Heterogeneity of 11β-hydroxysteroid dehydrogenase in rat tissues

Using specific antisera to purified rat liver 11β-hydroxysteroid dehydrogenase (11-HSD), we showed that the antigen is widely distributed in rat organs. Enzyme activity and immunoreactivity generally corresponded. Highest by both criteria were liver, testis, kidney and lung. In some tissues (epididymis, pancreas and duodenum) activity was found, but antigen corresponding to 11-HSD at a M_w of 34 kDa was absent. It is suggested that these tissues have alternate enzyme forms. The 11-HSD of brain and liver were compared. Brain enzyme may control selective binding of aldosterone to Type I receptors in the hippocampus and other regions. Rat brain 11-HSD resembled that of liver or kidney in most characteristics. It differed in (a) its steroid specificity: cortisol was a good substrate for liver 11-HSD, and a poor substrate for brain enzyme; (b) stability of 11-oxoreductase (11-OR) component. Brain 11-OR was not readily inactivated; 11-OR from other tissues lost activity rapidly and spontaneously. The variations in properties of 11-HSD in specific tissues may reflect aspects of its various specific functions.     

Placental 11β-hydroxysteroid dehydrogenase and the programming of hypertension

Excessive foetal exposure to glucocorticoids retards growth and “programmes” adult hypertension in rats. Placental 11β-hydroxysteroid dehydrogenase (11β-HSD), which catalyses the conversion of corticosterone and cortisol to inert 11 keto-products, normally protects the foetus from excess maternal glucocorticoids. In both rats and humans there is considerable natural variation in placental 11β-HSD, and enzyme activity correlates with birth weight. Moreover, inhibition of placental 11β-HSD in the rat reduces birth weight and produces hypertensive adult offspring, many months after prenatal treatment with enzyme inhibitors; these effects are dependent upon maternal adrenal products. These data suggest that placental 11β-HSD, by regulating foetal exposure to maternal glucocorticoids, crucially determines foeto-placental growth and the programming of hypertension. Maternal protein restriction during pregnancy also produces hypertensive offspring and selectively attenuates placental 11β-HSD activity. Thus, deficiency of the placental barrier to maternal glucocorticoids may represent a common pathway between the maternal environment and foeto-placental programming of later disease. These data may, at least in part, explain the human epidemiological observations linking early life events to the risk of subsequent hypertension. The recent characterization, purification and cDNA cloning of a distinct human placental 11β-HSD (type 2) will aid the further study of these intriguing findings.     

Molecular basis of human 3β-hydroxysteroid dehydrogenase deficiency

The enzyme 3β-hydroxysteroid dehydrogenase (3β-HSD) catalyses an essential step in the biosynthesis of all classes of steroid hormones. Classical 3β-HSD deficiency is responsible for CAHII, a severe form of congenital adrenal hyperplasia (CAH) that impairs steroidogenesis in both the adrenals and gonads. Newborns affected by 3β-HSD deficiency exhibit signs and symptoms of adrenal insufficiency of varying degrees associated with pseudohermaphroditism in males, whereas females exhibit normal sexual differentiation or mild virilization. Elevated ratios of 5-ene-to 4-ene-steroids appear as the best biological parameter for the diagnosis of 3β-HSD deficiency. The nonclassical form has been suggested to be related to an allelic variant of the classical form of 3β-HSD as described for steroid 21-hydroxylase deficiency. To elucidate the molecular basis of the classical form of 3β-HSD deficiency, we have analysed the structure of the highly homologous type I and II 3β-HSD genes in 12 male pseudohermaphrodite 3β-HSD deficient patients as well as in four female patients. 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 finding of a normal type I 3β-HSD gene provides the explanation for the intact peripheral intracrine steroidogenesis in these patients and increased androgen manifestations at puberty. The influence of the detected mutations on enzymatic activity was assessed by in vitro expression analysis of mutant enzymes generated by site-directed mutagenesis in COS-1 cells. The mutant type II 3β-HSD enzymes carrying mutations detected in patients affected by the salt-losing form exhibit no detectable activity in intact tranfected cells, whereas those with mutations found in nonsalt-loser index cases have some residual activity ranging from 1–10% compared to the wild-type enzyme. Although in general, our findings provide a molecular explanation for the enzymatic heterogeneity ranging from the severe salt-losing form to the clinically inapparent salt-wasting form of the disease, we have observed that the mutant L108W or P186L enzymes found in a compound heterozygote male presenting the salt-wasting form of the disease, has some residual activity (1%) similar to that observed for the mutant N100S enzyme detected in an homozygous male patient suffering from a nonsalt-losing form of this disorder. Unlike the classical 3β-HSD deficiency, our study in women presenting nonclassical 3β-HSD deficiency strongly suggests that this disorder is not due to a mutant type II 3β-HSD.     

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.     

Expression of human 17β-hydroxysteroid dehydrogenase in mammalian cells

The insert of 1278 bp containing the entire coding region of cDNA encoding human 17β-hydroxysteroid dehydrogenase (17β-HSD) was inserted into a pHS1 vector and expressed in HeLA human cervical carcinoma cells and COS-1 monkey kidney tumor cells. Western blot analysis indicated that the expressed protein migrates at the same position as the purified enzyme and is recognized by the antibody raised against purified human placental 17β-HSD. The expressed enzyme efficiently catalyzes the interconversion of estrone and estradiol while dehydroepiandrosterone and 5-androstene-3β,17β-diol are interconverted at a lower rate. The present data suggest the existence of two 17β-HSDs.     

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.     

Molecular cloning of multiple cDNAs encoding human enzymes structurally related to 3α-hydroxysteroid dehydrogenase

Rat liver 3α-hydroxysteroid dehydrogenase cDNA was previously cloned by us. In this study, we used the rat cDNA as the probe to screen a human liver lambda gt11 cDNA library. A total of four different cDNAs were identified and sequenced. The sequence of one of the cDNAs is identical to that of the human chlordecone reductase cDNA except that our clone contains a much longer 5′-coding sequence than previously reported. The other three cDNAs display high degrees of sequence homology to those of both rat 3α-hydroxysteroid dehydrogenase and human chlordecone reductase. Because 3α-hydroxysteroid dehydrogenase and human chlordecone reductase belong to the aldo-keto reductase superfamily, we named these human clones HAKRa to HAKRd. Northern blot analysis showed that the liver expresses the highest levels of all four clones. Expression of all four clones was also detected in the brain, kidney, lung, and testis, whereas the placenta expressed only the messenger RNA for HAKRb. Genomic blot analysis using HAKRb as the probe detected multiple DNA fragments hybridized to the probe and a high degree of restriction fragment length polymorphism, suggesting the complexity of this supergene family.     

Distribution of 17β-hydroxysteroid dehydrogenase gene expression and activity in rat and human tissues

The interconversion of estrone (E₁) and 17β-estradiol (E₂), androstenedione (4-ene-dione) and testosterone (T), as well as dehydroepiandrosterone and androst-5-ene-3β,17β-diol is catalyzed by 17β-hydroxysteroid dehydrogenase (17β-HSD). The enzyme 17β-HSD thus plays an essential role in the formation of all active androgens and estrogens in gonadal as well as extragonadal tissues. The present study investigates the tissue distribution of 17β-HSD activity in the male and female rat as well as in some human tissues and the distribution of 17β-HSD mRNA in some human tissues. Enzymatic activity was measured using ¹⁴C-labeled E₁, E₂, 4-ene-dione and T as substrates. Such enzymatic activity was demonstrated in all 17 rat tissues examined for both androgenic and estrogenic substrates. While the liver had the highestlevel of 17β-HSD activity, low but significant levels of E₂ as well as T formation were found in rat brain, heart, pancreas and thymus. The oxidative pathway (E₂→E₁, T→4-ene-dione) was favored over the reverse reaction in almost all rat tissues while in the human, almost equal rates were found in most of the 15 tissues examined. The widespread distribution of 17β-HSD in rat and human tissues clearly indicates the importance of this enzyme in peripheral sex steroid formation or intracrinology.     

Selective inhibition of 11β-hydroxysteroid dehydrogenase 1 by 18α-glycyrrhetinic acid but not 18β-glycyrrhetinic acid

Elevated cortisol concentrations have been associated with metabolic diseases such as diabetes type 2 and obesity. 11β-hydroxysteroid dehydrogenase (11β-HSD) type 1, catalyzing the conversion of inactive 11-ketoglucocorticoids into their active 11β-hydroxy forms, plays an important role in the regulation of cortisol levels within specific tissues. The selective inhibition of 11β-HSD1 is currently considered as promising therapeutic strategy for the treatment of metabolic diseases. In recent years, natural compound-derived drug design has gained considerable interest. 18β-glycyrrhetinic acid (GA), a metabolite of the natural product glycyrrhizin, is not selective and inhibits both 11β-HSD1 and 11β-HSD2. Here, we compare the biological activity of 18β-GA and its diastereomer 18α-GA against the two enzymes in lysates of transfected HEK-293 cells and show that 18α-GA selectively inhibits 11β-HSD1 but not 11β-HSD2. This is in contrast to 18β-GA, which preferentially inhibits 11β-HSD2. Using a pharmacophore model based on the crystal structure of the GA-derivative carbenoxolone in complex with human 11β-HSD1, we provide an explanation for the differences in the activities of 18α-GA and 18β-GA. This model will be used to design novel selective derivatives of GA.     

Differential estrogenic 17β-hydroxysteroid dehydrogenase activity and type 12 17β-hydroxysteroid dehydrogenase expression levels in preadipocytes and differentiated adipocytes

Estradiol (E2) is produced locally in adipose tissue and could play an important role in fat distribution and accumulation, especially in women. It is well recognized that aromatase is expressed in adipose tissue; however the identity of its estrogenic 17β-hydroxysteroid dehydrogenase (17β-HSD) partner is not identified. To gain a better knowledge about the enzyme responsible for the conversion of estrone into estradiol, we determined the activity and expression levels of known estrogenic 17β-HSDs, namely types 1, 7 and 12 17β-HSD in preadipocytes before and after differentiation into mature adipocytes using an adipogenic media. Estrogenic 17β-HSD activity was assessed using [¹⁴C]-labelled estrone, while mRNA expression levels of types 1, 7 and 12 17β-HSD were quantified using real-time PCR and protein expression levels of type 12 17β-HSD was determined using immunoblot analysis. The data indicate that there is a low conversion of E1 into E2 in preadipocytes; however this activity is increased 5-fold (p < 0.0001) in differentiated adipocytes. The increased estrogenic 17β-HSD activity is consistent with the increase in protein expression levels of 17β-HSD12.     

Molecular characterization of mouse 17β-hydroxysteroid dehydrogenase IV

17β-hydroxysteroid dehydrogenases (17β-HSD) catalyze the conversion of estrogens and androgens at the C17 position. The 17β-HSD type I, II, III and IV share less than 25% amino acid similarity. The human and porcine 17β-HSD IV reveal a three-domain structure unknown among other dehydrogenases. The N-terminal domains resemble the short chain alcohol dehydrogenase family while the central parts are related to the C-terminal parts of enzymes involved in peroxisomal β-oxidation of fatty acids and the C-terminal domains are similar to sterol carrier protein 2. We describe the cloning of the mouse 17β-HSD IV cDNA and the expression of its mRNA. A probe derived from the human 17β-HSD IV was used to isolate a 2.5 kb mouse cDNA encoding for a protein of 735 amino acids showing 85 and 81% similarity with human and porcine 17β-HSD IV, respectively. The calculated molecular mass of the mouse enzyme amounts to 79,524 Da. The mRNA for 17β-HSD IV is a single species of about 3 kb, present in a multitude of tissues and expressed at high levels in liver and kidney, and at low levels in brain and spleen. The cloning and molecular characterization of murine, human and porcine 17β-HSD IV adds to the complexity of steroid synthesis and metabolism. The multitude of enzymes acting at C17 might be necessary for a precise control of hormone levels.     

The (+)- and (−)-gossypols potently inhibit human and rat 11β-hydroxysteroid dehydrogenase type 2

Gossypol has been proven to be a very effective male contraceptive. However, clinical trials showed that the major side effect of gossypol was hypokalemia. Gossypol occurs naturally as enantiomeric mixtures of (+)-gossypol and (−)-gossypol. The (−)-gossypol is found to be the active component of antifertility. 11β-Hydroxysteroid dehydrogenase 2 (11βHSD2) has been demonstrated to be a mineralocorticoid receptor (MR) protector by inactivating active glucocorticoids including corticosterone (CORT) in rats, and therefore mutation or suppression of 11βHSD2 causes hypokalemia and hypertension. In the present study, the potency of gossypol enantiomers was tested for the inhibition of 11βHSD1 and 2 in rat and human. Both (+) and (−)-gossypols showed a potent inhibition of 11βHSD2 with the half maximal inhibitory concentration (IC₅₀) of 0.61 and 1.33 μM for (+) and (−)-gossypols, respectively in rats and 1.05 and 1.90 μM for (+) and (−)-gossypols, respectively in human. The potency of gossypol to inhibit 11βHSD1 was far less; the IC₅₀ was ≥100 μM for racemic gossypol. The gossypol-induced hypokalemia is likely associated with its potent inhibition of kidney 11βHSD2.     

Structures important in mammalian 11β- and 17β-hydroxysteroid dehydrogenases

We have used the X-ray crystallographic structures of rat and human dihydropteridine reductase and Streptomyces hydrogenans 20β-hydroxysteroid dehydrogenase to model parts of the 3-dimensional structure of human 11β- and 17β-hydroxysteroid dehydrogenases. We use this information along with previous results from studies of Drosophila alcohol dehydrogenase mutants to analyze the structures in binding sites for NAD(H) and NADP(H) in 11β-hydroxysteroid dehydrogenase-types 1 and 2. We also examine the structure of an -helix at catalytic site of 17β-hydroxysteroid dehydrogenase-types 1, 2, 3, and 4. This -helix contains a highly conserved tyrosine and lysine. Adjacent to the carboxyl side of this lysine is a site proposed to be important in subunit association. We find that 11β- and 17β-hydroxysteroid dehydrogenases-type 1 have the same residues at the “anchor site” and conserve other stabilizing features, despite only 20% sequence identity between their entire sequences. Similar conservation of stabilizing structures is found in the 11β- and 17β-hydroxysteroid dehydrogenases-type 2. We suggest that interactions of the dimerization surface of -helix F with proteins or membranes may be important in regulating activity of hydroxysteroid dehydrogenases.     

Unusual evolution of 11β- and 17β-hydroxysteroid and retinol dehydrogenases

11β-hydroxysteroid dehydrogenases regulate glucocorticoid concentrations and 17β-hydroxysteroid dehydrogenases regulate estrogen and androgen concentrations in mammals. Phylogenetic analysis of the sequences from two 11β-hydroxysteroid dehydrogenases and four mammalian 17β-hydroxysteroid dehydrogenases indicates unusual evolution in these enzymes. Type 1 11β- and 17β-hydroxysteroid dehydrogenases are on the same branch; Type 2 enzymes cluster on another branch with β-hydroxybutyrate dehydrogenase, 11-cis retinol dehydrogenase and retinol dehydrogenase; Type 3 17β-hydroxysteroid dehydrogenase is on a third branch; while the pig dehydrogenase clusters with a yeast multifunctional enzyme on a fourth branch. Pig 17β-hydroxysteroid dehydrogenase appears to have evolved independently from the other three 17β-hydroxysteroid dehydrogenase dehydrogenases; in which case, the evolution of 17β-hydroxysteroid dehydrogenase activity is an example of functional convergence. The phylogeny also suggests that independent evolution of specificity toward C11 substituents on glucocorticoids and C17 substituents on androgens and estrogens has occurred in Types 1 and 2 11β- and 17β-hydroxysteroid dehydrogenases.     

3β-hydroxysteroid dehydrogenase activity in tissues of the human fetus determined with 5α-androstane-3β,17β-diol and dehydroepiandrosterone as substrates

3β-Hydroxysteroid dehydrogenase (3β-HSD)/Δ^5→4-isomerase activity in steroidogenic tissues is required for the synthesis of biologically active steroids. Previously, by use of dehydroepiandrosterone (3β-hydroxy-5-androsten-17-one, DHEA) as substrate, it was established that in addition to steroidogenic tissues 3β-HSD/Δ^5→4-isomerase activity also is expressed in extraglandular tissues of the human fetus. In the present study, we attempted to determine whether the C-5,C-6-double bond of DHEA serves to influence 3β-HSD activity. For this purpose, we compared the efficiencies of a 3β-hydroxy-5-ene steroid (DHEA) and a 3β-hydroxy-5α-reduced steroid (5α-androstane-3β,17β-diol, 5α-A-diol) as substrates for the enzyme. The apparent Michaelis constant (K_m) for 5α-A-diol in midtrimester placenta, fetal liver, and fetal skin tissues was at least one order of magnitude higher than that for DHEA, viz the apparent K_m of placental 3β-HSD for 5α-A-diol was in the range of 18 to 40 μmol/l (n = 3) vs 0.45 to 4 μmol/l for DHEA (n = 3); for the liver enzyme, 17 μmol/l for 5α-A-diol and 0.60 μmol/l for DHEA, and for the skin enzyme 14 and 0.18 μmol/l, respectively. Moreover, in 13 human fetal tissues evaluated the maximal velocities obtained with 5α-A-diol as substrate were higher than those obtained with DHEA. A similar finding in regard to K_ms and rates of product formation was obtained by use of purified placental 3β-HSD with DHEA, pregnenolone, and 3β-hydroxy-5α-androstan-17-one (epiandrosterone) as substrates: the K_m of 3β-HSD for DHEA was 2.8 μmol/l, for pregnenolone 1.9 μmol/l, and for epiandrosterone 25 μmol/l. The specific activity of the purified enzyme with pregnenolone as substrate was 27 nmol/mg protein·min and, with epiandrosterone, 127 nmol/mg protein·min. With placental homogenate as the source of 3β-HSD, DHEA at a constant level of 5 μmol/l behaved as a competitive inhibitor when the radiolabeled substrate, [³H]5α-A-diol, was present in concentrations of 20 to 60 μmol/l, but a lower substrate concentrations the inhibition was of the mixed type; similar results were obtained with [³H]DHEA as the substrate at variable concentrations in the presence of a fixed concentration of 5α-A-diol (40 μmol/l). These findings are indicative that both steroids bind to a common site on the enzyme, however, the binding affinity for these steroids appear to differ markedly as suggested by the respective K_ms. Studies of inactivation of purified placental 3β-HSD/Δ^5→4-isomerase by an irreversible inhibitor, viz 5,10-secoestr-4-yne-3,10,17-trione, were suggestive that the placental protein adopts different conformations depending on whether the steroidal substrate has a 5α-configuration, e.g. epiandrosterone, or a C-5,C-6-double bond e.g. DHEA or pregnenolone. The lower rates of product formation obtained with placenta and fetal tissues by use of 3β-hydroxy-5-ene steroids as substrates when compared with those obtained with 3β-hydroxy-5α-reduced steroids may be explained by a combination of factors, including: (i) inhibition of 3β-HSD activity by end products of metabolism of 3β-hydroxy-5-ene steroids, e.g. 4-androstene-3,17-dione formed with DHEA as substrate; (ii) higher binding affinity of the enzyme for 3β-hydroxy-5-ene steroids—and possibly for their 3-oxo-5-ene metabolites; (iii) lack of a requirement for the isomerization step with 5α-reduced steroids as substrates, and (iv) the possible presence in fetal tissues of an enzyme with 3β-HSD activity only (i.e. no Δ^5→4-isomerase).     

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.     

Luteolytic action of RU486: Modulation of luteal 3β-hydroxysteroid dehydrogenase and 20α-hydroxysteroid dehydrogenase activities in late pregnant rats

The effect of the synthetic antiprogestin RU486 on luteal function in late pregnant rats was studied by evaluating the activities of the enzymes 3β-hydroxysteroid dehydrogenase (3β-HSD) and 20α-hydroxysteroid dehydrogenase (20α-HSD). RU486 (2 mg/kg) administered to rats on day 18 of pregnancy at 10.00 h induced preterm delivery 26.4 ± 0.35 h (n = 8) after treatment. Luteal 3β-HSD activity increased 24 and 34 h after RU486 injection, but a significant and progressive decrease started at 48 h with the maximal reduction 72 h after RU486 treatment, when compared with controls. Serum progesterone concentration decreased at the time of 3β-HSD activity reduction. Interestingly, 20α-HSD activity started to increase 58 h after RU486 injection. The administration of the cyclooxygenase inhibitor, diclofenac (1.3 mg/kg), on days 17–19 of pregnancy to RU486-treated rats, delayed abortion and the duration of delivery, and prevented the decrease in 3β-HSD and the increase in 20α-HSD activities observed 58 h after antiprogesterone treatment. RU486 administered intrabursally (1 μg per ovary) on day 20 (14.00–15.00 h) increased 3β-HSD and decreased 20α-HSD luteal activities at 18.00 h on day 21 of pregnancy, without modifying serum progesterone concentration, when compared with normal pregnant rats. In conclusion, the luteolytic process after preterm delivery induced by RU486 administration in late pregnant rats is characterized by a decrease in luteal 3β-HSD activity and circulating progesterone, which may trigger the increase in luteal 20α-HSD activity. Prostaglandins seems to be involved in the increase of 20α-HSD activity and therefore, in the demise of corpora lutea.     

The human 11β-hydroxysteroid dehydrogenase type II enzyme: Comparisons with other species and localization to the distal nephron

Effective glucocorticoid inactivation is currently thought to be an indispensable feature of mineralocorticoid target cells. The enzyme 11β-hydroxysteroid dehydrogenase (11β-HSD) inactivates glucocorticoids and prevents them from binding to the non-selective mineralocorticoid receptor. In the kidney it is the NAD dependent high affinity isoform (11β-HSD2) which is thought to endow specificity on the receptor. The recet cloning of the human, sheep and rabbit 11β-HSD2 enzymes permits a comparison of the enzyme from the three species. Human and rabbit enzymes are 87% identical and of similar length, while the human and sheep enzymes have only 75% identity. The last 12 residues in all three species were found to be highly divergent, but most of the ovine dishomology can be accounted for by the deletion of a single nucleotide toward the C-terminus of the protein resulting in a shift in reading frame generating a protein 27 residues longer than the human isoform. Numerous other deletions were also observed in this region of the sheep cDNA sequence. Furthermore, the rabbit cDNA also displayed a large degree of dishomology with the human sequence a short distance downstream from the termination codon. Conserved overlapping cytoplasmic translocation signals were observed in all three species, suggesting a topology whereby the enzyme is anchored into the endoplasmic reticulum by multiple hydrophobic regions in the N-terminus and the bulk of the 11β-HSD2 peptide is sited in the cytoplasm. A polyclonal antibody generated against the C-terminus of human 11β-HSD2 was used to localize the enzyme within the kidney. A high level of immunoreactivity was observed in distal tubules and collecting ducts, localizing the enzyme to the same part of the nephron as the mineralocorticoid receptor. Moderate levels of staining were also seen in vascular smooth muscle cells. These results support the notion that 11β-HSD2 is an autocrine protector of the mineralocorticoid receptor and that it plays an important role in cardiovascular homeostatic mechanisms.     

Dehydroepiandrosterone 7α-hydroxylation in human tissues: Possible interference with type 1 11β-hydroxysteroid dehydrogenase-mediated processes

Dehydroepiandrosterone (DHEA) is 7α-hydroxylated by the cytochome P450 7B1 (CYP7B1) in the human brain and liver. This produces 7α-hydroxy-DHEA that is a substrate for 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) which exists in the same tissues and carries out the inter-conversion of 7α- and 7β-hydroxy-DHEA through a 7-oxo-intermediary. Since the role of 11β-HSD1 is to transform the inactive cortisone into active cortisol, its competitive inhibition by 7α-hydroxy-DHEA may support the paradigm of native anti-glucocorticoid arising from DHEA. Therefore, our objective was to use human tissues to assess the presences of both CYP7B1 and 11β-HSD1. Human skin was selected then and used to test its ability to produce 7α-hydroxy-DHEA, and to test the interference of 7α- and 7β-hydroxy-DHEA and 7-oxo-DHEA with the 11β-HSD1-mediated oxidoreduction of cortisol and cortisone. Immuno-histochemical studies showed the presence of both CYP7B1 and 11β-HSD1 in the liver, skin and tonsils. DHEA was readily 7α-hydroxylated when incubated using skin slices. A S9 fraction of dermal homogenates containing the 11β-HSD1 carried out the oxidoreduction of cortisol and cortisone. Inhibition of the cortisol oxidation by 7α-hydroxy-DHEA and 7β-hydroxy-DHEA was competitive with a K_i at 1.85 ± 0.495 and 0.255 ± 0.005 μM, respectively. Inhibition of cortisone reduction by 7-oxo-DHEA was of a mixed type with a K_i at 1.13 ± 0.15 μM. These findings may support the previously proposed native anti-glucocorticoid paradigm and suggest that the 7α-hydroxy-DHEA production is a key for the fine tuning of glucocorticoid levels in tissues.