Regulation Of Aromatase Research Articles

Aromatase and COX in breast cancer: Enzyme inhibitors and beyond

Aromatase expression and enzyme activity in breast cancer patients is greater in or near the tumor tissue compared with the normal breast tissue. Complex regulation of aromatase expression in human tissues involves alternative promoter sites that provide tissue-specific control. Previous studies in our laboratories suggested a strong association between aromatase ( CYP19) gene expression and the expression of cyclooxygenase ( COX) genes. Additionally, nonsteroidal anti-inflammatory drugs (NSAIDs) and COX selective inhibitors can suppress CYP19 gene expression and decrease aromatase activity. Our current hypothesis is that pharmacological regulation of aromatase and/or cyclooxygenases can act locally to decrease the biosynthesis of estrogen and may provide additional therapy options for patients with hormone-dependent breast cancer. Two pharmacological approaches are being developed, one involving mRNA silencing by selective short interfering RNAs (siRNA) molecules and the second utilizing small molecule drug design. In the first approach, short interfering RNAs were designed against either human aromatase mRNA or human COX-2 mRNA. Treatment of breast cancer cells with siAROMs completely masked the aromatase enzyme activity. Treatment with COX-2 siRNAs decreased the expression of COX-2 mRNA; furthermore, the siCOX-2-mediated decrease also resulted in suppression of CYP19 mRNA. The small molecule drug design approach focuses on the synthesis and biological evaluation of a novel series of sulfonanilide analogs derived from the COX-2 selective inhibitors. The compounds suppress aromatase enzyme activity in SK-BR-3 breast cancer cells in a dose and time-dependent manner, and structure activity analysis does not find a correlation between aromatase suppression and COX inhibition. Real-time PCR analysis demonstrates that the sulfonanilide analogs decrease aromatase gene transcription in breast cells. Thus, these results suggest that the siRNAs and novel sulfonanilides targeting aromatase expression may be valuable tools for selective regulation of aromatase in breast cancer.

Brain aromatase: Roles in reproduction and neuroprotection

It is well established that aromatization constitutes an essential part of testosterone's signaling pathway in brain and that estrogen metabolites, often together with testosterone, organize and activate masculine neural circuits. This paper summarizes the current understanding regarding the distribution, regulation and function of brain aromatase in mammals. Data from our laboratory are presented that highlight the important function of aromatase in the regulation of androgen feedback sensitivity in non-human primates and the possible role that aromatase plays in determining the brain structure and sexual partner preferences of rams. In addition, new data is presented indicating that the capacity for aromatization in cortical astrocytes is associated with cell survival and may be important for neuroprotection. It is anticipated that a better appreciation of the physiological and pathophysiological functions of aromatase will lead to important clinical insights.

Mechanisms in the regulation of aromatase in developing ovary and placenta

During human gestation, the placental syncytiotrophoblast develops the capacity to synthesize large amounts of estrogen from C 19-steroids secreted by the fetal adrenals. The conversion of C 19-steroids to estrogens is catalyzed by aromatase P450 (P450arom), product of the CYP19 gene. The placenta-specific promoter of the hCYP19 gene lies ∼100,000 bp upstream of the translation initiation site in exon II. In studies using transgenic mice and transfected human trophoblast cells we have defined a 246-bp region upstream of placenta-specific exon I.1 that mediates placental cell-specific expression. Using transgenic mice, we also observed that as little as 278 bp of DNA flanking the 5′-end of ovary-specific hCYP19 exon IIa was sufficient to target ovary-specific expression. This ovary-specific promoter contains response elements that bind cAMP-response element-binding protein (CREB) and the orphan nuclear receptors SF-1 and LRH-1, which are required for cAMP-mediated stimulation of CYP19 expression in granulosa and luteal cells during the estrous cycle and pregnancy. In this article, we review our studies to define genomic regions and response elements that mediate placenta-specific expression of the hCYP19 gene. The temporal and spatial expression of LRH-1 versus SF-1 in the developing gonad during mouse embryogenesis and in the postnatal ovary also will be considered.

Role of the progesterone receptor (PR) in the regulation of inflammatory response pathways and aromatase in the breast

There is convincing evidence to suggest that estrogen and inflammatory mediators play important roles in growth and progression of breast cancer. Moreover, local conversion of androgens to estrogens by aromatase (product of CYP19 gene) occurs in 70% of all breast cancers. The actions of aromatase in both the breast tumor and in surrounding adipose stromal and endothelial cells can result in high local levels of estrogen production that stimulate tumor growth. The efficacy of current endocrine therapies is predicted only if the tumor contains significant amounts of ER. Presence of PR in the tumor also is an important predictor of tumor aggressiveness and responsiveness to endocrine therapy. Immunoreactivity for aromatase in human breast tumors is highly correlated with that for cyclooxygenase 2 (COX-2), the rate-determining enzyme in prostanoid biosynthesis. COX-2 expression also is correlated with expression of HER-2/neu, an oncogene expressed in >30% of breast tumors. In this manuscript, we will review findings to suggest that induction of COX-2 by inflammatory cytokines acting through NF-κB contributes to the increase in CYP19 expression and breast cancer progression, and that PR plays a dominant protective role in breast cancer cells by antagonizing NF-κB activation of COX-2.

Aromatase Gene Expression and Regulation in the Female Rat Pituitary

Aromatase cytochrome P450, the key enzyme of estrogen biosynthesis from androgens, is encoded by CYP19. Its structure shows some peculiarities: exons II to X encode the protein, while multiple alternative exons I encode unique 5'-untranslated regions of the aromatase mRNA transcripts. Immunohistochemistry studies in the rat have shown that pituitary aromatase expression is sex-dependent and varies across the estrous cycle, suggesting that estrogens might be involved in the regulation of aromatase activity and might act locally as a paracrine or autocrine factor in the pituitary. In the present study, we used RT-PCR to characterize aromatase transcripts and real-time PCR to quantify the expression of the total aromatase mRNA at the different stages of the estrous cycle and from an ovariectomy and estradiol replacement model. We identified the two previously described aromatase transcripts with a specific 5'untranslated region of the brain 1f and the gonadal PII transcripts. Total aromatase mRNA expression in the pituitary varied significantly during the estrous cycle, with the highest level occurring on the day of metestrus. After ovariectomy, we observed an increase of aromatase mRNA levels, and this effect was completely prevented by estradiol administration. These results suggest that pituitary aromatase mRNA expression is downregulated by estrogens.

In Men, Peripheral Estradiol Levels Directly Reflect the Action of Estrogens at the Hypothalamo-Pituitary Level to Inhibit Gonadotropin Secretion

Estradiol inhibits gonadotropin release in men by an action at the hypothalamus and pituitary. Because of the tissue-specific regulation of aromatase, peripheral estradiol levels may not reflect brain estradiol concentrations. We evaluated whether local aromatization of testosterone in the hypothalamus or pituitary is important for gonadotropin release and to what extent circulating estrogens affect gonadotropin levels and peripheral testosterone levels. DESIGN, SUBJECTS, AND INTERVENTIONS: We suppressed aromatase activity in 10 young healthy men with letrozole 2.5 mg once daily, restored plasma estradiol levels with estradiol patches (100 microg/d for the first week, 50 microg/d the second week, 25 microg/d the third week, and no estradiol patch the fourth week) and measured plasma testosterone, estradiol, LH, FSH, and SHBG levels. The mean estradiol and testosterone levels during the study ranged between 68.6 +/- 38.3 and 12.6 +/- 7.21 pg/ml for estradiol and 179 +/- 91 and 955 +/- 292 ng/dl (mean +/- sd) for testosterone. Levels of testosterone, LH, and FSH were inversely related to peripheral estradiol levels. During letrozole use, the mean plasma estradiol level needed to restore testosterone, LH, and FSH levels to baseline levels was not significantly different from the baseline mean estradiol level. Local aromatization of testosterone in the hypothalamo-pituitary compartment is not a prerequisite for expression of the inhibitory action of estrogens on gonadotropin secretion in men. Peripheral estradiol levels directly reflect the inhibitory tone exerted by estrogens on gonadotropin release and are a major determinant of peripheral testosterone, LH, and FSH levels.

Feedback regulation of SREBP and aromatase in A beta(25-35)-supplemented human neuroblastoma cells.

1. The analogies between the processing of amyloid precursor protein (APP) and other transmembrane sterol regulatory element binding proteins (SREBPs) inspired us to conduct further studies on whether beta-amyloid (Abeta) affects aromatase by interacting with APP and SREBP. 2. In this study, cultured human neuroblastoma cells (SHSY-5Y) were incubated in experimental media (media without FBS, the main cholesterol source) in the presence or absence of Abeta (1 microM) for 24 h. 3. Cellular extracts were subjected to immunoblot analysis using anti-APP, anti-aromatase and anti-SREBP-1. In these cell lines, we detected aromatase (55 kDa), SREBP cleavage product (68 kDa) and APP precursor (100-95 kDa) and cleavage product (60 kDa) by immunoblotting. Aromatase and SREBP levels were elevated in the cells incubated 24 h in experimental media and were attenuated in Abeta-supplemented experimental media. 4. The disturbance of cholesterol homeostasis appears to be an important factor in the pathogenesis of Alzheimer's disease. These findings may have important implications for understanding the mechanisms of the aromatase enzyme gene in disease states such as Alzheimer's.

Sex steroids and leptin regulate 11β-hydroxysteroid dehydrogenase I and P450 aromatase expressions in human preadipocytes: Sex specificities

Adipose tissue is an important site of steroid hormone biosynthesis, as type I 11β-hydroxysteroid dehydrogenase (HSD1), the enzyme responsible for the conversion of cortisone into cortisol and the P450 aromatase, the enzyme catalysing androgens aromatization into estrogens, are both expressed in human adipose tissue. In the present report, we have investigated the possibility that sex steroids and leptin could regulate these two enzymes in cultured preadipocytes from men and women intra-abdominal fat depots. In women preadipocytes, human recombinant leptin down-regulates HSD1 mRNA expression (−58%) and P450 aromatase activity (−26%). Conversely, leptin up-regulates the HSD1 (2.4-fold) and the P450 aromatase (1.6-fold) mRNA expression in men preadipocytes. In women preadipocytes, 17β-estradiol strongly stimulates HSD1 mRNA expression (10-fold) and, in contrast, decreases by half the P450 aromatase expression. In men, 17β-estradiol has no influence on HSD1 expression but up-regulates P450 aromatase mRNA expression (2.4-fold). Finally, androgens increase by a factor of 2.5–5 the mRNA expression of both enzymes in men. These findings suggest that sex steroids and leptin either increase or decrease local cortisol and estrogens productions in men or in women preadipocytes, respectively. They also indicate that steroid metabolism in adipose tissue is controlled by a coordinated regulation of P450 aromatase and HSD1 expressions. Finally, the important sex-specific differences described herein may also contribute to explain the sexual dimorphism of body fat distribution in humans.

Effects of ovarian theca cells on granulosa cell differentiation during gonadotropin‐independent follicular growth in cattle

We investigated the effects of theca cells or FSH on granulosa cell differentiation and steroid production during bovine early follicular growth, using a co-culture system in which granulosa and theca cells were cultured on opposite sides of a collagen membrane. Follicular cells were isolated from early antral follicles (2-4 mm) that were assumed to be in gonadotropin-independent phase and just before recruitment into a follicular wave. Granulosa cells were cultured under serum-free conditions with and without theca cells or recombinant human FSH to test their effects on granulosa cell differentiation. Messenger RNA levels for P450 aromatase (aromatase), P450 cholesterol side chain cleavage (P450scc), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), LH receptor (LHr), and steroidogenic acute regulatory protein (StAR) in granulosa cells were measured by real-time quantitative RT-PCR analysis. FSH enhanced aromatase mRNA expression in granulosa cells, but did not alter estradiol production. FSH also enhanced mRNA expression for P450scc, LHr, and StAR in granulosa cells, resulting in an increase in progesterone production. In contrast, theca cells enhanced aromatase mRNA expression in granulosa cells resulting in an increase in estradiol production. Theca cells did not alter progesterone production and mRNA expression in granulosa cells for P450scc, 3beta-HSD, LHr, and StAR. The results of the present study indicate that theca cells are involved in both rate-limiting steps in estrogen production, i.e., androgen substrate production and aromatase regulation, and that theca cell-derived factors regulate estradiol and progesterone production in a way that reflects steroidogenesis during the follicular phase of the estrous cycle.

Endogenous testicular D-aspartic acid regulates gonadal aromatase activity in boar

D-aspartic acid (D-Asp), aromatase enzyme activity and the putative D-Asp involvement on aromatase induction have been studied in the testis of mature boars. The peroxidase-antiperoxidase and the indirect immunofluorescence methods, applied to cryostat and paraffin sections, were used to evaluate D-Asp and aromatase distributions. D-Asp level was dosed by an enzymatic method performed on boar testis extracts. Biochemical aromatase activity was determined by in vitro experiments carried out on testis extracts. D-Asp immunoreactivity was found in Leydig cells, and, to a lesser extent, in germ cells. Analogously, aromatase immunoreactivity was present in Leydig cells, but absent from seminiferous tubule elements. In vitro experiments showed that the addition of D-Asp to testicular tissue acetone powder induced a significant increase of aromatase activity, as assessed by testosterone conversion to 17beta-estradiol. Enzyme Km was not affected by D-Asp (about 25 nM in control and D-Asp added tests). These findings suggest that D-Asp could be involved in the local regulation of aromatase in boar Leydig cells and intervenes in this organ's production of estrogens.

Relationship between brain and ovary aromatase activity and isoform-specific aromatase mRNA expression in the fathead minnow ( Pimephales promelas)

There is growing evidence that some chemicals present in the environment have the capacity to inhibit, or potentially induce, aromatase activity. This study compared aromatase activities and isoform-specific mRNA expression in brain and ovary tissue from non-exposed fathead minnows representing three different ages and stages of reproductive activity, and from fathead minnows exposed to the aromatase inhibitor fadrozole for 7 d. The goal was to determine whether measures of a single aromatase endpoint in either brain or ovary tissue would be sufficient to understand and predict system-wide effects of endocrine disrupting chemicals on aromatase activity and transcript levels. Aromatase activity in the ovary, but not brain, varied significantly with age/reproductive category, with adults held in non-reproductive conditions showing significantly lower activity than juveniles and reproductively-active adults. Significant correlations between isoform-specific transcript levels and aromatase activity were observed for ovary tissue, but those relationships were not robust for all age/reproductive categories, nor were they sustained in fadrozole-treated fish. In vitro, fadrozole inhibited the aromatase activity of brain and ovary post-mitochondrial supernatants with similar potency (IC50s = 8.82 ± 1.58 and 6.93 ± 0.80 μM for brain and ovary, respectively), despite large differences in the magnitude of activity. In vivo, fadrozole altered aromatase activity and isoform-specific transcript levels in both brain and ovary tissue, but concentration-response relationships were different for each tissue. Aromatase activity and P450aromB mRNA expression in brain showed a dose-dependent decrease at concentrations greater than 5.55 μg/L. In contrast, ovary activity showed an inverted U-shaped concentration-response consistent with the interplay between increased P450aromA transcript levels in ovary and competitive inhibition of the aromatase enzyme. As a whole, results of this study did not reveal any robust correlations between brain and ovary aromatase activity and/or isoform-specific mRNA expression. However, they were consistent with the current body of evidence related to teleost aromatase regulation, suggesting that increased understanding of the biology of aromatase may facilitate system-wide understanding of effects on aromatase based on relatively few measured endpoints.

Endocrine management of breast cancer—biology and current practice

Breast cancer frequency is related to age. There are impressive advances in the diagnostic armament and surgical techniques of breast cancer, and yet, it has continued its deadly impact. In women with operable breast cancer, the histologic status of the axillary lymphnodes remains the most useful prognostic information. Today, breast cancer is viewed as a systemic disease with spreads to local and distant sites at the same time. There is a high rate of occult disease. Factors of major influence on breast cancer include reproductive experience, ovarian activity, benign breast disease, familial tendency, genetic differences, dietary considerations and specific endocrine factors. Tamoxifen still is the standard endocrine treatment for hormone-receptor-positive breast cancer both in the adjuvant and metastatic settings. Because of its weak oestrogenicity, tamoxifen may not be optimally effective and can increase the risk of endometrial cancer and stroke. Patients may also be refractory or become resistant to tamoxifen treatment. Aromatase inhibitors block the synthesis of oestrogen, have low intrinsic oestrogenic activity and, thereby, offer the potential of being more effective than tamoxifen. The challenge of the coming years is to identify women who are best treated with an aromatase inhibitor upfront rather than a tamoxifen-to-aromatase inhibitor sequence. Third-generation aromatase inhibitors offer greater benefits in oestrogen receptor (ER)-positive, progesterone-negative and HER-2 overexpressing tumours. Overall, no more than 2–3% of breast cancer occurrences can be attributed to the inherited mutation of either maternal or paternal origin. Autocrine and paracrine regulation of local oestrogen biosynthesis in normal and tumour breast tissue is via growth factors acting upon aromatase activity, which is preferably expressed in the tumour-bearing quadrant. Aromatase regulation operates against a concentration gradient of oestrogens, comparing peripheral plasma to local tissue levels in both premenopausal and postmenopausal women. Clinical observations suggest to routinely determine both ER subtypes and its variants, by which, the prediction of the response of the breast tumours to endocrine therapy may improve. Polymorphisms of cytochrome CYP-17, CYP-1A1 and COMT are found to be associated with an increased risk of breast cancer. To identify high-risk genotypes in women may delineate the individual at increased risk of breast cancer. Arguments in favour of an impact of postmenopausal oestrogen–progestin therapy on pre-existing tumours derive from observations in epidemiologic studies showing no increase in non-invasive breast cancer with hormone therapy; on the other hand, several studies point to a lower grade and earlier stage of disease in hormone users, with subsequently better survival rates. The older a postmenopausal woman, the greater her risk of developing an increase in breast density with hormone therapy. This is considered a good reason to recommend discontinuation of hormone therapy for at least 2 weeks prior to mammography in women older than age 65 who have dense breasts. As yet, there is no endocrine or biological evidence as to a sequential use of non-cross-resistant endocrine therapies. After progression on adjuvant and first-line tamoxifen, ovarian ablation is an appropriate second-line therapy. For premenopausal women with ovarian ablation, the use of a third-line therapy with an aromatase inhibitor appears feasible. In postmenopausal women, a wide choice of endocrine treatment options is available and an optimal sequence has yet to be determined. The widening range of adjuvant endocrine options represents an opportunity to prolong patient benefits in the treatment of hormone-receptor-positive breast cancer. Further refinement is currently investigated. For example, tamoxifen therapy followed by an aromatase inhibitor may lead to a reduction in endometrial pathologies associated with tamoxifen. New adjuvant treatments with chemotherapy or endocrine agents is being used increasingly to down-stage locally advanced and large operable breast cancers, which often become fully resectable; in addition, initially operable tumours requiring mastectomy may be successfully removed by breast-conserving surgery. Patient selection is important to optimise new adjuvant endocrine therapy; only patients with oestrogen-receptor-rich breast cancer are candidates, and postmenopausal women are likely to benefit the most.

Translational studies on aromatase, cyclooxygenases, and enzyme inhibitors in breast cancer

Aromatase expression and enzyme activity in breast cancer patients is greater in or near the tumor tissue compared with the normal breast tissue. Regulation of aromatase expression in human tissues is quite complex, involving alternative promoter sites that provide tissue-specific control. Previous studies in our laboratories suggested a strong association between aromatase ( CYP19) gene expression and the expression of cyclooxygenase ( COX) genes. Our hypothesis is that higher levels of COX expression result in higher levels of prostaglandin E 2 (PGE 2), which in turn increases CYP19 expression through increases in intracellular cyclic AMP levels. This biochemical mechanism may explain the beneficial effects of non-steroidal anti-inflammatory drugs (NSAIDs) on reducing the risks of breast cancer. The effects of NSAIDs (ibuprofen, piroxicam, and indomethacin), a COX-1 selective inhibitor (SC-560), and COX-2 selective inhibitors (celecoxib, niflumic acid, nimesulide, NS-398, and SC-58125) on aromatase activity and CYP19 expression were investigated in breast cancer cell culture systems. Dose-dependent decreases in aromatase activity were observed following treatment with an NSAID or COX inhibitor, with the most effective agents being COX selective inhibitors. Real time PCR analysis of aromatase gene expression showed a significant decrease in mRNA levels in treated cells when compared to vehicle control. These results suggest that the effect of COX inhibitors on aromatase occurs at the transcriptional level. To further probe these interactions, short interfering RNAs (siRNA) were designed against either human CYP19 mRNA or human COX-2 mRNA. Treatment of breast cancer cells with aromatase siRNAs suppressed CYP19 mRNA and aromatase enzyme activity. Finally, treatment with COX-2 siRNAs downregulated the expression of COX-2 mRNA; furthermore, the siCOX-2-mediated suppression of COX-2 also resulted in suppression of aromatase mRNA. In summary, pharmacological regulation of aromatase and cyclooxygenases can act locally in an autocrine fashion to decrease the biosynthesis of estrogen and may provide additional therapy options for patients with hormone-dependent breast cancer.

Analysis of spatiotemporal regulation of aromatase in the brain using transgenic mice

Brain aromatase is widely distributed in the vertebrates, from fish to mammals, and plays important roles in functional reproductive behavior through production of estrogen as a neurosteroid. It is expressed only in the nerve cells of specific brain regions with a transient peak in the neonatal period when sexual behavior becomes organized, and therefore provides a good model system to study regulatory mechanism of cell-specific, brain region-specific, and developmental stage-specific expression. To elucidate spatiotemporal regulation of brain aromatase, we prepared transgenic mice carrying a reporter gene under the promoter of brain-specific exon 1f of the mouse aromatase gene. The reporter transgene carrying a 6.5 kb upstream region of the brain-specific promoter accurately reproduced the spatiotemporal expression patterns of aromatase in mouse brain, whereas transgenes carrying smaller fragments of the promoter showed ambiguous or inconsistent expression patterns. The binding sites of Aro-AI, Aro-AII, and Aro-B for nuclear factors were also identified in the proximal region of the exon 1f brain-specific promoter. Introduction of a mutation into the Aro-AII site in the reporter transgene carrying −6.5 kb promoter region of exon 1f caused complete alteration of the spatiotemporal expression pattern of the reporter gene in the transgenic mice. These results indicate that the −6.5 kb promoter region of exon 1f is the minimal essential element for brain-specific regulation, with both proximal and distal promoter regions required for accurate spatiotemporal expression of aromatase in the mouse brain.

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 O 2 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 17α-hydroxylase/17,20-lyase mRNAs, suggest an important role of LRH-1 together with SF-1 in ovarian steroidogenesis.

Regulation of aromatase and 5α-reductase by 25-hydroxyvitamin D 3, 1α,25-dihydroxyvitamin D 3, dexamethasone and progesterone in prostate cancer cells

Estrogens and androgens are proposed to play a role in the pathogenesis of prostate cancer. The effective metabolites, estradiol and 5α-dihydrotestosterone are produced from testosterone by aromatase and 5α-reductase, respectively. Metabolites of vitamin D have shown to inhibit the growth of prostate cancer cells. The aim of the present study was to verify whether 25-hydroxyvitamin D 3 (25OHD 3), 1α,25-dihydroxyvitamin D 3 [1α,25-(OH) 2D 3], dexamethasone, and progesterone regulate the expression of aromatase and 5α-reductase in human prostate cancer cells. LNCaP and PC3 cells were treated with 25OHD 3, 1α,25-(OH) 2D 3, dexamethasone, or progesterone. Aromatase and 5α-reductase mRNA was quantified by real-time RT-PCR and aromatase enzyme activity was measured by the [ 3H] water assay. Aromatase enzyme activity in LNCaP and PC3 cells was increased by both 10 nM dexamethasone, 1–100 nM 1α,25-(OH) 2D 3 and 100 nM–10 μM progesterone. The induction was enhanced when hormones were used synergistically. Real-time RT-PCR analysis showed no regulation of the expression of aromatase mRNA by any steroids tested in either LNCaP or PC3 cells. The expression of 5α-reductase type I mRNA was not regulated by 1α,25-(OH) 2D 3 and no expression of 5α-reductase type II was detected in LNCaP.

Isolation of chicken homolog of the FOXL2 gene and comparison of its expression patterns with those of aromatase during ovarian development

Mutations in the forkhead transcription factor gene FOXL2 are involved in ovarian failure, which occurs in human BPES syndrome. This syndrome presents a sexually dimorphic expression, specific to the ovary in several vertebrates. We cloned the open reading frame of chicken FOXL2 (cFoxL2) and studied cFoxL2 expression in developing gonads and during adulthood to examine the role of FOXL2 in ovarian differentiation and function in birds. The spatial and temporal dynamics of cFoxL2 and aromatase expression were analyzed in parallel by using real-time quantitative reverse transcriptase-polymerase chain reaction and immunohistochemistry in attempt to investigate the possible role of cFoxL2 in the regulation of aromatase. The expression patterns of cFoxL2 and aromatase transcripts were highly correlated during the sex-differentiation period (4.7-12.7 days of incubation). Aromatase and cFoxL2 proteins were colocalized in the medullar part of female gonads on embryonic day 14. Fourteen days after hatching, cFoxL2 protein was mainly detected in granulosa cells of developing follicles. In adult ovary follicular envelopes, apart from granulosa cells, cFoxL2 transcript and protein were detected at lower levels in theca cells where aromatase was present. A high level of cFoxL2 transcription was also observed in maturing and ovulated oocytes. Our results confirm that FoxL2 is an early regulator of ovarian development in birds and may be involved in aromatase transcription regulation.

Activation of peroxisome proliferator-activated receptor-gamma and retinoid X receptor inhibits aromatase transcription via nuclear factor-kappaB.

Our previous studies demonstrated that a peroxisome proliferator-activated receptor (PPAR)-gamma ligand, troglitazone (TGZ),and/or a retinoid X receptor (RXR) ligand, LG100268 (LG), decreased the aromatase activity in both cultured human ovarian granulosa cells and human granulosa-like tumor KGN cells. In the present study, we further found that a combined treatment of TGZ+LG decreased aromatase promoter II (ArPII) activity in both ovarian KGN cells and fibroblast NIH-3T3 cells in a PPARgamma-dependent manner. Furthermore, the inhibition of both aromatase activity and the transcription of ArPII by TGZ+LG was completely eliminated when nuclear factor-kappaB (NF-kappaB) signaling was blocked by specific inhibitors, suggesting NF-kappaB, which is endogenously expressed in both fibroblast and granulosa cells, might be a mediator of this inhibition. Interestingly, activation of NF-kappaB by either forced expression of the p65 subunit or NF-kappaB-inducing kinase up-regulated ArPII activity. Positive regulation of aromatase by endogenous NF-kappaB was also suggested by the fact that NF-kappaB-specific inhibitors suppress basal activity of the aromatase gene. A concomitant formation of high-order complex between NF-kappaB p65 and ArPII was also observed by chromatin immunoprecipitation assay. Although activation of PPARgamma and RXR affected endogenous expression levels of neither inhibitory kappaBalpha nor p65, it impaired the interaction between NF-kappaB and ArPII and the p65 based transcription as well. Altogether, these results indicate that activation of a nuclear receptor system, constituted by PPARgamma and RXR, down-regulates aromatase expression through the suppression of NF-kappaB-dependent aromatase activation and thus provide a new insight in the mechanism of regulation of the aromatase gene.

A comparison of human H295R and rat R2C cell lines as in vitro screening tools for effects on aromatase

In this study we evaluated the rat Leydig cell carcinoma cell line R2C and the human adrenocorticocarcinoma cell line H295R for their suitability as in vitro screening tools for potential interference of xenobiotics with aromatase activity. A 24 h exposure to prochloraz (PRO), fadrozole (FAD) and epoxyconazole (EPO) resulted in complete catalytic inhibition in H295R and R2C cells. In H295R cells, PRO and FAD were mixed-type inhibitors with apparent K i values of 0.04 and 0.03 μM, and apparent K i ′ values of 0.33 and 0.06 μM, respectively. EPO was a competitive inhibitor with an apparent K i value of 0.51 μM. In R2C, all three compounds showed mixed type inhibition kinetics, with apparent K i values (μM) of 0.004, 0.003 and 0.07, and apparent K i ′ values (μM) of 0.41, 0.01 and 2.42, respectively. Exposure for 24 h of H295R cells to 8-bromo-cyclic adenosine monophosphate (8-Br-cAMP), prostaglandin E2 (PGE2), phorbol 12-myristate 13-acetate (PMA), or dexamethasone (DEX) resulted in 4.0, 2.8, 3.6 or 3.6-fold induction of aromatase activity, respectively, as well as in an increase of several human aromatase transcripts with promoter-specific 5′-ends (pII and I.3). In R2C cells, only PMA slightly induced aromatase activity. A 24 h exposure of H295R cells to atrazine (ATR), resulted in a three-fold induction of aromatase activity and a slight increase in pII and I.3 aromatase transcripts. However, ATR did not induce aromatase activity in R2C cells. We conclude that the H295R cell line contains aromatase promoter regions, which are responsive to the respective stimulants. They play a role in aromatase regulation in various tissues such as brain, placenta, healthy and diseased gonadal and breast tissue and therefore, they may play an important role in tumor genesis, development, behavior and reproduction. The H295R cell line may therefore be a relevant and useful tool in risk assessment of xenobiotics. The R2C cell line, although not suitable for studying induction, appears to be a more sensitive cell line for studying inhibitory effects of xenobiotics on aromatase activity.

WT1 and DAX-1 regulate SF-1-mediated human P450arom gene expression in gonadal cells

Binding activity of steroidogenic factor-1 (SF-1) to promoters of the majority of steroidogenic genes in response to gonadotropins is a critical mechanism that regulates steroidogenesis in gonads. Thus, the modulation of SF-1 action may be essential for the differential regulation of formation of sex steroids in the ovary. Aromatase P450 (P450arom) is the rate-limiting enzyme for estrogen formation. In this study, we characterize another nuclear receptor half site in the gonadal aromatase promoter which we show to be important for aromatase regulation. We also show herein that the stimulation of P450arom promoter activity by SF-1 in ovarian granulosa, testicular Sertoli and JEG-3 choriocarcinoma cells is inhibited by two transcription factors, Wilms’ tumor suppressor gene (WT1) and dosage sensitive sex reversal adrenal hypoplasia congenita critical region on the X chromosome gene 1 (DAX-1). Given the characterized roles of these transcription factors in gonadal development and function, modulation of SF-1 action by WT1 and DAX-1 may represent an important key mechanism in steroidogenesis.