Shinohara Yasutake, Ando Hiromi, Maekawa Masamitsu, Arai Maki, Horibata Yasuhiro, Satou Motoyasu, Jojima Teruo, Usui Isao, Aso Yoshimasa, Sugimoto Hiroyuki
Department of Biochemistry, Dokkyo Medical University School of Medicine, Japan; Department of Endocrinology and Metabolism, Dokkyo Medical University School of Medicine, Japan.
Department of Biochemistry, Dokkyo Medical University School of Medicine, Japan.
Biochim Biophys Acta Mol Cell Biol Lipids. 2022 Jun;1867(6):159136. doi: 10.1016/j.bbalip.2022.159136. Epub 2022 Mar 16.
Extracellular administration of side-chain oxysterols, such as 24S-hydroxycholesterol (24S-HC), 27-hydroxycholesterol (27-HC) and 25-hydroxycholesterol (25-HC) to cells suppresses HMG-CoA reductase (Hmgcr) and CTP:phosphoethanolamine cytidylyltransferase (Pcyt2) mRNA levels. Oxysterols are enzymatically produced in cells from cholesterol by cytochrome P450 46A1 (Cyp46A1), Cyp27A1, Cyp3A11 and cholesterol 25-hydroxylase (Ch25h). We analyzed which of these oxysterol-producing enzymes are expressed in NIH3T3 cells and found that only Cyp46A1 was expressed. When Cyp46A1 was overexpressed in NIH3T3 cells, intrinsic oxysterols increased in the order 24S-HC > 25-HC > 27-HC. We investigated the mechanism regulating the production of endogenous oxysterols in NIH3T3 cells by Cyp46A1 and found that the mRNA, relative protein levels and enzymatic activity of Cyp46A1, and the amounts of 24S-HC, 25-HC and 27-HC significantly increased under serum-starved conditions, and these increases were suppressed by FBS supplementation. The aqueous phase of FBS obtained by the Bligh & Dyer method significantly suppressed Cyp46A1 mRNA levels. Fractionation of the aqueous phase by HPLC and analysis of the inhibiting fractions by nanoLC and TripleTOF MS/MS identified insulin-like factor-II (IGF-II). Cyp46A1 mRNA levels in serum-starved NIH3T3 cells were significantly suppressed by the addition of IGFs and insulin and endogenous oxysterol levels were decreased. CYP46A1 mRNA levels in the T98G human glioblastoma cell line were also increased by serum starvation but not by FBS supplementation, and the aqueous phase did not inhibit the increase. These results suggest that mRNA levels of Cyp46A1 are regulated by factors in FBS.
向细胞外给予侧链氧化甾醇，如24S-羟基胆固醇（24S-HC）、27-羟基胆固醇（27-HC）和25-羟基胆固醇（25-HC），可抑制3-羟基-3-甲基戊二酰辅酶A还原酶（Hmgcr）和CTP：磷酸乙醇胺胞苷酰转移酶（Pcyt2）的mRNA水平。氧化甾醇是细胞内胆固醇通过细胞色素P450 46A1（Cyp46A1）、Cyp27A1、Cyp3A11和胆固醇25-羟化酶（Ch25h）酶促生成的。我们分析了这些产生氧化甾醇的酶中哪些在NIH3T3细胞中表达，发现只有Cyp46A1表达。当Cyp46A1在NIH3T3细胞中过表达时，内源性氧化甾醇以24S-HC>25-HC>27-HC的顺序增加。我们研究了Cyp46A1调节NIH3T3细胞内源性氧化甾醇产生的机制，发现血清饥饿条件下Cyp46A1的mRNA、相对蛋白水平和酶活性以及24S-HC、25-HC和27-HC的量显著增加，而补充胎牛血清（FBS）可抑制这些增加。通过布莱-戴尔方法获得的FBS水相显著抑制Cyp46A1 mRNA水平。通过高效液相色谱（HPLC）对水相进行分级分离，并通过纳升液相色谱（nanoLC）和三重四极杆飞行时间质谱（TripleTOF MS/MS）对抑制组分进行分析，鉴定出胰岛素样因子-II（IGF-II）。添加IGF和胰岛素可显著抑制血清饥饿的NIH3T3细胞中Cyp46A1 mRNA水平，并降低内源性氧化甾醇水平。T98G人胶质母细胞瘤细胞系中CYP46A1 mRNA水平也因血清饥饿而增加，但FBS补充未使其增加，且水相不抑制这种增加。这些结果表明Cyp46A1的mRNA水平受FBS中的因子调节。
