Characterization of rat 17β-hydroxysteroid dehydrogenase type 1 gene and mRNA transcripts

In the present study, the gene encoding rat 17β-hydroxysteroid dehydrogenase type 1 (rHSD17B1 gene) was cloned and characterized. Like the analogous human gene (hHSD17B1), rHSD17B1 contains six exons and five introns spanning approximately 2.2 kb. The identity between the exons and introns of the two genes ranges from 58% to 82% and 42% to 57%, respectively. In contrast to hHSD17B1, rHSD17B1 is not duplicated. The cap site for rHSD17B1 was localized to position −41 upstream of the ATG translation initiation codon. Sequence comparison of the first 200 bp upstream of the cap site showed 72% identity between the human and rat HSD17B1 genes, including a conserved GC-rich area. Further upstream, no significant identity between the two genes was observed and several, cis-acting elements known to modulate the expression of hHSD17B1 are not conserved in the rat gene. Rat HSD17B1 unlike hHSD17B1 with two cap sites, possesses two polyadenylation signals, thus resulting in two mRNAs.     

Inhibition of human and rat 3β-hydroxysteroid dehydrogenase and 17β-hydroxysteroid dehydrogenase 3 activities by perfluoroalkylated substances

Perfluoroalkylated substances (PFASs) including perfluorooctane acid (PFOA) and perfluorooctane sulfonate (PFOS) have been classified as persistent organic pollutants and are known to cause reduced testosterone production in human males. The objective of the present study was to compare the potencies of five different PFASs including PFOA, PFOS, potassium perfluorooctane sulfonate (PFOSK), potassium perfluorohexane sulfonate (PFHxSK) and potassium perfluorobutane sulfonate (PFBSK) in the inhibition of 3β-hydroxysteroid dehydrogenase (3β-HSD) and 17β-hydroxysteroid dehydrogenase 3 (17β-HSD3) activities in the human and rat testes. Human and rat microsomal enzymes were exposed to various PFASs. PFOS and PFOSK inhibited rat 3β-HSD activity with IC₅₀ of 1.35 ± 0.05 and 1.77 ± 0.04 μM, respectively, whereas PFHxSK and PFBSK had no effect at concentrations up to 250 μM. All chemicals tested weakly inhibited human 3β-HSD activity with IC₅₀s over 250 μM. On the other hand, PFOS, PFOSK and PFOA inhibited human 17β-HSD3 activity with IC₅₀s of 6.02 ± 1.02, 4.39 ± 0.46 and 127.60 ± 28.52 μM, respectively. The potencies for inhibition of 17β-HSD3 activity were determined to be PFOSK > PFOS > PFOA > PFHxSK = PFBSK for human 17β-HSD3 activity. There appears to be a species-dependent sensitivity to PFAS-mediated inhibition of enzyme activity because the IC₅₀s of PFOS(K) for inhibition of rat 17β-HSD3 activity was greater than 250 μM. In conclusion, the present study shows that PFOS and PFOSK are potent inhibitors of rat 3β-HSD and human 17β-HSD3 activity, and implies that inhibition of steroidogenic enzyme activity may be a contributing factor to the effects that PFASs exert on androgen secretion in the testis.     

Effects of phthalates on 3β-hydroxysteroid dehydrogenase and 17β-hydroxysteroid dehydrogenase 3 activities in human and rat testes

The 3β-hydroxysteroid dehydrogenase (3β-HSD) and 17β-hydroxysteroid dehydrogenase 3 (17β-HSD3) are involved in the reactions that culminate in androgen biosynthesis in Leydig cells. Human and rat testis microsomes were used to investigate the inhibitory potencies on 3β-HSD and 17β-HSD3 activities of 14 different phthalates with various carbon numbers in the ethanol moiety. The results demonstrated that the half-maximal inhibitory concentrations (IC(50)s) of dipropyl (DPrP), dibutyl (DBP), dipentyl (DPP), bis(2-butoxyethyl) (BBOP) and dicyclohexyl (DCHP) phthalate were 123.0, 24.1, 25.5, 50.3 and 25.5μM for human 3β-HSD activity, and 62.7, 30.3, 33.8, 82.6 and 24.7μM for rat 3β-HSD activity, respectively. However, only BBOP and DCHP potently inhibited human (IC(50)s, 23.3 and 8.2μM) and rat (IC(50)s, 30.24 and 9.1μM) 17β-HSD3 activity. Phthalates with 1-2 or 7-8 carbon atoms in ethanol moieties had no effects on both enzyme activities even at concentrations up to 1mM. The mode of action of DCHP on 3β-HSD activity was competitive with the substrate pregnenolone but noncompetitive with the cofactor NAD+. The mode of action of DCHP on 17β-HSD3 activity was competitive with the substrate androstenedione but noncompetitive with the cofactor NADPH. In summary, our results showed that there are clear structure-activity responses for phthalates in the inhibition of both 3β-HSD and 17β-HSD3 activities. The length of carbon chains in the ethanol moieties of phthalates may determine the potency to inhibit these two enzymes.     

Substrate characteristics of progesterone-albumin conjugates with 20β-hydroxysteroid dehydrogenase

Progesterone covalently conjugated to bovine serum albumin through the 21-position is a substrate for 20β-hydroxysteroid dehydrogenase (E.C.1.1.1.53) from S. hydrogenans. Varying the progesterone to albumin molar ratio in a range of 1:1 to 19:1 results in variations in the apparent Km and Vmax values. A maximum in substrate activity is obtained at a progesterone to albumin ratio of 6:1, while at either extreme in the range of these ratios the activity is a minimum. Progesterone-albumin conjugates attached at the steroid 2α-, 6β, or 11α-position in varying ratios did not produce measurable substrate activity. The results show that a steroid need not be free in solution to serve as a substrate for the steroid oxido-reductase.     

Molecular cloning and expression of 17β-hydroxysteroid dehydrogenase type 2 gene in Hu sheep

17β-Hydroxysteroid dehydrogenase type 2 (17β-HSD2) catalyzes the NADP+-dependent oxidation of the most potent estrogen 17β-estradiol into the weak estrogen estrone, and the conversion of testosterone to androstenedione. It has been reported that 17β-HSD2 was expressed in many tissues in human, rats, however, the full-length sequence of 17β-HSD2 gene and its expression in ewe were still unknown. In this study, we cloned the full-length cDNA sequence and investigated mRNA differential expression in 28 tissues of 12 adult Hu-Sheep which were fed with high- and low- dietary intake. The 1,317 bp full-length cDNA sequence was first cloned. The coding region was 1,167 bp in length, and the monomer was estimated to contain 389 amino acid residues. It shares high AA sequence identity with that of bos Taurus (96.13 %), sus scrofa (77.06 %), canis lupus familiaris (70.44 %), Callithrix jacchus (65.72 %), Nomascus leucogenys (65.46 %), pan troglodytes (65.21 %), human (64.69 %), mus musculus (58.35 %), and a comparatively lower identity to danio rerio (37.85 %). 17β-HSD2 gene was high expressed in gastrointestinal (GI) tract, liver, but weakly expressed in other tissues. No detected expression was examined in lung. 17β-HSD2 gene expression was significantly difference in rumen, omasum, duodenum, cecum, hypophysis after high- and low- dietary intake. Results from the present study suggested that 17β-HSD2 plays a crucial role in almost all tissues protecting against excessive levels of active steroid hormone, and GI tract maybe an important steroid hormone metabolizing organ in Hu-Sheep. This present study is the first to provide the primary foundation for further insight into this ovine gene.     

Cell-based assay for screening 11β-hydroxysteroid dehydrogenase 1 inhibitors

11β-Hydroxysteroid dehydrogenase 1 (11β-HSD1) is primarily responsible for intracellular biosynthesis of active glucocorticoid, and its tissue-specific dysregulation has been implicated in the development of metabolic syndromes. We have developed a cell-based assay for measuring 11β-HSD1 activities using murine skeletal muscle cell line C2C12. We found that the messenger RNA (mRNA) expression of 11β-HSD1 increased on differentiation with enhanced enzyme activity as determined by homogeneous time-resolved fluorescence (HTRF) assay. Carbenoxolone, a well-known 11β-HSD1 inhibitor, exhibited an IC₅₀ value similar to that in in vitro microsomal assay (IC₅₀ = 0.3 μM). Unlike in vitro microsomal assay, cosubstrate NADPH was not required in the cell-based assay, indicating that viable cells might provide a sufficient amount of endogenous NADPH to catalyze the enzymatic conversion of inactive cortisone to active cortisol. Treatment of C2C12 myotubes with cortisone concentration dependently transactivated and transrepressed glutamine synthase and interleukin-6, respectively, which were abrogated by carbenoxolone or RU-486 (mifepristone), a glucocorticoid receptor antagonist. Accordingly, a newly designed cell-based assay using differentiated skeletal muscle cells would be useful for high-throughput screening of 11β-HSD1 inhibitors as well as for understanding the molecular mechanisms of glucocorticoid action.     

The role of 11β-hydroxysteroid dehydrogenase type 2 in human hypertension

Cortisol and aldosterone have the same in vitro affinity for the mineralocorticoid receptor (MR), although in vivo only aldosterone acts as a physiologic agonist of the MR, despite circulating levels of cortisol in humans and corticosterone in rodents being three orders of magnitude higher than aldosterone levels. In mineralocorticoid target organs the enzyme 11β-hydroxysteroid dehydrogenase type 2 (11βHSD2) inactivates 11-hydroxy steroids, to their inactive keto-forms, thus protecting the nonselective MR from activation by glucocorticoids. The gene is highly expressed in all sodium-transporting epithelia, particularly in the kidney and colon, but also in human placenta and vascular wall. Mutations in the HSD11B2 gene cause a rare monogenic juvenile hypertensive syndrome called apparent mineralocorticoid excess (AME). In AME, compromised 11βHSD2 enzyme activity results in activation of the MR by cortisol, causing sodium retention, hypokalaemia, and salt-dependent hypertension. Whereas mutations or inhibition of 11βHSD2 by licorice have been clearly shown to produce a congenital or acquired syndrome of mineralocorticoid excess, the questions remaining are the extent to which subtle abnormalities in MR/11βHSD2 mechanisms may contribute to essential hypertension. Studies in patients with essential hypertension showed a prolonged half-life of cortisol and an increased ratio of urinary cortisol to cortisone metabolites, suggesting a deficient 11βHSD2 activity. These abnormalities may be genetically determined, as suggested by the association of a microsatellite flanking the HSD11B2 gene with hypertension in black patients with end-stage kidney disease and with salt sensitivity of blood pressure in healthy subjects. These findings indicate that variants of the HSD11B2 gene may contribute to the enhanced blood pressure response to salt and possibly to hypertension in humans.     

Molecular biology of 11β-hydroxylase and 11β-hydroxysteroid dehydrogenase enzymes

There are two steroid 11β-hydroxylase isozymes encoded by the CYP11B1 and CYP11B2 genes on human chromosome 8q. The first is expressed at high levels in the normal adrenal gland, has 11β-hydroxylase activity and is regulated by ACTH. Mutations in the corresponding gene cause congenital adrenal hyperplasia due to 11β-hydroxylase deficiency; thus, this isozyme is required for cortisol biosynthesis. The second isozyme is expressed at low levels in the normal adrenal gland but at higher levels in aldosterone-secreting tumors, and has 11β-hydroxylase, 18-hydroxylase and 18-oxidase activities. The corresponding gene is regulated by angiotensin II, and mutations in this gene are found in persons who are unable to synthesize aldosterone due to corticosterone methyloxidase II deficiency. Thus, this isozyme is required for aldosterone biosynthesis.

Cortisol and aldosterone are both effective ligands of the “mineralocorticoid” receptor in vitro, but only aldosterone is a potent mineralocorticoid in vivo. This apparent specificity occurs because 11β-hydroxysteroid dehydrogenase in the kidney converts cortisol to cortisone, which is not a ligand for the receptor. This enzyme is a “short-chain” dehydrogenase which is encoded by a single gene on human chromosome 1. It is possible that mutations in this gene cause a form of childhood hypertension called apparent mineralocorticoid excess, in which the mineralocorticoid receptor is not protected from high concentrations of cortisol.     

17β-hydroxysteroid dehydrogenase from the fungus Cochlioboluslunatus: structural and functional aspects

17β-Hydroxysteroid dehydrogenase (17β-HSD) activity has been described in all filamentous fungi tested, but until now only one 17β-HSD from Cochlioboluslunatus (17β-HSDcl) was sequenced. We examined the evolutionary relationship among 17β-HSDcl, fungal reductases, versicolorin reductase (Ver1), trihydroxynaphthalene reductase (THNR), and other homologous proteins. In the phylogenetic tree 17β-HSDcl formed a separate branch with Ver1, while THNRs reside in another branch, indicating that 17β-HSDcl could have similar function as Ver1. The structural relationship was investigated by comparing a model structure of 17β-HSDcl to several known crystal structures of the short chain dehydrogenase/reductase (SDR) family. A similarity was observed to structures of bacterial 7α-HSD and plant tropinone reductase (TR). Additionally, substrate specificity revealed that among the substrates tested the 17β-HSDcl preferentially catalyzed reductions of steroid substrates with a 3-keto group, Δ⁴ or 5α, such as: 4-estrene-3,17-dione and 5α-androstane-3,17-dione.     

A novel NADP-dependent dehydrogenase activity for 7α/β- and 11β-hydroxysteroids in human liver nuclei: A third 11β-hydroxysteroid dehydrogenase

Human tissue from uninvolved liver of cancer patients was fractionated using differential centrifugation and characterized for 11βHSD enzyme activity against corticosterone, dehydrocorticosterone, 7α- and 7β-hydroxy-dehydroepiandrosterone, and 7-oxo-dehydroepiandrosterone. An enzyme activity was observed in nuclear protein fractions that utilized either NADP⁺ or NAD⁺, but not NADPH and NADH, as pyridine nucleotide cofactor with K_m values of 12 ± 2 and 390 ± 2 μM, compared to the K_m for microsomal 11βHSD1 of 43 ± 8 and 264 ± 24 μM, respectively. The K_m for corticosterone in the NADP⁺-dependent nuclear oxidation reaction was 102 ± 16 nM, compared to 4.3 ± 0.8 μM for 11βHSD1. The K_cat values for nuclear activity with NADP⁺ was 1687 nmol/min/mg/μmol, compared to 755 nmol/min/mg/μmol for microsomal 11βHSD1 activity. Inhibitors of 11βHSD1 decreased both nuclear and microsomal enzyme activities, suggesting that the nuclear activity may be due to an enzyme similar to 11βHSD Type 1 and 2.     

Phenylbenzenesulfonates and -sulfonamides as 17β-hydroxysteroid dehydrogenase type 2 inhibitors: Synthesis and SAR-analysis

17β-Hydroxysteroid dehydrogenase type 2 (17β-HSD2) converts the potent estrogen estradiol into the weakly active keto form estrone. Because of its expression in bone, inhibition of 17β-HSD2 provides an attractive strategy for the treatment of osteoporosis, a condition that is often caused by a decrease of the active sex steroids. Currently, there are no drugs on the market targeting 17β-HSD2, but in multiple studies, synthesis and biological evaluation of promising 17β-HSD2 inhibitors have been reported. Our previous work led to the identification of phenylbenzenesulfonamides and -sulfonates as new 17β-HSD2 inhibitors by ligand-based pharmacophore modeling and virtual screening. In this study, new molecules representing this scaffold were synthesized and tested in vitro for their 17β-HSD2 activity to derive more profound structure-activity relationship rules.     

Effect of the lyotropic series of anions on denaturation and renaturation of 20β-hydroxysteroid dehydrogenase

The effect of the lyotropic series of anions on the stability and renaturation of tetrameric 20β-hydroxysteroid dehydrogenase (17,20β,21-trihydroxysteroid:NAD⁺ oxidoreductase, EC 1.1.1.53) was investigated. The variations in enzymatic activity were correlated with the changes in protein fluorescence, circular dichroism, reactivity of histidine residues and molecular weight. High concentrations of salting-out anions (phosphate, citrate, sulphate) were found to stabilize the enzyme markedly and increase the renaturation yield of the urea-denaturated enzyme. Phosphate, for instance, induced the highest stabilization at about 1.2 M and the maximum reactivation (66%) at 0.5 M. At low anion concentration (0.01 M), the reactivation was only 7%. The renaturation property of salting-out anions seems to be due to their stabilizing effect on the end-product, i.e., the assembled tetramer. Salting-in anions (perchlorate, thiocyanate, iodide) inactivated the enzyme. At moderate anion concentrations (no greater than 0.25 M) the inactivation, which occurred slowly, without tetramer dissociation and with minor modifications of enzyme conformation, was fully reversed by concentrated phosphate or by saturating concentrations of NADH. In contrast, the inactivation induced by high anion concentrations (1–2 M) was rapid, irreversible and linked to considerable modifications of enzyme conformation.     

Optimization of cyclic sulfamide derivatives as 11β-hydroxysteroid dehydrogenase 1 inhibitors for the potential treatment of ischemic brain injury

The 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), a cortisol regenerating enzyme that amplifies tissue glucocorticoid levels, plays an important role in diabetes, obesity, and glaucoma and is recognized as a potential therapeutic target for various disease conditions. Moreover, a recent study demonstrated that selective 11β-HSD1 inhibitor can attenuate ischemic brain injury. This prompted us to optimize cyclic sulfamide derivative for aiming to treat ischemic brain injury. Among the synthesized compounds, 6e has an excellent in vitro activivity with an IC₅₀ value of 1 nM toward human and mouse 11β-HSD1 and showed good 11β-HSD1 inhibition in ex vivo study using brain tissue isolated from mice. Furthermore, in the transient middle cerebral artery occlusion model in mice, 6e treatment significantly attenuated infarct volume and neurological deficit following cerebral ischemia/reperfusion injury. Additionally, binding modes of 6e for human and mouse 11β-HSD1 were suggested.     

Structural assessment and identification of 11β-hydroxysteroid dehydrogenase type 1 inhibitors

Communicated by Ramaswamy H. Sarma     

3α-Hydroxysteroid dehydrogenase and 3β-hydroxysteroid dehydrogenase in the ovary of young Mongolian gerbils

Summary The ovaries of sexually mature, pregnant mare serum gonadotropin (PMSG) stimulated, 12 week old Mongolian gerbils were investigated morphologically and enzyme histochemically for the appearance of the 3-hydroxysteroid and the 3-hydroxysteroid dehydrogenase during the estrous cycle. Up to ovulation, on day 3 of the estrous cycle, the number of vesicular follicles increases continuously. Primarily atretic follicles can be seen on day 4. On day 5 corpora lutea appear, but they degenerate already by day 6.During the entire estrous cycle, 3-hydroxysteroid dehydrogenase and 3-hydroxysteroid dehydrogenase activity can be found in the theca of tertiary follicles and in the interstitial cells, whereas the theca of secondary follicles and the granulosa of healthy follicles do not exhibit any enzyme activity. The activity decreases from day 1 till day 6. The granulosa of atretic follicles and the cells of corpora lutea show only weak activity. It may be significant that the intensity of enzyme activity in the ovary and the estrogen level in the plasma are differently correlated to the estrous cycle.This investigation was supported by the Deutsche Forschungsgemeinschaft     

Lipid-mediated unfolding of 3β-hydroxysteroid dehydrogenase 2 is essential for steroidogenic activity

For inner mitochondrial membrane (IMM) proteins that do not undergo N-terminal cleavage, the activity may occur in the absence of a receptor present in the mitochondrial membrane. One such protein is human 3β-hydroxysteroid dehydrogenase 2 (3βHSD2), the IMM resident protein responsible for catalyzing two key steps in steroid metabolism: the conversion of pregnenolone to progesterone and dehydroepiandrosterone to androstenedione. Conversion requires that 3βHSD2 serve as both a dehydrogenase and an isomerase. The dual functionality of 3βHSD2 results from a conformational change, but the trigger for this change remains unknown. Using fluorescence resonance energy transfer, we found that 3βHSD2 interacted strongly with a mixture of dipalmitoylphosphatidylglycerol (DPPG) and dipalmitoylphosphatidylcholine (DPPC). 3βHSD2 became less stable when incubated with the individual lipids, as indicated by the decrease in thermal denaturation (T(m)) from 42 to 37 °C. DPPG, alone or in combination with DPPC, led to a decrease in α-helical content without an effect on the β-sheet conformation. With the exception of the 20 N-terminal amino acids, mixed vesicles protected 3βHSD2 from trypsin digestion. However, protein incubated with DPPC was only partially protected. The lipid-mediated unfolding completely supports the model in which a cavity forms between the α-helix and β-sheet. As 3βHSD2 lacks a receptor, opening the conformation may activate the protein.     

Estradiol 17β-dehydrogenase and 20α-hydroxysteroid dehydrogenase from human placental cytosol: one enzyme with two activities?

The soluble enzyme estradiol 17β-dehydrogenase (17β-ED) from human term placental cytosol is reported to be a stereospecific oxidoreductase for estrogen substrates. A published purification scheme (heat treatment and affinity chromatography) yielded a homogeneous protein which had the reported characteristics of pure 17β-ED and also had 20α-hydroxysteroid dehydrogenase (20α-HSD) activity. Spectrophotometric assay when the buffer contained albumin, 8 mg/ml, masked the 20α-HSD activity observed in albumin-free conditions and may explain why this bifunctional activity has gone unrecognized. In human placenta, one enzyme may catalyze stereospecific oxidation/reduction of both estrogen and progesterone.