Increased Aromatase Activity Research Articles

Effects of Calmodulin on Aromatase Activity in the Preoptic Area

Oestrogens derived from the neural aromatisation of testosterone play a key role in the activation of male sexual behaviour in many vertebrates. Besides their slow action on gene transcription mediated by the binding to nuclear receptors, oestrogens have now been recognised to have more rapid membrane-based effects on brain function. Rapid changes in aromatase activity, and hence in local oestrogen concentrations, could thus rapidly modulate behavioural responses. We previously demonstrated that calcium-dependent kinases are able to down-regulate aromatase activity after incubations of 10-15 min in phosphorylating conditions. In the present study, in quail hypothalamic homogenates, we show that Ca2+ or calmodulin alone can very rapidly change aromatase activity. Preincubation with 1 mM EGTA or with a monoclonal antibody raised against calmodulin immediately increased aromatase activity. The presence of calmodulin on aromatase purified by immunoprecipitation and electrophoresis was previously identified by western blot and two consensus binding sites for Ca2+-calmodulin are identified here on the deduced amino acid sequence of the quail brain aromatase. The rapid control of brain aromatase activity thus appears to include two mechanisms: (i) an immediate regulatory process that involves the Ca2+-calmodulin binding site and (ii) a somewhat slower phosphorylation by several protein kinases (PKC, PKA but also possibly Ca2+-calmodulin kinases) of the aromatase molecule.

Tumor-Stromal Interaction through the Estrogen-Signaling Pathway in Human Breast Cancer

In postmenopausal breast cancers, locally produced estrogen by adipose stromal cells causes the progression of tumor growth. Although aromatase, a key enzyme of estrogen synthesis, is highly expressed in the adipose stromal cells, and aromatase inhibitors show greater efficacy in postmenopausal breast cancers, the mechanism of increasing aromatase activity in the stromal cells remains unclear. To analyze the estrogen signals and to detect the estrogen receptor (ER)-activating ability of adipose stromal cells for individual human breast cancers, we developed a new reporter cell system. To visualize the activation of ER, we first established a stable transformant, named E10, of human breast cancer MCF-7 cells by transfection with the estrogen-responsive element-green fluorescent protein (GFP) gene. E10 cells specifically express GFP when ER is activated by estrogen or by coculture with adipose stromal cells isolated from breast tumor tissues in the presence of testosterone, a substrate for aromatase. Treatment of adipose stromal cells with dexamethasone, a stimulator of aromatase gene expression, resulted in an increase in the expression of GFP in E10 cells in the coculture. Using this system, we characterized the adipose stromal cells of 67 human breast cancers and found that GFP expression levels vary among the cases, suggesting that the ability of adipose stromal cells to activate ERs is unique for individual breast cancers. High induction levels of GFP were observed more frequently in postmenopausal cases than in premenopausal cases, whereas they did not significantly correlate with the ER expression status. Aromatase inhibitors inhibited the induction of GFP expression in the coculture, but the sensitivities to the drugs varied among the individual cases. Aromatase gene expression levels in adipose stromal cells did not always correlate with their ability to induce GFP. These results suggest that this system to detect total ER activation based on the interaction with adipose stromal cells is a useful tool for analyzing local estrogen signals and for tumor-stromal interactions.

Estrogen Receptor-β Is Critical to Granulosa Cell Differentiation and the Ovulatory Response to Gonadotropins

The process of granulosa cell differentiation that occurs in preovulatory follicles is dependent on FSH but requires augmentation by estradiol. To determine which estrogen receptor (ER) form mediates the effects of estradiol during gonadotropin-induced follicle growth, differentiation, and rupture, we characterized the response of ERalpha- and ERbeta-null mice to gonadotropin-induced ovulation. Immature mice were treated with an ovulatory regimen of exogenous gonadotropins and tissues were collected at distinct time points for morphological, biochemical, gene expression, and immunohistochemical analyses. Granulosa cells of ERbeta knockout (ERKO) preovulatory follicles exhibited an attenuated response to FSH-induced differentiation, as evident by reduced aromatase activity and estradiol synthesis, and insufficient expression of LH receptor. As a result, betaERKO ovaries were unable to fully respond to an ovulatory bolus of gonadotropin, leading to a reduced rate of follicle rupture; insufficient induction of prostaglandin-synthase 2 and progesterone receptor; an aberrant increase in aromatase activity and plasma estradiol; and incomplete expansion of the cumulus-oocyte complex. Parallel characterization of alphaERKO females indicated a minimal role for ERalpha in granulosa cell differentiation, ovulation, and the concomitant changes in gene expression, although some abnormalities were revealed. These studies demonstrate that ERbeta-mediated estradiol actions are vital to FSH-induced granulosa cell differentiation; and in the absence of ERbeta, preovulatory follicles are deficient in the necessary cellular organization (i.e. antrum and cumulus oocyte complex), enzymatic activity (i.e. capacity to convert androgen precursor to estradiol), and receptor signaling pathways (i.e. LH receptor) to respond to a gonadotropin surge and expel a healthy oocyte.

Corals have already evolved the vertebrate-type hormone system in the sexual reproduction

Most broadcast spawning scleractinian corals synchronously release gametes during a brief annual spawning period. In southern Taiwan, the mass spawning of scleractinians occurs in lunar mid-March. Endocrine system has been proposed to play important roles in this annual phenomenon. A scleractinian coral, Euphyllia ancora has been selected as a model for the hormones and reproduction studies. We detected the presence of estradiol (E2), testosterone (T), glucuronided E2, glucuronided T, aromatase, immunoreactive (ir)GnRH in coral polyps E. ancora. Annual profiles of sex steroids, aromatase, and irGnRH have also been characterized. We found the parallel increases in irGnRH concentrations, aromatase activity and free E2 and glucuronided E2 concentrations at the time of coral mass spawning. The stimulation of mammalian (m)GnRH agonist in the increased aromatase activity and sex steroid concentrations was also observed in corals. Coral extracts (irGnRH) and mGnRH agonist had a similar dose-dependent effect on luteinizing hormone release in black porgy fish pituitary cells (in vitro). In conclusion, our data suggest that irGnRH and glucuronided E2 may play important roles in the control of reproduction and mass spawning in corals. Corals already evolved the vertebrate-type hormone system in the sexual reproduction.

Forskolin and dexamethasone synergistically induce aromatase (CYP19) expression in the human osteoblastic cell line SV-HFO

Recent progress supports the importance of local estrogen secretion in human bone tissues to increase and maintain bone mineral density. In a previous study, we reported that the expression of aromatase (CYP19) is dexamethasone (Dex)-dependent and oncostatin M (OSM) increases the expression synergistically with Dex. In the present study, we examined the effects of forskolin (FSK) as another potential synergistic factor. Co-administration of 100 nM Dex and 10 microM FSK increased aromatase activity 4-fold compared with Dex alone. The results of reverse transcriptase (RT)-PCR suggest that the amount of CYP19 gene transcript was also up-regulated by FSK synergistically with Dex, and that promoter I.4, which is not activated by FSK alone, is activated by FSK synergistically with Dex. The results of RT-PCR also suggest that promoter II, which responds to FSK, was not activated even in the presence of FSK in SV-HFO. The promoter I.4 sequence that was transfected into SV-HFO was activated by FSK synergistically with Dex. Synergistic up-regulation of aromatase activity, CYP19 gene transcript, and promoter I.4 activity were Dex-dependent and not up-regulated by FSK alone. The results of this work may form the basis of bone-specific estrogen-replacement therapy that increases the estrogen concentration in bone tissue only.

DDE-Induced Changes in Aromatase Activity in Endometrial Stromal Cells in Culture

Environmental toxicants are thought to play a role in several estrogen-dependent diseases including breast cancer and endometriosis. Toxicant-induced increased aromatase activity, an enzyme complex that catalyzes the final rate-limiting step in the conversion of androgens to estrogens, has been reported in assays using placental microsomes and cancer cells in vitro. These data suggest that environmental toxicants can increase aromatase activity and thus increase local tissue estrogen levels, which could have implications for estrogen- dependent functions in target tissues. The objective of this study was therefore to quantify the effect of the stable breakdown product of DDT, 2,2-bis(p-chlorophenyl)ethylene (p,p'-DDE), a toxicant broadly detected in human adipose tissue, serum and follicular fluid, on aromatase activity in the endometrium, an estrogen-sensitive target tissue. Specifically, the effect of increasing log concentrations of p,p'-DDE on aromatase activity was determined in cultures of endometrial stromal cells (ESC). Relative to controls p,p'-DDE treatment significantly increased aromatase activity in ESC (135%). Moreover, ESC cells treated with p,p'-DDE were immunopositive for aromatase, whereas no aromatase staining could be demonstrated in control cultures. Our data demonstrate that p,p'-DDE treatment can increase aromatase activity in ESC in culture.

Leptin Levels in Boys with Pubertal Gynecomastia

It has been reported that there is a relationship between circulating leptin and sex steroid hormones and leptin is able to stimulate estrogen secretion by increasing aromatase activity in adipose stromal cells and breast tissue. Leptin receptors have been also shown in mammary epithelial cells and it has been suggested that leptin is involved in the control of the proliferation of both normal and malignant breast cells. To investigate circulating leptin levels in boys with pubertal gynecomastia. Twenty boys with pubertal gynecomastia who were in early puberty and had no obesity, and 20 healthy individuals matched for age, pubertal stage and body mass index (BMI) with the study group, were enrolled in the study. Body weight, height and left midarm circumference (MAC) and left arm triceps skinfold thickness (TSF) were measured and BMI was calculated. A fasting blood sample was collected and routine hormonal parameters including prolactin, beta-human chorionic gonadotropin (betaHCG), total and free testosterone, estradiol, luteinizing hormone (LH), follicle stimulating hormone (FSH), prolactin, androstenedione (AS) and dehydroepiandrosterone sulfate (DHEAS) levels were studied. Serum leptin levels were analyzed using radioimmunoassay. The mean ages of the study and control group were not different (13.9 +/- 0.89 and 14.2 +/- 0.66, respectively). No significant difference was found for BMI, MAC and TSF values between the two groups. There was no significant difference for hormonal parameters including FSH, LH, total and free testosterone, estradiol, AS, DHEAS and estradiol/total testosterone ratio between boys with pubertal gynecomastia and the controls. Serum leptin levels were found significantly higher in the study group compared with the healthy controls (5.58 +/- 0.81 and 2.39 +/- 0.29 ng/ml, respectively; p <0.001). No correlation could be determined between serum leptin levels and hormonal parameters. The presence of higher leptin levels in boys with pubertal gynecomastia indicates that leptin may be involved in the pathogenesis of pubertal gynecomastia. The role of circulating leptin in pubertal gynecomastia is probably related to increase in estrogen and/or estrogen/ androgen ratio by the stimulating effect of leptin on aromatase enzyme activity in both adipose and breast tissues, or a direct growth stimulating effect of leptin on mammary epithelial cells, or increase in sensitivity of breast epithelial cells to estrogen with inducing functional activation of estrogen receptors by leptin in breast tissue.

The effects of dietary phytoestrogens on aromatase activity in human endometrial stromal cells

Dietary phytoestrogens have been reported to inhibit aromatase activity in placental microsomes, but the effects in the human endometrium are unknown. Aromatase, the rate-limiting enzyme in the conversion of androgens to estrogens, has recently been shown to be expressed in the endometrium of women with endometriosis and is thought to play a role in the pathophysiology of this disease. Therefore, the objective of this study was to screen dietary phytoestrogens for their ability to inhibit aromatase activity in human endometrial stromal cells (ESC) and identify potential novel therapeutic agents for the treatment of endometriosis. The inhibition of aromatase activity by direct interaction with the dietary phytoestrogens genistein, daidzein, chrysin, and naringenin was tested in a cell free assay. Furthermore, test compound effects on aromatase activity in ESC cultures were also examined. Genistein and daidzein were inactive in the human recombinant aromatase assay whereas naringenin and chrysin inhibited aromatase activity. However, genistein (1 nM to 1 mM) stimulated aromatase activity in ESC whereas other phytoestrogens had no effect. Immunopositive aromatase cells were demonstrated in genistein-treated ESC but not in untreated control cultures. Taken together, our data suggest that genistein can increase aromatase activity in ESC likely via increased enzyme expression.

Dominant transmission of prepubertal gynecomastia due to serum estrone excess: hormonal, biochemical, and genetic analysis in a large kindred.

Increased extraglandular aromatization has been reported to cause the rare entity of familial gynecomastia. Recently heterozygous inversions at the p450 aromatase gene promotor locus were detected in two different families with this syndrome. We studied a family in which seven affected males over three generations had inherited prepubertal gynecomastia in an autosomal dominant manner. The proband developed gynecomastia at 11.5 yr, entered puberty at 12.5 yr, but was incompletely virilized at 19 yr. A similar development was observed in his affected stepbrother and one first-degree cousin. All three boys had acceleration of prepubertal growth and bone age. The older two had a diminished pubertal growth spurt and precocious growth arrest, but their final heights were within the range of their target height. In addition, the maternal grandfather and three maternal uncles were affected, who all had been mastectomized. The mother of the proband had normal age at menarche and no macromastia. Estrone levels of the proband and the other affected boys were elevated, 17beta-estradiol levels were high-normal, and testosterone levels were low. Hormonal analyses of the affected adults, who had all fathered children, revealed pathologically low serum testosterone levels but normal to high-normal levels of estradiol and estrone. The mother of the proband had elevated estrone levels. Treatment of the proband was more effective with anastrozole than with testolactone and increased the initially reduced testes volume to normal size, promoted virilization, and normalized serum estrone and testosterone levels. Neither preadipocytes from breast fat tissue of the affected stepbrother nor peripheral lymphocytes of the affected boys exhibited increased aromatase activity in culture. Therefore, these cells can be excluded from being the source of estrone excess. In addition, serum of the proband and his stepbrother did not contain factors promoting aromatase activity as assayed using preadipocytes from control individuals.A repeat polymorphism of the p450 aromatase gene cosegregated with the disease phenotype in the family, making a mutation of the p450 aromatase gene likely. Single-strand conformational polymorphism analysis of the known alternative untranslated exons and all coding exons of the p450 aromatase gene did not indicate any mutation. In addition, fluorescent in situ hybridization analysis using four probes covering the promotor region did not reveal the presence of any major inversion at this locus. In conclusion, preadipocytes and blood cells were excluded as the cell source of increased aromatization. Fluorescent in situ hybridization and single-strand conformational polymorphism analyses did not reveal any mutation of the p450 aromatase gene, but an intragenic polymorphic marker cosegregated with the disease phenotype. Excess of serum estrone in the presence of normal 17beta-estradiol levels may be the only indicative serum parameter of this mild manifestation of aromatase excess syndrome, which includes prepubertal gynecomastia and moderate hypogonadism in men but not necessarily short stature. In women, this mode of aromatase excess may remain clinically inapparent.

Atrazine is a competitive inhibitor of phosphodiesterase but does not affect the estrogen receptor

Atrazine (ATR), 2-chloro-4-ethylamino-6-isopropylamino- s-triazine, has been implicated in numerous studies to act as an endocrine disruptor, specifically by altering estradiol signaling via increased aromatase activity. Fluorescence polarization (FP) was used to show that the binding equilibria between estrogen receptor-α or estrogen receptor-β, and estradiol were not affected by ATR and its metabolites: ATR-desethyl (ADE), ATR-desisopropyl (ADI), ATR-desethyldesisopropyl (ADD) and terbuthylazine (TBZ). Therefore, ATR and its degradation products were studied to determine their ability to inhibit phosphodiesterase (PDE), the enzyme responsible for hydrolyzing the second messenger cAMP to 5′-AMP. Using FP, it was found that ATR inhibited PDE with an IC 50 value of 1.8 μM. This was lower than the known PDE inhibitor isobutyl methylxanthine (IBMX), which had an IC 50 value of 4.6 μM. The ATR degradation products ADE, ADI, ADD and TBZ were less effective than ATR at inhibiting PDE when assayed using FP. Classical competitive binding assays, using radiolabeled 14C-cAMP in conjunction with thin layer chromatography (TLC), were used to determine that ATR was a competitive inhibitor of PDE with an association constant of 85 μM.

Inhibition of aromatase activity by methyl sulfonyl PCB metabolites in primary culture of human mammary fibroblasts

In this study, the effects on catalytic activity and mRNA levels of aromatase in primary human mammary fibroblasts were evaluated after exposure to promoter-specific modulators of aromatase expression and methyl sulfonyl polychlorinated biphenyl metabolites (MeSO 2-PCBs). A method for fibroblast isolation from primary breast tissue was developed and optimized, and aromatase activity and promoter-specific mRNA levels were assessed in these cells after exposure to test compounds. A 24-h exposure of fibroblasts to dexamethasone (DEX) (1–100 nM) increased aromatase activity to a maximum of 313-fold. DEX also elevated promoter I.4-specific RNA levels. A 24-h exposure of fibroblasts to 3-MeSO 2-PCB-132, 4-MeSO 2-PCB-132, 4-MeSO 2-PCB-91, or 4-MeSO 2-PCB-149 (0.1–10 μM) resulted in a concentration-dependent decrease of aromatase activity. Exposure of fibroblasts to MeSO 2-PCBs just for the limited duration (6 h) of the catalytic assay caused a concentration-dependent inhibition of aromatase enzyme activity. mRNA levels were not altered by a 24-h MeSO 2-PCB exposure nor was cytotoxicity observed. In aromatase-expressing human adrenocortical carcinoma H295R cells, a 24-h exposure to 3-MeSO 2-PCB-132, 4-MeSO 2-PCB-132, 4-MeSO 2-PCB-91, or 4-MeSO 2-PCB-149 (0.1–10 μM) also resulted in a concentration-dependent decrease of aromatase activity. Additionally, there were no changes in aromatase mRNA levels after 24-h exposure of H295R cells to MeSO 2-PCBs. We conclude that in primary human mammary fibroblasts as well as in H295R cells, aromatase inhibition by MeSO 2-PCBs is likely to be due to catalytic inhibition.

A benzimidazole fungicide, benomyl, and its metabolite, carbendazim, induce aromatase activity in a human ovarian granulose-like tumor cell line (KGN).

Endocrine disruptor chemicals are known to cause a range of abnormalities in sexual differentiation and reproduction. One mechanism underlying such effects may be via alteration of aromatase activity, which is responsible for estrogen production. A good screening system for identifying endocrine disruptors has long been desired. We have recently established a human ovarian granulosa-like tumor cell line, KGN, which possesses a relatively high level of aromatase expression and is considered a useful mammalian model for investigating the in vitro effects of various chemicals on aromatase activity. In this study we screened 55 different candidate chemicals for endocrine disruptors by assaying aromatase activity. Only benomyl, known as both a benzimidazole fungicide and a microtubule-interfering agent, was found to induce aromatase activity in association with increased levels of aromatase mRNA in KGN cells. The effect of benomyl was presumed to be mediated by its metabolite carbendazim, because it produced an effect equivalent to that of benomyl. The mechanism underlying the benomyl-induced increase in aromatase activity appears independent of the cAMP-protein kinase A pathway. Treatment with taxol, another class of microtubule-interfering agents, also caused induction of aromatase in KGN cells. Both benomyl and taxol changed KGN cell morphology, including the development of cell roundness and a disorganized network of microtubules. These results indicate that benomyl is a potential endocrine disruptor that provides a novel estrogenicity and operates through a microtubule-interfering mechanism.

Effect of gonadotropins on brain-derived neurotrophic factor secretion by human follicular cumulus cells

Effect of gonadotropins on brain-derived neurotrophic factor secretion by human follicular cumulus cells

Protein kinase B is obligatory for follicle-stimulating hormone-induced granulosa cell differentiation.

Although FSH receptors are linked to the cAMP second messenger system, additional intracellular signaling pathways appear to be required for the induction of aromatase and the LH receptor during granulosa cell differentiation. We employed adenovirus vectors to modulate specific intracellular signaling systems in undifferentiated granulosa cells to identify the signaling pathway(s) that may be involved in the FSH-mediated induction of aromatase and the LH receptor. Expression of either the constitutively activated human LH receptor D578H or the constitutively active human G(s)alpha Q227L resulted in increased cAMP production without increasing aromatase activity or mRNA levels for the LH receptor. To explore the contributions of other pathways, we expressed the constitutively activated forms MAPK kinase (MEK) and protein kinase B (PKB). Neither MEK nor PKB alone increased estrogen or progesterone production by undifferentiated granulosa cells. Stimulation of granulosa cells by FSH in the presence of the constitutively active PKB, but not MEK, led to an amplification of FSH-induced aromatase and LH receptor mRNA levels, whereas a dominant negative PKB vector completely abolished the actions of FSH. The expression of the constitutively active PKB in combination with the constitutively active LH receptor D578H, the constitutively active G(s)alpha Q227L, or 8-bromo-cAMP led to an induction of aromatase as well as LH receptor mRNA comparable to that seen in cells stimulated with FSH alone. These results demonstrate that PKB is an essential component of the FSH-mediated granulosa cell differentiation and that both PKB and G(s)alpha signaling pathways are required.

Aromatase and cyclooxygenases: enzymes in breast cancer

Aromatase (estrogen synthase) is the cytochrome P450 enzyme complex that converts C 19 androgens to C 18 estrogens. Aromatase activity has been demonstrated in breast tissue in vitro, and expression of aromatase is highest in or near breast tumor sites. Thus, local regulation of aromatase by both endogenous factors as well as exogenous medicinal agents will influence the levels of estrogen available for breast cancer growth. The prostaglandin PGE 2 increases intracellular cAMP levels and stimulates estrogen biosynthesis, and previous studies in our laboratories have shown a strong linear association between aromatase ( CYP19) expression and expression of the cyclooxygenases ( COX-1 and COX-2) in breast cancer specimens. To further investigate the pathways regulating COX and CYP19 gene expression, studies were performed in normal breast stromal cells, in breast cancer cells from patients, and in breast cancer cell lines using selective pharmacological agents. Enhanced COX enzyme levels results in increased production of prostaglandins, such as PGE 2. This prostaglandin increased aromatase activity in breast stromal cells, and studies with selective agonists and antagonists showed that this regulation of signaling pathways occurs through the EP 1 and EP 2 receptor subtypes. COX-2 gene expression was enhanced in breast cancer cell lines by ligands for the various peroxisome proliferator-activated receptors (PPARs), and differential regulation was observed between hormone-dependent and -independent breast cancer cells. Thus, the regulation of both enzymes in breast cancer involves complex paracrine interactions, resulting in significant consequences on the pathogenesis of breast cancer.

Aspectos neuroendócrinos da síndrome metabólica

A síndrome metabólica (SM) é caracterizada por alterações no metabolismo glicídico, obesidade, hipertensão e dislipidemia. Estas alterações metabólicas interrelacionam-se com diversos eixos endócrinos controlados pelo hipotálamo e pela hipófise. A obesidade central parece relacionar-se a uma hiperativação do eixo hipotálamo-hipófise-adrenal, como também do sistema nervoso simpático, que poderia levar a um quadro de hipercortisolismo sub-clínico e hipertensão arterial. A SM é também um estado de hipo-somatotropismo relativo relacionado à gordura visceral. Além disso, níveis elevados de ácidos graxos livres e a hiperinsulinemia, secundários à resistência insulínica, estão relacionadas a um bloqueio do eixo somatotrófico. Em homens, a SM relaciona-se a um hipogonadismo tanto por diminuição de gonadotrofinas como por inibição direta da produção de testosterona. Já nas mulheres, existe um excesso de produção de androgênios, principalmente relacionado à hiperinsulinemia, aumento da atividade da aromatase e da liberação de LH. Desta forma, a SM é um estado relacionado a importantes modificações nos mecanismos de feedback responsáveis pelo correto funcionamento dos eixos neuroendócrinos.

Prostaglandin E2 regulates aromatase activity and expression in human adipose stromal cells via two distinct receptor subtypes.

The aromatase enzyme complex, located primarily in the stromal cells of breast tumors, catalyzes estrogen biosynthesis and is fundamental to hormone-dependent growth of breast cancer. Although an important pharmacological target, the mechanisms by which aromatase is regulated are poorly understood. Thus, regulation of aromatase activity and expression in human breast stromal cells by prostaglandin E(2) (PGE(2)) was investigated. PGE(2) exerts its actions via four transmembrane receptors, EP(1), EP(2), EP(3), and EP(4), which coordinate different signal transduction pathways. Using selective receptor agonists and antagonists, the involvement of the EP(1), EP(2), and EP(3) subtypes was assessed. Enzyme activity levels in cultures of disease-free stromal cells were determined using a tritiated water-release assay. PGE(2) and agonists of EP(1) and EP(2) significantly increased aromatase activity levels, which were decreased by the corresponding antagonists. An agonist of EP(3), an inhibitory pathway, antagonized activity levels induced by PGE(2). These results were generally reflective of changes in aromatase protein expression, determined by Western blotting analysis and the pattern of mRNA expression determined by a competitive RT-PCR method. Collectively, the results demonstrate that regulation of aromatase by PGE(2) is complex and may influence the development and progression of hormone-dependent breast cancer.

Effect of tibolone (Org OD14) and its metabolites on aromatase and estrone sulfatase activity in human breast adipose stromal cells and in MCF-7 and T47D breast cancer cells

Tibolone (Org OD14) is a synthetic steroid used for post-menopausal hormone replacement therapy (HRT). Since HRT might increase breast cancer risk, it is important to determine the possible effects of tibolone on breast tissues. Tibolone and its metabolites Org 4094, Org 30126 and Org OM38 have been reported to inhibit estrone sulfatase activity in MCF-7 and T47D breast cancer cell lines, which suggest beneficial effects on hormone dependent breast cancer by reducing local production of free estrogens. Breast adipose stromal cells (ASCs) contain aromatase activity—an obligatory step in the biosynthesis of estrogens—and possibly contain sulfatase activity. We investigated the effects of tibolone, its metabolites and the pure progestin Org 2058 on PGE 2-stimulated aromatase activity and on sulfatase activity in human ASC primary cultures and on sulfatase activity in MCF-7 and T47D cell lines. In MCF-7, tibolone and metabolites, but not Org 2058, were found to inhibit sulfatase activity. In T47D, tibolone inhibited sulfatase only at 10 −6 M, although weakly. ASC had high sulfatase activity, which was inhibited by 10 −6 M of tibolone, Org 4094 and Org 30126, but not by Org OM38 or Org 2058. Surprisingly, aromatase activity in ASC was increased by both tibolone and Org 2058 at 10 −6 M. As ligand binding assay results and immunohistochemistry indicated the absence of progesterone and estrogen receptors in ASC, these effects on aromatase and sulfatase activity in ASC likely take place by other routes. Because tibolone and its metabolites inhibit sulfatase activity, and because tibolone only increases aromatase activity at a high concentration, we conclude that effects of tibolone on the breast are probably safe.

Overexpression of aromatase P450 in leiomyoma tissue is driven primarily through promoter I.4 of the aromatase P450 gene (CYP19).

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-1beta and dexamethasone, known as a potent inducer of promoter I.4-driven transcription, effectively increased aromatase activity. A combination of (Bt)(2)cAMP, 3-isobutyl-1-myethylxanthine, and PGE(2), 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-1beta. 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.

Pituitary adenylate cyclase-activating polypeptide induces testicular testosterone synthesis through PGE(2) mediation in crested newt, Triturus carnifex.

The aim of the present work was to study the possible role of adenylate cyclase-activating polypeptide (PACAP) 38 in the testicular intracellular mechanism regulating steroidogenesis of crested newt, Triturus carnifex. Gonads were incubated in vitro with PACAP 38 and prostaglandin (PG) E(2) alone or with inhibitors of cyclooxygenase (COX), adenylate cyclase (AC), and phospholipase C (PLC) for 30 min and 60 min. PGE(2), PGF(2 alpha), testosterone, and estradiol-17 beta were measured in the culture medium; aromatase (AR) activity and cAMP were assessed in the tissue. PACAP 38 increased PGE(2) (30 min and 60 min), estradiol-17 beta (60 min), cAMP (60 min), and AR (60 min) but decreased testosterone (60 min). PGE(2) increased estradiol-17 beta, cAMP, and AR and decreased testosterone at 30 and 60 min.PLC inhibitor counteracted the effects of PACAP 38, while AC inhibitor counteracted these effects except for PGE(2) increase. AC inhibitor counteracted the effects of PGE(2), while PLC did not. COX inhibitor decreased PGF(2 alpha) (30 min and 60 min), PGE(2) (30 min and 60 min), estradiol-17 beta (60 min), cAMP (60 min), and AR (60 min), but increased testosterone (60 min). These in vitro results suggest that, in newt testis, PACAP 38 acts on PLC, inducing the increase of PGE(2) which, in turn, acting on AC, increases AR activity with the consequent estradiol-17 beta increase and testosterone decrease.