Activation of acyl-coenzyme A:cholesterol acyltransferase activity by cholesterol is not due to altered mRNA levels in HepG2 cells.
Many studies have shown that sterols can stimulate acyl-coenzyme A:cholesterol acyltransferase (ACAT) activity in cells. To elucidate this mechanism, effects of sterol-mediated induction on both the enzyme activity of ACAT and its mRNA levels were studied in human hepatoblastoma cell line, HepG2 cells. When HepG2 cells were loaded with cholesterol and 25-hydroxycholesterol, both the whole-cell ACAT activity and the microsomal ACAT activity were increased by 85.1% and 41.3%. In contrast, cholesterol depletion of HepG2 cells with compactin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, resulted in a decrease in both the whole-cell and the microsomal ACAT activity by 46.4% and 58.3%. Under identical conditions, RT-PCR and Northern blotting analyses revealed that neither cholesterol loading nor cholesterol depletion of HepG2 cells altered the amounts of ACAT mRNA. Moreover, these treatments had no effect on the enzymatic ACAT activity determined by the reconstituted assay in which HepG2 cell homogenate had been supplemented in vitro with a saturating level of exogenous cholesterol. These results indicate that cholesterol-induced up-regulation of ACAT activity in HepG2 cells does not occur at the level of transcription, but rather at a posttranscriptional level.
Matsuda
H
H
Department of Biochemistry, Kumamoto University School of Medicine, Japan.
Hakamata
H
H
Miyazaki
A
A
Sakai
M
M
Chang
C C
CC
Chang
T Y
TY
Kobori
S
S
Shichiri
M
M
Horiuchi
S
S
D001483:Base Sequence; D015152:Blotting, Northern; D002784:Cholesterol / Q000494:pharmacology*; D018197:Hepatoblastoma / Q000201:enzymology; D006801:Humans; D008077:Lipoproteins, LDL / Q000378:metabolism; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis*; D002785:Sterol O-Acyltransferase / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D014407:Tumor Cells, Cultured
D008077:Lipoproteins, LDL; D012333:RNA, Messenger; D002784:Cholesterol; D002785:Sterol O-Acyltransferase