Human Cytosolic 3α-Hydroxysteroid Dehydrogenases of the Aldo-keto Reductase Superfamily Display Significant 3β-Hydroxysteroid Dehydrogenase Activity

Abstract

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 ...

Identification of the molecular switch that regulates access of 5α-DHT to the androgen receptor

“…a These steady state kinetic constants are consistent with the re-estimation of these values described by Steckelbroeck et al 2004.…”

“…Using more discriminating TLC systems the products of the 3-ketosteroid reductase activities of the four AKR1C isoforms were re-examined. Each human enzyme produced two isomeric products, 3α-diol and 3β-diol, in different ratios indicating that they were not stereospecific (Steckelbroeck, et al, 2004). By contrast rat 3α-HSD (AKR1C9) showed strict stereochemical preference and reduced 5α-DHT only to 3α-diol.…”

An alternate pathway for androgen regulation of brain function: Activation of estrogen receptor beta by the metabolite of dihydrotestosterone, 5α-androstane-3β,17β-diol

“…For example, dihydrotestostoerne can be further metabolized to 5α -androstane-3α, 17β-diol (3α-Diol) or 5α-androstane-3β, 17β-diol (3β-Diol) by the actions of a number of p450 enzymes including 3α hydroxysteroid dehydrogenase (3α-HSD) , 3β hydroxysteroid dehydrogenase (3β-HSD), and 17β-hydroxysteroid dehydrogenase (Jin and Penning, 2001;Weihua et al, 2002;Gangloff et al 2003, Torn et al 2003, Steckelbroeck et al, 2004. Of these enzymes, 5 alpha reductase and 3β-HSD are also involved in the pathways for synthesis and metabolism of other steroids (see figure 2).…”

“…As a result, 3α-Diol has been implicated in the regulation of a number of different behaviors (Rupprecht and Holsboer, 1999, Rosellini et al, 2001, Fernandez-Guasti and Martinez-Mota 2005, Reddy, 2004. In contrast, 3β-Diol cannot bind the benzodiazepine receptor (unpublished) but, if its actions are similar to other 3β-tetrahydrosteroids, it may be an antagonist of 3α-tetrahydrosteroids at the GABAa receptor (Steckelbroeck et al 2004). Importantly, 3β-Diol has been reported to preferentially bind ERbeta, whereas 3α-Diol has little affinity for ERbeta or ERalpha (Kuiper et al, 1998).…”

Estrogen receptors: Their roles in regulation of vasopressin release for maintenance of fluid and electrolyte homeostasis

“…Further support for such a hypothesis is provided by evidence that the enzymes required to metabolize testosterone to estrogen (aromatase), DHT (5α-reductase), and DHT to 3β-diol [17β hydroxysteroid dehydrogenase (17β-HSD) or 3α HSD [20,82,90]] are expressed in the hypothalamus [36]. [Note: The report that VP immunoreactivity was not altered in SON of aromatase knock out mice [52] does not detract from this hypothesis, because as mentioned previously ERβ is not present in the mouse SON [42]].…”