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.

Estrogen regulates expression of tumor necrosis factor receptors in breast adipose fibroblasts.

In breast cancer, a dense layer of undifferentiated fibroblasts is formed around malignant breast epithelial cells and referred to as desmoplastic reaction. These cells provide structural and functional support for tumor growth. Aromatase, the key enzyme in the biosynthesis of estrogen, is overexpressed in these undifferentiated fibroblasts, producing large quantities of estrogen, which in turn influences the growth and progression of malignant epithelial cells. We previously demonstrated that malignant epithelial cells produce large amounts of TNFalpha, which inhibit the differentiation of breast fibroblasts. TNF action is mediated by its two receptors (TNFRs), TNFR1, which mediates inhibition of adipocyte differentiation, and TNFR2, which was linked to the proliferation of thymocytes. We present evidence here that estrogen modulates the synthesis of receptors for TNF in human adipose fibroblasts (HAFs) from breast tissue in a paracrine fashion, which may serve as a mechanism for the inhibition of adipocyte differentiation in breast cancer. Estradiol (E(2)) treatment increased TNFR1 mRNA and protein levels in primary HAFs in a dose- and time-dependent manner, which could be reversed by the estrogen antagonist ICI182,780. Interestingly, higher concentration of E(2) inhibited whereas lower concentrations stimulated TNFR2 mRNA levels in HAFs. To investigate the specific roles of TNFRs in adipocyte differentiation, we incubated breast HAFs with receptor selective muteins of TNF. TNFR1-selective mutein decreased mRNA levels of aP2, a marker for adipogenic differentiation. This antiadipogenic effect was enhanced by cotreatment with E(2). We conclude that high levels of estrogen found in breast tumors promote the antiadipogenic action of TNF on breast adipose fibroblasts by selectively up-regulating TNFR1, which may be a critical mechanism for desmoplastic reaction.

Aromatase and endometriosis.

Aromatase p450 (p450arom) is the key enzyme for biosynthesis of estrogen, which is an essential hormone for the establishment and growth of endometriosis. There is no detectable aromatase enzyme activity in normal endometrium; therefore, estrogen is not locally produced in endometrium. Endometriosis tissue, however, contains very high levels of aromatase enzyme, which leads to production of significant quantities of estrogen. Moreover, one of the best-known mediators of inflammation and pain, prostaglandin E (2), strikingly induces aromatase enzyme activity and formation of local estrogen in this tissue. Additionally, estrogen itself stimulates cyclo-oxygenase-2 and therefore increases the formation of prostaglandin E (2) in endometriosis. We were able to target this positive feedback cycle in endometriosis using aromatase inhibitors. In fact, pilot trials showed that aromatase inhibitors could decrease pelvic pain associated with endometriosis.

Estrogen up-regulates cyclooxygenase-2 via estrogen receptor in human uterine microvascular endothelial cells.

OBJECTIVE: To investigate the effects of 17beta-estradiol (E(2)) on cyclooxygenase-2 (COX-2) expression and prostaglandin E(2) (PGE(2)) synthesis in primary human uterine microvascular endothelial cells (HUMEC). DESIGN: Prospective study. SETTING: Basic research laboratory at an academic medical center. PATIENT(S): Primary HUMEC of three women donors and primary human dermal microvascular endothelial cells of three women donors (as control), purchased from a third-party source. INTERVENTION(S): The HUMEC were cultured in specific media in a humidified atmosphere with 5% CO(2) at 37 degrees C. MAIN OUTCOME MEASURE(S): Measures of COX-2 mRNA and protein, PGE(2) production, and estrogen receptor alpha and beta mRNA and protein. RESULT(S): Treatment with E(2) (10(-10) to 10(-6) M) increased COX-2 mRNA levels by 2.3-fold to 2.4-fold in HUMEC. Treatment of HUMEC with E(2) (10(-8) M) resulted in a time-dependent increase of COX-2 mRNA levels. This was accompanied by a 2.8-fold increase in COX-2 protein level and a 1.5-fold increase in PGE(2) synthesis. Pretreatment of HUMEC with a selective COX-2 inhibitor, NS-398, abolished E(2)-induced PGE(2) synthesis, suggesting that E(2) specifically up-regulates COX-2 activity. The estrogen receptor antagonist ICI 182,780 fully reversed the stimulation of COX-2 mRNA and protein levels and PGE(2) synthesis by E(2). Interestingly, estrogen receptor beta mRNA and protein were abundant in HUMEC, whereas estrogen receptor alpha mRNA or protein was barely detectable. CONCLUSION(S): We conclude that various levels of E(2) can significantly increase COX-2 expression and PGE(2) synthesis in HUMEC via the estrogen receptor.

A novel role of sodium butyrate in the regulation of cancer-associated aromatase promoters I.3 and II by disrupting a transcriptional complex in breast adipose fibroblasts.

The aromatase gene encodes the key enzyme for estrogen formation. Aromatase enzyme inhibitors eliminate total body estrogen production and are highly effective therapeutics for postmenopausal breast cancer. A distal promoter (I.4) regulates low levels of aromatase expression in tumor-free breast adipose tissue. Two proximal promoters (I.3/II) strikingly induce in vivo aromatase expression in breast fibroblasts surrounding malignant cells. Treatment of breast fibroblasts with medium conditioned with malignant breast epithelial cells (MCM) or a surrogate hormonal mixture (dibutyryl (Bt2)cAMP plus phorbol diacetate (PDA)) induces promoters I.3/II. The mechanism of promoter-selective expression, however, is not clear. Here we reported that sodium butyrate profoundly decreased MCM- or Bt2cAMP + PDA-induced promoter I.3/II-specific aromatase mRNA. MCM, Bt2cAMP + PDA, or sodium butyrate regulated aromatase mRNA or activity only via promoters I.3/II but not promoters I.1 or I.4 in breast, ovarian, placental, and hepatic cells. Mechanistically, recruitment of phosphorylated ATF-2 by a CRE (-211/-199, promoter I.3/II) conferred inductions by MCM or Bt2cAMP + PDA. Chromatin immunoprecipitation-PCR and immunoprecipitation-immunoblotting assays indicated that MCM or Bt2cAMP + PDA stabilized a complex composed of phosphorylated ATF-2, C/EBPbeta, and cAMP-response element-binding protein (CREB)-binding protein in the common regulatory region of promoters I.3/II. Overall, histone acetylation patterns of promoters I.3/II did not correlate with sodium butyrate-dependent silencing of promoters I.3/II. Sodium butyrate, however, consistently disrupted the activating complex composed of phosphorylated ATF-2, C/EBPbeta, and CREB-binding protein. This was mediated, in part, by decreased ATF-2 phosphorylation. Together, these findings represent a novel mechanism of sodium butyrate action and provide evidence that aromatase activity can be ablated in a signaling pathway- and cell-specific fashion.

Regulation of aromatase expression in estrogen-responsive breast and uterine disease: from bench to treatment.

A single gene encodes the key enzyme for estrogen biosynthesis termed aromatase, inhibition of which effectively eliminates estrogen production. Aromatase inhibitors successfully treat breast cancer and endometriosis, whereas their roles in endometrial cancer, uterine fibroids, and aromatase excess syndrome are less clear. Ovary, testis, adipose tissue, skin, hypothalamus, and placenta express aromatase normally, whereas breast and endometrial cancers, endometriosis, and uterine fibroids overexpress aromatase and produce local estrogen that exerts paracrine and intracrine effects. Tissue-specific promoters distributed over a 93-kilobase regulatory region upstream of a common coding region alternatively control aromatase expression. A distinct set of transcription factors regulates each promoter in a signaling pathway- and tissue-specific manner. Three mechanisms are responsible for aromatase overexpression in a pathologic tissue versus its normal counterpart. First, cellular composition is altered to increase aromatase-expressing cell types that use distinct promoters (breast cancer). Second, molecular alterations in stromal cells favor binding of transcriptional enhancers versus inhibitors to a normally quiescent aromatase promoter and initiate transcription (breast/endometrial cancer, endometriosis, and uterine fibroids). Third, heterozygous mutations, which cause the aromatase coding region to lie adjacent to constitutively active cryptic promoters that normally transcribe other genes, result in excessive estrogen formation owing to the overexpression of aromatase in many tissues.