7α- and 7β-hydroxy-epiandrosterone as substrates and inhibitors for the human 11β-hydroxysteroid dehydrogenase type 1

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Abstract

The human 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) catalyzes both the NADP(H)-dependent oxido-reduction of cortisol and cortisone and the inter-conversion of 7α- and 7β-hydroxy-dehydroepiandrosterone (DHEA) through a 7-oxo-DHEA intermediate. As shown with human liver and intestine fractions, 7α-hydroxy-epiandrosterone (7α-hydroxy-EpiA) and 7β-hydroxy-EpiA were readily inter-converted with no evidence for a 7-oxo-EpiA intermediate. Whether this inter-conversion resulted from action of the 11β-HSD1 or from an unknown epimerase is unresolved. Furthermore, whether these steroids could inhibit the cortisol–cortisone oxido-reduction remains a question. The recombinant human 11β-HSD1 was used to test these questions. NADP+ supplementation only provided the production of 7β-hydroxy-EpiA out of 7α-hydroxy-EpiA with a Vmax/KM ratio at 0.1. With NADPH supplementation, both 7α-hydroxy-EpiA and 7β-hydroxy-EpiA were formed in low amounts from 7β-hydroxy-EpiA and 7α-hydroxy-EpiA, respectively. These inter-conversions occurred without a trace of the putative 7-oxo-EpiA intermediate. In contrast, the 7-oxo-EpiA substrate was efficiently reduced into 7α-hydroxy-EpiA and 7β-hydroxy-EpiA, with Vmax/KM ratios of 23.6 and 5.8, respectively. Competitive and mixed type inhibitions of the 11β-HSD1-mediated cortisol oxidation were exerted by 7α-hydroxy-EpiA and 7β-hydroxy-EpiA, respectively. The 11β-HSD1-mediated cortisone reduction was inhibited in a competitive manner by 7-oxo-EpiA. These findings suggest that the active site of the human 11β-HSD1 may carry out directly the epimeric transformation of 7-hydroxylated EpiA substrates. The low amounts of these steroids in human do not support a physiological importance for modulation of the glucocorticoid status in tissues.

Introduction

In the human liver and brain, the cytochrome P4507B1 (CYP7B1) catalyzes the 7α-hydroxylation of 3β-hydroxysteroids [1]. Thus, 7α-hydroxy-dehydroepiandrosterone (7α-hydroxy-DHEA) and 7α-hydroxy-epiandrosterone (7α-hydroxy-EpiA) are produced out of DHEA and EpiA substrates, respectively. We showed that in addition to 7α-hydroxy-DHEA and 7α-hydroxy-EpiA, the human P4507B1 produced small quantities of the 7β-hydroxylated epimers that were assessed as a side production because of KM identical with that of 7α-hydroxylation but showing much lower Kcat [1]. The production yields of 7β-hydroxylated derivatives were not sufficient for a full explanation of the almost equivalent quantities of 7α- and 7β-hydroxy-DHEA found in the human body fluids [2], [3], [4], and several hypotheses were discussed. This and the report of Robinzon et al. [5] led us to consider the human 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) as responsible for the inter-conversion of 7α- and 7β-hydroxy-DHEA. Indeed the yeast-expressed human 11β-HSD1 carried out the inter-conversion of both steroids through an oxido-reduction process involving a 7-oxo-DHEA intermediate [6]. This was also confirmed in human liver S9 fractions where both 7α- and 7β-hydroxy-DHEA gave rise to inter-conversion through production of 7-oxo-DHEA [7]. Curiously, the use of 7α- and 7β-hydroxy-EpiA substrates with the same S9 fractions led to inter-conversion without a trace of the putative 7-oxo-EpiA intermediate [7], and this was also observed when human intestine microsomes were used [8]. This leads to question whether the 11β-HSD1-mediated inter-conversion process is identical for the DHEA and EpiA derivatives, and whether 7α- and 7β-hydroxy-EpiA could behave as well as 7α- and 7β-hydroxy-DHEA for inhibition of the 11β-HSD1-mediated cortisone/cortisol inter-conversion (unpublished data).

In this work, our aims were to use the previously produced yeast-expressed human 11β-HSD1 [6] and NADP+ or NADPH-regenerating systems for kinetic studies of the inter-conversion process with use of 7α-hydroxy-EpiA, 7β-hydroxy-EpiA and 7-oxo-EpiA substrates. Finally, we wanted to study their inhibition potencies on the 11β-HSD1-mediated cortisol/cortisone oxido-reduction.

Section snippets

Steroids and reagents

7β-Hydroxy-EpiA (HF0220) was a gift from Hunter-Fleming Ltd. (Bristol, UK). Custom chemical synthesis by Roowin S.A. (Romainville, France) provided milligram quantities of chemically pure 7α-hydroxy-EpiA, and 7-oxo-DHEA-17-ethylene-ketal. The latter steroid was reduced with hydrogen gas in the presence of Pd into 3β-hydroxy-5α-androstane-17-ethylene-ketal,7-one. After HCl treatment, the 3β-hydroxy-5α-androstane-7,17-dione was obtained and purified by preparative HPLC. Identity and purity of the

11β-HSD1-mediated transformation of 7-oxygenated EpiA

The three 7-oxygenated steroids, namely 7-oxo-EpiA, 7α-hydroxy-EpiA and 7β-hydroxy-EpiA, were used as substrates for the recombinant 11β-HSD1 after supplementation with either the NADPH- or the NADP+-regenerating system. The substrate and metabolite extracted were separated by HPLC and their major ions detected by the coupled MS (Table 1). Ions with m/z 287.6 and 289.6 were selected for the detection of 7-oxo-EpiA and 7-hydroxy-EpiA, respectively. Under oxidative conditions (NADP+-regenerating

Discussion

In this study, the recombinant human 11β-HSD1 enzyme expressed in S. cerevisiae was the same as the one previously used to compare of its action on the classical cortisol–cortisone substrates with its inter-converting properties for 7α- and 7β-hydroxy-DHEA substrates [6]. We stated that other authors had over-expressed the human 11β-HSD1 in yeast with use of Pichia pastoris strains [16] and that microsomes of human liver and the recombinant yeast provided the same KMs when cortisone reduction

Acknowledgements

This work was supported by a grant from Hunter-Fleming Inc. (UK). We thank Patrice Rool (Roowin S.A., Romainville, France) for the gift of EpiA derivatives.

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