Amounts Of P450scc Research Articles

Progesterone attenuates the inhibitory effects of cardiotonic digitalis on pregnenolone production in rat luteal cells.

Previous studies have shown that digoxin decreases testosterone secretion in testicular interstitial cells. However, the effect of digoxin on progesterone secretion in luteal cells is unclear. Progesterone is known as an endogenous digoxin-like hormone (EDLH). This study investigates how digitalis affected progesterone production and whether progesterone antagonized the effects of digitalis. Digoxin or digitoxin, but not ouabain, decreased the basal and human chorionic gonadotropin (hCG)-stimulated progesterone secretion as well as the activity of cytochrome P450 side chain cleavage enzyme (P450scc) in luteal cells. 8-Br-cAMP and forskolin did not affect the reduction. Neither the amount of P450scc, the amount of steroidogenic acute regulatory (StAR) protein, nor the activity of 3beta-hydroxysteroid dehydrogenase (3beta-HSD) was affected by digoxin or digitoxin. Moreover, in testicular interstitial and luteal cells, progesterone partially attenuated the reduction of pregnenolone by digoxin or digitoxin and the progesterone antagonist, RU486, blocked this attenuation. These new findings indicated that (1) digoxin or digitoxin inhibited pregnenolone production by decreasing the activity of P450scc enzyme, but not Na(+)-K(+)-ATPase, resulting in a decrease on progesterone secretion in rat luteal cells, and (2) the inhibitory effect on pregnenolone production by digoxin or digitoxin was reversed partially by progesterone. In conclusion, digoxin or digitoxin decreased progesterone production via the inhibition of pregnenolone by decreasing P450scc activity. Progesterone, an EDLH, could antagonize the effects of digoxin or digitoxin in luteal cells.

Sandwich Configuration of Type I Collagen Suppresses Progesterone Production in Primary Cultured Porcine Granulosa Cells by Reducing Gene Expression of Cytochrome P450 Cholesterol Side-Chain Cleavage Enzyme

When porcine granulosa cells were cultured on type I collagen (TIC)-coated dishes, progesterone was continuously secreted in the culture medium. However, when cells were overlaid with a TIC gel, progesterone production was decreased to 34% (day 3) and 16% (day 4) of the value measured for cells without the overlay. The effect of TIC gel overlay on cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc), which catalyzes the conversion of cholesterol to pregnenolone and is thought to be the rate-limiting enzyme in the conversion of cholesterol to progesterone, was examined. P450scc gene expression in cells overlaid with a TIC gel was decreased to 62% (day 3) and 36% (day 4) of the value measured for cells without the overlay. Amounts of P450scc were also reduced in the cells overlaid with a TIC gel. When pregnenolone, the direct precursor of progesterone, was added to the culture medium, the increase in progesterone production by cells overlaid with a TIC gel was much greater than that for cells without a TIC gel and a statistical difference in progesterone production was no longer observed between the two groups of cells. Treatment of the cells with human FSH (hFSH) enhanced progesterone production in a dose-dependent manner, irrespective of the presence of a TIC gel overlay. Moreover, hFSH induced P450scc gene expression in cells with and without a TIC gel overlay. These results indicate that a TIC gel overlay reduces progesterone production in granulosa cells via the suppression of P450scc gene expression. This supports the possibility that the existence of a TIC gel on the apical side of granulosa cells prevents the spontaneous luteinization of granulosa cells cultured on TIC-coated dishes. The fact that hFSH overcomes the suppressive effect of the TIC gel overlay on progesterone production may explain the mechanism for the subtle rise in serum progesterone concentration in the late follicle phase of the “in vitro fertilization” program.

Molecular pathology and mechanism of action of the steroidogenic acute regulatory protein, StARProceedings of Xth International Congress on Hormonal Steroids, Quebec, Canada, 17–21 June 1998.

The first and rate-limiting step in the synthesis of all steroid hormones is the conversion of cholesterol to pregnenolone by the mitochondrial enzyme, P450scc. Tropic hormones such ACTH and gonadotropins induce steroidogenesis via cAMP by elaborating intracellular cAMP which stimulates P450scc activity in two distinct ways. Chronic stimulation (h to days) occurs through the induction of P450scc gene transcription leading to increased P450scc protein and consequent increased steroidogenic capacity. Acute regulation, over minutes, occurs through the phosphorylation of preexisting StAR and the rapid synthesis of new StAR protein. StAR, the steroidogenic acute regulatory protein, increases the flow of cholesterol into mitochondria, thus regulating substrate availability to whatever amount of P450scc is available. In the absence of StAR, up to 14% of maximal StAR-induced level of steroidogenesis persists as StAR-independent steroidogenesis. Congenital lipoid adrenal hyperplasia, an autosomal recessive disorder in which conversion of cholesterol to pregnenolone is severely impaired, results in female genitalia in 46,XY genetic males, variable onset of a severe salt-losing crisis in the first months of life, but normal feminization and cyclical vaginal bleeding in 46,XX females. Lipoid CAH was once thought to be due to P450scc mutations, but in fact homozygous P450scc mutations cannot exist in human beings as they would prohibit placental progesterone production, causing spontaneous abortion of the affected fetus. Lipoid CAH is caused by StAR mutations, which result in tropic hormone-induced intracellular accumulation of cholesterol in the adrenals and gonads. Our two-hit model, which considers the persistence of StAR-independent steroidogenesis and the differences in the fetal and postnatal ages at which the testis, adrenal zona glomerulosa, adrenal zona fasciculata and ovary are stimulated, predicts and explains all of the various clinical manifestations of lipoid CAH. Structure–function studies of StAR show that the critical domains for biological activity reside in the protein's carboxy-terminus. When the N-terminal mitochondrial targeting sequences are deleted and the resulting N-62 StAR remains in the cytoplasm, it retains the ability to stimulate steroidogenesis both in intact cells or when added to isolated mitochondria in vitro. These observations suggest that StAR acts on the outer mitochondrial membrane to promote sterol translocation to P450scc, and that the importation of StAR into mitochondria terminates its action. Data from circular dichroism and Fourier-transform infrared spectroscopy show that the mutant StAR proteins in lipoid CAH are misfolded, suggesting disrupted interaction with another protein. Preliminary data suggest that StAR facilitates cholesterol desorption from membranes, stimulating transfer from the outer mitochondrial (donor) membrane to the inner mitochondrial (acceptor) membrane.

Reconstitution of the steroidogenic pathway from cholesterol to aldosterone in liposome membranes

A steroidogenic pathway from cholesterol to aldosterone was reconstituted in liposome membranes using cytochromes P-450 scc, P-450 C21 and P-450 11 β , and 3β-hydroxysteroid dehydrogenase /gD 5-Δ 4 isomerase (3βHSD/I) with their electron transfer systems. All of the enzymes were purified from bovine adrenocortical mitochondria and microsomes. The cholesterol metabolism in the liposomal reconstituted system was compared with that in the combined organella system composed of bovine adrenocortical mitochondria and microsomes, where the activity of P-450 17 α,lyase was inhibited by bifonazole. The metabolic activities in these two systems were similar except for aldosterone production. Aldosterone was produced in the liposomal system but not in the combined organella system. 4-fold increase in the amount of P-450 scc in the liposomal system enhanced the activity of 3β HSD/I, P-450 C21 and 11 β-hydroxylase of P-450 11 β but decreased 18-hydroxycorticosterone and aldosterone production by P-450 11 β , supporting our previous findings describing the regulation mechanism of aldosterone synthesis (Kominami, S., Harada, D. and Takemori, S. (1994) Biochim. Biophys. Acta 1192, 234). It was demonstrated using the liposomal reconstituted system that the increase in the amount of one enzyme did not only increase the metabolizing activity of that enzyme but also affect other enzyme in various ways.

Effect of transforming growth factor beta-1 on the cholesterol side-chain cleavage system in the adrenal gland of sheep fetuses and newborns.

The present work examined the effect of transforming growth factor-beta1 (TGFbeta1) on the cholesterol side-chain cleavage system of sheep fetal and neonatal adrenal glands. Freshly isolated fetal adrenal cells produced 3-fold less pregnenolone from 22R- hydroxycholesterol than neonatal cells. Also, the relative amounts of immunoreactive cytochrome P450 side-chain cleavage (P450scc), adrenodoxin, and adrenodoxin reductase were 1.5- to 2-fold lower in mitochondria from fetal than neonatal cells. However, during culture under control conditions, the cholesterol side-chain cleavage activity and the amounts of P450scc, adrenodoxin, and adrenodoxin reductase of fetal cells increased after 48 h to reach neonatal values. A 5-day treatment with TGFbeta1 (1ng/ml) decreased significantly both the cholesterol side-chain cleavage activity and the amounts of immunoreactive P450scc, adrenodoxin, and adrenodoxin reductase in sheep fetal and neonatal adrenal cells in culture. Immunoreactive TGFbeta1- like material was present in freshly isolated adrenal cells from both fetuses and newborn lambs. After 2 days of culture, the amount of TGFbeta1-like protein was 2-fold lower than in freshly isolated fetal cells. No change was observed for neonatal cells. Finally, TGFbeta1 encoding messenger RNAs and TGFbeta-like immunoreactive protein were much higher in adrenal cortices from fetuses than from neonates. Taken together, these results have made TGFbeta1 a potentially attractive candidate for being an auto/paracrine negative regulator of the cholesterol side-chain cleavage system in the sheep fetal adrenal gland.

Regulation of cholesterol side-chain cleavage cytochrome P450 in mouse testis Leydig cell line I-10.

We have characterized regulation of steroidogenesis in a mouse testis Leydig cell line, I-10. Progesterone secretion was increased in a time- and dose-dependent fashion by 8-Br-cAMP treatment. The amount of cholesterol side-chain cleavage enzyme (P450scc), the first and rate-limiting enzyme for the synthesis of steroids, as detected by immunoblotting, was also increased. The calcium ionophore A23187 decreased the amount of P450scc, but it did not interfere with cAMP stimulation of the accumulation of P450scc. This regulation of P450scc expression by cAMP and A23187 is at the post-transcriptional level because the amount of P450scc mRNA was not affected by either treatment. This result was further confirmed by direct measurement of transcription in the presence or absence of forskolin treatment. I-10, however, supported cAMP-dependent transcriptional activation of the exogenous gene, as shown by the increased expression of a reporter gene under the control of the -600 to -2,500 fragment of the P450scc gene. The ability for transcriptional activation of the exogenous but not endogenous P450scc gene makes I-10 a unique steroidogenic cell line.

Mechanisms of action for an androgen-mediated autoregulatory process in rat thecal-interstitial cells.

In rat thecal-interstitial cells (TIC), treatment with the synthetic androgen mibolerone has led to the documentation of an autoregulatory process for androgen production. In the present study, accumulated evidence has provided insight into the mechanisms of mibolerone action that control this process. Investigations using the nonsteroidal antiandrogen hydroxyflutamide were conducted to characterize mibolerone's mode of action. Hydroxyflutamide had differential effects on hCG action, the 1-microM dose stimulating hCG-induced androsterone synthesis by 27% and the 10-microM concentration decreasing the androgen levels by 84%. In addition, treatment with 1 microM hydroxyflutamide was effective in partially reversing the inhibitory action of mibolerone on hCG-stimulated androsterone production. Thus, the data indicated that mibolerone's mode of action may be mediated, at least in part, via the androgen receptor. The possibility that mibolerone had multiple sites of action prompted studies on the effectiveness of this androgen to alter various signaling pathways. Treatment with increasing concentrations (0.01-100 nM) of the phorbol ester 12-0-tetradecanoylphorbol 13-acetate (TPA), which activates protein kinase C, resulted in a 75% decrease in hCG-stimulated androgen production at a dose of 100 nM TPA. Treatment with mibolerone (100 nM) was unable to alter the action of TPA on androgen synthesis when doses of 1 and 10 nM TPA were employed. It was also found that Ca2+ can serve as a mediator of mibolerone action. Treatment with a 0.01-microM dose of A23187, a Ca2+ ionophore known to increase intracellular Ca2+, was ineffective in altering hCG-stimulated androsterone synthesis. The concurrent treatment of mibolerone (100 nM) and A23187 (0.01 microM) resulted in the potentiation of mibolerone's inhibitory effects on hCG-stimulated androgen production, thereby suggesting that mibolerone can stimulate Ca2+ influx. Additional studies revealed that the administration of a 1-microM dose of the L-type Ca2+ channel blocker verapamil to TIC cultures was able to partially block the inhibitory effect of mibolerone on androgen synthesis. Evidence for an additional site of mibolerone action was revealed through an analysis of the mRNA levels of P450scc and P450(17) alpha enzymes. Although hCG and insulin-like growth factor I treatment resulted in 20- and 32-fold increases in the amount of P450scc and P450(17) alpha mRNA, respectively, the addition of mibolerone (100 nM) reduced only P450(17) alpha mRNA levels by 91%. Overall, the evidence indicates that mibolerone has multiple sites of action in exerting its regulatory effect on androgen synthesis.

Ontogenesis of cholesterol side-chain cleavage activity in the ovine adrenal during late gestation

The present study examined the activity of the cholesterol side-chain cleavage system, and the amount of cytochrome P450scc in adrenal glands of sheep fetuses and newborn lambs as well as the in vitro regulation of these parameters. Freshly isolated fetal adrenal cells incubated in the presence of 1 mM 8Br-cAMP or 25 μM 22R-OH cholesterol, produced 4- to 5-fold less pregnenolone than neonatal cells under similar conditions. Likewise, pregnenolone production by isolated fetal adrenal mitochondria was lower than that of neonatal mitochondria when endogenous cholesterol was used as a substrate or when 22R-OH cholesterol was added to the incubation medium. Also, the amount of P450scc, determined by immunoblot, was lower in fetal mitochondria than in neonatal mitochondria. In culture, ACTH, despite enhancing both the production of pregnenolone and the incorporation of [ 14C]acetate in cholesterol and its end-products by fetal adrenal cells, neither increased the amount of pregnenolone formed from 22R-OH cholesterol nor the amount of immunoreactive P450scc. By contrast, during the first 48 h of culture under standard conditions, there was a “spontaneous” increase in the activity of P450scc which reached values observed in neonatal adrenal cells. Such a development was inhibited when 5% ovine fetal serum was added to the culture medium. These results reinforce the view that in the ovine fetal adrenal gland, the development of P450scc is not ACTH-dependent but involves most probably a decrease in inhibitory factors present in fetal blood.

Isolation, characterization, and culture of cell subpopulations forming the pregnant rat corpus luteum.

The aim of this investigation was to isolate, characterize, and culture the small and large luteal cell subpopulations forming the corpus luteum of the pregnant rat. Since the large luteal cells are extremely fragile and do not survive standard cell dispersion, a method which allows the survival and the long-term culture in serum-free media of small and large cells was developed. The two luteal cell populations differed not only by their size but also by their morphology in culture. The small luteal cells (12-20 mu in diameter) are characterized by a large oval nucleus, contain few lipid droplets and have a stellate shape. In contrast, the large luteal cells have a smaller spherical nucleus, high lipid content, and do not flatten out completely in culture, most probably due to the abundance of lipid droplets. Both luteal cell types express 3 beta HSD and the cytochrome P450 enzymes involved in steroidogenesis. However, it is the lipid filled large luteal cells that secrete the most progesterone, androgen, and estradiol; express greater amounts of P450scc and P450AROM; and possess more PRL and LH receptors. Despite the greater expression of LH receptor in the large luteal cells, small and large luteal cells responded to LH with equal increase in steroidogenic output. In serum free culture, luteal cells produced progesterone for up to 20 days; however, an exogenous source of cholesterol was a prerequisite for maximal progesterone secretion. The pattern of progesterone secretion by cultures of small and large luteal cells differed remarkably from that of mixed cell population. When nonsteroidogenic corpus luteum cells were cocultured with the large luteal cells, a severalfold increase in progesterone secretion was observed. This stimulation occurred even when cells were cocultured in the absence of exogenous source of cholesterol. In summary, a successful method was developed to disperse, isolate, and independently culture the two luteal cell populations forming the rat corpus lutem. The results indicate that the marked difference in the steroidogenic capacity of these two cell populations is due, in large part, to the difference in their size rather than to their origin in the follicle. In addition, the results have revealed an important effect of the nonsteroidogenic cells forming the corpus luteum on luteal cell steroidogenesis.

Quantification of P450scc, P45017α, and Iron Sulfur Protein Reductase in Leydig Cells and Adrenals of Inbred Strains of Mice*

The relationship of maximal testosterone production to the amounts of cholesterol side-chain cleavage (P450scc), 17 alpha-hydroxylase/C17-20 lyase (P450(17) alpha), and iron sulfur protein (ISP) reductase was determined in Leydig cells from four inbred strains of mice (RF/J, SWR/J, C3H/He, and DBA/2). The amounts of P450scc, P450(17) alpha, and ISP reductase were also determined in adrenal glands of the same mice. cAMP-stimulated testosterone production and P450scc protein were high in RF/J and SWR/J compared to C3H/He and DBA/2 Leydig cells. A significant correlation between the amount of this enzyme and the capacity for testosterone production was found (r = 0.89; P less than 0.0005). ISP reductase was highest in RF/J, SWR/J, and C3H/He Leydig cells, which are significantly different from DBA/2. No significant differences in the amount of P450(17) alpha in Leydig cells from the four strains could be detected, and neither ISP reductase nor P450(17) alpha correlated with testosterone production. To ascertain if tissue-specific factors affect the expression of these enzymes, P450scc, ISP reductase, and P450(17) alpha were quantitated in adrenals from the same mice. P450scc and ISP reductase were expressed differently in adrenals compared to Leydig cells; levels of both proteins were high in C3H/He and RF/J adrenals compared to SWR/J and DBA/2. P450scc and ISP reductase were coordinately expressed in the adrenal, unlike in Leydig cells. P450(17) alpha was not detected in mouse adrenal glands. The results of this study suggest that strain-related differences in the capacity of Leydig cells for testosterone production may be determined by the amount of P450scc per Leydig cell. The expression of P450scc and ISP reductase in Leydig cells and adrenal glands appears to be influenced by both genetic and tissue-specific factors.

Hormonal regulation of messenger ribonucleic acids for P450scc (cholesterol side-chain cleavage enzyme) and P450c17 (17 alpha-hydroxylase/17,20-lyase) in cultured human fetal adrenal cells.

ACTH has acute and long term effects on adrenal steroidogenesis by week 14 of fetal life. We used human fetal adrenal cells to investigate the long term effect of physiological doses of ACTH on mRNAs for P450scc (the cholesterol side-chain cleavage enzyme) and P450c17 (17 alpha-hydroxylase/17,20-lyase). Monolayer cultures of 18- to 24-week gestation fetal zone adrenal cells were maintained in the presence and absence of 10(-9) or 10(-8) M ACTH for up to 12 days. As assessed by RNA dot blots probed with cloned homologous human cDNAs, ACTH increased P450scc and P450c17 mRNAs 4- and 9-fold, respectively, over control values on day 7 of culture. ACTH-mediated stimulation was slightly less on day 12 of culture. The ACTH-mediated accumulation of those mRNAs were time dependent. When cells were exposed to a single 10(-8)-M dose of ACTH, the amount of P450scc and P450c17 mRNA was increased by 24 h, reaching a maximum at 48 h and diminishing by 72 h. When cells were maintained in 10(-8) M ACTH continuously, mRNA for both enzymes accumulated in a similar pattern, reaching a peak at 48 h but remaining at nearly maximal values thereafter, up to 96 h. Dibutyryl cAMP (10(-3) M) mimicked these stimulatory actions of ACTH, although its effect was greater at 24 h and more stable up to 96 h. Angiotensin II (1-100 ng/mL) and hCG (1-100 ng/mL) had no effect on accumulation of P450scc and P450c17 mRNAs. The production of both dehydroepiandrosterone sulfate and cortisol also was stimulated by ACTH, suggesting that the increased mRNAs were translated into active enzymes. These results indicate that ACTH induces human fetal adrenal cells to accumulate mRNAs for both P450scc and P450c17; this effect of ACTH is probably mediated by cAMP. Chronic 96-h stimulation of human fetal adrenal cells did not diminish their responsiveness to ACTH. Together with our earlier studies of the human fetal adrenal, these data indicate that fetal adrenal tissue does not exhibit the desensitization to trophic hormone stimulation characteristic of adult tissue.