Gut microbiota-derived short-chain fatty acids protect against the progression of endometriosis.

Worldwide, ∼196 million are afflicted with endometriosis, a painful disease in which endometrial tissue implants and proliferates on abdominal peritoneal surfaces. Theories on the origin of endometriosis remained inconclusive. Whereas up to 90% of women experience retrograde menstruation, only 10% develop endometriosis, suggesting that factors that alter peritoneal environment might contribute to endometriosis. Herein, we report that whereas some gut bacteria promote endometriosis, others protect against endometriosis by fermenting fiber to produce short-chain fatty acids. Specifically, we found that altered gut microbiota drives endometriotic lesion growth and feces from mice with endometriosis contained less of short-chain fatty acid and n-butyrate than feces from mice without endometriosis. Treatment with n-butyrate reduced growth of both mouse endometriotic lesions and human endometriotic lesions in a pre-clinical mouse model. Mechanistic studies revealed that n-butyrate inhibited human endometriotic cell survival and lesion growth through G-protein-coupled receptors, histone deacetylases, and a GTPase activating protein, RAP1GAP. Our findings will enable future studies aimed at developing diagnostic tests, gut bacteria metabolites and treatment strategies, dietary supplements, n-butyrate analogs, or probiotics for endometriosis.

Estrogen receptor alpha (Esr1) regulates aromatase (Cyp19a1) expression in the mouse brain.

OBJECTIVES: Local estrogen production in the brain regulates critical functions including neuronal development, gonadotropin secretion and sexual behavior. In the mouse brain, a 36 kb distal promoter (l.f) regulates the Cyp19a1 gene that encodes aromatase, the key enzyme for estrogen biosynthesis. In vitro, promoter l.f interacts with estrogen receptor alpha (Esr1) to mediate Cyp19a1 mRNA expression and enzyme activity in mouse hypothalamic neuronal cell lines. The in vivo mechanisms that control mammalian brain aromatase expression during fetal and adult development, however, are not thoroughly understood. Our aim was to elucidate the basis of the in vivo connection between Esr1 and Cyp19a1. METHODS: Pregnant mice were sacrificed at gestational days 9, 11, 13, 15, 16, 19, 21 and the brain tissues of the fetuses were harvested along with five newborns at the age of postnatal day 2. Esr1KO (female) were also sacrificed and their hypothalamus were excised out. Then both fetuses and adults RNA were isolated, reverse transcribed and amplified employing primers specific for Esr1 and Cyp19a1 with Real time PCR. RESULTS: In the fetal mouse brain, Cyp19a1 mRNA levels are inversely correlated with estrogen receptor alpha (Esr1) mRNA levels in a temporal manner. Moreover, Cyp19a1 mRNA levels increased in the hypothalamus of estrogen receptor-alpha knockout female mice (Esr1KO). CONCLUSION: Taken together, our findings might indicate that Esr1 has crucial roles in the in vivo regulation of aromatase expression in the brain during fetal and adult life.

Expression of estrogen-related gene markers in breast cancer tissue predicts aromatase inhibitor responsiveness.

Aromatase inhibitors (AIs) are the most effective class of drugs in the endocrine treatment of breast cancer, with an approximate 50% treatment response rate. Our objective was to determine whether intratumoral expression levels of estrogen-related genes are predictive of AI responsiveness in postmenopausal women with breast cancer. Primary breast carcinomas were obtained from 112 women who received AI therapy after failing adjuvant tamoxifen therapy and developing recurrent breast cancer. Tumor ERα and PR protein expression were analyzed by immunohistochemistry (IHC). Messenger RNA (mRNA) levels of 5 estrogen-related genes-AKR1C3, aromatase, ERα, and 2 estradiol/ERα target genes, BRCA1 and PR-were measured by real-time PCR. Tumor protein and mRNA levels were compared with breast cancer progression rates to determine predictive accuracy. Responsiveness to AI therapy-defined as the combined complete response, partial response, and stable disease rates for at least 6 months-was 51%; rates were 56% in ERα-IHC-positive and 14% in ERα-IHC-negative tumors. Levels of ERα, PR, or BRCA1 mRNA were independently predictive for responsiveness to AI. In cross-validated analyses, a combined measurement of tumor ERα and PR mRNA levels yielded a more superior specificity (36%) and identical sensitivity (96%) to the current clinical practice (ERα/PR-IHC). In patients with ERα/PR-IHC-negative tumors, analysis of mRNA expression revealed either non-significant trends or statistically significant positive predictive values for AI responsiveness. In conclusion, expression levels of estrogen-related mRNAs are predictive for AI responsiveness in postmenopausal women with breast cancer, and mRNA expression analysis may improve patient selection.

Aromatase promoter I.f is regulated by progesterone receptor in mouse hypothalamic neuronal cell lines.

Aromatase catalyzes the conversion of C(19) steroids to estrogens. Aromatase and progesterone, both of which function at different steps of steroidogenesis, are crucial for the sexually dimorphic development of the fetal brain and the regulation of gonadotropin secretion and sexual interest in adults. The aromatase gene (Cyp19a1) is selectively expressed in distinct neurons of the mouse hypothalamus through a distal brain-specific promoter, I.f, located ∼40 kb upstream of the coding region. However, the regulation of aromatase expression in the brain is not well understood. In this study, we investigated a short feedback effect of progesterone analogues on aromatase mRNA expression and enzyme activity in estrogen receptor α (Esr1)-positive or -negative mouse embryonic hypothalamic neuronal cell lines that express aromatase via promoter I.f. In a hypothalamic neuronal cell line that highly expresses aromatase, progesterone receptor (Pgr), and Esr1, a progesterone agonist, R5020, inhibited aromatase mRNA level and enzyme activity. The inhibitory effect of R5020 was reversed by its antagonist, RU486. Deletion mutants of promoter I.f suggested that inhibition of aromatase expression by progesterone is conferred by the nt -1000/-500 region, and R5020 enhanced binding of Pgr to the nt -800/-600 region of promoter I.f. Small interfering RNA knockdown of Pgr eliminated progesterone-dependent inhibition of aromatase mRNA and enzyme activity. Taken together, progesterone enhances recruitment of Pgr to specific regions of the promoter I.f of Cyp19a1 and regulates aromatase expression in hypothalamic neurons.

Aromatase promoter I.f is regulated by estrogen receptor alpha (ESR1) in mouse hypothalamic neuronal cell lines.

Aromatase (CYP19A1) catalyzes the conversion of C(19) steroids to estrogens. Aromatase and its product estradiol (E(2)) are crucial for the sexually dimorphic development of the fetal brain and the regulation of gonadotropin secretion and sexual interest in adults. The regulation of aromatase expression in the brain is not well understood. The aromatase (Cyp19a1) gene is selectively expressed in distinct neurons of the hypothalamus through a distal brain-specific promoter I.f located approximately 36 kb upstream of the coding region. Here, we investigated a short feedback effect of E(2) on aromatase mRNA expression and enzyme activity using estrogen receptor alpha (ESR1; also known as ER alpha)-positive or ESR1-negative mouse embryonic hypothalamic neuronal cell lines that express aromatase via promoter I.f. Estradiol regulated aromatase mRNA expression and enzyme activity in a time- and dose-dependent manner, whereas an E(2) antagonist reversed these effects. The nucleotide -200/-1 region of promoter I.f conferred E(2) responsiveness. Two activator protein 1 (AP-1) elements in this region were essential for induction of promoter activity by E(2). ESR1 and JUN (c-Jun) bound to these AP-1 motifs in intact cells and under cell-free conditions. The addition of an ESR1 mutant that interacts with JUN but not directly with DNA enhanced E(2)-dependent promoter I.f activity. Independently, we demonstrated an interaction between ESR1 and JUN in hypothalamic cells. Knockdown of ESR1 abolished E(2)-induced aromatase mRNA and enzyme activity. Taken together, E(2) regulates Cyp19a1 expression via promoter I.f by enhanced binding of an ESR1/JUN complex to distinct AP-1 motifs in hypothalamic cells. We speculate that this mechanism may, in part, regulate gonadotropin secretion and sexual activity.

Novel promoter I.8 and promoter usage in the CYP19 (aromatase) gene.

To date, 10 promoters were reported to regulate the expression of the human aromatase (CYP19) gene, giving rise to transcripts with an identical coding region but tissue-specific first exons comprising unique 5'-untranslated regions. We describe the identification and characterization of a new CYP19 exon I, designated exon I.8, in a 5'-rapid amplification of complementary DNA ends-generated library of human THP-1 monocytic cells. A construct containing exon I.8 and its 5'-flanking sequence was sufficient to drive transcription in THP-1 cells. This novel promoter was located approximately 2-kb upstream of promoter I.4 and approximately 75-kb upstream of the common splice junction. We detected several I.8-containing splice variants, 2 of which also contained a sequence from exon I.4. Analysis of human tissues revealed a unique pattern of promoter I.8 usage. The placenta contained the highest level of I.8-specific transcripts. This work underscores the complexity of the mechanisms that regulate normal and pathologic aromatase expression.

Aromatase expression in uterine leiomyomata is regulated primarily by proximal promoters I.3/II.

CONTEXT: Uterine leiomyomata are common tumors that cause irregular uterine bleeding and pregnancy loss and depend on estrogen for growth. Aromatase catalyzes the conversion of androgens to estrogens. Aromatase expression is regulated via alternatively used promoters in the placenta (I.1 and I.2a), fat (I.4, I.3, and II), bone (I.6), and gonads (II). A prostaglandin E(2)/cAMP-dependent pathway regulates coordinately the proximal promoters I.3/II, whereas glucocorticoids and cytokines regulate the distal promoter I.4. Use of each promoter gives rise to a population of aromatase mRNA species with unique 5'-untranslated regions (5'-UTRs). Uterine leiomyoma tissue, but not normal myometrium, overexpresses aromatase leading to estrogen-stimulated cell proliferation. Aromatase inhibitor treatment shrank uterine leiomyomata in a few women. OBJECTIVE AND DESIGN: Promoter I.4 was reported to regulate aromatase expression in uterine leiomyomata from a group of Japanese women. Here, we used two independent techniques to identify the promoters that regulate aromatase expression in uterine leiomyomata (n = 30) from 23 African-American, Hispanic, and white women. RESULTS: Rapid amplification of 5'-cDNA ends of aromatase mRNA species revealed the following distribution of promoter usage in leiomyomata: promoters I.3/II, 61.5%; I.2a, 15.4%; I.6, 15.4%; and I.4, 7.7%. Real-time PCR, which quantifies mRNA species with promoter-specific 5'-UTRs, revealed the following distribution for each 5'-UTR as a fraction of total aromatase mRNA: I.3/II, 69.6%; I.4, 7.3%; and other promoters, 23.1%. CONCLUSIONS: The primary in vivo aromatase promoter in leiomyoma tissues in non-Asian U.S. women is the prostaglandin E(2)/cAMP-responsive I.3/II region. Alternative signals may stimulate aromatase expression that is a common biological phenotype in uterine leiomyomata.

Paracrine-stimulated gene expression profile favors estradiol production in breast tumors.

Paracrine interactions between adipose fibroblasts and malignant epithelial cells are essential for structural and hormonal support of breast tumors. Factors derived from malignant epithelial cells inhibit adipogenic differentiation of fibroblasts and upregulate expression of aromatase, which stimulates estrogen synthesis and creates a localized, growth-stimulatory environment. Here, we characterized the gene expression profile of breast adipose fibroblasts in an in vitro model of malignancy to identify other paracrine interactions that support tumor growth. Primary breast adipose fibroblasts from cancer-free women were treated with conditioned media from malignant breast epithelial cells or normal breast epithelial cells, and differences in gene expression were identified by microarray. A total of 79 differentially regulated genes encoding cytokines, enzymes, angiogenic factors, cytoskeletal proteins, extra-cellular matrix remodeling proteins, signal transduction proteins and cell surface receptors were identified, and 6 of these were verified by real-time PCR. Among these, the expression of aldo-keto reductase family 1, member C3 (AKR1C3) was upregulated. AKR1C3 has multiple enzymatic properties, including conversion of estrone to estradiol and androstenedione to testosterone. Immunoreactive AKR1C3 was detected in epithelial and stromal components of benign lesions and ductal carcinomas in situ, and in 59.8% of epithelial and 69.6% of stromal cells in invasive breast carcinomas. AKR1C3 expression was significantly higher in myoepithelial cells surrounding the neoplastic epithelium of ductal carcinoma in situ compared with those surrounding benign epithelial lesions. Importantly, AKR1C3 and aromatase mRNA levels correlated positively in 61 malignant breast tumors (R=0.3967, p=0.00156). Malignant epithelial cell-conditioned medium significantly increased formation of testosterone and estradiol from androstenedione in breast adipose fibroblasts. In conclusion, malignant epithelial cell-derived factors significantly upregulate the enzymes AKR1C3 and aromatase that catalyze a series of complementary reactions to convert the circulating precursor androstenedione to biologically active estradiol in vitro in the stromal fibroblasts, and in vivo, in stromal component of breast tumors.