The Journal of Steroid Biochemistry and Molecular Biology, 2001
Fetal human osteoblast-like cells and the THP-1 cell line that differentiates into macrophage/ost... more Fetal human osteoblast-like cells and the THP-1 cell line that differentiates into macrophage/osteoblast-like cells in the presence of Vitamin D3 and which possesses high aromatase activity, constitute a useful model with which to study the regulation of aromatase in bone. We showed that dexamethasone (DEX)-induced aromatase activity in the THP-1 cell line is completely suppressed by forskolin and by dibutyryl cAMP. We therefore investigated the contribution of mitogen-activated protein kinase (MAPK) to the regulation of aromatase, because cAMP inhibits MAPK in many cells. We examined the role of MAPK on aromatase activity using PD98059, a selective inhibitor of MEK-1. PD98059 (100 microM) reduced DEX+interleukin (IL)-1beta-induced aromatase activity in human osteoblast-like cells by more than 90%, whereas 50% of the aromatase mRNA concentration was retained compared with the control incubated with DEX+IL-1beta. PD98059 (50 microM) reduced the activity of aromatase in THP-1 cells by 80% without significantly affecting the mRNA level. These results indicated that MAPK plays an important role in aromatase activation at the post-transcriptional level.
In leiomyoma of the uterus, both aromatase and 17beta-hydroxysteroid dehydrogenase (17beta-HSD) t... more In leiomyoma of the uterus, both aromatase and 17beta-hydroxysteroid dehydrogenase (17beta-HSD) type I are overexpressed compared with myometrium. This suggests that leiomyoma cells convert circulating androstenedione into estrone (via aromatase), then into the active form of estrogen, estradiol (via 17beta-HSD type I). In vitro experiments and several clinical findings support the notion that in situ estrogen plays a role in leiomyoma growth under hypoestrogenemic conditions, such as natural menopause and therapy with gonadotropin-releasing hormone (GnRH) agonists. GnRH agonists abolish estrogen production both in situ in leiomyoma and in the ovary, leading to quick and profound regression of the leiomyoma. Aromatase inhibitors also inhibit estrogen synthesis in both leiomyoma and the ovary and may be used therapeutically. Certain doses of competitive aromatase inhibitors would completely inhibit estrogen production in leiomyoma, whereas ovarian production of estrogen would continue at reduced levels. This may lead to advantageous therapeutic conditions in which leiomyoma regresses without adverse symptoms related to estrogen depletion because levels of ovarian estrogen would be insufficient to support leiomyoma growth but sufficient to prevent symptoms associated with deficiency. This article discusses the potential uses of aromatase inhibitors.
Reliable leak-proof aspiration of cyst contents is required for treatment of large ovarian cysts ... more Reliable leak-proof aspiration of cyst contents is required for treatment of large ovarian cysts by minilaparotomy. Through a small abdominal wound a transparent plastic bag was instantly mounted onto the cyst surface using an ethyl-2-cyanoacrylate adhesive. A 1-2-cm-wide cut was made in the consolidated cyst wall through the inside of the bag and the contents directly aspirated. The fluid was trapped inside the bag without leaking into the abdominal cavity. This method can also be applied to relatively small cysts by holding the cyst just beneath the wound. We used this method in 30 patients with unilateral ovarian cysts and in one patient with an ovarian cyst associated with an ipsilateral paraovarian cyst. All patients were successfully treated without spillage, although in one case a large mucinous ovarian cyst ruptured before surgery. Minilaparotomy using the instant adhesive is cost-effective, safe, reliable, and easily implemented. This procedure is also applicable to relatively small cysts and is a viable alternative to laparoscopic surgery for treatment of dermoid cysts showing considerable calcification.
It may be difficult to differentiate the consecutive occurrence of two independent molar pregnanc... more It may be difficult to differentiate the consecutive occurrence of two independent molar pregnancies from gestational trophoblastic neoplasia after the initial molar pregnancy, especially when the interval between them is short. A 25-year-woman who had had a complete hydatidiform mole 6 months earlier presented with a 6-week history of secondary amenorrhea. Serum human chorionic gonadotropin had increased to 19,857 micro-international units/mL, with no gestational sac demonstrated. Dilation and curettage was performed. Pathologic examination identified a tiny amount of hydropic villi compatible with complete hydatidiform mole. Analysis of short tandem repeat polymorphisms revealed that the molar tissues of the first and second complete hydatidiform moles were of different genetic origin. The patient went into remission spontaneously without chemotherapy. Genetic profiling was useful to discriminate a recurrent mole from suspected gestational trophoblastic neoplasia.
Gynecomastia of prepubertal onset may result from increased estrogen owing to excessive aromatase... more Gynecomastia of prepubertal onset may result from increased estrogen owing to excessive aromatase activity in extraglandular tissues. A gene in chromosome 15q21.2 encodes aromatase, the key enzyme for estrogen biosynthesis. Several physiologic tissue-specific promoters regulate the expression of aromatase, giving rise to messenger RNA (mRNA) species with an identical coding region but tissue-specific 5'-untranslated regions in placenta, gonads, brain, fat, and skin. We studied skin, fat, and blood samples from a 36-year-old man, his 7-year-old son, and an unrelated 17-year-old boy with severe gynecomastia of prepubertal onset and hypogonadotropic hypogonadism caused by elevated estrogen levels. Aromatase activity and mRNA levels in fat and skin and whole-body aromatization of androstenedione were severely elevated. Treatment with an aromatase inhibitor decreased serum estrogen levels and normalized gonadotropin and testosterone levels. The 5'-untranslated regions of aromatase mRNA contained the same sequence (FLJ) in the father and son and another sequence (TMOD3) in the unrelated boy; neither sequence was found in control subjects. These 5'-untranslated regions normally make up the first exons of two ubiquitously expressed genes clustered in chromosome 15q21.2-3 in the following order (from telomere to centromere): FLJ, TMOD3, and aromatase. The aromatase gene is normally transcribed in the direction opposite to that of TMOD3 and FLJ. Two distinct heterozygous inversions reversed the direction of the TMOD3 or FLJ promoter in the patients. Heterozygous inversions in chromosome 15q21.2-3, which caused the coding region of the aromatase gene to lie adjacent to constitutively active cryptic promoters that normally transcribe other genes, resulted in severe estrogen excess owing to the overexpression of aromatase in many tissues.
Intratumoral expression of aromatase P450 (P450arom) promotes the growth of breast tumors via inc... more Intratumoral expression of aromatase P450 (P450arom) promotes the growth of breast tumors via increased local estrogen concentration. We cloned a novel 101-bp untranslated first exon (I.7) that comprises the 5'-end of 29-54% of P450arom transcripts isolated from breast cancer tissues (n = 7). The levels of P450arom transcripts with exon I.7 were significantly increased in breast tumor tissues and adipose tissue adjacent to tumors. We identified a promoter immediately upstream of exon I.7 and mapped this to about 36 kb upstream of ATG translation start site of the CYP19 (aromatase cytochrome P450) gene. Sequence analysis of I.7 revealed a TATA-less promoter containing an initiator, two consensus GATA sites, and cis-regulatory elements found in megakaryocytes and endothelial type promoters. Luciferase activity directed by the promoter I.7 sequence (-299/+81 bp) was 4-fold greater than a minimum length promoter sequence (-35/+81 bp) in human microvascular endothelial cells (HMEC-1), but only 2-fold greater in MCF-7 breast malignant epithelial cells. There was no promoter activity in primary breast adipose fibroblasts. Site-directed mutations demonstrated that maximal basal promoter activity required two GATA motifs at -146/-141 bp and -196/-191 bp. Gel shift and deoxyribonuclease I footprinting assays demonstrated the binding of GATA-2 transcription factor but not GATA-1 to the -196/-191-bp region. Overexpression of GATA-2 in HMEC-1 cells increases promoter I.7 activity by 5-fold. In conclusion, promoter I.7 is a GATA-2-regulated endothelial promoter of the human CYP19 gene and may increase estrogen biosynthesis in vascular endothelial cells of breast cancer. The activity of this promoter may also be important for intracrine and paracrine effects of estrogen on blood vessels.
Recent evidence has shown that bone is not only a target of estrogen action but also a source of ... more Recent evidence has shown that bone is not only a target of estrogen action but also a source of local estrogen production. Bone cells such as osteoblasts express aromatase (P450arom) and the expression of P450arom in osteoblasts is positively regulated in a tissue specific fashion, as in the case of other tissues which express P450arom. To clarify the physiological factors regulating expression of P450arom in bone, we tested TGF-β1 using osteoblast-like cells obtained from human fetuses as well as THP-1 cells. TGF-β1 increased IL-1β+DEX- induced aromatase activity in osteoblast-like cells, while it inhibited activity in skin fibroblasts. Similar enhancement of aromatase activity by TGF-β1 was found in DEX-stimulated THP-1 cells and this cell line was used for further experiments. In THP-1 cells, TGF-β1 enhanced DEX-induced aromatase activity almost linearly by 12 h and thereafter. Increased levels of P450arom transcripts were also demonstrated by RT-PCR at 3 h of TGF-β1 treatment and thereafter. Cyclohexamide abolished enhancement of activity but did not inhibit the accumulation of P450arom transcripts induced by TGF-β1. Increase in P450arom expression by TGF-β1 was attributable to expression driven by promoter I.4. TGF-β1 did not change the half life of P450arom transcripts. To identify the cis-acting elements responsible for TGF-β1 action on aromatase expression, transient transfection assays were performed using a series of deletion constructs for promoter I.4 (P450-I.4/Luc). Two constructs (−410/+14 and−340/+14) that contain a functional glucocorticoid response element (GRE) and downstream sequence showed significant increase of luciferase activity in response to TGF-β1. Deletion and mutation of the GRE in P450-I.4/Luc (−340/+14) abolished the TGF-β1. The luciferase activity of a (GRE)1-SV40/Luc construct was also stimulated by TGF-β1. These results indicate that TGF-β1 increases the expression of P450arom at the level of transcription through promoter I.4, at least in part via an enhancement of transactivation activity of the GR in THP-1 cells. TGF-β1 is suggested to be one of the physiological up-regulatory factors of bone aromatase.
Estrogen plays a major role in bone mineral homeostasis, maintaining a balance between bone forma... more Estrogen plays a major role in bone mineral homeostasis, maintaining a balance between bone formation and bone resorption not only in women but also in men. Extraglandular aromatization of circulating androgen is the major source of estrogen in post-menopausal women and men. In order to assess the capacity of bone cells as a local source of estrogen, osteoblast-like cells (OLCs) were obtained from human fetal bone in mid-trimester by the explant method and by mechanical disaggregation. The integrity of OLCs was confirmed by their ability to produce alkaline phosphatase and osteocalcin in response to vitamin D3 and also by their ability to deposit mineral. Aromatase activity was assessed by the formation of estrone from [1,2,6,7-3H]androstenedione and by the release of tritium from [1beta-3H]androstenedione into [3H]water. Formation of estrone was confirmed by thin layer chromatography (TLC) in OLCs stimulated with dexamethasone (DEX) + oncostatin M. The aromatase activity was 10 x higher in non-passaged OLCs than in passaged cells in the presence or absence of the stimulants (DEX + IL-1beta). The apparent Km and Vmax estimated by the release of [3H]water was 5.8+/-0.6 nM and 10.8+/-1.4 pmol/mg per 6 h in the presence of DEX + IL-1beta. The effects of several stimulants on aromatase activity in OLCs were examined: serum, IL-1beta, TNFalpha and type I cytokines stimulated activity in the presence of DEX, while PMA and PMA + dibutyryl cAMP did not. To confirm the expression of aromatase in OLCs, cells prepared from periosteal membranes were also examined: These cells in culture possessed aromatase activity corresponding to OLCs prepared from bone specimens. Moreover, the fresh periosteum expressed aromatase at higher levels than that of metaphyseal specimens. The aromatase gene employs several different promoters (I.1, 1.2, I.3, I.4, I.5, I.6, 2a, 1f and PII) and the usage of these promoters is known to be controlled in a tissue-specific fashion. Accordingly, promoter usage in OLCs and fetal long bone (tibia) tissue was examined using the 5' rapid amplification of cDNA ends (RACE) technique. The major promoter used was I.4, not only in stimulated and non-stimulated OLCs, but also in fetal tibia. Some minor transcripts were also found: 1f (brain-specific promoter), PII and I.6 in OLCs stimulated by DEX + IL-1beta, and PII and I.3 in OLCs stimulated by DEX + serum. Fetal tibia also expressed I.3 (15%) and I.6 (10%). Thus, regulation and promoter usage in OLCs was quite different from other tissues known as estrogen sources including adipose tissue, ovary and placenta. These results suggest that bone is an extraglandular source of local estrogen which plays an important role in bone mineral metabolism through autocrine and paracrine actions.
Journal of the American Academy of Dermatology, 1994
The pyloric atresia--junctional epidermolysis bullosa (PA-JEB) syndrome is an autosomal recessive... more The pyloric atresia--junctional epidermolysis bullosa (PA-JEB) syndrome is an autosomal recessive disorder with a poor prognosis. Electron microscopy of fetal skin has been the only reliable method for prenatal diagnosis. The purpose of this study was to make the prenatal diagnosis of PA-JEB syndrome with a more reliable method by means of immunocytochemical probes. Expression of a range of basement membrane antigens was examined in different types of JEB. On the basis of the results, a fetal skin biopsy specimen was obtained for prenatal diagnosis. In PA-JEB syndrome (n = 2), GB3 antigen (BM600) was normally expressed; the 19-DEJ-1 antigen was completely absent. In fetal skin at risk for PA-JEB syndrome, the 19-DEJ-1 antigen was normally expressed, and no ultrastructural abnormality was found by electron microscopy. A normal male infant was delivered at 38 weeks of pregnancy. 19-DEJ-1 monoclonal antibody serves as a useful probe for the prenatal diagnosis of PA-JEB syndrome.
Journal of Obstetrics and Gynaecology Research, 2011
Metastasis of ovarian carcinoma to the small bowel parenchyma without peritoneal dissemination is... more Metastasis of ovarian carcinoma to the small bowel parenchyma without peritoneal dissemination is uncommon. A 63-year-old woman underwent surgery for a clear cell adenocarcinoma of the ovary and received adjuvant chemotherapy. Eighteen months after the operation, she presented with recurrent occult bowel hemorrhage without evidence of an abdominal mass. Nine months later, a rapidly growing abdominal mass was detected. Laparoscopy revealed a solitary tumor of the ileum covered with an intact serosal layer. Partial ileectomy was performed for tumor resection. Histological examination revealed cells resembling the primary ovarian tumor in the mucosal surface of the small bowel along with an intact serosa. The tumor cells were positive for cytokeratin 7 and negative for cytokeratin 20, suggesting an ovarian origin. This is the first report of solitary metastasis of an ovarian carcinoma to the small bowel parenchyma without peritoneal dissemination. Metastasis to the small bowel should be considered in ovarian carcinoma patients with occult gastrointestinal hemorrhage.
Journal of Obstetrics and Gynaecology Research, 2004
A number of studies for the measurement of cell-free fetal DNA in maternal blood have been report... more A number of studies for the measurement of cell-free fetal DNA in maternal blood have been reported; however, their clinical significance has remained unclear. We proposed to clarify the relationship between fetal DNA levels and obstetrical disorders. One hundred and eighty-five cases of normal pregnancy, ranging from 8 to 40 weeks' gestation, and 70 cases of abnormal pregnancy were included. SRY levels in maternal plasma were quantified with a real-time quantitative polymerase chain reaction. Sex-determining region Y (SRY) levels and the number of patients with positive levels peaked at 33-36 weeks in normal pregnancy. The SRY levels in threatened abortion (11.6 +/- 4.8 copies/mL to 0 +/- 0, P < 0.05) and threatened preterm labor (44.6 +/- 16.1 copies/mL to 15.9 +/- 6.2, P < 0.01) were significantly higher than those of the normal group. In pre-eclamptic patients, SRY levels were markedly higher than those of the normal group (173.2 +/- 94.8 copies/mL to 22.4 +/- 8.9, P < 0.05). Patients with premature separation of the placenta (266.8 +/- 137.1 copies/mL to 4.9 +/- 3.7, P < 0.05) and placenta previa (167.7 +/- 32.4 copies/mL to 37.0 +/- 17.3, p <0.01) also showed elevated SRY levels. Sex-determining region Y levels in maternal plasma were elevated in patients with an abnormal pregnancy, particularly those with placental injury of damage. These results suggested that increased SRY levels are consistently caused by the leak of fetal components, and thus the measurement of SRY levels in maternal plasma is useful for the evaluation of placental injuries.
The Journal of Clinical Endocrinology & Metabolism, 2004
ABSTRACT Expression of 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) was compared between le... more ABSTRACT Expression of 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) was compared between leiomyoma and myometrium. Cytosolic fractions from leiomyoma homogenate displayed 5-fold higher activity (estrone to estradiol), compared with surrounding myometrium (n = 6, P < 0.05), whereas microsomal fractions showed no difference. Oxidative activity (estradiol to estrone) did not differ between leiomyoma and myometrium. Levels of mRNA for 17beta-HSDs were then measured using real-time PCR techniques. Among the eight different types of 17beta-HSDs (types 1-5, 7, 8, and 10), type 1 was the only enzyme displaying differential expression between leiomyoma and myometrium. Mean concentration of type 1 17beta-HSD mRNA was 4-fold higher in leiomyoma than in surrounding myometrium (n = 20, P < 0.05). Type 1 transcript levels correlated significantly with reductive activity in individual samples (n = 6, P < 0.05). Northern blot analysis of leiomyoma and myometrium tissues detected 2.3- and 1.0-kb transcripts of type 1 enzyme, whereas the major 1.3-kb transcript for 17beta-HSD in placenta-derived JEG-3 cells was not detected. None of the factors increasing mRNA levels for type 1 enzyme in placenta increased mRNA levels in leiomyoma. These results indicate that leiomyoma tissues overexpress type 1 17beta-HSD, resulting in high conversion of estrone to estradiol. In situ expression of type 1 17beta-HSD may play a role in self-supported growth of leiomyoma cells.
The Journal of Clinical Endocrinology & Metabolism, 2002
The CYP19 gene encoding aromatase P450 (estrogen synthetase) is expressed in several extragonadal... more 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.
The Journal of Clinical Endocrinology & Metabolism, 2001
We have shown that in situ estrogen synthesized in leiomyoma of the uterus plays a possible role ... more We have shown that in situ estrogen synthesized in leiomyoma of the uterus plays a possible role in the promotion of leiomyoma cell growth via an autocrine/paracrine mechanism. In the present study, we demonstrated that leuprorelin acetate, a GnRH agonist widely used for treatment of uterine leiomyoma by down-regulation of pituitary-ovarian function, suppressed the expression of aromatase P450 (an estrogen synthetase) in leiomyoma cells. Given the role of in situ estrogen in leiomyoma cell growth, the inhibition of in situ estrogen synthesis may play a role in GnRH agonist-induced rapid regression of leiomyomas. Quantitative RT-PCR revealed that in women receiving no medication uterine leiomyomas express aromatase P450 mRNA at levels 20 times higher than that in the surrounding myometrium. Leuprorelin acetate treatment (1.88 mg every 4 wk, sc injection) for 12-24 wk reduced the expression of aromatase P450 mRNA in leiomyoma tissue as well as in the myometrium, to approximately one tenth of that in the myometrium of untreated women. Suppression of aromatase P450 expression was also demonstrated by Western blot analysis and aromatase activity assay of microsomal fractions prepared from leiomyomas. On the other hand, no differences in the levels of activity and mRNA of aromatase P450 were observed between leiomyoma cells obtained from women treated with and without leuprorelin acetate injections when cells were cultured ex vivo and stimulated by various combinations of stimulants such as dexamethasone + IL-1beta. The addition of various concentrations of E2 did not affect the aromatase activity of leiomyoma cells, suggesting that deprivation of circulating (ovarian) estrogen is not a cause of decreased expression of aromatase during leuprorelin acetate therapy. On the other hand, 8-d treatment with leuprorelin acetate (100 nmol/liter) reduced dexamethasone + IL-1beta-induced activity and a mRNA level of aromatase by 28% and 42%, respectively. These results indicated that leuprorelin acetate inhibits the expression of aromatase P450 in leiomyoma cells, which contributes to the rapid regression of leiomyoma during leuprorelin acetate therapy.
The Journal of Clinical Endocrinology & Metabolism, 1991
A description is presented of the first documented case of placental aromatase deficiency. The de... more A description is presented of the first documented case of placental aromatase deficiency. The deficiency caused maternal virilization during pregnancy and pseudohermaphroditism of the female fetus. A 24-yr-old primigravida showed progressive virilization during the third trimester. Urinary excretion of estrogen was less than 14 mumol/day between 35-38 weeks of pregnancy, although nonstress tests showed reactive patterns and serum levels of human placental lactogen were above 460 nmol/L. Maternal serum levels of estrogens were low, and those of androgens were high in the third trimester. A dehydroepiandrosterone sulfate loading test induced a marked increase in maternal serum levels of androgens, whereas no such increase was observed in estrogens. The woman delivered vaginally a live full-term infant who exhibited female pseudohermaphroditism. Cord serum levels of estrogens were extremely low, while those of androgens were high. The aromatase activity of the placenta, determined by the conversion of [7-3H]androstenedione to 17 beta-[7-3H]estradiol and [7-3H]estrone, were less than 0.03 fmol/microgram protein.min (control, 9.6 +/- 2.2 fmol/microgram protein.min). The sulfatase activity of the placenta was 0.63 pmol/microgram protein.min compared to 0.46 +/- 0.16 pmol/microgram protein.min in controls. The rate of aromatization by normal control placentas was the same as that obtained during coincubation of samples of normal placentas and that of the patient. Thus, the presence of aromatase inhibitor in the patient's placenta was excluded.
The Journal of Steroid Biochemistry and Molecular Biology, 2001
Fetal human osteoblast-like cells and the THP-1 cell line that differentiates into macrophage/ost... more Fetal human osteoblast-like cells and the THP-1 cell line that differentiates into macrophage/osteoblast-like cells in the presence of Vitamin D3 and which possesses high aromatase activity, constitute a useful model with which to study the regulation of aromatase in bone. We showed that dexamethasone (DEX)-induced aromatase activity in the THP-1 cell line is completely suppressed by forskolin and by dibutyryl cAMP. We therefore investigated the contribution of mitogen-activated protein kinase (MAPK) to the regulation of aromatase, because cAMP inhibits MAPK in many cells. We examined the role of MAPK on aromatase activity using PD98059, a selective inhibitor of MEK-1. PD98059 (100 microM) reduced DEX+interleukin (IL)-1beta-induced aromatase activity in human osteoblast-like cells by more than 90%, whereas 50% of the aromatase mRNA concentration was retained compared with the control incubated with DEX+IL-1beta. PD98059 (50 microM) reduced the activity of aromatase in THP-1 cells by 80% without significantly affecting the mRNA level. These results indicated that MAPK plays an important role in aromatase activation at the post-transcriptional level.
In leiomyoma of the uterus, both aromatase and 17beta-hydroxysteroid dehydrogenase (17beta-HSD) t... more In leiomyoma of the uterus, both aromatase and 17beta-hydroxysteroid dehydrogenase (17beta-HSD) type I are overexpressed compared with myometrium. This suggests that leiomyoma cells convert circulating androstenedione into estrone (via aromatase), then into the active form of estrogen, estradiol (via 17beta-HSD type I). In vitro experiments and several clinical findings support the notion that in situ estrogen plays a role in leiomyoma growth under hypoestrogenemic conditions, such as natural menopause and therapy with gonadotropin-releasing hormone (GnRH) agonists. GnRH agonists abolish estrogen production both in situ in leiomyoma and in the ovary, leading to quick and profound regression of the leiomyoma. Aromatase inhibitors also inhibit estrogen synthesis in both leiomyoma and the ovary and may be used therapeutically. Certain doses of competitive aromatase inhibitors would completely inhibit estrogen production in leiomyoma, whereas ovarian production of estrogen would continue at reduced levels. This may lead to advantageous therapeutic conditions in which leiomyoma regresses without adverse symptoms related to estrogen depletion because levels of ovarian estrogen would be insufficient to support leiomyoma growth but sufficient to prevent symptoms associated with deficiency. This article discusses the potential uses of aromatase inhibitors.
Reliable leak-proof aspiration of cyst contents is required for treatment of large ovarian cysts ... more Reliable leak-proof aspiration of cyst contents is required for treatment of large ovarian cysts by minilaparotomy. Through a small abdominal wound a transparent plastic bag was instantly mounted onto the cyst surface using an ethyl-2-cyanoacrylate adhesive. A 1-2-cm-wide cut was made in the consolidated cyst wall through the inside of the bag and the contents directly aspirated. The fluid was trapped inside the bag without leaking into the abdominal cavity. This method can also be applied to relatively small cysts by holding the cyst just beneath the wound. We used this method in 30 patients with unilateral ovarian cysts and in one patient with an ovarian cyst associated with an ipsilateral paraovarian cyst. All patients were successfully treated without spillage, although in one case a large mucinous ovarian cyst ruptured before surgery. Minilaparotomy using the instant adhesive is cost-effective, safe, reliable, and easily implemented. This procedure is also applicable to relatively small cysts and is a viable alternative to laparoscopic surgery for treatment of dermoid cysts showing considerable calcification.
It may be difficult to differentiate the consecutive occurrence of two independent molar pregnanc... more It may be difficult to differentiate the consecutive occurrence of two independent molar pregnancies from gestational trophoblastic neoplasia after the initial molar pregnancy, especially when the interval between them is short. A 25-year-woman who had had a complete hydatidiform mole 6 months earlier presented with a 6-week history of secondary amenorrhea. Serum human chorionic gonadotropin had increased to 19,857 micro-international units/mL, with no gestational sac demonstrated. Dilation and curettage was performed. Pathologic examination identified a tiny amount of hydropic villi compatible with complete hydatidiform mole. Analysis of short tandem repeat polymorphisms revealed that the molar tissues of the first and second complete hydatidiform moles were of different genetic origin. The patient went into remission spontaneously without chemotherapy. Genetic profiling was useful to discriminate a recurrent mole from suspected gestational trophoblastic neoplasia.
Gynecomastia of prepubertal onset may result from increased estrogen owing to excessive aromatase... more Gynecomastia of prepubertal onset may result from increased estrogen owing to excessive aromatase activity in extraglandular tissues. A gene in chromosome 15q21.2 encodes aromatase, the key enzyme for estrogen biosynthesis. Several physiologic tissue-specific promoters regulate the expression of aromatase, giving rise to messenger RNA (mRNA) species with an identical coding region but tissue-specific 5'-untranslated regions in placenta, gonads, brain, fat, and skin. We studied skin, fat, and blood samples from a 36-year-old man, his 7-year-old son, and an unrelated 17-year-old boy with severe gynecomastia of prepubertal onset and hypogonadotropic hypogonadism caused by elevated estrogen levels. Aromatase activity and mRNA levels in fat and skin and whole-body aromatization of androstenedione were severely elevated. Treatment with an aromatase inhibitor decreased serum estrogen levels and normalized gonadotropin and testosterone levels. The 5'-untranslated regions of aromatase mRNA contained the same sequence (FLJ) in the father and son and another sequence (TMOD3) in the unrelated boy; neither sequence was found in control subjects. These 5'-untranslated regions normally make up the first exons of two ubiquitously expressed genes clustered in chromosome 15q21.2-3 in the following order (from telomere to centromere): FLJ, TMOD3, and aromatase. The aromatase gene is normally transcribed in the direction opposite to that of TMOD3 and FLJ. Two distinct heterozygous inversions reversed the direction of the TMOD3 or FLJ promoter in the patients. Heterozygous inversions in chromosome 15q21.2-3, which caused the coding region of the aromatase gene to lie adjacent to constitutively active cryptic promoters that normally transcribe other genes, resulted in severe estrogen excess owing to the overexpression of aromatase in many tissues.
Intratumoral expression of aromatase P450 (P450arom) promotes the growth of breast tumors via inc... more Intratumoral expression of aromatase P450 (P450arom) promotes the growth of breast tumors via increased local estrogen concentration. We cloned a novel 101-bp untranslated first exon (I.7) that comprises the 5'-end of 29-54% of P450arom transcripts isolated from breast cancer tissues (n = 7). The levels of P450arom transcripts with exon I.7 were significantly increased in breast tumor tissues and adipose tissue adjacent to tumors. We identified a promoter immediately upstream of exon I.7 and mapped this to about 36 kb upstream of ATG translation start site of the CYP19 (aromatase cytochrome P450) gene. Sequence analysis of I.7 revealed a TATA-less promoter containing an initiator, two consensus GATA sites, and cis-regulatory elements found in megakaryocytes and endothelial type promoters. Luciferase activity directed by the promoter I.7 sequence (-299/+81 bp) was 4-fold greater than a minimum length promoter sequence (-35/+81 bp) in human microvascular endothelial cells (HMEC-1), but only 2-fold greater in MCF-7 breast malignant epithelial cells. There was no promoter activity in primary breast adipose fibroblasts. Site-directed mutations demonstrated that maximal basal promoter activity required two GATA motifs at -146/-141 bp and -196/-191 bp. Gel shift and deoxyribonuclease I footprinting assays demonstrated the binding of GATA-2 transcription factor but not GATA-1 to the -196/-191-bp region. Overexpression of GATA-2 in HMEC-1 cells increases promoter I.7 activity by 5-fold. In conclusion, promoter I.7 is a GATA-2-regulated endothelial promoter of the human CYP19 gene and may increase estrogen biosynthesis in vascular endothelial cells of breast cancer. The activity of this promoter may also be important for intracrine and paracrine effects of estrogen on blood vessels.
Recent evidence has shown that bone is not only a target of estrogen action but also a source of ... more Recent evidence has shown that bone is not only a target of estrogen action but also a source of local estrogen production. Bone cells such as osteoblasts express aromatase (P450arom) and the expression of P450arom in osteoblasts is positively regulated in a tissue specific fashion, as in the case of other tissues which express P450arom. To clarify the physiological factors regulating expression of P450arom in bone, we tested TGF-β1 using osteoblast-like cells obtained from human fetuses as well as THP-1 cells. TGF-β1 increased IL-1β+DEX- induced aromatase activity in osteoblast-like cells, while it inhibited activity in skin fibroblasts. Similar enhancement of aromatase activity by TGF-β1 was found in DEX-stimulated THP-1 cells and this cell line was used for further experiments. In THP-1 cells, TGF-β1 enhanced DEX-induced aromatase activity almost linearly by 12 h and thereafter. Increased levels of P450arom transcripts were also demonstrated by RT-PCR at 3 h of TGF-β1 treatment and thereafter. Cyclohexamide abolished enhancement of activity but did not inhibit the accumulation of P450arom transcripts induced by TGF-β1. Increase in P450arom expression by TGF-β1 was attributable to expression driven by promoter I.4. TGF-β1 did not change the half life of P450arom transcripts. To identify the cis-acting elements responsible for TGF-β1 action on aromatase expression, transient transfection assays were performed using a series of deletion constructs for promoter I.4 (P450-I.4/Luc). Two constructs (−410/+14 and−340/+14) that contain a functional glucocorticoid response element (GRE) and downstream sequence showed significant increase of luciferase activity in response to TGF-β1. Deletion and mutation of the GRE in P450-I.4/Luc (−340/+14) abolished the TGF-β1. The luciferase activity of a (GRE)1-SV40/Luc construct was also stimulated by TGF-β1. These results indicate that TGF-β1 increases the expression of P450arom at the level of transcription through promoter I.4, at least in part via an enhancement of transactivation activity of the GR in THP-1 cells. TGF-β1 is suggested to be one of the physiological up-regulatory factors of bone aromatase.
Estrogen plays a major role in bone mineral homeostasis, maintaining a balance between bone forma... more Estrogen plays a major role in bone mineral homeostasis, maintaining a balance between bone formation and bone resorption not only in women but also in men. Extraglandular aromatization of circulating androgen is the major source of estrogen in post-menopausal women and men. In order to assess the capacity of bone cells as a local source of estrogen, osteoblast-like cells (OLCs) were obtained from human fetal bone in mid-trimester by the explant method and by mechanical disaggregation. The integrity of OLCs was confirmed by their ability to produce alkaline phosphatase and osteocalcin in response to vitamin D3 and also by their ability to deposit mineral. Aromatase activity was assessed by the formation of estrone from [1,2,6,7-3H]androstenedione and by the release of tritium from [1beta-3H]androstenedione into [3H]water. Formation of estrone was confirmed by thin layer chromatography (TLC) in OLCs stimulated with dexamethasone (DEX) + oncostatin M. The aromatase activity was 10 x higher in non-passaged OLCs than in passaged cells in the presence or absence of the stimulants (DEX + IL-1beta). The apparent Km and Vmax estimated by the release of [3H]water was 5.8+/-0.6 nM and 10.8+/-1.4 pmol/mg per 6 h in the presence of DEX + IL-1beta. The effects of several stimulants on aromatase activity in OLCs were examined: serum, IL-1beta, TNFalpha and type I cytokines stimulated activity in the presence of DEX, while PMA and PMA + dibutyryl cAMP did not. To confirm the expression of aromatase in OLCs, cells prepared from periosteal membranes were also examined: These cells in culture possessed aromatase activity corresponding to OLCs prepared from bone specimens. Moreover, the fresh periosteum expressed aromatase at higher levels than that of metaphyseal specimens. The aromatase gene employs several different promoters (I.1, 1.2, I.3, I.4, I.5, I.6, 2a, 1f and PII) and the usage of these promoters is known to be controlled in a tissue-specific fashion. Accordingly, promoter usage in OLCs and fetal long bone (tibia) tissue was examined using the 5' rapid amplification of cDNA ends (RACE) technique. The major promoter used was I.4, not only in stimulated and non-stimulated OLCs, but also in fetal tibia. Some minor transcripts were also found: 1f (brain-specific promoter), PII and I.6 in OLCs stimulated by DEX + IL-1beta, and PII and I.3 in OLCs stimulated by DEX + serum. Fetal tibia also expressed I.3 (15%) and I.6 (10%). Thus, regulation and promoter usage in OLCs was quite different from other tissues known as estrogen sources including adipose tissue, ovary and placenta. These results suggest that bone is an extraglandular source of local estrogen which plays an important role in bone mineral metabolism through autocrine and paracrine actions.
Journal of the American Academy of Dermatology, 1994
The pyloric atresia--junctional epidermolysis bullosa (PA-JEB) syndrome is an autosomal recessive... more The pyloric atresia--junctional epidermolysis bullosa (PA-JEB) syndrome is an autosomal recessive disorder with a poor prognosis. Electron microscopy of fetal skin has been the only reliable method for prenatal diagnosis. The purpose of this study was to make the prenatal diagnosis of PA-JEB syndrome with a more reliable method by means of immunocytochemical probes. Expression of a range of basement membrane antigens was examined in different types of JEB. On the basis of the results, a fetal skin biopsy specimen was obtained for prenatal diagnosis. In PA-JEB syndrome (n = 2), GB3 antigen (BM600) was normally expressed; the 19-DEJ-1 antigen was completely absent. In fetal skin at risk for PA-JEB syndrome, the 19-DEJ-1 antigen was normally expressed, and no ultrastructural abnormality was found by electron microscopy. A normal male infant was delivered at 38 weeks of pregnancy. 19-DEJ-1 monoclonal antibody serves as a useful probe for the prenatal diagnosis of PA-JEB syndrome.
Journal of Obstetrics and Gynaecology Research, 2011
Metastasis of ovarian carcinoma to the small bowel parenchyma without peritoneal dissemination is... more Metastasis of ovarian carcinoma to the small bowel parenchyma without peritoneal dissemination is uncommon. A 63-year-old woman underwent surgery for a clear cell adenocarcinoma of the ovary and received adjuvant chemotherapy. Eighteen months after the operation, she presented with recurrent occult bowel hemorrhage without evidence of an abdominal mass. Nine months later, a rapidly growing abdominal mass was detected. Laparoscopy revealed a solitary tumor of the ileum covered with an intact serosal layer. Partial ileectomy was performed for tumor resection. Histological examination revealed cells resembling the primary ovarian tumor in the mucosal surface of the small bowel along with an intact serosa. The tumor cells were positive for cytokeratin 7 and negative for cytokeratin 20, suggesting an ovarian origin. This is the first report of solitary metastasis of an ovarian carcinoma to the small bowel parenchyma without peritoneal dissemination. Metastasis to the small bowel should be considered in ovarian carcinoma patients with occult gastrointestinal hemorrhage.
Journal of Obstetrics and Gynaecology Research, 2004
A number of studies for the measurement of cell-free fetal DNA in maternal blood have been report... more A number of studies for the measurement of cell-free fetal DNA in maternal blood have been reported; however, their clinical significance has remained unclear. We proposed to clarify the relationship between fetal DNA levels and obstetrical disorders. One hundred and eighty-five cases of normal pregnancy, ranging from 8 to 40 weeks' gestation, and 70 cases of abnormal pregnancy were included. SRY levels in maternal plasma were quantified with a real-time quantitative polymerase chain reaction. Sex-determining region Y (SRY) levels and the number of patients with positive levels peaked at 33-36 weeks in normal pregnancy. The SRY levels in threatened abortion (11.6 +/- 4.8 copies/mL to 0 +/- 0, P < 0.05) and threatened preterm labor (44.6 +/- 16.1 copies/mL to 15.9 +/- 6.2, P < 0.01) were significantly higher than those of the normal group. In pre-eclamptic patients, SRY levels were markedly higher than those of the normal group (173.2 +/- 94.8 copies/mL to 22.4 +/- 8.9, P < 0.05). Patients with premature separation of the placenta (266.8 +/- 137.1 copies/mL to 4.9 +/- 3.7, P < 0.05) and placenta previa (167.7 +/- 32.4 copies/mL to 37.0 +/- 17.3, p <0.01) also showed elevated SRY levels. Sex-determining region Y levels in maternal plasma were elevated in patients with an abnormal pregnancy, particularly those with placental injury of damage. These results suggested that increased SRY levels are consistently caused by the leak of fetal components, and thus the measurement of SRY levels in maternal plasma is useful for the evaluation of placental injuries.
The Journal of Clinical Endocrinology & Metabolism, 2004
ABSTRACT Expression of 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) was compared between le... more ABSTRACT Expression of 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) was compared between leiomyoma and myometrium. Cytosolic fractions from leiomyoma homogenate displayed 5-fold higher activity (estrone to estradiol), compared with surrounding myometrium (n = 6, P < 0.05), whereas microsomal fractions showed no difference. Oxidative activity (estradiol to estrone) did not differ between leiomyoma and myometrium. Levels of mRNA for 17beta-HSDs were then measured using real-time PCR techniques. Among the eight different types of 17beta-HSDs (types 1-5, 7, 8, and 10), type 1 was the only enzyme displaying differential expression between leiomyoma and myometrium. Mean concentration of type 1 17beta-HSD mRNA was 4-fold higher in leiomyoma than in surrounding myometrium (n = 20, P < 0.05). Type 1 transcript levels correlated significantly with reductive activity in individual samples (n = 6, P < 0.05). Northern blot analysis of leiomyoma and myometrium tissues detected 2.3- and 1.0-kb transcripts of type 1 enzyme, whereas the major 1.3-kb transcript for 17beta-HSD in placenta-derived JEG-3 cells was not detected. None of the factors increasing mRNA levels for type 1 enzyme in placenta increased mRNA levels in leiomyoma. These results indicate that leiomyoma tissues overexpress type 1 17beta-HSD, resulting in high conversion of estrone to estradiol. In situ expression of type 1 17beta-HSD may play a role in self-supported growth of leiomyoma cells.
The Journal of Clinical Endocrinology & Metabolism, 2002
The CYP19 gene encoding aromatase P450 (estrogen synthetase) is expressed in several extragonadal... more 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.
The Journal of Clinical Endocrinology & Metabolism, 2001
We have shown that in situ estrogen synthesized in leiomyoma of the uterus plays a possible role ... more We have shown that in situ estrogen synthesized in leiomyoma of the uterus plays a possible role in the promotion of leiomyoma cell growth via an autocrine/paracrine mechanism. In the present study, we demonstrated that leuprorelin acetate, a GnRH agonist widely used for treatment of uterine leiomyoma by down-regulation of pituitary-ovarian function, suppressed the expression of aromatase P450 (an estrogen synthetase) in leiomyoma cells. Given the role of in situ estrogen in leiomyoma cell growth, the inhibition of in situ estrogen synthesis may play a role in GnRH agonist-induced rapid regression of leiomyomas. Quantitative RT-PCR revealed that in women receiving no medication uterine leiomyomas express aromatase P450 mRNA at levels 20 times higher than that in the surrounding myometrium. Leuprorelin acetate treatment (1.88 mg every 4 wk, sc injection) for 12-24 wk reduced the expression of aromatase P450 mRNA in leiomyoma tissue as well as in the myometrium, to approximately one tenth of that in the myometrium of untreated women. Suppression of aromatase P450 expression was also demonstrated by Western blot analysis and aromatase activity assay of microsomal fractions prepared from leiomyomas. On the other hand, no differences in the levels of activity and mRNA of aromatase P450 were observed between leiomyoma cells obtained from women treated with and without leuprorelin acetate injections when cells were cultured ex vivo and stimulated by various combinations of stimulants such as dexamethasone + IL-1beta. The addition of various concentrations of E2 did not affect the aromatase activity of leiomyoma cells, suggesting that deprivation of circulating (ovarian) estrogen is not a cause of decreased expression of aromatase during leuprorelin acetate therapy. On the other hand, 8-d treatment with leuprorelin acetate (100 nmol/liter) reduced dexamethasone + IL-1beta-induced activity and a mRNA level of aromatase by 28% and 42%, respectively. These results indicated that leuprorelin acetate inhibits the expression of aromatase P450 in leiomyoma cells, which contributes to the rapid regression of leiomyoma during leuprorelin acetate therapy.
The Journal of Clinical Endocrinology & Metabolism, 1991
A description is presented of the first documented case of placental aromatase deficiency. The de... more A description is presented of the first documented case of placental aromatase deficiency. The deficiency caused maternal virilization during pregnancy and pseudohermaphroditism of the female fetus. A 24-yr-old primigravida showed progressive virilization during the third trimester. Urinary excretion of estrogen was less than 14 mumol/day between 35-38 weeks of pregnancy, although nonstress tests showed reactive patterns and serum levels of human placental lactogen were above 460 nmol/L. Maternal serum levels of estrogens were low, and those of androgens were high in the third trimester. A dehydroepiandrosterone sulfate loading test induced a marked increase in maternal serum levels of androgens, whereas no such increase was observed in estrogens. The woman delivered vaginally a live full-term infant who exhibited female pseudohermaphroditism. Cord serum levels of estrogens were extremely low, while those of androgens were high. The aromatase activity of the placenta, determined by the conversion of [7-3H]androstenedione to 17 beta-[7-3H]estradiol and [7-3H]estrone, were less than 0.03 fmol/microgram protein.min (control, 9.6 +/- 2.2 fmol/microgram protein.min). The sulfatase activity of the placenta was 0.63 pmol/microgram protein.min compared to 0.46 +/- 0.16 pmol/microgram protein.min in controls. The rate of aromatization by normal control placentas was the same as that obtained during coincubation of samples of normal placentas and that of the patient. Thus, the presence of aromatase inhibitor in the patient's placenta was excluded.
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