大鼠海马神经元内11β-HSD1和GR的共存及其意义

本研究旨在探讨糖皮质激素代谢酶-11β-羟基类固醇脱氢酶Ⅰ型（11β-HSD1）和糖皮质激素受体（GR）在大鼠海马神经元内的共同分布及其意义。用免疫细胞化学方法研究显示,海马神经元内不仅存在11β-HSD1免疫反应物质,还存在GR免疫反应物质,而且11β-HSD1与GR共存于同一个海马神经元内,用Western印迹杂交和薄层层析（TLC）方法研究表明,地塞米松（DEX）可以促进11β-HSD1与GR共存于同一个海马神经元内,用Western印迹杂交和薄层层析（TLG）方法研究表明,地塞米松（DEX）可以促进11β-HSD1蛋白表达及其酶的活性,利用11β-HSD1基因启动子区序列构建的以CAT酶为报告基因的pBLCAT6质粒转染PC12细胞,证实DEX能够促进CAT酶的表达。以上糖皮质激素的作用均可为GR受体阻断剂RU38486所阻断,结果提示;糖皮质激素（GC）与GR结合后,可以作用于与其共存的11β-HSD1基因启动子区,使11β-HSD1表达增加,从而使更多的GC代谢产物转化为有活性的GC,此机制可能与保证GC在海马神经元内与亲和力较低的GR结合有关。     

The intracellular localization of the mineralocorticoid receptor is regulated by 11beta-hydroxysteroid dehydrogenase type 2

11beta-hydroxysteroid dehydrogenase (11beta-HSD) type 2 has been considered to protect the mineralocorticoid receptor (MR) by converting 11beta-hydroxyglucocorticoids into their inactive 11-keto forms, thereby providing specificity to the MR for aldosterone. To investigate the functional protection of the MR by 11beta-HSD2, we coexpressed epitope-tagged MR and 11beta-HSD2 in HEK-293 cells lacking 11beta-HSD2 activity and analyzed their subcellular localization by fluorescence microscopy. When expressed alone in the absence of hormones, the MR was both cytoplasmic and nuclear. However, when coexpressed with 11beta-HSD2, the MR displayed a reticular distribution pattern, suggesting association with 11beta-HSD2 at the endoplasmic reticulum membrane. The endoplasmic reticulum membrane localization of the MR was observed upon coexpression only with 11beta-HSD2, but not with 11beta-HSD1 or other steroid-metabolizing enzymes. Aldosterone induced rapid nuclear translocation of the MR, whereas moderate cortisol concentrations (10-200 nm) did not activate the receptor, due to 11beta-HSD2-dependent oxidation to cortisone. Compromised 11beta-HSD2 activity (due to genetic mutations, the presence of inhibitors, or saturating cortisol concentrations) led to cortisol-induced nuclear accumulation of the MR. Surprisingly, the 11beta-HSD2 product cortisone blocked the aldosterone-induced MR activation by a strictly 11beta-HSD2-dependent mechanism. Our results provide evidence that 11beta-HSD2, besides inactivating 11beta-hydroxyglucocorticoids, functionally interacts with the MR and directly regulates the magnitude of aldosterone-induced MR activation.     

脑内皮质激素受体及其受体前代谢酶的作用

中枢神经系统的发生、分化、发育成熟和退化中均有糖皮质激素的参与。糖皮质激素对神经系统的影响是在11β－羟基类固醇脱氢酶的调节下,通过与糖皮质激素受体和盐皮质激素受体结合,调节靶基因的转录而实现的。本文介绍了脑内糖皮质激素受体和11β－羟基类固醇脱氢酶的类型、分布、功能,以及二者在糖皮质激素发挥作用中的意义。     

Differential expression of 11beta-hydroxysteroid dehydrogenase types 1 and 2 mRNA and glucocorticoid receptor protein during mouse embryonic development

Accumulating evidence suggests that the actions of glucocorticoids in target tissues are critically determined by the expression of not only the glucocorticoid receptor (GR) but also the glucocorticoid-metabolizing enzymes, known as 11β-hydroxysteroid dehydrogenase types 1 and 2 (11β-HSD1 and 11β-HSD2). To gain insight into the role of glucocorticoids in fetal development, the expression patterns of the two distinct 11β-HSD isozymes and GR were studied in the mouse embryo from embryonic day 12.5 (E12.5, TERM = E19) to postnatal day 0.5 (P0.5) by in situ hybridization and immunohistochemistry, respectively. 11β-HSD1 mRNA was detected in the heart as early as E12.5 and maintained thereafter. In the lung and liver, 11β-HSD1 mRNA was first detected between E14.5 and E16.5, increased to high levels towards term and maintained after birth. Relatively low levels of 11β-HSD1 mRNA were also detected in the kidney, adrenal glands and gastrointestinal tract at E18.5. However, the mRNA for 11β-HSD1 was undetectable in all other embryonic tissues including the brain. In contrast, kidney was the only organ that expressed appreciable levels of 11β-HSD2 mRNA during embryonic life. The level of 11β-HSD2 mRNA in the kidney increased dramatically in the newborn, which coincided with expression of 11β-HSD2 mRNA in the whisker follicle, tooth and salivary gland. Distinct from the profiles of 11β-HSD1 and 11β-HSD2 mRNA, GR protein was detectable in all tissues at all ages studied except for the thymus, salivary gland, and bone. Taken together, the present study demonstrates that tissue- and developmentally-stage specific expression of 11β-HSD1 and 11β-HSD2 as well as GR occurs in the developing mouse embryo, thus highlighting the importance of these two enzymes and GR in regulating glucocorticoid-mediated maturational events in specific tissues during murine embryonic development.     

Hexose-6-phosphate dehydrogenase knock-out mice lack 11 beta-hydroxysteroid dehydrogenase type 1-mediated glucocorticoid generation

The local generation of active glucocorticoid by NADPH-dependent, 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) oxoreductase activity, has emerged as an important factor in regulating hepatic glucose output and visceral adiposity. We have proposed that this NADPH is generated within the endoplasmic reticulum by the enzyme hexose-6-phosphate dehydrogenase. To address this hypothesis, we generated mice with a targeted inactivation of the H6PD gene. These mice were unable to convert 11-dehydrocorticosterone (11-DHC) to corticosterone but demonstrated increased corticosterone to 11-DHC conversion consistent with lack of 11beta-HSD1 oxoreductase and a concomitant increase in dehydrogenase activity. This increased corticosterone clearance in the knock-out mice resulted in a reduction in circulating corticosterone levels. Our studies define the critical requirement of hexose-6-phosphate dehydrogenase for 11beta-HSD1 oxoreductase activity and add a new dimension to the investigation of 11beta-HSD1 as a therapeutic target in patients with the metabolic syndrome.     

The human gene for 11 beta-hydroxysteroid dehydrogenase. Structure, tissue distribution, and chromosomal localization

The Type I (mineralocorticoid) receptor has identical affinities in vitro for cortisol and aldosterone. It has been suggested that the selective role of aldosterone in regulating sodium homeostasis relies on the microsomal enzyme 11 beta-hydroxysteroid dehydrogenase (11-HSD). This enzyme converts cortisol to its inactive metabolite, cortisone, preventing cortisol from binding to the Type I receptor. We have isolated human cDNA clones encoding 11-HSD from a human testis cDNA library by hybridization with a previously isolated rat 11-HSD cDNA clone. The cDNA contains an open reading frame of 876 bases, which predicts a protein of 292 amino acids. The sequence is 77% identical at the amino acid level to rat 11-HSD cDNA. The mRNA is widely expressed, but the level of expression is highest in the liver. Hybridization of the human 11-HSD cDNA to a human-hamster hybrid cell panel localized the single corresponding HSD11 gene to chromosome 1. This gene was isolated from a chromosome 1 specific library using the cDNA as a probe. HSD11 consists of 6 exons and is at least 9 kilobases long. The data developed in this study should be applicable to the study of patients with hypertension due to apparent mineralocorticoid excess, a deficiency in 11-HSD activity.     

Cloning of chicken 11beta-hydroxysteroid dehydrogenase type 1 and its tissue distribution

11beta-Hydroxysteroid dehydrogenase type 1 (11HSD1) is an enzyme that interconverts active 11-hydroxy glucocorticoids (cortisol, corticosterone) and their inactive 11-oxo derivatives (cortisone, 11-dehydrocorticosterone). Although bidirectional, it is considered to operate in vivo as an 11-reductase that regenerates active glucocorticoids and thus amplifies their local activity in mammals. Here we report the cloning, characterization and tissue distribution of chicken 11HSD1 (ch11HSD1). Its cDNA predicts a protein of 300 amino acids that share 51-56% sequence identity with known mammalian 11HSD1 proteins, while in contrast to most mammals, ch11HSD1 contains only one N-linked glycosylation site. Analysis of the tissue distribution pattern by RT-PCR revealed that ch11HSD1 is expressed in a large variety of tissues, with high expression in the liver, kidney and intestine, and weak in the gonads, brain and heart. 11-Reductase activity has been found in the liver, kidney, intestine and gonads with low or almost zero activity in the brain and heart. These results provide evidence for a role of 11HSD1 as a tissue-specific regulator of glucocorticoid action in non-mammalian vertebrates and may serve as a suitable model for further analysis of 11HSD1 evolution in vertebrates.     

Characterization and localization of human type10 17beta-hydroxysteroid dehydrogenase.

The tissue distribution, subcellular localization, and metabolic functions of human 17beta-hydroxysteroid dehydrogenase type 10/short chain L-3-hydroxyacyl-CoA dehydrogenase have been investigated. Human liver and gonads are abundant in this enzyme, but it is present in only negligible amounts in skeletal muscle. Its N-terminal sequence is a mitochondrial targeting sequence, but is not required for directing this protein to mitochondria. Immunocytochemical studies demonstrate that this protein, which has been referred to as ER-associated amyloid beta-binding protein (ERAB), is not detectable in the ER of normal tissues. We have established that protocols employed to investigate the subcellular distribution of ERAB yield ER fractions rich in mitochondria. Mitochondria-associated membrane fractions believed to be ER fractions were employed in ERAB/Abeta-binding alcohol dehydrogenase studies. The present studies establish that in normal tissues this protein is located in mitochondria. This feature distinguishes it from all known 17beta-hydroxysteroid dehydrogenases, and endows mitochondria with the capability of modulating intracellular levels of the active forms of sex steroids.     

11 beta-hydroxysteroid dehydrogenase activity in the mammary gland

Levels of 11 beta-hydroxysteroid dehydrogenase activity in mammary gland homogenates from pregnant and lactating Sprague-Dawley rats were determined by incubation with [3H]corticosterone under standard conditions, followed by thin-layer chromatography of incubated media. Enzyme activity was high in virgin and pregnant rats, but fell soon after parturition, suggesting a possible role for this enzyme in the co-ordinate regulation of glucocorticoid effects on milk protein synthesis.     

Glycyrrhetinic acid bound to 11 beta-hydroxysteroid dehydrogenase in rat liver microsomes.

A binding protein which exhibits high affinity to [3H]glycyrrhetinic-acid in the rat liver microsomal fraction was solubilized with 0.2% Triton DF-18 and then purified to homogeneity. The equilibrium dissociation constant of the [3H]glycyrrhetinic-acid binding reaction and the maximal concentration for the binding of the purified protein, as determined by Scatchard plot analysis, were 27.6 nM and 7.79 nmol/mg protein, respectively. The molecular mass of the subunit (34 kDa) and 30 amino acids of N-terminal sequence of the purified protein were entirely the same as those of the reported 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD). In each purification step, the recovery and purification (fold) of the glycyrrhetinic-acid binding activity corresponded to the values of 11 beta-HSD activity. These results show that the purified [3H]glycyrrhetinic-acid binding protein is 11 beta-HSD. From the molecular mass of 11 beta-HSD (135 kDa) and the maximal concentration of the binding site, it was calculated that one glycyrrhetinic acid molecule binds to one 11 beta-HSD molecule. The inhibitory effects of various glycyrrhetinic-acid derivatives on [3H]glycyrrhetinic acid binding and 11 beta-HSD activity indicate that the C30-carboxyl and C11-carbonyl groups of glycyrrhetinic acid are the principal structures for the 11 beta-HSD inhibition.     

Changes in expression of 11beta-hydroxysteroid dehydrogenase type-1, type-2 and glucocorticoid receptor mRNAs in porcine corpus luteum during the estrous cycle

The objective of the present study was to determine whether glucocorticoid (GC) and its receptor (GC-R) are expressed in the porcine corpus luteum (CL), and whether GC influences porcine luteal hormone production. The gene expressions of 11beta-hydroxysteroid dehydrogenase type 1 (11-HSD1), type 2 (11-HSD2), GC-R, and the concentrations of GC were determined in the CL of Chinese Meishan pigs during the estrous cycle. Moreover, the effects of GC on progesterone (P(4)), estradiol-17beta (E(2)), and prostaglandin (PG) F2alpha secretion by cultured luteal cells were investigated. Messenger RNAs of the 11-HSD1, 11-HSD2, and GC-R were clearly expressed in the CL throughout the estrous cycle. The 11-HSD1 mRNA level in the CL was higher at the regressed stage than at the other stages (P < 0.05), whereas 11-HSD2 mRNA was lower at the regressed stage than at the other stages (P < 0.05). GC-R mRNA level was higher at the regressed stages than at the other stages (P < 0.01). Concentrations of GC were lower in the regressed CL than in the other stages (P < 0.01). When the cultured luteal cells obtained from mid-stage CL (Days 8-11) were exposed to GC (50-5,000 ng/ml), P(4) and PGF2alpha secretion by the cells were reduced (P < 0.05), whereas GC had no effect on E(2) secretion by the cells. The overall results suggest that GC is regulated locally by 11-HSD1 and 11-HSD2 in the porcine CL. GC inhibits P(4) and PGF2alpha production from luteal cells via their specific receptors, implying GC plays some roles in regulating porcine CL function throughout the estrous cycle.     

11beta-hydroxysteroid dehydrogenase and glucocorticoid receptor messenger RNA expression in porcine placentae: effects of stage of gestation,breed, and uterine environment

Glucocorticoids are known to influence many aspects of prenatal development. Three important regulators of glucocorticoid actions at the cellular level are the enzymes 11beta-hydroxysteroid dehydrogenase type 1 (11betaHSD-1), 11beta-hydroxysteroid dehydrogenase type 2 (11betaHSD-2), and glucocorticoid receptors (GR). The present study was conducted to determine the presence of these regulators in porcine placentae during early gestation (Days 24-40; term = 114 days) and to examine the influence of breed and uterine environment. Three pig models differing in uterine environment as reflected by embryonic survival from Days 24 to 40 were used: intact white cross-bred gilts (WC-INT); white cross-bred gilts that had been unilaterally hysterectomized-ovariectomized before puberty (WC-UHO); and intact Meishan gilts (ME). Porcine-specific partial cDNAs for 11betaHSD-1 and 11betaHSD-2 and a cRNA for GRalpha were developed and used to produce 32P-labeled probes for Northern blot analyses. The 11betaHSD dehydrogenase activity was measured in vitro at saturating concentrations of substrate and coenzyme. At Day 24 of gestation, 11betaHSD-2 mRNA, dehydrogenase activity, and GR mRNA were present, but 11betaHSD-1 mRNA was absent. All three mRNAs and dehydrogenase activity increased (P < 0.01) by Day 40. On Day 30, placental 11betaHSD-2 mRNA was decreased (P = 0.03) by 47% in WC-UHO versus WC-INT. Placental 11betaHSD dehydrogenase activity was 2-fold greater (P < 0.01) in ME versus WC-INT on Day 24 of gestation. These results demonstrate, to our knowledge for the first time, the presence of 11betaHSD-1, 11betaHSD-2, and GR mRNA as well as 11betaHSD dehydrogenase activity in the porcine placenta during early pregnancy. Moreover, a role for glucocorticoids in porcine embryonic development is suggested.     

The type I and type II 11beta-hydroxysteroid dehydrogenase enzymes.

Local tissue concentrations of glucocorticoids are modulated by the enzyme 11β-hydroxysteroid dehydrogenase which interconverts cortisol and the inactive glucocorticoid cortisone in man, and corticosterone and 11-dehydrocorticosterone in rodents. The type I isoform (11β-HSD1) is a bidirectional enzyme but acts predominantly as a oxidoreductase to form the active glucocorticoids cortisol or corticosterone, while the type II enzyme (11β-HSD2) acts unidirectionally producing inactive 11-keto metabolites. There are no known clinical conditions associated with 11β-HSD1 deficiency, but gene deletion experiments in the mouse indicate that this enzyme is important both for the maintenance of normal serum glucocorticoid levels, and in the activation of key hepatic gluconeogenic enzymes. Other important sites of action include omental fat, the ovary, brain and vasculature. Congenital defects in the 11β-HSD2 enzyme have been shown to account for the syndrome of apparent mineralocorticoid excess (AME), a low renin severe form of hypertension resulting from the overstimulation of the non-selective mineralocorticoid receptor by cortisol in the distal tubule of the kidney. Inactivation of the 11β-HSD2 gene in mice results in a phenotype with similar features to AME. In addition, these mice show high neonatal mortality associated with marked colonic distention, and remarkable hypertrophy and hyperplasia of the distal tubule epithelia. 11β-HSD2 also plays an important role in decreasing the exposure of the fetus to the high levels of maternal glucocorticoids. Recent work suggests a role for 11β-HSD2 in non-mineralocorticoid target tissues where it would modulate glucocorticoid access to the glucocorticoid receptor, in invasive breast cancer and as a mechanism providing ligand for the putative 11-dehydrocorticosterone receptor. While previous homologies between members of the SCAD superfamily have been of the order of 20–30% phylogenetic analysis of a new branch of retinol dehydrogenases indicates identities of >60% and overlapping substrate specificities. The availability of crystal structures of family members has allowed the mapping of conserved 11β-HSD domains A–D to a cleft in the protein structure (cofactor binding domain), two parallel β-sheets, and an -helix (active site), respectively.     

Corticosteroids, receptors, and the organ-specific functions of 11 beta-hydroxysteroid dehydrogenase.

Reversible oxidation of the biologically active corticosteroids to the inactive 11-dehydrocorticosteroids is catalyzed by 11 beta-hydroxysteroid dehydrogenase (11 beta HSD). The properties of the enzyme based on clinical observations of individuals with defective 11 beta HSD expression, and laboratory studies of the properties and behavior of the enzyme, are consistent with separate 11 beta-dehydrogenase and 11-oxoreductase species. However, recombinant enzyme expressed in mammalian cells retain both activities, leading to the conclusion that 11 beta HSD is a unique, reversible enzyme. 11 beta HSD is present in most tissues, but its specific functions in most tissues are unknown. How the enzyme may mediate corticosteroid-receptor interaction is illustrated by studies using kidney, testis, and brain. In kidney, 11 beta HSD prevents glucocorticoids from competing inappropriately with aldosterone for mineralocorticoid receptor (MR). Lack of enzyme in humans due to natural causes or inhibition by pharmacological agents results in maximum activation of MR by glucocorticoids, leading to the clinical symptoms of apparent mineralocorticoid excess. Leydig cells of the testes synthesize testosterone, a process that is suppressed by events initiated by the binding of corticosteroid to glucocorticoid receptors (GR). Depletion of active steroid mediated by 11 beta HSD may initiate testosterone production at puberty and affect testosterone production during adult life, as for example during periods of stress. The heterogeneous distribution of MR and GR in the brain reflects the specific regional effects of glucocorticoids and mineralocorticoids on neural function. Colocalization of 11 beta HSD and corticosteroid receptors in brain may be important in controlling the specificity of corticosteroid interaction with GR and MR. The patterns of 11 beta HSD-steroid-receptor interaction illustrated with these three tissues may provide models applicable to other tissues in which corticosteroid receptors and 11 beta HSD coexist.     

Pathophysiological roles of vascular 11beta-hydroxysteroid dehydrogenase and aldosterone

Mineralocorticoid receptor (MR) binding is tightly regulated by the enzyme 11β-hydroxysteroid dehydrogenase type 2 (11β-HSDII) which selectively metabolizes glucocorticoids to inactive metabolites, thus allowing for MR activation by aldosterone. To examine whether this enzyme is involved in the pathophysiology of salt-sensitive hypertension, 11β-HSDII activity and messenger RNA (mRNA) levels were determined in blood vessels of Dahl Iwai salt-sensitive (DS) and salt-resistant (DR) rats. Decreased 11β-HSDII activity and mRNA levels in mesenteric arteries were observed in 8-week-old DS rats on a high-salt diet, indicating that 11β-HSDII may play a significant role in salt sensitivity and hypertension. It has been suggested that mineralocorticoids act on blood vessels, leading to increased vasoreactivity and peripheral resistance. We present direct evidence that blood vessels are aldosteronogenic. The production of aldosterone in blood vessels was compared between stroke-prone spontaneously hypertensive rats (SHRSP) and Wistar-Kyoto (WKY) rats. Vascular aldosterone and CYP11B2 mRNA levels were significantly increased in 2-week-old SHRSP versus WKY rats. However, the vascular aldosterone levels in 4- and 9-week-old SHRSP and WKY rats were similar. High sodium intake further increased both blood pressure and vascular aldosterone synthesis in the SHRSPs. Both the local renin–angiotensin–aldosterone system (RAAS) and the vascular 11β-HSDII level are critically important in the pathophysiology of cardiovascular disorders.     

Histochemical localization of 3 beta-hydroxysteroid dehydrogenase in the testes of chicken-pheasant hybrids.

Synopsis Histochemical studies on the activity of 3-hydroxysteroid dehydrogenase (3-HSD) in the testes of adult chicken-pheasant hybrids and domestic fowl of similar age were carried out using dehydroepiandrosterone as the substrate. The reaction for 3-HSD was positive in the interstitial tissue and negative within seminiferous tubules of domestic fowl. In chicken-pheasant hybrids, a strong positive reaction for 3-HSD was observed in the interstitial tissue and within the seminiferous tubules suggesting that, in hybrid testes, both Leydig cells and cells of seminiferous tubules may be capable of steroid biosynthesis. Since the plasma testosterone levels in these birds were found to be extremely low, it is hypothesized that either the chicken-pheasant hybrids do not release testosterone in sufficient amounts in the circulation or the type of steroid produced by the 3HSD-positive cells may be different from testosterone that is required for the maintenance of normal fertility and the development of secondary sexual characteristics.