Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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Steroid hormones, which are ubiquitous regulators of physiologic processes, are produced primarily in the adrenals, gonads, and placenta. Each steroidogenic cell type produces different steroids due to cell-specific expression of various steroidogenic enzymes, but all steroidogenesis is initiated by P450scc, the mitochondrial enzyme that converts cholesterol to pregnenolone. We previously showed the unique segments of the P450scc promoter that are responsible for basal and cAMP-induced expression of this gene in the placenta are not employed for expression in the adrenal (C.C.D. Moore, D.W. Hum, and W.L. Miller, Mol. Endocrinol. 6, 2045-2058, 1992). We now show that sequences between -142 and -153 exhibit placental-specific activator activity. Sequences between -131 and -155 can confer activator activity to a 32-bp promoter from the thymidine kinase gene of herpes simplex virus in an orientation-independent fashion. Two protein complexes, termed IV and VII, interact specifically with DNA from -131 to -155. Mutating bases -142 to -151 abolishes formation of complex VII and partially inhibits complex IV, suggesting that the proteins forming these complexes bind neighboring segments of DNA. Mutating only two cytosines at bases 141 and 142 also eliminates the formation of complex VII and reduces the transcriptional activity of the activator by about 75-80%, indicating that complex VII is important for placental expression of P450scc. The sequence from -140 to -149 on the antisense strand resembles an NF-kappa B binding site. Antibodies to NF-kappa B subunit p50, but not to p52, p65, or c-Rel, will supershift some but not all of complex IV, whereas none of these antibodies interact with complex VII. A consensus NF-kappa B oligonucleotide does not form complex IV, suggesting that p50 interacts with the protein component, but not the DNA component of complex IV. Photoaffinity UV cross-linking yielded single bands of cross-linked DNA-protein complexes at approximately 85 kD for complex IV and approximately 70 kD for complex VII, indicating that separate proteins form complexes IV and VII. Southwestern blotting identified a single protein of 55 kD forming complex VII but did not identify the protein forming complex IV. Bandshifts and Southwestern blots with nuclear extracts from steroidogenic human placental JEG-3 cells and human adrenal NCI-H295 cells show that this 55-kD protein is found in placental but not adrenal cells. This 55-kD nuclear protein appears to be a trans-acting factor necessary for placental but not adrenal expression of P450scc.
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PMID:Characterization of placental transcriptional activation of the human gene for P450scc. 774 95

The rate-limiting step in luteal biosynthesis of progesterone consists of cleavage of the side chain of cholesterol by mitochondrial cytochrome P450 side-chain cleavage enzyme (P450scc) to form pregnenolone. Luteal mRNA encoding P450scc, quantitated on selected days of the 16-day ovine estrous cycle, was similar on days 3 and 6, increased by 2-fold on day 9 (P < 0.05) and remained elevated on day 15. Levels of P450scc mRNA on day 15 of pregnancy were not different from those found on any day of the cycle (P < 0.05). To determine whether levels of mRNA encoding P450scc are hormonally regulated, ewes on day 10 of the estrous cycle were injected with hCG or prostaglandin F2 alpha (PGF2 alpha). P450scc mRNA was not increased for up to 36 h after injection of hCG, nor decreased within 8 h after injection of PGF2 alpha (P < 0.05). An assay for P450scc activity was developed which utilized ovine small and large luteal cells in the presence of 22R-hydroxycholesterol and ovine high density lipoprotein. Enzyme activity was quantitated by measurement of progesterone production. In small luteal cells activation of the protein kinase A (PKA) second-messenger system by treatment with LH resulted in 910% increase in progesterone production without altering activity of P450scc. Activation of the protein kinase C (PKC) second-messenger system with phorbol 12-myristate 13-acetate caused a 51% reduction in progesterone secretion from large luteal cells but did not alter activity of P450scc. These findings suggest that in mature luteal tissue steady state levels of mRNA encoding P450scc, and enzyme activity are independent of acute regulation by activation of PKA or PKC second-messenger systems.
J Steroid Biochem Mol Biol 1994 Dec
PMID:Regulation of cytochrome P450scc synthesis and activity in the ovine corpus luteum. 782 90

Using cultured human fetal adrenal cells, we have investigated the basal secretion of cortisol and dehydroepiandrosterone sulfate (DHAS) and the effect of corticotropin (ACTH), angiotensin-II (A-II) and transforming growth factor beta 1 (TGF beta 1) on the secretion of these steroids and on the mRNA levels of ACTH receptor (ACTHR), cytochrome P-450scc (cholesterol side-chain cleavage), P450 17 alpha (17 alpha-hydroxylase/17-20 lyase) and 3 beta-HSD (3 beta-hydroxysteroid dehydrogenase). The basal DHAS/cortisol ratio declined progressively between 12.5 and 21 weeks. ACTH treatment enhanced the secretion of cortisol and to a lesser extent that of DHAS, and increased the steroidogenic response to an acute stimulation with ACTH. These changes were associated with increased mRNA levels of ACTHR and of the steroidogenic enzymes. A-II treatment also increased the secretion of both DHAS and cortisol, but less than ACTH, enhanced the responsiveness to ACTH and increased ACTHR, P450scc and P450 17 alpha mRNA levels. In contrast, TGF beta 1 alone or together with ACTH decreased DHAS secretion, but not cortisol secretion. Moreover, TGF beta 1 had no effect on ACTHR and P450scc mRNA levels, decreased by about 50% the mRNA levels of P450 17 alpha both in the absence or presence of ACTH, but enhanced the stimulatory effects of ACTH on 3 beta-HSD mRNA. These results, along with those previously reported, suggest that both A-II and TGF beta may play a role in fetal adrenal function. In addition, they show that the effects of both peptides are qualitatively different from, even sometimes opposite to, those previously reported in bovine and ovine adrenal cells.
Mol Cell Endocrinol 1994 Dec
PMID:Regulation of corticotropin and steroidogenic enzyme mRNAs in human fetal adrenal cells by corticotropin, angiotensin-II and transforming growth factor beta 1. 789 1

We have previously identified and purified a splenocyte-derived factor (PSF) that stimulates the accumulation of progesterone and 20 alpha-dihydroprogesterone (20 alpha-OH-P) in rat ovarian granulosa cells independently of FSH. In the present study, time course experiments comparing the response to PSF with that to FSH revealed that PSF-stimulated progesterone accumulation was slower than that of FSH, but PSF-stimulated 20 alpha-OH-P accumulation had a time course similar to that of FSH. To determine the basis for the slower progesterone response to PSF, the effect of these two agents on each step of the steroidogenic pathway was assessed. First, to examine whether PSF-stimulated cholesterol mobilization was limiting, cultured granulosa cells were treated with 22(R)-hydroxycholesterol. While both FSH- and PSF-stimulated progesterone and 20 alpha-OH-P accumulation approximately doubled, the overall time courses did not change indicating that cholesterol availability was not the factor limiting the response to PSF. Next, PSF and FSH induction of steroidogenic enzyme activities and messenger RNAs were compared. While FSH-stimulated cytochrome P450 side chain cleavage enzyme (SCC) activity rapidly increased (peaking at 2 days) and then slowly declined, PSF-stimulated SCC activity gradually increased over 5 days to approximately 35% of the maximal activity stimulated by FSH. PSF also induced slower increases in P450scc mRNA levels than did FSH. In addition, PSF stimulated 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity more slowly than did FSH, but after 3 days of culture, PSF-stimulated activity was significantly higher than that induced by FSH.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Cell Endocrinol 1994 Dec
PMID:Induction of rat granulosa cell steroidogenic enzyme activities and their messenger ribonucleic acids by a splenocyte-derived factor. 789 3

LHRH and its analogues are known to exert direct effects on the ovary. Herein we have described a direct inhibitory effect of an LHRH antagonist (Nal-Lys antagonist: antide) on the basal progesterone (P4) and pregnenolone (P5) production by luteal cells obtained from the day-8 pregnant rat. Luteal cells incubated with two doses of antide (10(-4) and 10(-7) M) for 24 or 48 h showed suppression of P4 production. P5 production was suppressed by both doses of antide within 12 h of incubation. Neither dose of antide interfered with P5 production when the duration of incubation was extended beyond 12 h. The 20 alpha-dihydroprogesterone yield from the luteal cells treated with these doses of antide remained unaffected. We estimated the activities of the cholesterol side-chain cleavage (P450scc) enzyme (which is a key enzyme involved in the conversion of cholesterol to P5) and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) (which catalyses the conversion of P5 to P4) in the luteal cells treated with different doses of antide. Both doses of antide suppressed the activity of the P450scc enzyme after 12 h of incubation and the 3 beta-HSD content of the luteal cells after 48 h of incubation. These observations indicate that antide exerts a direct inhibitory effect at the level of the corpus luteum, that differential suppression of P5 and P4 during different periods of incubation with antide is due to a defect in either the P450scc or the 3 beta-HSD enzyme system, or both.
J Mol Endocrinol 1994 Aug
PMID:Suppression of luteal steroidogenesis by an LHRH antagonist (Nal-Lys antagonist: antide) in vitro during early pregnancy in the rat. 799 57

Several substances with different inhibitory effects on adrenal steroid biosynthesis were investigated in patients with Cushing's syndrome. It has been shown that trilostane, a 3 beta-hydroxysteroid-dehydrogenase inhibitor, is not potent enough to block cortisol biosynthesis in patients with hypercortisolism. Aminoglutethimide inhibits side chain cleavage of cortisol synthesis, but it has been demonstrated that the blocking effect on cortisol secretion is not strong enough to normalize urinary cortisol excretion in patients with Cushing's disease. For metyrapone, an inhibitor of adrenal 11 beta-hydroxylase, promising results were reported for the treatment of Cushing's syndrome. However, the drug has several side effects and depending on the definition of the desired reduction of cortisol secretion a true remission was only found in a minority of patients. The antifungal drug ketoconazole in vitro predominantly blocks 17,20-desmolase (IC50 1 microM) and to a lesser extent 17 alpha-hydroxylase (IC50 10 microM) and 11 beta-hydroxylase (IC50 15-40 microM). Therefore, ketoconazole in vivo most potently suppresses androgen secretion and only to a lesser extent cortisol biosynthesis. Several therapeutic trials with ketoconazole treatment in patients with pituitary Cushing's disease showed various remission rates between 30 and 90%. In contrast, in almost all patients with benign, primary adrenal Cushing's syndrome cortisol levels were normalized. In patients with ectopic ACTH syndrome ketoconazole was effective in about 50% of all reported cases, while cortisol hypersecretion due to adrenocortical carcinoma was only rarely inhibited by ketoconazole. The main side effect of ketoconazole treatment was liver toxicity which occurred in 12% of all treated patients. In contrast to ketoconazole, the narcotic drug etomidate shows a strong inhibitory effect on 11 beta-hydroxylase (IC50 0.03-0.15 microM) but only a weak inhibition of 17,20 desmolase (IC50 380 microM). This correlates with in vivo studies where even low, non-hypnotic doses of etomidate induced a pronounced fall in serum cortisol levels in normals and in patients with Cushing's syndrome. However, its clinical use is limited by its mandatory intravenous application and its sedative effects. In conclusion, ketoconazole remains the only available steroid-inhibitory drug for a therapeutic trial in patients with Cushing's syndrome who cannot be treated definitively by surgery.
J Steroid Biochem Mol Biol 1994 Jun
PMID:Therapy of Cushing's syndrome with steroid biosynthesis inhibitors. 804 88

In adrenal cortex and other steroidogenic tissues including glial cells, the conversion of cholesterol into pregnenolone is catalyzed by the cytochrome P450scc located in the inner mitochondrial membrane. A complex mechanism operative in regulating cholesterol access to P450scc limits the rate of pregnenolone biosynthesis. Participating in this mechanism are DBI (diazepam binding inhibitor), an endogenous peptide that is highly expressed in steroidogenic cells and some of the DBI processing products including DBI 17-50 (TTN). DBI and TTN activate steroidogenesis by binding to a specific receptor located in the outer mitochondrial membrane, termed mitochondrial DBI receptor complex (MDRC). MDRC is a hetero-oligomeric protein: only the subunit that includes the DBI and benzodiazepine (BZD) recognition sites has been cloned. Several 2-aryl-3-indoleacetamide derivatives (FGIN-1-X) with highly selective affinity (nM) for MDRC were synthesized which can stimulate steroidogenesis in mitochondrial preparations. These compounds stimulate adrenal cortex steroidogenesis in hypophysectomized rats but not in intact animals. Moreover, this steroidogenesis is inhibited by the isoquinoline carboxamide derivative PK 11195, a specific high affinity ligand for MDRC with a low intrinsic steroidogenic activity. Some of the FGIN-1-X derivatives stimulate brain pregnenolone accumulation in adrenalectomized-castrated rats. The FGIN-1-X derivatives that increase brain pregnenolone content, elicit antineophobic activity and antagonize punished behavior in the Vogel conflict test in rats. These actions of FGIN-1-X are resistant to inhibition by flumazenil, a specific inhibitor of BZD action in GABAA receptors but are antagonized by PK 11195, a specific blocker of the steroidogenesis activation via MDRC stimulation. It is postulated that the pharmacological action of FGIN-1-X depends on a positive modulation of the GABA action on GABAA receptors mediated by the stimulation of brain neurosteroid production.
J Steroid Biochem Mol Biol 1994 Jun
PMID:The pharmacology of neurosteroidogenesis. 804 4

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. Although steroidogenic enzyme expression was maintained throughout gestation in both the fetal adrenals and the testes, adrenal P450c17 expression was higher in the early and late stages when compared with the intermediate stages of fetal development. The stimulation of fetal adrenal steroidogenic enzyme expression in the later stage fetuses was accompanied by increased expression of P450c17 in both the fetal testes and placenta. The expression of 3 beta-HSD by porcine fetal testes was low compared with that of the fetal adrenal gland at all stages of development. Adrenal explants and cultured cells secreted cortisol and androstenedione but much lower amounts of corticosterone, dehydroepiandrosterone and aldosterone. Secretion of cortisol and androstenedione by adrenal explants was maintained by ACTH for 5 days of culture but declined in controls. In cultured porcine fetal adrenal cells, ACTH and angiotensin II stimulated the secretion of multiple steroids. Porcine fetal testis explants and cultured cells secreted testosterone, dehydroepiandrosterone and androstenedione, but were only moderately responsive to trophic stimulation by LH. In general, the data suggest that the fetal adrenal glands and the fetal testes have the potential to contribute significantly to the production of steroids during pregnancy in pigs.
J Mol Endocrinol 1994 Apr
PMID:Ontogeny of steroidogenic enzyme expression in the porcine conceptus. 806 Apr 80

The co-ordinated biosynthesis of progesterone and oestradiol in the human ovary is critical for reproductive cyclicity and eventual pregnancy. The crucial regulatory enzymes for progesterone and oestradiol biosynthesis in granulosa cells are the cholesterol side-chain cleavage (P450scc) and aromatase (P450arom) enzymes respectively. We utilized the cDNA sequences encoding P450arom and P450scc to examine the roles of FSH and LH, and their intracellular second messenger, cyclic AMP (cAMP), in regulating steroidogenic gene expression. Mature granulosa cells (aspirated before the onset of the endogenous LH surge) and granulosa lutein cells (obtained after an ovulatory dose of human chorionic gonadotrophin) were cultured for 4 days with FSH, LH or dibutyryl cAMP (dbcAMP). After the period of culture, total RNA was extracted from granulosa cells and Northern analyses were performed utilizing 32P-labelled cDNAs encoding P450arom and P450scc. Spent culture media were analysed for steroid and cAMP content. Both FSH and LH strongly stimulated P450arom mRNA expression and oestradiol production in mature granulosa cells. On the other hand, P450scc mRNA expression and progesterone biosynthesis were weakly induced by FSH; maximal synthesis occurred only in the presence of LH. With both gonadotrophins at equivalent concentrations, LH generated a 30-fold higher level of cAMP than FSH. Furthermore, the differential effects of FSH and LH on P450 mRNA expression were reproduced by the presence of low and high concentrations of dbcAMP respectively. LH (and high levels of dbcAMP) increased P450arom mRNA expression in mature granulosa cells but inhibited its accumulation in granulosa lutein cells. In contrast, it stimulated P450scc mRNA expression and progesterone synthesis in both mature granulosa and granulosa lutein cells. Therefore, FSH/low cAMP levels stimulated P450arom gene expression and oestradiol production, while LH/high cAMP levels maximally induced P450scc gene expression and function, in a development-related manner consistent with steroid production in vivo. These findings support the hypothesis that one set of genes (like P450arom) in human granulosa cells is regulated by FSH/low cAMP levels and another (like P450scc) by LH/high cAMP levels.
J Mol Endocrinol 1994 Apr
PMID:Differential regulation of cholesterol side-chain cleavage (P450scc) and aromatase (P450arom) enzyme mRNA expression by gonadotrophins and cyclic AMP in human granulosa cells. 806 Apr 87

Turosteride was tested in a series of studies for its effect on 5 alpha-reductase and for its possible influence on other steroidogenic enzymes and on steroid receptors. The compound was found to inhibit human and rat prostatic 5 alpha-reductases with IC50 values of 55 and 53 nM, respectively, whereas it caused a less marked inhibition of the dog enzyme (IC50 2.2 microM). Turosteride showed no relevant effect on rat adrenal C20,22-desmolase (IC50 254 microM) and human placental aromatase (IC50 > 100 microM), and only at relatively high concentrations it caused inhibition of human placental 5-ene-3 beta-hydroxysteroid dehydrogenase-isomerase (3 beta-HSD-I) (IC50 2.5 microM). Turosteride was found to be a selective 5 alpha-reductase inhibitor showing no noteworthy binding to receptors for androgens (relative binding affinity, RBA, 0.004%), estrogens (< or = 0.005%), progesterone (< 0.005%), glucocorticoids (< 0.01%) and mineralocorticoids (< 0.03%). Its biochemical profile was similar to that of finasteride, whereas 4-MA (17 beta-N,N-diethyl-carbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one) was confirmed to be a non-selective 5 alpha-reductase inhibitor, showing a degree of binding affinity to the androgen receptor (RBA 0.1%) and a marked inhibition of 3 beta-HSD-I (IC50 32 nM). When given orally in immature castrated rats together with subcutaneous testosterone propionate (TP) for 7 consecutive days, turosteride reduced the ventral prostate and seminal vesicle growth promoting effect of TP, with IC50 values of approximately 5 and 6.7 mg/kg/day, whereas levator ani weight was unchanged. In comparison, 4-MA was approx. 3-fold less potent than turosteride in reducing the prostate and seminal vesicle weights and caused a marked reduction of levator ani weight, thus showing its unselectivity.
J Steroid Biochem Mol Biol 1994 Feb
PMID:Endocrine properties of the testosterone 5 alpha-reductase inhibitor turosteride (FCE 26073). 814 1


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