Characterization of 17alpha-hydroxysteroid dehydrogenase activity (17alpha-HSD) and its involvement in the biosynthesis of epitestosterone

Abstract

Background: Epi-testosterone (epiT) is the 17alpha-epimer of testosterone. It has been found at similar level as testosterone in human biological fluids. This steroid has thus been used as a natural internal standard for assessing testosterone abuse in sports. EpiT has been also shown to accumulate in mammary cyst fluid and in human prostate. It was found to possess antiandrogenic activity as well as neuroprotective effects. So far, the exact pathway leading to the formation of epiT has not been elucidated.

Results: In this report, we describe the isolation and characterization of the enzyme 17alpha-hydroxysteroid dehydrogenase. The name is given according to its most potent activity. Using cells stably expressing the enzyme, we show that 17alpha-HSD catalyzes efficienty the transformation of 4-androstenedione (4-dione), dehydroepiandrosterone (DHEA), 5alpha-androstane-3,17-dione (5alpha-dione) and androsterone (ADT) into their corresponding 17alpha-hydroxy-steroids : epiT, 5-androstene-3beta,17alpha-diol (epi5diol), 5alpha-androstane-17alpha-ol-3-one (epiDHT) and 5alpha-androstane-3alpha,17alpha-diol (epi3alpha-diol), respectively. Similar to other members of the aldo-keto reductase family that possess the ability to reduce the keto-group into hydroxyl-group at different position on the steroid nucleus, 17alpha-HSD could also catalyze the transformation of DHT, 5alpha-dione, and 5alpha-pregnane-3,20-dione (DHP) into 3alpha-diol, ADT and 5alpha-pregnane-3alpha-ol-20-one (allopregnanolone) through its less potent 3alpha-HSD activity. We also have over-expressed the 17alpha-HSD in Escherichia coli and have purified it by affinity chromatography. The purified enzyme exhibits the same catalytic properties that have been observed with cultured HEK-293 stably transfected cells. Using quantitative Realtime-PCR to study tissue distribution of this enzyme in the mouse, we observed that it is expressed at very high levels in the kidney.

Conclusion: The present study permits to clarify the biosynthesis pathway of epiT. It also offers the opportunity to study gene regulation and function of this enzyme. Further study in human will allow a better comprehension about the use of epiT in drug abuse testing; it will also help to clarify the importance of its accumulation in breast cyst fluid and prostate, as well as its potential role as natural antiandrogen.

Figure 1

Alignment of the amino acid sequence of mouse 17α-HSD with those of related enzymes. Amino acid sequence of mouse 17α-HSD was aligned with sequences of mouse (m), rat (r), rabbit (rb) and human (h) enzyme members of the AKR1c subfamily. The corresponding name according to the nomenclature for aldo-keto reductase (AKR) family members are: m17α-HSD, AKR1C21; m3α-HSD, AKR1C14; m20α-HSD, AKR1C18; m17β-HSD5, AKR1C6; r3α-HSD, AKR1C9; r20α-HSD, AKR1C8; h3α-HSD1, AKR1C4; h3α-HSD3, AKR1C2; h17β-HSD5 or 3α-HSD2, AKR1C3; and h20α-HSD, AKR1C1. Amino acids are given in conventional single letter code and numbered on the right. Dashes and dots, respectively, represent identical and missing amino acids.

Figure 2

Identification by HPLC of epiT produced from HEK-293 cells stably expressing 17α-HSD. (A) Elution profile of non labeled steroids; 4-dione (1^st peak), testosterone (2^nd peak) and epitestosterone (3^rd peak). (B) Products extracted from enzymatic assay done with cells stably expressing 17α-HSD, substrate is 4-dione. (C) Products extracted from enzymatic assay done with purified enzyme; substrate is 4-dione. Separation and identification of metabolites were performed as described in Materials and Methods.

Figure 3

Identification of the 17α-HSD activity by TLC. A- Incubation of [¹⁴C]-4-dione with cells stably expressing mouse 17β-HSD type 5 (1) and 17α-HSD (2) activity. Standards of 4-dione (3), T (4) and epiT (5) have been deposited and co-migrated. B- Incubation of [¹⁴C]-ADT with cells cells stably expressing mouse 17β-HSD type 5 (1) and 17α-HSD (2) activity. Standards of ADT (3), 3α-diol (4) and epi3α-diol (5) have been deposited and co-migrated.

Figure 4

Substrate specificity of mouse 17α-HSD activity of HEK-293 cells stably transfected with pCMVneo-m17α-HSD. The experiments were performed using HEK-293 cells stably expressing 17α-HSD in culture. 0,1 μM of the indicated [¹⁴C]-and [³H]-labeled steroid was added to culture medium for one hour. Testo, conversion of testosterone to 4-dione and vice-versa; E1, conversion of estrone to estradiol and vice-versa; DHEA, conversion of dehydroepiandrosterone to 5-diol; 5α-dione, conversion of androstanedione to androsterone (ADT) and vice-versa; DHT, conversion of dihydrotestosterone to 3α-diol and vice-versa; DHP, conversion of dihydroprogesterone to allopregnanolone and vice-versa; Preg and Prog, conversion of pregnenolone and progesterone to 20α-OHPreg and 20α-OHProg. The error bar indicates mean ± SEM of triplicate assays. Incubation, extraction, separation and quantification were performed as described in Materials and Methods.

Figure 5

Diagram illustrating the 2 putative pathways for the conversion of DHEA to epiT via 17α-HSD and 3β-HSD. The thickness of the arrows indicates the relative importance of each pathway

Figure 6

SDS-PAGE of fractions obtained during the purification process of 17α-HSD. MW, molecular weight standards; CE, cell extract of 100000 g; FP, fusion protein; DP, protein obtained after digestion with thrombin; 17α-HSD, purified.