Abstract
Regulation of squalene epoxidase (SE) gene expression was studied in comparison with those of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase and low density lipoprotein (LDL) receptor. An increased expression of SE mRNA and protein content in mouse L929 cells grown in 10% lipoprotein-deficient fetal bovine serum (LPDS) for 48 h was found by performing immunoblot and Northern blot analyses when compared with the culture in the presence of fetal bovine serum (FBS). The same results in mRNA levels were seen using human cell lines HepG2, HeLa, and Chang liver cells. The increase of SE mRNA in HeLa cells grown in LPDS was preventable in a dose-dependent manner by feeding cells with 25-hydroxycholesterol or cholesterol. When an SE inhibitor, NB-598, was fed to HeLa cells grown in LPDS, it caused further increases in mRNA levels of SE, HMG-CoA reductase, and LDL receptor. In contrast, NB-598 had no effect on the message levels of these genes when fed to HeLa cells grown in FBS. These results suggest that sterol produced endogenously can also regulate SE expression at the level of transcription.
Highlights
Since cholesterol is an essential structural component of cytoplasmic membranes, it is crucial for cells to maintain intracellular cholesterol homeostasis
Effect of fetal bovine serum (FBS) and lipoprotein-deficient fetal bovine serum (LPDS) Media on squalene epoxidase (SE) in L929 Cells—To establish whether LPDS and FBS have effects on the expression of SE, we analyzed the levels of SE protein and mRNA in L929 cells grown in 10% LPDS or FBS
Mouse L929 cells were harvested after incubation in medium with either LPDS or FBS for 48 h
Summary
Materials—[␣-32P]dCTP (3000 Ci/mmol) was purchased from DuPont NEN. Cholesterol and 25-hydroxycholesterol were purchased from Nakarai Tesque Co. (Tokyo, Japan) and Sigma, respectively. Fetal bovine serum (FBS) was purchased from Bioserum (Canterbury, Australia). HepG2 and Chang liver cells were gifts from Dr J. Cell culture medium was purchased from Nissui Pharmaceutical Co. Cell Culture and Induction—Cells were grown in Dulbecco’s modified essential medium (DMEM) containing 10% fetal bovine serum (FBS medium). 5.0 ϫ 106 cells were seeded in 100-mm dishes containing 10 ml of FBS medium. The medium was supplemented with cholesterol, 25-hydroxycholesterol, lovastatin, NB-598, or ethanol vehicle alone (1%) and cultured for 48 h.
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