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The Journal of Clinical Endocrinology & Metabolism Vol. 82, No. 1 70-74
Copyright © 1997 by The Endocrine Society

Experimental Studies

Alternatively Spliced Transcripts of the Aromatase Cytochrome P450 (CYP19) Gene in Adipose Tissue of Women1

Veena R. Agarwal2, Christy I. Ashanullah, Evan R. Simpson and Serdar E. Bulun

Cecil H. and Ida Green Center for Reproductive Biology Sciences, Departments of Obstetrics and Gynecology and Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9051

Address all correspondence and requests for reprints to: Dr. Veena R. Agarwal, Cecil H. and Ida Green Center for Reproductive Biology Sciences, Departments of Obstetrics and Gynecology and Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75235-9051.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Estrogen biosynthesis in adipose tissue has assumed great significance in terms of a number of estrogen-related diseases. The biosynthesis of estrogens from C19 steroids is catalyzed by a specific form of cytochrome P450, namely aromatase cytochrome P450 (P450arom; the product of the CYP19 gene). The human CYP19 gene comprises nine coding exons, II–X, and its transcripts are expressed in the ovary, placenta, testes, adipose tissue, and brain. Tissue-specific expression of the CYP19 gene is determined at least in part by the use of tissue-specific promoters, which give rise to transcripts with unique 5'-noncoding termini. Thus, the distal promoter I.1 is responsible for expression uniquely in placenta. On the other hand, the proximal promoter II, which regulates expression via a cAMP-dependent signaling pathway, is responsible for expression in the gonads. Transcripts in breast adipose tissue contain 5'-termini corresponding to expression derived from promoters I.4, II, and I.3, with I.4-specific termini predominating. The latter are derived from promoter I.4, which contains a glucocorticoid response element and an interferon-{gamma} activation site element and is responsible for expression in the presence of glucocorticoids and members of the class I cytokine family. The object of the present study was to determine the distribution of these various transcripts in adipose tissue from abdomen, buttocks, and thighs of women, as this would provide important clues to the factors regulating aromatase expression in these sites. To achieve this, we employed competitive reverse transcription-PCR to amplify unique 5'-ends of each of the transcripts of the CYP19 gene that are expressed in adipose tissue as well as for the coding region to evaluate total CYP19 gene (P450arom) transcript levels. We observed that exon I.4-specific transcripts were predominantly present in adipose tissue samples obtained from women regardless of the tissue site or the age of the individual. In these tissues, promoter II- and exon I.3-specific transcripts were present in lower copy numbers. We also demonstrated that in these sites total or exon-specific P450arom transcripts levels increased in direct proportion to advancing age and that transcript levels were the highest in buttocks, followed by thighs, and lowest in the abdomen. These results suggest that in normal human adipose tissue, aromatase expression is mainly under local control by a number of cytokines via paracrine and autocrine mechanisms in the presence of systemic glucocorticoids.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ESTROGENS have diverse actions at different body sites of women. Estradiol is produced in the ovarian granulosa cells of premenopausal women, whereas estriol and estradiol are secreted by the placenta (1). In both women and men, estrone is produced in the adipose tissue (2, 3, 4), and a substantial fraction of this estrone is further converted to estradiol in the periphery (5). Adipose tissue is the major site of estrogen biosynthesis in postmenopausal women (4, 6). Increased estrogen production in elderly obese women is believed to play a role in the pathogenesis of endometrial cancer (7). Furthermore, estrogen produced by adipose tissue within the breast may act locally to promote the growth of breast carcinomas (8, 9).

Estrogen biosynthesis is catalyzed by an enzyme known as aromatase cytochrome P450 (P450arom; the product of the CYP19 gene) (10, 11, 12). In the human, aromatase expression occurs in a number of human tissues and cell types, including the syncytiotrophoblast of the placenta (13), ovarian granulosa cells (14, 15), testicular Leydig cells (16, 17, 18), various sites in the brain (19, 20), as well as adipose tissue (3, 6, 21, 22, 23). Hemsell and co-workers first addressed the significance of human adipose tissue as a major source of estrogen production and demonstrated that in both women and men, there is a progressive increase in the efficiency with which circulating androstenedione is converted to estrone with advancing age (2). Subsequently, we have shown that with aging, there is an increase in the specific activity of the aromatase enzyme in adipose stromal cells, and we concluded that this may result in increased estrone production associated with aging (24, 25). Recently, we determined that this age-related increase in aromatase activity in adipose tissue is a result of increased levels of P450arom transcripts in various body sites of women, including buttocks, thighs, and abdomen (26). Moreover, expression was highest in buttocks, with lower levels of expression in thighs and abdomen.

The coding region of the CYP19 gene spans nine exons beginning with exon II, but 5'-termini (exon I) of aromatase transcripts differ from one another in a tissue-specific fashion (27). These 5'-termini correspond to untranslated exons spliced into the P450arom transcripts due to the use of tissue-specific promoters.

Analysis of P450arom transcripts in samples of breast adipose tissue revealed that in addition to exon I.4-specific transcripts, promoter II (PII)-specific as well as exon I.3 (I.3)-specific transcripts are present, albeit in lower copy number (28, 29). Interestingly, when adipose fibroblasts are placed in culture, the proportions of these exon-specific transcripts depend on the stimulatory factors present in the culture medium (28). Thus, when aromatase expression is stimulated by members of the class I cytokine family, such as IL-6 or IL-11, in the presence of glucocorticoids, the transcripts that are present are those derived from promoter I.4 (30). On the other hand, when expression is stimulated by dibutyryl cAMP in the presence or absence of phorbol esters, the transcripts that are present are those specific for PII and I.3 (28).

An important question then arises as to the nature of the 5'-termini (exon I) of the aromatase transcripts present in body sites other than the breast, as aromatase expression varies with age and in a site-specific fashion. In the present study we report determination of the various exon-specific aromatase transcripts in adipose tissue of buttocks, thighs, and abdomen obtained from normal women of various ages.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissue acquisition and processing

Our studies were carried out in 11 healthy women who ranged in age from 23–61 yr and in body weight from 50–84 kg. Body mass index [defined as body weight (kilograms)/height (meters)2] varied from 18–31 (Table 1Go). The average adult has a body mass index of 25. Subcutaneous fat samples (n = 33) from buttocks, thighs, and abdomen of these women were obtained by needle aspiration biopsies, as previously described (26). The samples (wet weight ranging from 0.3–0.4 g) were immediately placed in guanidinium thiocyanate solution, and RNA was isolated within 2 h (31). Written consent was obtained before all surgical procedures, employing a consent form and protocol approved by the institutional review board for human research of the University of Texas Southwestern Medical Center.


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  		Table 1. Clinical parameters of 11 normal women involved in this study

 
Reverse transcription-PCR (RT-PCR)

RT-PCR was performed according to a recently standardized competitive RT-PCR method that we developed for this purpose (32). RNA was initially treated with deoxyribonuclease I to remove any contaminating DNA. Total RNA was then reverse transcribed using random hexamers. Complementary DNA was used in subsequent PCR amplifications for 25 cycles. Specific sense 5'-end primers were used to amplify the various 5'-termini. The 3'-end primer was identical in all samples (32). A trace amount of [32P]deoxy-CTP was added to each sample. The reaction products were analyzed on 4% nondenaturing polyacrylamide gels, and radioactivity on the gels was visualized by exposure to x-ray film. To check the integrity and comparative quantity of RNA used in amplification of P450arom transcripts, transcripts of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a housekeeping gene, were amplified by the RT-PCR method as described previously (32). Specific transcript levels were expressed as arbitrary units normalized by GAPDH.

Statistical analyses

Data were analyzed on a VAX-8800 computer with the UTSTAT program (ACS Data Group, Dallas, TX). Regression analysis was performed to calculate simple correlation coefficient (Pearson’s R) to determine whether adipose tissue P450arom transcript levels correlated with the ages of the subjects. A two-way parametric repeated measures ANOVA was applied to determine whether there were significant differences between levels of specific transcripts (PII specific, I.3, and I.4) at each body site and between different body sites (buttocks, thighs, and abdomen). A Newman-Keuls multiple comparisons test was then applied to evaluate levels of significance for differences between individual groups.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Figure 1Go depicts a representative experiment employing three samples (buttocks, thighs, and abdomen) from one individual and shows the amplification of the coding region (total transcripts) and transcripts with three unique 5'-termini: PII, I.3, and I.4. The amplification products were of the expected size (coding, 194 bp; PII, 305 bp; I.3, 289 bp; I.4, 294 bp). The value in arbitrary units for each P450arom transcript level was obtained from the quantified radioactivity of the amplification products (Fig. 2Go). These values were normalized to total RNA quantity and to GAPDH amplification products in each sample. With advancing age, there was a progressive increase in total P450arom transcript levels in samples obtained from the buttocks, thighs, and abdomen. This age-dependent increase was statistically significant in the buttocks and thighs (correlation coefficients: Pearson’s R = 0.889; P < 0.002 and R = 0.817; P < 0.05 respectively; Fig. 2Go, A and B), whereas this linear trend (R = 0.704) did not reach statistical significance (P < 0.10) in the abdomen (Fig. 2CGo). The P450arom transcript levels were highest in the buttocks, followed by the thighs, and lowest in the abdomen. Statistically significant differences were found among these three body sites using parametric repeated measures ANOVA [p(F) < 0.001]. This was followed by a Newman-Keuls multiple comparisons test, which showed statistically significant differences between buttocks and thighs (P < 0.005) and buttocks and abdomen (P < 0.005). Although transcript levels in the thighs were higher than those in the abdomen, this difference did not reach a level of statistical significance.



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  		Figure 1. I) Amplification of coding region and specific 5'-termini of P450arom transcripts in RNA isolated from adipose tissue of women. Complementary DNA from 1 µg RNA was used for each reaction. The experiment shown is representative of several. A, Buttocks; B, thighs; C, abdomen. II) GAPDH amplification.

 


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  		Figure 2. Amplification of specific 5'-termini of P450arom transcripts in complementary DNA from 1 µg RNA isolated from different body sites of normal women. A, Buttocks; B, thighs; C, abdomen. Data are normalized to GAPDH transcripts. PII, PII-specific transcripts; I.3, I.3-specific transcripts; I.4, I.4-specific transcripts.

 
When the levels of the exon-specific transcripts (PII, I.3, and I.4) were added together, the sum was approximately equal to the measured quantity of total P450arom transcript levels (i.e. common coding region) in each case. This indicates that these three species accounted for essentially all of the P450arom transcripts present in the RNA (Fig. 2Go). I.4-specific transcripts comprised the majority of P450arom mRNA in all adipose tissue samples regardless of age and body site, followed by I.3-specific transcripts, and PII-specific transcripts were present in lowest quantities. A repeated measures parametric ANOVA revealed statistically significant differences among these three types of transcripts at each body site [p(F) < 0.001, 0.005, and 0.001 in buttocks, thighs, and abdomen, respectively]. A Newman-Keuls multiple comparisons test revealed statistically significant differences between I.4 and PII (P < 0.001, 0.025, and 0.001 in buttocks, thighs, and abdomen, respectively). The differences between I.4 and I.3 were significant (P < 0.005) in buttocks and abdomen, but not in thighs. Significant differences were also observed between PII and I.3 in all three body sites (P < 0.001, 0.025, and 0.001 in buttocks, thighs, and abdomen, respectively). With advancing age, there was a progressive increase in exon-specific transcripts in samples obtained from the buttocks, thighs, and abdomen (Fig. 2Go). The age-dependent increase was statistically significant for I.4- and I.3-specific transcripts in samples obtained from buttocks and thighs (Fig. 2Go, A and B). This linear trend, however, did not reach statistical significance for any exon-specific transcript in the abdomen (Fig. 2CGo). This may be due to the presence of low levels of P450arom transcripts in abdominal adipose tissue.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Recognition of the importance of adipose tissue as a source of estrogens in postmenopausal women came from the pioneering studies of MacDonald and co-workers in the 1970s, who demonstrated that the fractional conversion of androstenedione to estrone in humans increases as a function of obesity and aging (2, 4, 33). This parameter was shown to be correlated positively with excess body weight in both pre- and postmenopausal women and may be increased as much as 10-fold in morbidly obese postmenopausal women. Furthermore, the fractional conversion of androstenedione to estrone is also increased with aging. This increase with both obesity and aging bears a striking relationship to the incidence of endometrial cancer, which is a disease primarily of elderly obese women. It is now generally accepted that the continuous production of estrogen by the adipose tissue in such women is a major causative factor in the etiology of this condition. Evidence is also accumulating suggesting a role of estrogen produced by adipose tissue in the pathogenesis of breast cancer. A number of studies have attempted to relate tumor site to either aromatase activity or expression (8, 9, 29, 34, 35, 36, 37) in breast adipose tissue. In most cases (8, 9, 29, 34, 36), a direct relationship has been found between the presence of a tumor and aromatase expression in the tumor-bearing quadrant.

Previously, we have shown that the positive effect of aging on estrogen biosynthesis is due to a progressive increase in adipose tissue P450arom transcript levels in different body sites (buttocks, thighs, and abdomen) of normal women (26). Additionally, P450arom transcript levels were highest in the buttocks, followed by the thighs, and lowest in the abdomen. To gain insight into the factors that stimulate aromatase expression in adipose tissue, we recently studied the expression of the various exon-specific transcripts of P450arom in breast adipose tissue of cancer-free reduction mammoplasty patients and patients with breast cancer (29). We found that in breast adipose tissue of cancer-free patients, I.4-specific transcripts were present predominantly, whereas I.3-specific and PII-specific transcripts were present in lower copy numbers. Interestingly, in the breast adipose tissue of patients with cancer, promoter switching takes place. PII- and I.3-specific transcripts were present in high copy numbers compared to I.4-specific transcripts. In the present study, we observed that I.4-specific transcripts were present in highest copy numbers in adipose tissue of buttocks, abdomen, and thighs, whereas I.3-specific and PII-specific transcripts were present in low copy numbers. There was no difference in the expression pattern of exon-specific transcripts of P450arom with advancing age. Thus, it seems likely that the same promoters are being used for the expression of P450arom transcripts in adipose tissue regardless of the body site. This suggests that similar mechanisms of transcriptional regulation of the CYP19 gene are involved in its expression in adipose tissue of the buttocks, thighs, abdomen, and breast of women as well as in the age-dependent increase in expression.

It is well established that a number of cytokines are produced by adipose fibroblasts, such as interleukin-6, leukemia inhibitory factor, and interleukin-11 (38). Furthermore, tumor necrosis factor-{alpha} is believed to be produced by adipocytes (39, 40). These factors have been shown to stimulate aromatase expression via promoter I.4 in the presence of glucocorticoids in adipose fibroblasts (30) Thus, when adipose fibroblasts are placed in culture in the presence of one of these stimulatory factors plus dexamethasone, most of the P450arom transcripts expressed contain I.4 at the 5'-terminus. On the other hand, when expression is stimulated by dibutyryl cAMP in the presence or absence of phorbol esters or by PGE2, the transcripts present are those specific for PII and I.3. In fact, circulating IL-6 levels were shown to increase with advancing age (41). This explains at least in part why adipose tissue aromatase activity (25), P450arom transcript levels (26), and promoter I.4-specific transcripts (current study) also increase with age. We conclude that in normal human adipose tissue aromatase expression is mainly under local control by a number of cytokines via paracrine and autocrine mechanisms in the presence of systemic glucocorticoids.


   Acknowledgments
 
The authors gratefully acknowledge the skilled editorial assistance of Susan Hepner.


   Footnotes
 
1 This work was supported in part by USPHS Grant R37-AG-08174 and Texas Higher Education Coordinating Board ARP Grant 003660–046 (to E.R.S.), and by U.S. Army Medical Research and Development Command Grant DAMD17-94J-4188 and NCI Grant R29-CA-67167 (to S.E.B.). Back

2 Supported in part by USPHS Training Grant 5-T32-HD-07190. Back

Received July 3, 1996.

Revised August 28, 1996.

Accepted September 10, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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