Selective activation of liver X receptor alpha by 6α-hydroxy bile acids and analogs

doi:10.1016/S0039-128X(00)00127-6

Steroids

Volume 65, Issue 8, August 2000, Pages 423-427

https://doi.org/10.1016/S0039-128X(00)00127-6 Get rights and content

Abstract

We have found that certain natural 6α-hydroxylated bile acids are receptor-specific activators of nuclear liver X receptor alpha (LXRα) (NR1H3), a nuclear receptor regulating the expression of the cholesterol 7α-hydroxylase gene, coding for the rate-limiting enzyme in the major pathway of bile acid synthesis. The LXR homolog, ubiquitous nuclear receptor (UR/LXRβ) (NR1H2), was also activated by these bile acids, but at higher concentrations than for LXRα. Synthetic 6α-hydroxylated bile acid analogs were synthesized with LXRα-selective agonistic activity, with potential to modulate cholesterol catabolism in hypercholesterolemia.

Introduction

Bile acids are synthesized from cholesterol by two pathways [1], [2]. The first is the classic or neutral pathway in which cholesterol 7α-hydroxylase catalyzes the first step. In the second, known as the acidic pathway, cholesterol is first oxidized to form 27-hydroxycholesterol and later by an oxysterol 7α-hydroxylase [3], [4]. Another alternative step involves 6-hydroxylation of primary or secondary bile acids. In the acidic pathway of bile acid synthesis, cholesterol is first hydroxylated and oxidized at the side chain C-27 position. The bile acids ultimately formed can then be hydroxylated at the 6α position by a cytochrome P450 enzyme coded by the CYP3A4 gene [5]. Natural 6α-hydroxylated steroids with 24-keto side chains include free and conjugated 3α, 6α-dihydroxy-5β-cholanoic (hyodeoxycholic) [6], [7] and 3α,6α,7α-trihydroxy-5β-cholanoic (hyocholic) acids [8].

Recently a role for certain nuclear receptors in cholesterol catabolism was defined [9], [10], [11], [12], [13], [14]. RLD-1/LXR (LXRα) [15], [16] was identified as a transcriptional activator of the cholesterol 7α-hydroxylase gene. Genetically engineered mice with a LXRα deficiency maintain basal expression of cholesterol 7α-hydroxylase, but fail to up regulate 7α-hydroxylase expression in response to a high-cholesterol diet [10]. The nuclear receptor UR/NER/OR-1/RIP15 (UR/LXRβ) [17], [18], [19], [20], which shares high structural homology and oxycholesterol ligand specificity with LXRα and is ubiquitously expressed, did not compensate for the loss of LXRα expression [10]. This paper describes selective activation of LXRα by 6α-hydroxy bile acids and their analogs.

Section snippets

Transactivation assay

Human embryonic kidney 293 cells were seeded into 48-well culture plates at 10⁵ cells per well in DMEM supplemented with 10% fetal bovine serum. After 24 h, cells were transfected by a calcium phosphate coprecipitation method with 250 ng of the pGL3/UREluc reporter gene that consists of three copies of AGGTCAagccAGGTCA fused to nucleotides −56 to +109 of the human c-fos promoter in front of the firefly luciferase gene in the plasmid basic pGL3 (Promega, Madison, WI, USA), 40 ng pSG5/hRXRα, 40

Results

In this study, we co-transfected human embryonic kidney 293 cells with a LXRα/UR reporter vector (pGL3/UREluc), along with expression vectors for LXRα or UR, RXRα, and the co-activator Grip1. Test compounds were added to the culture media and reporter gene expression was measured. Using this assay system, we determined the ED₅₀ for oxysterol ligands (e.g. 100 nM for N,N-dimethyl-3β-hydroxy-5-cholenamide, Fig. 2j) for both receptors and these values were in agreement with the published K_i of

Discussion

The acidic bile acid synthesis pathway has been proposed to have regulatory roles for cholesterol catabolism [28]. Based on the data presented here, it remains a possibility that 6α-hydroxy bile acids, products of the acidic bile acid pathway, which are normally not present in large quantity, but accumulate in 7α-hydroxylation blockage, are an endogenous signal serving as natural ligands for LXRα regulating expression of the cholesterol 7α-hydroxylase, the rate-limiting enzyme of the neutral

Acknowledgements

The authors would like to thank Dr J. Y. Chiang for the rat pGL2/7αluc reporter plasmid and human PH/hCYP7A-135 7α-hydroxylase reporter plasmids, Dr D. J. Mangelsdorf for CMV/hLXRα, Dr P. A. Dawson for pCMV/hIBAT, Dr M. Stallcup for pSG5/hGrip1, and Dr Fangjie Zhang for advice on chemical synthesis. This work was supported by NIH grants.

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Rapid communicationSelective activation of liver X receptor alpha by 6α-hydroxy bile acids and analogs

Abstract

Introduction

Section snippets

Transactivation assay

Results

Discussion

Acknowledgements

J Lipid Res

J Steroid Biochem

Biochim Biophys Acta

J Biol Chem

J Biol Chem

Cell

J Lipid Res

Mol Cell

Gene

J Biol Chem

J Chromatogr B Biomed Sci Appl

J Biol Chem

J Lipid Res

J Biol Chem

Regulation of bile acid synthesis [In Process Citation]

Front Biosci

Rapid communication
Selective activation of liver X receptor alpha by 6α-hydroxy bile acids and analogs