11β-Hydroxysteroid dehydrogenases control access of 7β,27-dihydroxycholesterol to retinoid-related orphan receptor γ

Katharina R Beck,Silvia G Inderbinen,Sharavan Kanagaratnam,Denise V Kratschmar,Anton M Jetten,Hideaki Yamaguchi,Alex Odermatt

doi:10.1194/jlr.m092908

Abstract

Oxysterols previously were considered intermediates of bile acid and steroid hormone biosynthetic pathways. However, recent research has emphasized the roles of oxysterols in essential physiologic processes and in various diseases. Despite these discoveries, the metabolic pathways leading to the different oxysterols are still largely unknown and the biosynthetic origin of several oxysterols remains unidentified. Earlier studies demonstrated that the glucocorticoid metabolizing enzymes, 11β-hydroxysteroid dehydrogenase (11β-HSD) types 1 and 2, interconvert 7-ketocholesterol (7kC) and 7β-hydroxycholesterol (7βOHC). We examined the role of 11β-HSDs in the enzymatic control of the intracellular availability of 7β,27-dihydroxycholesterol (7β27OHC), a retinoid-related orphan receptor γ (RORγ) ligand. We used microsomal preparations of cells expressing recombinant 11β-HSD1 and 11β-HSD2 to assess whether 7β27OHC and 7-keto,27-hydroxycholesterol (7k27OHC) are substrates of these enzymes. Binding of 7β27OHC and 7k27OHC to 11β-HSDs was studied by molecular modeling. To our knowledge, the stereospecific oxoreduction of 7k27OHC to 7β27OHC by human 11β-HSD1 and the reverse oxidation reaction of 7β27OHC to 7k27OHC by human 11β-HSD2 were demonstrated for the first time. Apparent enzyme affinities of 11β-HSDs for these novel substrates were equal to or higher than those of the glucocorticoids. This is supported by the fact that 7k27OHC and 7β27OHC are potent inhibitors of the 11β-HSD1-dependent oxoreduction of cortisone and the 11β-HSD2-dependent oxidation of cortisol, respectively. Furthermore, molecular docking calculations explained stereospecific enzyme activities. Finally, using an inducible RORγ reporter system, we showed that 11β-HSD1 and 11β-HSD2 controlled RORγ activity. These findings revealed a novel glucocorticoid-independent prereceptor regulation mechanism by 11β-HSDs that warrants further investigation.

Highlights

Oxysterols previously were considered intermediates of bile acid and steroid hormone biosynthetic pathways
The family of oxysterols comprises a large number of compounds; their abundance in biological systems is low compared with cholesterol [1]
We showed two novel alternative biosynthetic pathways, i.e., the 11 -HSD1-dependent generation of 7 27OHC and the 11 -HSD2-dependent formation of 7k27OHC

Summary

MATERIALS AND METHODS

Chemicals and reagents 7k27OHC, 7 27OHC, 7 27OHC, and 7 27OHC-d6 were obtained from Avanti Polar Lipids, Inc. (Alabaster, AL), [1,2-3H] cortisone, [1,2,6,7-3H]cortisol, and [1,2,6,7-3H] corticosterone from American Radiolabeled Chemicals Microsomes (1–20 g) expressing 11 -HSD1 and H6PDH were incubated for 20 min with 1 mM of NADPH, 2 mM of glucose-6-phosphate, 0.1 units of glucose-6-phosphate dehydrogenase (from Saccharomyces cerevisiae), and different concentrations (62.5–2,000 nM) of 7k27OHC, adapted to a total assay volume of 50 l with TS2 buffer [100 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1 mM MgCl2, 250 mM sucrose, 20 mM Tris/HCl (pH 7.4)]. For 11 -HSD2 activity measurements, 0.8 g of HEK-293 cell lysate stably expressing 11 -HSD2 was incubated with different concentrations (50–800 nM) of 7 27OHC and 500 M of NAD+ for 10–30 min. The enzymatic reaction was assessed using 0.075 mg/ml total protein of the kidney homogenate in incubation buffer [300 mM NaCl, 20 mM Tris/HCl, 1 mM EDTA, 10% glycerol (pH 7.7)], different concentrations of oxysterols, 50 nM of corticosterone (containing 50 nCi [1,2,6,7-3H] corticosterone), and 500 M NAD+ at 37°C for 20 min.

RESULTS

Findings

DISCUSSION