Regulation of 3 beta-hydroxysteroid dehydrogenase expression in human adrenocortical H295R cells.

Previous studies of the effects of angiotensin II (All), alone or in combination with activators of the protein kinase. A signalling pathway, have yielded inconsistent findings on the expression of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD and 17 alpha-hydroxylase cytochrome P450 (P450c17) as well as the corresponding responses on steroid secretory products in human adrenocortical cells. We have used the human adrenocortical carcinoma H295R cell further to evaluate this question, as well as to determine the role of protein kinase C in each of these responses to All.

Differential regulation of 11 beta-hydroxylase and aldosterone synthase in human adrenocortical H295R cells.

In humans the last steps in the synthesis of aldosterone and cortisol rely on the activity of two cytochrome P450 genes termed CYP11B2 (aldosterone synthase; P450aldo) and CYP11B1 (11 beta hydroxylase; P450cl1). The mechanisms which lead to differential expression of these two genes within the adrenal cortex are not well-defined. The human adrenocortical cell line.

Role of calmodulin-dependent protein kinase II in the acute stimulation of aldosterone production.

Acute aldosterone production in adrenocortical cells is highly dependent on calcium (Ca2+) and calmodulin (CaM) activation. To determine the role of calmodulin-dependent protein kinase II (CaM kinase II) in human adrenal aldosterone production, the action of KN93 (a specific CaM kinase II inhibitor) on human adrenocortical H295R cells was examined. The stimulation of aldosterone, production by angiotensin II (Ang II) and potassium (K+) were inhibited by KN93 in a concentration-dependent manner with an IC50 of approximately 0.

Differential control of 17 alpha-hydroxylase and 3 beta-hydroxysteroid dehydrogenase expression in human adrenocortical H295R cells.

Previous studies of human adrenocortical cells have given inconsistent findings concerning the effects of angiotensin II (AII) alone or in combination with activators of the protein kinase A-signaling pathway on expression of cholesterol side-chain cleavage cytochrome P450 (P450scc), 17 alpha-hydroxylase cytochrome P450 (P450c17), and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), as well as the corresponding effects on adrenocortical cell steroid secretory products. We have used the human adrenocortical carcinoma H295R cell to evaluate further this question and determine the role of protein kinase C in each of these responses to AII. Treatment with AII alone (10 nmol/L, 48 h) resulted in a significant increase in cortisol production (1.

The H295R human adrenocortical cell line contains functional atrial natriuretic peptide receptors that inhibit aldosterone biosynthesis.

The inhibitory effect of atrial natriuretic peptide (ANP) on angiotensin II (AII)-stimulated aldosterone secretion has been previously studied in rat and bovine adrenal zona glomerulosa cells in primary culture. However the understanding of the mode of action of ANP at the molecular level has been hampered by limitations of those primary cell culture systems and by the lack of cell lines from human adrenal cortex. Here we demonstrate the presence of fully functional ANP receptors in the recently characterized AII-responsive adrenocortical carcinoma cell line H295R.

17 beta-hydroxysteroid dehydrogenase in common epithelial ovarian tumors.

Type 1 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) is an estrogen-metabolizing enzyme that catalyzes the conversion of estrone to the more biologically potent estradiol. We examined the immunolocalization of 17 beta-HSD in five specimens of normal human ovary and in 51 specimens of common epithelial tumors of the ovary to study the expression of 17 beta-HSD in these ovarian neoplasms. 17 beta-HSD immunoreactivity was detected in granulosa cells of dominant preantral follicles but not in the surface epithelium of the normal cycling human ovary.

Inhibin-B: a likely candidate for the physiologically important form of inhibin in men.

Inhibin is a glycoprotein hormone that is defined on the basis of inhibition of pituitary FSH production, However, previous data have not shown any correlation between RIA measurements of inhibin and FSH in men. New enzyme-linked immunosorbent assays, specific for inhibin A, inhibin B, and inhibin pro-alphaC-related immunoreactivity, were applied to the measurement of inhibin in 32 healthy men. Further measurements of inhibin B and pro-alphaC-RI were carried out on groups of men exhibiting a wide range of FSH concentrations, including semen donors, infertile men, and men with elevated FSH concentrations.

The effect of transforming growth factor-beta on steroidogenesis and expression of key steroidogenic enzymes with a human ovarian thecal-like tumor cell model.

Objective: Our purpose was to determine the effects of transforming growth factor-beta on steroidogenesis and regulation of steroidogenic enzyme expression by use of a human ovarian thecal-like tumor cell culture system.

Study Design: Human ovarian thecal-like tumor cells were treated in serum-free medium in the presence or absence of forskolin and transforming growth factor-beta 1. The accumulation of progesterone and androstenedione in the culture medium was evaluated by radioimmunoassay.

Inhibin and activin differentially regulate androgen production and 17 alpha-hydroxylase expression in human ovarian thecal-like tumor cells.

Activin and inhibin are structurally related dimeric glycoproteins belonging to the transforming growth factor-beta superfamily of proteins which are synthesized and secreted by the granulosa cells of the ovary. Although initially characterized by their ability to influence FSH secretion from pituitary cells, paracrine regulatory roles of these factors on neighboring ovarian theca interna have been suggested. While inhibin has been shown to increase and activin to decrease the production of androgens, the mechanisms of action are not well defined, partly due to difficulties in obtaining adequate numbers of thecal cells from individual patients or animal models.

Telomerase activity in human germline and embryonic tissues and cells.

Telomerase is a ribonucleoprotein that synthesizes telomere repeats onto chromosome ends and is involved in maintaining telomere length in germline tissues and in immortal and cancer cells. In the present study, the temporal regulation of expression of telomerase activity was examined in human germline and somatic tissues and cells during development. Telomerase activity was detected in fetal, newborn, and adult testes and ovaries, but not in mature spermatozoa or oocytes.

The effect of insulin and insulin-like growth factors on the expression of steroidogenic enzymes in a human ovarian thecal-like tumor cell model.

Objective: To determine the effects of insulin and insulin-like growth factors (IGF-I and IGF-II) on steroidogenesis and steroidogenic enzyme expression in a human ovarian thecal-like tumor cell culture model system.

Design: Human ovarian thecal-like tumor cells treated with forskolin and insulin IGF-I or IGF-II were evaluated for media accumulation of P and androstenedione (A) as well as 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) and cytochrome P450 17 alpha-hydroxylase (P450c17) enzyme activity. Northern analysis of cytochrome P450 side chain cleavage (P450scc), P450c17, and 3 beta HSD messenger RNA (mRNA) also was performed.

Human ovarian tumor cells: a potential model for thecal cell steroidogenesis.

Ovarian thecal cell production of C19 steroids (i.e. dehydroepiandrosterone, androstenedione, and testosterone) is necessary to provide substrate for granulosa cell biosynthesis of estrogen; however, excessive production of C19 steroids can lead to disorders associated with androgen excess.

The steroidogenic acute regulatory protein is induced by angiotensin II and K+ in H295R adrenocortical cells.

Adrenal steroid hormone biosynthesis can be activated by the protein kinase A pathway by ACTH, the protein kinase C pathway by angiotensin II (AII), or by increasing intracellular Ca2+ levels by AII or K+. Although their mechanisms of action are not known, each of these pathways is dependent upon the de novo synthesis of a protein that is required for the acute production of steroids. We have recently proposed the steroidogenic acute regulatory (StAR) protein as this required protein, therefore, we examined the effect of different agonists on StAR's expression in H295R human adrenocortical carcinoma cells.

Adrenal androgen biosynthesis with special attention to P450c17.

In vitro studies of human adrenal androgen synthesis are limited because of the difficulties in obtaining adrenals. We describe the use of the human adrenocortical tumor H295 cell line as a model to evaluate mechanisms controlling C19-steroid production. The cells were characterized with regard to responsiveness to a variety of agents as measured by steroid secretion and induction of 17 alpha-hydroxylase cytochrome P450 (P450c17) expression, a key enzyme in C19-steroid production.

Ca(2+)-regulated expression of steroid hydroxylases in H295R human adrenocortical cells.

Although changes in the expression of key steroidogenic enzymes such as cytochrome P450 cholesterol side-chain cleavage, 17 alpha-hydroxylase (P450c17), aldosterone synthase, and 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) in the human adrenal cortex are known to be controlled by factors activating the protein kinase A or protein kinase C signaling pathways, little is known concerning the effects of increased intracellular Ca2+. In this study we describe the effects of K+, an agent known to increase intracellular Ca2+ through the opening of voltage-sensitive Ca2+ channels, on steroidogenesis in H295R human adrenocortical cells and corresponding changes in expression of these vital steroidogenic enzymes. Treatment of cells for 48 h with K+ (14 mM) resulted in an increase in aldosterone (3.

Potassium negatively regulates angiotensin II type 1 receptor expression in human adrenocortical H295R cells.

We have previously shown that the human adrenocortical H295R cell line expresses the type 1 angiotensin II receptor (AT1-R) and that expression of this receptor is downregulated at the level of mRNA by forskolin or dibutyryl-cAMP as well as by angiotensin II (Ang II). In this study we examine the effects of K+ on both AT1-R mRNA and receptors, as monitored through 125I-Ang II binding in the presence of PD 123319. After treatment with a maximal stimulatory steroidogenic dose of K+ (14 mmol/L), H295R cells showed an increase in cytosolic free Ca2+ from 113 to 212 nmol/L.

Regulation of ferredoxin gene in steroidogenic and nonsteroidogenic cells.

Ferredoxin is an electron transport intermediate for all the mitochondrial cytochromes P450. It is especially abundant in steroidogenic organs where it functions in steroid biosynthesis. The regulation of ferredoxin gene expression was studied in both steroidogenic and nonsteroidogenic cell lines.

The effects of insulin on 3 beta-hydroxysteroid dehydrogenase expression in human luteinized granulosa cells.

Objective: We determined the relative effects of insulin and FSH on progesterone accumulation as well as activity, protein content, and mRNA expression of 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) in human luteinized granulosa cells.

Methods: Luteinized granulosa cells obtained from women undergoing in vitro fertilization were plated and grown to near confluence and treated with FSH, insulin, or a combination of insulin and FSH. Progesterone production as well as enzyme activity, protein content, and mRNA expression for 3 beta HSD were evaluated.

The effects of KN62, a Ca2+/calmodulin-dependent protein kinase II inhibitor, on adrenocortical cell aldosterone production.

The effects of KN62 on aldosterone secretion have been studied using an angiotensin II (AII)- and K(+)-responsive human adrenocortical tumor cell line (H295R). Basal aldosterone secretion (measured by RIA) was 0.57 +/- 0.

Hormonal regulation of angiotensin II type 1 receptor expression and AT1-R mRNA levels in human adrenocortical cells.

Human adrenocortical H295R cells express AII receptors which are predominantly of the AT1 but not AT2 subclass. These receptors are functionally coupled to phosphoinositidase C in a manner similar to that seen in fetal human, sheep and bovine adrenocortical cells. Treatment of H295R cells with forskolin or dbcAMP to activate the protein kinase A pathway caused a rapid (maximal by 3 h) and sustained decrease in AT1-R mRNA levels which in turn preceded a time-dependent (maximal by 12 h) and dose-dependent loss of [125I]AII binding and phosphoinositidase C activation on subsequent AII challenge.

Immunohistochemical and biochemical analysis of a human Sertoli-Leydig cell tumor: autonomous steroid production characteristic of ovarian theca cells.

Objective: To ascertain the steroidogenic profile and location of steroidogenic enzymes in a steroid-secreting Sertoli-Leydig cell tumor of the ovary.

Methods: Steroid levels from peripheral, left ovarian (tumor), and right ovarian venous blood were measured. Tumor tissue was examined for the steroidogenic enzymes 17 alpha-hydroxylase (P450c17) and 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) by immunohistochemistry.

Regulatory effects of multifunctional cytokines and steroid hormones on apolipoprotein B production by human fetal hepatocytes.

Objective: The purpose of the present investigation was to determine the effects of certain multifunctional cytokines (tumor necrosis factor-alpha [TNF-alpha], interleukin-1 beta [IL-1 beta], and IL-6) and steroid hormones (estradiol, testosterone, progesterone, and cortisol) on the production of apolipoprotein B (Apo B) by cultured human fetal hepatocytes. We conducted these experiments because of our recent observations that purified human fetal Kupffer cells produce TNF-alpha, IL-1 beta, and IL-6.

Methods: Human fetal hepatocytes, specifically depleted of hematopoietic precursors and Kupffer cells, were cultured in defined medium.

Regulation of type 1 angiotensin II receptor messenger ribonucleic acid expression in human adrenocortical carcinoma H295 cells.

We have studied the hormonal regulation of type 1 angiotensin-II receptor (AT1-R) mRNA expression and [125I]angiotensin-II ([125I]AII) binding in human adrenocortical carcinoma H295 cells, which exhibit predominantly AT1-subtype receptors. Activation of the cAMP signaling pathway with forskolin or (Bu)2cAMP caused a rapid decrease in AT1-R mRNA levels (decreased 65% within 3 h). This preceded a time-dependent (maximal, 70% within 12 h) and dose-dependent (IC50, 2 microM forskolin) loss of [125I]AII binding together with decreased phosphoinositidase-C activation (72% decrease) on subsequent AII challenge.

Ontogeny of steroidogenic enzyme expression in the porcine conceptus.

This study examined fetal steroidogenic enzyme expression and function during pregnancy in the pig. Northern and Western analyses were performed to detect the cytochrome P450 enzyme 17 alpha-hydroxylase/17-20 lyase (P450c17) and that for cholesterol side-chain cleavage (P450scc), as well as 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) expression in several porcine fetal tissues. The data demonstrate higher steroidogenic enzyme expression in the fetal adrenal glands and testes than in the placenta at all stages of development examined.

The NCI-H295 cell line: a pluripotent model for human adrenocortical studies.

The human adrenal cortex is a complex endocrine organ that secretes mineralocorticoids, glucocorticoids and adrenal androgens. These steroids arise from morphologically and biochemically distinct zones of the adrenal gland. Studying secretion of these distinct steroid hormones has, in the past, required the isolation of cells from each of the adrenocortical zones.