Hep G2 cells: a model for studies on regulation of human cholesterol 7alpha-hydroxylase at the molecular level.
The present study examines the feedback control governing human cholesterol 7alpha-hydroxylase mRNA expression in the human hepatoblastoma cell line, Hep G2. Glycochenodeoxycholate (GCDC) and glycodeoxycholate, hydrophobic bile salts, decreased cholesterol 7alpha-hydroxylase mRNA levels and bile acid synthesis in a concentration-dependent (76 +/- 8%, P<0.001, and 48 +/- 3%, P<0.01, respectively) and time-dependent manner. Cholesterol 7alpha-hydroxylase mRNA levels were repressed with a half-maximal inhibitory concentration of <12.5 microM by GCDC and a half-life of 30 min by 100 microM of the bile acid. The addition of actinomycin D (10 microgram/ml) alone or in combination with GCDC (100 microM) led to similar concentration-and time-dependent suppression of cholesterol 7alpha-hydroxylase mRNA. Glycocholate (100 microM), not internalized based on lack of uptake of a fluorescent cholate analogue, had no effect on cholesterol 7alpha-hydroxylase mRNA or total bile acid synthesis. In cultures transfected with a rat cholesterol 7alpha-hydroxylase promoter construct, reporter gene activity was decreased (31%, P<0.01) by GCDC (100 microM). Hep G2 cells maintain the intracellular machinery to express and rapidly regulate human cholesterol 7alpha-hydroxylase by hydrophobic bile acids. These data suggest that Hep G2 cells will support functional studies of the human cholesterol 7alpha-hydroxylase gene.
Pandak
W M
WM
Department of Medicine, Medical College of Virginia, Virginia Commonwealth University, Richmond 23298-0711.
Stravitz
R T
RT
Lucas
V
V
Heuman
D M
DM
Chiang
J Y
JY
D000818:Animals; D001647:Bile Acids and Salts / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D002790:Cholesterol 7-alpha-Hydroxylase / Q000096:biosynthesis*; D003609:Dactinomycin / Q000494:pharmacology; D005246:Feedback; D005456:Fluorescent Dyes; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D005999:Glycochenodeoxycholic Acid / Q000494:pharmacology; D006002:Glycodeoxycholic Acid / Q000494:pharmacology; D018197:Hepatoblastoma; D006801:Humans; D007700:Kinetics; D008081:Liposomes; D008113:Liver Neoplasms; D008156:Luciferases / Q000096:biosynthesis; D008954:Models, Biological; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis; D014162:Transfection; D014407:Tumor Cells, Cultured
D001647:Bile Acids and Salts; D005456:Fluorescent Dyes; D008081:Liposomes; D012333:RNA, Messenger; D011994:Recombinant Proteins; D003609:Dactinomycin; D006002:Glycodeoxycholic Acid; D005999:Glycochenodeoxycholic Acid; D008156:Luciferases; D002790:Cholesterol 7-alpha-Hydroxylase