Human Cytosolic 3 -Hydroxysteroid Dehydrogenases of the Aldo-keto Reductase Superfamily Display Significant 3 -Hydroxysteroid Dehydrogenase Activity IMPLICATIONS FOR STEROID HORMONE METABOLISM AND ACTION*

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 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.