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Aromatase Expression in Uterine Leiomyomata Is Regulated Primarily by Proximal Promoters I.3/II

Division of Reproductive Biology Research (A.G.I., Z.L., P.Y., S.D., B.Y., S.E.B.), Department of Obstetrics and Gynecology, Northwestern University, Chicago, Illinois 60611; and Department of Obstetrics and Gynecology (M.C., A.C.), Cumhuriyet University School of Medicine, 58140 Sivas, Turkey

Address all correspondence and requests for reprints to: Serdar E. Bulun, M.D., Department of Obstetrics and Gynecology, Northwestern University, 303 East Superior Street, Suite 4-123, Chicago, Illinois 60611. E-mail: s-bulun{at}northwestern.edu.

	Abstract

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Context: Uterine leiomyomata are common tumors that cause irregular uterine bleeding and pregnancy loss and depend on estrogen for growth. Aromatase catalyzes the conversion of androgens to estrogens. Aromatase expression is regulated via alternatively used promoters in the placenta (I.1 and I.2a), fat (I.4, I.3, and II), bone (I.6), and gonads (II). A prostaglandin E₂/cAMP-dependent pathway regulates coordinately the proximal promoters I.3/II, whereas glucocorticoids and cytokines regulate the distal promoter I.4. Use of each promoter gives rise to a population of aromatase mRNA species with unique 5'-untranslated regions (5'-UTRs). Uterine leiomyoma tissue, but not normal myometrium, overexpresses aromatase leading to estrogen-stimulated cell proliferation. Aromatase inhibitor treatment shrank uterine leiomyomata in a few women.

Objective and Design: Promoter I.4 was reported to regulate aromatase expression in uterine leiomyomata from a group of Japanese women. Here, we used two independent techniques to identify the promoters that regulate aromatase expression in uterine leiomyomata (n = 30) from 23 African-American, Hispanic, and white women.

Results: Rapid amplification of 5'-cDNA ends of aromatase mRNA species revealed the following distribution of promoter usage in leiomyomata: promoters I.3/II, 61.5%; I.2a, 15.4%; I.6, 15.4%; and I.4, 7.7%. Real-time PCR, which quantifies mRNA species with promoter-specific 5'-UTRs, revealed the following distribution for each 5'-UTR as a fraction of total aromatase mRNA: I.3/II, 69.6%; I.4, 7.3%; and other promoters, 23.1%.

Conclusions: The primary in vivo aromatase promoter in leiomyoma tissues in non-Asian U.S. women is the prostaglandin E₂/cAMP-responsive I.3/II region. Alternative signals may stimulate aromatase expression that is a common biological phenotype in uterine leiomyomata.

	Introduction

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UTERINE LEIOMYOMATA, or fibroids, cause irregular uterine bleeding, pelvic pressure symptoms, and recurrent pregnancy loss and are the most common indication for the approximately 600,000 hysterectomies performed annually in the United States (1, 2). Symptomatic leiomyomata are detected in approximately 30% of reproductive-age women in the United States (3, 4). Uterine fibroids are three to nine times more common in African-American women than in white women (3).

Monoclonal proliferation of transformed smooth muscle cells of myometrium was proposed to give rise to leiomyomata (4). Leiomyoma growth is dependent on estrogen (5, 6). The ovary represents the most obvious source of estrogen, although other extraovarian sources may also contribute to estrogen production. Aromatase catalyzes the conversion of C₁₉ steroids to estrogens and is expressed physiologically in a number of human cells, including the ovarian granulosa cell, placental syncytiotrophoblast, testicular Leydig cell, and adipose fibroblast (7, 8). Aromatase activity in peripheral tissues, such as the adipose tissue and skin, gives rise to small but significant increases in circulating estrogen that may contribute to leiomyoma growth (8).

Leiomyoma tissue was found to contain strikingly higher levels of aromatase mRNA and activity and estrogen levels in vivo compared with surrounding myometrial tissue, and conversion of androstenedione to estrone occurs in leiomyoma tissues and leiomyoma smooth muscle cells but not in normal myometrial tissues or cells (3, 9, 10). It was also shown that local aromatase activity driving in situ estrogen production within leiomyoma tissue stimulated leiomyoma cell growth in an intracrine manner (5). The clinical significance of these observations was exemplified by recent reports that describe the shrinkage of uterine leiomyomata in women treated with aromatase inhibitors (11, 12, 13).

A single gene (CYP19) encodes aromatase, the inhibition of which effectively eliminates estrogen production in the entire body. The downstream 30-kb region comprises nine coding exons (II–X) (7, 8). The upstream 93-kb portion of the gene contains multiple promoters that direct transcription of alternative first exons giving rise to aromatase mRNA species with unique 5'-untranslated regions (5'-UTRs). The most proximal promoter (II) and two other proximal promoters, I.3 (used in adipose tissue and breast cancer) and I.6 (bone), are located within the 1-kb region upstream of the translation start site and may be coordinately regulated (8). Aromatase promoters active in the brain (I.f), endothelial cells (I.7), fetal tissues (I.5), fat (I.4), placenta minor (I.2a), and placenta major (I.1) are located approximately 33, 36, 43, 73, 78, and 93 kb, respectively, upstream of the translation start site in exon II (13), and a distinct signaling pathway regulates each promoter (14). For example, prostaglandin E₂ (PGE₂) or cAMP analogs coordinately activate promoters I.3/II, whereas a glucocorticoid plus a class I cytokine activate promoter I.4 (7). The unique 5'-UTR(s) at aromatase mRNA isolated from a tissue indicate the particular promoter(s) used in that tissue.

Determination of alternative aromatase promoter usage in an estrogen-dependent tissue provides critical biological information regarding the promoter-related signaling mechanisms that regulate aromatase expression in that particular tissue. In leiomyoma tissues (n = 21) from a population of Japanese women (n = 13), promoter I.4 was reported to primarily direct aromatase expression (15). We conducted the current study to determine the promoter usage in leiomyoma tissues (n = 30) from a larger population of non-Asian (African-American, Hispanic, and white; n = 23) women employing two independent methodologies. First, we analyzed aromatase mRNA species in leiomyoma tissue using an unbiased method rapid amplification of 5'-complementary DNA ends (5'-RACE) to sequence previously known or unknown promoter-specific 5'-UTRs. Then, we verified these promoter-specific aromatase mRNA species by TaqMan-based real-time PCR using cRNA standards.

	Subjects and Methods

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Tissue samples, RNA isolation, and 5'-RACE

Leiomyomata (n = 30) and normal myometria (n = 8) sampled 2 cm proximal to a leiomyoma were obtained at the time of surgery from 23 women undergoing hysterectomy or myomectomy at Prentice Women’s Hospital affiliated with Northwestern University. Patients were African-American (44%), Hispanic (30%), or white (26%). Control myometrial tissues were obtained from disease-free uteri of African-American (n = 2) and white (n = 3) women undergoing hysterectomy for benign disorders other than leiomyoma. All patients (age range, 31–45 yr) were premenopausal and were not receiving any hormonal therapy at the time of tissue collection. Menstrual cycles were estimated based on last menstrual periods and histological findings of the endometrium. Written informed consent was obtained from each patient before the surgical procedure as per a protocol approved by the Institutional Review Board of Northwestern University. Subjects with adenomyosis or endometriosis were excluded. Histologically confirmed leiomyomata measuring 3–8 cm in largest diameter were sampled consistently adjacent to the capsule. The two largest tumors were sampled in seven subjects with multiple leiomyomata.

Snap-frozen tissue samples (0.2–0.5 g) stored at –80 C were homogenized. RNA was extracted using TRIzol reagent (Invitrogen Corp., Carlsbad, CA) according to the manufacturer’s instructions. RNA was treated by RNase-free DNase (RQ1 RNase-Free DNase; Promega, Madison, WI) followed by phenol/chloroform extraction. The RNA concentration was determined spectrophotometrically. 5'-RACE was performed using SmartRACE cDNA Amplification Kit (BD Biosciences, Rockville, MD) following the instructions provided by the manufacturer with minor modifications (16, 17). The promoter-specific first exons that comprised the 5'-end of the aromatase transcripts were cloned, sequenced, and mapped to the human genome, as described previously (16, 17).

Real-time PCR

Levels of promoter-specific aromatase transcripts were measured by real-time PCR. In the RT reaction, RNA (5 µg) were reverse transcribed employing Superscript II enzyme (SuperScript First-Strand Synthesis System; Invitrogen) using oligo dT as the primer. Real-time PCR was carried out in triplicate using an ABI Prism 7900 apparatus (Applied Biosystems, Foster City, CA). cDNA corresponding to 240 ng of RNA input was amplified using 500 nM forward and reverse primers and 100 nM probes for the aromatase coding region, a promoter-specific aromatase mRNA species (promoters I.3, II, and I.4), and a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) reference gene (table published as supplemental data on The Endocrine Society’s Journals Online web site at http://jcem.endojournals.org). A specific 5'-primer and a specific probe were used for each transcript. The TaqMan primer pairs were designed to recognize two exons separated by one intron, whereas each probe was complementary to the exon-exon junction for additional specificity. See supplemental data for list of primers and probes.

PCR conditions were denaturing (50 C for 2 min) followed by a hold (95 C for 10 min) and amplification for 50 cycles (95 C for 15 sec and 60 C for 1 min). Each PCR product was sequenced for fidelity during the initial experiment. A specific standard curve for each promoter-specific aromatase mRNA was generated employing a serial of six dilutions of cRNA of each sequence of interest. Based on these curves, the levels of total and promoter-specific transcripts were calculated after normalization of the aromatase product to GAPDH.

We analyzed differences in percent total aromatase promoter utilization by 5'-RACE using the ² method and by real-time PCR using the Kruskal-Wallis ANOVA with post hoc Tukey test. A P < 0.05 was considered to be significant. Each experiment was repeated three times using tissues obtained from the same subject.

	Results

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Promoter usage by 5'-RACE

Using 5'-RACE, aromatase mRNA sequences were cloned in 19 of 30 leiomyoma samples (63%) from 18 subjects. This yield was expected because, in contrast to real-time PCR, 5'-RACE may not detect mRNA species in low copy number. The clinical parameters of patients and promoter use are listed in Table 1. The percentages of promoter-specific aromatase mRNA species in the total number of aromatase-positive samples were as follows: promoters I.3/II, 16 (61.5%); promoter I.2a, 4 (15.4%); promoter I.6, 4 (15.4%); and promoter I.4, 2 (7.7%) (Fig. 1A). Leiomyoma nodules did not show any significant difference in the expression of promoter-specific aromatase with respect to age, race, phase of menstrual cycle, and the size of nodule (Table 1) (18). In eight leiomyoma nodules, we amplified more than one promoter-specific aromatase mRNA sequence. No amplification of aromatase mRNA was observed in the myometrial samples from patients with leiomyoma or from disease-free uteri.

View larger version (45K): [in this window] [in a new window]   FIG. 1. A, Percentage of aromatase transcripts with promoter-specific 5'-untranslated ends (exon I) determined by 5'-RACE. Pr, Promoter; Pr I.3/II, sum of promoters I.3- and II-specific transcripts. a, P < 0.05, Pr I.3/II vs. Pr. I.2a or Pr I.6 or Pr I.4. B, Percentage of aromatase transcripts with promoter-specific 5'-untranslated ends determined by real-time PCR. Other Prs, Sum of promoters other than II, I.3, or I.4. a, P < 0.01, Pr. I.3/II vs. Pr I.4; b, P < 0.05, Pr. I.3/II vs. sum of other promoters.

Exon-specific real-time PCR assay was employed using internal cRNA standards to quantify the aromatase mRNA species containing II-, I.3-, and I.4-specific exons and total aromatase transcripts in all 30 leiomyoma samples. The usage of each promoter-specific aromatase transcript was reported as a percentage of total aromatase mRNA level that was determined independently (Fig. 1B). The median GAPDH-normalized moles of aromatase in 1 µg RNA were as follows: aromatase coding region, 53.6 (16.3–224); promoter II-specific, 31.7 (3.5–99.5); promoter I.3-specific, 12.3 (0.7–54.3); and promoter I.4-specific, 4.9 (0.7–27.6). The combined median levels of promoter II-specific, I.3-specific (69.6%), and I.4-specific (7.3%) transcripts comprised 76.9% of the median total aromatase mRNA level determined by amplification of the coding region. Thus, promoters II, I.3, or I.4 did not regulate transcription of 23.1% of aromatase mRNA species, and possibly other promoters (e.g. I.2a and I.6) accounted for these transcripts. Promoter II-, I.3-, and I.4-specific aromatase mRNA levels correlated highly and significantly with total aromatase mRNA levels (Pearson’s r = 0.949, P = 0.000029; r = 0.893, P = 0.000040; r = 0.943, P = 0.000001, respectively). Aromatase mRNA was not detected in myometrial samples from uteri with or without leiomyomata.

	Discussion

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Our findings reveal that the PGE₂-responsive promoter I.3/II region is primarily responsible for regulating aromatase expression in leiomyoma tissue from non-Asian subjects. Consistent with previous reports, we did not find any correlation between the utilization of tissue-specific aromatase promoters and leiomyoma size, localization, race (African-American, Hispanic, or white), or age.

Shozu et al. (15) previously reported that promoter I.4-specific mRNA (originally cloned from adipose and skin fibroblasts) was predominant in leiomyoma samples obtained from Japanese women. In contrast, we found a limited use of promoter I.4, which differs from promoters I.3/II in terms of its regulation, tissue specificity, and sequence, in leiomyomata from women in the United States. The discrepancy between these results and ours may be attributed to race-dependent differences, because our samples were obtained from African-American, Hispanic, and white patients, whereas all patients in the Japanese study were of Asian origin. It should be noted that all three (II, I.3, and I.4) promoter-specific mRNA levels correlated significantly with total mRNA levels. This suggests that all three promoters contribute to aromatase expression to varying degrees in most tumors.

A number of neoplastic tissues including cancers of the breast, endometrium, adrenal, and liver and endometriosis were shown to express high levels of aromatase compared with nonneoplastic tissue (8, 19). Thus, it is not surprising that promoters I.3/II also serve as the major regulators of aromatase expression in leiomyoma tissue (8, 19). In fibroblasts isolated from endometriosis and breast tumors, PGE₂ or cAMP analogs coordinately activate promoters II and I.3 located within 215 bp from each other (7, 8). In leiomyoma-derived smooth muscle cells, treatment with PGE₂ or dibutyryl cAMP robustly increased aromatase activity (3). These findings and our unpublished observations support the in vivo model that PGE₂ regulates aromatase expression via the promoter I.3/II region.

Several transcription factors including steroidogenic factor-1, liver receptor homologue-1, cAMP response element-binding protein-1, activating transcription factor-2, and CCAAT/enhancer binding protein-ß mediate cAMP or PGE₂-dependent activation of aromatase promoters II and I.3 in the ovary, endometriosis, and breast cancer (8). Future studies will investigate the transcription factors involved in regulation of aromatase expression in leiomyoma cells.

	Footnotes

 
This work was supported by grants from the National Institutes of Health (HD46260) and Friends of Prentice.

Disclosure: A.G.I., Z.L., P.Y., S.D., B.Y., M.C., and A.C. have nothing to disclose. S.E.B. consults for Serono and Novartis and received lecture fees from Serono.

First Published Online March 6, 2007

Abbreviations: GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; PGE₂, prostaglandin E₂; 5'-RACE, rapid amplification of 5'-complementary DNA ends; 5'-UTR, 5'-untranslated region.

Received November 13, 2006.

Accepted February 23, 2007.

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