Regulation of cholesterol 7 alpha-hydroxylase gene expression in Hep-G2 cells. Effect of serum, bile salts, and coordinate and noncoordinate regulation with other sterol-responsive genes.
Regulation of cholesterol 7 alpha-hydroxylase mRNA level in Hep-G2 cells was studied and compared with that of two other sterol-responsive genes, those for the low density lipoprotein (LDL) receptor and 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. In culture medium containing 10% fetal bovine serum (complete medium) for up to 24 h, the mRNA for cholesterol 7 alpha-hydroxylase gradually increased to 2-fold of the time 0 control. Culture of Hep-G2 cells in serum-free medium for 24 h resulted in stimulation of mRNA levels for LDL receptor (5-fold) and HMG-CoA reductase (6-fold). Surprisingly, the mRNA level for cholesterol 7 alpha-hydroxylase also increased 5-fold at 8 h and 4-fold at 24 h compared with the time 0 control. The addition of beta-migrating very low density lipoprotein (beta-VLDL) (40 micrograms/ml) and 25-hydroxycholesterol (5 micrograms/ml) prevented the increase in mRNA level for the LDL receptor, and HMG-CoA reductase and the levels were 10-26% of the control at 8 h. The effect with beta-VLDL was sustained for 24 h. With 25-hydroxycholesterol, both LDL receptor and HMG-CoA reductase mRNA returned to base line by 24 h. In contrast, beta-VLDL increased cholesterol 7 alpha-hydroxylase mRNA level above the serum-free control within 8 h (+32%), and this was sustained for 24 h (+47%). There was a slight induction of cholesterol 7 alpha-hydroxylase mRNA levels by 25-hydroxycholesterol at 8 h (+18%); but by 24 h, its level was below that of the control (-47%). There was no induction of cholesterol 7 alpha-hydroxylase mRNA levels by beta-VLDL or 25-hydroxycholesterol when the cells were grown in complete medium. As determined by nuclear run-on assay, the increase in the transcriptional rate of the cholesterol 7 alpha-hydroxylase gene in cells grown in serum-free medium (3.9-fold of the rate in complete medium) and incubated with beta-VLDL (+68% above serum-free control) at 8 h, was comparable with the increase in mRNA levels (3.5-fold and +32%, respectively). When bile salts were added to serum-free medium and cells cultured for up to 24 h, chenodeoxycholate and glycochenodeoxycholate caused a marked suppression of the level of cholesterol 7 alpha-hydroxylase mRNA, while cholate and its conjugates did not.(ABSTRACT TRUNCATED AT 400 WORDS)
Taniguchi
T
T
Department of Medicine, Stanford University School of Medicine, California 94305.
Chen
J
J
Cooper
A D
AD
D001647:Bile Acids and Salts / Q000494:pharmacology*; D006528:Carcinoma, Hepatocellular; D002460:Cell Line; D002790:Cholesterol 7-alpha-Hydroxylase / Q000096:biosynthesis* / Q000235:genetics; D003470:Culture Media; D016895:Culture Media, Serum-Free; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D006888:Hydroxycholesterols / Q000494:pharmacology; D006903:Hydroxymethylglutaryl CoA Reductases / Q000096:biosynthesis; D007700:Kinetics; D008074:Lipoproteins / Q000494:pharmacology; D008079:Lipoproteins, VLDL / Q000096:biosynthesis; D008113:Liver Neoplasms; D012333:RNA, Messenger / Q000096:biosynthesis / Q000378:metabolism; D011973:Receptors, LDL / Q000096:biosynthesis; D013997:Time Factors; D014407:Tumor Cells, Cultured
D001647:Bile Acids and Salts; D003470:Culture Media; D016895:Culture Media, Serum-Free; D006888:Hydroxycholesterols; D008074:Lipoproteins; D008079:Lipoproteins, VLDL; D012333:RNA, Messenger; D011973:Receptors, LDL; C007997:25-hydroxycholesterol; D006903:Hydroxymethylglutaryl CoA Reductases; D002790:Cholesterol 7-alpha-Hydroxylase