Molecular genetics of androgenic 17β-Hydroxysteroid dehydrogenases

17β-Hydroxysteroid dehydrogenase (17β-HSD) type 2 catalyzes the NAD⁺-dependent oxidation of androgens, estrogens and progestins, predominantly in the secretory endometrium, placenta, liver and small intestine. 17β-HSD type 3 catalyzes the NADPH-dependent conversion of androstenedione to testosterone in the testis, and the genetic disease 17β-HSD deficiency is caused by mutations in the 17β-HSD3 gene.     

Structure-function relationships and molecular genetics of the 3β-hydroxysteroid dehydrogenase gene family

The isoenzymes of the 3β-hydroxysteroid dehydrogenase/5-ene-4-ene-isomerase (3β-HSD) gene family catalyse the transformation of all 5-ene-3β-hydroxysteroids into the corresponding 4-ene-3-keto-steroids and are responsible for the interconversion of 3β-hydroxy- and 3-keto-5-androstane steroids. The two human 3β-HSD genes and the three related pseudogenes are located on the chromosome 1p13.1 region, close to the centromeric marker D1Z5. The 3β-HSD isoenzymes prefer NAD⁺ to NADP⁺ as cofactor with the exception of the rat liver type III and mouse kidney type IV, which both prefer NADPH as cofactor for their specific 3-ketosteroid reductase activity due to the presence of Tyr³⁶ in the rat type III and of Phe³⁶ in mouse type IV enzymes instead of Asp³⁶ found in other 3β-HSD isoenzymes. The rat types I and IV, bovine and guinea pig 3β-HSD proteins possess an intrinsic 17β-HSD activity psecific to 5-androstane 17β-ol steroids, thus suggesting that such “secondary” activity is specifically responsible for controlling the bioavailability of the active androgen DHT. To elucidate the molecular basis of classical form of 3β-HSD deficiency, the structures of the types I and II 3β-HSD genes in 12 male pseudohermaphrodite 3β-HSD deficient patients as well as in four female patients were analyzed. The 14 different point mutations characterized were all detected in the type II 3β-HSD gene, which is the gene predominantly expressed in the adrenals and gonads, while no mutation was detected in the type I 3β-HSD gene predominantly expressed in the placenta and peripheral tissues. The mutant type II 3β-HSD enzymes carrying mutations detected in patients affected by the salt-losing form exhibit no detectable activity in intact transfected cells, at the exception of L108W and P186L proteins, which have some residual activity (1%). Mutations found in nonsalt-loser patients have some residual activity ranging from 1 to 10% compared to the wild-type enzyme. Characterization of mutant proteins provides unique information on the structure-function relationships of the 3β-HSD superfamily.     

Glycyrrhizic acid suppresses type 2 11 beta-hydroxysteroid dehydrogenase expression in vivo

Licorice-derivatives such as glycyrrhizic acid (GA) competitively inhibit 11β-hydroxysteroid dehydrogenase(11β-HSD) type 2 (11-HSD2) enzymatic activity, and chronic clinical use often results in pseudoaldosteronism. Since the effect of GA on 11-HSD2 expression remains unknown, we undertook in vivo and in vitro studies. Male Wistar rats were given 30, 60 or 120 mg/kg of GA twice a day for 2 weeks. Plasma corticosterone was decreased in those given the 120 mg dose, while urinary corticosterone excretion was increased in those given the 30 and 60 mg doses but decreased in those given 120 mg GA. NAD⁺-dependent dehydrogenase activity in kidney microsomal fraction was decreased in animals receiving doses of 60 and 120 mg GA. The 11-HSD2 protein and mRNA levels were decreased in those given 120 mg GA. In contrast, in vitro studies using mouse kidney M1 cells revealed that 24 h treatment with glycyrrhetinic acid did not affect the 11-HSD2 mRNA expression levels. Thus, in addition to its role as a competitive inhibitor of 11-HSD2, the chronic high dose of GA suppresses mRNA and protein expression of 11-HSD2 possibly via indirect mechanisms. These effects may explain the prolonged symptoms after cessation of GA administration in some pseudoaldosteronism patients.     

Dual subcellular localization of the 3β-hydroxysteroid dehydrogenase isomerase: Characterization of the mitochondrial enzyme in the bovine adrenal cortex

The enzyme 3β-hydroxysteroid dehydrogenase isomerase (3β-HSD/I) in an essential step in the biosynthesis of steroid such as progesterone, mineralo- and gluco-corticoids, estrogens and androgens in steroidogenic tissues. It is considered to be mainly localized in microsomes; however, 3β-HSD/I activity has also been described to be associated with mitochondrial preparations. In this study, we examined the subcellular distribution of 3β-HSD/I in bovine adrenocortical tissue and we characterized the catalytic properties of the enzyme present in the various cell compartments. About 30% of the total 3β-HSD/I activity was found to remain tightly associated with the purified mitochondrial pellet. The 3β-HSD/I and 3-ketoreductase activities were found in microsomes as well as in mitochondria. The 3β-HSD/I associated with the mitochondrial fraction did not required addition of exogenous NAD⁺. When the pyridine nucleotide was reduced ollowing addition of substrate of the tricarboxyllic acids cycle, the mitochondrial 3β-HSD/I activity decreased, suggesting that the enzyme utilizes NAD⁺ available from the matrix space. By contrast, the microsomal enzyme was inactive in the absence of exogenous NAD⁺. Submitochondrial fraction disclosed that 3β-HSD/I was associated (i) with the inner membrane and (ii) with a particulate fraction sedimenting in a density gradient between inner and outer membranes. This fraction was characterized as contact sites between the two membranes. 3β-HSD/I specific activity was much higher in this fraction than in the inner mitochondrial membrane. Altogether, these observations suggest that these mitochondrial intermembrane contact sites may represent a spacial organization of functional significance, facilitating both the access of cholesterol to the inner membrane where cytochrome P-450_scc is located and the rapid transformation of its product, pregnenolone, to progesterone, through 3β-HSD/I activity.     

Type 2 11β-hydroxysteroid dehydrogenase in foetal and adult life

Two isoforms of 11β-hydroxysteroid dehydrogenase (11β-HSD) catalyse the interconversion of active cortisol to inactive cortisone; 11β-HSD1 is a low affinity, NADP(H)-dependent dehydrogenase/oxo-reductase, and 11β-HSD2 a high affinity, NAD-dependent dehydrogenase. Because of the importance of 11β-HSD in regulating corticosteroid hormone action, we have analysed the distribution of the 11β-HSD isoforms in human adult and foetal tissues (including placenta), and, in addition have performed a series of substrate specificity studies on the novel, kidney 11β-HSD2 isoform. Using an RT-PCR approach, we failed to detect 11β-HSD1 mRNA in any human mid-gestational foetal tissues. In contrast 11β-HSD2 mRNA was present in foetal lung, adrenal, colon and kidney. In adult tissues 11β-HSD2 gene expression was confined to the mineralocorticoid target tissues, kidney and colon, whilst 11β-HSD1 was expressed predominantly in glucocorticoid target tissues, liver, lung, pituitary and cerebellum. In human kidney homogenates, 11-hydroxylated progesterone derivatives, glycyrrhetinic acid, corticosterone and the “end products” cortisone and 11-dehydrocorticosterone were potent inhibitors of the NAD-dependent conversion of cortisol to cortisone. Finally high levels of 11β-HSD2 mRNA and activity were observed in term placentae, which correlated positively with foetal weight. The tissue-specific distribution of the 11β-HSD isoforms is in keeping with their differential roles, 11β-HSD1 regulating glucocorticoid hormone action and 11β-HSD2 mineralocorticoid hormone action. The correlation of 11β-HSD2 activity in the placenta with foetal weight suggests, in addition, a crucial role for this enzyme in foetal development, possibly in mediating ontogeny of the foetal hypothalamo-pituitary-adrenal axis.     

Endogenous inhibitors (GALFs) of 11beta-hydroxysteroid dehydrogenase isoforms 1 and 2: derivatives of adrenally produced corticosterone and cortisol

Two isoforms of 11β-HSD exist; 11β-HSD1 is bi-directional (the reductase usually being predominant) and 11β-HSD2 functions as a dehydrogenase, conferring kidney mineralocorticoid specificity. We have previously described endogenous substances in human urine, “glycyrrhetinic acid-like factors (GALFs)”, which like licorice, inhibit the bi-directional 11β-HSD1 enzyme as well as the dehydrogenase reaction of 11β-HSD2.

Many of the more potent GALFs are derived from two major families of adrenal steroids, corticosterone and cortisol. For example, 35-tetrahydro-corticosterone, its derivative, 35-tetrahydro-11β-hydroxy-progesterone (produced by 21-deoxygenation of corticosterone in intestinal flora); 35-tetrahydro-11β-hydroxy-testosterone (produced by side chain cleavage of cortisol); are potent inhibitors of 11β-HSD1 and 11β-HSD2-dehydrogenase, with IC₅₀'s in range 0.26–3.0 μM, whereas their 11-keto-35-tetrahydro-derivatives inhibit 11β-HSD1 reductase, with IC₅₀'s in range 0.7–0.8 μM (their 35β-derivatives being completely inactive).

Inhibitors of 11β-HSD2 increase local cortisol levels, permitting it to act as a mineralocorticoid in kidney. Inhibitors of 11β-HSD1 dehydrogenase/11β-HSD1 reductase serve to adjust the set point of local deactivation/reactivation of cortisol in vascular and other glucocorticoid target tissues, including adipose, vascular, adrenal tissue, and the eye. These adrenally derived 11-oxygenated C₂₁- and C₁₉-steroidal substances may serve as 11β-HSD1- or 11β-HSD2-GALFs. We conclude that adrenally derived products are likely regulators of local cortisol bioactivity in humans.     

Kinetic analysis of enzymic activities: Prediction of multiple forms of 17β-hydroxysteroid dehydrogenase

An overview of the application of kinetic methods to the delineation of 17β-hydroxysteroid dehydrogenase (17β-HSD) heterogeneity in mammalian tissues is presented. Early studies of 17β-HSD activity in animal liver and kidney subcellular fractions were suggestive of multiple forms of the enzyme. Subsequently, detailed characterization of activity in cytosol and subcellular membrane fractions of human placenta, with particular emphasis on inhibition kinetics, yielded evidence of two kinetically-differing forms of 17β-HSD in that organ. Gene cloning and transfection experiments have confirmed the identity of these two proteins as products of separate genes. 17β-HSD type 1 is a cytosolic enzyme highly specific for C₁₈ steroids such as 17β-estradiol (E₂) and estrone (E₁). 17β-HSD type 2 is a membrane bound enzyme reactive with testosterone (T) and androstenedione (A), as well as E₂ and E₁. Useful parameters for the detection of multiple forms of 17β-HSD appear to be the E₂/T activity ratio, NAD/NADP activity ratios, steroid inhibitor specificity and inhibition patterns over a wide range of putative inhibitor concentrations. Evaluation of these parameters for microsomes from samples of human breast tissue suggests the presence of 17β-HSD type 2. The 17β-HSD enzymology of human testis microsomes appears to differ from placenta. Analysis of human ovary indicates granulosa cells are particularly enriched in the type 1 enzyme with type 2-like activity in stroma/theca. Mouse ovary appears to contain forms of 17β-HSD which differ from 17β-HSD type 1 and type 2 in their kinetic properties.     

The molecular biology of androgenic 17β-hydroxysteroid dehydrogenases

The enzyme 17β-hydroxysteroid dehydrogenase (17β-HSD) catalyzes the 17β-oxidation/reduction of C₁₈- and C₁₉-steroids in a variety of tissues. Three human genes encoding isozymes of 17β-HSD, designated 17β-HSD types 1, 2 and 3 have been cloned. 17β-HSD type 1 (also referred to as estradiol 17β-dehydrogenase) catalyzes the conversion of estrone to estradiol, primarily in the ovary and placenta. The 17β-HSD type 2 is expressed to high levels in the liver, secretory endometrium and placenta. The type 2 isozyme catalyzes the oxidation of androgens and estrogens equally efficiently. Also, the enzyme possesses 20-HSD activity demonstrated by its ability to convert 20-dihydro-progesterone to progesterone. Testicular 17β-HSD type 3 catalyzes the conversion of androstenedione to testosterone, dehydroepiandrosterone to 5-androstenediol and estrone to estradiol. The 17β-HSD3 gene is mutated in male pseudohermaphrodites with the genetic disease 17β-HSD deficiency.     

Regulation of expression of the 3β-hydroxysteroid dehydrogenases of human placenta and fetal adrenal

The appropriate expression of 3β-hydroxysteroid dehydrogenase/Δ^5→4-isomerase (3β-HSD) is vital for mammalian reproduction, fetal growth and life maintenance. Several isoforms of 3β-HSD, the products of separate genes, have been identified in various species including man. Current investigations are targeted toward defining the processes that regulate the levels of specific isoforms in various steroidogenic tissues of man. High levels of expression of 3β-HSD were observed in placental tissues. It has been generally considered that the multinucleated syncytiotrophoblastic cells are the principal sites of 3β-HSD expression and, moreover, that 3β-HSD expression is intimately associated with cyclic AMP-promoted formation of syncytia. Herein we report the presence of 3β-HSD immunoreactive and mRNA species in uninucleate cytotrophoblasts in the chorion laeve, similar to that in syncytia but not cytotrophoblast placenta. In vitro, 3β-HSD levels in chorion laeve cytotrophoblasts were not increased with time nor after treatment with adenylate cyclase activators, whereas villous cytotrophoblasts spontaneously demonstrated progressive, increased 3β-HSD expression. Moreover, 3β-HSD synthesis appeared to precede morphologic syncytial formation. Thus high steroidogenic enzyme expression in placenta is not necessarily closely linked to formation of syncytia. Both Western immunoblot and enzymic activity analyses also indicated that the 3β-HSD expressed in these cytotrophoblastic populations was the 3β-HSD type I gene product (M_r, 45K) and not 3β-HSD type II (M_r, 44K) expressed in fetal testis. In cultures of fetal zone and definitive zone cell of human fetal adrenal, 3β-HSD expression was not detected until ACTH was added. ACTH, likely acting in a cyclic AMP-dependent process, induced 3β-HSD type II activity and mRNA expression. The higher level of 3β-HSD mRNA in definitive zone compared with fetal zone cells was associated with parallel increases in cortisol secretion relative to dehydroepiandrosterone sulfate formation.     

Site-directed Mutagenesis Identifies Amino Acid Residues Associated with the Dehydrogenase and Isomerase Activities of Human Type I (Placental) 3β-Hydroxysteroid Dehydrogenase/Isomerase

3β-Hydroxysteroid dehydrogenase/steroid Δ^{5 → 4}-isomerase (3β-HSD/isomerase) was expressed by baculovirus in Spodoptera fungiperda (Sf9) insect cells from cDNA sequences encoding human wild-type I (placental) and the human type I mutants - H261R, Y253F and Y253,254F. Western blots of SDS-polyacrylamide gels showed that the baculovirus-infected Sf9 cells expressed the immunoreactive wild-type, H261R, Y253F or Y253,254F protein that co-migrated with purified placental 3β-HSD/isomerase (monomeric M_r=42,000 Da). The wild-type, H261R and Y253F enzymes were each purified as a single, homogeneous protein from a suspension of the Sf9 cells (5.01). In kinetic studies with purified enzyme, the H261R mutant enzyme had no 3β-HSD activity, whereas the K_m and V_max values of the isomerase substrate were similar to the values obtained with the wild-type and native enzymes. The V_max (88 nmol/min/mg) for the conversion of 5-androstene-3,17-dione to androstenedione by the Y253F isomerase activity was 7.0-fold less than the mean V_max (620 nmol/min/mg) measured for the isomerase activity of the wild-type and native placental enzymes. In microsomal preparations, isomerase activity was completely abolished in the Y253,254F mutant enzyme, but Y253,254F had 45% of the 3β-HSD activity of the wild-type enzyme. In contrast, the purified Y253F, wild-type and native enzymes had similar V_max values for substrate oxidation by the 3β-HSD activity. The 3β-HSD activities of the Y253F, Y253,254F and wild-type enzymes reduced NAD⁺ with similar kinetic values. Although NADH activated the isomerase activities of the H261R and wild-type enzymes with similar kinetics, the activation of the isomerase activity of H261R by NAD⁺ was dramatically decreased. Based on these kinetic measurements, His²⁶¹ appears to be a critical amino acid residue for the 3β-HSD activity, and Tyr²⁵³ or Tyr²⁵⁴ participates in the isomerase activity of human type I (placental) enzyme.     

Lack of effect of nicotine or ethanol on the activity of 11β-hydroxysteroid dehydrogenase type 2

Low birth weight in combination with a large placenta predicts human hypertension. The pathophysiological link remains unclear, but glucocorticoid excess impairs fetal growth and leads to offspring hypertension. A key controller of fetal glucocorticoid exposure and local tissue availability is 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2). The activity of placental 11β-HSD2 correlates with fetal growth in animals and humans. Ethanol abuse and smoking are known to retard fetal growth which may relate to altered glucocorticoid action or dynamics. This study has examined whether nicotine or ethanol modulate glucocorticoid action in the placenta or fetus by inhibiting 11β-HSD2, using clonal cell cultures, freshly isolated dually perfused intact human placentas and placentas from in vivo treated rats. No significant effect on the activity of 11β-HSD2 by pathophysiologically relevant nicotine or ethanol concentrations was observed. The mechanism of action of nicotine and ethanol relevant to reduced fetal growth requires further study.     

Expression and characterization of isoforms of 3β-hydroxysteroid dehydrogenase/Δ-isomerase in the hamster

The enzyme 3β-hydroxysteroid dehydrogenase/Δ^5→4-isomerase (3β-HSD) is essential for the production of all classes of steroid hormones. Multiple isozymes of this enzyme have been demonstrated in the kidney and liver of both the rat and the mouse, although the function of the enzyme in these tissues is unknown. We have characterized three isozymes of 3β-HSD expressed in various tissues of the hamster. Both western and northern blot analyses demonstrated very high levels of 3β-HSD in the adrenal, kidney and male liver. Conversely, there were extremely low levels of enzyme expression in the female liver. cDNA libraries prepared from RNA isolated from hamster adrenal, kidney and liver were screened with a full-length cDNA encoding human type 1 3β-HSD. Separate cDNAs encoding three isoforms of 3β-HSD were isolated from these libraries. To examine the properties of the isoforms, the cDNAs were ligated into expression vectors for over-expression in 293 human fetal kidney cells. The type 1 isoform, isolated from an adrenal cDNA library, was identified as a high-affinity 3β-hydroxysteroid dehydrogenase. A separate isoform, designated type 2, was isolated from the kidney, and this was also a high-affinity dehydrogenase/isomerase. Two cDNAs were isolated from the liver, one identical in sequence to type 2 of the kidney, and a distinct cDNA encoding an isoform designated type 3. The type 3 3β-HSD possessed no steroid dehydrogenase activity but was found to function as a 3-ketosteroid reductase. Thus male hamster liver expresses a high-affinity 3β-HSD (type 2) and a 3-ketosteroid reductase (type 3), whereas the kidney of both sexes express the type 2 3β-HSD isoform. These differ from the type 1 3β-HSD expressed in the adrenal cortex.     

Progestin regulation of 11beta-hydroxysteroid dehydrogenase expression in T-47D human breast cancer cells

This study examined the enzymatic characteristics and steroid regulation of the glucocorticoid-metabolizing enzyme 11β-hydroxysteroid dehydrogenase (11β-HSD) in the human breast cancer cell line T-47D. In cell homogenates, exogenous NAD significantly increased the conversion of corticosterone to 11-dehydrocorticosterone, while NADP was ineffective. There was no conversion of 11-dehydrocorticosterone to corticosterone either with NADH or NADPH demonstrating the lack of reductase activity. In keeping with these results, RT-PCR analysis indicated a mRNA for 11β-HSD2 in T-47D cells, while 11β-HSD1 mRNA levels were undetectable. In T-47D cells treated for 24 h with medroxyprogesterone acetate (MPA), 11β-HSD catalytic activity was elevated 11-fold, while estrone (E₁), estradiol (E₂) and the synthetic glucocorticoid dexamethasone (DEX) were ineffective. The antiprogestin mifepristone (RU486) acted as a pure antagonist of the progestin-enhanced 11β-HSD activity, but did not exert any agonistic effects of its own. In addition, RT-PCR analysis demonstrated that MPA was a potent inducer of 11β-HSD2 gene expression, increasing the steady-state levels of 11β-HSD2 mRNA. Taken together, these results demonstrate that 11β-HSD2 is the 11β-HSD isoform expressed by T-47D cells under steady-state conditions and suggest the existence of a previously undocumented mechanism of action of progestins in breast cancer cells.     

The tissue distribution of porcine 17β-estradiol dehydrogenase and its induction by progesterone

Porcine 17β-estradiol dehydrogenase (EDH) was recently purified and cloned. It catalyzes the NAD⁺-dependent oxidation of estradiol to estrone 360-fold more efficiently than the back reaction with NADPH. The 32 kDa EDH is cut from an 80 kDa primary translation product with a multidomain structure unknown for other hydroxysteroid dehydrogenases. The highest EDH activities and strongest immunoreactions are found in liver (hepatocytes) and kidney (proximal tubuli) followed by uterus (luminal and glandular epithelium), lung (bronchial epithelium). Progesterone treatment of ovariectomized gilts stimulates oxidative EDH activity in uterus, anterior pituitary, skeletal muscle (diaphragm) and kidney. Constitutive levels of EDH activity were seen in the adrenals, the lung and the liver.     

The murine 3β-hydroxysteroid dehydrogenase multigene family: Structure, function and tissue-specific expression

The classical form of the enzyme 5-ene-3β-hydroxysteroid dehydrogenase/isomerase (3βHSD), expressed in adrenal glands and gonads, catalyzes the conversion of 5-ene-3β-hydroxysteroids to 4-ene-3-ketosteroids, an essential step in the biosynthesis of all active steroid hormones. To date, four distinct mouse 3βHSD cDNAs have been isolated and characterized. These cDNAs are expressed in a tissue-specific manner and encode proteins of two functional classes. Mouse 3βHSD I and III function as 3β-hydroxysteroid dehydrogenases and 5-en→4-en isomerases using NAD⁺ as a cofactor. The enzymatic function of 3βHSD II has not been completely characterized. Mouse 3βHSD IV functions only as a 3-ketosteroid reductase using NADPH as a cofactor. The predicted amino acid sequences of the four isoforms exhibit a high degree of identity. Forms II and III are 85 and 83% homologous to form I. Form IV is most distant from the other three with 77 and 73% sequence identity to I and III, respectively. 3βHSD I is expressed in the gonads and adrenal glands of the adult mouse. 3βHSD II and III are expressed in the kidney and liver with the expression of form II greater in kidney and form III greater in liver. Form IV is expressed exclusively in the kidney. Although the amino acid composition of forms I, III and IV predicts proteins of the same molecular weight, the proteins have different mobilities on SDS-polyacrylamide gel electrophoresis. This characteristic allows for differential identification of the expressed proteins. The four structural genes encoding the different isoforms are closely linked within a segment of mouse chromosome 3 that is conserved on human chromosome 1.     

Inhibition of human and rat 11beta-hydroxysteroid dehydrogenase type 1 by 18beta-glycyrrhetinic acid derivatives

11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) plays an important role in regulating the cortisol availability to bind to corticosteroid receptors within specific tissue. Recent advances in understanding the molecular mechanisms of metabolic syndrome indicate that elevation of cortisol levels within specific tissues through the action of 11β-HSD1 could contribute to the pathogenesis of this disease. Therefore, selective inhibitors of 11β-HSD1 have been investigated as potential treatments for metabolic diseases, such as diabetes mellitus type 2 or obesity. Here we report the discovery and synthesis of some 18β-glycyrrhetinic acid (18β-GA) derivatives (2–5) and their inhibitory activities against rat hepatic11β-HSD1 and rat renal 11β-HSD2. Once the selectivity over the rat type 2 enzyme was established, these compounds’ ability to inhibit human 11β-HSD1 was also evaluated using both radioimmunoassay (RIA) and homogeneous time resolved fluorescence (HTRF) methods. The 11-modified 18β-GA derivatives 2 and 3 with apparent selectivity for rat 11β-HSD1 showed a high percentage inhibition for human microsomal 11β-HSD1 at 10 μM and exhibited IC₅₀ values of 400 and 1100 nM, respectively. The side chain modified 18β-GA derivatives 4 and 5, although showing selectivity for rat 11β-HSD1 inhibited human microsomal 11β-HSD1 with IC₅₀ values in the low micromolar range.     

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.     

Novel non-steroidal inhibitors of human 11beta-hydroxysteroid dehydrogenase type 1

11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) regulates glucocorticoid action at the pre-receptor stage by converting cortisone to cortisol. 11β-HSD1 is selectively expressed in many tissues including the liver and adipose tissue where metabolic events are important. Metabolic syndrome relates to a number of metabolic abnormalities and currently has a prevalence of >20% in adult Americans. 11β-HSD1 inhibitors are being investigated by many major pharmaceutical companies for type 2 diabetes and other abnormalities associated with metabolic syndrome. In this area of intense interest a number of structural types of 11β-HSD1 inhibitor have been identified. It is important to have an array of structural types as the physicochemical properties of the compounds will determine tissue distribution, HPA effects, and ultimately clinical utility. Here we report the discovery and synthesis of three structurally different series of novel 11β-HSD1 inhibitors that inhibit human 11β-HSD1 in the low micromolar range. Docking studies with 1–3 into the crystal structure of human 11β-HSD1 reveal how the molecules may interact with the enzyme and cofactor and give further scope for structure based drug design in the optimisation of these series.