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Query: UNIPROT:P06889 (Mol)
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Testicular androgens induce formation of the male urogenital tract in all mammals. In marsupials male development occurs after birth and over a prolonged period. For example, in the tammar wallaby virilization of the Wolffian ducts begins by day 20, prostate formation begins about day 25, and phallic development starts after day 80 of pouch life. Between days 20 and 40 5alpha-androstane-3alpha,17beta-diol (5alpha-adiol) is formed in tammar testes and secreted into plasma. Administration of 5alpha-adiol to pouch young females induces urogenital sinus virilization by day 40 and formation of a mature male prostate and phallus by day 150. 5alpha-Adiol is synthesized in pouch young testes by two pathways, one involving testosterone and dihydrotestosterone and the other 5alpha-pregnane-3alpha,17alpha-diol-20-one and androsterone as intermediates, both utilizing steroid 5alpha-reductase. In target tissues 5alpha-adiol acts via the androgen receptor after conversion to dihydrotestosterone but may have other actions as well. Whether 5alpha-adiol plays a role in male development in placental mammals is uncertain.
Mol Cell Endocrinol 2003 Dec 15
PMID:Unsolved problems in male physiology: studies in a marsupial. 1465 73

In vitro enzyme assays have demonstrated that human type 10 17beta-hydroxysteroid dehydrogenase (17beta-HSD10) catalyzes the oxidation of 5alpha-androstane-3alpha,17beta-diol (adiol), an almost inactive androgen, to dihydrotestosterone (DHT) rather than androsterone or androstanedione. To further investigate the role of this steroid-metabolizing enzyme in intact cells, we produced stable transfectants expressing 17beta-HSD10 or its catalytically inactive Y168F mutant in human embryonic kidney (HEK) 293 cells. It was found that DHT levels in HEK 293 cells expressing 17beta-HSD10, but not its catalytically inactive mutant, will dramatically increase if adiol is added to culture media. Moreover, certain malignant prostatic epithelial cells have more 17beta-HSD10 than normal controls, and can generate DHT, the most potent androgen, from adiol. This event might promote prostate cancer growth. Analysis of the 17beta-HSD10 sequence shows that this enzyme does not have any ER retention signal or transmembrane segments and has not originated by divergence from a retinol dehydrogenase. The data suggest that the unique mitochondrial location of this HSD [Eur. J. Biochem. 268 (2001) 4899] does not prevent it from oxidizing the 3alpha-hydroxyl group of a C19 sterol in living cells. The experimental results lead to the conclusion that mitochondrial 17beta-HSD10 plays a significant part in a non-classical androgen synthesis pathway along with microsomal retinol dehydrogenases.
J Steroid Biochem Mol Biol 2003 Nov
PMID:Oxidative 3alpha-hydroxysteroid dehydrogenase activity of human type 10 17beta-hydroxysteroid dehydrogenase. 1467 39

Human constitutive androstane (or active) receptor (hCAR), a member of the nuclear receptor superfamily NR1I3, regulates the expression of several genes that are mainly involved in the metabolism of endogenous and xenobiotic compounds (e.g., CYP2B6, CYP3A4, and UGT1A1). We found four novel splice variants in the ligand-binding domain (LBD) of hCAR (NCBI reference sequence, NM_005122; designated SV0 herein). The variants designated SV1 and SV2 contained in-frame 12- and 15-base pair (bp) insertions, respectively. SV3 carried both of the insertions, and SV4 contained an in-frame 117-bp deletion. The insertion site of SV1 is located in the alpha6 helix of hCAR LBD, which makes up the ligand-binding cavity, and that of SV2 is located in the highly conserved loop between helices alpha8 and alpha9. SYBR Green real-time reverse transcription-polymerase chain reaction analysis of each splice variant revealed that the hepatic expression of SV2 was almost comparable with that of SV0 (approximately 40%), whereas other variants accounted for 6 to 10% of the total hCAR transcripts. In the reporter gene assays employing the phenobarbital-responsible enhancer module (PBREM) from CYP2B6 and UGT1A1 genes, the splice variants, except for SV1, were inactive, whereas SV1 transactivated the CYP2B6 PBREM but not the UGT1A1 PBREM reporter. A nuclear translocation assay in rat hepatocytes revealed that all the splice variants lack the responsiveness toward phenobarbital and 6-(4-chloropheny-l)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO) in terms of the ligand-dependent nuclear translocation. Further characterization, such as the identification of specific ligands, will help elucidate physiological implication of these hCAR splice variants.
Mol Pharmacol 2004 Mar
PMID:Identification of novel alternative splice variants of human constitutive androstane receptor and characterization of their expression in the liver. 1497 27

Many cytochrome P450 isoforms are known to be drug-inducible. The anticonvulsant phenytoin has been reported to be an inducer of human CYP2B6, CYP3A4, and murine CYP2C29. However, the molecular mechanism mediating phenytoin induction remains unclear. Herein, we used in vivo and in vitro gene reporter assays of the Cyp2c29 promoter to delineate the phenytoin-response activity to a phenytoin-responsive module located at -1371 kb upstream of the Cyp2c29 translation start site. The phenytoin-responsive module, consisting of two motifs of two imperfect direct repeat hexamers spaced by four nucleotides and a putative CCAAT/enhancer-binding protein-binding site, mediated luciferase reporter induction by phenytoin in mouse livers in vivo and was activated by CAR in HepG2 cells. Hepatic CYP2C29 mRNA was induced by phenytoin in wild-type but not in CAR-null mice, indicating that constitutive active or androstane receptor (CAR) regulates phenytoin-induced transcription of the Cyp2c29 gene. Furthermore, the constitutive levels of CYP2C29 mRNA were reduced approximately 77-fold in CAR-null mice compared with those in the wild-type mice, suggesting that CAR may also regulate the constitutive expression of the Cyp2c29 gene either directly or indirectly.
Mol Pharmacol 2004 Jun
PMID:The constitutive active/androstane receptor regulates phenytoin induction of Cyp2c29. 1515 33

Oestrogens have a major impact on reproductive function in both males and females. Two oestrogen receptor genes known as ERalpha (ESR1NR3A1) and ERbeta (ESR2NR3A2) have been cloned. Splice variant isoforms of the ERbeta gene have been identified in human, bovine and rodents and it has been suggested that the existence of these forms can influence oestrogen responsiveness. In the human, splicing of an alternative eighth exon results in the formation of a C-terminal variant called hERbetacx, or hERbeta2, but this isoform has not been identified in other species. The aim of the present study was to clone ERbeta cDNAs from primates so as to determine how closely they resembled the ERbeta isoforms found in the human. The two species studied were the stump-tailed macaque (Macaca arctoides), an Old World primate, and the common marmoset (Callithrix jacchus jacchus), a New World primate. Full length ERbeta (wild type, ERbeta1) cDNAs were cloned from macaque and marmoset; they encoded proteins of similar size to those found in human (59 and 54 kDa, long and short forms respectively) and shared significant sequence homology (97.5% in macaque and 93.8% in marmoset) with the human peptide sequence. Full length cDNAs homologous to the hERbeta2 variant were identified in both primates. Marmoset ERbeta2 was slightly shorter than that of human ERbeta2 (54 kDa compared with 55 kDa) and did not contain the peptide sequence used to raise an anti-hERbeta2 antibody. All the macaque ERbeta2 cDNAs contained 56 bp of intronic sequence which included an in-frame stop codon resulting in translation of a truncated protein ( approximately 35 kDa). In all three species, truncated, alternatively spliced mRNAs lacking exon 5 were isolated on multiple occasions from all tissue extracts. In transient transfection assays, ERbeta2-containing constructs were unable to induce transcription of an oestrogen response element (ERE) reporter plasmid in the presence of oestradiol. ERbeta1 from human, macaque and marmoset exhibited minor differences in their ability to induce transcription of the ERE reporter when incubated with different ligands (oestradiol, PPT, DPN, 5-alpha-androstane-3-beta, 17beta-diol (3betaAdiol), genistein) and this may be due to amino acid substitutions within their ligand binding domains. In conclusion, we have identified and cloned wild type ERbeta (ERbeta1) from macaque and marmoset and demonstrated that splice variant mRNAs homologous to hERbeta2 are formed in both species. The marmoset monkey, therefore, provides a suitable animal model in which to investigate the impact of ERbeta variant expression on tissue responsiveness to oestrogens.
J Mol Endocrinol 2004 Jun
PMID:Cloning of oestrogen receptor beta from Old and New World primates: identification of splice variants and functional analysis. 1517 10

The growth and development of the prostate gland are regulated by androgens. Despite our understanding of molecular actions of 5alpha-dihydrotestosterone (5alpha-DHT) in the prostate through the trans-activation of the androgen receptor (AR), comprehensive analysis of androgen responsive genes (ARGs) has just been started. Moreover, expression changes induced by the androgen effects of 5alpha-androstane-3alpha,17beta-diol (3alpha-diol), a metabolite of 5alpha-DHT through the action of 3alpha-hydroxysteroid dehydrogenases (3alpha-HSDs), remain undefined. We demonstrated that both 5alpha-DHT and 3alpha-diol stimulated similar levels of androgen sensitive human prostate cancer LNCaP cell proliferation. However, consistent with the fact that 3alpha-diol has low affinity toward the AR, 3alpha-diol did not elicit the same levels of AR trans-activation activity as that of 5alpha-DHT. Since LNCaP cells respond to androgen stimulation by transcriptionally activating the AR downstream genes, gene expression patterns between 0 and 48 h following 3alpha-diol and 5alpha-DHT stimulation were analyzed using cDNA-based membrane arrays to define the temporal regulation of ARGs. Array analysis identified 217 and 219 androgen-modulated genes in at least one time point following 3alpha-diol and 5alpha-DHTstimulation, respectively, including key regulators of cell proliferation. Only a subset of these genes (143) was regulated by both androgens. These data suggest that 3alpha-diol exerts androgenic effects independent of the action of 5alpha-DHT in steroid target tissues. Accordingly, 3alpha-diol might activate cell proliferation cascades independent of AR pathway in the prostate.
J Steroid Biochem Mol Biol 2004 Jul
PMID:Partitioning of 5alpha-dihydrotestosterone and 5alpha-androstane-3alpha, 17beta-diol activated pathways for stimulating human prostate cancer LNCaP cell proliferation. 1527 23

The cytochrome P4507B1 (P4507B1) in the human hippocampus is responsible for the production of 7alpha-hydroxylated derivatives of dehydroepiandrosterone (DHEA) and other 3beta-hydroxylated neurosteroids. Minor quantities of the 7beta-hydroxylated derivatives are also produced. Neuroprotective action of these 7-hydroxysteroids was reported. Recombinant human P4507B1 was prepared from yeast coexpressing the human hippocampal P450 cDNA and the human P450 reductase genes. Microsomal P4507B1 activity was tested in the presence of NADPH and (14)C-labeled steroid substrates to deduce kinetic parameters and to study inhibitor responses. The K(M) values obtained for DHEA, pregnenolone, epiandrosterone, 5alpha-androstane-3beta,17beta-diol and estrone were 1.90 +/- 0.06, 1.45 +/- 0.03, 1.05 +/- 0.12, 0.8 +/- 0.04 and 1.20 +/- 0.26 microM, respectively. Production of limited amounts of 7beta-hydroxylated derivatives was also observed, but only with DHEA, 5alpha-androstane-3beta,17beta-diol and epiandrosterone. K(M) values determined for 7beta-hydroxylation were identical to those for 7alpha-hydroxylation. The DHEA 7alpha-hydroxylation was inhibited by estrone and estradiol (mixed type inhibition) and by the [25-35] beta-amyloid peptide (non-competitive inhibition). These results indicate that in human, the 7-hydroxylation catalysed by P4507B1 preferentially takes place on DHEA, 5alpha-androstane-3beta,17beta-diol and epiandrosterone with major and minor formation of 7alpha- and 7beta-hydroxylated derivatives, respectively. Both estrogens and a beta-amyloid component inhibit the P4507B1-mediated production of the 7-hydroxysteroid metabolites.
J Steroid Biochem Mol Biol 2004 Dec
PMID:The human cytochrome P4507B1: catalytic activity studies. 1569 43

The solid-phase synthesis of 16alpha-derivatives of 5alpha-androstane-3alpha, 17beta-diol with one, two or three levels of molecular diversity was accomplished using the diethylsilyloxy linker. Libraries with one level of diversity (10 members) and two levels of diversity (40 members) were synthesized in a parallel fashion in good yields and acceptable HPLC purities for the majority of library members. Compounds with three levels of diversity (15 pools) were realized in a split and pool fashion to allow further deconvolution by the positional scanning method. The screening of the generated model libraries revealed interesting preliminary structure-activity relationships related to their antiproliferative activities on androgen-sensitive Shionogi cells. In the case of the two-level library, the presence of a hydrophobic amino acid at R1 (isoleucine (Ile) or phenylalanine (Phe)) and a six-membered ring (aromatic or not) at R2 seems an important requirement for activity. In the three-level library, the amino acid residues isoleucine and phenylalanine clearly provided a better antiproliferative activity than glycine (Gly) and proline (Pro). These model libraries will serve as basis for the generation of larger libraries of peptidosteroids toward the development of therapeutic agents.
Mol Divers 2005
PMID:Solid-phase synthesis of model libraries of 3alpha,17beta-dihydroxy-16alpha-(aminoethyl-N-substituted)-5alpha-androstanes for the development of steroidal therapeutic agents. 1578 54

Type 7 17beta-HSD catalyzes the transformation of estrone (E1) into estradiol (E2) and dihydrotestosterone (DHT) into 5alpha -androstane-3beta,17beta-diol (3beta-diol) as well as zymosterone into zymosterol. This suggests that in addition to cholesterol metabolism, the enzyme could play a critical role in estrogen-sensitive cells, since it inactivates DHT that generally shows antagonistic effect in the cells, while producing active E2 for cell proliferation. In this report, we describe the cloning and characterization of a second form of type 7 17beta-HSD (17beta-HSD7_2) that shares 95.6% identity with 17beta-HSD7_1. Using a 7.5kb genomic DNA fragment of 17beta-HSD7_1 as probe, we have obtained 7 BAC clones: three clones containing the 17beta-HSD7_1 gene and four containing the 17beta-HSD7_2 gene. The corresponding 17beta-HSD7_2 cDNA fragments of the coding region were obtained by amplification using RT-PCR and subcloned into pCMV expression vector and stably transfected into human embryonic kidney (HEK-293) cells. The overexpressed 17beta-HSD7_2 catalyzes efficiently the transformation of E1 into E2 and of DHT into 3beta-diol. Ribonuclease protection assays (RPA) indicate that 17beta-HSD7_2 is expressed in the liver, prostate, uterus and placenta. FISH mapping using the 7.5kb genomic DNA fragment as well as 2 BAC clones of each form allowed us to map the 17beta-HSD7_1 gene on chromosome band 1q23, and 17beta-HSD7_2 on band 10p11.2. These results contrast with a previous report that the 17beta-HSD7_1 gene was mapped to chromosomal band 10p11.2. This newly identified form of 17beta-HSD7 could have a significant role by modulating active hormone levels in estrogen-sensitive cells or tissues.
J Steroid Biochem Mol Biol 2005 Feb
PMID:Cloning and characterization of human form 2 type 7 17beta-hydroxysteroid dehydrogenase, a primarily 3beta-keto reductase and estrogen activating and androgen inactivating enzyme. 1586 63

Androgen-dependent prostate diseases initially require 5alpha-dihydrotestosterone (DHT) for growth. The DHT product 5alpha-androstane-3alpha,17beta-diol (3alpha-diol), is inactive at the androgen receptor (AR), but induces prostate growth, suggesting that an oxidative 3alpha-hydroxysteroid dehydrogenase (HSD) exists. Candidate enzymes that posses 3alpha-HSD activity are type 3 3alpha-HSD (AKR1C2), 11-cis retinol dehydrogenase (RODH 5), L-3-hydroxyacyl coenzyme A dehydrogenase , RODH like 3alpha-HSD (RL-HSD), novel type of human microsomal 3alpha-HSD, and retinol dehydrogenase 4 (RODH 4). In mammalian transfection studies all enzymes except AKR1C2 oxidized 3alpha-diol back to DHT where RODH 5, RODH 4, and RL-HSD were the most efficient. AKR1C2 catalyzed the reduction of DHT to 3alpha-diol, suggesting that its role is to eliminate DHT. Steady-state kinetic parameters indicated that RODH 4 and RL-HSD were high-affinity, low-capacity enzymes whereas RODH 5 was a low-affinity, high-capacity enzyme. AR-dependent reporter gene assays showed that RL-HSD, RODH 5, and RODH 4 shifted the dose-response curve for 3alpha-diol a 100-fold, yielding EC(50) values of 2.5 x 10(-9) M, 1.5 x 10(-9) M, and 1.0 x 10(-9) M, respectively, when compared with the empty vector (EC(50) = 1.9 x 10(-7) M). Real-time RT-PCR indicated that L-3-hydroxyacyl coenzyme A dehydrogenase and RL-HSD were expressed more than 15-fold higher compared with the other candidate oxidative enzymes in human prostate and that RL-HSD and AR were colocalized in primary prostate stromal cells. The data show that the major oxidative 3alpha-HSD in normal human prostate is RL-HSD and may be a new therapeutic target for treating prostate diseases.
Mol Endocrinol 2006 Feb
PMID:Identification of the major oxidative 3alpha-hydroxysteroid dehydrogenase in human prostate that converts 5alpha-androstane-3alpha,17beta-diol to 5alpha-dihydrotestosterone: a potential therapeutic target for androgen-dependent disease. 1617 81


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