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The Journal of Clinical Endocrinology & Metabolism Vol. 87, No. 6 2540-2548
Copyright © 2002 by The Endocrine Society

The Impact of the Human Genome on Endocrinology: Original Articles

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

Makio Shozu, Hiroshi Sumitani, Tomoya Segawa, Hei-Juan Yang, Kouichi Murakami, Tadayuki Kasai and Masaki Inoue

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.

LEIOMYOMA OF THE uterus is the most common disease in women of reproductive age and is composed of monoclonal proliferation of a transformed myocyte derived from myometrium. However, the pathogenesis of the transformation remains unknown, although cytogenetic studies have implicated involvement of several genes that may function as tumor suppressor genes in some leiomyomas, such as HMGI-C on chromosome 12q15 (1, 2, 3, 4, 5, 6, 7, 8, 9). On the other hand, it is widely accepted that the growth of leiomyoma of the uterus is up-regulated by circulating sex steroids, namely estrogen and progesterone secreted from the ovary (10). Leiomyoma develops only after the commencement of menstrual cyclicity and becomes symptomatic usually in the 30s and 40s. At the time of menopause, the leiomyomas start to regress, and the symptoms resolve. Therefore, the ovary is thought to be the major source of sex steroids for leiomyoma growth.

In addition to endocrine estrogen from the ovary, recent works by other researchers and ourselves have reported the possible contribution of in situ estrogen to leiomyoma growth, namely estrogen synthesized in leiomyoma cells. Leiomyomas per se express estrogen synthetase (aromatase P450) at a higher level than the surrounding myometrium and are able to synthesize estrogens (11, 12, 13, 14). In fact, the tissue concentrations of estrogens are elevated in leiomyoma nodules compared with levels in the surrounding myometrium (15, 16). In a previous in vitro experiment we confirmed that estrogen synthesized in leiomyoma cells in culture, albeit at too low a level to exert endocrine effect on the distant organs, is sufficient to promote leiomyoma cell growth in a paracrine/autocrine/intracrine fashion (14).

More recently, we found clinical evidence supporting the importance of in situ estrogen in leiomyoma growth. Preoperative treatment with leuprorelin acetate, a GnRH agonist widely used for the treatment of uterine leiomyoma cells, profoundly suppresses the expression of aromatase P450 in leiomyoma cells (17). The basic rationale of GnRH agonist therapy is that GnRH agonists desensitize pituitary gonadotrophs and reduce ovarian steroids. Thus, GnRH agonist therapy abolishes both endocrine (ovarian) estrogen and in situ (leiomyoma) estrogen, simultaneously. This combined suppression of estrogen synthesis may explain why GnRH agonists induce shrinkage of leiomyomas more rapidly and more profoundly than natural menopause does (18, 19, 20). In postmenopausal women, leiomyomas continue to synthesize in situ estrogen through the conversion of plasma androstenedione, which possibly prevents the leiomyoma from rapid regression at early menopause, which would be viewed as a result of the complete depletion of ovarian estrogen (17). The role of estrogen in maintaining postmenopausal leiomyoma size is also suggested by the finding that administration of raloxifene, an estrogenic antagonist for leiomyomas, reduces leiomyoma size in postmenopausal women (21).

There is further evidence to support the relative importance of in situ estrogen in leiomyoma growth during GnRH agonist therapy. Even women who continue to have significant circulating estrogen levels (150 pmol/liter or higher) show regression of leiomyomas during GnRH agonist therapy (22, 23). Moreover, low dose estrogen add-back does not induce regrowth of leiomyomas during GnRH agonist therapy (24). Therefore, complete down-regulation of the ovary and hypoestrogenemia are not necessary for successful shrinkage of leiomyomas. Suppression of in situ estrogen instead is most likely important for the reduction of leiomyoma size in these borderline hypoestrogenemic conditions, such as menopause and GnRH agonist therapy.

The next question that arises is the mechanism by which aromatase P450 is overexpressed in leiomyoma tissues. Aromatase P450, a product of a single copy gene CYP19 on chromosome 15q21, is expressed in several tissues and cells and is regulated differently in a tissue-dependent manner. The tissue-specific regulation is realized by alternative use of seven different promoters of aromatase P450 (Fig. 1Go), each of which has different cis-acting elements function. Thus, identification of the promoter of aromatase P450 used in leiomyomas is the first step in understanding the mechanism of overexpression of aromatase P450 in leiomyoma tissues.



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  		Figure 1. Schematic representation of the human CYP19 gene. The gray bars represent untranslated first exons; arrows upstream of each untranslated first exon indicate promoters for each exon 1-specific transcript. The closed bars represent coding sequences. The 3'-acceptor splice junction of the untranslated first exons is located in exon II just downstream of the PII-specific sequence (gray bar). The white bar in exon II just downstream of this common acceptor site represents untranslated exon II sequenced, which is common to all human CYP 19 transcripts. The white bar in exon X is the 3'- untranslated region.

 
In the present study we examined the promoter use of aromatase P450 in leiomyoma cells and found that promoter I.4, originally identified as a promoter for skin and adipose tissues, mainly drives the transcription of aromatase P450 in leiomyoma tissue. We also confirmed that the promoter I.4 sequences were transcriptionally active and that a glucocorticoid-responsive element (GRE) within them was functioning in leiomyoma tissue.

Materials and Methods

Tissue acquisition

Uterine specimens were obtained from women undergoing hysterectomies or myomectomies for uterine leiomyoma. Forty-one leiomyoma nodules obtained from 31 women (age range, 28–49 yr) were examined. This study was approved by the medical ethics committee of Kanazawa University, Japan (no. 067), and informed consent was obtained before the study. Women with evidence of adenomyosis and/or endometriosis at the time of laparotomy were excluded. Intramural and subserosal nodules, but not submucosal nodules, were included in this study. Tissue samples were dissected immediately after surgery, snap-frozen in liquid nitrogen, and then stored at -74 C. Leiomyoma specimens were obtained from leiomyoma nodules 3 cm or larger in maximum diameter, just beneath the capsule of the nodule. All tissues samples used for this study were confirmed as histologically ordinary leiomyomas, with no cellular, epithelioid, bizarre, or plexiform variants present. Menstrual cycles were estimated based on histological findings of endometrium (25). Because leiomyoma nodules did not show any significant difference in the expression level of aromatase P450 among menstrual cycles (menstrual, secretory, and proliferative) or among the size of nodules (range, 3–15 cm in maximum diameter), statistical analysis was performed regardless of menstrual cycle and nodular size.

Cell culture

Leiomyoma cells were isolated from leiomyoma tissues using an enzymatic digestion method and were characterized as described previously (14, 26). Briefly, leiomyoma nodules were minced and digested in digestion medium for 4–6 h with vigorous shaking at 37 C. The digestion medium (HBSS) contained 0.1% (wt/vol) collagenase type B (Roche, Mannheim, Germany) and 0.015% (wt/vol) deoxyribonuclease I (Sigma, St. Louis, MO). After complete suspension, digestion medium was filtered through two layers of sterile gauze and then centrifuged at 700 x g for 10 min. Cell pellets were suspended, washed, and cultured in F-12/DMEM (1:1) medium supplemented with 10% FBS (Sigma), 100 IU/ml penicillin (Life Technologies, Inc., Gaithersburg, MD), 100 µg/ml streptomycin (Life Technologies, Inc.), and 100 µg/ml kanamycin (Life Technologies, Inc.). Cells obtained from approximately 5 g were divided into three to five 9-cm dishes, and cells reached confluence within 1–2 wk. Cells in culture were confirmed to have the characteristic features of uterine muscle cells: a fusiform shape, expression of smooth muscle-specific {alpha}-actin, and estrogen responsiveness (14). More than 95% of cells stained positively for smooth muscle-specific {alpha}-actin ({alpha}-smooth muscle actin immunohistology kit, Sigma) (14). Experiments were conducted on the first or second passage cells.

KW cells were previously established from myometrial cells and were provided by Dr. O. Matsuo (Kinki University, Osaka, Japan) (27). The cells were cultured in DMEM with 10% FBS. The cells have the characteristic features of myometrial cells: positive for vimentin, smooth muscle-specific {alpha}-actin, latent TGFß1, tissue-type plasminogen activator, and plasminogen activator inhibitor 1 and negative for desmin, endoglin, and cytokeratin 9 (27, 28). Preliminary experiments showed that this cell line does not express aromatase P450, but that transfected promoter constructs of aromatase P450 were still transcriptionally active.

5'-Rapid amplification of cDNA ends (5'-RACE)

5'-Untranslated sequences of aromatase P450 were amplified using the 5'-RACE kit (Life Technologies, Inc.) as described previously (29). The gene-specific primer used for cDNA synthesis was oligo17 (5'-ACTTGCTGATAATGAGTGTT-3'), which was located at the 3'-end of exon 5. The resulting cDNAs were tailed at the 3'-end with poly(C)+ using terminal transferase and then amplified by PCR using a 5'-RACE-abridged anchor primer and an antisense primer (oligoIIIb, 5'-CCAGAGATCCAGACTCGCATGAAT-3') specific for exon III of aromatase P450. The PCR product was reamplified using a universal amplification primer and a nested primer (oligo24, 5'-TAATACTCCCGTGTAGGAGTTATGGTC-3') specific for the 3'-end of exon II of aromatase P450. The reamplified PCR products were subcloned into pCR2.1 vector (Invitrogen, Carlsbad, CA) and sequenced using an automatic sequencer, the ABI PRISM 310 genetic analyzer (PE Applied Biosystems, Foster City, CA).

Preparation of total RNA

Total RNA was extracted from snap-frozen tissue samples using an Ultraspec RNA isolation kit (Biotecx, Houston, TX) according to the manufacturer’s instructions. The RNA concentration was determined spectrophotometrically.

Synthesis of internal standard RNAs and their DNA templates

For quantitative detection of exon 1 sequences of aromatase P450, two plasmids (pCR&Dgr;aromI.4 and pCR&Dgr;aromPII) containing one exon I copy (exon I.4- and promoter II-specific sequence (PII), respectively) and one exon II–IV copy (that had 107 bp of wild-type sequence deleted between exons II and III) was constructed by two-step PCR amplification as described previously (14). The PCR primers used for introduction of an internal deletion were arom[/{delta}]1 (5'-ATCCTCTGAGTCGACCCTCATAATTCCACACCA-3') and arom[/{delta}]2 (5'-AGGGTCGACTCAGAGGATTTCATGCGAGTCTGG3'). The exon 1-specific sense primers were oligo69 (5'-GGTTTGATGGGCTGACCAG-3') for exon I.4 sequence and oligo2.79 (5'-GCACCCTCTGAAGCAACAGGA-3') for PII-specific sequence, and the antisense primer was arom202 (5'-CTCCAACCTGTCCAGATGTGT-3') for the coding sequence of aromatase.

Internal standard RNAs were synthesized in vitro with T7-RNA polymerase using HindIII digests of pCR&Dgr;aromI.4 and pCR&Dgr;aromPII plasmids as templates (14). The synthetic RNA was extracted twice with acid-phenol and chloroform, treated twice with deoxyribonuclease I (Life Technologies, Inc.), and then purified using an anion exchange column (RNeasy kit, QIAGEN, Valencia, CA). RNA was quantified by spectrophotometry, and purity and size were confirmed by PAGE.

Quantification of aromatase exon 1-specific mRNA

Aromatase exon 1-specific mRNA in the total RNA samples was quantified by competitive RT-PCR analysis using synthetic RNAs as internal standards, as previously described (14) with some modifications. Briefly, total RNA (1 µg) and a mixture of internal standard RNAs [2 amol for exon I.4, 2 amol for PII, and 1 pmol for glyceraldehyde-3-phosphate dehydrogenase (G3PDH)] were reverse transcribed by RevatraAce (Toyobo, Tokyo, Japan) and random hexamer (PE Applied Biosystems) at 42 C for 40 min. One tenth of the resulting cDNAs were amplified by PCR using one 6-FAM-labeled sense primer, arom111 (5'-CACTGGTCAGCCCATCAA-3') for exon I.4 or arom2.79 for PII, and one antisense primer, arom203 (5'-GCCGAATCGAGAGCTGTAAT-3'). The amplification products were separated by capillary electrophoresis under denaturing conditions and detected using the ABI PRISM 310 genetic analyzer (Fig. 2AGo). The data were analyzed using GeneScan analysis software (PE Applied Biosystems). The amount of aromatase mRNA in the total RNA was calculated from the ratio of the peak height of the target to that of the internal standards. The standard curves obtained by serial dilution of a known amount of target (0.01–50 amol) were reproducible and linear over the 3 orders of magnitude tested (Fig. 2BGo).



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  		Figure 2. Analysis of aromatase P450 mRNA level by exon 1-specific quantitative RT-PCR. A representative electropherogram of the fluorescent PCR product (A) and the standard curves of competitive RT-PCR using a constant amount of competitor RNA (B) are shown. A, An aliquot (1:5 to 1:10) of exon 1-specific PCR products was denatured in deionized formamide and subjected to capillary electrophoresis using POP4 polymer (PE Applied Biosystems). A ROX-labeled size standard (GeneScan-500, PE Applied Biosystems) was used for determination of fragment lengths. Two peaks derived from an internal standard and from a target were identified based on calculated sizes as 305 and 412 nucleotides for exon I.4 transcript and 361 and 468 nucleotides for PII transcript. B, To validate the semiquantitative analysis using 2 amol competitors, a series of 4-fold dilutions of target RNA (0.01–50 amol) was reverse transcribed in the presence of a constant amount of competitor RNA (2 amol). Logarithmically transformed rations of the target intensity to the competitor intensity were plotted against the logarithmically transformed amount of target RNA added. The data are representative of two independent experiments of each.

 
Similarly, G3PDH mRNA was quantified to monitor the quality of the total RNA samples as described previously (26). The PCR primers used were 6-fluorescein-labeled G3PDH1 (5'-CTGAGAACGGGAAGCTTGTCATCAATGG-3') and unlabeled G3PDH2 (5'-TGTGGTCATGAGTCCTTCCACGATACCA-3').

Aromatase activity

The aromatase activity of primary cells was assayed by detecting the formation of tritiated water from [1ß-3H]androstenedione (NEN Life Science Products, Boston, MA) as described previously (17). Aromatase activity was expressed as the incorporation rate of tritium into water per mg protein/6 h. Protein concentration was determined using a bicinchoninic acid protein assay kit (Pierce Chemical Co., Rockford, IL).

Transient transfection assay

Reporter constructs containing promoter I.4 sequence and a mutant thereof were synthesized in a previous study using PCR-based methods, and the fidelity was confirmed by sequencing (30). These constructs carry various lengths of the 5'-flanking sequence of exon I.4 of aromatase P450 and a firefly luciferase gene as a reporter downstream of the promoter sequence.

For primary cells obtained from leiomyoma nodules, cells cultured in 12-well plates (5 x 105 cells/well) were transfected with 0.5 µg promoter constructs and 0.05 µg pSV-ß-galactosidase control vector (Promega Corp., Madison, WI) using the calcium phosphate method. Leiomyoma cells were incubated with calcium/DNA complexes for 16 h and then recovered in complete medium for 6 h. For KW cells, cells plated on 12-well plates (2 x 105 cell/well) were transfected for 2.5 h with 1.0 µg promoter I.4 constructs and 0.1 µg pSV-ß-galactosidase using 1 µl Superfect transfection reagent (QIAGEN) according to the manufacturer’s instructions. After 2.5-h incubation, cells were washed twice in PBS and then refed with complete medium. After 24-h recovery in complete medium, cells were stimulated with dexamethasone (Dex) for 24 h in medium supplemented with 0.5% serum. Cells were then lysed, and luciferase activity was measured (luciferase reporter gene assay, Roche) and normalized to ß-galactosidase activity (ß-galactosidase enzyme assay system, Promega Corp.).

Western blotting

Western blotting and detection of signals were conducted as previously described (14). Antibody for GR was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Statistical analysis

Differences in levels of two species of aromatase P450 transcripts were evaluated using the Wilcoxon signed rank test. Differences in levels of relative luciferase activities between two groups were evaluated using the Mann-Whitney U test. Statistical significance was established at the P < 0.05 level.

Results

Distribution of aromatase transcripts with specific 5'-untranslated ends (exon 1s) in a RACE-generated library

To identify the promoter used for aromatase expression, 5'-untranslated exon 1 sequence was amplified by 5'-RACE and subcloned into a pCR2.1 vector, followed by sequencing. Table 1Go summarizes the distribution of the 5'-termini in randomly selected clones generated by 5'-RACE. Only two species of 5'-terimni were found in the six different leiomyoma nodules obtained from six women: clones containing I.4-specific sequence were exclusively found in four leiomyoma nodules, clones containing PII-specific sequence were exclusively found in one nodule, and the remaining nodule contained both clones carrying exon I.4 sequence and those carrying PII-specific sequences.


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  		Table 1. Distribution of exon 1 sequences of aromatase P450 found in the RACE-generated library

 
Quantification of aromatase mRNA by exon 1-specific competitive RT-PCR

The result of the 5'-RACE experiment suggested that the major promoter responsible for overexpression of aromatase P450 in leiomyoma nodules is promoter I.4. To confirm this, we next conducted a quantitative analysis of promoter use in 21 leiomyoma nodules obtained from another 13 patients who were not included in the 5'-RACE analysis. Based on the results of 5'-RACE, we developed quantitative RT-PCR specific for exon I.4 sequence and for PII-specific sequence of aromatase P450.

The level of aromatase P450 expression showed wide variability among nodules, and the promoter use was different between nodules even from the same individuals. Thus, the results for all leiomyoma nodules (n = 21) were equally included in the analyses regardless of individuals. Of 21 nodules tested, aromatase P450 expression was detected by RT-PCR specific for coding sequence in 20 nodules. Sixteen of the 20 nodules were exclusively positive for I.4-specific transcript and were negative for PII-specific transcript. The remaining 4 leiomyoma nodules were positive for both PII-specific transcript and I.4-specific transcript simultaneously; 2 of them contained much more abundant I.4-specific transcript than PII-specific transcript, with the converse true in the remaining 2. There was no nodule that expressed PII-specific transcript alone. Accordingly, 18 of 20 nodules contained much more I.4-specific transcript than PII-specific transcript. The average and SEM of exon 1-specific mRNA levels are shown in Fig. 3Go (two right columns), and the difference between I.4-specific transcripts and PII-specific transcript was statistically significant (P < 0.05) in leiomyoma tissues. This result demonstrated that promoter I.4 was predominantly used in the most leiomyoma nodules. For comparison, we determined the promoter use of aromatase P450 in the myometrium surrounding leiomyoma nodules. As shown in Fig. 3Go (two left columns), myometrium contained much more I.4-specific transcript than PII-specific transcript, although this difference failed to reach statistical significance (P = 0.11). No correlation was observed between promoter use and specific histological features.



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  		Figure 3. Levels of transcripts containing I.4- and PII-specific sequence in leiomyoma and surrounding myometrium. Data represent the mean ± SEM of 21 leiomyomas obtained from 13 patients and 7 myometrium obtained from 7 patients. Myometrial samples were obtained at hysterectomy from normal-appearing myometrium surrounding leiomyoma nodule, more than 2 cm away from the leiomyoma capsule. *, P < 0.05 compared with the PII-specific transcripts of the same group (by Wilcoxon signed-rank test).

 
Inducibility of aromatase activity by Dex and IL-1ß, (Bt)2cAMP, and PGE2 in leiomyoma cells

In various cells and tissues, specific factors and their combinations up-regulate the expression of aromatase P450 through alternative use of corresponding promoters. A combination of Dex and IL-1ß increases the transcription of aromatase P450 through promoter I.4 in all cells in which this has been examined, such as adipose stromal cells, osteoblast-like cells, skin fibroblasts, and THP-1 cells (31, 32, 33). On the other hand, PGE2 and cAMP have been known to be the most potent inducers of promoter II-driven transcription of aromatase P450 in cells such as adipose stromal cells, endometrial stromal cells, and granulosa cells (34, 35, 36).

To determine which promoter is more active in leiomyoma cells in terms of aromatase activity, we treated leiomyoma cells with these stimulatory factors and compared the induced levels of aromatase activity. As shown in Fig. 4Go, combined treatment with Dex and IL-1ß for 24 h increases aromatase activity 20-fold compared with that in the nonstimulated control, whereas the increase induced by PGE2 and (Bt)2cAMP was 3-fold at best. Competitive RT-PCR analysis confirmed exclusive use of promoter I.4 in Dex- and IL-1ß-stimulated leiomyoma cells (Fig. 5Go, B and C). These results again support the idea that transcription of aromatase through promoter I.4 is dominant in leiomyoma cells. In the experiment shown in Fig. 5Go, we also determined the effect of Dex alone on the level of aromatase mRNA. As shown in Fig. 5BGo, Dex alone induced a small, but statistically significant, increase in the level of I.4-specific transcripts. The increase was reproducible even when cells were cultured in serum-free medium after extensive washing in PBS. In contrast, IL-1ß alone did not increase the level of mRNA aromatase in the absence of Dex.



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  		Figure 4. Aromatase activity of leiomyoma cells treated with combinations of several stimulants. Leiomyoma cells plated in 12-well plates were preincubated in medium containing 0.2% BSA for 4–6 h and stimulated with PGE2 (100 nM), (Bt)2cAMP (50 µM) plus 3-isobutyl-1-myethylxanthine (IBMX; 100 µM), or IL-1ß (1 ng/ml) plus Dex (25 nM) for 24 h. Six hours before the end of incubation, [1ß-3H]androstenedione was added to the medium, and incorporation of tritium into the water phase was measured as described in Materials and Methods. Results are the mean ± SEM of three independent experiments. *, P < 0.05 compared with the control group (by Wilcoxon signed-rank test).{dagger}, P < 0.05 compared with PGE2-treated group and (Bt)2cAMP- plus IBMX-treated group.

 


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  		Figure 5. Effect of IL-1ß, Dex, or their combination on aromatase activity (A), I.4-specific transcript (B), and PII-specific transcript (C). Serum-starved (6 h) leiomyoma cells in 12-well plates were incubated for 24 h in serum-free medium in the presence of Dex (25 nM), IL-1ß (1 ng/ml), or a combination thereof. Aromatase activity was measured as described in Materials and Methods. The levels of transcripts were determined by exon 1-specific RT-PCR on cell lysates from 9-cm dishes in the collateral experiment. Data are the mean ± SEM of three independent experiments.

 
Next we tested the effect of intrinsic stimulatory factors on aromatase activity using the supernatant of homogenates obtained from leiomyoma nodules. As shown in Fig. 6Go, addition of the supernatant mimicked IL-1ß in the presence of Dex, but not in the absence of Dex. The necessity for Dex is again suggestive of the use of promoter I.4. Promoter I.4-driven transcription was confirmed by exon 1-specific RT-PCR (data not shown).



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  		Figure 6. Aromatase activity of leiomyoma cells treated with homogenates of leiomyoma tissue (B). Leiomyoma tissue was homogenized in cold phosphate buffer and centrifuged at 20,000 x g for 15 min, and the supernatant was used for the experiment. Leiomyoma cells were prepared as described above, and 50 µl supernatant were added with or without Dex (25 nM). After 24 h of stimulation, aromatase activity was measured. For control experiments, supernatants from tissue-free digestion medium were used. Results are the mean ± SEM of two independent experiments.

 
Transcriptional activity of promoter I.4 sequence

To confirm that promoter I.4 sequence is transcriptionally active in leiomyoma cells, luciferase vectors containing promoter I.4 sequence were transfected into leiomyoma cells. After recovery in complete medium, cells were treated with Dex for 24 h, and luciferase activity was measured. As shown in Fig. 7AGo, P-450-I.4(-340/+14), which contains a putative GRE, showed a 9-fold increase in luciferase activity in response to Dex, whereas P-450-I.4(310/+14), which lacks the GRE, did not. The transcriptional activity of the promoter I.4 sequence and the GRE within it was also examined in KW cells. A series of constructs containing various lengths of promoter I.4 sequence was transfected into KW cells and treated with Dex for 24 h. As shown in Fig. 7BGo, the P-450-I.4(-340/+14, or longer) constructs that contain the putative GRE responded to 25 nM Dex, whereas shorter constructs (-310/+14 or shorter) lacking the GRE did not. The function of the putative GRE was further confirmed using a GRE mutant of promoter I.4, P-450-I.4(-340/+14)mGRE. As shown in Fig. 7CGo, mutation of the putative GRE site abolished the responsiveness to Dex. Collectively, promoter I.4 sequence (-340/+14 or longer) was transcriptionally active in leiomyoma cells, and the GRE site was functioning. The presence of the GR was confirmed by Western blotting in leiomyoma cells and KW cells (data not shown).



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  		Figure 7. Transient transfection assay of constructs containing the 5'-flanking sequence of exon I.4 in leiomyoma cells (A) and KW cells (B and C). A, P-450-I.4(-340/+14) and P-450-I.4(-310/+14) were transfected into leiomyoma cells as described in Materials and Methods. After 6-h recovery from transfection, cells were treated with Dex (25 nM) for 16 h, and luciferase activity was measured. Data are the mean ± SEM of triplicate samples from one experiment of three independent experiments that yielded essentially similar results. *, P < 0.05 compared with the corresponding control. B, A series of deletion constructs of promoter I.4 were transfected into KW cells as described in Materials and Methods. After 24-h incubation for recovery, cells were treated with Dex (25 nM) for 24 h, and luciferase activity was measured. Data are mean ± SEM of triplicate samples from one experiment of three independent experiments that yielded similar results. *, P < 0.05 compared with the corresponding control. A diagram of putative binding sites of transcription factors in I.4 sequence is included. C, P-450-I.4(-340/+14) and P-450-I.4(-340/+14)mGRE were transfected into KW cells, and luciferase activity was assayed as outlined above. P-450-I.4(-340/+14)mGRE carries a GRE mutation (AGAAGATTCTGTTCT to AGAAGATTCTGgctT). Data are the mean ± SEM of triplicate samples from one experiment of three independent experiments that yielded similar results.

 
Discussion

Many extragonadal tissues, including leiomyoma of the uterus, which used to be considered merely as targets of estrogen have recently been shown to possess the ability to metabolize circulating androgen into active estrogen (37). The amount of estrogen synthesized in these extragonadal sites is usually too small to increase the serum level of estrogen, but is sufficient to exert significant biological influence on the tissue in situ (37, 38). One possible explanation is that the local tissue concentrations of estrogen in that compartment become so high that in situ estrogen acts on its own cell or on nearby cells in an autocrine and paracrine fashion. Moreover, estrogen may act directly on its own cell through binding to the ER of the same cell without diffusion outside the cell and without inactivation by binding to SHBG, namely in an intracrine fashion. Recent studies have focused on the roles of these extragonadal estrogens in health and disease and revealed unanticipated roles of local estrogen (37, 39). Breast cancer is a good example in which the pathological role of local estrogen synthesis has been well documented (40, 41, 42, 43, 44, 45, 46, 47). Aromatase P450 is overexpressed, and the local production of estrogen is elevated in breast cancer tissue, in particular in adipose tissue surrounding the cancer (40, 41, 42, 43, 44, 45, 46, 47). Local estrogen production plays an important role in the progression of breast cancer in postmenopausal women and oophorectomized women and is a target of hormonal therapy by aromatase inhibitors (48, 49). Interestingly, overexpression of aromatase P450 is realized through the promoter switching of aromatase P450 from an adipose/skin type promoter, promoter I.4, to a gonadal type of promoter, promoters II and I.3 (50, 51). Identification of the mechanism of how this promoter switching accompanies malignant transition is a major focus of current research (34, 51, 52, 53).

Recently, we and other researchers have shown that leiomyomas overexpress aromatase P450 and that leiomyoma cells in culture actually synthesize significant amounts of estrogen from androgen, which promotes their own cell growth in vitro (13, 14). As in the case of breast cancer, local production of estrogen most likely plays a role in the promotion of leiomyoma cell growth over that of the surrounding myometrium in vivo (14, 17). As described above, GnRH agonist abolished ovarian estrogen as well as in situ estrogen simultaneously, causing leiomyomas to show rapid and profound shrinkage (17). Moreover, the administration of small doses of endocrine estrogen (estrogen add-back) does not induce regrowth of leiomyoma cells during GnRH agonist therapy, as in situ estrogen remains virtually ablated (24). These findings indicate the relative importance of in situ estrogen over ovarian (endocrine) estrogen in leiomyoma growth under these conditions.

In the present study we determined the promoter use of aromatase P450 in leiomyoma nodules. Based on the results of complementary approaches, we concluded that promoter I.4 is the major promoter of aromatase P450 responsible for overexpression of aromatase P450 in leiomyoma tissue. Unlike breast cancer, promoter switching was not detected in leiomyoma nodules. Both leiomyoma nodules and myometrium bearing them used mainly promoter I.4, followed by promoter II. In a previous study promoter II was found to be more predominantly used than promoter I.4 in leiomyoma tissue (13), a discrepancy that may be explained by the comparatively larger number of specimens and/or the fully quantitative approaches employed in the present study.

We next attempted to determine the mechanism by which transcription from promoter I.4 is overdriven in leiomyoma cells. We found compelling evidence for a role for the GRE in transcription through promoter I.4. An apparent EC50 for Dex was 2 nM in leiomyoma cells in terms of aromatase activity (data not shown). Given that cortisol is 1/20th as potent as Dex, the circulating level of cortisol in normal subjects (100–400 nM) is probably sufficient for sustained activation of the transcription through the GRE. Thus, a factor(s) other than cortisol that acts in synergy with cortisol must be a major determinant(s) of transcriptional levels of aromatase P450 in situ. This up-regulatory factor(s) is most likely a local factor(s) that is secreted and acts in situ, as surrounding myometrium, which is able to use the same promoter (promoter I.4) of aromatase P450, does not overexpress aromatase. This factor(s) was apparently lost in the cell culture techniques employed in the present study. Once cells are cultured in vitro, the level of aromatase transcripts in leiomyoma cells drastically decreases (13, 14). The level of aromatase transcripts in normal myometrium, 1/10–1/20th of that in leiomyoma, also decreases when cells are maintained in vitro, and then no difference in the level of aromatase transcript is found between primary cells obtained from leiomyoma tissue and those from surrounding myometrium (17). Conversely, the decreased level of aromatase P450 transcript in cultured cells quickly returns to the level comparable to that in myometrial tissues in vivo in the presence of IL-1ß and Dex (17). These observations fit well with the idea that a transcriptional level of aromatase P450 is maintained by local up-regulatory factor(s) and that that factor(s) is present to a much higher degree in leiomyoma than in myometrium. In fact, we found that homogenate of leiomyoma tissue contained some humoral factor(s) that increased aromatase activity in the presence of Dex. Among the cytokines, IL-1ß is a candidate for these up-regulatory factors, as IL-1ß is secreted from leiomyoma cells or from another cell component(s) found in leiomyoma nodules. Myometrium per se has been shown to synthesize and secrete IL-1{alpha} and IL-1ß in response to serotonin and lipopolysaccharide (54, 55). Some leiomyomas contain numerous mast cells compared with homologous myometrium (56, 57). Mast cells secrete IL-1ß directly, and more likely stimulate IL-1ß synthesis of leiomyoma cells via serotonin release (54). Therefore, IL-1ß may be a physiological up-regulatory factor of aromatase P450 in leiomyoma cells. The relationship between aromatase P450 mRNA levels and IL-1ß is currently under investigation.

IL-1ß has been reported to be a potent stimulator of aromatase P450 in adipose tissue and human osteoblast-like cells (31, 32, 33). In these cells IL-1ß induces the transcription of aromatase P450 primarily through promoter I.4 (32), as in the leiomyoma cells shown here. However, the mechanism of transcriptional activation is unknown. There are several putative nuclear factor-{kappa}B-binding sites in promoter I.4, which may mediate a signal downstream of IL-1ß. Cis-acting elements remain to be identified.

In the experiment shown in Fig. 5Go, some dissociation in the induced level of mRNA and aromatase enzyme activity was observed. Dex alone resulted in an increase in aromatase mRNA levels without a corresponding increase in enzyme activity. In contrast, coadministration of IL-1ß provoked a profound increase in the Dex-induced level of enzyme activity compared with the increase in the mRNA level. This dissociation may be due to an as yet unidentified posttranslational modulation of the enzyme activity of aromatase P450, as previously proposed (58, 59). This possibility is currently under investigation.

In conclusion, we demonstrated that promoter I.4 is the main promoter used for the expression of aromatase P450 in leiomyoma tissue, and that a putative GRE within promoter I.4 is essential for promoter activity. Given the role of in situ estrogen in leiomyoma growth, inhibition of in situ expression of aromatase is a possible adjuvant therapy of conservative management, as in the case of breast cancer. Selective aromatase inhibitors are a choice for treatment under suppression of the hypothalamus-pituitary-ovarian axis. The suppression of the axis is then obligatory; otherwise, hypoestrogenemia induced by aromatase inhibitors reactivates the axis, which, in turn, increases ovarian estrogen. The other possible strategy targeting in situ aromatase is disruption of an up-regulatory factor(s) of aromatase P450 specific for leiomyoma cells, if it exists. For this reason, further investigation is required to identify transcriptional factors/mechanisms responsible for promoter I.4-driven overexpression of aromatase P450 in leiomyoma nodules.

Acknowledgments

Footnotes

This work was supported by Grants-in-Aid for Scientific Research (B12557136, B13470348, and C11671602) from the Ministry of Education, Science, Sports, and Culture of Japan.

Abbreviations: Dex, Dexamethasone; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; GRE, glucocorticoid-responsive element; 5'-RACE, 5'-rapid amplification of cDNA ends.

Received November 15, 2001.

Accepted February 12, 2002.

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