The source of
NADPH-dependent cytosolic 3beta-hydroxysteroid
dehydrogenase (3beta-HSD) activity is unknown to date. This important reaction leads e.g. to the reduction of the potent
androgen 5alpha-dihydrotestosterone (DHT) into inactive 3beta-androstanediol (3beta-Diol). Four human cytosolic
aldo-keto reductases (AKR1C1-AKR1C4) are known to act as non-positional-specific 3alpha-/17beta-/20alpha-HSDs. We now demonstrate that AKR1Cs catalyze the reduction of DHT into both 3alpha- and 3beta-Diol (established by (1)H NMR spectroscopy). The rates of 3alpha- versus 3beta-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 3alpha[17beta]-hydroxysteroid
oxidase activity with 3alpha-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 3beta-HSD/Delta(5-4) ketosteroid
isomerase mRNA expression, but express AKR1C1-AKR1C3) were able to convert DHT into 3alpha- and 3beta-Diol. This conversion was inhibited by
flufenamic acid establishing the in vivo significance of the 3alpha/3beta-HSD activities of the AKR1C
enzymes. Molecular docking simulations using available crystal structures of AKR1C1 and AKR1C2 demonstrated how 3alpha/3beta-HSD activities are achieved. The observation that AKR1Cs are a source of 3beta-tetrahydrosteroids is of physiological significance because: (i) the formation of 3beta-Diol (in contrast to 3alpha-Diol) is virtually irreversible, (ii) 3beta-Diol is a pro-apoptotic
ligand for
estrogen receptor beta, and (iii) 3beta-tetrahydrosteroids act as
gamma-aminobutyric acid type A receptor antagonists.