Margaret M. Hinshelwood

Mechanisms in tissue-specific regulation of estrogen biosynthesis in humans.

In humans, aromatase P450, which catalyses conversion of C(19)-steroids to estrogens, is expressed in several tissues, including gonads, brain, adipose tissue, skin and placenta, and is encoded by a single-copy gene (CYP19); however, this does not hold true for all species. The human gene is approximately 130 kb and its expression is regulated, in part, by tissue-specific promoters and by alternative splicing mechanisms. Using transgenic mouse technology, it was observed that ovary-, adipose tissue- and placenta-specific expression of human CYP19 is directed by relatively small segments of DNA within 500 bp upstream of each of the tissue-specific first exons. Thus, the use of alternative promoters allows greater versatility in tissue-specific regulation of CYP19 expression. Characterization and identification of transcription factors and crucial cis-acting elements within genomic regions that direct tissue-specific expression will contribute to improved understanding of the regulation of CYP19 expression in the tissues that synthesize estrogens under both physiological and pathophysiological conditions.

Cytochromes P450 11: expression of the CYP19 (aromatase) gene: an unusual case of alternative promoter usage.

Family 19 of the P450 super family is responsible for the conversion of C19 androgenic steroids to the corresponding estrogens, a reaction known as aromatization because it involves conversion of the Δ4‐3‐one A‐ring of the androgens to the corresponding phenolic A‐ring characteristic of estrogens. The gene encoding human aromatase has been cloned and characterized and shown to be unusual compared to genes encoding other P450 enzymes, because there are numerous untranslated first exons that occur in aromatase transcripts in a tissue‐specific fashion due to differential splicing as a consequence of the use of tissue‐specific promoters. Thus, expression in the ovary uses a proximal promoter that is regulated primarily by cAMP. On the other hand, expression in the placenta uses a distal promoter located at least 40 kb upstream of the start of transcription that is regulated by retinoids. Other promoters are used in brain and adipose tissue. In the latter case, class I cytokines such as IL‐6 and IL‐11, as well as TNF‐α, are important regulatory factors. A common 3‘‐splice junction located upstream of the start of translation is used in all of the splicing events involved in the use of these various promoters. Thus, the coding region of the transcripts, and hence the protein, are identical regardless of the tissue site of expression; what differs in a tissue‐specific fashion is the 5‘‐end of the transcripts. This pattern of expression has great significance both from a phylogenetic and ontogenetic standpoint, as well as for the physiology and pathophysiology of estrogen formation, as will be discussed in thie review.—Simpson, E. R., Michael, M. D., Agarwal, V. R., Hinshelwood, M. M., Bulun, S. E., Zhao, T. Expression of the CYP19 (aromatase) gene: an unusual case of alternative promoter usage. FASEB J. 11, 29‐36 (1997)

Endocrine disorders associated with inappropriately high aromatase expression

Aromatase P450 (P450arom) is responsible for conversion of C19 steroids to estrogens in a number of human tissues, such as the placenta, gonads, adipose tissue, skin and the brain. Aromatase expression in human tissues is regulated by use of alternative promoters in the placenta (promoter I.1), adipose tissue (promoters I.4, I.3 and II) and gonads (promoter II). Aromatase expression is absent in the disease-free adult liver, adrenal and uterine tissues. Excessive or inappropriate aromatase expression in adipose fibroblasts and endometriosis-derived stromal cells, as well as in testicular, hepatic, adrenal and uterine tumors, is associated with abnormally high circulating estrogen levels and/or with increased local estrogen concentrations in these tissues. Whether systemically delivered or locally produced, elevated estrogen levels will in turn promote the growth of hormone-responsive tissues. We recently studied aromatase expression in testicular tumor and adipose tissue samples from prepubertal boys with gynecomastia, in hepatocellular cancer and adrenocortical tumor samples from adult men with gynecomastia, in breast adipose tissue samples proximal to breast tumors, and in endometrial cancer, leiomyoma and endometriosis tissues. Excessive aromatase activity and P450arom transcript levels were found in these tissue samples or in cultured cells derived from these tissues. In these neoplastic or non-neoplastic tissues or cells, the regulation of aromatase expression was studied in terms of alternative promoter use, both in vivo and in response to various hormonal stimuli. Our results were suggestive of a common metabolic abnormality associated with activation of a cyclic AMP-dependent signalling pathway that gives rise to transcriptional transactivation of aromatase expression via promoters I.3 and II in all of the above tissues. This article describes the common pathophysiological and molecular features of excessive aromatase expression in these disease states.

Expression of LRH-1 and SF-1 in the mouse ovary: localization in different cell types correlates with differing function.

Steroid biosynthesis in ovary is enhanced by the orphan nuclear receptor, steroidogenic factor-1 (SF-1); however, we reported that liver receptor homolog-1 (LRH-1), a closely related receptor to SF-1, is also expressed in mouse ovary. To further investigate the role of LRH-1 in mouse ovary, we used in situ hybridization to identify the cell types that express LRH-1 versus SF-1, and carried out functional studies to determine the role of LRH-1 in the regulation of the human (h) ovary-specific CYP19 promoter. LRH-1 expression was found to be abundant and highly restricted to cells involved in estrogen biosynthesis-granulosa cells during the estrous cycle, and in corpora lutea (CL) of pregnancy. In contrast, SF-1 was expressed most highly in C(19)-steroid-producing theca cells and interstitium, and at low levels in granulosa and luteal cells. Transfection studies using granulosa cells demonstrated that LRH-1 is a potent regulator of both basal and forskolin-induced transcription of the ovary-specific hCYP19 promoter. This activity was dependent upon two nuclear receptor half-sites within the proximal hCYP19 promoter. Based on these findings, we propose that LRH-1 plays an important role as a competence factor in regulating aromatase, and thus estrogen biosynthesis, in ovary.

Transcriptional regulation of aromatase in placenta and ovary.

Our goal is to define the cellular and molecular mechanisms for tissue- and cell-specific, developmental and hormonal regulation of the human CYP19 (aromatase P450/P450arom) gene in estrogen-producing cells. In this article, we review studies using transgenic mice and transfected cells to identify genomic regions and response elements that mediate CYP19 expression in placenta and ovary, as well as to define the molecular mechanisms for O2 regulation of differentiation and CYP19 gene expression in human trophoblast cells in culture. We also highlight recent findings regarding LRH-1 versus SF-1 mRNA expression and cellular localization in the mouse ovary during the estrous cycle and various stages of pregnancy. Spatial and temporal expression patterns of mRNAs encoding these orphan nuclear receptors in comparison to those of P450arom and 17alpha-hydroxylase/17,20-lyase mRNAs, suggest an important role of LRH-1 together with SF-1 in ovarian steroidogenesis.

Temporal and spatial expression of liver receptor homologue-1 (LRH-1) during embryogenesis suggests a potential role in gonadal development.

Liver receptor homologue‐1 (LRH‐1), an orphan member of the nuclear receptor family highly expressed in adult mouse ovary, is closely related to steroidogenic factor 1 (SF‐1), known to be important in gonadal formation. To analyze the potential role of LRH‐1 in gonadal differentiation, we compared LRH‐1 and SF‐1 expression during mouse embryonic and postnatal development. LRH‐1 expression was first detected in the urogenital ridge before sexual determination, in primordial germ cells and surrounding somatic cells; expression persisted after differentiation into testes and ovaries. Of interest, LRH‐1 expression declined in the developing ovary and testis at embryonic day 15.5 but increased again just after birth in the ovary in granulosa cells and transiently in oocytes of developing follicles. By comparing and contrasting LRH and SF‐1 expression with the two tissue‐specific steroidogenic markers, cytochromes P450 aromatase and P450 17α‐hydroxylase/17,20 lyase, we provide evidence for a potential role for LRH‐1 in gonadal development, the initiation of folliculogenesis and regulation of estrogen biosynthesis within the ovary. Developmental Dynamics 234:159–168, 2005.

The farnesoid X receptor regulates transcription of 3β-hydroxysteroid dehydrogenase type 2 in human adrenal cells

Recent studies have shown that the adrenal cortex expresses high levels of farnesoid X receptor (FXR), but its function remains unknown. Herein, using microarray technology, we tried to identify candidate FXR targeting genes in the adrenal glands, and showed that FXR regulated 3beta-hydroxysteroid dehydrogenase type 2 (HSD3B2) expression in human adrenocortical cells. We further demonstrated that FXR stimulated HSD3B2 promoter activity and have defined the cis-element responsible for FXR regulation of HSD3B2 transcription. Transfection of H295R adrenocortical cells with FXR expression vector effectively increased FXR expression levels and additional treatment with chenodeoxycholic acid (CDCA) caused a 25-fold increase in the mRNA for organic solute transporter alpha (OSTalpha), a known FXR target gene. HSD3B2 mRNA levels also increased following CDCA treatment in a concentration-dependent manner. Cells transfected with a HSD3B2 promoter construct and FXR expression vector responded to CDCA with a 20-fold increase in reporter activity compared to control. Analysis of constructs containing sequential deletions of the HSD3B2 promoter suggested a putative regulatory element between -166 and -101. Mutation of an inverted repeat between -137 and -124 completely blocked CDCA/FXR induced reporter activity. Chromatin immunoprecipitation assays further confirmed the presence of a FXR response element in the HSD3B2 promoter. In view of the emerging role of FXR agonists as therapeutic treatment of diabetes and certain liver diseases, the effects of such agonists on other FXR expressing tissues should be considered. Our findings suggest that in human adrenal cells, FXR increases transcription and expression of HSD3B2. Alterations in this enzyme would influence the capacity of the adrenal gland to produce corticosteroids.

Chapter 5 Tissue-specific expression of the CYP19 (aromatase) gene

Publisher Summary This chapter focuses on the tissue specific expression of the CYP19 (aromatase) gene. CYP19 is a member of the P450 superfamily of genes, which currently contains over 300 members in some 36 gene families. The physiological significance of estrogen biosynthesis in the placenta and adipose of humans is unclear at this time. The C 18 steroid produced in each tissue site of biosynthesis is quite tissue-specific. For example, the human ovary synthesizes primarily estradiol, whereas the placenta synthesizes estriol and adipose synthesizes estrone. This appears to reflect primarily the nature of the C 19 steroid presented to the estrogen-synthesizing enzyme in each tissue site. Estrogen biosynthesis by adipose tissue not only increases as a function of body weight but also as a function of age and has been correlated directly with the incidence of endometrial cancer as well as with postmenopausal breast cancer. While this may be, in part, the consequence of the bones of the latter being subject to load-bearing exercise, nonetheless it seems likely that the increased production of estrogens by the adipose of obese women is a significant factor.