RESUMEN
In an effort to understand the origin of blood-pressure lowering effects observed in recent clinical trials with 11ß-HSD1 inhibitors, we examined a set of 11ß-HSD1 inhibitors in a series of relevant in vitro and in vivo assays. Select 11ß-HSD1 inhibitors reduced blood pressure in our preclinical models but most or all of the blood pressure lowering may be mediated by a 11ß-HSD1 independent pathway.
Asunto(s)
11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Hipertensión/tratamiento farmacológico , Hipertensión/enzimología , Triazoles/farmacología , Animales , Humanos , Ratones , Ratones Noqueados , Ratas , Ratas Endogámicas SHRRESUMEN
Cerebral 3α-hydroxysteroid dehydrogenase (3α-HSD) activity was suggested to be responsible for the local directed formation of neuroactive 5α,3α-tetrahydrosteroids (5α,3α-THSs) from 5α-dihydrosteroids. We show for the first time that within human brain tissue 5α-dihydroprogesterone and 5α-dihydrotestosterone are converted via non-stereo-selective 3-ketosteroid reductase activity to produce the respective 5α,3α-THSs and 5α,3ß-THSs. Apart from this, we prove that within the human temporal lobe and limbic system cytochrome P450c17 and 3ß-HSD/Δ(5-4) ketosteroid isomerase are not expressed. Thus, it appears that these brain regions are unable to conduct de novo biosynthesis of Δ(4)-3-ketosteroids from Δ(5)-3ß-hydroxysteroids. Consequently, the local formation of THSs will depend on the uptake of circulating Δ(4)-3-ketosteroids such as progesterone and testosterone. 3α- and 3ß-HSD activity were (i) equally enriched in the cytosol, (ii) showed equal distribution between cerebral neocortex and subcortical white matter without sex- or age-dependency, (iii) demonstrated a strong and significant positive correlation when comparing 46 different specimens and (iv) exhibited similar sensitivities to different inhibitors of enzyme activity. These findings led to the assumption that cerebral 3-ketosteroid reductase activity might be catalyzed by a single enzyme and is possibly attributed to the expression of a soluble AKR1C aldo-keto reductase. AKR1Cs are known to act as non-stereo-selective 3-ketosteroid reductases; low AKR1C mRNA expression was detected. However, the cerebral 3-ketosteroid reductase was clearly refractory to inhibition by AKR1C inhibitors indicating the expression of a currently unidentified enzyme. Its lack of stereo-selectivity is of physiological significance, since only 5α,3α-THSs enhance the effect of GABA on the GABA(A) receptor, whereas 5α,3ß-THSs are antagonists.
Asunto(s)
20-Hidroxiesteroide Deshidrogenasas/genética , Encéfalo/metabolismo , Regulación Enzimológica de la Expresión Génica , 20-Hidroxiesteroide Deshidrogenasas/antagonistas & inhibidores , 3-Hidroxiesteroide Deshidrogenasas/metabolismo , Adulto , Anciano , Encéfalo/patología , Línea Celular Tumoral , Cromatografía en Capa Delgada/métodos , Femenino , Cromatografía de Gases y Espectrometría de Masas/métodos , Humanos , Lactante , Persona de Mediana Edad , Esteroide 17-alfa-Hidroxilasa/genética , Esteroides/química , Lóbulo Temporal/patologíaRESUMEN
25-Hydroxycholesterol is produced in mammalian tissues. The function of this oxysterol is unknown. Here we describe a central role for 25-hydroxycholesterol in regulating the immune system. In initial experiments, we found that stimulation of macrophage Toll-like receptors (TLR) induced expression of cholesterol 25-hydroxylase and the synthesis of 25-hydroxycholesterol. Treatment of naïve B cells with nanomolar concentrations of 25-hydroxycholesterol suppressed IL-2-mediated stimulation of B cell proliferation, repressed activation-induced cytidine deaminase (AID) expression, and blocked class switch recombination, leading to markedly decreased IgA production. Consistent with these findings, deletion of the mouse cholesterol 25-hydroxylase gene caused an increase in serum IgA. Conversely, inactivation of the CYP7B1 oxysterol 7alpha-hydroxylase, which degrades 25-hydroxycholesterol, decreased serum IgA. The suppression of IgA class switching in B cells by a macrophage-derived sterol in response to TLR activation provides a mechanism for local and systemic negative regulation of the adaptive immune response by the innate immune system.
Asunto(s)
Hidroxicolesteroles/metabolismo , Inmunoglobulina A/biosíntesis , Macrófagos/metabolismo , Receptores Toll-Like/metabolismo , Animales , Linfocitos B/metabolismo , Citocinas/metabolismo , Ratones , Ratones Transgénicos , Esteroide Hidroxilasas/genética , Esteroide Hidroxilasas/metabolismoRESUMEN
The CYP7B1 cytochrome P450 enzyme hydroxylates carbons 6 and 7 of the B ring of oxysterols and steroids. Hydroxylation reduces the biological activity of these substrates and facilitates their conversion to end products that are readily excreted from the body. CYP7B1 is expressed in the liver, reproductive tract, and brain and performs different physiological functions in each tissue. Hepatic CYP7B1 activity is crucial for the inactivation of oxysterols and their subsequent conversion into bile salts. Loss of CYP7B1 activity is associated with liver failure in children. In the reproductive tract, the enzyme metabolizes androgens that antagonize estrogen action; mice without CYP7B1 have abnormal prostates and ovaries. The role of CYP7B1 in brain is under investigation; recent studies show that spastic paraplegia type 5, a progressive neuropathy, is caused by loss-of-function mutations in the human gene.
Asunto(s)
Fallo Hepático/genética , Enfermedades del Sistema Nervioso/genética , Esteroide Hidroxilasas/genética , Esteroide Hidroxilasas/fisiología , Animales , Aspergillus niger/metabolismo , Niño , Familia 7 del Citocromo P450 , Humanos , Inmunoglobulinas/metabolismo , Ligandos , Fallo Hepático/diagnóstico , Ratones , Modelos Químicos , Enfermedades del Sistema Nervioso/diagnóstico , ARN Mensajero/metabolismo , Ratas , Esteroides/metabolismoRESUMEN
The human androgen receptor (AR) is a ligand-activated nuclear transcription factor and mediates the induction of genes involved in the development of the male phenotype and male secondary sex characteristics, as well as the normal and abnormal growth of the prostate. We have identified the pair of hydroxysteroid dehydrogenases (HSDs) that regulate ligand access to the AR in human prostate. We find that type 3 3alpha-HSD (aldo-keto reductase (AKR)1C2) catalyzes the NADPH dependent reduction of the potent androgen 5alpha-dihydrotestosterone (5alpha-DHT) to yield the inactive androgen 3alpha-androstanediol (3alpha-diol). We also find that RoDH like 3alpha-HSD (RL-HSD) catalyzes the NAD(+) dependent oxidation of 3alpha-diol to yield 5alpha-DHT. Together these enzymes are involved in the pre-receptor regulation of androgen action. Inhibition of AKR1C2 would be desirable in cases of androgen insufficiency and inhibition of RL-HSD might be desirable in benign prostatic hyperplasia.
Asunto(s)
Regulación de la Expresión Génica , Receptores Androgénicos/genética , Oxidorreductasas de Alcohol/metabolismo , Oxidorreductasas de Alcohol/fisiología , Aldehído Reductasa , Aldo-Ceto Reductasas , Andrógenos/biosíntesis , Andrógenos/metabolismo , Regulación Enzimológica de la Expresión Génica , Humanos , Hidroxiesteroide Deshidrogenasas/genética , Hidroxiesteroide Deshidrogenasas/metabolismo , Hidroxiesteroide Deshidrogenasas/fisiología , Ligandos , Masculino , Modelos Biológicos , Modelos Moleculares , Próstata/metabolismoRESUMEN
Pairs of hydroxysteroid dehydrogenases (HSDs) govern ligand access to steroid receptors in target tissues and act as molecular switches. By acting as reductases or oxidases, HSDs convert potent ligands into their cognate inactive metabolites or vice versa. This pre-receptor regulation of steroid hormone action may have profound effects on hormonal response. We have identified the HSDs responsible for regulating ligand access to the androgen receptor (AR) in human prostate. Type 3 3alpha-hydroxysteroid dehydrogenase (aldo-keto reductase 1C2) acts solely as a reductase to convert 5alpha-dihydrotestosterone (DHT), a potent ligand for the AR (K(d)=10(-11)M for the AR), to the inactive androgen 3alpha-androstanediol (K(d)=10(-6)M for the AR); while RoDH like 3alpha-HSD (a short-chain dehydrogenase/reductase (SDR)) acts solely as an oxidase to convert 3alpha-androstanediol back to 5alpha-DHT. Our studies suggest that aldo-keto reductase (AKRs) and SDRs function as reductases and oxidases, respectively, to control ligand access to nuclear receptors.
Asunto(s)
Dihidrotestosterona/metabolismo , Hidroxiesteroide Deshidrogenasas/metabolismo , Receptores Androgénicos/metabolismo , Andrógenos/metabolismo , Animales , Humanos , Isoenzimas/metabolismo , Masculino , Próstata/metabolismoRESUMEN
Human prostate adenocarcinoma (CaP) and benign prostatic hyperplasia (BPH) have epithelial and stromal cell origins, respectively. To determine whether the androgen signal is processed differently in these cell types the expression of transcripts for enzymes that control ligand access to the androgen receptor (AR) were measured. Transcripts for type 2 5alpha-reductase, ketosteroid reductases [aldo-keto reductase (AKR)1C1-AKR1C4], the major oxidative 3alpha-hydroxysteroid dehydrogenase (HSD) retinol dehydrogenase (RODH)-like 3alpha-HSD (RL-HSD) and nuclear receptors [AR, estrogen receptor (ER)alpha, and ERbeta] were determined in whole human prostate and in cultures of primary epithelial cells (PEC) and primary stromal cells (PSC) from normal prostate, CaP and BPH by real-time RT-PCR. Normal PEC (n=14) had higher levels of AKR1C1 (10-fold, P<0.001), AKR1C2 (115-fold, P<0.001) and AKR1C3 (6-fold, P<0.001) than normal PSC (n=15), suggesting that reductive androgen metabolism occurs. By contrast, normal PSC had higher levels of AR (8-fold, P<0.001) and RL-HSD (21-fold, P<0.001) than normal PEC, suggesting that 3alpha-androstanediol is converted to 5alpha-dihydrotestosterone to activate AR. In CaP PEC (n=14), no significant changes in transcript levels vs. normal PEC were observed. In BPH PSC (n=21) transcripts for AR (2-fold, P<0.001), AKR1C1 (4-fold, P<0.001), AKR1C2 (10-fold P<0.001), AKR1C3 (4-fold, P<0.001) and RL-HSD (3-fold, P<0.003) were elevated to increase androgen response. Differences in the AR:ERbeta transcript ratios (eight in normal PEC vs. 280 in normal PSC) were maintained in PEC and PSC in diseased prostate. These data suggest that CaP may be more responsive to an ERbeta agonist and BPH may be more responsive to androgen ablation.
Asunto(s)
Andrógenos/metabolismo , Carcinoma/metabolismo , Perfilación de la Expresión Génica/métodos , Próstata/metabolismo , Hiperplasia Prostática/metabolismo , Neoplasias de la Próstata/metabolismo , 20-Hidroxiesteroide Deshidrogenasas/metabolismo , Células Epiteliales/metabolismo , Humanos , Hidroxiesteroide Deshidrogenasas/metabolismo , Masculino , Modelos Biológicos , Especificidad de Órganos , Receptores Androgénicos/metabolismo , Receptores de Estrógenos/metabolismo , Transducción de Señal , Células Tumorales CultivadasRESUMEN
The current arsenal of tools and methods for the continuous monitoring and imaging of redox metabolic pathways in the context of intact cells is limited. Fluorogenic substrates allow for direct measurement of enzyme activity in situ; however, in contrast to proteases and exo-glycosidases, there are no simple guidelines for the design of selective probes for redox metabolic enzymes. Here, we introduce redox probe 1 and demonstrate its high selectivity in living cells for human hydroxysteroid dehydrogenases (HSDs) of the aldo-keto reductase (AKR) superfamily. AKR1C isoforms perform multiple functions among which the metabolism of potent steroid hormones is well documented. Moreover, expression of these enzymes is responsive to cellular stress and pathogenesis, including cancer. Our probe design is based on redox-sensitive optical switches, which couple a ketone-alcohol redox event to a profound change in fluorescence. The high selectivity of phenyl ketone 1 for AKR1C2 over the many endogenous reductases present in mammalian cells was established by a quantitative comparison of the metabolic rates between null control cells (COS-1) and AKR1C2-transfected cells. Phenyl ketone 1 is a cell-permeable fluorogenic probe that permits a direct, real-time, and operationally simple readout of AKR1C2 enzyme activity in intact mammalian cells. Furthermore, it was demonstrated that probe 1 enables the quantitative examination of physiological substrate 5alpha-dihydrotestosterone ("dark substrate") in situ by means of a two-substrate competitive assay. Similarly, inhibitor potency of physiological (ursodeoxycholate) and synthetic inhibitors (flufenamic acid, ibuprofen, and naproxen) was also readily evaluated.
Asunto(s)
Colorantes Fluorescentes/metabolismo , Hidroxiesteroide Deshidrogenasas/análisis , Animales , Células COS , Chlorocebus aethiops , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/química , Humanos , Hidroxiesteroide Deshidrogenasas/química , Hidroxiesteroide Deshidrogenasas/metabolismo , Cinética , Estructura Molecular , Oxidación-Reducción , Sensibilidad y Especificidad , Espectrometría de Fluorescencia , Especificidad por SustratoRESUMEN
Human aldo-keto reductases (AKR) of the 1A, 1B, 1C and 1D subfamilies are involved in the pre-receptor regulation of nuclear (steroid hormone and orphan) receptors by regulating the local concentrations of their lipophilic ligands. AKR1C3 is one of the most interesting isoforms. It was cloned from human prostate and the recombinant protein was found to function as a 3-, 17- and 20-ketosteroid reductase with a preference for the conversion of Delta4-androstene-3,17-dione to testosterone implicating this enzyme in the local production of active androgens within the prostate. Using a validated isoform specific real-time RT-PCR procedure the AKR1C3 transcript was shown to be more abundant in primary cultures of epithelial cells than stromal cells, and its expression in stromal cells increased with benign and malignant disease. Using a validated isoform specific monoclonal Ab, AKR1C3 protein expression was also detected in prostate epithelial cells by immunoblot analysis. Immunohistochemical staining of prostate tissue showed that AKR1C3 was expressed in adenocarcinoma and surprisingly high expression was observed in the endothelial cells. These cells are a rich source of prostaglandin G/H synthase 2 (COX-2) and vasoactive prostaglandins (PG) and thus the ability of recombinant AKR1C enzymes to act as PGF synthases was compared. AKR1C3 had the highest catalytic efficiency (kcat/Km) for the 11-ketoreduction of PGD2 to yield 9alpha,11beta-PGF2 raising the prospect that AKR1C3 may govern ligand access to peroxisome proliferator activated receptor (PPARgamma). Activation of PPARgamma is often a pro-apoptotic signal and/or leads to terminal differentiation, while 9alpha,11beta-PGF2 is a pro-proliferative signal. AKR1C3 is potently inhibited by non-steroidal anti-inflammatory drugs suggesting that the cancer chemopreventive properties of these agents may be mediated either by inhibition of AKR1C3 or COX. To discriminate between these effects we developed potent AKR1C inhibitors based on N-phenylanthranilic acids that do not inhibit COX-1 or COX-2. These compounds can now be used to determine the role of AKR1C3 in producing two proliferative signals in the prostate namely testosterone and 9alpha,11beta-PGF2.
Asunto(s)
3-Hidroxiesteroide Deshidrogenasas/antagonistas & inhibidores , Antiinflamatorios no Esteroideos/farmacología , Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Hidroxiprostaglandina Deshidrogenasas/antagonistas & inhibidores , Enfermedades de la Próstata/enzimología , 3-Hidroxiesteroide Deshidrogenasas/análisis , 3-Hidroxiesteroide Deshidrogenasas/fisiología , Miembro C3 de la Familia 1 de las Aldo-Ceto Reductasas , Antiinflamatorios no Esteroideos/síntesis química , Antiinflamatorios no Esteroideos/química , Dinoprost/biosíntesis , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Hormonas Esteroides Gonadales/biosíntesis , Hormonas Esteroides Gonadales/metabolismo , Humanos , Hidroxiprostaglandina Deshidrogenasas/análisis , Hidroxiprostaglandina Deshidrogenasas/fisiología , Masculino , Próstata/enzimología , Enfermedades de la Próstata/genética , Relación Estructura-Actividad , Testosterona/biosíntesis , Transcripción GenéticaRESUMEN
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.
Asunto(s)
3-alfa-Hidroxiesteroide Deshidrogenasa (B-Específica)/metabolismo , Andrógenos/metabolismo , Androstano-3,17-diol/metabolismo , Dihidrotestosterona/metabolismo , Próstata/enzimología , Enfermedades de la Próstata/enzimología , 3-alfa-Hidroxiesteroide Deshidrogenasa (B-Específica)/antagonistas & inhibidores , 3-alfa-Hidroxiesteroide Deshidrogenasa (B-Específica)/genética , Animales , Células Cultivadas , Ácido Graso Sintasas/genética , Humanos , Masculino , NADH NADPH Oxidorreductasas/genética , Próstata/metabolismo , Enfermedades de la Próstata/tratamiento farmacológico , Enfermedades de la Próstata/metabolismo , Receptores Androgénicos/genética , Activación Transcripcional , TransfecciónRESUMEN
Human aldo-keto reductases (AKRs) regulate nuclear receptors by controlling ligand availability. Enzymes implicated in regulating ligand occupancy and trans-activation of the nuclear receptors belong to the AKR1C family (AKR1C1-AKR1C3). Nuclear receptors regulated by AKR1C members include the steroid hormone receptors (androgen, estrogen, and progesterone receptors) and the orphan peroxisome proliferator-activated receptor (PPARgamma). In human myeloid leukemia (HL-60) cells, ligand access to PPARgamma is regulated by AKR1C3, which diverts PGD(2) metabolism away from J-series prostanoids (Desmond et al., 2003). Inhibition of AKR1C3 by indomethacin, a nonsteroidal anti-inflammatory drug (NSAID), caused PPARgamma-mediated terminal differentiation of the HL-60 cells. To discriminate between antineoplastic effects of NSAIDs that are mediated by either AKR1C or cyclooxygenase (COX) isozymes, selective inhibitors are required. We report a structural series of N-phenylanthranilic acid derivatives and steroid carboxylates that selectively inhibit recombinant AKR1C isoforms but do not inhibit recombinant COX-1 or COX-2. The inhibition constants, IC(50), K(I) values, and inhibition patterns were determined for the NSAID analogs and steroid carboxylates against AKR1C and COX isozymes. Lead compounds, 4-chloro-N-phenylanthranilic acid and 4-benzoyl-benzoic acid for the N-phenylanthranilic acid analogs and most steroid carboxylates, exhibited IC(50) values that had greater than 500-fold selectivity for AKR1C isozymes compared with COX-1 and COX-2. Crystallographic and molecular modeling studies showed that the carboxylic acid of the inhibitor ligand was tethered by the catalytic Tyr55-OH(2)(+) and explained why A-ring substituted N-phenylanthranilates inhibited only AKR1C enzymes. These compounds can be used to dissect the role of the AKR1C isozymes in neoplastic diseases and may have cancer chemopreventive roles independent of COX inhibition.
Asunto(s)
Oxidorreductasas de Alcohol/metabolismo , Antiinflamatorios no Esteroideos/farmacología , Ácidos Carboxílicos/farmacología , Isoenzimas/metabolismo , Oxidorreductasas de Alcohol/efectos de los fármacos , Aldehído Reductasa , Aldo-Ceto Reductasas , Antiinflamatorios no Esteroideos/síntesis química , Sitios de Unión , Ácidos Carboxílicos/síntesis química , Inhibidores de la Ciclooxigenasa/farmacología , Ácido Flufenámico/química , Ácido Flufenámico/farmacología , Células HL-60 , Humanos , Isoenzimas/efectos de los fármacos , Cinética , Ligandos , Modelos Moleculares , Prostaglandina-Endoperóxido Sintasas/metabolismo , Esteroides/síntesis química , Esteroides/farmacologíaRESUMEN
The human aldo-keto reductase 1C (AKR1C) isozymes are implicated in the pre-receptor regulation of steroid receptors, nuclear orphan receptors and membrane-bound ligand-gated ion channels. Human AKR members that may regulate the local concentration of steroid hormones include: AKR1C1, AKR1C2, AKR1C3, AKR1C4 and AKR1D1. Since, these enzymes are pluripotent, the physiological role for the human AKR1C isozymes is determined by their tissue-specific expression patterns and their substrate availability in target tissues. AKRs work in concert with short-chain dehydrogenases/reductases as switches to control ligand access to nuclear receptors. Consequently, they are potential targets in treating prostate cancer, breast cancer, endometriosis and endometrial cancer.
Asunto(s)
Oxidorreductasas de Alcohol/fisiología , Hormonas Esteroides Gonadales/farmacología , Oxidorreductasas de Alcohol/efectos de los fármacos , Oxidorreductasas de Alcohol/farmacología , Aldehído Reductasa , Aldo-Ceto Reductasas , Animales , Hormonas Esteroides Gonadales/fisiología , Humanos , Isoenzimas/química , Isoenzimas/farmacología , Isoenzimas/fisiologíaRESUMEN
Human aldo-keto reductases (AKRs) of the AKR1C subfamily function in vitro as 3-keto-, 17-keto-, and 20-ketosteroid reductases or as 3alpha-, 17beta-, and 20alpha-hydroxysteroid oxidases. These AKRs can convert potent sex hormones (androgens, estrogens, and progestins) into their cognate inactive metabolites or vice versa. By controlling local ligand concentration AKRs may regulate steroid hormone action at the prereceptor level. AKR1C2 is expressed in prostate, and in vitro it will catalyze the nicotinamide adenine dinucleotide (NAD(+))-dependent oxidation of 3alpha-androstanediol (3alpha-diol) to 5alpha-dihydrotestosterone (5alpha-DHT). This reaction is potently inhibited by reduced NAD phosphate (NADPH), indicating that the NAD(+): NADPH ratio in cells will determine whether AKR1C2 makes 5alpha-DHT. In transient COS-1-AKR1C2 and in stable PC-3-AKR1C2 transfectants, 5alpha-DHT was reduced by AKR1C2. However, the transfected AKR1C2 oxidase activity was insufficient to surmount the endogenous 17beta-hydroxysteroid dehydrogenase (17beta-HSD) activity, which eliminated 3alpha-diol as androsterone. PC-3 cells expressed retinol dehydrogenase/3alpha-HSD and 11-cis-retinol dehydrogenase, but these endogenous enzymes did not oxidize 3alpha-diol to 5alpha-DHT. In stable LNCaP-AKR1C2 transfectants, AKR1C2 did not alter androgen metabolism due to a high rate of glucuronidation. In primary cultures of epithelial cells, high levels of AKR1C2 transcripts were detected in prostate cancer, but not in cells from normal prostate. Thus, in prostate cells AKR1C2 acts as a 3-ketosteroid reductase to eliminate 5alpha-DHT and prevents activation of the androgen receptor. AKR1C2 does not act as an oxidase due to either potent product inhibition by NADPH or because it cannot surmount the oxidative 17beta-HSD present. Neither AKR1C2, retinol dehydrogenase/3alpha-HSD nor 11-cis-retinol dehydrogenase is a source of 5alpha-DHT in PC-3 cells.
Asunto(s)
Hidroxiesteroide Deshidrogenasas/metabolismo , Próstata/enzimología , Testosterona/metabolismo , 17-Hidroxiesteroide Deshidrogenasas/metabolismo , Adenocarcinoma , Androstano-3,17-diol/química , Androstano-3,17-diol/metabolismo , Animales , Neoplasias Óseas , Células COS , Dihidrotestosterona/química , Dihidrotestosterona/metabolismo , Células Epiteliales/citología , Células Epiteliales/enzimología , Regulación Enzimológica de la Expresión Génica , Humanos , Hidroxiesteroide Deshidrogenasas/genética , Técnicas In Vitro , Isoenzimas/metabolismo , Masculino , Próstata/citología , Neoplasias de la Próstata , Testosterona/química , Células Tumorales CultivadasRESUMEN
The aldo-keto reductases (AKRs) are one of the three enzyme superfamilies that perform oxidoreduction on a wide variety of natural and foreign substrates. A systematic nomenclature for the AKR superfamily was adopted in 1996 and was updated in September 2000 (visit www.med.upenn.edu/akr). Investigators have been diligent in submitting sequences of functional proteins to the Web site. With the new additions, the superfamily contains 114 proteins expressed in prokaryotes and eukaryotes that are distributed over 14 families (AKR1-AKR14). The AKR1 family contains the aldose reductases, the aldehyde reductases, the hydroxysteroid dehydrogenases and steroid 5beta-reductases, and is the largest. Other families of interest include AKR6, which includes potassium channel beta-subunits, and AKR7 the aflatoxin aldehyde reductases. Two new families include AKR13 (yeast aldose reductase) and AKR14 (Escherichia coli aldehyde reductase). Crystal structures of many AKRs and their complexes with ligands are available in the PDB and accessible through the Web site. Each structure has the characteristic (alpha/beta)(8)-barrel motif of the superfamily, a conserved cofactor binding site and a catalytic tetrad, and variable loop structures that define substrate specificity. Although the majority of AKRs are monomeric proteins of about 320 amino acids in length, the AKR2, AKR6 and AKR7 family may form multimers. To expand the nomenclature to accommodate multimers, we recommend that the composition and stoichiometry be listed. For example, AKR7A1:AKR7A4 (1:3) would designate a tetramer of the composition indicated. The current nomenclature is recognized by the Human Genome Project (HUGO) and the Web site provides a link to genomic information including chromosomal localization, gene boundaries, human ESTs and SNPs and much more.