Masafumi Haji

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.

Aromatase in bone cell: Association with osteoporosis in postmenopausal women

To clarify the possible action of adrenal androgen on bone cell, the existence, characteristics and regulation of aromatase in human osteoblast-like osteosarcoma cells (HOS) and primary cultured osteoblast-like cells from normal human bones (HO) were examined in this study. 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 (E2). In subset analysis, strongly positive correlation of serum DHEA-S and estrone (E1) with BMD was observed in postmenopausal women aged less than 69 years old. Administration of DHEA to ovariectomized rat significantly increased BMD and decreased relative osteoid volume in femur. These in vivo findings strongly suggested that serum adrenal androgen may be converted to estrogen in peripheral organ, especially, osteoblast and be important steroids to maintain BMD. [3H]DHEA was converted to [3H]androstenedione and [3H]androstenedione to [3H]estrone in primary cultured human osteoblast. Osteoblast-like cells showed aromatase activity, and an apparent Km and the Vmax were 4.74 +/- 0.78 nM (mean +/- SD, n = 3) and 0.83 +/- 0.79 fmol/mg protein/h for HOS, and 4.6 +/- 2.9 nM and 279 +/- 299 fmol/mg protein/h (mean +/- SD, n = 19) for HO, respectively. The aromatase activity was significantly increased by dexamethasone in a dose-dependent manner. Reverse transcription-polymerase chain reaction analysis revealed that dexamethasone increased the transcript of P450AROM gene. Osteoblast-specific promoters were also determined. Dexamethasone and 1 alpha,25-dihydroxyvitamin D3 synergistically enhanced aromatase activity and P450AROM mRNA expression. These results demonstrate that adrenal androgen, DHEA, is converted to E1 in osteoblast by P450AROM which is positively regulated by glucocorticoid and 1 alpha,25-dihydroxyvitamin D3 and important to maintain BMD in the 6 to 7th decade, after menopause.

Molecular cloning, sequence analysis and expression of a cDNA encoding human type-1 angiotensin II receptor

We isolated a cDNA encoding type-1 angiotensin II receptor from a human liver cDNA library. The cDNA had an open reading frame encoding a protein of 359 amino acid residues with a relative Mr of 41,060. The deduced amino acid sequence of the human angiotensin II (Ang II) receptor was 95.3% and 94.2% identical to those of bovine and rat type-1 Ang II receptors, respectively, and had a significant similarity with the G protein-coupled receptor. The rank order of the binding to the receptor expressed in COS-7 cells was Ang II greater than Ang III greater than Ang I. The expression of the Ang II receptor mRNA was detected in human liver, lung, adrenal and adrenocortical adenomas but not in adrenomedullary tumor, pheochromocytoma, by Northern blot analysis.

Aromatase activity in human osteoblast-like osteosarcoma cell

SummaryAromatase activity was studied in cultured human osteosarcoma cell (HOS). HOS was incubated from 12 to 72 hours with 10-10 M-10-5 M dexamethasone. Aromatase activity was determined by measuring [3H]H2O released upon the conversion of [1β-3H]androstenedione to estrone. HOS showed aromatase activity, and apparent km for [1β-3H]androstenedione was 4.46±0.98 nm (mean±SD). The aromatase activity was significantly increased by 10-9 M-10-5 M dexamethasone in a dose-dependent manner. Dexamethasone increased Vmax of aromatase activity but did not change its km value. These results suggest that osteoblastic cells have aromatase activity which is regulated by glucocorticoid, and directly convert androgen to estrogen in itself.

Age-related changes in the concentrations of cytosol receptors for sex steroid hormones in the hypothalamus and pituitary gland of the rat

Estrogen and androgen receptors were measured in cytosols from hypothalamus, pituitary and uterus or prostate of rats at 3 stages in life, from 90 to 650 days old in females and from 90 to 550 days old in males. Saturation analysis of cytosol 17 beta-estradiol receptors in females demonstrated a significant age-associated reduction in maximum binding capacity in hypothalamus and uterus already at 300-330 days of life, but there was no significant change in pituitary gland. However, there was no difference in binding affinity, steroid specificity, sedimentation coefficient, chemical nature and heat lability of cytosol 17 beta-estradiol binding of these tissues among the 3 age groups. In males, each receptor for 17 beta-estradiol, testosterone and 5 alpha-dihydrotestosterone was isolated by sucrose density gradient centrifugation from hypothalamic, pituitary and prostate cytosols. These receptors showed the same sedimentation coefficient of 8-9S in all age groups. Androgen binding by cytosols already decreased at 300-330 days of life, but estrogen binding was lower at 500-550 days of life than in younger adults. The increase in the serum luteinizing hormone level after gonadectomy was significantly depressed with aging in both females and males. These findings suggested that the age-associated reduction in cytosol sex steroid hormone receptors was ascribable to changes of numbers of binding sites. These age-related changes may be concerned with feedback system dysfunction of hypothalamic-pituitary-gonadal axis in aged rats.

Effect of dehydroepiandrosterone on glucose uptake in cultured human fibroblasts

Dehydroepiandrosterone (DHEA) and its sulfate derivative (DHEA-S) reportedly have antidiabetic and antiobesity effects. The effect of DHEA on glucose uptake in cultured human fibroblasts was examined. Incubation of cells with supraphysiologic concentrations of DHEA (10(-5) mol/L) for > or = 10 hours enhanced 2-deoxyglucose (2-DG) uptake significantly (P < .05). Supraphysiologic concentrations of insulin (10(-7) mol/L) increased the sensitivity of glucose uptake to DHEA. Conversely, the sensitivity of glucose uptake to insulin was increased by incubating cells with 10(-6) mol/L DHEA. Both the abundance of transcripts encoding glucose transporter-1 (Glut-1) and the maximal velocity (Vmax) of 2-DG transport were increased in cultured fibroblasts incubated with DHEA. Cultured fibroblasts expressed a specific binding factor with low affinity for [3H]DHEA (maximal number of binding sites, 18,496 sites per cell; Kd, 298 nmol/L). Other androgen hormones exerted a less-marked effect on glucose uptake; DHEA-S had no effect. These results suggested that DHEA increases Glut-1 mRNA through binding to a specific factor in cultured human fibroblasts and thereby stimulates glucose uptake in these cells.

Atrial and brain natriuretic peptide in adrenal steroidogenesis

We elucidated the role of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) in human and bovine adrenocortical steroidogenesis. The urinary volume, sodium excretion and cyclic GMP (cGMP) excretion and plasma cGMP were markedly increased by the synthetic alpha-human ANP (alpha-hANP) infusion in healthy volunteers. Plasma arginine vasopressin (AVP) and aldosterone levels were significantly suppressed. Both ANP and BNP inhibited aldosterone, 19-OH-androstenedione, cortisol and DHEA secretion dose-dependently and increased the accumulation of intracellular cGMP in cultured human and bovine adrenal cells. alpha-hANP significantly suppressed P450scc-mRNA in cultured bovine adrenal cells stimulated by ACTH. Autoradiography and affinity labeling of [125I]hANP, and Scatchard plot demonstrated a specific ANP receptor in bovine and human adrenal glands. Purified ANP receptor from bovine adrenal glands identified two distinct types of ANP receptors, one is biologically active, the other is silent. A specific BNP receptor was also identified on the human and bovine adrenocortical cell membranes. The binding sites were displaced by unlabelled ANP as well as BNP. BNP showed an effect possibly via a receptor which may be shared with ANP. The mean basal plasma alpha-hANP level was 25 +/- 5 pg/ml in young men. We confirmed the presence of ANP and BNP in bovine and porcine adrenal medulla. Plasma or medullary ANP or BNP may directly modulate the adrenocortical steroidogenesis. We demonstrated that the lack of inhibitory effect of alpha-hANP on cultured aldosterone-producing adenoma (APA) cells was due to the decrease of ANP-specific receptor, which caused the loss of suppression of aldosterone and an increase in intracellular cGMP.

Sertoli cell function declines earlier than Leydig cell function in aging Japanese men

In order to evaluate the age-related changes in Leydig cell and Sertoli cell function, we measured serum levels of total testosterone (TT), free testosterone (FT), inhibin, LH and FSH in 116 healthy Japanese men, aged 24-92 years. Serum TT remained constant up to the age of 80 years and decreased thereafter. Serum FT declined linearly with aging and was significantly lower in men in their forties than in younger men (24-39 years old). Serum inhibin levels also declined with aging, with serum concentrations significantly lower in men older than 40 and markedly lower in men over 80 years old. LH and FSH were elevated in men over 60 and 40 years old, respectively. We also examined relationships between gonadotropins and gonadal hormones in these men divided into three age groups, young (24-39 years old), middle aged (40-59 years old) and aged (60-92 years old) men. Although there was a significant inverse correlation between LH and TT or FT for the entire population, subset analysis demonstrated that this inverse correlation was confined to men over 60 years old. In fact, in young men, TT and FT were positively correlated with LH. Overall, there was also an inverse correlation between FSH and inhibin. In subset analysis this relationship was present in both middle aged and aged men. These findings suggest that in Japanese men testicular endocrine functions decline after the fourth decade of life, and that Sertoli cell function declines earlier than Leydig cell function.

In-vitro evidence for the regulation of 17,20-lyase activity by cytochrome b5 in adrenocortical adenomas from patients with Cushing's syndrome

OBJECTIVE The electron transfer system molecules, NADPH‐cytochrome P450 reductase (Red) and cytochrome b5 (bS) are known to increase the relative activity of 17,20‐lyase to 17α‐hydroxylase in vitro. Consistent with this hypothesis, we have reported recently that adrenocortical adenomas from patients with Cushings syndrome that produced exceptionally high concentrations of androgens also contained more b5 mRNA as well as greater 17,20‐lyase activity than adenomas that produced low concentrations of androgens. This finding was suggestive but inconclusive in linking b5 functionally to this difference in adenoma 17,20‐lyase activity. In the present study, we have extended this finding by examining the effect of bS on microsomal 17,20‐lyase activity using an antibody against cytochrome b5.

Markedly increased expression of cytochrome P-450 17α-hydroxylase (P-450c17) mRNA in adrenocortical adenomas from patients with Cushing's syndrome

We studied the contents of cortisol (F) and dehydroepiandrosterone (DHEA) and the expression of mRNA of cytochrome P-450 for side-chain cleavage (P-450scc), 17 alpha-hydroxylase (P-450c17), 21 alpha-hydroxylase (P-450c21) and 11 beta-hydroxylase (P-450c11) in adrenocortical adenomas from three patients with Cushings syndrome. The F content was significantly higher in adrenocortical adenomas than in normal adrenal glands, while the DHEA level was similar to that in normal adrenal glands. The adrenal adenomas showed a markedly higher level of P-450c17 mRNA, and a slightly but not significantly increased level of P-450c21 mRNA, compared with normal adrenal glands. The expression of P-450scc and P-450c11 mRNA in the adenomas was similar to that in normal adrenal glands. These results suggest that the overproduction of cortisol in adrenocortical adenomas associated with Cushings syndrome results from an increased expression of P-450c17 and P-450c21 mRNA.