Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.1.1.3 (HSD)
3,464 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The development of long term culture conditions with which to study the regulation of expression of aromatase, cholesterol side-chain cleavage enzyme, and 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) in human granulosa-lutein cells is described in this report. Conditions have been established for the dispersal, growth, freezing, and storage of functional human granulosa cells isolated from preovulatory follicles of women undergoing laparoscopy for gamete intrafallopian tube transfer and in vitro fertilization procedures. Optimal growth conditions for human granulosa-lutein cells were determined by plating cells at a low density and testing the capacity of a variety of culture conditions to support growth. A combination of fetal bovine serum (FBS), horse serum, and the serum substitute UltroSer G was found to increase cell number to maximal levels, 8- to 10-fold higher than with sera alone. Human granulosa-lutein cells grown under these conditions had a doubling rate of 36-40 h and were morphologically distinct from human theca interna cells grown under similar conditions. Human granulosa-lutein cells treated with forskolin retracted and rounded up, whereas cultures of human ovarian theca interna cells or human fibroblasts treated similarly did not retract. Human granulosa-lutein cells were grown for successive passages and transferred to serum-free medium containing forskolin, LH, hCG, or cholera toxin. Addition of these agents resulted in a time- and dose-dependent increase in aromatase activity and progesterone secretion. In these studies FSH treatment was found not to increase aromatase activity. In a study of the time course of 3 beta HSD activity in the absence of forskolin under serum-free conditions, it was found that 3 beta HSD activity increased 3-fold during the 72-h treatment period. Forskolin-stimulated 3 beta HSD activity also increased in a time-dependent manner, with levels in treated cells 3-fold higher than those in control cells. Northern analysis performed on total RNA obtained from forskolin- or hCG-stimulated granulosa-lutein cells confirmed that the increase in aromatase activity was associated with a corresponding increase in levels of mRNA specific for aromatase cytochrome P-450. Levels of mRNA encoding cholesterol side-chain cleavage cytochrome P-450 were similarly increased in cells treated with forskolin compared with unstimulated values at each of the time points investigated. Under serum-free conditions in the absence of stimulation, the 3.4-kilobase band of aromatase cytochrome P-450 mRNA was detectable.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Proliferating human granulosa-lutein cells in long term monolayer culture: expression of aromatase, cholesterol side-chain cleavage, and 3 beta-hydroxysteroid dehydrogenase. 237 Feb 96

The relationship between cAMP and protein kinase C in the regulation of 3 beta-hydroxysteroid dehydrogenase (3 beta HSD), 17 alpha-hydroxylase, and sulfotransferase was examined in human fetal adrenocortical cells under defined serum-free conditions in culture. Forskolin induced 3 beta HSD and 17 alpha-hydroxylase in a dose-dependent manner, with maximal effects at 10 microM. 12-O-Tetradecanoyl phorbol 13-acetate (TPA) at 1 nM depressed the induction of 17 alpha-hydroxylase activity by forskolin by more than 95% and increased the stimulation of 3 beta HSD activity by forskolin by 4- to 5-fold. Increases were maximal at 48-72 h of incubation. Dehydroepiandrosterone sulfotransferase activity increased over 48 h when cells were transferred to serum-free defined medium. Addition of 10 microM forskolin stimulated sulfotransferase activity only when cells remained in 10% serum. TPA at 1 nM inhibited the increase in sulfotransferase activity. The concentration of TPA required for inhibition of forskolin-stimulated 17 alpha-hydroxylase and sulfotransferase activity was similar to that required for enhancement of forskolin-induced 3 beta HSD activity, suggesting that comparable levels of C kinase activation are involved in these events. Angiotensin II, carbachol, epidermal growth factor, and fibroblast growth factor had actions similar to those of TPA on one or more of these enzyme activities. TPA also had similar actions on enzyme activities when they were stimulated by cAMP analogs rather than by forskolin. These studies suggest that adrenal steroid biosynthesis is under dual regulation by cAMP and protein kinase C. cAMP induces enzymes required for synthesis of 17 alpha-hydroxylated steroids, including the adrenal androgens. Activation of protein kinase C may play a complementary role by enhancing the induction of enzymes required for non-17 alpha-hydroxylated steroid biosynthesis and inhibiting those involved in the synthesis of androgens.
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PMID:Dual regulation of 3 beta-hydroxysteroid dehydrogenase, 17 alpha-hydroxylase, and dehydroepiandrosterone sulfotransferase by adenosine 3',5'-monophosphate and activators of protein kinase C in cultured human adrenocortical cells. 283 83

Intracellular effector systems which utilize PKA and PKC can be pharmacologically activated by forskolin and phorbol 12-myristate 13-acetate (PMA) and appear to be important for regulation of steroidogenesis by cells of the corpus luteum. In this study the effect of pharmacologic activation of PKA (forskolin) or PKC (PMA) on the activity of adenylate cyclase, cholesterol esterase, P450 cholesterol side chain cleavage (P450scc) and 3 beta-hydroxysteroid dehydrogenase/delta 5, delta 4 isomerase (3 beta HSD) was determined. Basal adenylate cyclase activity (as measured by intracellular and secreted cAMP) was extremely low in both large and small luteal cells. Forskolin stimulated adenylate cyclase activity in both large and small luteal cells but progesterone production was increased only in small cells. PMA inhibited progesterone production by large and forskolin-stimulated small cells without altering adenylate cyclase activity. Basal cholesterol esterase activity was greater in small than in large cells and was stimulated by forskolin only in small cells. PMA did not significantly alter cholesterol esterase activity in either cell type. Activity of P450scc or 3 beta HSD was measured by conversion of hydroxylated cholesterol derivatives (P450scc) or pregnenolone (3 beta HSD) to progesterone. Although basal progesterone production was 47 times greater in large than small cells, there was only 5.1 (P450scc) and 6.4 (3 beta HSD) times greater enzyme activity in large than in small luteal cells. Activation of PKA and/or PKC did not alter the activity of P450scc or 3 beta HSD in either cell type.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Steroidogenic enzyme activity after acute activation of protein kinase (PK) A and PKC in ovine small and large luteal cells. 814 91

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. Using a unique human ovarian thecal-like tumor (HOTT) cell culture model system we investigated the biochemical and molecular mechanisms controlling C19 steroidogenesis and the effects of activin and inhibin on the activity and expression of key ovarian thecal steroidogenic enzymes, cholesterol side-chain cleavage cytochrome P450 (P450scc), 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) and 17 alpha-hydroxylase/17,20 lyase cytochrome P450 (P450c17). Steroid production, level of steroidogenic enzyme mRNA expression, and enzyme activity following treatment with forskolin, inhibin-A and activin-A were examined. Basal steroid production, enzyme activities, and steroidogenic enzyme mRNA levels were not markedly different following treatment with activin (25 ng/ml) or inhibin (25 ng/ml) alone. Forskolin (10 microM) markedly increased production of both androstenedione (fivefold) and progesterone (threefold) as well as the activity of 3 beta HSD (sevenfold), and P450c17 (sevenfold) over basal. Forskolin stimulated the expression of mRNA for P450scc (fourfold), 3 beta HSD (threefold), and P450c17 (eightfold) over basal. Androstenedione accumulation was decreased by 60% in the forskolin plus activin group compared with forskolin alone, while progesterone production was maintained. This was attributed to a reduction of P450c17 mRNA (45% of forskolin alone) and activity (45% of forskolin alone). In contrast, co-treatment with forskolin and inhibin increased androstenedione production by 40% while decreasing progesterone by 40% compared with forskolin alone. Concomitantly, this was associated with a higher P450c17 mRNA expression (1.5-fold) and activity (twofold) but with minimal effects on the mRNA for 3 beta HSD and P450scc. HOTT cell responses to activin (0.05-50 ng/ml) and inhibin (0.05-50 ng/ml) in the presence of forskolin demonstrated dose-dependent effects on the steroid accumulation, enzymatic activity and mRNA expression of P450c17. Additionally, the differences seen on mRNA expression of steroidogenic enzymes in response to these factors were time-dependent. In summary, forskolin stimulated C19 steroid production from HOTT cells by increasing the expression of all steroidogenic enzymes examined. Inhibin and activin exerted differential effects on the expression of these enzymes which resulted in alterations in the steroid profile toward production of C19 steroids in the case of inhibin and away from C19 steroids in the case of activin. The influence of these important intraovarian factors on the expression of P450c17, a pivotal enzyme in thecal cell production of C19 steroids, could impact greatly on the follicular milieu of a normal developing follicle as well as in pathophysiological disorders such as polycystic ovarian syndrome.
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PMID:Inhibin and activin differentially regulate androgen production and 17 alpha-hydroxylase expression in human ovarian thecal-like tumor cells. 869 35

The physiological importance of adrenal 21-hydroxylase cytochrome P450 (CYP21) expression is clearly demonstrated by 21-hydroxylase deficiency, which results in adrenal hyperplasia and over-production of C19 steroids, leading to virilization. The mechanisms regulating normal expression of this key enzyme in human adrenocortical cells are ill defined. Herein we examine the role of the calcium, protein kinase C, and protein kinase A signaling pathways in the expression of CYP21 messenger ribonucleic acid (mRNA) using the H295R human adrenocortical cell model. Forskolin (10 mumol/L) treatment caused a progressive increase in CYP21 mRNA levels (maximum, 4-fold; P < 0.05) over 36 h of treatment, whereas angiotensin II (AII; 10 nmol/L) produced a smaller, biphasic rise (maximum, 1.8-fold at 12 h; P < 0.05). K+ (14 mmol/L) also induced a time-dependent (maximal, 1.5-fold at 12 h; P < 0.05) and dose-dependent (P < 0.05 12 mmol/L or above at 20 h) rise in CYP21 mRNA levels. The action of forskolin was reproduced by dibutyryl cAMP, confirming the involvement of cAMP in this response. The action of AII was greater than that of K+ or the calcium channel agonist BAYK8644, suggesting that AII action was not solely through the Ca2+ signaling pathway. The action of AII was reproduced and indeed exceeded by the protein kinase C activator 12-O-tetradecanoylphorbol 13-acetate (TPA; 10 nmol/L; 5.5-fold increase; P < 0.05). The actions of forskolin alone were not significantly increased by combined treatment with AII, suggesting neither synergy nor attenuation of the effects of protein kinase A activation. This was further demonstrated at the level of mRNA and 21-hydroxylase activity by the observation that the effect of forskolin and TPA in combination did not exceed that of TPA alone. Inhibition of protein synthesis with cycloheximide blocked induction of CYP21 as well as type II 3 beta-hydroxysteroid dehydrogenase (3 beta HSDII) mRNA expression in response to AII, forskolin, and dibutyryl cAMP, but had no effect on 17 alpha-hydroxylase cytochrome P450 (CYP17) or cholesterol side-chain cleavage cytochrome P450 (CYP11A) mRNA. Together, these findings were remarkably similar to those of our previous studies regarding mechanisms regulating 3 beta HSDII expression and underline the existence of a subset of steroidogenic enzymes regulated positively (CYP21 and 3 beta HSDII) as opposed to negatively (CYP17 and CYP11A) by the protein kinase C signaling pathway. The additional finding of a small induction of CYP21 expression in response to increased Ca2+, as previously reported for CYP17, but not 3 beta HSDII, expression, also demonstrates that the mechanisms of control of CYP21 and 3 beta HSDII are not identical. This latter finding may also relate to how CYP21 as well as CYP17 expression continues in the zona reticularis after adrenarche, whereas 3 beta HSD expression declines.
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PMID:Protein kinase A, protein kinase C, and Ca(2+)-regulated expression of 21-hydroxylase cytochrome P450 in H295R human adrenocortical cells. 958 61

Since a discrepancy concerning the effects of phytoestrogens on steroidogenesis exists in the literature we investigated the effects of genistein and daidzein on progesterone and estradiol synthesis in cultured primary granulosa cells derived from follicles of porcine ovaries. In this context, the investigation was performed to test the hypothesis that isoflavones can reduce hydroxysteroid dehydrogenase/isomerase (3beta-HSD) activity by down-regulation of its transcription. We found that daidzein did not impair the viability of cultured granulosa cells in the concentration range from 0.1 to 100 microM, but genistein inhibited the cell viability at 50 microM compared to the unexposed controls. Forskolin (10 microM) and pregnenolone (2.5 microM) enhanced the basal progesterone secretion in the absence of both phytoestrogens. Daidzein or genistein at non-toxic concentrations alone or combined with forskolin or pregnenolone significantly reduced progesterone synthesis. This reduction was not due to changes of the abundance of P450scc protein, but the gene hydroxysteroid dehydrogenase/isomerase (3beta-HSD) was significantly decreased at a non-toxic concentration of daidzein (50 microM) in non-stimulated and pregnenolone-stimulated cells. Moreover, genistein (1, 10 microM) significantly inhibited the 3beta-HSD-mRNA only in pregnenolone-stimulated granulosa cells. It can be suggested that the effect of genistein on steroidogenesis only partly results from the impairment of 3beta-HSD gene expression. In non-toxic concentrations daidzein and genistein did not change the androstenedione- or testosterone-stimulated estradiol-17beta synthesis. In summary, genistein and daidzein have direct effects on porcine granulosa cell progesterone synthesis which involve the inhibition of 3beta-HSD enzyme activity across the post-cyclic AMP pathway.
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PMID:In vitro exposure of porcine granulosa cells to the phytoestrogens genistein and daidzein: effects on the biosynthesis of reproductive steroid hormones. 1772 1

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