Biosynthesis of the regulatory oxysterol, 5-cholesten-3β,25-diol 3-sulfate, in hepatocytes

Xiaobo Li,William M Pandak,Sandra K Erickson,Yongjie Ma,Lianhua Yin,Phillip Hylemon,Shunlin Ren

doi:10.1194/jlr.m700301-jlr200

Abstract

Cellular cholesterol homeostasis is maintained through coordinated regulation of cholesterol synthesis, degradation, and secretion. Nuclear receptors for oxygenated cholesterol derivatives (oxysterols) are known to play key roles in the regulation of cholesterol homeostasis. We recently identified a sulfated oxysterol, 5-cholesten-3beta,25-diol 3-sulfate (25HC3S), that is localized to liver nuclei. The present study reports a biosynthetic pathway for 25HC3S in hepatocytes. Assays using mitochondria isolated from rats and sterol 27-hydroxylase (Cyp27A1) gene knockout mice indicated that 25-hydroxycholesterol (25HC) is synthesized by CYP27A1. Incubation of cholesterol or 25HC with mitochondrial and cytosolic fractions in the presence of 3'-phosphoadenosyl 5'-phosphosulfate resulted in the synthesis of 25HC3S. Real-time RT-PCR and Western blot analysis showed the presence of insulin-regulated hydroxycholesterol sulfotransferase 2B1b (SULT2B1b) in hepatocytes. 25HC3S, but not 25HC, decreased SULT2B1b mRNA and protein levels. Specific small interfering RNA decreased SULT2B1b mRNA, protein, and activity levels. These findings demonstrate that mitochondria synthesize 25HC, which is subsequently 3beta-sulfated to form 25HC3S.

Highlights

Cellular cholesterol homeostasis is maintained through coordinated regulation of cholesterol synthesis, degradation, and secretion
We provide evidence that CYP27A1 is responsible for the synthesis of 25HC in hepatocyte mitochondria and that the insulin-regulated cytosolic SULT2B1b is responsible for its sulfation
To confirm that mitochondrial CYP27A1 is responsible for the synthesis of 25HC, mitochondria were isolated from livers of wild-type and Cyp27A1 gene knockout mice

Summary

Introduction

Cellular cholesterol homeostasis is maintained through coordinated regulation of cholesterol synthesis, degradation, and secretion. A novel oxysterol, 5-cholesten-3b,25-diol 3sulfate (25HC3S), was found in human liver nuclei, and its levels were increased dramatically in nuclei after overexpression of a mitochondrial cholesterol delivery protein, StarD1, in primary rat hepatocytes [15]. This sulfated oxysterol was believed to be initially synthesized by CYP27A1 in mitochondria [15]. It has been reported that the addition of 25HC3S to primary human hepatocytes decreases cholesterol and fatty acid biosynthesis by blocking the activation of sterol-regulatory element binding protein-1c and inhibiting its expression [16] These results suggest that 25HC3S plays an important role in the maintenance of intracellular cholesterol and lipid homeostasis. It is likely that SULT2B1b is involved in oxysterol sulfation, which plays an important role in lipid metabolism in liver [16]

Methods

Results

Conclusion