Aromatase Stimulation Research Articles

CC chemokine ligand 2 (CCL2) stimulates aromatase gene expression in mammary adipose tissue.

Obesity is a risk factor for postmenopausal breast cancer. Obesity-related inflammation upregulates aromatase expression, the rate-limiting enzyme for estrogen synthesis, in breast adipose tissue (BAT), increasing estrogen production and cancer risk. The regulation of aromatase gene (CYP19A1) in BAT is complex, and the mechanisms linking obesity and aromatase dysregulation are not fully understood. An obesity-associated factor that could regulate aromatase is the CC chemokine ligand (CCL) 2, a pro-inflammatory factor that also activates signaling pathways implicated in CYP19A1 transcription. By using human primary breast adipose stromal cells (ASCs) and aromatase reporter (hARO-Luc) mouse mammary adipose explants, we demonstrated that CCL2 enhances the glucocorticoid-mediated CYP19A1 transcription. The potential mechanism involves the activation of PI.4 via ERK1/2 pathway. We also showed that CCL2 contributes to the pro-inflammatory milieu and aromatase expression in obesity, evidenced by increased expression of CCL2 and CYP19A1 in mammary tissues from obese hARO-Luc mice, and subcutaneous adipose tissue from obese women. In summary, our results indicate that postmenopausal obesity may promote CCL2 production in BAT, leading to exacerbation of the menopause-related inflammatory state and further stimulation of local aromatase and estrogens. These results provide new insights into the regulation of aromatase and may aid in finding approaches to prevent breast cancer.

Gonadotropin Treatment for the Male Hypogonadotropic Hypogonadism.

Hypogonadotropic hypogonadism (HH) is caused by a dysfunction in the hypothalamus and/or the pituitary gland and it can be congenital or acquired. This condition is biochemically characterized by low or inappropriately normal gonadotropin levels along with low total testosterone levels. If fertility is not an issue, testosterone therapy is the treatment of choice to induce and maintain secondary sexual characteristics and sexual function. Spermatogenesis is frequently impaired in patients with HH, but usually responsive to hormonal therapy such as gonadotropin therapy or GnRH supplementary/replacement therapy. When gonadotropins are the choice of treatment, conventional therapy includes human chorionic gonadotropin (hCG) along with different FSH formulations: human menopausal gonadotropins (hMG), highly purified urinary FSH preparations (hpFSH) (e.g., urofollitropin) or recombinant FSH (rFSH). The combination of FSH and hCG demonstrated to be associated with better outcomes than single compounds, whereas similar results were obtained with different FSH preparations in male individuals; both regarding the ability to stimulate spermatogenesis and eventually inducing physiology pregnancy. Gonadotropins can be administered either subcutaneously or intramuscularly. The combination therapy with hCG and FSH for a period of 12-24 months was found to promote testicular growth in almost all patients, spermatogenesis in approximately 80% and pregnancy rates in the range of 50%. Gynecomastia is the most common side effect of gonadotropin therapy and is due to hCG stimulation of aromatase causing increased secretion of estradiol. The therapeutic success is higher in patients with post-pubertal HH, in those without previously undescended testes, in patients with higher baseline testicular volume, who underwent repeated cycles of therapy and in patients with higher baseline inhibin B serum concentrations. Reversal of hypogonadism can occur in up to 10% of patients but its physiopathologic mechanism has yet to be elucidated. In conclusion, gonadotropin therapy is effective in promoting puberty and in supporting spermatogenesis onset and preservation in HH patients with either hypothalamic or pituitary conditions.

Oocyte-Secreted Factors Synergize With FSH to Promote Aromatase Expression in Primary Human Cumulus Cells.

The role of growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) on aromatase regulation is poorly understood in humans. Determine GDF9 and BMP15 effects on FSH stimulation of estradiol production in primary human cumulus granulosa cells (GCs). We hypothesized that the combination of GDF9 and BMP15 potentiates FSH-induced aromatase expression. Primary human cumulus GCs in culture. University infertility center. GCs of 60 women undergoing in vitro fertilization were collected. Cells were treated with GDF9 and/or BMP15 (GB) in the presence or absence of FSH, dibutyryl cAMP, or SMAD inhibitors. Promoter activity, mRNA, protein, and estradiol levels were quantified. FSH and GB treatment increased CYP19A1 promoter activity, mRNA, and protein levels as well as estradiol when compared with cells treated with FSH only. GB treatment potentiated cAMP stimulation of aromatase and IGF2 stimulation by FSH. GB effects were inhibited by SMAD3 inhibitors and IGF1 receptor inhibitors. GB, but not FSH, stimulates SMAD3 phosphorylation. The combination of GDF9 and BMP15 potently stimulates the effect of FSH and cAMP on CYP19a1 promoter activity and mRNA/protein levels. These effects translate into an increase in estradiol production. This potentiation seems to occur through activation of the SMAD2/3 and SMAD3 signaling pathway and involves, at least in part, the effect of the IGF system.

Leptin regulation of the p53-HIF1α/PKM2-aromatase axis in breast adipose stromal cells: a novel mechanism for the obesity-breast cancer link.

Background/ObjectivesObesity (body mass index (BMI) ≥ 30) is associated with an increased risk of estrogen-dependent breast cancer after menopause. Levels of aromatase, the rate-limiting enzyme in estrogen biosynthesis, are elevated in breast tissue of obese women. Recently, the regulation of aromatase by the p53-HIF1α/PKM2 axis was characterized in adipose stromal cells (ASCs) of women with Li-Fraumeni Syndrome, a hereditary cancer syndrome that predisposes to estrogen-dependent breast cancer. The current study aimed to determine whether stimulation of aromatase by obesity-associated adipokine leptin involves the regulation of the p53-HIF1α/PKM2 axis.Subjects/MethodsHuman breast ASCs were used to characterize the p53-HIF1α/PKM2-aromatase axis in response to leptin. The effect of pharmacological or genetic modulation of PKC, MAPK, p53, Aha1, Hsp90, HIF1α and PKM2 on aromatase promoter activity, expression and enzyme activity was examined. Semi-quantitative immunofluorescence and confocal imaging were used to assess ASC-specific protein expression in FFPE sections of breast of women and mammary tissue of mice following a low fat (LF) or high fat (HF) diet for 17 weeks.ResultsLeptin-mediated induction of aromatase was dependent on PKC/MAPK signaling and the suppression of p53. This, in turn, was associated with an increase in Aha1 protein expression, activation of Hsp90 and the stabilization of HIF1α and PKM2, known stimulators of aromatase expression. Consistent with these findings, ASC-specific immunoreactivity for p53 was inversely associated with BMI in breast tissue, while HIF1α, PKM2 and aromatase were positively correlated with BMI. In mice, HF feeding was associated with significantly lower p53 ASC-specific immunoreactivity compared to LF feeding, while immunoreactivity for HIF1α, PKM2 and aromatase were significantly higher.ConclusionsOverall, findings demonstrate a novel mechanism for the obesity-associated increase in aromatase in ASCs of the breast and support the study of lifestyle interventions, including weight management, which may reduce breast cancer risk via effects on this pathway.

Des-acyl ghrelin inhibits the capacity of macrophages to stimulate the expression of aromatase in breast adipose stromal cells

Des-acyl ghrelin is the unacylated form of the well-characterized appetite-stimulating hormone ghrelin. It affects a number of physiological processes, including increasing adipose lipid accumulation and inhibiting adipose tissue inflammation. Breast adipose tissue inflammation in obesity is associated with an increase in the expression of the estrogen biosynthetic enzyme, aromatase, and is hypothesized to create a hormonal milieu conducive to tumor growth. We previously reported that des-acyl ghrelin inhibits the expression and activity of aromatase in isolated human adipose stromal cells (ASCs), the main site of aromatase expression in the adipose tissue. The current study aimed to examine the effect of des-acyl ghrelin on the capacity of mouse macrophages (RAW264.7 cells) and human adipose tissue macrophages (ATMs) to stimulate aromatase expression in primary human breast ASCs. RAW264.7 cells were treated with 0, 10 and 100pM des-acyl ghrelin following activation with phorbol 12-myristate 13-acetate, and cells and conditioned media were collected after 6 and 24h. The effect of des-acyl ghrelin on macrophage polarization was examined by assessing mRNA expression of pro-inflammatory M1-specific marker Cd11c and anti-inflammatory M2-specific marker Cd206, as well as expression of Tnf and Ptgs2, known mediators of the macrophage-dependent stimulation of aromatase. TNF protein in conditioned media was assessed by ELISA. The effect of RAW264.7 and ATM-conditioned media on aromatase expression in ASCs was assessed after 6h. Results demonstrate des-acyl ghrelin significantly increases the expression of Cd206 and suppresses the expression of Cd11c, Tnf and Ptgs2 in activated RAW264.7 cells. Treatment of RAW264.7 and ATMs with des-acyl ghrelin also significantly reduces the capacity of these cells to stimulate aromatase transcript expression in human breast ASCs. Overall, these findings suggest that in addition to direct effects on aromatase in ASCs, des-acyl ghrelin also has the capacity to inhibit the macrophage-dependent induction of aromatase, and provides a novel mechanism for potential effects of des-acyl ghrelin to break the linkage between obesity and breast cancer.

Seasonal changes and endocrine regulation of pejerrey (Odontesthes bonariensis) oogenesis in the wild

The goal of this study was to evaluate the essential components controlling the brain–pituitary–gonad axis during pejerrey (Odontesthes bonariensis) oogenesis in the wild. Ovarian developmental stages from vitellogenesis up to ovulation were associated with increasing day length and water temperatures below 21°C (winter and beginning of spring). Gonadal regression was observed when water temperature exceeded this value or when photoperiod decreased. Most females were arrested at primary growth stage during summer (high temperature) or at cortical alveoli stage between autumn and beginning of winter (short photoperiod). Plasma E2 and transcript levels of fshr, cyp19a1b and cyp19a1a increased during vitellogenesis, while fshb remained high at all vitellogenic stages. A significant correlation between plasma sex steroids (T and E2) and cyp19a1b as well as lhcgr transcript levels was observed during vitellogenesis, suggesting a steroid positive feedback. Gnrh-I, Gth subunits and lhcgr transcript levels increased significantly during late vitellogenesis and final maturation. Present results suggest that pejerrey vitellogenesis is controlled by Fsh/Fshr, stimulating gonadal aromatase and estradiol synthesis. Moreover, the increase of testosterone and estradiol during final vitellogenesis could induce coordinately the functioning of the Gnrh/Lh system (perhaps through brain P450 aromatase stimulation and brain estradiol increase) and the gonadal Lhcgr synthesis to promote the final maturation of oocytes. All these stimulation mechanisms of gonadal development would be possible only under permissive environmental conditions.

Melatonin and aromatase stimulating activity of human seminal plasma

Melatonin concentrations and aromatase stimulating activity were determined in human seminal plasma and correlated with sperm density and motility. Aromatase stimulating activity was determined with an in vitro rat granulosa cell system and melatonin by radioimmunoassay. Compared to normal semen, aromatase stimulating activity was lower in azoospermic individuals, while melatonin was higher in oligospermic and azoospermic samples. Aromatase stimulating activity correlated positively with sperm concentrations and a negative correlation was found between melatonin and sperm progression. These findings suggest that low sperm production is associated with low aromatase stimulating bioactivity in seminal plasma; and melatonin may have an effect upon both sperm production and motility.

Cloning and differential expression of estrogen receptor and aromatase genes in the self-fertilizing hermaphrodite and male mangrove rivulus, Kryptolebias marmoratus

The mechanisms underlying sex determination and differentiation in fishes are labile in response to environmental parameters. Sex-specific phenotypes are largely regulated by sex steroids, and the inhibition or the stimulation of aromatase can reverse sex as well as alter secondary sexual characteristics in fishes. Among vertebrates, the mangrove rivulus is the only known self-fertilizing hermaphrodite. Throughout most of its range, rivulus appear to exist as clonally reproducing hermaphrodites. However, outcrossing has been documented in Belize, where up to 25% of rivulus collected are males. The direct development of (primary) males occurs when embryos are incubated at 18 degrees C and hermaphrodites develop into secondary males when held at 28 degrees C. Given the importance of sex steroids, their receptors, and aromatase in sex determination and differentiation of fishes, we cloned, sequenced, and quantified the expression of estrogen receptors (ERalpha, ERbeta) and ovarian (AroA) and brain (AroB) aromatase genes. Hermaphrodites had increased ERalpha, ERbeta, AroA, and AroB gene expression in the liver, gonad, gonad, and brain respectively, compared to males. These data are consistent with the gene expression data reported for other species and are reflective of the presence of ovarian tissue in the hermaphrodites. Interestingly, we show the elevated expression of brain aromatase in the hermaphrodite brain. The role of the dimorphic expression of brain aromatase in the regulation of sex-specific characteristics is intriguing and requires further research. Because of the uniqueness of its reproductive biology, rivulus is an excellent model for elucidating the mechanisms regulating vertebrate sex determination and sexual differentiation.

No evidence of mutations in the P450 aromatase gene in patients with polycystic ovary syndrome

Etiology and inheritance pattern in polycystic ovary syndrome (PCOS) remain uncertain. Granulosa cells from follicles of women with PCOS have little, if any, aromatase (encoded by the CYP19 gene) activity; follicles contain low levels of estradiol, P450arom mRNA and aromatase stimulating bioactivity. Mice with targeted disruption of the CYP19 gene present cystic follicles. It has been proposed that chronic exposure to high levels of LH, because of aromatase deficiency, determines the development of ovarian cysts. Herein, we investigated if mutations in the CYP19 gene and/or its ovary promoter are causal in patients with PCOS. Twenty-five patients with PCOS and 50 control women were studied. PCR analysis of genomic DNA and complete sequence of all exons of the aromatase gene and its ovary promoter were performed. No heterozygous or homozygous mutant alleles were present in any of the patients studied. In the population studied, mutations of the P450arom gene or its promoter are not the cause of PCOS. However, these findings do not preclude the possible importance of an aromatase disorder in PCOS etiology. Variations in aromatase complex function could play a role in PCOS etiology, but the determinants of such variations might be located in other genes.

Inhibition of IL-6+IL-6 soluble receptor-stimulated aromatase activity by the IL-6 antagonist, Sant 7, in breast tissue-derived fibroblasts.

Interleukin 6 (IL-6) and its soluble receptor (IL-6sR) can markedly stimulate aromatase activity in cultured fibroblasts derived from normal or malignant breast tissues. IL-6 acts by binding to a low-affinity membrane-spanning receptor (IL-6R), which must associate with a high-affinity receptor (gp130) for signal transduction to occur. Sant 7 is a mutated form of IL-6 that can bind to the IL-6R, but inhibits its ability to interact with the gp130 signal transducing protein. In this study, we have used Sant 7 to examine its ability to inhibit IL-6+IL-6 soluble receptor (IL-6sR)-stimulated aromatase activity in breast tissue-derived fibroblasts. As previously observed, IL-6+IL-6sR markedly stimulated aromatase activity (7.7–20.8-fold) in fibroblasts derived from reduction mammoplasty tissue, tissue proximal to tumours and breast tumours. Sant 7 inhibited basal aromatase activity in some fibroblasts by 25–30% that had a high basal activity, but almost completely blocked the ability of IL-6+IL-6sR to stimulate aromatase activity. The IC50 for the inhibition of IL-6+IL-6sR-stimulated aromatase activity by Sant 7 was 60 ng ml−1. A comparison of the effects of prostaglandin E2 (PGE2), which can also regulate aromatase activity, and IL-6+IL-6sR revealed a greater degree of aromatase stimulation by IL-6+IL-6sR. Sant 7, however, inhibited PGE2-stimulated aromatase activity by 70% suggesting that PGE2 acts, in part, by stimulating IL-6 production. Much of the IL-6 and IL-6sR available to stimulate breast tumour aromatase activity may originate from infiltrating macrophages and lymphocytes. The ability to block aromatase stimulation by these factors may offer a novel therapeutic strategy for reducing oestrogen synthesis in breast tumours.

Aromatase regulation and breast cancer.

Aromatase regulation and breast cancer.

The regulation of aromatase activity in breast fibroblasts: the role of interleukin-6 and prostaglandin E2.

Prostaglandin E2 (PGE2) and cytokines, such as interleukin-6 (IL-6) or tumour necrosis factor a (TNFalpha) can regulate aromatase activity. In the present study we have compared their abilities to stimulate aromatase activity in fibroblasts derived from 'normal' breast adipose tissue proximal to a tumour or breast tumours. PGE2, TNFalpha and IL-6 plus its soluble receptor (IL-6sR) all increased aromatase activity in these cells. Basal aromatase activity and the degree of aromatase stimulation by these factors were greater in fibroblasts derived from 'normal' breast tissue than from breast tumours. The ability of IL-6+IL-6sR to increase aromatase activity was only marginally reduced by the PG synthesis inhibitor, indomethacin, indicating that IL-6+IL-6sR does not appear to act via induction of PG synthesis. The ability of PGE2 to stimulate aromatase activity in fibroblasts derived from 'normal' breast tissue was potentiated by IL-6sR suggesting that PGE2 may act via induction of IL-6. This was confirmed by measurement of IL-6 in conditioned medium collected from these cells. A significant increase in IL-6 concentrations was detected in conditioned medium collected from cells treated with PGE2. It is concluded that in some fibroblasts PGE2 may exert part of its regulatory effect on breast tissue aromatase activity via induction of IL-6.

Involvement of growth factors in the pathophysiology of polycystic ovary syndrome

The etiology of polycystic ovary syndrome (PCOS) has not yet been fully elucidated but involves a disruption of normal ovarian function and multisystem sequelae. A combination of abnormally functioning genes whose expression is influenced by environmental, extra-ovarian factors determines the symptoms. Growth factors are heavily involved in the pathophysiology, either contributing to or as a consequence of the arrested development of follicles, abnormal steroidogenesis and hyperinsulinemia. Hyperactivity of a-transforming growth factor (TGFα) and epidermal growth factor (EGF) may block stimulation of aromatase and attenuate apoptosis of follicles and other factors may interface with the insulin-like growth factor (IGF) system preventing arrested follicles from becoming atretic and preventing the selection of a dominant follicle. IGF-binding protein concentrations are decreased by insulin, freeing biologically active IGF-I which augments the action of luteinizing hormone (LH) by inducing LH receptors, hyperactivating the enzymes P450c17α and 17,20 lyase resulting in hyperandrogenism. Growth hormone itself may be involved in the pathophysiology, as in normoinsulinemic PCOS patients it is hypersecreted and its actions on growth factors and their binding proteins are similar to those of insulin.

Aromatase mRNA expression in individual follicles from polycystic ovaries.

Polycystic ovary syndrome (PCOS) is a common reproductive disorder characterized by arrested follicular development prior to selection of a dominant follicle. Dominant follicles produce large amounts of oestradiol but PCOS follicles do not. With several potential aromatase (P450AROM) inhibitors in follicular fluid, the question arises whether P450AROM is expressed in PCOS granulosa cells, but the activity is inhibited, or whether P450AROM is not expressed in PCOS. The purpose of the present study was to determine whether P450AROM mRNA expression is altered in PCOS and to correlate P450AROM mRNA expression in individual follicles with aromatase stimulatory bioactivity and oestradiol in the follicular microenvironments. P450AROM mRNA was measured in individual follicles from 16 PCOS and 48 regularly cycling control women by quantitative polymerase chain reaction (PCR) and correlated with follicular fluid oestradiol concentrations and aromatase stimulating bioactivity measured by the rat granulosa cells aromatase bioassay. Follicular fluid oestradiol was low in all control follicles <7 mm in diameter. Some follicles > or = 7 mm contained elevated oestradiol values (P < 0.01) and all had an androstenedione:oestradiol ratio of <4. Only in granulosa cells from follicles > or = 7 mm with an androstenedione:oestradiol ratio of <4 were P450AROM mRNA levels increased (P < 0.05). These same follicles also contained increased levels of aromatase stimulating bioactivity whereas follicles <7 mm or with androstenedione:oestradiol ratio of >4 contained little or no bioactivity. All PCOS follicles contained low levels of oestradiol, P450AROM mRNA and aromatase stimulating bioactivity similar to size-matched control follicles. These data indicate that P450AROM mRNA expression and oestradiol production begin in developing follicles when they reach approximately 7 mm in diameter. Oestradiol production is low in PCOS follicles because there is insufficient aromatase stimulating bioactivity to increase P450AROM mRNA expression.

Regulation of arachidonic acid metabolism, aromatase activity and growth in human breast cancer cells by interleukin-1beta and phorbol ester: dissociation of a mediatory role for prostaglandin E2 in the autocrine control of cell function.

Prostaglandin E2 (PGE2) levels are elevated in malignant human breast tissue. However, the cellular mechanisms regulating this arachidonate metabolism and the autocrine influence PGE2 production may have on breast cancer cell growth and function are unclear. In the present study, we have investigated the effects of 2 putative cyclo-oxygenase inducers, interleukin-1beta (IL-1beta) and the protein kinase C agonist 12-O-tetradecanoyl-phorbol-13-acetate (TPA), on PGE2 production, growth and aromatase activity in the MDA MB 231 breast cancer cell line. TPA stimulated a dose-dependent increase in PGE2 production, inhibited cell growth and stimulated aromatase activity. Although IL-1beta alone had no effect on any of these breast cancer cell functions, the cytokine greatly potentiated PGE2 production in the presence of TPA. Similarly, growth inhibition and aromatase stimulation in response to TPA were both further enhanced by IL-1beta treatment. Indomethacin and dexamethasone both prevented PGE2 production in response to IL-Ibeta and TPA but had no effect on the anti-proliferative action of the cytokine and phorbol ester. While indomethacin had no effect on induction of aromatase activity by IL-1beta and TPA, dexamethasone exhibited a temporally biphasic action. Dexamethasone alone stimulated aromatase activity and demonstrated a permissive action on aromatase stimulation by IL-1beta and TPA. However, pre-treatment of cells with dexamethasone prevented subsequent induction of aromatase activity by IL-1beta and TPA. Our study describes a novel synergistic interaction in response to protein kinase C activation and IL-1beta during the regulation of arachidonate metabolism, cell growth and aromatase activity in human breast cancer cells. We conclude that the cyclooxygenase pathway does not play a mediatory role during the inhibition of cell growth and the induction of aromatase activity by IL-1beta and TPA.

Estradiol production by granulosa cells of normal and polycystic ovaries: relationship to menstrual cycle history and concentrations of gonadotropins and sex steroids in follicular fluid.

The underlying cause of anovulation in polycystic ovary syndrome is unknown. Circulating levels of immuno- and bioactive FSH are within the normal range, and the follicles contain measurable levels of bioactive FSH. The aim of this study was to compare estradiol (E2) production in response to FSH by granulosa cells from normal ovaries with those from polycystic ovaries derived from both anovulatory (anovPCO) and ovulatory subjects (ovPCO). Intrafollicular levels of immunoactive FSH, E2, and androstenedione in follicles of less than 12 mm were also measured. Follicular fluid steroid concentrations were obtained from 41 pairs of normal ovaries and 23 pairs of polycystic ovaries (8 anovPCO and 15 ovPCO). In size-matched follicles from each group there were no significant differences in follicular fluid FSH or E2 concentrations, but androstenedione levels were significantly higher in 5- to 11-mm follicles from ovPCO than in corresponding follicles from normal ovaries. Dose responses to FSH were determined in granulosa cells derived from 9 pairs of normal ovaries, 7 anovPCO, and 8 ovPCO. Cells from anovPCO produced 6- to 10-fold more E2 in response to FSH than normal cells, although there was no significant difference in the ED50 values. The response in cells from ovPCO was reduced compared to normal, but this difference did not reach significance. In summary, as judged by their FSH and E2 contents, polycystic ovaries do not have a higher proportion of atretic follicles than normal. Indeed, cells from anovPCO are hyperesponsive to FSH in vitro. This could be explained by stimulation of aromatase in vivo by either paracrine or, more probably, by endocrine factors, of which insulin is an arguable candidate.

Molecular composition of two different batches of urofollitropin: analysis by immunofluorimetric assay, radioligand receptor assay and in vitro bioassay.

The molecular heterogeneity of two different batches of commercially available urofolitropin was analyzed after fractionation by isoelectric focusing (IEF). FSH was measured before and after IEF by a highly specific time-resolved immunofluorimetric assay (IFMA), by a radioligand receptor assay (RRA) employing a preparation of calf testis FSH receptors, and by the in vitro bioassay based on FSH-dependent aromatase stimulation in immature rat Sertoli cells. An overall good correspondence between the results obtained with the three different methods was observed. However, the RRA and the in vitro bioassay appeared to be more suitable than the IFMA in resolving individual FSH isoforms. The mean isoelectric points of the two FSH preparations analyzed were slightly different, due to different molecular composition. These differences, however, seem too minute to be considered as cause of the different pharmacokinetics of FSH described in the literature or to explain the inconsistent therapeutical results seen in patients treated with FSH of urinary origin.

Abnormal Induction of Aromatase Activity by Dexamethasone in Fibroblasts from a Patient with Cortisol Resistance*

Cortisol resistance is a rare condition due to abnormal glucocorticoid receptor function. Stimulation of aromatase activity by dexamethasone (DEX) in cultured human skin fibroblasts provides a model for studying the biological activity of glucocorticoid receptors in cells. Skin fibroblasts derived from an affected father and his less severely affected son with cortisol resistance were used for this study. Saturation analysis of DEX receptor binding was performed after incubation of cells with various DEX concentrations (1-50 nmol/L). In normal cells, the mean maximal binding capacity (Bmax) and dissociation constant (Kd) were 24 +/- 3 pmol/mg DNA and 14 +/- 3 nmol/L, respectively. Although the Bmax in cells of the father (33 +/- 2 pmol/mg DNA) was normal, the Kd (31 +/- 7 nmol/L) was abnormally elevated. By contrast, both the Bmax and Kd in cells of the son were normal. The dose response of aromatase activity to DEX stimulation was determined by assay of aromatase activity after incubation of cells in the absence or presence of DEX (0.25-500 nmol/L) for 14 h. In three strains of normal fibroblasts, the mean concentration of DEX that produced a half-maximal response was 6 +/- 1 nmol/L). In cells from the father, the mean concentration of DEX that produced half-maximal stimulation of aromatase (27 +/- 4 nmol/L) was abnormally elevated. By contrast, the concentration of DEX that half-maximally stimulated aromatase activity (6.0 and 5.9 nmol/L) was normal in cells from the son. These data provide additional evidence of abnormal glucocorticoid action in the father, but not in his son, and demonstrate the potential usefulness of determining aromatase induction by DEX as a means of assessing the biological activity of the glucocorticoid receptor.

Down-regulation of the glucocorticoid receptor by dexamethasone in cultured human skin fibroblasts: implications for the regulation of aromatase activity.

Human genital skin fibroblasts grown in cell culture contain aromatase activity and glucocorticoid receptors, providing a model for studying the relationship between changes in levels of dexamethasone (DEX) receptor binding and changes in the response of aromatase to DEX. Incubation of cells with media containing DEX produced a time-dependent decline in receptor number, with a nadir at 18-24 h. When DEX was removed from the media, only 40-60% of the lost receptor content was recovered. Binding of DEX to its receptor declined in a dose-dependent fashion after 24-h exposure to this glucocorticoid; the concentration of DEX that produced a half-maximal decline (2.2 nmol/L) was in the same range as the Kd for the receptor-DEX complex (14 nmol/L). Incubation of cells with DEX for 20 h also reduced the level of DEX receptor binding in purified nuclei and nuclear matrix by 76% and 89%, respectively. When subcellular fractions were prepared after incubation of cells with DEX for 1-24 h, whole cell, cytosolic and nuclear DEX receptor binding declined in parallel with time. Incubation of skin fibroblasts with DEX resulted in progressive stimulation of aromatase activity, with a peak at 24 h followed by a return to baseline at 72 h. The initial stimulation of aromatase was mediated by DEX receptors. However, the decline in aromatase activity after prolonged DEX exposure did not appear to be due to a decline in the level of DEX receptor binding. The data supporting this last conclusion included the following. When cells were washed free of DEX after 48 h, DEX receptor binding recovered within 24 h, whereas aromatase activity could not be maximally restimulated until 36 h; when cells were incubated with media containing DEX and cycloheximide for 1-48 h, DEX receptor binding declined to a nadir by 24 h, whereas aromatase activity rose continuously up to 48 h. These findings are consistent with the concept that the aromatase gene in skin fibroblasts is subject to both positive and negative regulation.

Time-dependent biphasic response of aromatase to dexamethasone in cultured human skin fibroblasts.

Human genital skin fibroblasts grown in cell culture possess aromatase activity and, therefore, provide a model to investigate the molecular mechanisms that control aromatase in extraglandular tissues. Following the observation by other investigators that glucocorticoids stimulated aromatase activity in cultured stromal-vascular cells from adipose tissue, we examined the influence of dexamethasone (DEX) on aromatase in cultured skin fibroblasts. Preincubation of skin fibroblasts with DEX stimulated aromatase expression in all cell strains. In time-course studies, aromatase activity showed a biphasic curve, with peak levels at 12 h and a return to baseline levels by 72 h. When DEX was removed after 12 h, aromatase activity could be completely restimulated by DEX only after a period of 60-72 h. The DEX stimulation appeared to involve glucocorticoid receptor function, since the concentration of DEX required for half-maximal stimulation of aromatase activity (4.2 nM) was similar to the dissociation constant (Kd, 4.3 nM) of the receptor (for DEX). Actinomycin D and cycloheximide (CHX) inhibited DEX stimulation of aromatase when they were present in the preincubation and assay media. When cells were preincubated with DEX and CHX and then washed free of CHX and DEX before the assay, superinduction of aromatase activity occurred. Our data concerning the time course and superinduction of aromatase activity by DEX are in contrast to the findings reported by others for adipose tissue stromal-vascular cells and suggest that the mechanisms for the control of aromatase in extraglandular tissue may vary significantly in different tissues.