TY - JOUR
T1 - Transcription of cytochrome P450 46A1 in NIH3T3 cells is negatively regulated by FBS
AU - Shinohara, Yasutake
AU - Ando, Hiromi
AU - Maekawa, Masamitsu
AU - Arai, Maki
AU - Horibata, Yasuhiro
AU - Satou, Motoyasu
AU - Jojima, Teruo
AU - Usui, Isao
AU - Aso, Yoshimasa
AU - Sugimoto, Hiroyuki
N1 - Funding Information:
We would like to thank Dr. Takashi Namatame of the Clinical Research Center for DNA Sequencing, Dokkyo Medical University. This work was supported in part by Japan Society for the Promotion of Science KAKENHI Grants 15K08284 (H.S.), 20K07314 (H.S.), and 20K07329 (H.A.).
Funding Information:
We would like to thank Dr. Takashi Namatame of the Clinical Research Center for DNA Sequencing, Dokkyo Medical University. This work was supported in part by Japan Society for the Promotion of Science KAKENHI Grants 15K08284 (H.S.), 20K07314 (H.S.), and 20K07329 (H.A.). (a) NIH3T3 cells were cultured in 10% FBS (white bars) or serum-starved medium (0.5% FBS) (black bars) for 24 h, then treated with or without actinomycin D (1 ?M) for 16 h. The mRNA levels of Cyp46A1 were quantified relative to Gapdh mRNA levels. (B) NIH3T3 cells were cultured in medium with 10% FBS (white bars) or serum-starved medium (0.5% FBS) (black bars). At 0, 10 and 24 h incubation, the mRNA levels of Cyp46A1 were quantified relative to Gapdh mRNA levels. (C) NIH3T3 cells were cultured in serum-starved medium (0.5% FBS) for 48 h, then FBS was added at concentrations ranging from 0.5% to 10%. After 12 h incubation, the mRNA level of Cyp46A1 was quantified relative to Gapdh mRNA levels. Values shown are means ? S.D. from three independent culture dishes. Each experiment was repeated at least three times with similar results. * and ** indicate significant differences as compared to cells cultured in 0.5% FBS medium (p < 0.05 and p < 0.01, respectively). For A and B, statistical significance was assessed using the Student's t-test. For C, one-way ANOVA with Tukey test was performed.
Publisher Copyright:
© 2022 Elsevier B.V.
PY - 2022/6
Y1 - 2022/6
N2 - 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.
AB - 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.
KW - 24S-Hydroxycholesterol
KW - 25-Hydroxycholesterol
KW - 27-Hydroxycholesterol
KW - CTP: phosphoethanolamine cytidylyltransferase (Pcyt2)
KW - Cholesterol
KW - Cytochrome P450 46A1
KW - Fetal bovine serum (FBS)
KW - HMG-CoA reductase (HMGCR)
KW - Insulin-like growth factor-II (IGF-II)
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U2 - 10.1016/j.bbalip.2022.159136
DO - 10.1016/j.bbalip.2022.159136
M3 - Article
C2 - 35306146
AN - SCOPUS:85126509547
SN - 1388-1981
VL - 1867
JO - BBA - Specialised Section On Lipids and Related Subjects
JF - BBA - Specialised Section On Lipids and Related Subjects
IS - 6
M1 - 159136
ER -