Molecular and neurochemical evidence for the biosynthesis of dehydroepiandrosterone in the adult rat spinal cord

Various studies have indicated that exogenous dehydroepiandrosterone (DHEA) modulates several mechanisms in the CNS of rodents. As adult rodent glands do not secrete significant amounts of DHEA, its role as endogenous modulator of the CNS remains possible only if DHEA is produced by nerve cells. Therefore, the last decade has been marked by diverse unsuccessful investigations aiming to demonstrate the activity of cytochrome P450c17 (P450c17), the key DHEA-synthesizing enzyme, in adult rodent CNS. Here, we combined molecular, anatomical, cellular and neurochemical approaches to provide the first demonstration of the existence of P450c17 and bioactivity in adult rat spinal cord (SC). Real-time RT-PCR revealed P450c17 gene expression in all SC segments. Western blot analyses allowed identification of a specific P450c17 protein in the SC and immunohistochemical studies localized P450c17 in neurones and glial cells. Pulse-chase experiments combined with HPLC and radioactive steroid detection showed that SC slices converted [3H]pregnenolone into [3H]DHEA, a conversion markedly reduced by ketoconazole, a P450c17 inhibitor. Kinetics studies revealed accumulation of [3H]DHEA newly synthesized by SC slices in the incubation medium as its amount declined slowly. This first cellular mapping of an active P450c17 in adult rodent SC suggests that endogenous DHEA synthesized in spinal neural networks may control various spinally-mediated activities.     

Assessment of porcine and human 16-ene-synthase, a third activity of P450c17, in the formation of an androstenol precursor. Role of recombinant cytochrome b5 and P450 reductase.

Recently, we have shown that the biosynthesis of androstenol, a potential endogenous ligand for the orphan receptors constitutive androstane receptor and pregnane-X-receptor, requires the presence of enzymes of the steroidogenic pathway, such as 3 beta-hydroxysteroid dehydrogenase, 5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase. In this report, we examine at the molecular level whether the enzyme 17 alpha-hydroxylase/17,20-lyase (P450c17), which possesses dual 17 alpha-hydroxylase and 17,20-lyase activities and catalyzes the production of precursors for glucocorticoids and sex steroids, is also able to catalyze the formation of a third class of active steroids, 16-ene steroids (including androstenol). The role of components of the P450 complex is also assessed. We transfected human embryonic kidney (HEK-293) cells with various amounts of vectors expressing P450c17, NADPH-cytochrome P450 reductase, and cytochrome b5. Our results showed that P450c17 possesses a 16-ene-synthase activity able to transform pregnenolone into 5,16-androstadien-3 beta-ol, without the formation of the precursor 17-hydroxypregnenolone. Cytochrome b5 has a much stronger effect on the 16-ene-synthase activity than on the 17 alpha-hydroxylase/17,20-lyase activities. On the other hand, P450reductase has a drastic effect on the latter, but a negligible one on 5,16-androstadien-3 beta-ol synthesis. Our results therefore demonstrate that human P450c17, as other enzymes of the classical steroidogenic pathway, is involved in the biosynthetic pathway leading to the formation of androstenol.     

Androgen and estrogen treatments alter steady state messengers RNA (mRNA) levels of testicular steroidogenic enzymes in the rainbow trout, Oncorhynchus mykiss

Recent investigations have shown that estrogens have profound inhibitory effects on steroidogenic enzyme gene expressions before and after testicular differentiation in the rainbow trout, Oncorhynchus mykiss. This present study bring new data on juvenile rainbow trout treated with estrogens and androgens. Following a 8 days oral treatment of juvenile male with 17alpha-ethynyl-estradiol (EE2, 20 mg/kg diet) or 11beta-hydroxyandrostenedione (11betaOHDelta4, 10 mg/kg diet), we observed a fast and marked decrease of steady-state mRNA levels for 3betaHSD, P450scc, P450c17, and P450c11 enzymes in the testis. After completion of these treatments, mRNA levels of these enzymes remained low in EE2 treated males whereas in 11betaOHDelta4 treated males they recovered their initial levels in 8 days. This demonstrate that both androgen and estrogen treatments have profound effects on testicular steroidogenesis by decreasing steroid enzymes steady-state mRNA. After in vitro incubation of testicular explants with 17beta-estradiol (E2, 600 ng/ml of medium), we also observed a decrease of mRNA levels for 3betaHSD and P450c11. This suggest that estrogens effects could be triggered, at least to some extend, directly on the testis. We also investigated the hypothesis of a negative feedback of steroids on follicle stimulating hormone (FSH) secretion, but FSH plasmatic levels in treated fish did not showed any significant decrease. This demonstrate that FSH is not implied in this steroids inhibition of steroidogenic enzymes gene expression.     

Expression of steroidogenic enzymes and synthesis of sex steroid hormones from DHEA in skeletal muscle of rats

The functional importance of sex steroid hormones (testosterone and estrogens), derived from extragonadal tissues, has recently gained significant appreciation. Circulating dehydroepiandrosterone (DHEA) is peripherally taken up and converted to testosterone by 3beta-hydroxysteroid dehydrogenase (HSD) and 17beta-HSD, and testosterone in turn is irreversibly converted to estrogens by aromatase cytochrome P-450 (P450arom). Although sex steroid hormones have been implicated in skeletal muscle regulation and adaptation, it is unclear whether skeletal muscles have a local steroidogenic enzymatic machinery capable of metabolizing circulating DHEA. Thus, here, we investigate whether the three key steroidogenic enzymes (3beta-HSD, 17beta-HSD, and P450arom) are present in the skeletal muscle and are capable of generating sex steroid hormones. Consistent with our hypothesis, the present study demonstrates mRNA and protein expression of these enzymes in the skeletal muscle cells of rats both in vivo and in culture (in vitro). Importantly, we also show an intracellular formation of testosterone and estradiol from DHEA or testosterone in cultured muscle cells in a dose-dependent manner. These findings are novel and important in that they provide the first evidence showing that skeletal muscles are capable of locally synthesizing sex steroid hormones from circulating DHEA or testosterone.     

Steroid and xenobiotic effects on the adrenal cortex: mediation by oxidative and other mechanisms

Because steroids reach high concentrations within the adrenal cortex, effects of the direct interaction of steroids and cytochrome P450 enzymes are possible and may involve oxidative damage. Steroid pseudosubstrate effects studied in cultured adrenocortical cells show that these effects are probably not mediated by steroid receptors. Release of oxidants during pseudosubstrate interaction with cytochrome P450s may be responsible for loss of enzymatic activity observed; enzyme activity can be protected by cytochrome P450 inhibitors, antioxidants, and lowered oxygen concentration. There may be pathological effects of pseudosubstrates in the adrenal cortex. Cytochrome P450/pseudosubstrate effects could be involved in the aging and death of adrenocortical cells in vivo, and necrosis of the adrenal cortex due to excessive ACTH stimulation or due to the action of adrenolytic chemicals could result from damage by oxygen radicals originating from cytochrome P450s. The possible mechanism of damage to the adrenal cortex by the xenobiotics dimethylbenzanthracene, TCDD, 3-methylcholanthrene, and o', p'-DDD are reviewed.     

Onset of steroidogenic enzyme gene expression during ovarian follicular development in sheep

Steroidogenesis is a major function of the developing follicle. However, little is known about the stage of onset of steroid regulatory proteins during follicular development in sheep. In this study, several steroidogenic enzymes were studied by immunohistochemistry and/or in situ hybridization; cytochrome P450 side chain cleavage (P450(scc)), cytochrome P450 17alpha-hydroxylase (17alphaOH), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), cytochrome P450 aromatase (P450(arom)), steroidogenic factor 1 (SF-1), steroidogenic acute regulatory protein (StAR), and LH receptor (LH-R). To define the stages of follicular growth, ovarian maps were drawn from serial sections of ovine ovaries, and follicles were located and classified at specific stages of growth based on morphological criteria. In this way, the precise onset of gene expression with respect to stages of follicular growth for all these proteins could be observed. The key findings were that ovine oocytes express StAR mRNA at all stages of follicular development and that granulosa cells in follicle types 1-3 express 3beta-HSD and SF-1. Furthermore, the onset of expression in theca cells of StAR, P450(scc), 17alphaOH, 3beta-HSD, and LH-R occurred in large type 4 follicles just before antrum formation. This finding suggests that although the theca interna forms from the type 2 stage, it does not become steroidogenically active until later in development. These studies also confirm that granulosa cells of large type 5 follicles express SF-1, StAR, P450(scc), LH-R, and P450(arom) genes. These findings raise new questions regarding the roles of steroidogenic regulatory factors in early follicular development.     

Cellular distribution and bioactivity of the key steroidogenic enzyme,cytochrome P450side chain cleavage,in sensory neural pathways

Neurosteroids are steroids produced within the nervous system. Based on behavioural responses evoked in animals by synthetic steroid injections, several studies suggested neurosteroid involvement in important neurophysiological processes. These observations should be correlated only to neuroactive effects of the injected steroids. Neurosteroids mostly control the CNS activity through allosteric modulation of neurotransmitter receptors within concentration ranges used by neurotransmitters themselves. Therefore, neurosteroid production within pathways controlling a neurophysiological process is necessary to consider neurosteroid involvement in that process. Because of the increasing speculation about pain modulation by neurosteroids based on pharmacological observations, we decided to clarify the situation by investigating neurosteroidogenesis occurrence in sensory pathways, particularly in nociceptive structures. We studied the presence and activity of cytochrome P450side chain cleavage (P450scc) in rat pain pathways. P450scc-immunoreactive cells were localized in dorsal root ganglia (DRG), spinal cord (SC) dorsal horn, nociceptive supraspinal nuclei (SSN) and somatosensory cortex. Incubation of DRG, SSN or SC tissue homogenates with [3H]cholesterol yielded the formation of radioactive metabolites including [3H]pregnenolone of which the synthesis was reduced in presence of aminogluthetimide, a P450scc inhibitor. These first neuroanatomical and neurochemical results demonstrate the occurrence of neurosteroidogenesis in nociceptive pathways and strongly suggest that neurosteroids may control pain mechanisms.     

Steroid hormone hydroxylase specificities of eleven cDNA-expressed human cytochrome P450s

Steroid hydroxylation specificities were determined for 11 forms of human cytochrome P450, representing four gene families and eight subfamilies, that were synthesized in human hepatoma Hep G2 cells by means of cDNA-directed expression using vaccinia virus. Microsomes isolated from the P450-expressing Hep G2 cells were isolated and then assayed for their regioselectivity of hydroxylation toward testosterone, androstenedione, and progesterone. Four of the eleven P450s exhibited high steroid hydroxylase activity (150-900 pmol hydroxysteroid/min/mg Hep G2 microsomal protein), one was moderately active (30-50 pmol/min/mg) and six were inactive. In contrast, 10 of the P450s effectively catalyzed O-deethylation of 7-ethoxycoumarin, a model drug substrate, while only one (P450 2A6) catalyzed significant coumarin 7-hydroxylation. Human P450 4B1, which is expressed in lung but not liver, catalyzed the 6 beta-hydroxylation of all three steroids at similar rates and with only minor formation of other hydroxylated products. Three members of human P450 family 3A, which are expressed in liver and other tissues, also catalyzed steroid 6 beta-hydroxylation as their major activity but, additionally, formed several minor products that include 2 beta-hydroxy and 15 beta-hydroxy derivatives in the case of testosterone. These patterns are similar to those exhibited by rat family 3A P450s. Although several rodent P450s belonging to subfamilies 2A, 2B, 2C, 2D are active steroid hydroxylases, four of five human P450s belonging to these subfamilies exhibited very low activity or were inactive, as were the human 1A and 2E P450s examined in the present study. These studies demonstrate that individual human cytochrome P450 enzymes can hydroxylate endogenous steroid hormones with a high degree of stereospecificity and regioselectivity, and that some, but not all of the human cytochromes exhibit metabolite profiles similar to their rodent counterparts.     

CYP11A1 stimulates the hydroxylase activity of CYP11B1 in mitochondria of recombinant yeast in vivo and in vitro.

In mammals, hydrocortisone synthesis from cholesterol is catalyzed by a set of five specialized enzymes, four of them belonging to the superfamily of cytochrome P-450 monooxygenases. A recombinant yeast expression system was recently developed for the CYP11B1 (P45011beta) enzyme, which performs the 11beta hydroxylation of steroids such as 11-deoxycortisol into hydrocortisone, one of the three mitochondrial cytochrome P-450 proteins involved in steroidogenesis in mammals. This heterologous system was used to test the potential interaction between CYP11B1 and CYP11A1 (P450scc), the mitochondrial cytochrome P-450 enzyme responsible for the side chain cleaving of cholesterol. Recombinant CYP11B1 and CYP11A1 were targeted to Saccharomyces cerevisiae mitochondria using the yeast cytochrome oxidase subunit 6 mitochondrial presequence fused to the mature form of the two proteins. In yeast, the presence of CYP11A1 appears to improve 11beta hydroxylase activity of CYP11B1 in vivo and in vitro. Fractionation experiments indicate the presence of the two proteins in the same membrane fractions, i.e. inner membrane and contact sites of mitochondria. Thus, yeast mitochondria provide interesting insights to study some molecular and cellular aspects of mammalian steroid synthesis. In particular, recombinant yeast should permit a better understanding of the mechanism permitting the synthesis of steroids (sex steroids, mineralocorticoids and glucocorticoids) with a minimal set of enzymes at physiological level, thus avoiding disease states.     

Estrogen-producing steroidogenic pathways in parietal cells of the rat gastric mucosa

Recently we demonstrated the presence of aromatase (P450(arom)), estrogen synthetase, and the active production of estrogen in parietal cells of the rat stomach. We therefore investigated the steroidogenic pathways of estrogen and also other steroid metabolites in the gastric mucosa of male rats, by showing the mRNA expression of steroidogenic enzymes using RT-PCR and in situ hybridization histochemistry, and by measuring the blood concentration of steroids in the artery and the portal vein. RT-PCR analysis showed the strong mRNA expression of 17alpha-hydroxylase/17,20-lyase (P450(17alpha)), 17beta-hydroxysteroid dehydrogenase (HSD) type III and P450(arom), and the weak mRNA expression of 17beta-HSD type II, 5alpha-reductase type I and 3alpha-HSD. The other mRNAs of steroidogenic enzymes examined were not detected. In situ hybridization histochemistry demonstrated the localization of mRNAs for P450(17alpha), 17beta-HSD type III and P450(arom) in the parietal cells. Higher levels of progesterone and testosterone were found in the artery compared with the portal vein. Higher amounts of estrone and 17beta-estradiol, by contrast, were present in the portal vein compared with the artery. These results indicate that parietal cells of rat stomach convert circulating progesterone and/through androstenedione and testosterone to synthesize both estrone and 17beta-estradiol, which then enter the liver via the portal vein.     

Ontogeny of steroidogenesis in the fetal sheep gonad.

The aim of this study was to determine 1) the time of onset and cellular localization of gene expression for steroidogenic factor-1 (SF-1), steroidogenic acute regulatory protein, 3beta-hydroxysteroid dehydrogenase/Delta(5),Delta(4) isomerase (3beta-HSD), and the cytochrome P450 enzymes for cholesterol side-chain cleavage (P450(scc)), 17alpha-hydroxylase (P450(17alphaOH)), and aromatase (P450(arom)) during gonadal development; and 2) the amount of progesterone, androstenedione, testosterone, and 17beta-estradiol present in the fetal sheep gonad. Fetuses were collected on Days 24, 26, 28, 30, 32, 35, 40, 55, and 75 of gestation, and gene expression was determined by in situ hybridization. The steroid content of gonads collected on Days 30, 35, 55, and 75 of gestation was determined by RIA. Developing gonads collected from both male and female fetuses were steroidogenically active around the time of morphological sexual differentiation. In the female, the steroidogenic cells were initially located at the boundary of the cortex and medulla but become increasingly restricted to the mesonephric-derived cell streams. In the male, once tubules were identifiable, steroidogenesis was restricted to the interstitial regions. Interestingly, expression of both SF-1 and 3beta-HSD was observed prior to morphological sexual differentiation. In addition, expression of both of these genes was more widespread than the other genes in both males and females.     

Aldosterone synthase activity in the Y-1 adrenal cell line

The Y-1 adrenal cell line was shown to produce 20-dihydroaldosterone from deoxycorticosterone. This compound was identified by GC-MS by comparison with the previously synthesized reference compound. Two other 18-hydroxylated metabolites were identified as 11β,18-dihydroxy-20-dihydroprogesterone from endogenous cholesterol and 18-hydroxy-20-dihydro-11-dehydrocorti-costerone from DOC. The conditions necessary for the synthesis of these compounds are culturing in 20% serum-supplemented medium and repeated incubations with the substrate. The production of 11β-hydroxylated steroids and that of 18-oxygenated steroids is stimulated differently by ACTH and angiotensin II suggesting the expression of two different enzymes, cytochrome P-450_11β and cytochrome P-450_aldo The Y-1 cell line can secrete either 11β-hydroxylated steroids characteristic of the glucocorticoid pathway or 18-oxygenated steroids characteristic of the mineralocorticoid pathway, which in vivo are generally produced in two different zones of the adrenal cortex. This cell line should be an interesting model for the study of the molecular mechanisms regulating the expression of these two enzymes involved in the final steps of the steroidogenic pathways.     

Immunolocalization of cholesterol side-chain-cleavage cytochrome P-450 and 17 alpha-hydroxylase cytochrome P-450 in bovine ovarian follicles

Follicles were collected from cows and processed for electron microscopy and for immunofluorescent staining at the light microscope level. Key regulatory steroidogenic enzymes cholesterol side-chain-cleavage cytochrome P-450 (P-450scc) and 17 alpha-hydroxylase cytochrome P-450 (P-45017 alpha) were immunolocalized using specific IgG fractions raised against these enzymes. In larger follicles in which the theca interna had differentiated, positive staining for cytochromes P-450scc and P-450(17) alpha was observed in the cells of the theca interna. Electron microscopic examination showed that these cells were rich in endoplasmic reticulum, mainly rough, and had moderate numbers of mitochondria with tubular and lamellar cristae. Positive staining was also present in the theca of follicles undergoing atresia. Positive staining for cytochrome P-450(17) alpha was not observed in the membrana granulosa but cytochrome P-450scc was present in the membrana granulosa in some follicles, particularly in the larger antral follicles. By contrast, positive staining for both enzymes was not observed in stroma, surface epithelium or in small preantral follicles in which the theca interna had not differentiated. These results indicate good agreement between the type(s) of steroidogenic enzyme(s) present in tissues and the type(s) of steroid hormone(s) produced. It is concluded that regulation of steroid hormone production involves, at least in part, regulation of the levels of steroidogenic enzymes.     

Neurosteroidogenesis in Rat Retinas

Abstract: Neurosteroids (steroids synthesized in the CNS) function by modulating neurotransmission. To establish an experimental model for investigation of neurosteroid synthesis and regulation, independent of blood-borne steroids, we examined the steroidogenic activity of isolated rat retinas. We identified progesterone, pregnenolone, dehydroepiandrosterone, desoxycorticosterone, 3α,5α-tetrahydrodesoxycorticosterone, 3α-hydroxy-5α-dihydroprogesterone, 17-hydroxyprogesterone, and 17-hydroxypregnenolone together with their esterified forms. As pregnenolone is the precursor of all steroids, its formation was studied in detail as an index of a steroid-synthesizing tissue. Pregnenolone was identified further by gas chromatography coupled to mass spectrometry, and its in vitro synthesis was inhibited by lovastatin, an inhibitor of mevalonolactone and cholesterol biosynthesis. We then examined pregnenolone synthesis in the presence of mevalonolactone as a precursor of sterol formation together with lovastatin, which reduces endogenous mevalonolactone synthesis, as well as with inhibitors of pregnenolone metabolism. The incorporation of mevalonolactone into pregnenolone and its sulfate ester was time- and concentration-dependent and blocked by aminoglutethimide, a competitive inhibitor of cytochrome P450 side-chain cleavage (P450_scc) enzyme. Immunocytochemical studies with a specific antibody to P450_scc revealed a primary localization of the enzyme at the retinal ganglion cell layer. A less pronounced immunostaining was also seen at cells of the inner nuclear layer. Compounds known to stimulate cyclic AMP content also stimulated pregnenolone formation by rat retinas. These results demonstrate that rat retinas synthesize steroids and, for the first time, they reveal the steroidogenic ability of neuronal cells. We propose rat retinas as an in vitro model system to study neurosteroidogenesis in the CNS.