Masashi Demura

Aromatase excess in cancers of breast, endometrium and ovary

Pathogenesis and growth of three common womens cancers (breast, endometrium and ovary) are linked to estrogen. A single gene encodes the key enzyme for estrogen biosynthesis named aromatase, inhibition of which effectively eliminates estrogen production in the entire body. Aromatase inhibitors successfully treat breast cancer, whereas their roles in endometrial and ovarian cancers are less clear. Ovary, testis, adipose tissue, skin, hypothalamus and placenta express aromatase normally, whereas breast, endometrial and ovarian cancers overexpress aromatase and produce local estrogen exerting paracrine and intracrine effects. Tissue-specific promoters distributed over a 93-kb 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. In cancers of breast, endometrium and ovary, aromatase expression is primarly regulated by increased activity of the proximally located promoter I.3/II region. Promoters I.3 and II lie 215 bp from each other and are coordinately stimulated by PGE(2) via a cAMP-PKA-dependent pathway. In breast adipose fibroblasts exposed to PGE(2) secreted by malignant epithelial cells, PKC is also activated, and this potentiates cAMP-PKA-dependent induction of aromatase. Thus, inflammatory substances such as PGE(2) may play important roles in inducing local production of estrogen that promotes tumor growth.

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.

Cardiac Aldosterone Production in Genetically Hypertensive Rats

Aldosterone is synthesized in extra-adrenal tissues, both blood vessels and brain. We undertook the present study to determine whether the rat heart produces aldosterone and to investigate the effects of adrenalectomy, ACE inhibition, and angiotensin II on aldosterone synthesis in the heart. To clarify the pathophysiological role of cardiac aldosterone in the hypertensive heart, we compared the synthesis of aldosterone in the hearts of stroke-prone spontaneously hypertensive rats (SHRSP) with that in Wistar-Kyoto rats. The effects of the aldosterone antagonist spironolactone on myocardial hypertrophy in adrenalectomized SHRSP were also studied. Isolated rat hearts were perfused for 2 hours, and the perfusate was analyzed with HPLC and mass spectrometry. The activity of aldosterone synthase was estimated on the basis of the conversion of [14C]deoxycorticosterone to [14C]aldosterone. The levels of aldosterone synthase gene (CYP11B2) mRNA were determined with competitive polymerase chain reaction. Aldosterone production, the activity of aldosterone synthase, and the expression of CYP11B2 mRNA were increased in hearts from adrenalectomized rats and rats treated with angiotensin II. ACE inhibitors decreased cardiac aldosterone synthesis. Cardiac aldosterone, aldosterone synthase activity, and CYP11B2 mRNA levels in hearts from 2- and 4-week-old SHRSP were significantly greater than those of age-matched Wistar-Kyoto rats. Spironolactone prevented cardiac hypertrophy in adrenalectomized SHRSP. These results suggest that the rat heart produces aldosterone and that endogenous cardiac aldosterone may affect cardiac function and hypertrophy in hypertension in rats.

High Aromatase Expression in Uterine Leiomyoma Tissues of African-American Women

CONTEXT Symptomatic uterine leiomyoma is associated with irregular uterine bleeding, anemia, and recurrent pregnancy loss. African-American women develop uterine leiomyomas at an earlier age and with higher frequency compared with Caucasian-American women or other races; however, the underlying mechanism for this discrepancy is unknown. OBJECTIVE Our objective was to determine whether gene targets of emerging leiomyoma therapeutics such as aromatase inhibitors and antiprogestins, which reduce tumor size and symptoms, are differentially expressed in tissues of African-American (n = 31), Caucasian-American (n = 34), and Japanese women (n = 36). RESULTS We found strikingly higher aromatase mRNA levels in leiomyoma compared with adjacent myometrium in African-American (83 fold), Caucasian-American (38 fold), and Japanese women (33 fold). Among the four major promoters that regulate aromatase expression in leiomyoma, the proximal promoter II accounted for higher aromatase mRNA levels in tissues from African-American women. Estrogen receptor subtype alpha mRNA levels were significantly, and 1.8- to 2.6-fold, higher in leiomyoma compared with adjacent myometrium in all groups, whereas leiomyoma estrogen receptor subtype beta mRNA levels were significantly elevated only in Japanese women. Leiomyoma progesterone receptor mRNA levels were significantly higher in Japanese women compared with African-American or Caucasian-American women. CONCLUSIONS Leiomyoma tissues from African-American women contained the highest level of aromatase expression, which may result in elevated tissue concentrations of estrogen, and account for the higher prevalence and earlier incidence. Analysis of leiomyoma tissue for biomarkers may predict the response to hormonal treatments such as aromatase inhibitors.

Effects of high sodium intake on cardiovascular aldosterone synthesis in stroke-prone spontaneously hypertensive rats.

Objectives Aldosterone is synthesized in extra-adrenal tissues such as the vasculature, heart and brain. The mechanisms underlying the effect of high salt intake on the development and acceleration of vascular injury and cardiac hypertrophy in the stroke-prone spontaneously hypertensive rats (SHRSP) are still not clear. The goal of this study was to determine whether high salt intake increases cardiovascular aldosterone synthesis in SHRSP. Methods Four-week-old SHRSP were given tap water or 0.9% NaCl solution for hydration for 4 weeks in addition to a normal salt diet. Isolated rat mesenteric arteries and hearts were perfused for 2 h, and the perfusate was analysed by high-performance liquid chromatography. The concentrations of aldosterone synthase gene (CYP11B2) mRNA and angiotensin II receptor (AT1R) mRNA were determined by competitive polymerase chain reaction. Results Salt-loaded SHRSP had higher blood pressures than SHRSP with normal salt intake. Plasma aldosterone concentrations and plasma renin activity were decreased by high salt intake. Aldosterone production, the expression of CYP11B2 mRNA and AT1R mRNA in mesenteric arteries and hearts were significantly increased by high salt intake. Conclusions These results suggest that high salt intake increases aldosterone production and expression of the AT1R mRNA in the cardiovascular tissue in SHRSP, which may contribute to the development of malignant hypertension in salt-loaded SHRSP.

CpG dinucleotide methylation of the CYP19 I.3/II promoter modulates cAMP-stimulated aromatase activity.

Aromatase expression varies in a tissue-specific manner and among individuals. Aromatase promoter I.3/II, regulated by a cAMP response element (CRE), is normally quiescent in human skin fibroblasts, whereas its hyperactivity may cause local or systemic estrogen excess. We describe the methylation status of 6 CpG dinucleotides within a 571-bp fragment of promoter I.3/II containing a CRE in cAMP-responsive (n=1) or nonresponsive (n=3) primary skin fibroblasts cultured from healthy volunteers. Four out of 6 CpG dinucleotides were unmethylated in cAMP-responsive fibroblasts, whereas all 6 CpG dinucleotides were hypermethylated in cAMP-nonresponsive fibroblasts. Basal and cAMP-stimulated aromatase activity and promoter I.3/II activation were significantly higher in the presence of unmethylated DNA. Furthermore, methylation at the CRE interfered with CREB binding. Thus, methylation of CpG dinucleotides within promoter I.3/II regulates aromatase expression and may be one source of inter-individual variability. Furthermore, abnormal methylation of the aromatase promoter may contribute to aromatase overexpression in breast cancer.

Effect of mineralocorticoid receptor blockade on the renal renin-angiotensin system in Dahl salt-sensitive hypertensive rats.

Background The (pro)renin receptor exists in the kidney, blood vessels and the heart. (Pro)renin binds to the receptor and induces tissue injuries directly, completely independent of angiotensin II (Ang II). The renal renin–angiotensin–aldosterone system is activated in salt-sensitive hypertensive rats with in-vitro studies showing aldosterone increases angiotensin-converting enzyme (ACE) activity, renin production and angiotensin II type 1 receptor (AT1R) activity. However, the effect of blockade of mineralocorticoid receptor on the renal (pro)renin receptor, angiotensinogen, ACE and AT1R in Dahl salt-sensitive rats is unknown. Methods The following parameters were measured in Dahl salt-sensitive rats and in Dahl salt-resistant rats fed high-salt or low-salt diets and treated for 8 weeks with or without eplerenone (100 mg/kg per day, orally): blood pressure, plasma renin activity, plasma aldosterone concentration, kidney weight and Ang II contents, urinary protein excretion, glomerular injury (assessed by semiquantitative morphometric analysis) and levels of expression in the kidney of (pro)renin receptor protein and messenger RNA (mRNA) for angiotensinogen, ACE and AT1R. Results Dahl salt-sensitive rats fed a high-salt diet had increased kidney/body weight (175%) and urinary protein excretion (886%) and decreased plasma renin activity and plasma aldosterone concentration. The rats developed progressive sclerotic and proliferative glomerular changes, concomitant with increased expression of renal (pro)renin receptor protein and mRNA levels of angiotensinogen, ACE and AT1R and kidney Ang II content. Treatment with eplerenone in Dahl salt-sensitive rats was associated with significant improvements in kidney to body weight ratio, urinary protein excretion and renal injury scores and decreased renal (pro)renin receptor protein expression and angiotensinogen and AT1R mRNA levels and kidney Ang II content. Conclusion A high salt diet increased the renal renin–angiotensin system, whereas blockade of mineralocorticoid receptors attenuated renal injuries by decreasing the activity of tissue renin–angiotensin system in Dahl salt-sensitive rats.

Genetic analysis of the cytochrome P-450c17alpha (CYP17) and aldosterone synthase (CYP11B2) in Japanese patients with 17alpha-hydroxylase deficiency.

OBJECTIVE To determine the clinical and molecular genetic characterization of two Japanese patients with 17α‐hydroxylase deficiency, we analysed the 17α‐hydroxylase/17,20‐lyase gene (CYP17). Next, to clarify the mechanism of hypoaldosteronism in 17α‐hydroxylase deficiency, we analysed the expression of aldosterone synthase (CYP11B2) messenger RNA and sequenced CYP11B2 in these patients.

Dynamic CCAAT/Enhancer Binding Protein–Associated Changes of DNA Methylation in the Angiotensinogen Gene

DNA methylation patterns are maintained in adult somatic cells. Recent findings, however, suggest that all methylation patterns are not preserved. We demonstrate that stimulatory signals can change the DNA methylation status at a CCAAT/enhancer binding protein (CEBP) binding site and a transcription start site and activate expression of the angiotensinogen gene (AGT). A CEBP binding site in the human AGT promoter was hypomethylated in tissues with high expression of AGT, but not in those with low expression. The transcriptional activity of AGT promoter sequences cloned into a reporter plasmid depended on DNA methylation. In cultured human cells, interleukin 6 stimulation caused DNA demethylation around a CEBP binding site and a transcription start site; demethylation was accompanied by increased CEBP-&bgr; recruitment and chromatin accessibility of the AGT promoter. DNA methylation activity decreased in the nucleus. Excess circulating aldosterone upregulated AGT expression and was accompanied by DNA hypomethylation around a CEBP binding site and a transcription start site in human visceral adipose tissue. High salt intake led to upregulation of Agt expression, DNA hypomethylation around 2 CEBP binding sites and a transcription start site, and decreased DNA methylation activity in rat visceral adipose tissue. Taken together, CEBP binding initiates chromatin relaxation and transcription, which are followed by DNA demethylation around a CEBP binding site and a transcription start site in the AGT promoter. Decreased DNA methylation activity in the nucleus may play a role in DNA demethylation. DNA demethylation switches the phenotype of AGT expression from an inactive to an active state.

Unilateral Primary Aldosteronism with Spontaneous Remission after Long-Term Spironolactone Therapy

CONTEXT Medical treatment with a mineralocorticoid receptor (MR) antagonist, which has produced spontaneous remission of bilateral primary aldosteronism (PA), may also produce spontaneous remission of unilateral PA, for which laparoscopic adrenalectomy is recommended. However, few reports exist regarding spontaneous remission after MR antagonist therapy in unilateral PA. OBJECTIVE The aim of this paper is to report a case of unilateral PA with spontaneous remission and reduction of cardiac hypertrophy after long-term spironolactone (SP) therapy. RESULTS A 41-yr-old Japanese male was treated for hypertension and hypokalemia for 5 yr. Primary aldosteronism was diagnosed by a furosemide and upright posture test and a captopril challenge test. Computed tomography imaging showed a 5-mm left-sided adrenal mass. Adrenal vein sampling demonstrated overproduction of aldosterone from the left adrenal gland. Long-term treatment with SP normalized the plasma aldosterone concentration. After discontinuation of SP, the patients blood pressure, serum potassium level, and plasma aldosterone concentration remained in the normal range. The associated cardiac hypertrophy also improved and continued to resolve even after discontinuation of SP. Although the left adrenal gland tumor was still present on computed tomography after treatment, a furosemide and upright posture test, a captopril challenge test, and a saline loading test produced no evidence of PA. Adrenal vein sampling demonstrated no sign of lateralization. CONCLUSION These results demonstrate that SP not only antagonizes the MR, but also decreases aldosterone synthetic activity, which may produce remission in some patients with unilateral PA.