The source of NADPH-dependent cytosolic 3␤-hydroxysteroid dehydrogenase (3␤-HSD) activity is unknown to date. This important reaction leads e.g. to the reduction of the potent androgen 5␣-dihydrotestosterone (DHT) into inactive 3␤-androstanediol (3␤-Diol). Four human cytosolic aldo-keto reductases (AKR1C1-AKR1C4) are known to act as non-positional-specific 3␣-/ 17␤-/20␣-HSDs. We now demonstrate that AKR1Cs catalyze the reduction of DHT into both 3␣-and 3␤-Diol (established by 1 H NMR spectroscopy). The rates of 3␣-versus 3␤-Diol formation varied significantly among the isoforms, but with each enzyme both activities were equally inhibited by the nonsteroidal anti-inflammatory drug flufenamic acid. In vitro, AKR1Cs also expressed substantial 3␣[17␤]-hydroxysteroid oxidase activity with 3␣-Diol as the substrate. However, in contrast to the 3-ketosteroid reductase activity of the enzymes, their hydroxysteroid oxidase activity was potently inhibited by low micromolar concentrations of the opposing cofactor (NADPH). This indicates that in vivo all AKR1Cs will preferentially work as reductases. Human hepatoma (HepG2) cells (which lack 3␤-HSD/⌬ 5-4 ketosteroid isomerase mRNA expression, but express AKR1C1-AKR1C3) were able to convert DHT into 3␣-and 3␤-Diol. This conversion was inhibited by flufenamic acid establishing the in vivo significance of the 3␣/3␤-HSD activities of the AKR1C enzymes. Molecular docking simulations using available crystal structures of AKR1C1 and AKR1C2 demonstrated how 3␣/3␤-HSD activities are achieved. The observation that AKR1Cs are a source of 3␤-tetrahydrosteroids is of physiological significance because: (i) the formation of 3␤-Diol (in contrast to 3␣-Diol) is virtually irreversible, (ii) 3␤-Diol is a pro-apoptotic ligand for estrogen receptor ␤, and (iii) 3␤-tetrahydrosteroids act as ␥-aminobutyric acid type A receptor antagonists.Two classes of 3␤-hydroxysteroids, i.e. the ⌬ 5 -3␤-hydroxysteroids and the fully saturated 3␤-tetrahydrosteroids, represent pivotal intermediates in steroid hormone metabolism. In steroidogenic glands, ⌬ 5 -3␤-hydroxysteroid precursors are converted into ⌬ 4 -3-ketosteroids to produce active steroid hormones (1, 2), whereas 3-ketosteroid reduction of 5␣/5␤-dihydrosteroids into 3␤-tetrahydrosteroids is an important catabolic step in steroid hormone transformation.Human steroid hormone target tissues like the prostate express membrane attached and/or cytosolic 3␣-HSD 1 and 3␤-HSD activity (3-9). One key example of the catabolic function of these HSDs is the 3-ketosteroid reduction of the potent androgen 5␣-dihydrotestosterone (DHT, 17␤-hydroxy-5␣-androstan-3-one) into the inactive androgens 5␣-androstane-3␣,17␤-diol (3␣-Diol; Fig. 1) and 5␣-androstane-3␤,17␤-diol (3␤-Diol) (10 -12). In vivo, the formation of 3␤-Diol is virtually irreversible, whereas 3␣-Diol can be converted back to DHT via 3␣-hydroxysteroid oxidase activity (13-17). Reformation of DHT from 3␤-Diol is prevented, because 3␤-Diol is either irreversibly hydroxylated at the C-6 and/or C-7 position or ...

Aldo-keto reductase (AKR) 1C3 (type 2 3α-HSD, type 5 17β-HSD, and prostaglandin F synthase) regulates ligand access to steroid hormone and prostaglandin receptors and may stimulate proliferation of prostate and breast cancer cells. NSAIDs are known inhibitors of AKR1C enzymes. An NSAID analogue that inhibits AKR1C3 but is inactive against the cyclooxygenases and the other AKR1C family members would provide an important tool to examine the role of AKR1C3 in proliferative signaling. We tested NSAIDs and NSAID analogues for inhibition of the reduction of 9,10-phenanthrenequinone (PQ) catalyzed by AKR1C3 and the closely related isoforms AKR1C1 and AKR1C2. Two of the compounds initially screened, indomethacin and its methyl ester, were specific for AKR1C3 versus the other AKR1C isoforms. Based on these results and the crystal structure of AKR1C3, we predicted that N-(4-chlorobenzoyl)-melatonin (CBM), an indomethacin analogue that does not inhibit the cyclooxygenases, would selectively inhibit AKR1C3. CBM inhibited the reduction of PQ by AKR1C3, but did not significantly inhibit AKR1C1 or AKR1C2. Indomethacin and CBM also inhibited the AKR1C3-catalyzed reduction of Δ 4 -androstene-3,17-dione but did not significantly inhibit the reduction of steroid hormones catalyzed by AKR1C1 or AKR1C2. The pattern of inhibition of AKR1C3 by indomethacin and CBM was uncompetitive versus PQ, but competitive versus Δ 4 -androstene-3,17-dione, indicating that two different inhibitory complexes form during the ordered bi-bi reactions. The identification of CBM as a specific inhibitor of AKR1C3 will aid the investigation of its roles in steroid hormone and prostaglandin signaling and the resultant effects on cancer development.

Human aldo-keto reductases (AKRs) of the AKR1C subfamily function in vitro as 3-keto-, 17-keto-, and 20-ketosteroid reductases or as 3␣-, 17␤-, and 20␣-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 3␣-androstanediol (3␣-diol) to 5␣-dihydrotestosterone (5␣-DHT). This reaction is potently inhibited by reduced NAD phosphate (NADPH), indicating that the NAD ؉ : NADPH ratio in cells will determine whether AKR1C2 makes 5␣-DHT. In transient COS-1-AKR1C2 and in stable PC-3-AKR1C2 transfectants, 5␣-DHT was reduced by AKR1C2. However, the transfected AKR1C2 oxidase activity was insufficient to surmount the endogenous 17␤-hydroxysteroid dehydrogenase (17␤-HSD) activity, which eliminated 3␣-diol as androsterone. PC-3 cells expressed retinol dehydrogenase/3␣-HSD and 11-cis-retinol dehydrogenase, but these endogenous enzymes did not oxidize 3␣-diol to 5␣-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 5␣-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 17␤-HSD present. Neither AKR1C2, retinol dehydrogenase/3␣-HSD nor 11-cis-retinol dehydrogenase is a source of 5␣-DHT in PC-3 cells.

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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.