Transcription of cytochrome P450 46A1 in NIH3T3 cells is negatively regulated by FBS
Transcription of cytochrome P450 46A1 in NIH3T3 cells is negatively regulated by FBS
复制标题
NIH3T3 细胞中细胞色素 P450 46A1 的转录受到 FBS 的负调控
DOI：
10.1016/j.bbalip.2022.159136
复制
发表时间：
2022
期刊：
影响因子：
--
通讯作者：
Sugimoto H.
中科院分区：
文献类型：
--
作者：
Shinohara Y;Ando H;Maekawa M;Arai M;Horibata Y;Satou M;Jojima T;Usui I;Aso Y;Sugimoto H.
细胞外给予侧链氧固醇，如24 S-羟基胆固醇（24 S-HC）、27-羟基胆固醇（27-HC）和25-羟基胆固醇（25-HC），抑制Hmgcr和Pcyt 2 mRNA水平。氧化固醇也是通过细胞中的细胞色素P450 46 A1（Cyp 46 A1）、Cyp 27 A1、Cyp 3A 11和胆固醇25-羟化酶（Ch 25 h）从胆固醇酶促产生的。由于我们主要用NIH 3 T3细胞进行了先前的实验，我们分析了哪种类型的这些氧固醇产生酶在NIH 3 T3细胞中表达。在NIH 3 T3细胞中，仅Cyp 46 A1表达。当Cyp 46 A1在NIH 3 T3细胞中过表达时，内源性氧化固醇的增加为24 S-HC> 25-HC> 27-HC，并且d-胆固醇被代谢为24 S-HC。因此，我们感兴趣的机制，调节生产的内源性氧化固醇在细胞Cyp 46 A1。在此，我们发现，在血清饥饿条件下，NIH 3 T3细胞中Cyp 46 A1的mRNA和相对蛋白水平和酶活性以及24 S-HC、25-HC和27-HC的量显著增加，并且这些增加被FBS补充抑制。为了在FBS中鉴定该因子，通过Bligh & Dyer方法获得的水相显著抑制Cyp 46 A1 mRNA水平，并通过HPLC分级。通过nanoLC和TripleTOF MS/MS分析抑制级分，然后鉴定胰岛素样因子-II（IGF-II）。在NIH 3 T3细胞中加入IGF-II、IGF-I和胰岛素可显著抑制血清饥饿细胞中Cyp 46 A1 mRNA的表达，显著提高Hmgcr mRNA的表达，并降低内源性氧固醇的水平。这些结果表明，Cyp 46 A1的mRNA和蛋白质水平的调控因素，如IGF-II在FBS。
Extracellular administration of side-chain oxysterols, such as 24S-hydroxycholesterol (24S-HC), 27-hydroxycholesterol (27-HC) and 25-hydroxycholesterol (25-HC), to cells suppresses Hmgcr and Pcyt2 mRNA levels. Oxysterols are also enzymatically produced from cholesterol by Cytochrome P450 46A1 (Cyp46A1), Cyp27A1, Cyp3A11 and cholesterol 25-hydroxylase (Ch25h) in cells. As we performed previous experiments mainly with NIH3T3 cells, we analyzed which type of these oxysterol producing enzymes were expressed in NIH3T3 cells. In NIH3T3 cells, only Cyp46A1 was expressed. When Cyp46A1 was overexpressed in NIH3T3 cells, intrinsic oxysterols were increased as 24S-HC> 25-HC> 27-HC, and d-cholesterol was metabolized to 24S-HC. We are therefore interested in the mechanism regulating the production of endogenous oxysterols in cells by Cyp46A1. Here, we showed that the mRNA and relative protein levels and enzymatic activity of Cyp46A1, and the amount of 24S-HC, 25-HC and 27-HC in NIH3T3 cells, significantly increased under serum starved conditions, and these increases are suppressed by FBS supplementation. To identify this factor in FBS, the aqueous phase obtained by Bligh & Dyer method significantly suppressed Cyp46A1 mRNA levels, and was fractionated by HPLC. The inhibiting fractions were analyzed by nanoLC and TripleTOF MS/MS and then insulin like factor-II (IGF-II) was identified. Cyp46A1 mRNA levels in serum-starved cells were significantly suppressed and Hmgcr mRNA levels were significantly enhanced by the addition of IGF-II, IGF-I and insulin in NIH3T3 cells, and endogenous oxysterol levels were decreased. These results suggest that mRNA and protein levels of Cyp46A1 are regulated by factors such as IGF-II in FBS.