生水 真紀夫

BACKGROUND: Gynecomastia of prepubertal onset may result from increased estrogen owing to excessive aromatase activity in extraglandular tissues. A gene in chromosome 15q21.2 encodes aromatase, the key enzyme for estrogen biosynthesis. Several physiologic tissue-specific promoters regulate the expression of aromatase, giving rise to messenger RNA (mRNA) species with an identical coding region but tissue-specific 5'-untranslated regions in placenta, gonads, brain, fat, and skin. METHODS: We studied skin, fat, and blood samples from a 36-year-old man, his 7-year-old son, and an unrelated 17-year-old boy with severe gynecomastia of prepubertal onset and hypogonadotropic hypogonadism caused by elevated estrogen levels. RESULTS: Aromatase activity and mRNA levels in fat and skin and whole-body aromatization of androstenedione were severely elevated. Treatment with an aromatase inhibitor decreased serum estrogen levels and normalized gonadotropin and testosterone levels. The 5'-untranslated regions of aromatase mRNA contained the same sequence (FLJ) in the father and son and another sequence (TMOD3) in the unrelated boy; neither sequence was found in control subjects. These 5'-untranslated regions normally make up the first exons of two ubiquitously expressed genes clustered in chromosome 15q21.2-3 in the following order (from telomere to centromere): FLJ, TMOD3, and aromatase. The aromatase gene is normally transcribed in the direction opposite to that of TMOD3 and FLJ. Two distinct heterozygous inversions reversed the direction of the TMOD3 or FLJ promoter in the patients. CONCLUSIONS: Heterozygous inversions in chromosome 15q21.2-3, which caused the coding region of the aromatase gene to lie adjacent to constitutively active cryptic promoters that normally transcribe other genes, resulted in severe estrogen excess owing to the overexpression of aromatase in many tissues.

Objective: To assess the management of symptomatic leiomyomas using a nonsteroidal aromatase inhibitor in perimenopausal women. Design: Case report. Setting: Academic clinical practice. Patient(s): A 53-year-old woman suffering from recurrent urinary retention secondary to a uterine leiomyoma. Intervention(s): Fadrozole, orally, 2 mg daily for 8 weeks and then 1 mg daily for 4 weeks. Main Outcome Measure(s): Measurements of leiomyoma volume, and levels of serum E-2, LH, and FSH. Result(s): Urinary retention resolved after 2 weeks of treatment and did not recur. Leiomyoma volume estimated by ultrasonography revealed a 71% reduction after 8 weeks of treatment. Conclusion(s): Fadrozole was useful for the management of a symptomatic leiomyoma without transient deterioration of symptoms. Clinical trials are warranted. (Fertil Steril((R)) 2003;79:628-31. (C) 2003 by American Society for Reproductive Medicine.).

Intratumoral expression of aromatase P450 (P450arom) promotes the growth of breast tumors via increased local estrogen concentration. We cloned a novel 101-bp untranslated first exon (1.7) that comprises the 5'-end of 29-54% of P450arom transcripts isolated from breast cancer tissues (n = 7). The levels of P450arom transcripts with exon 1.7 were significantly increased in breast tumor tissues and adipose tissue adjacent to tumors. We identified a promoter immediately upstream of exon 1.7 and mapped this to about 36 kb upstream of ATG translation start site of the CYP19 (aromatase cytochrome P450) gene. Sequence analysis of 1.7 revealed a TATA-less promoter containing an initiator, two consensus GATA sites, and cis-regulatory elements found in megakaryocytes and endothelial type promoters. Luciferase activity directed by the promoter 1.7 sequence (-299/+81 bp) was 4-fold greater than a minimum length promoter sequence (-35/+81 bp) in human microvascular endothelial cells (HMEC-1), but only 2-fold greater in MCF-7 breast malignant epithelial cells. There was no promoter activity in primary breast adipose fibroblasts. Site-directed mutations demonstrated that maximal basal promoter activity required two GATA motifs at -146/-141 bp and -196/-191 bp. Gel shift and deoxyribonuclease I footprinting assays demonstrated the binding of GATA-2 transcription factor but not GATA-1 to the -196/-191-bp region. Overexpression of GATA-2 in HMEC-1 cells increases promoter 1.7 activity by 5-fold. In conclusion, promoter 1.7 is a GATA-2-regulated endothelial promoter of the human CYP19 gene and may increase estrogen biosynthesis in vascular endothelial cells of breast cancer. The activity of this promoter may also be important for intracrine and paracrine effects of estrogen on blood vessels.

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-1beta) 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-1beta 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 heterogenous 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.

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 1.4- 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 1.4 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 1.4 sequence eliminated promoter activity. These results indicate that promoter 1.4 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.