Selective loss of AKR1C1 and AKR1C2 in breast cancer and their potential effect on progesterone signaling.
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Progesterone plays an essential role in breast development and cancer formation. The local metabolism of progesterone may limit its interactions with the progesterone receptor (PR) and thereby act as a prereceptor regulator. Selective loss of AKR1C1, which encodes a 20alpha-hydroxysteroid dehydrogenase [20alpha-HSD (EC 1.1.1.149)], and AKR1C2, which encodes a 3alpha-hydroxysteroid dehydrogenase [3alpha-HSD (EC 1.1.1.52)], was found in 24 paired breast cancer samples as compared with paired normal tissues from the same individuals. In contrast, AKR1C3, which shares 84% sequence identity, and 5alpha-reductase type I (SRD5A1) were minimally affected. Breast cancer cell lines T-47D and MCF-7 also expressed reduced AKR1C1, whereas the breast epithelial cell line MCF-10A expressed AKR1C1 at levels comparable with those of normal breast tissues. Immunohistochemical staining confirmed loss of AKR1C1 expression in breast tumors. AKR1C3 and AKR1C1 were localized on the same myoepithelial and luminal epithelial cell layers. Suppression of ARK1C1 and AKR1C2 by selective small interfering RNAs inhibited production of 20alpha-dihydroprogesterone and was associated with increased progesterone in MCF-10A cells. Suppression of AKR1C1 alone or with AKR1C2 in T-47D cells led to decreased growth in the presence of progesterone. Overexpression of AKR1C1 and, to a lesser extent, AKR1C2 (but not AKR1C3) decreased progesterone-dependent PR activation of a mouse mammary tumor virus promoter in both prostate (PC-3) and breast (T-47D) cancer cell lines. We speculate that loss of AKR1C1 and AKR1C2 in breast cancer results in decreased progesterone catabolism, which, in combination with increased PR expression, may augment progesterone signaling by its nuclear receptors. (+info)
Expression and activity of steroid aldoketoreductases 1C in omental adipose tissue are positive correlates of adiposity in women.
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We examined expression and activity of steroid aldoketoreductase (AKR) 1C enzymes in adipose tissue in women. AKR1C1 (20alpha-hydroxysteroid dehydrogenase; 20alpha-HSD), AKR1C2 (3alpha-HSD-3), and AKR1C3 (17beta-HSD-5) are involved mainly in conversion of progesterone to 20alpha-hydroxyprogesterone and inactivation of dihydrotestosterone to 5alpha-androstane-3alpha,17beta-diol. Abdominal subcutaneous and omental adipose tissue biopsies were obtained during abdominal hysterectomies in seven women with low visceral adipose tissue (VAT) area and seven age- and total body fat mass-matched women with visceral obesity. Women with elevated VAT areas were characterized by significantly higher omental adipose tissue 20alpha-HSD and 3alpha-HSD-3 mRNA abundance compared with women with low VAT accumulations (1.4- and 1.6-fold differences, respectively; P < 0.05). Omental and subcutaneous adipose tissue 3alpha-HSD activities were significantly higher in women with high vs. low VAT areas (P < 0.05 for both comparisons). Total and visceral adiposities were positively associated with omental 20alpha-HSD mRNA level (r = 0.75, P < 0.003 for fat mass; r = 0.57, P < 0.04 for VAT area) and omental 3alpha-HSD-3 mRNA level (r = 0.68, P < 0.01 for fat mass; r = 0.74, P < 0.003 for VAT area). Enzyme activities in both depots were also positively correlated with adiposity measures. Omental adipose tissue enzyme expression and activity were positively associated with omental adipocyte size and LPL activity. In conclusion, mRNA abundance and activity of AKR1C enzymes in abdominal adipose tissue compartments are positive correlates of adiposity in women. Increased progesterone and/or dihydrotestosterone reduction in abdominal adipose tissue may impact locally on fat cell metabolism. (+info)
Tibolone metabolism in human liver is catalyzed by 3alpha/3beta-hydroxysteroid dehydrogenase activities of the four isoforms of the aldo-keto reductase (AKR)1C subfamily.
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Tibolone [[7alpha,17alpha]-17-hydroxy-7-methyl-19-norpregn-5(10)-en-20-yn-3-one] is used to treat climacteric symptoms and prevent osteoporosis. It exerts tissue-selective effects via site-specific metabolism into 3alpha- and 3beta-hydroxymetabolites and a Delta4-isomer. Recombinant human cytosolic aldo-keto reductases 1C1 and 1C2 (AKR1C1 and AKR1C2) produce 3beta-hydroxytibolone, and the liver-specific AKR1C4 produces predominantly 3alpha-hydroxytibolone. These observations may account for the appearance of 3beta-hydroxytibolone in target tissues and 3alpha-hydroxytibolone in the circulation. Using liver autopsy samples (which express AKR1C1-AKR1C4), tibolone was reduced via 3alpha- and 3beta-hydroxysteroid dehydrogenase (HSD) activity. 3beta-Hydroxytibolone was exclusively formed in the cytosol and was inhibited by the AKR1C2-specific inhibitor 5beta-cholanic acid-3alpha, 7alpha-diol. The cytosolic formation of 3alpha-hydroxytibolone was inhibited by an AKR1C4-selective inhibitor, phenolphthalein. The ratio of these stereoisomers was 4:1 in favor of 3beta-hydroxytibolone. In HepG2 cell cytosol and intact cells (which do not express AKR1C4), tibolone was exclusively reduced to 3beta-hydroxytibolone and was blocked by the AKR1C1-AKR1C3 inhibitor flufenamic acid. In primary hepatocytes (which express AKR1C1-AKR1C4), time-dependent reduction of tibolone into 3beta- and 3alpha-hydroxytibolone was observed again in a 4:1 ratio. 3beta-HSD activity was inhibited by both 5beta-cholanic acid-3alpha,7alpha-diol and flufenamic acid, implicating a role for AKR1C2 and AKR1C1. By contrast, the formation of 3alpha-hydroxytibolone was exclusively inhibited by phenolphthalein implicating AKR1C4 in this reaction. 3beta- and 3alpha-Hydroxytibolone were rapidly metabolized into polar metabolites (>85%). The formation of minor amounts of tibolone was also observed followed by AKR1C-catalyzed epimerization. The low hepatic formation of 3alpha-hydroxytibolone suggests that AKR1C4 is not the primary source of this metabolite and instead it maybe formed by an intestinal or enterobacterial 3alpha-HSD. (+info)
Human chorionic gonadotropin-dependent induction of an equine aldo-keto reductase (AKR1C23) with 20alpha-hydroxysteroid dehydrogenase activity during follicular luteinization in vivo.
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Aldo-keto reductases (AKRs) are multifunctional enzymes capable of acting on a wide variety of substrates, including sex steroids. AKRs having 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD) activity can reduce progesterone to 20alpha-hydroxy-4-pregnen-3-one (20alpha-DHP), a metabolite with lower affinity for the progesterone receptor. The objective of this study was to investigate the regulation of equine AKR1C23 during human chorionic gonadotropin (hCG)-induced ovulation/luteinization. The equine AKR1C23 cDNA was cloned and shown to encode a 322 amino acid protein that is conserved (71-81% identity) when compared with mammalian orthologs. RT-PCR/Southern blotting analyses were performed to study the regulation of AKR1C23 transcripts in equine preovulatory follicles isolated between 0 and 39 h after hCG treatment (ovulation occurring 39-42 h post-hCG). Results showed the presence of low AKR1C23 expression before hCG treatment, but a marked increase was observed in follicles obtained 12 h after hCG (P<0.05). Analyses of isolated preparations of granulosa and theca interna cells identified low mRNA expression in both cell types prior to hCG treatment, with granulosa cells clearly being the predominant site of follicular AKR1C23 mRNA induction. A specific polyclonal antibody was raised against a fragment of the equine protein and immunoblotting analyses showed an increase in AKR1C23 protein in granulosa cell extracts when comparing follicles isolated at 36 h post-hCG vs those collected prior to treatment, in keeping with mRNA results. Immunohistochemical data confirmed the induction of the enzyme in follicular cells after hCG treatment. The enzyme was tested for 20alpha-HSD activity and was shown to exhibit a K(M) of 3.12 microM, and a V(max) of 0.86 pmol/min per 10 microg protein towards progesterone. The levels of 20alpha-DHP measured in follicular fluid reflected this activity. Collectively, these results demonstrate for the first time that the gonadotropin-dependent induction of follicular luteinization is accompanied by an increase in AKR1C23 expression. Considering the 20alpha-HSD activity of AKR1C23, its regulated expression in luteinizing preovulatory follicles may provide a biochemical basis for the increase in ovarian 20alpha-DHP observed during gonadotropin-induced luteinization/ovulation. (The nucleotide sequence reported in this paper has been submitted to GenBank with accession number AY955082.). (+info)
Changes in mRNAs encoding steroidogenic acute regulatory protein, steroidogenic enzymes and receptors for gonadotropins during spermatogenesis in rainbow trout testes.
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In vertebrates, sperm development and maturation are directly regulated by gonadal steroid hormone secretion. The relationships among the expression of genes encoding steroidogenic proteins and receptors for gonadotropins, and testicular steroid production have not yet been comprehensively determined in male teleosts. In this study, the changes in levels of mRNAs encoding follicle-stimulating hormone (FSH) receptor, luteinizing hormone (LH) receptor, steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side-chain cleavage, 3beta-hydroxysteroid dehydrogenase/delta5-4-isomerase, cytochrome P450 17alpha-hydroxylase/17,20-lyase, cytochrome P450 11beta-hydroxylase, 11beta-hydroxysteroid dehydrogenase and 20beta-hydroxysteroid dehydrogenase were determined by real-time, quantitative PCR assays and related to changes in serum steroid levels throughout the reproductive cycle in male rainbow trout. Serum 11-ketotestosterone and 17alpha,20beta-dihydroxy-4-pregnen-3-one levels were measured by RIA. Although the pattern of change in the mRNA levels for the enzymes was variable, the increases in steroidogenic enzyme mRNAs started prior to a significant increase of serum steroid levels. The patterns of transcript levels of FSH and LH receptors suggest that changes in StAR and steroidogenic enzyme transcripts are largely mediated by the FSH receptor during early and mid-spermatogenesis and by the LH receptor during late spermatogenesis and spermiation. Levels of StAR (10-fold) and P450 17alpha-hydroxylase/17,20-lyase (sevenfold) transcripts changed with the greatest magnitude and were closely related to the changes in serum steroids, suggesting that changes in StAR and P450 17alpha-hydroxylase/17,20-lyase abundance are likely to be the major influences on overall steroidogenic output during the reproductive cycle in male rainbow trout. (+info)
Cloning and expression of chicken 20-hydroxysteroid dehydrogenase.
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The ligand specificity and activation of steroid receptors depend considerably on the enzymatic activities involved in local pre-receptor synthesis and the metabolism of the steroids. Several enzymes in particular, steroid dehydrogenases have been shown to participate in this process. Here we report the isolation of 20-hydroxysteroid dehydrogenase (ch20HSD) cDNA from chicken intestine and the distribution of ch20HSD mRNA and 20-reductase activity in various avian tissues. Using a reverse transcription PCR and comparison with the known sequences of mammalian 20betaHSDs, we have isolated a new ch20HSD cDNA. This cDNA predicted 276 amino acid residues that shared about 75% homology with mammalian 20betaHSD. Sequences specific to the short-chain dehydrogenase/reductase superfamily (SDR) were found, the Gly-X-X-X-Gly-X-Gly cofactor-binding motif (residues 11-17) and the catalytic activity motif Tyr-X-X-X-Lys (residues 193-197). The cDNA coding for ch20HSD was expressed in Escherichia coli by placing it under isopropylthiogalactoside (IPTG) inducible control. Both the IPTG cells of E. coli and the isolated recombinant protein reduced progesterone to 20-dihydroprogesterone, corticosterone to 20-dihydrocorticosterone and 5alpha-dihydrotestosterone to its 3-ol derivative. The 20-reductase and 3-reductase activities of ch20HSD catalyzed both 3alpha/beta- and 20alpha/20beta-epimers. The mRNA transcripts of ch20HSD were found in the kidney, colon, and testes; weaker expression was also found in the heart, ovaries, oviduct, brain, liver, and ileum. 20-Reductase activity has been proven in tissue slices of kidney, colon, ileum, liver, oviduct, testis, and ovary; whereas the activity was nearly absent in the heart and brain. A similar distribution of 20-reductase activity was found in tissue homogenates measured under V(max) conditions. These results suggest that chicken 20HSD is the latest member of the SDR superfamily to be found, is expressed in many avian tissues and whose precise role remains to be determined. (+info)
Hypothyroidism prolongs corpus luteum function in the pregnant rat.
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It has been shown that hypothyroidism in the rat produces a prolongation of pregnancy associated with a delay in the fall of circulating progesterone (P4) at term. The aim of the present work is to determine whether the delayed P4 decline in hypothyroid mother rats is due to a retarded induction of P4 degradation to 20alphaOH P4 or to a stimulation of its synthesis, and to investigate the possible mechanisms that may underlie the altered luteal function. We determined by RIA the circulating profile of the hormones (TSH, PRL, LH, P4, PGF2alpha, and PGE2) involved in luteal regulation at the end of pregnancy and, by semiquantitative RT-PCR, the expression of factors involved in P4 synthesis (CytP450scc, StAR, 3betaHSD, PRLR) and metabolism (20alphaHSD, PGF2alphaR, iNOS and COX2). Our results show that the delay in P4 decline and parturition is the resultant of retarded luteal regression, caused by a combination of decreases in luteolytic factors, mainly luteal PGF2alpha, iNOS mRNA expression and also circulating LH, and increased synthesis or action of luteotrophic factors, such as luteal and circulating PGE2 and circulating PRL. All these changes may be direct causes of the decreased 20alphaHSD mRNA and protein (measured by western blot analysis) expression, which in the presence of unchanged expression of the factors involved in P4 synthesis results in elevated luteal and circulating P4 that prolonged pregnancy and also may favor longer survival of the corpus luteum. (+info)
Impaired dihydrotestosterone catabolism in human prostate cancer: critical role of AKR1C2 as a pre-receptor regulator of androgen receptor signaling.
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We previously reported the selective loss of AKR1C2 and AKR1C1 in prostate cancers compared with their expression in paired benign tissues. We now report that dihydrotestosterone (DHT) levels are significantly greater in prostate cancer tumors compared with their paired benign tissues. Decreased catabolism seems to account for the increased DHT levels as expression of AKR1C2 and SRD5A2 was reduced in these tumors compared with their paired benign tissues. After 4 h of incubation with benign tissue samples, (3)H-DHT was predominantly catabolized to the 5alpha-androstane-3alpha,17beta-diol metabolite. Reduced capacity to metabolize DHT was observed in tumor samples from four of five freshly isolated pairs of tissue samples, which paralleled loss of AKR1C2 and AKR1C1 expression. LAPC-4 cells transiently transfected with AKR1C1 and AKR1C2, but not AKR1C3, were able to significantly inhibit a dose-dependent, DHT-stimulated proliferation, which was associated with a significant reduction in the concentration of DHT remaining in the media. R1881-stimulated proliferation was equivalent in all transfected cells, showing that metabolism of DHT was responsible for the inhibition of proliferation. PC-3 cells overexpressing AKR1C2 and, to a lesser extent, AKR1C1 were able to significantly inhibit DHT-dependent androgen receptor reporter activity, which was abrogated by increasing DHT levels. We speculate that selective loss of AKR1C2 in prostate cancer promotes clonal expansion of tumor cells by enhancement of androgen-dependent cellular proliferation by reducing DHT metabolism. (+info)