Spatially heterogeneous expression of aromatase P450 through promoter II is closely correlated with the level of steroidogenic factor-1 transcript in endometrioma tissues.

Endometriosis is an estrogen-dependent disease of women of reproductive age. Recent studies demonstrate that endometriosis per se express high levels of estrogen synthetase (aromatase P450). The resulting estrogen synthesized in situ may play a role in the development and exacerbation of the disease. For ovarian endometrioma, previous studies have been conducted ex vivo using cells obtained from endometrioma and have demonstrated that steroidogenic factor-1 is involved in the expression of aromatase. The aim of the present study was to provide in vivo evidence that steroidogenic factor-1 plays an important role in the regulation and overexpression of aromatase P450 in situ. First, promoter use of aromatase P450 in endometrioma tissue was determined using quantitative methods. Ovarian endometrioma tissue was chopped into small pieces, and two exon 1-specific transcripts of aromatase P450 (PII-specific and I.4-specific transcripts) were quantified using competitive RT-PCR. PII-specific transcript was more abundant than the I.4-specific transcript in 13 of the 15 endometriomas and less abundant in the remaining two. Spatial distribution of aromatase P450 transcripts in these endometrioma tissues revealed heterogeneous expression in the cyst wall, demonstrating wide variability even in the same endometrioma. Two possible regulators of aromatase expression (steroidogenic factor-1 and IL-1 beta) were then measured in all endometrioma samples and the correlation between aromatase P450 transcripts and these possible regulators in the endometrioma samples were tested using Spearman's rank order correlation test. Levels of steroidogenic factor-1 transcript were found to correlate closely with levels of PII-specific transcript in eight of nine endometriomas examined. On the other hand, the level of IL-1 beta weakly correlated with I.4-specific transcripts in three of the nine endometriomas. We next histologically examined samples of four endometriomas in which complete sets of tissue samples corresponded to the RNA samples. We could not identify any specific pathology to explain the heterogeneous expression of PII-specific transcripts of aromatase P450, although the number of CD-68 positive macrophages in the tissue sections weakly correlated with the level of I.4-specific transcript in two of four endometriomas. These results provide strong evidence that promoter II is the predominant promoter of aromatase P450 in endometrioma tissues in vivo and that steroidogenic factor-1 in situ is a major determinant of aromatase P450 overexpression in endometrioma tissues in vivo.

Overexpression of aromatase P450 in leiomyoma tissue is driven primarily through promoter I.4 of the aromatase P450 gene (CYP19).

The CYP19 gene encoding aromatase P450 (estrogen synthetase) is expressed in several extragonadal sites and regulated in a tissue-specific fashion, which is achieved by alternative use of the seven different promoters (and corresponding exons 1) of the CYP19 gene. Previously, we demonstrated that aromatase P450 is overexpressed in leiomyoma tissue and that in situ estrogen synthesized in leiomyoma tissues possibly plays a role in leiomyoma growth. To elucidate the mechanism of overexpression of aromatase P450, we determined the promoter use of aromatase P450 in leiomyomas. 5'-Rapid amplification of cDNA ends analysis revealed that of six leiomyoma nodules tested, four nodules contained I.4-specific transcript of aromatase P450 alone, one nodule contained PII-specific transcript alone, and the remaining nodule contained both I.4- and PII-specific transcripts simultaneously. The levels of aromatase transcripts were then quantified by competitive RT-PCR assay. Among 21 leiomyomas, I.4-specific transcript and PII-specific transcript were predominant in 18 and 2 leiomyomas, respectively, whereas the remaining leiomyoma was negative for aromatase P450 expression. We next compared the aromatase activity of leiomyoma cells stimulated by promoter-specific regulatory factors. A combination of IL-1beta and dexamethasone, known as a potent inducer of promoter I.4-driven transcription, effectively increased aromatase activity. A combination of (Bt)(2)cAMP, 3-isobutyl-1-myethylxanthine, and PGE(2), known as inducers of promoter II-driven transcription, also increased aromatase activity, but the increases found were smaller than that induced by dexamethasone and IL-1beta. The transcriptional ability of the promoter I.4 sequence was confirmed by transient transfection assay using primary cells released from leiomyomas and established cells from normal myometrium (KW cells). Luciferase vectors containing promoter I.4 sequence (-340/+14 or longer) showed a significant increase in luciferase activity in response to dexamethasone. Deletion or mutation of a putative glucocorticoid-responsive element in the promoter I.4 sequence eliminated promoter activity. These results indicate that promoter I.4 is the major promoter responsible for overexpression of aromatase P450 in leiomyomas and that a glucocorticoid-responsive element within it plays a substantial role in the expression of aromatase P450.