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

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.     

Oxidative 3alpha-hydroxysteroid dehydrogenase activity of human type 10 17beta-hydroxysteroid dehydrogenase

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.     

Characterization of the oxidative 3alpha-hydroxysteroid dehydrogenase activity of human recombinant 11-cis-retinol dehydrogenase.

11-cis-Retinol dehydrogenase catalyzes the oxidation of cis-retinols, a rate-limiting step in the biosynthesis of 9-cis-retinoic acid. It is also active toward 3alpha-hydroxysteroids, and thus might be involved in steroid metabolism. To better understand the role of this enzyme, we produced stable transfectants expressing 11-cis-retinol dehydrogenase in human embryonic kidney 293 cells. In vitro enzymatic assays have demonstrated that, with an appropriate exogenous cofactor, the enzyme catalyzes the interconversion of 5alpha-androstane-3alpha,17beta-diol and dihydrotestosterone and that of androsterone and androstanedione. However, using intact transfected cells, we found that the enzyme catalyzes reactions only in the oxidative direction. Thus, it is possible that 5alpha-androstane-3alpha,17beta-diol (an inactive androgen) can be converted into dihydrotestosterone, the most potent androgen, by the action of 11-cis-retinol dehydrogenase. This reaction could constitute a non-classical pathway of production of active androgens in the peripheral tissues. We also showed that all-trans-, 9-cis- and 13-cis-retinol inhibit the oxidative 3alpha-hydroxysteroid steroid activity of 11-cis-retinol dehydrogenase with similar K(i) values. Since all-trans-retinol is a precursor of cis-retinols, its inhibitory effect on the activity suggests that it could play an important role in modulating the formation of 9-cis-retinoic acid. In addition, we examined the effect of several known enzyme modulators, namely carbenoxolone, phenylarsine oxide and phosphatidylcholine, on 11-cis-retinol dehydrogenase activity. Taken together, our results suggest that, in humans, this enzyme might play a role in the biosynthesis of both 9-cis-retinoic acid and dihydrotestosterone.     

Androgen metabolism in rat L6 myoblast cells; high formation of 5 alpha-androstane-3 alpha,17 beta-diol from testosterone

We have studied androgen metabolism in L6 rat myoblasts. 4-androstene-3,17-dione (Adione), testosterone, 5 alpha-dihydrotestosterone (DHT), and 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-diol) were used for substrates and the amounts of metabolites formed from the respective substrates in the medium were determined. Conversion of Adione to testosterone was dominant over the reverse conversion. DHT formation from testosterone was low and did not change with the duration of incubation, whereas 3 alpha-diol formation increased in a time-dependent manner. Major metabolite of testosterone was not DHT but 3 alpha-diol. A large amount of 3 alpha-diol was formed from DHT, however, DHT formation from 3 alpha-diol was very low. These data indicate that L6 cells have high 5 alpha-reductase activity and suggest that DHT formed from testosterone is rapidly metabolized to 3 alpha-diol in these cells.     

Isolation and characterization of a cDNA encoding mouse 3alpha-hydroxysteroid dehydrogenase: an androgen-inactivating enzyme selectively expressed in female tissues

3alpha-Hydroxysteroid dehydrogenase catalyzes the transformation of 3-ketosteroids into 3alpha-hydroxysteroids, thus playing an important role in androgen and progesterone metabolism. So far, mouse cDNA and gene encoding 3alpha-HSD has not been reported. In this report, we describe the isolation of a mouse 3alpha-HSD cDNA and the characterization of its substrate specificity and tissue distribution. Sequence analysis indicates that m3alpha-HSD shares 87% amino acid identity with rat 3alpha-HSD. Cells stably transfected with this enzyme catalyze the transformation of dihydrotestosterone (DHT), 5alpha-androstanedione (5alpha-dione) and dihydroprogesterone (DHP) into 5alpha-androstane-3alpha,17beta-diol (3alpha-diol), androsterone (ADT) and 5alpha-pregnan-3alpha-ol-20-one (allopregnanolone), respectively. Quantification of mRNA expression levels of this enzyme was determined in male and female mouse sex-specific tissues using quantitative Realtime PCR. We show that this enzyme is mainly expressed in female-specific tissues while being almost absent from male-specific tissues. In the liver, the same expression level is seen in both male and female, while there is 6-fold higher expression level in female pituitary than in male. These results strongly suggest that m3alpha-HSD could play an important role in the female mouse physiology similar to that of type 1 5alpha-reductase with which it works in tandem. This role could be related to the inactivation of excess of androgen and progesterone that are more severely regulated than in man.     

Nicotine and cotinine effects on 3 alpha hydroxysteroid dehydrogenase in canine prostate

We have recently observed that cigarette smoking affects plasma androgen concentrations. The effects of nicotine and cotinine, two products of cigarette smoking, on testosterone metabolism were determined. The activity of delta 4 steroid 5 alpha-reductase, which converts testosterone to 5 alpha-dihydrotestosterone (DHT) was measured in isolated dog prostate nuclei using testosterone (0-200 nM) as substrate and NADPH as cofactor. Activity of 3 alpha-hydroxysteroid dehydrogenase (HSD), which converts DHT to 3 alpha-androstanediol (3 alpha-diol) and is a reversible enzyme, was measured in isolated dog prostate microsomes with DHT (0-20 microM) as substrate and NADPH as cofactor. When microsomal fractions were incubated for 1 hour with and without nicotine (0-50 microM) and cotinine (0-100 microM), enzyme activity of HSD was significantly suppressed (p less than 0.001). The Vmax was not affected significantly (p greater than 0.60) and Km increased with increasing concentrations of nicotine and cotinine (p less than 0.05). Both nicotine and cotinine are competitive inhibitors of HSD in dog prostate microsomes with Ki's of 61 and 89 microM, respectively. The apparent 5 alpha-reductase activity was unaffected by nicotine and cotinine. The inhibitors produced a marked effect on activity of HSD when used in concentrations achieved in humans who smoke cigarettes. The results suggest that nicotine and cotinine are competitive inhibitors of the HSD, an important enzyme involved in the metabolism of DHT and produce an accumulation of DHT. These products of cigarette smoking could alter androgen action in tissue such as skin and prostate.     

Opposing changes in 3alpha-hydroxysteroid dehydrogenase oxidative and reductive activities in rat leydig cells during pubertal development

The enzyme 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) has an important role in androgen metabolism, catalyzing the interconversion of dihydrotestosterone (DHT) and 5alpha-androstane-3alpha,17beta-diol (3alpha-DIOL). The net direction of this interconversion will affect the amount of biologically active ligand available for androgen receptor binding. We hypothesize that in Leydig cells, differential expression of 3alpha-HSD enzymes favoring one of the two directions is a mechanism by which DHT levels are controlled. In order to characterize 3alpha-HSD in rat Leydig cells, the following properties were analyzed: rates of oxidation (3alpha-DIOL to DHT) and reduction (DHT to 3alpha-DIOL) and preference for the cofactors NADP(H) and NAD(H) (i.e., the oxidized and reduced forms of both pyridine nucleotides) in Leydig cells isolated on Days 21, 35, and 90 postpartum. Levels of 3alpha-HSD protein were measured by immunoblotting using an antibody directed against the liver type of the enzyme. Levels of 3alpha-HSD protein and rates of reduction were highest on Day 21 and lowest on Day 90. The opposite was true for the rate of 3alpha-HSD oxidation, which was barely detectable on Day 21 and highest on Day 90 (59.08 +/- 6.35 pmol/min per 10(6) cells, mean +/- SE). Therefore, the level of 3alpha-HSD protein detectable by liver enzyme was consistent with reduction but not with oxidation. There was a clear partitioning of NADP(H)-dependent activity into the cytosolic fraction of Leydig cells, whereas on Days 35 and 90, Leydig cells also contained a microsomal NAD(H)-activated 3alpha-HSD. We conclude that 1) the cytosolic 3alpha-HSD in Leydig cells on Day 21 behaves as a unidirectional NADPH-dependent reductase; 2) by Day 35, a microsomal NAD(H)-dependent enzyme activity is present and may account for predominance of 3alpha-HSD oxidation over reduction and the resultant high capacity of Leydig cells on Day 90 to synthesize DHT from 3alpha-DIOL.     

Bioinorganic and bioorganic studies of liver alcohol dehydrogenase

Liver alcohol dehydrogenase (E.C.1.1.1.1) is an NAD(+)/NADH dependent enzyme with a broad substrate specificity being active on an assortment of primary and secondary alcohols. It catalyzes the reversible oxidation of a wide variety of alcohols to the corresponding aldehydes and ketones as well as the oxidation of certain aldehydes to their related carboxylic acids. Although the bioinorganic and bioorganic aspects of the enzymatic mechanism, as well as the structures of various ternary complexes, have been extensively studied, the kinetic significance of certain intermediates has not been fully evaluated. Nevertheless, the availability of computer-assisted programs for kinetic simulation and molecular modeling make it possible to describe the biochemical mechanism more completely. Although the true physiological substrates of this zinc metalloenzyme are unknown, alcohol dehydrogenase effectively catalyzes not only the interconversion of all-trans-retinol and all-trans-retinal but also the oxidation of all-trans-retinal to the corresponding retinoic acid. Retinal and related vitamin A derivatives play fundamental roles in many physiological processes, most notably the vision process. Furthermore, retinoic acid is used in dermatology as well as in the prevention and treatment of different types of cancer. The enzyme-NAD(+)-retinol complex has an apparent pK(a) value of 7.2 and loses a proton rapidly. Proton inventory modeling suggests that the transition state for the hydride transfer step has a partial negative charge on the oxygen of retinoxide. Spectral evidence for an intermediate such as E-NAD(+)-retinoxide was obtained with enzyme that has cobalt(II) substituted for the active site zinc(II). Biophysical considerations of water in these biological processes coupled with the inverse solvent isotope effect lead to the conclusion that the zinc-bound alkoxide makes a strong hydrogen bond with the hydroxyl group of Ser48 and is thus activated for hydride transfer. Moderate pressure accelerates enzyme action indicative of a negative volume of activation. The data with retinol is discussed in terms of enzyme stability, mechanism, adaptation to extreme conditions, as well as water affinities of substrates and inhibitors. Our data concern all-trans, 9-cis, 11-cis, and 13-cis retinols as well as the corresponding retinals. In all cases the enzyme utilizes an approximately ordered mechanism for retinol-retinal interconversion and for retinal-retinoic acid transformation.     

Ethanol directly increases dihydrotestosterone conversion to 5 alpha-androstan-3 beta,17 beta-diol and 5 alpha-androstan-3 alpha,17 beta-diol in rat Leydig cells

The direct effect of ethanol on dihydrotestosterone (DHT) conversion to 5 alpha-androstan-3 beta,17 beta-diol (3 beta-diol) and 5 alpha-androstan-3 alpha,17 beta-diol (3 alpha-diol) by adult rat Leydig cells was examined. Concentrations of ethanol comparable to blood levels of alcoholic men (2.2 - 65 mM) increased DHT conversion to 3 beta - and 3 alpha-diol, in direct relation to the dose of ethanol added; a 2-fold or greater stimulation was observed. Because this effect was blocked by 4-methylpyrazole or a saturating NADH concentration, these results suggest that this action is mediated by Leydig cell alcohol dehydrogenase activity. These results may have significant impact in the testis and/or other DHT sensitive tissues because ethanol may decrease the availability of the proposed active androgen.     

Evidence for a granulosa cell site of dihydrostestosterone metabolism in the adult rat ovary

The ovarian enzyme 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) converts dihydrotestosterone (DHT) to 5 alpha-androstan-3 alpha,17 beta-diol (3 alpha-diol), a reduced androgen that does not bind to the granulosa cell androgen receptor. To determine the relative contribution of the granulosa cells to total ovarian 3 alpha-HSD activity, adult rats treated with either medroxyprogesterone acetate (MPA) or vehicle underwent ovarian microdissection. 3 alpha-Hydroxysteroid dehydrogenase is primarily located in excised follicles and corpora lutea, and is inhibited in the follicles but not corpora lutea by MPA (P less than 0.05). Elimination of healthy granulosa cells while maintaining healthy theca cells by irradiation of the exteriorized ovaries with 6000 rads resulted in a marked reduction in 3 alpha-HSD to 19% of control levels on a per-organ basis (P less than 0.01). The granulosa cell is the major ovarian site for 3 alpha-hydroxylation of ring A-reduced C19 steroids in the adult rat.     

Reductive metabolism of 5 alpha-dihydrotestosterone by rat ventral and dorsolateral prostate: kinetic parameters of the enzymes

In male sex accessory organs the active androgen 5 alpha-dihydrotestosterone (DHT) is metabolized to 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-diol) and 5 alpha-androstane-3 beta, 17 beta-diol (3 beta-diol) by the reductase activities of 3 alpha-hydroxysteroid oxidoreductase (3 alpha-HSOR; EC 1.1.1.50) and 3 beta-hydroxysteroid oxidoreductase (3 beta-HSOR; EC 1.1.1.51). After separation of radiosubstrate and products by HPLC, these enzymes activities in subcellular preparations of rat ventral and dorsolateral prostate were determined from the conversion of [3H]DHT to the radiometabolites 3 alpha-diol and 3 beta-diol and 3 beta-triols (5 alpha-androstane-3 beta, 6 alpha, 17 beta-triol plus 5 alpha-androstane-3 beta, 7 alpha, 17 beta-triol). Whereas both enzymes were found in the dorsolateral prostate, 3 beta-HSOR reductase activity was near the limit of detection in ventral prostate. Unlike the equal distribution of 3 alpha-HSOR reductase between the microsomal and cytosol fractions of the ventral prostate, both 3 alpha- and 3 beta-HSOR reductase activities of the dorsolateral prostate are mainly confined to its cytosol fraction. Km and Vmax of the 3 alpha- and 3 beta-HSOR reductases in dorsolateral prostate cytosol were 1.8 microM, 24.6 pmol.mg-1 min-1 and 25.4 microM, 45.7 pmol.mg-1 min-1, respectively. We surmise from these and earlier studies that 3 beta-HSOR reductase is the rate-limiting prostatic enzyme in the catabolic disposition of intracellular DHT.     

3 alpha-hydroxysteroid dehydrogenase in the cytosol of rat submandibular gland

We examined the in vitro shuttle metabolism between dihydrotestosterone (DHT) and 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-diol) by 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD, E.C. 1.1.1.50) in rat submandibular gland (SMG) and ventral prostate (VP). The protein having molecular weight of 30 kDa, which was revealed by Sephacryl S-200 column chromatography, had 3 alpha-HSD activity to produce 3 alpha-diol from DHT, and also showed an oxidative 3 alpha-HSD (3 alpha-HSDO) ability to produce DHT from 3 alpha-diol. From the kinetic studies, the apparent Km and Vmax values of 3 alpha-HSD for DHT and NADPH were 6.4 microM, 1429 pmol/mg protein per min and 33.0 microM, 1205 pmol in SMG, and 9.3 microM, 377 pmol and 34.0 microM, 192 pmol in VP. The corresponding values of 3 alpha-HSDO for 3 alpha-diol and NADP+ were 18.0 microM, 714 pmol and 14.0 microM, 445 pmol in SMG, and 14.0 microM, 417 pmol and 36.0 microM, 77 pmol in VP. The affinities for DHT and 3 alpha-diol and the cosubstrate requirements of this enzyme in SMG were similar to those in VP. However, higher capacities of 3 alpha-HSD and 3 alpha-HSDO in SMG than in VP were shown. This suggests that there may be more 3 alpha-HSD in the SMG.     

Testosterone and progesterone metabolism in the human neuroblastoma cell line SH-SY5Y

The ability of the human neuroblastoma cell line SH-SY5Y to metabolize androgens and progesterone was studied by incubating the cells in the presence of labeled testosterone (T) or progesterone (P) to measure, respectively, the formation of dihydrotestosterone (DHT) or dihydroprogesterone (DHP) (5-reductase activitiy). The 3-hydroxysteroid dehydrogenase activity was studied by evaluating the conversion of labeled DHT into 5-androstan-3, 17β-diol (3-diol). The results show that undifferentiated neuroblastoma cells posses a significant 5-reductase activity, as shown by the considerable conversion of T into DHT; moreover, this enzymatic activity seems to be significantly stimulated following cell differentiation induced by the phorbol ester TPA, but not after differentiation induced by retinoic acid (RA). The 5-reductase(s) present in SH-SY5Y cells is also able to convert P into DHP. In undifferentiated cells, this conversion was about 8 times higher than that of T into DHT. Under the influences of TPA and RA, the formation of DHP followed the same pattern observed for the formation of DHT. SH-SY5Y cells also appear to possess the enzyme 3-hydroxysteroid dehydrogenase, since they are able to convert DHT into 3-diol. This enzymatic activity is not altered following TPA-induced differentiation and appears to be decreased following treatment with RA. It is suggested that the SH-SY5Y cell line may represent a useful “in vitro” model for the study of the mechanisms involved in the control of androgen and P metabolism in nervous cells.     

Retinol forms retinoic acid via retinal.

Hepatic cytosol from normal deermice having cytosolic alcohol dehydrogenase (ADH+) also displays retinol dehydrogenase activity and converts retinol to retinoic acid, whereas cytosol from ADH- deermice lacks these enzyme activities and does not produce retinoic acid. Furthermore, microsomes from either strain do not convert retinol to retinoic acid. However, when cytosol from ADH- animals is added to the microsomes, retinoic acid is produced. The obligatory role of retinal as an intermediary step in retinoic acid formation is further shown by isotopic dilution of retinoic acid formed from labeled retinol upon addition of unlabeled retinal. Microsomal retinol dehydrogenase also catalyzes the reduction of retinal to retinol, thereby explaining the decrease in retinoic acid production from retinol in liver cytosol of ADH+ deermice when microsomes are added. Thus, the results of this study indicate that retinal is an obligatory intermediate in the hepatic production of retinoic acid from retinol and that cytosolic and microsomal retinol dehydrogenases play a key role in this process.     

Androgen metabolism by hepatic and renal tissues of the fetal rhesus monkey

Liver and kidney from fetal monkeys (day 125 of gestation) were fractionated into low speed pellets, microsomal and cytosolic fractions. Liver cytosols converted as much testosterone (T) to 5 beta-androstane-3 alpha,17 beta-diol (5 beta-diol) at 0 degrees C as at 4 degrees-45 degrees C without exogenous cofactors. The principal product formed from 5 alpha-dihydrotestosterone (5 alpha-DHT) was 5 alpha-diol. A 1000-fold molar excess of radioinert 5 beta- or 5 alpha-DHT inhibited 5 beta-diol formation from [3H]T by cytosols and increased 5 beta-DHT formation. Similarly, using 5 alpha-DHT as substrate, 5 alpha-diol formation was inhibited. Microsomal and low speed pellets with added cofactors formed products which recrystallized with either etiocholanolone or androsterone from [3H]T or [3H]DHT, respectively. Little product was formed without cofactor. Whole liver homogenates produced 5 beta-reduced products from [3H]T in the presence of an NADPH generating system whereas kidney homogenates produced 5 alpha-reduced products. These data provide new information on the capacity of fetal monkey liver and kidney to metabolize androgens. The 3 alpha-reductases are cytosolic. The 5 alpha- and 5 beta-reductases are mostly in the low speed pellet but are sufficiently represented in cytosols to mediate diol formation. The 17-hydroxysteroid dehydrogenases are in the microsomal fraction. Our results suggest that 5 alpha-DHT is the active androgen in fetal liver since testosterone is metabolized to 5 beta-DHT and 5 beta-diol which are inactive androgens.     

DHT formation and degradation in cultured human skin fibroblasts: DHT accumulation in the genital skin

The conversion of testosterone to dihydrotestosterone (DHT) by 5 alpha-reductase and the interconversion between DHT and 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-diol) by 3 alpha-hydroxy-steroid oxidoreductase (3 alpha-HSOR) were studied in fibroblasts derived from the genital skin of 22 males and 6 females, and from the nongenital skin of 19 males and 9 females with normal gonadal function. The formation of DHT from testosterone (5 alpha-reduction) was significantly greater in fibroblasts from genital skin than in those from nongenital skin in both males (2.15 +/- 1.43 vs 0.81 +/- 0.46 pmol/mg protein/h, mean +/- SD, P less than 0.001) and females (2.52 +/- 1.99 vs 0.69 +/- 0.18, P less than 0.01). Furthermore, DHT formation from 3 alpha-diol (3 alpha-HSOR oxidation) was also significantly greater in genital skin fibroblasts than in nongenital skin fibroblasts of males (5.47 +/- 3.37 vs 2.52 +/- 1.74 pmol/mg protein/h, P less than 0.01). However, the degradation of DHT to 3 alpha- and/or 3 beta-diol (3 alpha- and/or 3 beta-HSOR reductions) was not different between genital and nongenital skin fibroblasts of either males or females. Respective ratios of DHT formation to DHT degradation (5 alpha-reduction/3 alpha-HSOR reduction, 3 alpha-HSOR oxidation/3 alpha-HSOR reduction) were also significantly greater (P less than 0.002) in genital skin fibroblasts than in nongenital skin fibroblasts of males. On the other hand, both DHT formation and degradation were not different between male and female genital skin fibroblasts. These results suggest that the increased production of DHT in genital compared to nongenital skin results from increased 5 alpha-reduction and 3 alpha-HSOR oxidation.     

Androgen regulation of aldehyde dehydrogenase 1A3 (ALDH1A3) in the androgen-responsive human prostate cancer cell line LNCaP

Previous gene array data from our laboratory identified the retinoic acid (RA) biosynthesis enzyme aldehyde dehydrogenase 1A3 (ALDH1A3) as a putative androgen-responsive gene in human prostate cancer epithelial (LNCaP) cells. In the present study, we attempted to identify if any of the three ALDH1A/RA synthesis enzymes are androgen responsive and how this may affect retinoid-mediated effects in LNCaP cells. We demonstrated that exposure of LNCaP cells to the androgen dihydrotestosterone (DHT) results in a 4-fold increase in ALDH1A3 mRNA levels compared with the untreated control. The mRNA for two other ALDH1A family members, ALDH1A1 and ALDH1A2, were not detected and not induced by DHT in LNCaP cells. Inhibition of androgen receptor (AR) with both the antiandrogen bicalutamide and small interfering RNA for AR support that ALDH1A3 regulation by DHT is mediated by AR. Furthermore, specific inhibition of the extracellular signal-regulated kinase and Src family of kinases with PD98059 and PP1 supports that AR's regulation of ALDH1A3 occurs by the typical AR nuclear-translocation cascade. Consistent with an increase in ALDH1A3 mRNA, DHT-treated LNCaP cells showed an 8-fold increase in retinaldehyde-dependent NAD(+) reduction compared with control. Lastly, treatment of LNCaP with all-trans retinal (RAL) in the presence of DHT resulted in significant up-regulation of the RA-inducible, RA-metabolizing enzyme CYP26A1 mRNA compared with RAL treatment alone. Taken together, these data suggest that (i) the RA biosynthesis enzyme ALDH1A3 is androgen responsive and (ii) DHT up-regulation of ALDH1A3 can increase the oxidation of retinal to RA and indirectly affect RA bioactivity and metabolism.     

Isomer-specific retinoic acid biosynthesis in HeLa cells expressing recombinant class I aldehyde dehydrogenases

Retinal dehydrogenase type 1 (RALDH1) catalyzes the oxidation of all-trans and 9-cis retinal to the respective retinoic acids (RAs), whereas another member of the aldehyde dehydrogenase family, the phenobarbital-induced aldehyde dehydrogenase (PB-ALDH), is very poorly active. We have previously generated chimeras between these two enzymes that displayed selectivity for retinal isomers in crude bacterial extracts. To examine whether the selectivity of the recombinant enzymes is retained in intact cells, we first assessed whether retinoid-isomerizing activity is present in cultured eukaryotic cells. Our results demonstrate that the only RA isomers detected in RALDH1-expressing or non-expressing cells corresponded to the same steric conformation as the supplied retinoids, indicating a lack of measurable 9-cis/all-trans retinoid-isomerizing activity. Finally, HeLa cells transfected with RALDH1 derivatives that were retinal isomer-selective in vitro produced only the corresponding RA isomers, establishing these enzymes as useful tools to assess the respective roles of the two RA isomers in vivo.