Li Xiaobo, Pandak William M, Erickson Sandra K, Ma Yongjie, Yin Lianhua, Hylemon Phillip, Ren Shunlin
Department of Medicine Veterans Affairs McGuire Medical Center, Virginia Commonwealth University, Richmond, VA 23249, USA.
J Lipid Res. 2007 Dec;48(12):2587-96. doi: 10.1194/jlr.M700301-JLR200. Epub 2007 Sep 21.
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.
细胞胆固醇稳态通过胆固醇合成、降解和分泌的协调调节得以维持。含氧胆固醇衍生物（氧化甾醇）的核受体在胆固醇稳态调节中发挥关键作用。我们最近鉴定出一种硫酸化氧化甾醇，即5-胆甾烯-3β,25-二醇3-硫酸盐（25HC3S），它定位于肝细胞核。本研究报告了肝细胞中25HC3S的生物合成途径。使用从大鼠和甾醇27-羟化酶（Cyp27A1）基因敲除小鼠分离的线粒体进行的测定表明，25-羟胆固醇（25HC）由CYP27A1合成。在3'-磷酸腺苷5'-磷酸硫酸存在下，将胆固醇或25HC与线粒体和胞质部分一起孵育会导致25HC3S的合成。实时逆转录聚合酶链反应（RT-PCR）和蛋白质印迹分析表明肝细胞中存在胰岛素调节的羟胆固醇硫酸转移酶2B1b（SULT2B1b）。25HC3S而非25HC降低了SULT2B1b的mRNA和蛋白质水平。特异性小干扰RNA降低了SULT2B1b的mRNA、蛋白质和活性水平。这些发现表明线粒体合成25HC，随后25HC被3β-硫酸化形成25HC3S。
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