生水 真紀夫

子宮内膜症はエストロゲン依存性の発育を示す疾患である.このエストロゲンは主として卵巣から供給されているが,最近内膜症組織自体が自らエストロゲンを産生していることが明らかになった.そこで,この子宮内膜症局所で合成されるエストロゲンが内膜症細胞自身の発育を促進している可能性について検討し,エストロゲン合成阻害剤による新たな治療戦略についての可能性を模索した.内膜症腹膜病変およびチョコレート嚢胞では,主として同質細胞にアロマターゼ蛋白・mRNAの発現が認められた.同質・上皮共培養系を用いて検討したところ,同質細胞が血中のアンドロステンジオンをエストロゲンに転換し,このエストロゲンが内膜症上皮細胞の増殖を促進している(パラクライン効果)可能性が強く示唆された.つぎに,内膜症におけるアロマターゼの発現プロモーターについて検討を行った.内膜症腹膜病変ではI.4が主なアロマターゼ発現プロモーターであり術前GnRHa投与によりその発現量が著減したのに対し,チョコレート嚢胞ではPIIが主要な発現プロモーターとなっており術前GnRHa投与によっても発現量の減少を示さなかった.このことは,腹膜病変ではGnRHa投与により局所でのエストロゲン産生が停止するのに対し,チョコレート嚢胞ではGnRHa投与中にも局所でのエストロゲン産生が継続することを意味する.したがって,腹膜病変はGnRHa療法によく反応するが,チョコレート嚢胞はGnRHa療法に抵抗性を示すという臨床的観察によく一致するものであった.つぎに,アロマターゼのプロモーター構造(PH)について検討を行った.EMSAによる検討などから,PHのTATA box上流にはnuclear half siteとCREがあり,チョコレート嚢胞ではそれぞれの部位にNR5A1(SF1)とCREBが結合していると考えられた.組織内では,サイトカインなどのシグナルを受けて活性化されるA-kinaseによりリン酸化されたCREBがconstitutiveにCREに結合しているものと考えられた.一方,NR5A1の発現量は局所的に制御されており,この増減が局所アロマターゼの発現量のおもな決定因子となっていた.この局所アロマターゼの発現は,IL-1bのシグナル伝達を阻止するBAY-11-7082,dexamethasone,NS-398などにより抑制された.アロマターゼを分子標的とする治療法のうちアロマターゼ阻害剤の効果についてラット子宮内膜症モデルなどを用いて検討した.アロマターゼ阻害剤は内膜症病変の縮小に有効であることが確認された.以上より,アロマターゼ阻害剤は,内膜症局所でのエストロゲン産生をも抑制する点でGnRHaに勝る治療効果を上げる可能性があると考えられる.しかしながら実際の投与にあたっては,骨などへの影響にも十分配慮する必要がある.

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 muM) 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 muM) 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. (C) 2002 Elsevier Science Ltd. All tights reserved.

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 muM) 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 muM) 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. (C) 2002 Elsevier Science Ltd. All tights reserved.

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, se 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-1 beta. 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-1 beta -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.

Background: Reliable leak-proof aspiration of cyst contents is required for treatment of large ovarian cysts by minilaparotomy. Technique: Through a small abdominal wound a transparent plastic bag was instantly mounted onto the cyst surface using an ethyl-2-cyanoacrylate adhesive. A l-P-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. Experience: 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. Conclusion: 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. (Obstet Gynecol 2001;97:1007-10. (C) 2001 by The American College of Obstetricians and Gynecologists.).

Background: Reliable leak-proof aspiration of cyst contents is required for treatment of large ovarian cysts by minilaparotomy. Technique: Through a small abdominal wound a transparent plastic bag was instantly mounted onto the cyst surface using an ethyl-2-cyanoacrylate adhesive. A l-P-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. Experience: 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. Conclusion: 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. (Obstet Gynecol 2001;97:1007-10. (C) 2001 by The American College of Obstetricians and Gynecologists.).

In the present study we characterized in detail the expression of aromatase P450 in leiomyomas to determine the role of in situ estrogen in the growth advantage of leiomyomas. The levels of aromatase P450 transcripts were determined by quantitative RT-PCR to be significantly higher in leiomyomas than in corresponding myometrium. The overexpression of aromatase P450 in leiomyomas was also confirmed by Western blot analysis. The estimated size of immunoreactive aromatase was 58 kDa, similar to that in placenta. To identify a cell type that express aromatase P450 in leiomyomas, histological specimens were stained for aromatase P450 using a polyclonal antibody. Strong immunoreactivity was detected in the cytoplasm of leiomyoma cells, whereas surrounding normal myometrium displayed weak or negative staining. Smooth muscle-like cells in culture obtained from leiomyomas, positive for actin D fiber, possessed immunoreactive granules of aromatase in the cytoplasm. Conversion of androgen to estrogen was effectively stimulated by phorbol myristate acetate and dexamethasone plus interleukin-1 beta and was completely abolished by selective inhibitors of aromatase P450 (fadrozole and TZA-2209), but not by inhibitors of 5 alpha-reductase (finasteride and flutamide). The apparent K-m of androstenedione was 3 nM in the presence of dexamethasone and interleukin-1 beta, corresponding to the plasma concentration of androstenedione in women of reproductive age. To determine whether endogenous aromatase P450 plays a role in the growth promotion of leiomyoma cells, we evaluated the cell growth of smooth muscle-like cells treated with various concentrations of estrogen and androgen using a WST-1 assay. Treatment with testosterone (10(-8) and 10(-7) M) and androstenedione (10(-8) and 10(-7) M) stimulated the growth of smooth muscle-like cells obtained from leiomyomas to the same extent as estradiol (10(-1)4-10(-7) M) whereas dihydrotestosterone (10(-11)-10(-8) M) did not. The stimulatory effect of testosterone on cell growth was again abolished by cotreatment with fadrozole. The level of estradiol in the medium of testosterone (10(-8) M)-treated smooth muscle-like cells was 10(-11) M, which was 1 order lower than the minimum concentration of estradiol necessary to promote cell growth (10(-10) M). This indicates that estradiol synthesized in leiomyomas promotes their growth via an autocrine/intracrine mechanism. We conclude that myometrial cells of leiomyomas overexpress aromatase P450 and are able to synthesize sufficient estrogen to accelerate their own cell growth. Overexpression of aromatase P450 may play a role in the growth advantage of leiomyoma tissue over surrounding myometrium via an autocrine/intracrine mechanism.

In the present study we characterized in detail the expression of aromatase P450 in leiomyomas to determine the role of in situ estrogen in the growth advantage of leiomyomas. The levels of aromatase P450 transcripts were determined by quantitative RT-PCR to be significantly higher in leiomyomas than in corresponding myometrium. The overexpression of aromatase P450 in leiomyomas was also confirmed by Western blot analysis. The estimated size of immunoreactive aromatase was 58 kDa, similar to that in placenta. To identify a cell type that express aromatase P450 in leiomyomas, histological specimens were stained for aromatase P450 using a polyclonal antibody. Strong immunoreactivity was detected in the cytoplasm of leiomyoma cells, whereas surrounding normal myometrium displayed weak or negative staining. Smooth muscle-like cells in culture obtained from leiomyomas, positive for actin D fiber, possessed immunoreactive granules of aromatase in the cytoplasm. Conversion of androgen to estrogen was effectively stimulated by phorbol myristate acetate and dexamethasone plus interleukin-1 beta and was completely abolished by selective inhibitors of aromatase P450 (fadrozole and TZA-2209), but not by inhibitors of 5 alpha-reductase (finasteride and flutamide). The apparent K-m of androstenedione was 3 nM in the presence of dexamethasone and interleukin-1 beta, corresponding to the plasma concentration of androstenedione in women of reproductive age. To determine whether endogenous aromatase P450 plays a role in the growth promotion of leiomyoma cells, we evaluated the cell growth of smooth muscle-like cells treated with various concentrations of estrogen and androgen using a WST-1 assay. Treatment with testosterone (10(-8) and 10(-7) M) and androstenedione (10(-8) and 10(-7) M) stimulated the growth of smooth muscle-like cells obtained from leiomyomas to the same extent as estradiol (10(-1)4-10(-7) M) whereas dihydrotestosterone (10(-11)-10(-8) M) did not. The stimulatory effect of testosterone on cell growth was again abolished by cotreatment with fadrozole. The level of estradiol in the medium of testosterone (10(-8) M)-treated smooth muscle-like cells was 10(-11) M, which was 1 order lower than the minimum concentration of estradiol necessary to promote cell growth (10(-10) M). This indicates that estradiol synthesized in leiomyomas promotes their growth via an autocrine/intracrine mechanism. We conclude that myometrial cells of leiomyomas overexpress aromatase P450 and are able to synthesize sufficient estrogen to accelerate their own cell growth. Overexpression of aromatase P450 may play a role in the growth advantage of leiomyoma tissue over surrounding myometrium via an autocrine/intracrine mechanism.

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-beta 1 using osteoblast-like cells obtained from human fetuses as well as THP-1 cells. TGF-beta 1 increased IL-1 beta + DEX- induced aromatase activity in osteoblast-like cells, while it inhibited activity in skin fibroblasts. Similar enhancement of aromatase activity by TGF-PI was found in DEX-stimulated THP-1 cells and this cell line was used for further experiments. In THP-1 cells, TGF-beta 1 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-beta 1 treatment and thereafter. Cyclohexamide abolished enhancement of activity but did not inhibit the accumulation of P450arom transcripts induced by TGF-beta 1. Increase in P450arom expression by TGF-beta 1 was attributable to expression driven by promoter 1.4. TGF-beta 1 did not change the half life of P450arom transcripts. To identify the cis-acting elements responsible for TGF-beta 1 action on aromatase expression, transient transfection assays were performed using a series of deletion constructs for promoter 1.4 (P450-I.4/Luc). Two constructs (-4101 + 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-PI. Deletion and mutation of the GRE in P450-I.4/Luc ( - 340/ + 14) abolished the TGF-beta 1. The luciferase activity of a (GRE)(1)-SV40/Luc construct was also stimulated by TGF-beta 1. These results indicate that TGF-beta 1 increases the expression of P450arom at the level of transcription through promoter 1.4, at least in part via an enhancement of transactivation activity of the GR in THP-1 cells. TGF-beta 1 is suggested to be one of the physiological up-regulatory factors of bone aromatase. (C) 2000 Published by Elsevier Science Ireland Ltd. All rights reserved.

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-beta 1 using osteoblast-like cells obtained from human fetuses as well as THP-1 cells. TGF-beta 1 increased IL-1 beta + DEX- induced aromatase activity in osteoblast-like cells, while it inhibited activity in skin fibroblasts. Similar enhancement of aromatase activity by TGF-PI was found in DEX-stimulated THP-1 cells and this cell line was used for further experiments. In THP-1 cells, TGF-beta 1 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-beta 1 treatment and thereafter. Cyclohexamide abolished enhancement of activity but did not inhibit the accumulation of P450arom transcripts induced by TGF-beta 1. Increase in P450arom expression by TGF-beta 1 was attributable to expression driven by promoter 1.4. TGF-beta 1 did not change the half life of P450arom transcripts. To identify the cis-acting elements responsible for TGF-beta 1 action on aromatase expression, transient transfection assays were performed using a series of deletion constructs for promoter 1.4 (P450-I.4/Luc). Two constructs (-4101 + 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-PI. Deletion and mutation of the GRE in P450-I.4/Luc ( - 340/ + 14) abolished the TGF-beta 1. The luciferase activity of a (GRE)(1)-SV40/Luc construct was also stimulated by TGF-beta 1. These results indicate that TGF-beta 1 increases the expression of P450arom at the level of transcription through promoter 1.4, at least in part via an enhancement of transactivation activity of the GR in THP-1 cells. TGF-beta 1 is suggested to be one of the physiological up-regulatory factors of bone aromatase. (C) 2000 Published by Elsevier Science Ireland Ltd. All rights reserved.

Objective: Age-related changes of steroid levels in the central nervous system (CNS) are not well understood. To investigate whether steroidal conditions in the CNS of women change with aging and menopause, steroid levels have been measured in serum and cerebrospinal fluid (CSF), and examined correlations with aging. Methods: Serum and CSF concentrations of estradiol (E2), cortisol, dehydroepiandrosterone (DHEA), DHEA sulfate (DHEAS) and albumin were measured in 80 female patients who underwent operations for benign gynecological diseases. They had no endocrinological or neurological disorders and were aged 17 - 71 years 62 patients were in premenopause and 18 were in postmenopause. Results: Serum levels of E2 decreased markedly after menopause, while levels of DHEA and DHEAS decreased gradually with age. There was no significant change with age of serum cortisol levels. The CSF concentrations of E2 (0.2-3 pg/ml) decreased with age [correlation coefficient (r) = 0.31, P &lt 0.01]. The CSF DHEA levels (0.1- 0.8 ng/ml) did not change with age although not significantly, but CSF cortisol levels (0.1-0.6 μg/dl) increased with age (r = 0.35, P &lt 0.01). The CSF DHEAS concentrations were below the sensitivity of the radioimmunoassay (RIA) (1 ng/ml). The CSF/serum ratios of cortisol increased with age (r = 0.30, P &lt 0.01), as did those of DHEA (r = 0.55, P &lt 0.01). Although serum albumin levels did not change throughout life, CSF albumin levels and CSF/serum albumin ratios increased gradually with age (r = 0.28, P = 0.052 r = 0.23, P = 0.114, respectively), but there was no significance. There were marked decreases of serum E2 and DHEA levels and CSF E2 levels in postmenopausal women (P &lt 0.05), but CSF cortisol levels increased (P &lt 0.05) and DHEA levels in CSF were maintained after menopause. Conclusion: These results indicate that steroids in CSF become cortisol dominated and deficient in estrogens with aging, especially after menopause.

Objective: Age-related changes of steroid levels in the central nervous system (CNS) are not well understood. To investigate whether steroidal conditions in the CNS of women change with aging and menopause, steroid levels have been measured in serum and cerebrospinal fluid (CSF), and examined correlations with aging. Methods: Serum and CSF concentrations of estradiol (E-2), cortisol, dehydroepiandrosterone (DHEA), DHEA sulfate (DHEAS) and albumin were measured in 80 female patients who underwent operations for benign gynecological diseases. They had no endocrinological or neurological disorders and were aged 17-71 years; 62 patients were in premenopause and 18 were in postmenopause. Results: Serum levels of E-2 decreased markedly after menopause, while levels of DHEA and DHEAS decreased gradually with age. There was no significant change with age of serum cortisol levels. The CSF concentrations of E-2 (0.2-3 pg/ml) decreased with age [correlation coefficient (r) = 0.31, P < 0.01]. The CSF DHEA levels (0.1-0.8 ng/ml) did not change with age although not significantly, but CSF cortisol levels (0.1-0.6 mu g/dl) increased with age (r = 0.35, P < 0.01). The CSF DHEAS concentrations were below the sensitivity of the radioimmunoassay (RIA) (1 ng/ml). The CSF/serum ratios of cortisol increased with age (r = 0.30, P < 0.01), as did those of DHEA (r = 0.55, P < 0.01). Although serum albumin levels did not change throughout life, CSF albumin levels and CSF/serum albumin ratios increased gradually with age (r = 0.28, P = 0.052; r = 0.23, P = 0.114, respectively), but there was no significance. There were marked decreases of serum E-2 and DHEA levels and CSF E2 levels in postmenopausal women (P < 0.05), but CSF cortisol levels increased (P < 0.05) and DHEA levels in CSF were maintained after menopause. Conclusion: These results indicate that steroids in CSF become cortisol dominated and deficient in estrogens with aging, especially after menopause. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.

In spite of the widespread use of assisted reproductive technology, there have been, to our knowledge, only two reported cases of molar pregnancies after gamete intra-fallopian transfer and five reported cases after in-vitro fertilization and embryo transfer. We report here a case of a complete hydatidiform mole in a twin pregnancy after gamete intra-fallopian transfer, as well as a case of a complete hydatidiform mole in a triplet: pregnancy after in-vitro fertilization and embryo transfer. The genetic constitution of each conceptus was determined by examination of the restriction fragment length polymorphism of the DNA with four different single-locus probes. This analysis revealed that both hydatidiform moles were of androgenetic origin and probably of monospermic origin. Moreover, the analysis confirmed that the pregnancies were dizygotic and trizygotic pregnancies respectively. The diagnostic utility of the analysis of DNA polymorphism is discussed in cases of a molar pregnancy with coexisting fetuses.

In spite of the widespread use of assisted reproductive technology, there have been, to our knowledge, only two reported cases of molar pregnancies after gamete intra-fallopian transfer and five reported cases after in-vitro fertilization and embryo transfer. We report here a case of a complete hydatidiform mole in a twin pregnancy after gamete intra-fallopian transfer, as well as a case of a complete hydatidiform mole in a triplet: pregnancy after in-vitro fertilization and embryo transfer. The genetic constitution of each conceptus was determined by examination of the restriction fragment length polymorphism of the DNA with four different single-locus probes. This analysis revealed that both hydatidiform moles were of androgenetic origin and probably of monospermic origin. Moreover, the analysis confirmed that the pregnancies were dizygotic and trizygotic pregnancies respectively. The diagnostic utility of the analysis of DNA polymorphism is discussed in cases of a molar pregnancy with coexisting fetuses.

The expression of aromatase is regulated in a tissue-specific fashion through alternative use of multiple promoter-specific first exons. To date, eight different first exons have been reported in human aromatase, namely I.1., I.2, I.3. I.4, I.5, PII, 2a, and If. Recently, we have found a new putative exon I in a RACE-generated Library of THP-1 cells and have conducted studies to characterize this new exon I. We confirmed that the constructs containing -1552/+17 or less flanking sequence of this exon function as a promoter in THP-1 cells, JEG-3 cells and osteoblast-like cells obtained from a human fetus. Results of transfection assays using a series of deletion constructs and mutation constructs indicate that a l-bp mismatch of the consensus TATA-like box (TTTAAT) and the consensus sequence of the initiator site, which is located 45 bp downstream of the putative TATA box, were functioning cooperatively as a core promoter. The putative transcription site was confirmed by the results of RT-PCR southern blot analysis. We examined the regulation and the expression of this exon, 1.6, in several human cells and tissues by RT-PCR Southern blot analysis. THP-1 cells (mononuclear leukemic origin) and JEG-3 cells (choriocarcinoma origin) expressed exon 1.6 in serum-free media. The level of expression was increased by serum and phorbol myristyl acetate (PMA) in both cell lines. Adipose stromal cells also expressed exon I.6 in the presence of PMA. In fetal osteoblasts, the expression of exon 1.6 was increased most effectively by serum and less so by dexamethasone (DEX) + IL-1 beta and DEX + IL-11, whereas induction by serum was suppressed by the addition of DEX. The level of expression was low in granulosa cells in culture and did not change with forskolin. On the other hand, dibutyryl cAMP suppressed PMA-stimulated expression of exon 1.6 in THP-1 cells and adipose stromal cells. This result supports the hypothesis that the expression of exon 1.6 is regulated mainly via an AP-1 binding site that is found upstream of the initiator site of the promoter region. Expression of exon I.6-specific transcripts was examined in several human tissues. Testis and bone obtained from normal adults expressed exon I.6. Testicular tumor and hepatic carcinoma expressed high levels of exon 1.6, whereas granulosa cell tumor did not. Fetal liver and bone also showed a significant level of exon 1.6 expression, but not so much as testicular tumor and hepatic tumor. Several splicing variants of exon I.6 were detected especially in THP-1 and JEG-3 cells, and to a lesser extent in primary cultures and tissue samples. These variants were identified as an unspliced form, a form spliced at the end of exon I.4, a form spliced at the end of exon I.3 (truncated) and a form spliced 220 bp downstream of the 3' end of exon I.6. The last variant revealed a new splicing site. Because most of the splicing variants contain the sequence specific for exon I.3, RT-PCR specific for exon I.3 can coamplify these splicing variants of exon I.6 transcripts. These results suggests that it is necessary to examine the expression of I.6 in tissues that are known to express exon I.3 such as breast adipose tissue, in which promoter usage of exon I of the aromatase gene switches from exon I.4 to I.3 in the course of malignant transformation.

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 D-3 and also by their ability to deposit mineral. Aromatase activity was assessed by the formation of estrone from [1,2,6,7-H-3]androstenedione and by the release of tritium from [1 beta-H-3]androstenedione into [H-3]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-1 beta). The apparent K-m and V-max estimated by the release of [H-3]water was 5.8 +/- 0.6 nM and 10.8 +/- 1.4 pmol/mg per 6 h in the presence of DEX + IL-1 beta. The effects of several stimulants on aromatase activity in OLCs were examined: serum, IL-1 beta, TNF alpha 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, I.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: If (brain-specific promoter), PII and I.6 in OLCs stimulated by DEX + IL-1 beta, 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 knows 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. (C) 1998 Elsevier Science Ireland Ltd. All rights reserved.

The expression of aromatase is regulated in a tissue-specific fashion through alternative use of multiple promoter-specific first exons. To date, eight different first exons have been reported in human aromatase, namely I.1., I.2, I.3. I.4, I.5, PII, 2a, and If. Recently, we have found a new putative exon I in a RACE-generated Library of THP-1 cells and have conducted studies to characterize this new exon I. We confirmed that the constructs containing -1552/+17 or less flanking sequence of this exon function as a promoter in THP-1 cells, JEG-3 cells and osteoblast-like cells obtained from a human fetus. Results of transfection assays using a series of deletion constructs and mutation constructs indicate that a l-bp mismatch of the consensus TATA-like box (TTTAAT) and the consensus sequence of the initiator site, which is located 45 bp downstream of the putative TATA box, were functioning cooperatively as a core promoter. The putative transcription site was confirmed by the results of RT-PCR southern blot analysis. We examined the regulation and the expression of this exon, 1.6, in several human cells and tissues by RT-PCR Southern blot analysis. THP-1 cells (mononuclear leukemic origin) and JEG-3 cells (choriocarcinoma origin) expressed exon 1.6 in serum-free media. The level of expression was increased by serum and phorbol myristyl acetate (PMA) in both cell lines. Adipose stromal cells also expressed exon I.6 in the presence of PMA. In fetal osteoblasts, the expression of exon 1.6 was increased most effectively by serum and less so by dexamethasone (DEX) + IL-1 beta and DEX + IL-11, whereas induction by serum was suppressed by the addition of DEX. The level of expression was low in granulosa cells in culture and did not change with forskolin. On the other hand, dibutyryl cAMP suppressed PMA-stimulated expression of exon 1.6 in THP-1 cells and adipose stromal cells. This result supports the hypothesis that the expression of exon 1.6 is regulated mainly via an AP-1 binding site that is found upstream of the initiator site of the promoter region. Expression of exon I.6-specific transcripts was examined in several human tissues. Testis and bone obtained from normal adults expressed exon I.6. Testicular tumor and hepatic carcinoma expressed high levels of exon 1.6, whereas granulosa cell tumor did not. Fetal liver and bone also showed a significant level of exon 1.6 expression, but not so much as testicular tumor and hepatic tumor. Several splicing variants of exon I.6 were detected especially in THP-1 and JEG-3 cells, and to a lesser extent in primary cultures and tissue samples. These variants were identified as an unspliced form, a form spliced at the end of exon I.4, a form spliced at the end of exon I.3 (truncated) and a form spliced 220 bp downstream of the 3' end of exon I.6. The last variant revealed a new splicing site. Because most of the splicing variants contain the sequence specific for exon I.3, RT-PCR specific for exon I.3 can coamplify these splicing variants of exon I.6 transcripts. These results suggests that it is necessary to examine the expression of I.6 in tissues that are known to express exon I.3 such as breast adipose tissue, in which promoter usage of exon I of the aromatase gene switches from exon I.4 to I.3 in the course of malignant transformation.

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 D-3 and also by their ability to deposit mineral. Aromatase activity was assessed by the formation of estrone from [1,2,6,7-H-3]androstenedione and by the release of tritium from [1 beta-H-3]androstenedione into [H-3]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-1 beta). The apparent K-m and V-max estimated by the release of [H-3]water was 5.8 +/- 0.6 nM and 10.8 +/- 1.4 pmol/mg per 6 h in the presence of DEX + IL-1 beta. The effects of several stimulants on aromatase activity in OLCs were examined: serum, IL-1 beta, TNF alpha 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, I.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: If (brain-specific promoter), PII and I.6 in OLCs stimulated by DEX + IL-1 beta, 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 knows 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. (C) 1998 Elsevier Science Ireland Ltd. All rights reserved.

The expression of aromatase, the enzyme responsible for estrogen biosynthesis, has been studied in THP-1 cells of human mononuclear leukemic origin, which exhibit high rates of aromatase activity. These cells have the capacity to differentiate in the presence of vitamin D into cells with osteoclast-like properties. Differentiated cells displayed higher rates of aromatase than undifferentiated cells, and, in both cases, activity was stimulated 10- to 20-fold by dexamethasone. Phorbol esters also increased aromatase activity, but the effect was the same in differentiated as in undifferentiated cells. In a similar fashion to adipose stromal cells, serum potentiated the response to dexamethasone but had no effect on phorbol ester-stimulated activity. By contrast to its action in adipose stromal cells, (Bu)(2)cAMP markedly inhibited aromatase activity of THP-1 cells, as did factors whose actions are mediated by cAMP, such as PTH and PTH-related peptide. This was true of control cells, as well as of dexamethasone-and phorbol ester-stimulated cells. Previously we have shown that type 1 cytokines as well as tumor necrosis factor-alpha stimulate aromatase activity of adipose stromal cells in the presence of dexamethasone. By contrast, interleukin-6, interleukin-ll, and leukemia-inhibitory factor had no effect on aromatase activity of THP-1 cells, whereas tumor were slightly inhibitory of aromatase activity. Exon-specific Southern analysis of rapid amplification of cDNA ends-amplified transcripts was employed to examine the distribution of the various 5'-termini of aromatase transcripts. In the control group, most of the clones contained transcripts specific for the proximal promoter IS, whereas in dexamethasone-treated cells, most transcripts contained exon I.4. In the phorbol ester-treated cells, a broader spectrum of transcripts was present, with equal proportions of I.4, II, and I.3-containing clones. Additionally, one clone containing a new sequence, exon I.6, was found. This was shown to be located about 1 kb upstream of exon II. By contrast, all clones from cells heated with (Bu)(2)cAMP contained promoter II specific sequences. In addition to these transcripts, two clones in the library from the dexamethasone-treated cells contained the sequence previously defined as the brain-specific sequence, If. In one of these, the If sequence was fused downstream of exon I.4, indicative that its expression likely employed promoter I.4. These results point to similarities and important differences between aromatase expression in THP-1 cells and other cells such as adipose stromal cells, indicative of unique regulatory pathways governing aromatase expression in these cells.

The expression of aromatase, the enzyme responsible for estrogen biosynthesis, has been studied in THP-1 cells of human mononuclear leukemic origin, which exhibit high rates of aromatase activity. These cells have the capacity to differentiate in the presence of vitamin D into cells with osteoclast-like properties. Differentiated cells displayed higher rates of aromatase than undifferentiated cells, and, in both cases, activity was stimulated 10- to 20-fold by dexamethasone. Phorbol esters also increased aromatase activity, but the effect was the same in differentiated as in undifferentiated cells. In a similar fashion to adipose stromal cells, serum potentiated the response to dexamethasone but had no effect on phorbol ester-stimulated activity. By contrast to its action in adipose stromal cells, (Bu)(2)cAMP markedly inhibited aromatase activity of THP-1 cells, as did factors whose actions are mediated by cAMP, such as PTH and PTH-related peptide. This was true of control cells, as well as of dexamethasone-and phorbol ester-stimulated cells. Previously we have shown that type 1 cytokines as well as tumor necrosis factor-alpha stimulate aromatase activity of adipose stromal cells in the presence of dexamethasone. By contrast, interleukin-6, interleukin-ll, and leukemia-inhibitory factor had no effect on aromatase activity of THP-1 cells, whereas tumor were slightly inhibitory of aromatase activity. Exon-specific Southern analysis of rapid amplification of cDNA ends-amplified transcripts was employed to examine the distribution of the various 5'-termini of aromatase transcripts. In the control group, most of the clones contained transcripts specific for the proximal promoter IS, whereas in dexamethasone-treated cells, most transcripts contained exon I.4. In the phorbol ester-treated cells, a broader spectrum of transcripts was present, with equal proportions of I.4, II, and I.3-containing clones. Additionally, one clone containing a new sequence, exon I.6, was found. This was shown to be located about 1 kb upstream of exon II. By contrast, all clones from cells heated with (Bu)(2)cAMP contained promoter II specific sequences. In addition to these transcripts, two clones in the library from the dexamethasone-treated cells contained the sequence previously defined as the brain-specific sequence, If. In one of these, the If sequence was fused downstream of exon I.4, indicative that its expression likely employed promoter I.4. These results point to similarities and important differences between aromatase expression in THP-1 cells and other cells such as adipose stromal cells, indicative of unique regulatory pathways governing aromatase expression in these cells.

The first case of Meckel-Gruber syndrome in a dizygotic twin pregnancy following in vitro fertilization and embryo transfer is reported. An ultrasound examination at 26 weeks of gestation revealed multiple malformations of the presenting twin: the combination of an occipital encephalomeningocele, bilateral polycystic kidneys and postaxial polydactyly was suggestive of Meckel-Gruber syndrome. Diagnosis was confirmed after birth. The importance of this case is stressed since particular care must be taken to avoid multiple pregnancies in couples at risk undergoing assisted reproduction.

The first case of Meckel-Gruber syndrome in a dizygotic twin pregnancy following in vitro fertilization and embryo transfer is reported. An ultrasound examination at 26 weeks of gestation revealed multiple malformations of the presenting twin: the combination of an occipital encephalomeningocele, bilateral polycystic kidneys and postaxial polydactyly was suggestive of Meckel-Gruber syndrome. Diagnosis was confirmed after birth. The importance of this case is stressed since particular care must be taken to avoid multiple pregnancies in couples at risk undergoing assisted reproduction.