Orphan Nuclear Receptor RORα Regulates Enzymatic Metabolism of Cerebral 24S-Hydroxycholesterol through CYP39A1 Intronic Response Element Activation.

Hiroshi Matsuoka,Akiho Shima,Yuya Nishimoto,Riki Tokunaga,Kazutake Hirooka,Ami Ohishi,Kaoruko Miya,Sou Kobayashi,Akihiro Michihara,Miyu Katayama

doi:10.3390/ijms21093309

Abstract

Oxysterols, important regulators of cholesterol homeostasis in the brain, are affected by neurodegenerative diseases. Early-onset Alzheimer’s disease is associated with higher levels of circulating brain-derived 24S-hydroxycholesterol (24S-OHC). Conversion of cholesterol to 24S-OHC is mediated by cholesterol 24S-hydroxylase in the brain, which is the major pathway for oxysterol elimination, followed by oxidation through hepatic first-pass metabolism by CYP39A1. Abnormal CYP39A1 expression results in accumulation of 24S-OHC, influencing neurodegenerative disease-related deterioration; thus, it is important to understand the normal elimination of 24S-OHC and the system regulating CYP39A1, a selective hepatic metabolic enzyme of 24S-OHC. We examined the role of transcriptional regulation by retinoic acid receptor-related orphan receptor α (RORα), a nuclear receptor that responds to oxysterol ligands. In humans, the promoter and first intronic regions of CYP39A1 contain two putative RORα response elements (ROREs). RORα binding and responses of these ROREs were assessed using electrophoretic mobility shift, chromatin immunoprecipitation, and luciferase reporter assays. CYP39A1 was upregulated by RORα overexpression in HEK293 cells, while RORα knockdown by siRNA significantly downregulated CYP39A1 expression in human hepatoma cells. Additionally, CYP39A1 was induced by RORα agonist treatment, suggesting that CYP39A1 expression is activated by RORα nuclear receptors. This may provide a way to increase CYP39A1 activity using RORα agonists, and help halt 24S-OHC accumulation in neurodegenerative illnesses.

Highlights

While the brain only accounts for 2% of the total body weight, it contains 25% of all the cholesterol in the body [1]
About 2–7 mg of cholesterol gets converted to 24S-hydroxycholesterol (24S-OHC), moving from the brain to the periphery every 24 h [4], where it is selectively transformed by the 7-α-hydroxylase activity of CYP39A1, a cytochrome P450 enzyme involved in hepatic first-pass metabolism [5]
27-OHC is the main oxysterol that flows from the periphery of the blood circulation into the brain through the blood-brain barrier (BBB) [6]

Summary

Introduction

While the brain only accounts for 2% of the total body weight, it contains 25% of all the cholesterol in the body [1]. We showed that CYP39A1 was induced following RORα agonist treatment (Figure 5), suggesting CYP39A1 expression can be upregulated, increasing elimination and inhibiting accumulation of 24S-OHC, reducing neuronal death. Another possibility is that 24S-OHC acts as an endogenous inverse agonist at RORα and RORγ [24] and an agonist at LXR [18]. We showed the RORα nuclear receptor directly bound to two ROREs located upstream of the promoter and downstream of the first intronic region of the CYP39A1 transcription start site, resulting in CYP39A1 expression regulation. RORα agonist administration induced CYP39A1 expression, suggesting RORα agonists could be used to suppress neuronal death

Cell Culture

Luciferase Reporter Assays

Western Blotting

Overexpression Analysis

Findings

RORα Agonist Treatments