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Overexpression of Aromatase P450 in Leiomyoma Tissue Is Driven Primarily through Promoter I.4 of the Aromatase P450 Gene (CYP19)

Department of Obstetrics and Gynecology, Kanazawa University School of Medicine, Kanazawa 920-0934, Japan

Address all correspondence and requests for reprints to: Makio Shozu, M.D., Ph.D., Department of Obstetrics and Gynecology, Kanazawa University School of Medicine, Kanazawa 920-0934, Japan. E-mail: . shozu{at}med.kanazawa-u.ac.jp

Abstract

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-1ß and dexamethasone, known as a potent inducer of promoter I.4-driven transcription, effectively increased aromatase activity. A combination of (Bt)₂cAMP, 3-isobutyl-1-myethylxanthine, and PGE₂, 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-1ß. 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.

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