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

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. 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. Oxysterols originate from cholesterol or cholesterol precursors and previously were mainly considered intermediates of bile acid and steroid hormone biosynthetic pathways. They display additional oxygen functionalities in the ring system and/or at the side chain of cholesterol, deriving through either enzymatic reactions and/or radical processes (1Schroepfer Jr., G.J. Oxysterols: modulators of cholesterol metabolism and other processes.Physiol. Rev. 2000; 80: 361-554Crossref PubMed Scopus (823) Google Scholar). Oxysterols are involved in several physiological processes including cholesterol, carbohydrate, and lipid homeostasis, immune system regulation, and neuronal development. Furthermore, recent research emphasized their role as bioactive lipids, contributing to the progression of various pathologies, such as neurodegenerative diseases, atherosclerosis, and cancer [reviewed in (2Guillemot-Legris O. Mutemberezi V. Muccioli G.G. Oxysterols in metabolic syndrome: from bystander molecules to bioactive lipids.Trends Mol. Med. 2016; 22: 594-614Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 3Zarrouk A. Vejux A. Mackrill J. O'Callaghan Y. Hammami M. O'Brien N. Lizard G. Involvement of oxysterols in age-related diseases and ageing processes.Ageing Res. Rev. 2014; 18: 148-162Crossref PubMed Scopus (127) Google Scholar, 4Jeitner T.M. Voloshyna I. Reiss A.B. Oxysterol derivatives of cholesterol in neurodegenerative disorders.Curr. Med. Chem. 2011; 18: 1515-1525Crossref PubMed Scopus (47) Google Scholar, 5Poli G. Biasi F. Leonarduzzi G. Oxysterols in the pathogenesis of major chronic diseases.Redox Biol. 2013; 1: 125-130Crossref PubMed Scopus (201) Google Scholar, 6Silvente-Poirot S. Dalenc F. Poirot M. The effects of cholesterol-derived oncometabolites on nuclear receptor function in cancer.Cancer Res. 2018; 78: 4803-4808Crossref PubMed Scopus (39) Google Scholar)]. Nevertheless, the metabolic pathways leading to the different oxysterols remain insufficiently understood and their biosynthetic origins need to be elucidated. 7-Ketocholesterol (7kC) is one of the most extensively studied oxysterols and particularly known for its pro-inflammatory and cytotoxic properties [reviewed in (1Schroepfer Jr., G.J. Oxysterols: modulators of cholesterol metabolism and other processes.Physiol. Rev. 2000; 80: 361-554Crossref PubMed Scopus (823) Google Scholar, 7Vejux A. Lizard G. Cytotoxic effects of oxysterols associated with human diseases: Induction of cell death (apoptosis and/or oncosis), oxidative and inflammatory activities, and phospholipidosis.Mol. Aspects Med. 2009; 30: 153-170Crossref PubMed Scopus (220) Google Scholar, 8Brown A.J. Jessup W. Oxysterols and atherosclerosis.Atherosclerosis. 1999; 142: 1-28Abstract Full Text Full Text PDF PubMed Scopus (762) Google Scholar)], and it was recently reported to be associated with cardiovascular outcomes and total mortality in patients with coronary artery disease (9Song J. Wang D. Chen H. Huang X. Zhong Y. Jiang N. Chen C. Xia M. Association of plasma 7-ketocholesterol with cardiovascular outcomes and total mortality in patients with coronary artery disease.Circ. Res. 2017; 120: 1622-1631Crossref PubMed Scopus (32) Google Scholar). However, it remains unclear whether 7kC itself or its metabolites caused these biological effects. In situ, 7kC is mainly generated by autoxidation of cholesterol during conditions of oxidative stress. In contrast to the low abundance of oxysterols in the circulation compared with cholesterol, 7kC is present at levels up to 10 μM in macrophage-derived foam cells in atherosclerotic lesions and in lenses of patients with cataract (10Girão H. Mota M.C. 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PubMed Scopus Google as was 7β27OHC was by 11β-HSD1 the conditions and be using microsomal preparations revealed an of and an of total for the oxoreduction of 7k27OHC to 7β27OHC by 11β-HSD1 showed the of 7k27OHC to 7β27OHC the total cell total of 1 μM was used for the M. A. metabolism of 7-ketocholesterol by dehydrogenase 1: the and Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A. for inhibitors of PubMed Scopus Google S. J. H. A. of the oxysterols and by dehydrogenase 1 and Mol. Biol. PubMed Scopus Google of was A. for inhibitors of PubMed Scopus Google S. J. H. A. of the oxysterols and by dehydrogenase 1 and Mol. Biol. PubMed Scopus Google of the oxoreduction of 7k27OHC and cortisone by 11β-HSD1 and the oxidation of 7β27OHC and by from of 1 μM was used for the of was in a of the oxoreduction of 7k27OHC and cortisone by 11β-HSD1 and the oxidation of 7β27OHC and by from The role of 11β-HSD2 in the metabolism of was in cells expressing the recombinant human for with of 7β27OHC or showed the oxidation of 7β27OHC to 7k27OHC and a of metabolism Apparent enzyme were as total for 7β27OHC and total for S. J. H. A. of the oxysterols and by dehydrogenase 1 and Mol. Biol. PubMed Scopus Google Furthermore, an an of 11β-HSD2 be of the oxidation reaction of 7β27OHC to 7k27OHC revealed a of and a of 10 total a was recently that 7β27OHC in a first an oxidation from to 7kC by by at by to 7k27OHC and of 7k27OHC to 7β27OHC by oxygen N. D. P. P. oxysterols 2018; Scholar). reactions by oxygen are we the of of and oxidation to 7k27OHC in cancer cells expressing and cells were with 1 μM of in the or of the 11β-HSD2 and the of 7β27OHC and 7k27OHC was 7β27OHC was generated in a from by and further by 11β-HSD2 to To assess the of to with the metabolism of of enzyme were in of cells expressing the enzymes. 7k27OHC the 11β-HSD1-dependent of cortisone to with an of for the human enzyme and of for the of human 11β-HSD1 was for the enzyme at up to 10 μM However, enzyme was by 7β27OHC with an of and 1 μM revealed enzyme 11β-HSD2-dependent of to cortisone was by 7β27OHC and 7k27OHC with of and 1 expressing levels of were used to the of oxysterols with the metabolism of to 7β27OHC the 7k27OHC was a with an of μM 11β-HSD2 has associated with effects and an of atherosclerotic lesions in to of the D. dehydrogenase and in the in 2011; PubMed Scopus Google reporter were cells expressing 11β-HSD2 were with for and a reporter to with and of 11β-HSD2 by 7β27OHC and was to cortisone and an of for 7β27OHC and for compared with the by be 7β27OHC 7k27OHC was to by itself docking calculations were to the of 7β27OHC and in the of 11β-HSDs a for the from the enzyme and were to to the ring of the as as to the and of human and 11β-HSD1 the enzymatic was the of the and the as the substrates to 11β-HSD1 of with a in the of human 11β-HSD1 showed to and particularly to However, docking of to a with as the substrates to the were used for docking to the of 11β-HSD2 7β27OHC and 7k27OHC were in to the and the in a with the of human 11β-HSD2 was the and the human 11β-HSD2 in to the and the the the of the and the In the human was of the with were docking and 7k27OHC to a for their or recent 7β27OHC as an RORγ 7k27OHC was to RORγ in reporter P. J. X. J. M. J. X. G. are of and cell 2014; PubMed Scopus Google Scholar). the of 11β-HSDs to as metabolic or the RORγ was To these cells expressing a RORγ and a reporter were with either an or 11β-HSD2 and to with 7k27OHC and 7k27OHC was of the of the RORγ in the as 7β27OHC of RORγ with 11β-HSD1 compared with the control 11β-HSD2 expressing RORγ cells with 7β27OHC RORγ and showed the as the cells with the as 7k27OHC expressing the control The of oxysterols a of their abundance in biological is low compared with cholesterol (1Schroepfer Jr., G.J. Oxysterols: modulators of cholesterol metabolism and other processes.Physiol. Rev. 2000; 80: 361-554Crossref PubMed Scopus (823) Google Scholar). several on a and the involved the of their biosynthetic pathways is oxysterols a for to (1Schroepfer Jr., G.J. Oxysterols: modulators of cholesterol metabolism and other processes.Physiol. Rev. 2000; 80: 361-554Crossref PubMed Scopus (823) Google Scholar). 7β27OHC be either by oxoreduction of 7kC through 11β-HSD1 to and further by to 7β27OHC through or of by We showed novel biosynthetic the 11β-HSD1-dependent of 7β27OHC and the 11β-HSD2-dependent of the of the 7kC and M. A. metabolism of 7-ketocholesterol by dehydrogenase 1: the and Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M. N. S. A. H. and 1 are involved in Mol. PubMed Scopus Google Scholar, S. D. A. of dehydrogenase 1 from Mol. PubMed Scopus Google Scholar, N. D. P. P. oxysterols 2018; the reported enzymatic reaction by 11β-HSD2 oxysterols the oxidation of to the and the reverse reaction through 11β-HSD1 M. P. A. Dalenc F. J. N. G. A. of a cholesterol in human through the glucocorticoid 2017; PubMed Scopus Google Scholar). is from metabolism by The for the first an of 11β-HSDs in the of side with for RORγ revealed affinities with in the of 11β-HSD1 and 11β-HSD2 for 7k27OHC and respectively. The of 11β-HSD1 for 7k27OHC in the present is higher than the affinities previously reported for other substrates and M. A. metabolism of 7-ketocholesterol by dehydrogenase 1: the and Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A. for inhibitors of PubMed Scopus Google M. A. metabolism of 7-ketocholesterol by dehydrogenase 1: the and Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and A. C. C. A. M. of the bile acid acid is by dehydrogenase J. 2011; PubMed Scopus Google the of 11β-HSD2 for 7β27OHC is higher than that of the previously reported A. for inhibitors of PubMed Scopus Google Scholar). from their metabolic by 7k27OHC and 7β27OHC other reactions by with these substrates at the and the In 7k27OHC the oxoreduction of cortisone to by human 11β-HSD1 of of physiological In the of 7k27OHC and 7β27OHC of and to with 11β-HSD2-dependent oxidation of to the most potent 11β-HSD2 inhibitors and in the 2017; PubMed Scopus Google Scholar). of is in higher availability and of the as in role for 11β-HSD2 function was by the an in the of essential cancer and in the 2017; PubMed Scopus Google Scholar). from the or the to as a leading to and and in the 2017; PubMed Scopus Google Scholar, S. Jiang dehydrogenase is a for of 2013; 1: PubMed Google Scholar). Furthermore, intracellular levels of and through different the physiological of plasma levels for or to the of oxysterols and the of metabolizing enzymes, their be by plasma to it is to assess the or of pathways and the involved and The using cells revealed a of 7k27OHC by and 11β-HSD2 from with an of 7β27OHC of of 7kC and be in the and in the the of 7kC and were reported to to the and the [reviewed in D. S. G. Biasi F. and cholesterol oxidation in and Biol. Med. 2017; PubMed Scopus Google Scholar, Oxysterols in and Med. PubMed Scopus Google Scholar)]. these effects are to remains to be elucidated. are by the and by the of the to to the regulation of the Oxysterols in and Med. PubMed Scopus Google Scholar, C. to the by 2016; Full Text Full Text PDF PubMed Scopus Google Scholar). However, a of these cells has to to C. to the by 2016; Full Text Full Text PDF PubMed Scopus Google Scholar). the nuclear RORγ and are by or by 7k27OHC are considered P. J. X. J. M. J. X. G. are of and cell 2014; PubMed Scopus Google present 11β-HSDs an role by either the 7β27OHC expressing or the from expressing that an of cells the a role for 11β-HSD2 by the RORγ 7β27OHC to 7k27OHC is 11β-HSD1 the Furthermore, recent that the a role in the of inflammatory disease F. and its in inflammatory 2011; PubMed Scopus Google Scholar). of was to in and with disease [reviewed in Oxysterols in and Med. PubMed Scopus Google Scholar)]. a of has associated with progression and with 11β-HSD1 and 11β-HSD2 P. J. J. I. J. of dehydrogenase 1 in during in the Mol. Biol. 2011; PubMed Scopus Google Scholar, M. G. S. M. D. The role and regulation of the dehydrogenase enzyme system in patients with inflammatory 2017; PubMed Scopus Google Scholar, of human 1 and in inflammatory 2009; PubMed Scopus Google Scholar, S. J. P. M. A. J. dehydrogenase 1 and in from patients with 22: PubMed Scopus (39) Google Scholar). This the the to the However, whether prereceptor regulation of RORγ and by 11β-HSDs is involved in the of further and investigation. to be that the is of to the regulation of that human and 11β-HSD1 7k27OHC as and 7k27OHC with cortisone at the 7β27OHC is a for human 11β-HSD2 to be to with for to the enzyme to Nevertheless, the regulation of by 11β-HSDs be further in human cell with of these However, in of the function of RORγ using cell is and cell is still unclear whether are or whether their are by including different oxysterols or intermediates of the cholesterol biosynthetic of the and of the cholesterol biosynthetic in a cell expressing RORγ control receptor and Furthermore, RORγ be with and/or the that to the on of orphan roles in and 2009; PubMed Scopus Google Scholar). The of RORγ be by and by other nuclear and that are and the of a cell RORγ with and either 11β-HSD1 or 11β-HSD2 remains cells be with a reporter to a To the of our knowledge, human cell levels of of our with human and and that their is and that 11β-HSD1 is these cell to RORγ to we in a of cells that RORγ and and of a previously reported Y. C. Wang J. A. W. and are in human and as for and J. 2014; PubMed Scopus Google Scholar). The potent RORγ was to the reporter at of 10 in the potent to of and/or with RORγ we RORγ in in a and a of the reporter by and an of μM for the showed that of 11β-HSD2 in these cells by in a RORγ compared with cells with 7β27OHC This that the of 7β27OHC to 7k27OHC by 11β-HSD2 RORγ from 7β27OHC that the was to 11β-HSD2-dependent of RORγ In of 11β-HSD2 by of in a The the to 11β-HSD2-dependent regulation of RORγ in our autoxidation of 7β27OHC and with the RORγ further metabolism of the oxysterols during the of other RORγ by the cell of from cholesterol by in reporter 7β27OHC is by and with the intracellular with the the cell has to the enzymatic of to it to the prereceptor regulation of on the of cell including to to the control of RORγ by the role of 11β-HSDs on control of 7β27OHC levels be studied in in the of these are In the present demonstrated the stereospecific oxoreduction of 7k27OHC to 7β27OHC by 11β-HSD1 and the reverse oxidation reaction from 7β27OHC to 7k27OHC by human 11β-HSD2 The affinities of 11β-HSD1 and 11β-HSD2 for the oxysterols were equal or higher than for the that are Furthermore, by either the of or 7β27OHC to a novel glucocorticoid-independent prereceptor regulation mechanism by 11β-HSDs be research to cell to the control of RORγ by 11β-HSDs and to assess the of 11β-HSDs in the metabolism of oxysterols is The of and for the for and for for to the with acid dehydrogenase 11β-hydroxysteroid dehydrogenase inflammatory disease cells 7-ketocholesterol 7β-hydroxycholesterol receptor retinoid-related orphan receptor γ known as