Characterisation of estrogenic 17beta-hydroxysteroid dehydrogenase (17beta-HSD) activity in the human brain

Estrogens play a crucial role in multiple functions of the brain and the proper balance of inactive estrone and active estradiol-17beta might be very important for their cerebral effects. The interconversion of estrone and estradiol-17beta in target tissues is known to be catalysed by a number of human 17beta-hydroxysteroid dehydrogenase (17beta-HSD) isoforms. The present study shows that enzyme catalysed interconversion of estrone and estradiol-17beta occurs in the human temporal lobe. The oxidative cerebral pathway preferred estradiol-17beta to Delta(5)-androstenediol and testosterone, whereas the reductive pathway preferred dehydroepiandrosterone (DHEA) to Delta(4)-androstenedione and estrone. An allosteric Hill kinetic for NAD-dependent oxidation of estradiol-17beta was observed, whereas a typical Michaelis-Menten kinetic was shown for NADPH-dependent reduction of estrone. Investigations of the interconversion of estrogens in cerebral neocortex (CX) and subcortical white matter (SC) preparations of brain tissue from 12 women and 10 men revealed no sex-differences, but provide striking evidence for the presence of at least one oxidative membrane-associated 17beta-HSD and one cytosolic enzyme that catalyses both the reductive and the oxidative pathway. Membrane-associated oxidation of estradiol-17beta was shown to be significantly higher in CX than in SC (P<0.05), whereas the cytosolic enzyme activities were significantly higher in SC than in CX (P<0.0005). Finally, real-time RT-PCR analyses revealed that besides 17beta-HSD types 4 and 5 also the isozymes type 7, 8, 10 and 11 show substantial expression in the human temporal lobe. The characteristics of the isozymes lead us to the conclusion that cytosolic 17beta-HSD type 5 is the best candidate for the observed cytosolic enzyme activities, whereas the data gave no clear answer to the question, which enzyme is responsible for the membrane-associated oxidation of estradiol-17beta. In conclusion, the study strongly suggests that different cell types and different isozymes are involved in the cerebral interconversion of estrogens, which might play a pivotal role in maintaining the functions of the central nervous system.     

Oxysterol 7 alpha-hydroxylase activity by cholesterol 7 alpha-hydroxylase (CYP7A)

A 7 alpha-hydroxylation is necessary for conversion of both cholesterol and 27-hydroxycholesterol into bile acids. According to current theories, cholesterol 7 alpha-hydroxylase (CYP7A) is responsible for the former and oxysterol 7 alpha-hydroxylase (CYP7B) for the latter reaction. CYP7A is believed to have a very high substrate specificity whereas CYP7B is active toward oxysterols, dehydroepiandrosterone, and pregnenolone. In the present study, 7 alpha-hydroxylation of various oxysterols in liver and kidney was investigated. Surprisingly, human cholesterol 7 alpha-hydroxylase, CYP7A, expressed as a recombinant in Escherichia coli and COS cells, was active toward 20(S)-hydroxycholesterol, 25-hydroxycholesterol, and 27-hydroxycholesterol. This enzyme has previously been thought to be specific for cholesterol and cholestanol. A partially purified and reconstituted cholesterol 7 alpha-hydroxylase enzyme fraction from pig liver showed 7 alpha-hydroxylase activity toward the same oxysterols as metabolized by expressed recombinant human and rat CYP7A. The 7 alpha-hydroxylase activity toward 20(S)-hydroxycholesterol, 25-hydroxycholesterol, and 27-hydroxycholesterol in rat liver was significantly increased by treatment with cholestyramine, an inducer of CYP7A. From the present results it may be concluded that CYP7A is able to function as an oxysterol 7 alpha-hydroxylase, in addition to the previously known human oxysterol 7 alpha-hydroxylase, CYP7B. These findings may have implications for oxysterol-mediated regulation of gene expression and for pathways of bile acid biosynthesis. A possible use of 20(S)-hydroxycholesterol as a marker substrate for CYP7A is proposed.     

Augmented androgen production is a stable steroidogenic phenotype of propagated theca cells from polycystic ovaries.

To test the hypothesis that the hyperandrogenemia associated with polycystic ovary syndrome (PCOS) results from an intrinsic abnormality in ovarian theca cell steroidogenesis, we examined steroid hormone production, steroidogenic enzyme activity, and mRNA expression in normal and PCOS theca cells propagated in long-term culture. Progesterone (P4), 17alpha-hydroxyprogesterone (17OHP4), and testosterone (T) production per cell were markedly increased in PCOS theca cell cultures. Moreover, basal and forskolin-stimulated pregnenolone, P4, and dehydroepiandrosterone metabolism were increased dramatically in PCOS theca cells. PCOS theca cells were capable of substantial metabolism of precursors into T, reflecting expression of an androgenic 17beta-hydroxysteroid dehydrogenase. Forskolin-stimulated cholesterol side chain cleavage enzyme (CYP11A) and 17alpha-hydroxylase/17,20-desmolase (CYP17) expression were augmented in PCOS theca cells compared with normal cells, whereas no differences were found in steroidogenic acute regulatory protein mRNA expression. Collectively, these observations establish that increased CYP11A and CYP17 mRNA expression, as well as increased CYP17, 3beta-hydroxysteroid dehydrogenase, and 17beta-hydroxysteroid dehydrogenase enzyme activity per theca cell, and consequently increased production of P4, 17OHP4, and T, are stable properties of PCOS theca cells. These findings are consistent with the notion that there is an intrinsic alteration in the steroidogenic activity of PCOS thecal cells that encompasses multiple steps in the biosynthetic pathway.     

Steroid sulfatase (STS) expression in the human temporal lobe: enzyme activity, mRNA expression and immunohistochemistry study

Dehydroepiandrosterone (DHEA) and its sulfate (DHEAS) are suggested to be important neurosteroids. We investigated steroid sulfatase (STS) in human temporal lobe biopsies in the context of possible cerebral DHEA(S) de novo biosynthesis. Formation of DHEA(S) in mature human brain tissue has not yet been studied. 17 alpha-Hydroxylase/C17-20-lyase and hydroxysteroid sulfotransferase catalyze the formation of DHEA from pregnenolone and the subsequent sulfoconjugation, respectively. Neither their mRNA nor activity were detected, indicating that DHEA(S) are not produced within the human temporal lobe. Conversely, strong activity and mRNA expression of DHEAS desulfating STS was found, twice as high in cerebral neocortex than in subcortical white matter. Cerebral STS resembled the characteristics of the known placental enzyme. Immunohistochemistry revealed STS in adult cortical neurons as well as in fetal and adult Cajal-Retzius cells. Organic anion transporting proteins OATP-A, -B, -D, and -E showed high mRNA expression levels with distinct patterns in cerebral neocortex and subcortical white matter. Although it is not clear whether they are expressed at the blood-brain barrier and facilitate an influx rather than an efflux, they might well be involved in the transport of steroid sulfates from the blood. Therefore, we hypothesize that DHEAS and/or other sulfated 3beta-hydroxysteroids might enter the human temporal lobe from the circulation where they would be readily converted via neuronal STS activity.     

Kinetic properties of the 5 alpha-reductase of testosterone in the purified myelin, in the subcortical white matter and in the cerebral cortex of the male rat brain

The 5 alpha-reductase, the enzyme which converts testosterone into dihydrotestosterone (DHT), is present in several CNS structures of the rat. Recent reports from this laboratory indicate that the subcortical white matter and the myelin possess a 5 alpha-reductase activity several times higher than that present in the cerebral cortex. Moreover, previous ontogenetic observations indicate that in all cerebral tissues examined (including the myelin) the 5 alpha-reductase has a higher activity in immature animals. This study was performed in order to verify whether the differences in the 5 alpha-reductase activity on the various brain components might be due to the presence of different concentrations of the same enzyme or to different isoenzymes. To this purpose, the kinetic properties Km and Vmax were measured in the purified myelin as well as in homogenates of the subcortical white matter and of the cerebral cortex, obtained from the brain of adult (60-90-day-old), immature (23-day-old), and aged (greater than 20-month-old) male rats. The results indicate that the enzymes present in the myelin, in the subcortical white matter and in the cerebral cortex of adult male rats possess a very similar apparent Km (1.93 +/- 0.2, 2.72 +/- 0.73 and 3.83 +/- 0.49 microM respectively). On the contrary, the Vmax values obtained in the myelin (34.40 +/- 5.54), in the white matter (19.57 +/- 2.36) and in the cerebral cortex (6.47 +/- 1.03 ng/h/mg protein) of adult animals have been found to be consistently different. Very similar Km values were found in the myelin obtained from the brain of immature and very old rats (2.14 +/- 0.11 and 3.39 +/- 0.75 microM respectively). The Vmax measured in the myelin purified from the immature rat brain (62.25 +/- 4.52) showed a value which was much higher than that found in the myelin of adult animals (34.40 +/- 5.54); a Vmax (34.31 +/- 9.41) almost identical to that of adult animals was found in the myelin prepared from the brain of aged rats.     

Expression of key enzymes in bile acid biosynthesis during development: CYP7B1-mediated activities show tissue-specific differences

The developmental variation of cytochrome P450 (CYP)7A1, CYP7B1, CYP27A1, and 3beta-hydroxy-Delta(5)-C(27)-steroid dehydrogenase, key enzymes in bile acid biosynthesis, were investigated in pigs of different ages. As part of these studies, peptide sequences from a purified pig liver oxysterol 7alpha-hydroxylase were analyzed. The sequences showed a high degree of identity with those of murine and human CYP7B1. Enzymatic activities and mRNA levels of CYP27A1 and 3beta-hydroxy-Delta(5)-C(27)-steroid dehydrogenase were similar in livers of newborn and 6-month-old pigs. Enzymatic activity mediated by CYP7A1 increased several-fold between infancy and adolescence. Hepatic CYP7A1 and CYP7B1 mRNA levels increased several-fold with age. Hepatic microsomal 7alpha-hydroxylation of 27-hydroxycholesterol and dehydroepiandrosterone, substrates typical for CYP7B1, increased about 5-fold between infancy and adolescence whereas the activities in kidney microsomes decreased at least 10-fold. In conclusion, the results indicate that the expression of CYP27A1 and 3beta-hydroxy-Delta(5)-C(27)-steroid dehydrogenase are similar in livers of newborn and 6-month-old pigs whereas the levels of CYP7A1 increase. The finding that the levels of CYP7B1 increase with age in the liver but decrease in the kidney suggest a tissue-specific developmental regulation of CYP7B1. The age-dependent variation in the liver and kidney suggests that hormonal factors are involved in the regulation of CYP7B1.     

A light and electron microscopic study of the iron transporter protein DMT-1 in the monkey cerebral neocortex and hippocampus

We have studied by immunocytochemistry, the distribution of DMT-1, a cellular iron transporter responsible for transport of metal irons from the plasma membrane to endosomes, in the normal monkey cerebral neocortex and hippocampus. Light to moderate DMT-1 staining was observed in glial cell bodies in the neocortex, the subcortical white matter, and the hippocampus. Despite light labeling of cell bodies, glial end feet around cortical and subcortical blood vessels were heavily labeled. In the neocortex, the glial cell bodies displayed the morphological features of protoplasmic astrocytes. Labeled glial cells in the subcortical white matter contained dense bundles of glial filaments and were identified as fibrous astrocytes. The observation that DMT-1 was present on astrocytic endfeet suggests that these cells are involved in uptake of iron from endothelial cells. It is possible that the iron could then be redistributed into the extracellular space in the brain parenchyma.     

The 5 alpha-reductase activity of the subcortical white matter, the cerebral cortex, and the hypothalamus of the rat and of the mouse: possible sex differences and effect of castration

Previous studies have shown that the central nervous system is able to convert testosterone into 17-beta-hydroxy-5-alpha-androstan-3-one (DHT), by the action of the enzyme 5-alpha-reductase. The data here presented show that, in the brain of the rat and the mouse of both sexes, the 5-alpha-reductase activity is more concentrated in the subcortical white matter than in the hypothalamus and in the cerebral cortex. The enzymatic activity is apparently higher in the rat than in the mouse brain. The formation of DHT in the subcortical white matter, in the hypothalamus and in the cerebral cortex of both rats and mice does not show any sexual difference. Moreover, in the rat no effect of short- or long-term castration or neonatal castration or testosterone replacement could be observed on the formation of DHT in the three brain structures considered (even in the subcortical white matter, the cerebral tissue more active in converting testosterone into DHT). The present data support the view that the 5-alpha-reductase present in the brain is not under androgenic control.     

Estrogen-mediated regulation of CYP7B1: a possible role for controlling DHEA levels in human tissues

The current study examines regulation of CYP7B1, a DHEA 7alpha-hydroxylase, by sex hormones. Transfection with estrogen receptor alpha and treatment with 17beta-estradiol in human embryonic kidney 293 cells significantly increased CYP7B1 catalytic activity and mRNA, and stimulated a human CYP7B1 reporter gene. Transfection with estrogen receptor beta showed similar but less significant effects. In the absence of receptors, 17beta-estradiol suppressed CYP7B1 activity, suggesting that estrogenic effects may be different in cells not expressing receptors. Quantitation of CYP7B1 mRNA in adult and fetal human tissues showed markedly higher CYP7B1 mRNA levels in fetal tissues compared with the corresponding adult ones, except in the liver. This indicates a tissue-specific, developmental regulation of CYP7B1 and suggests an important function for this enzyme in fetal life. DHEA secreted by fetal adrenals is an essential precursor for placental estrogen formation. Since CYP7B1 diverts DHEA from the sex hormone biosynthetic pathway, estrogen receptor-mediated up-regulation of CYP7B1 should lead to less DHEA available for sex hormone synthesis and may help to maintain normal levels of estrogens and androgens in human tissues, especially during fetal development. Regulation by estrogens may also be of importance in other processes where CYP7B1 is involved, including cholesterol homeostasis, cellular proliferation, and CNS function.     

Regulatory mechanism of Cordyceps sinensis mycelium on mouse Leydig cell steroidogenesis

We demonstrate the mechanism by which Cordyceps sinensis (CS) mycelium regulates Leydig cell steroidogenesis. Mouse Leydig cells were treated with forskolin, H89, phorbol 12-myristate 13-acetate, staurosporine, or steroidogenic enzyme precursors with or without 3 mg/ml CS; then testosterone production was determined. H89, but not phorbol 12-myristate 13-acetate or staurosporine, decreased CS-treated Leydig cell steroidogenesis. CS inhibited Leydig cell steroidogenesis by suppressing the activity of P450scc enzyme, but not 3beta-hydroxysteroid dehydrogenase, 17alpha-hydroxylase, 20alpha-hydroxylase, or 17beta-hydroxysteroid dehydrogenase enzymes. Thus, CS activated the cAMP-protein kinase A signal pathway, but not protein kinase C, and attenuated P45scc enzyme activity to reduce human chorionic gonadotropin-stimulated steroidogenesis in purified mouse Leydig cells.     

Inter-conversion of 7alpha- and 7beta-hydroxy-dehydroepiandrosterone by the human 11beta-hydroxysteroid dehydrogenase type 1

The dehydroepiandrosterone (DHEA) 7alpha-hydroxylation in humans takes place in the liver, skin, and brain. These organs are targets for the glucocorticoid hormones where 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) activates cortisone through its reduction into cortisol. The putative interference of 7alpha-hydroxy-DHEA with the 11beta-HSD1-catalyzed reduction of cortisone into cortisol has been confirmed in preliminary works with human liver tissue preparations of the enzyme demonstrating the transformation of 7alpha-hydroxy-DHEA into 7-oxo-DHEA and 7beta-hydroxy-DHEA. However, the large production of 7beta-hydroxy-DHEA could not be explained satisfactorily. Therefore our objective was to study the role in the metabolism of oxygenated DHEA by recombinant human 11beta-HSD1 expressed in yeast. The 7alpha- and 7beta-hydroxy-DHEA were each oxidized into 7-oxo-DHEA with quite dissimilar K(M) (70 and 9.5 microM, respectively) but at equivalent V(max). In contrast, the 11beta-HSD1-mediated reduction of 7-oxo-DHEA led to the production of both 7alpha- and 7beta-hydroxy-DHEA with equivalent K(M) (1.1 microM) but with a 7beta-hydroxy-DHEA production characterized by a significantly greater V(max). The 7alpha-hydroxy-DHEA produced by the cytochrome CYP7B1 in tissues may exert anti-glucocorticoid effects through interference with the 11beta-HSD1-mediated cortisone reduction.     

Differential distribution of the 5-alpha-reductase in the central nervous system of the rat and the mouse: are the white matter structures of the brain target tissue for testosterone action?

In the brain of several animal species testosterone is converted into a series of 5-alpha-reduced metabolites, and especially into 17-beta-hydroxy-5-alpha-androstan-3-one (DHT), by the action of the enzyme 5-alpha-reductase. The formation of DHT has never been evaluated in the white matter structures of the brain, which are composed mainly of myelinated axons. The experiments here described were performed in order to study, in the rat and the mouse, the DHT forming activity of several white matter structures, in comparison with that of the cerebral cortex and of the hypothalamus. Two sampling techniques were used in the rat: microdissection under a stereo-microscope from frozen brain sections of fragments of corpus callosum, optic chiasm and cerebral cortex; fresh tissue macrodissection of subcortical white matter, cerebral cortex and hypothalamus. Only macrodissection was used in the mice. The data show that, independently from the sampling technique used, there are considerable quantitative differences in the distribution pattern of the 5-alpha-reductase activity within different brain structures. Both in the rat and in the mouse, the enzyme appears to be present in higher concentrations in the white matter structures, than in the cerebral cortex and in the hypothalamus. The present results clearly show that the subcortical white matter and the corpus callosum are at least three times as potent as the cerebral cortex in converting testosterone into DHT. An even higher 5-alpha-reductase activity has been found in the optic chiasm. Further work is needed in order to understand the possible physiological role of DHT formation in the white matter structures.     

THE DISTRIBUTION AND PROPERTIES OF THE NONSPECIFIC N-METHYLTRANSFERASE IN BRAIN

Abstract— The distribution and properties of a nonspecific N -methyltransferase in the rat brain are described. The enzyme N -methylates tryptamine and N -methyltryptamine as well as β-phenylethylamine, phenylethanolamine, tyramine and octopamine. The enzyme exhibits a pH optimum of 7·9 with phosphate buffer and has a K_m for tryptamine of 28 μM. There are potent inhibitors to the enzyme that can be removed by dialysis. Enzymatic activity is present in the brains of a number of species including man, rat, mouse, guinea-pig and frog. Its activity is unevenly distributed in the brain with the highest activity in the cerebral cortex and striaturn of the rat and in the subcortical white matter in man. Studies of its subcellular distribution indicate that most of the N -methylation activity is released into the soluble fraction. Enzymatic activity is also present in a number of peripheral tissues of the rat.     

Constitutive expression and localization of cytochrome P-450 1A1 in rat and human brain: presence of a splice variant form in human brain

Cytochrome P-450 function as mono-oxygenases and metabolize xenobiotics. CYP1A1, a cytochrome P-450 enzyme, bioactivates polycyclic aromatic hydrocarbons to reactive metabolite(s) that bind to DNA and initiate carcinogenesis. Northern and immunoblot analyses revealed constitutive expression of Cyp1a1 and CYP1A1 in rat and human brain, respectively. CYP1A1 mRNA and protein were localized predominantly in neurons of cerebral cortex, Purkinje and granule cell layers of cerebellum and pyramidal neurons of CA1, CA2, and CA3 subfields of the hippocampus. RT-PCR analyses using RNA obtained from autopsy human brain samples demonstrated the presence of a splice variant having a deletion of 87 bp of exon 6. This splice variant was present in human brain, but not in the liver from the same individual, and was absent in rat brain and liver. Structural modeling indicated broadening of the substrate access channel in the brain variant. The study demonstrates the presence of a unique cytochrome P-450 enzyme in human brain that is generated by alternate splicing. The presence of distinct cytochrome P-450 enzymes in human brain that are different from well-characterized hepatic forms indicates that metabolism of xenobiotics including drugs could occur in brain by pathways different from those known to occur in liver.     

Metabolism of [4-14C]testosterone and precursors by homogenates of rat testes after chronic LHRH-treatment

Adult male rats were injected daily for 8 days with an LHRH agonist. Twenty-four hours after the last injection testes-homogenates were incubated in the presence of a 4-14C-labeled steroid, either progesterone, 17 alpha-hydroxyprogesterone, dehydroepiandrosterone, androstenedione or testosterone. The activity of several enzymes involved in the androgen biosynthetic pathway was inferred from the amount of metabolites produced under these conditions. After LHRH-treatment a significant increase in the 17,20-lyase activity was observed without any significant change in the activity of 17 alpha-hydroxylase, 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase and 17 beta-hydroxysteroid dehydrogenase. The results of the experiments indicate that the decreased testosterone secretion observed in rats after chronic LHRH-administration is not due to an inhibition of the enzyme-systems studied.     

Rv1106c from Mycobacterium tuberculosis is a 3beta-hydroxysteroid dehydrogenase

New approaches are required to combat Mycobacterium tuberculosis (Mtb), especially the multi-drug resistant and extremely drug resistant organisms (MDR-TB and XDR-TB). There are many reports that mycobacteria oxidize 3beta-hydroxysterols to 3-ketosteroids, but the enzymes responsible for this activity have not been identified in mycobacterial species. In this work, the Rv1106c gene that is annotated as a 3beta-hydroxysteroid dehydrogenase in Mtb has been cloned and heterologously expressed. The purified enzyme was kinetically characterized and found to have a pH optimum between 8.5 and 9.5. The enzyme, which is a member of the short chain dehydrogenase superfamily, uses NAD+ as a cofactor and oxidizes cholesterol, pregnenolone, and dehydroepiandrosterone to their respective 3-keto-4-ene products. The enzyme forms a ternary complex with NAD+ binding before the sterol. The enzyme shows no substrate preference for dehydroepiandrosterone versus pregnenolone with second-order rate constants (kcat/Km) of 3.2 +/- 0.4 and 3.9 +/- 0.9 microM-1 min-1, respectively, at pH 8.5, 150 mM NaCl, 30 mM MgCl2, and saturating NAD+. Trilostane is a competitive inhibitor of dehydroepiandrosterone with a Ki of 197 +/- 8 microM. The expression of the 3beta-hydroxysteroid dehydrogenase in Mtb is intracellular. Disruption of the 3beta-hydroxysteroid dehydrogenase gene in Mtb abrogates mycobacterial cholesterol oxidation activity. These data are consistent with the Rv1106c gene being the one responsible for 3beta-hydroxysterol oxidation in Mtb.     

Xenopus laevis CYP17 regulates androgen biosynthesis independent of the cofactor cytochrome b5

The enzyme CYP17 primarily regulates androgen production by mediating four reactions: conversion of pregnenolone and progesterone to 17-hydroxypregnenolone and 17-hydroxyprogesterone, respectively (17alpha-hydroxylase activity), followed by conversion of the 17-hydroxylated steroids to dehydroepiandrosterone and androstenedione, respectively (17,20-lyase activity). Most mammalian CYP17 isoforms have high 17alpha-hydroxylase relative to 17,20-lyase activities and preferentially mediate one of the two 17,20-lyase reactions. In contrast, Xenopus laevis CYP17 potently regulates all four reactions in the frog ovary. CYP17 isoforms generally rely on the cofactor cytochrome b(5) for the 17,20-lyase reaction, suggesting that the high lyase activity of Xenopus CYP17 might be due to a lesser dependence on b(5). The kinetics of Xenopus CYP17 expressed in yeast microsomes were therefore examined in the absence and presence of Xenopus on human b(5). Xenopus CYP17 mediated both 17,20-lyase reactions in the absence of b(5), confirming that the activity did not require b(5). However, both Xenopus and human b(5) slightly enhanced Xenopus CYP17-mediated lyase activity, indicating that the enzyme was still at least partially responsive to b(5). Surprisingly, only the human b(5) cofactor enhanced human CYP17-mediated lyase activity, implying that the human enzyme had more specific cofactor requirements than Xenopus CYP17. Studies using human/Xenopus chimeric b(5) proteins revealed that human b(5) residues 16-41 were important for the specific regulation of the lyase activity of HuCYP17, possibly serving as an interacting domain with the enzyme. CYP17 may therefore have evolved from a general producer of sex steroids in lower vertebrates to a more tightly regulated producer of both sex steroids and glucocorticoids in mammals.