17 beta-hydroxysteroid dehydrogenases and cancers

17β-Hydroxysteroid dehydrogenases (17HSDs) catalyze the interconversions between active 17β-hydroxysteroids and less-active 17-ketosteroids thereby affecting the availability of biologically active estrogens and androgens in a variety of tissues. The enzymes have different enzymatic properties and characteristic cell-specific expression patterns, suggesting differential physiological functions for the enzymes. Epidemiological and endocrine evidence indicate that estrogens play a key role in the etiology of breast cancer while androgens are involved in mechanisms controlling the growth of prostatic cells, both normal and malignant. Recently, we have developed, using LNCaP prostate cancer cell lines, a cell model to study the progression of prostate cancer. In the model LNCaP cells are transformed in culture condition to more aggressive cells, able to grow in suspension cultures. Our results suggest that substantial changes in androgen and estrogen metabolism occur in the cells during the process. These changes lead to increased production of active estrogens during transformation of the cells. Data from studies of breast cell lines and tissues suggest that the oxidative 17HSD type 2 may predominate in human non-malignant breast epithelial cells, while the reductive 17HSD type 1 activity prevails in malignant cells. Deprivation of an estrogen response by using specific 17HSD type 1 inhibitors is a tempting approach to treat estrogen-dependent breast cancer. Our recent studies demonstrate that in addition to sex hormone target tissues, estrogens may be important in the development of cancer in some other tissues previously not considered as estrogen target tissues such as colon. Our data show that the abundant expression of 17HSD type 2 present in normal colonic mucosa is significantly decreased during colon cancer development.     

Characterization of fungal 17beta-hydroxysteroid dehydrogenases

To promote understanding of the evolution of the steroid hormone signalling and hydroxysteroid dehydrogenases (HSDs), comparative characterization of fungal 17beta-HSDs was performed. Constitutive 17beta-HSD activity was determined in cytosols of the fungi: Cochliobolus lunatus, Pleospora herbarum, Fusarium lini, Trichoderma viride, Mucor spinosus, Rhizopus nigricans and Pleurotus ostreatus. The reaction equilibrium in all species except P. ostreatus was shifted towards reduction. The preferential coenzyme for reduction of androstenedione was NADPH, while for oxidation of testosterone, NAD4 was preferred. The highest enzyme activities were found in the Ascomycete C. lunatus (152.4 nmol mg(-1) h(-1)) and in the Basidiomycete P. ostreatus (69.1 nmol mg(-1) h(-1)). No similarities on the protein and mRNA level between fungal 17beta-HSDs and the purified enzyme from C. lunatus were observed. To investigate the nature of these enzymes, 17beta-HSD was purified from P. ostreatus using ammonium sulphate precipitation, hydrophobic interaction chromatography, and affinity chromatography. The purified enzyme has an apparent molecular mass of approximately 35 kDa and is probably a dimer as determined by gel filtration. Chemical modifications exposed Lys, His and Tyr as important for enzyme activity. Additionally, no similarities of C. lunatus and P. ostreatus enzymes were found to bacterial 3alpha,20beta-HSD from Streptomyces hydrogenans, 3beta,17beta-HSD from Comamonas testosteroni and mammalian 17beta-HSD types 1 and 4. The results thus suggest that there are most probably different enzymes responsible for 17beta-HSD activity in filamentous fungi.     

Instability of serum 17 beta-hydroxysteroid dehydrogenases of placental origin

The stabilization by glycerol of the serum estradiol 17 beta dehydrogenase has been investigated here in terms of time and temperature. The protected enzyme is stable for a month at least at --20 degrees C. Its activity reaches a maximum at 60 degrees C. In another connection, effect of reduced glutathione, of N. acetyl-cysteine and of various mineral ions on the enzymic conversion has been studied.     

Kinetic and stereochemical studies on reaction mechanism of mouse liver 17 beta-hydroxysteroid dehydrogenases

The kinetic mechanism of two major monomeric 17 beta-hydroxysteroid dehydrogenases from mouse liver cytosol was studied at pH 7 in both directions with NADP(H) and three steroid substrates: testosterone, 5 beta-androstane-3 alpha, 17 17 beta-diol, and estradiol-17 beta. In each case the reaction mechanism of the two enzymes was sequential, and inhibition patterns by-products and dead-end inhibitors were consisted with an ordered bi bi mechanism with the coenzyme binding to the free enzyme, although there was difference in affinity and maximum velocity for the steroidal substrates between the two enzymes. Binding studies of the coenzyme and substrate indicate the binding of coenzyme to the free enzyme, in which 1 mol of NADPH binds to 1 mol of each monomeric enzyme. The 4-pro-R-hydrogen atom of NADPH was transferred to the alpha-face of the steroid molecule by the two enzymes.     

Two 17beta-hydroxysteroid dehydrogenases (17HSDs) of estradiol biosynthesis: 17HSD type 1 and type 7.

Two 17β-hydroxysteroid dehydrogenases (17HSDs), type 1 and type 7, are enzymes of estradiol biosynthesis, in addition to which rodent type 1 enzymes are also able to catalyze androgens. Both of the 17HSDs are abundantly expressed in ovaries, the type 1 enzyme in granulosa cells and type 7 in luteinized cells. The expression of 17HSD7, which has also been described as a prolactin receptor-associated protein (PRAP), is particularly up-regulated in corpus luteum during the second half of rodent pregnancy. A moderate or slight signal for mouse 17HSD7/PRAP mRNA has also been demonstrated in samples of placenta and mammary gland, for example. Human, but not rodent, 17HSD1 is expressed in placenta, breast epithelium and endometrium in addition to ovaries. A cell-specific enhancer, silencer and promoter in the hHSD17B1 gene participate in the regulation of type 1 enzyme expression. The enhancer consists of several subunits, including a retinoic acid response element, the silencer has a binding motif for GATA factors, and the proximal promoter contains adjacent and competing AP-2 and Sp binding sites.     

Histochemical distribution of delta5-3beta- and 17beta-hydroxysteroid dehydrogenases in hamster trophoblast.

The histochemical distribution of delta5-3beta- and 17beta-hydroxysteroid dehydrogenases was demonstrated in hamster trophoblast between Days 8 and 15 of pregnancy. The delta5-3beta-hydroxysteroid dehydrogenase activity in the ectoplacental trophoblast of 8-day embryos was demonstrated by use of delta5-pregnenolone and dehydroepiandrosterone as substrates; between Days 11 and 15, activity was demonstrated in the trophoblastic giant cells of the placenta and in the intra-arterial trophoblast cells when delta5-pregnenolone was the substrate. Between Days 11 and 15, 17beta-hydroxysteroid activity was present in the spongiotrophoblast, labyrinth, placental giant cells and intra-arterial trophoblast cells, as shown by use of testosterone and oestradiol as substrates. Both enzymes were demonstrated in ectopic trophoblast cells, indicating that these activities are autonomous.     

Structure-based phylogenetic analysis of short-chain alcohol dehydrogenases and reclassification of the 17beta-hydroxysteroid dehydrogenase family.

Short-chain alcohol dehydrogenases (SCAD) constitute a large and diverse family of ancient origin. Several of its members play an important role in human physiology and disease, especially in the metabolism of steroid substrates (e.g., prostaglandins, estrogens, androgens, and corticosteroids). Their involvement in common human disorders such as endocrine-related cancer, osteoporosis, and Alzheimer disease makes them an important candidate for drug targets. Recent phylogenetic analysis of SCAD is incomplete and does not allow any conclusions on very ancient divergences or on a functional characterization of novel proteins within this complex family. We have developed a 3D structure-based approach to establish the deep-branching pattern within the SCAD family. In this approach, pairwise superpositions of X-ray structures were used to calculate similarity scores as an input for a tree-building algorithm. The resulting phylogeny was validated by comparison with the results of sequence-based algorithms and biochemical data. It was possible to use the 3D data as a template for the reliable determination of the phylogenetic position of novel proteins as a first step toward functional predictions. We were able to discern new patterns in the phylogenetic relationships of the SCAD family, including a basal dichotomy of the 17beta-hydroxysteroid dehydrogenases (17beta-HSDs). These data provide an important contribution toward the development of type-specific inhibitors for 17beta-HSDs for the treatment and prevention of disease. Our structure-based phylogenetic approach can also be applied to increase the reliability of evolutionary reconstructions in other large protein families.     

Hydroxysteroid dehydrogenases of Pseudomonas testosteroni. Separation of a 17 beta-hydroxysteroid dehydrogenase from the 3(17) beta-hydroxysteroid dehydrogenase and comparison of the two enzymes.

When a crude extract of Pseudomonas testosteroni induced with testosterone was subjected to polyacrylamide gel electrophoresis, six bands that stained for 17 beta-hydroxysteroid dehydrogenase activity was observed. A protein fraction containing the enzyme corresponding to the fastest migrating band and devoid of the other hydroxysteroid dehydrogenase activities has been obtained. This preparation appears to be distinct from the previously isolated 3(17) beta-hydroxysteroid dehydrogenase (EC 1.1.1.51) in its chromatography properties on DEAE-cellulose, substrate and cofactor specificity, immunological properties and heat stability. The preparation appears devoid of 3alpha-, 3beta-, 11beta-, 17alpha-, 20alpha-, and 20beta-hydroxysteroid dehydrogenase activities. The enzyme transfers th 4-pro-S-hydrogen of NADH from estradiol-17beta (1,3,5(10)estratriene-3,17beta-diol) to estrone (3-hydroxy-1,3,5(10)-estratriene-17-one).     

Genetic manipulation of 11beta-hydroxysteroid dehydrogenases in mice

11beta-hydroxysteroid dehydrogenases (HSDs) interconvert active 11-hydroxy glucocorticoids (cortisol, corticosterone) and their inert 11-keto derivatives (cortisone, 11-dehydrocorticosterone). 11beta-HSD type 1 is a predominant reductase that regenerates active glucocorticoids in expressing cells, thus amplifying local glucocorticoid action, whereas 11beta-HSD type 2 catalyzes rapid dehydrogenation, potently inactivating intracellular glucocorticoids. Both isozymes thus regulate receptor activation by substrate availability. Spatial and temporal regulation of expression are important determinants of the physiological roles of 11beta-HSDs, with each isozyme exhibiting a distinct, tissue-restricted pattern together with dynamic regulation during development and in response to environmental challenges, including diet and stress. Transgenic approaches in the mouse have contributed significantly toward an understanding of the importance of these prereceptor regulatory mechanisms on corticosteroid receptor activity and have highlighted its potential relevance to human health and disease. Here we discuss current ideas of the physiological roles of 11beta-HSDs, with emphasis on the key contributions made by studies of 11beta-HSD gene manipulation in vivo.     

Study on mesophilic and thermophilic alcohol dehydrogenases in gas-phase reaction

The initial reaction rate and the thermostability of the mesophilic alcohol dehydrogenase (ADH) from Lactobacillus brevis (LBADH), and the thermophilic ADH from Thermoanaerobacter sp. (ADH T) in gas-phase reaction were compared. The effects of water activity, cofactor-to-protein molar ratio, and reaction temperature on the reduction of acetophenone to 1-phenylethanol were studied. An optimal water activity of 0.55 in terms of productivity was found for both ADHs. The cofactor-to-protein molar ratio was chosen slightly higher than equimolar to increase both activity and thermostability. An excellent optimal productivity of 1,000 g x L(-1) x d(-1) for LBADH and 600 g x L(-1) x d(-1)for ADH T was found at 60 degrees C, while the highest total turnover numbers with respect to the enzyme were achieved at 30 degrees C and amounted to 4.2 million for LBADH and 1.7 million for ADH T, respectively. Interestingly, the ADH from the mesophilic L. brevisshowed the higher thermostability in the nonconventional medium gas phase.     

Characterisation of 11 beta-hydroxysteroid dehydrogenases in feline kidney and liver

11 Beta-hydroxysteroid dehydrogenases type 1 and 2 (11 beta-HSD1 and 11 beta-HSD2) are microsomal enzymes responsible for the interconversion of cortisol into the inactive form cortisone and vice versa. 11 beta-HSD1 is mainly present in the liver, and has predominantly reductase activity although its function has not yet been elucidated. 11 beta-HSD2, present in mineralocorticoid target tissues such as the kidney, converts cortisol into cortisone. Reduced activity due to inhibition or mutations of 11 beta-HSD2 leads to hypertension and hypokalemia resulting in the Apparent Mineralocorticoid Excess Syndrome (AMES). Like humans, cats are highly susceptible for hypertension. As large species differences exist with respect to the kinetic parameters (K(m) and V(max)) and amino acid sequences of both enzymes, we determined these characteristics in the cat. Both enzyme types were found in the kidneys. 11 beta-HSD1 in the feline kidney showed bidirectional activity with predominantly dehydrogenase activity (dehydrogenase: K(m) 1959+/-797 nM, V(max) 766+/-88 pmol/mg*min; reductase: K(m) 778+/-136 nM, V(max) 112+/-4 pmol/mg*min). 11 beta-HSD2 represents a unidirectional dehydrogenase with a higher substrate affinity (K(m) 184+/-24 nM, V(max) 74+/-3 pmol/mg*min). In the liver, only 11 beta-HSD1 is detected exerting reductase activity (K(m) 10462 nM, V(max) 840 pmol/mg*min). Sequence analysis of conserved parts of 11 beta-HSD1 and 11 beta-HSD2 revealed the highest homology of the feline enzymes with the correspondent enzymes found in man. This suggests that the cat may serve as a suitable model species for studies directed to the pathogenesis and treatment of human diseases like AMES and hypertension.     

人17β羟类固醇脱氢酶（17β—HSD）基因在家蚕系统中的表达（简报）

Estrogenic 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) plays a crucial role in the synthesis of estrogens, but it also produces negative action of estrogens in promoting the growth of hormone-sensitive cancers, especially in breast and prostate. The high specific activity can be taken as an important signal for the diagnosis of cancers. Recombinant rAcBm-NPV/17 beta-HSD virus which contains the human 17 beta-HSD cDNA under the control of polyhedron gene promoter is generated by cotransfection of the BmN cells with the transfer plasmid pVL/17 beta-HSD and wild BmNPV genomic DNA. 17 beta-HSD is maximally expressed 72 h and 120 h post infection in BmN cells and the 5th instar silkworm larvae respectively. At those time interval, intracellular and hemolymphic enzymatic activity reach 0.12 U/mg and 0.15 U/mg of protein which produced total activity of 0.97 U/1.5 x 10(6) cells and 4.7 U/larva. The expressed quantities in female larvae are a little higher than that in male larvae. The present data shows that Silkworm/BmNPV expression system can express 3-5 times higher than that of the richest human placenta. It also indicates that there is an apparent band with a molecular mass of 35 kDa using SDS-PAGE method, the size of which is similar to that of the crude enzyme from placenta.     

Isolation of novel microbial 3 alpha-, 3 beta-, and 17 beta-hydroxysteroid dehydrogenases. Purification, characterization, and analytical applications of a 17 beta-hydroxysteroid dehydrogenase from an Alcaligenes sp

By selecting for growth on testosterone or estradiol-17 beta as the only source of organic carbon, we have isolated a number of soil microorganisms which contain highly active and novel, inducible, NAD-linked 3 alpha-, 3 beta-, and 17 beta-hydroxysteroid dehydrogenases. Such enzymes are suitable for the microanalysis of steroids and of steroid-transforming enzymes, as well as for performing stereoselective oxidations and reductions of steroids. Of particular interest among these organisms is a new species of Alcaligenes containing 17 beta-hydroxysteroid dehydrogenase, easily separable from 3 beta-hydroxysteroid dehydrogenase. Unlike any of the other isolated organisms, this Alcaligenes sp. contained no 3 alpha-hydroxysteroid dehydrogenase activity. A large-scale purification (763-fold) to homogeneity of the major induced 17 beta-hydroxysteroid dehydrogenase was achieved by ion-exchange, hydrophobic, and affinity chromatographies. The enzyme has high specific activity for the oxidation of testosterone (Vmax = 303 mumol/min/mg of protein; Km = 3.6 microM) and reacts almost equally well with estradiol-17 beta (Vmax = 356 mumol/min/mg; Km = 6.4 microM). It consists of apparently identical subunits (Mr = 32,000) and exists in polymeric form under nondenaturing conditions (Mr = 68,000 by gel filtration and 86,000 by polyacrylamide gel electrophoresis). The isoelectric point is pH 5.1. The enzyme is almost completely specific for 17 beta-hydroxysteroids which may be delta 5-olefins or ring A phenols or have cis or trans A/B ring fusions. Substituents at other positions are tolerated, although the presence of a 16 alpha- or 16 beta-hydroxyl group blocks the oxidation of the 17 beta-hydroxyl function. 3 beta-Hydroxysteroids (A/B ring fusion trans, but not cis, or delta 5-olefins) are very poor substrates. The application of this highly active, specific, and stable 17 beta-hydroxysteroid dehydrogenase to the microestimation of steroids by enzymatic cycling of nicotinamide nucleotides and for the stereospecific oxidation of steroids is demonstrated.     

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。     

Expression of 17beta-hydroxysteroid dehydrogenease in testis of the ascidian Ciona intestinalis [corrected

Ascidians have been employed as model organisms in investigating spermatogenesis. 17beta-hydroxysteroid dehydrogenase (HSD) is a steroidogenic enzyme essential for invertebrate spermatogenesis. A homologue of HSD was found in the EST database of Ciona intestinalis and cloned. Sequence analysis showed significant homology to zebra fish, sea urchin and human 17beta-HSD. The gene has an open reading frame (ORF) of 918 nucleotides coding for a polypeptide of 306 amino acids and a calculated mass of 35-kDa. Immunoblotting with an antibody raised against HSD recognized a 35-kDa protein purified from the C. intestinalis testis. The HSD protein was localized in steroidogenic cells in the Ciona testis. These results suggest that C. intestinalis 17beta-HSD is equivalent to the enzyme of vertebrate Leydig cells and that 17beta-HSD could be a phylogenetic marker for organisms producing steroids.