for vs. Dexamethasone treatment. †p < 0.05 for vs. FBS treatment.
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Figure 6: Dexamethasone or dexamethasone plus FBS induced adipose-specific Cyp19a1 mRNA expression in primary MAF. Following overnight serum starvation, cells were incubated in serum-free DMEM/F-12 medium or DMEM/F-12 supplemented with 10% FBS in the absence or presence of 250 nM dexamethasone for 24 hours. Adipose-specific, ovarian-specific or total C yp19a1 mRNA levels were analyzed by real-time RT-PCR. *p < 0.05 for vs. vehicle treatment. #p < 0.05 for vs. Dexamethasone treatment. †p < 0.05 for vs. FBS treatment.

Mentions: The literature indicates that hormonal treatments with broad actions such as PKA stimulators (cAMP analogs), PKC stimulators (phorbol esters), glucocorticoids and serum, alone and in combination, regulated aromatase expression in human adipose fibroblasts via different promoters[26,27]. To determine aromatase expression and regulation in MAFs, mouse gonadal fat pads were harvested and primary MAFs were cultured. Based on our results regarding the activity of the adipose tissue promoter (Figure 5), we treated these cells with vehicle, dexamethasone, FBS, or dexamethasone plus FBS. We quantitated the levels of various promoter-specific Cyp19a1 mRNA expressed using exon-specific real-time RT-PCR. Adipose-specific Cyp19a1 mRNA levels were significantly increased by 2.5 fold upon dexamethasone treatment and further significantly increased to 7.8 fold following dexamethasone plus FBS treatment. Dexamethasone, FBS, or dexamethasone plus FBS treatment did not significantly alter ovarian-specific Cyp19a1 mRNA levels. Although total Cyp19a1 mRNA levels were increased by 4.5 fold following dexamethasone treatment as compared to vehicle treatment, statistically this result did not rise above the level of significance. FBS alone had no effect on Cyp19a1 expression, but significantly augmented the effect of dexamethasone to cause a 15-fold increase in Cyp19a1 expression (Figure 6).


A novel promoter controls Cyp19a1 gene expression in mouse adipose tissue.

Zhao H, Innes J, Brooks DC, Reierstad S, Yilmaz MB, Lin Z, Bulun SE - Reprod. Biol. Endocrinol. (2009)

Dexamethasone or dexamethasone plus FBS induced adipose-specific Cyp19a1 mRNA expression in primary MAF. Following overnight serum starvation, cells were incubated in serum-free DMEM/F-12 medium or DMEM/F-12 supplemented with 10% FBS in the absence or presence of 250 nM dexamethasone for 24 hours. Adipose-specific, ovarian-specific or total C yp19a1 mRNA levels were analyzed by real-time RT-PCR. *p < 0.05 for vs. vehicle treatment. #p < 0.05 for vs. Dexamethasone treatment. †p < 0.05 for vs. FBS treatment.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2684739&req=5

Figure 6: Dexamethasone or dexamethasone plus FBS induced adipose-specific Cyp19a1 mRNA expression in primary MAF. Following overnight serum starvation, cells were incubated in serum-free DMEM/F-12 medium or DMEM/F-12 supplemented with 10% FBS in the absence or presence of 250 nM dexamethasone for 24 hours. Adipose-specific, ovarian-specific or total C yp19a1 mRNA levels were analyzed by real-time RT-PCR. *p < 0.05 for vs. vehicle treatment. #p < 0.05 for vs. Dexamethasone treatment. †p < 0.05 for vs. FBS treatment.
Mentions: The literature indicates that hormonal treatments with broad actions such as PKA stimulators (cAMP analogs), PKC stimulators (phorbol esters), glucocorticoids and serum, alone and in combination, regulated aromatase expression in human adipose fibroblasts via different promoters[26,27]. To determine aromatase expression and regulation in MAFs, mouse gonadal fat pads were harvested and primary MAFs were cultured. Based on our results regarding the activity of the adipose tissue promoter (Figure 5), we treated these cells with vehicle, dexamethasone, FBS, or dexamethasone plus FBS. We quantitated the levels of various promoter-specific Cyp19a1 mRNA expressed using exon-specific real-time RT-PCR. Adipose-specific Cyp19a1 mRNA levels were significantly increased by 2.5 fold upon dexamethasone treatment and further significantly increased to 7.8 fold following dexamethasone plus FBS treatment. Dexamethasone, FBS, or dexamethasone plus FBS treatment did not significantly alter ovarian-specific Cyp19a1 mRNA levels. Although total Cyp19a1 mRNA levels were increased by 4.5 fold following dexamethasone treatment as compared to vehicle treatment, statistically this result did not rise above the level of significance. FBS alone had no effect on Cyp19a1 expression, but significantly augmented the effect of dexamethasone to cause a 15-fold increase in Cyp19a1 expression (Figure 6).

Bottom Line: Subcutaneous and brown adipose tissue did not contain detectable Cyp19a1 mRNA.Dexamethasone significantly induced activity of this adipose-specific promoter region.These results expand the known 5'-regulatory region of the murine Cyp19a1 gene to 75 kb upstream of the translation start site.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Obstetrics and Gynecology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, USA. h-zhao@northwestern.edu

ABSTRACT

Background: Aromatase, the key enzyme in estrogen biosynthesis, is encoded by the Cyp19a1 gene. Thus far, 3 unique untranslated first exons associated with distinct promoters in the mouse Cyp19a1 gene have been described (brain, ovary, and testis-specific). It remains unknown whether aromatase is expressed in other mouse tissues via novel and tissue-specific promoters.

Methods: Real-time PCR was used to examine the aromatase expression levels in various C57BL/6 mouse tissues. 5'-rapid amplification of cDNA ends (5'-RACE) was used to determine the transcriptional start sites of Cyp19a1 transcripts. Promoter activity was measured using serial deletion mutants of DNA fused to the luciferase reporter gene. Primary mouse adipose fibroblasts were isolated and cultured from 16-week-old mouse gonadal fat pads.

Results: We systematically analyzed Cyp19a1 expression in a large number of mouse tissues, and demonstrated for the first time that aromatase was expressed in the male but not female gonadal fat pad. Subcutaneous and brown adipose tissue did not contain detectable Cyp19a1 mRNA. We used 5'-RACE to clone a novel gonadal fat-specific untranslated first exon, which is spliced onto a common junction 15 bp upstream of the translation start site. This adipose-specific first exon was mapped to approximately 75 kb upstream of the translation start site. Transfection of luciferase reporter gene plasmids containing the promoter region upstream of the adipose-specific first exon into murine 3T3-L1 adipose fibroblasts demonstrated significant basal promoter activity conferred primarily by the sequence located at -343/-1 bp. Dexamethasone significantly induced activity of this adipose-specific promoter region. Adipose-specific Cyp19a1 mRNA was expressed in primary mouse adipose fibroblasts and significantly induced by dexamethasone alone or serum plus dexamethasone.

Conclusion: Taken together, this research identified a novel, adipose-specific first exon of Cyp19a1 and its hormonally regulated promoter region in male murine gonadal fat. These results expand the known 5'-regulatory region of the murine Cyp19a1 gene to 75 kb upstream of the translation start site. Cyp19a1 expression in mouse adipose tissue may play an important role in reproductive biology and lipid metabolism.

Show MeSH
Related in: MedlinePlus