Kiminobu Goto

Establishment and Characterization of a Steroidogenic Human Granulosa-Like Tumor Cell Line, KGN, That Expresses Functional Follicle-Stimulating Hormone Receptor

We established a steroidogenic human ovarian granulosa-like tumor cell line, designated KGN, from a patient with invasive ovarian granulosa cell carcinoma. KGN had a relatively long population doubling time of about 46.4 h and had an abnormal karyotype of 45,XX, 7q-, -22. A steroid analysis of the cultured medium by RIA performed 5 yr after the initiation of culture showed that KGN was able to secrete pregnenolone and progesterone, and both dramatically increased after stimulation with (Bu)(2)cAMP. However, little or no secretion of 17alpha-hydroxylated steroids, dehydroepiandrosterone, androstenedione, or estradiol was observed. The aromatase activity of KGN was relatively high and was further stimulated by (Bu)(2)cAMP or FSH. These findings showed a pattern similar to that of steroidogenesis in human granulosa cells, thus allowing analysis of naturally occurring steroidogenesis in human granulosa cells. Fas-mediated apoptosis of KGN was also observed, which mimicked the physiological regulation of apoptosis in normal human granulosa cells. Based on these findings, this cell line is considered to be a very useful model for understanding the regulation of steroidogenesis, cell growth, and apoptosis in human granulosa cells.

Combined treatment with specific ligands for PPARγ:RXR nuclear receptor system markedly inhibits the expression of cytochrome P450arom in human granulosa cancer cells

Our previous study demonstrated that PPARgamma specific ligand troglitazone (TGZ) or RXR specific ligand LG100268 (LG) alone decreased the aromatase activity in cultured human ovarian granulosa cells from pre-ovulatory follicles, and combined treatment caused an even greater reduction in this activity. Since similar manners of effects of TGZ or/and LG on the aromatase activity in human ovarian granulosa cancer cell line were observed, we performed the detailed analysis of the mechanisms of these effects using this cell line. The changes in the aromatase activity were associated with comparable changes in the P450arom mRNA levels based on a RNase protection assay. A nuclear run-on assay indicated the P450arom transcript to decrease by 40 and 66% at 24 and 48 h, respectively, after TGZ plus LG treatment. An RNA stability analysis showed the half-life of P450arom mRNA to decrease from 13 to 9 h after the TGZ plus LG treatment. The inhibitory effect of TGZ plus LG on the aromatase activity and P450arom mRNA may not be mediated by the cAMP-PKA pathway that is usually implicated in the regulation of aromatase activity in granulosa cells: because (1) the aromatase activity stimulated by forskolin was not inhibited by TGZ plus LG; (2) the specific PKA inhibitor H89 could not block the inhibitory effect of TGZ plus LG on the aromatase activity; and (3) the luciferase activity of P450arom promoter II did not decrease by the addition of TGZ and LG in transfected cells either at a basic state or in the states stimulated by forskolin or PGE2, respectively. Taken together, these results indicate that TGZ plus LG inhibited the aromatase activity and also decreased the P450arom mRNA level in granulosa cancer cells, and the loss of P450arom mRNA expression was considered to be due to both the decreased transcription and rapid degradation of its RNA.

Mechanism of action of anti-aging DHEA-S and the replacement of DHEA-S

The plasma ACTH and cortisol levels do not change during aging. On the other hand, the plasma dehydroepiandrosterone sulfate (DHEA-S) changes remarkably during aging. Before puberty, the plasma DHEA-S level both in males and females is very low, however, it rapidly increases at puberty, and thereafter significantly decreases both linearly and age-dependently. Cytochrome P450c17 has two enzyme activities, 17-alpha-hydroxylase and 17,20-lyase. Cortisol is synthesized by 17-alpha-hydroxylase, and DHEA is synthesized by 17,20-lyase. The mechanism of dissociation of cortisol and DHEA synthesis in aging depends on another regulator of 17,20-lyase of cytochrome P450c17 such as cytochrome P450 reductase. We demonstrated significant decrease in cytochrome P450 reductase activity in bovine aged adrenal glands. We clarified the beneficial effects of DHEA as an anti-aging steroid based on both in vitro and in vivo experiments, such as the stimulatory effect of immune system, anti-diabetes mellitus, anti-atherosclerosis, anti-dementia (neurosteroid), anti-obesity and anti-osteoporosis. It is very important to identify the mechanism of action of DHEA. We clarified the conversion of DHEA to estrone by cytochrome P450 aromatase in primary cultured human osteoblasts. We indentified high affinity of DHEA binding with K(d)=6.6 nM in antigen and DHEA stimulated human T lymphocytes. We searched for the target genes that are specifically induced in activated T lymphocytes in the presence of DHEA by subtractive hybridization screening for differentially expressed transcripts. The double blind, randomized human replacement therapies utilizing DHEA are also reviewed.

Aromatase in bone: roles of Vitamin D3 and androgens☆

We have mainly focused on the regulatory mechanism of cytochrome P450 aromatize in bone cells. Our previous study demonstrated a strong positive correlation of serum dehydroepiandrosterone sulfate (DHEA-S) and estrone (E1) with BMD in postmenopausal women but no correlation between serum estradiol (E2) and BMD in the same group. In addition, administration of DHEA to ovariectomized rat significantly increased BMD. These in vivo findings strongly suggested that circulating adrenal androgen may be converted to estrogen in osteoblast and may contribute to BMD maintenance. Actually, in cultured human osteoblast cells, DHEA was found to convert to androstenedione by 3beta-hydroxysteroid dehydrogenase (3beta-HSD) activity and then androstenedione to estrone through the apparent aromatase activity. The aromatase activity in cultured human osteoblast cells was significantly increased by dexamethasone (DEX). Interestingly, DEX and 1alpha,25-dihydroxyvitamin D3 (VD3) synergistically enhanced aromatase activity as well as P450arom mRNA expression. A little stronger induction of aromatase activity by DEX and VD3 was observed in cultured human fibroblasts. The increase of the aromatase activity by DEX and VD3 was accompanied with the increase of luciferase activity of fibroblast cells transfected with Exon 1b-promoter-luciferase construct, but not of osteoblasts transfected with the same construct, suggesting a different regulatory mechanism of aromatase by DEX and 1alpha,25-dihydroxyvitamin D3 (VD3) between these two cells despite the same promotor usage. In human bone cells, intracrine mechanism through aromatase activity, together with a positive regulation of aromatase activity by glucocorticoid and VD3, may contribute to the local production of estrogens, thus leading to protective effect against osteoporosis especially after menopause. The effect of sex steroids (estrogen versus testosterone) in bone remodeling was also briefly reviewed based on several recent findings in this field.

SF-1/Ad4BP transforms primary long-term cultured bone marrow cells into ACTH-responsive steroidogenic cells

Bone marrow stem cells develop into haematopoietic and mesenchymal lineages, but have not been known to participate in steroidogenic cell production. Steroidogenic factor 1 (SF‐1), also designated adrenal 4 binding protein (Ad4BP), is an essential orphan nuclear receptor for steroidogenesis as well as for adrenal and gonadal gland development. In the present study, we revealed that the adenovirus‐mediated forced expression of SF‐1 can transform cultured primary long‐term cultured bone marrow cells into steroidogenic cells, showing the de novo synthesis of multiple steroid hormones in response to adrenocorticotropic hormone (ACTH). This finding may provide an initial step in innovative autograft cell transfer therapy for steroid hormone deficiencies.

Activation Function-1 Domain of Androgen Receptor Contributes to the Interaction between Subnuclear Splicing Factor Compartment and Nuclear Receptor Compartment IDENTIFICATION OF THE p102 U5 SMALL NUCLEAR RIBONUCLEOPROTEIN PARTICLE-BINDING PROTEIN AS A COACTIVATOR FOR THE RECEPTOR

In the androgen receptor (AR), most of its transactivation activity is mediated via the activation function-1 (AF-1). By employing yeast two-hybrid assay, we isolated a cDNA sequence encoding a protein binding to AR-AF-1. This protein, named ANT-1 (AR N-terminal domain transactivating protein-1), enhanced the ligand-independent autonomous AF-1 transactivation function of AR or glucocorticoid receptor but did not enhance that of estrogen receptor α. In contrast, the ANT-1 did not enhance any ligand-dependent AF-2 activities. Furthermore, the ligand-independent interaction between AR-AF-1 and ANT-1 was confirmedin vivo and in vitro. The ANT-1 sequence was identical to that of a protein that binds to U5 small nuclear ribonucleoprotein particle, a human homologue of yeast splicing factor Prp6p, involved in spliceosome. ANT-1 was compartmentalized into 20–40 coarse splicing factor compartment speckles against the background of the diffuse reticular distribution. AR colocalized with ANT-1 only in the diffusely distributed area, whereas the ANT-1 speckles were spatially distinct from but surrounded by the AR compartments. The active gene transcription has been shown to couple simultaneously with pre-mRNA processing at the periphery of the splicing factor compartment. The molecular interaction between two spatially distinct subnuclear compartments mediated by ANT-1 may therefore recruit AR into the transcription-splicing-coupling machinery.

Sexually dimorphic expression of Dax-1 in the adrenal cortex

Background: The DAX‐1 (also known as AHC) gene encodes an unusual member of the nuclear receptor superfamily, and the gene product plays a pivotal role during gonadal and adrenal differentiation. Mutations of the human DAX‐1 gene cause X‐linked adrenal hypoplasia congenita associated with hypogonadotropic hypogonadism. However, the molecular mechanisms underlying the transcriptional regulation of the gene are not well understood.

Dehydroepiandrosterone (DHEA) as a possible source for estrogen formation in bone cells: correlation between bone mineral density and serum DHEA-sulfate concentration in postmenopausal women, and the presence of aromatase to be enhanced by 1,25-dihydroxyvitamin D3 in human osteoblasts.

A significant positive correlation between bone mineral density (BMD) and serum dehydroepiandrosterone sulfate (DHEA-S) was found in 120 postmenopausal women (51-99 years old) but no correlation was seen between BMD and serum estradiol. In subset analysis, strong positive correlation of serum DHEA-S and estrone with BMD was observed in postmenopausal women aged less than 69 years old. To study a possible role of DHEA-S in preventing osteoporosis, we characterized aromatase activity converting androgens to estrogens in human osteoblasts, because postmenopausal women maintain considerable levels of adrenal androgens. Glucocorticoids at 10(-9) to 10(-7) M induced transiently the expression of and the enzymatic activity of aromatase cytochrome P450 (P450AROM) in primary cultured osteoblasts. 1,25-Dihydroxyvitamin D3 (1,25-(OH)(2)D(3)) alone did not induce the aromatase activity, but enhanced and maintained the glucocorticoid-induced P450AROM gene expression. Analysis of the activity of P450AROM gene 1b (I.4) promoter, which is used dominantly in human osteoblasts, indicated that the region from -888 bp to -500 bp, which does not contain a typical vitamin D responsive element, is responsible for the enhancing effect of 1,25-(OH)(2)D(3). These results may suggest that adrenal androgen, DHEA, is converted to estrone in osteoblast by P450AROM, which is positively regulated by glucocorticoid and 1,25-(OH)(2)D(3), and is important in maintaining BMD in the sixth to the seventh decade, after menopause.

Isolation and characterization of the rat catalase-encoding gene

Using cDNA clones coding for rat liver catalase (hydrogen peroxide:hydrogen peroxide oxidoreductase, EC 1.11.1.6), overlapping genomic clones were isolated. By Southern blotting analysis and nucleotide sequencing, the gene was characterized to be about 33 kb in length and to have 13 exons and twelve introns. S1 mapping and primer extension analyses showed the presence of multiple transcription start points (tsp) located between 66 bp and 105 bp upstream from the translational start codon. Nucleotide sequence immediately upstream from the tsp lacks the TATA box but contains multiple CCAAT boxes and GC-like boxes.

Regulation of aromatase by nuclear receptors.

We investigated the effects of a nuclear receptor system constituted by retinoid X receptor (RXR) and its heterodimer partner on the aromatase activity in a cultured MCF-7 human breast cancer cell line and also in human ovarian granulosa cells, using each selective ligand for retinoic acid receptor, RAR (TTNPB), retinoid X receptor, RXR (LG100268), PPARgamma (troglitazone), and vitamin D3 receptor (cholecalciferol). In MCF-7 cells, the combined treatment with TTNPB and LG100268 caused a dramatic stimulation of the aromatase activity. The combined treatment with other ligand and LG100268 had little or no effect on the aromatase activity. The increase in the aromatase activity by TTNPB plus LG100268 was accompanied by an increase in the P450arom mRNA levels, which was also found to be related to the specific usage of promoter 1a of the CYP19 gene. These results suggest that a nuclear receptor system constituted by a RAR:RXR heterodimer is involved in the regulation of aromatase activity in MCF-7 breast cancer cells. In cultured human ovarian granulosa cells obtained from patients who underwent in vitro fertilization, troglitazone or LG100268 alone decreased the aromatase activity, while the combined treatment caused an even greater reduction in this activity. Little effect of other specific ligands for RXR heterodimer partners may support the notion that the effects of troglitazone and/or LG100268 in human granulosa cells may be mediated through the specific activation of PPARgamma:RXR heterodimer system. Since similar manners of effects of several PPARgamma ligands and/or LG100268 on the aromatase activity were observed in a newly established human ovarian granulosa cancer cell line, KGN, we performed the detailed analysis of the mechanisms of these effects using this cell line. As a result, the inhibitory effect of aromatase activity by troglitazone plus LG100268 was accompanied by the decrease of P450arom mRNA level. Furthermore, the loss of P450arom expression was considered to be due to both the decreased transcription and rapid degradation of its RNA based on the studies of nuclear run-on assay and RNA stability assay. In conclusion, RAR:RXR and PPARgamma:RXR heterodimer nuclear receptor systems may be other important modulators of estrogen production in human breast cancer cells and ovarian granulosa cells, respectively.