Hep G2 cells: a model for studies on regulation of human cholesterol 7alpha-hydroxylase at the molecular level

Abstract

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.

Complex feedback regulation of bile acid synthesis in the hamster: The role of newly synthesized cholesterol

“…63 Because the amount of deoxycholate infused represented only 24% of the cholate dose, a superior suppressive capacity of this more hydrophobic bile acid is likely, at least under physiological conditions. Recent studies in human HepG2 cells support 15,16,64 the role of hydrophobicity for regulation of bile acid synthesis. Our studies in healthy volunteers 19 clearly support such an assumption, although a reduced capability of conjugated cholate uptake in HepG2 cells 15 may have contributed to these observations in this experimental system.…”

“…Recent studies in human HepG2 cells support 15,16,64 the role of hydrophobicity for regulation of bile acid synthesis. Our studies in healthy volunteers 19 clearly support such an assumption, although a reduced capability of conjugated cholate uptake in HepG2 cells 15 may have contributed to these observations in this experimental system. With respect to de novo cholesterol in the short term, bile acid uptake by periportal hepatocytes, known for their high capacity to synthesize cholesterol 65 and cholate, 66 may have stimulated cholate formation from de novo cholesterol by increasing the availability of newly formed cholesterol.…”

“…[9][10][11][12] Thus, we suggested that the more hydrophobic secondary bile acids may be more effective than the primary bile acids. 10 In general, with increasing hydrophobicity, bile acids gain a higher capacity to suppress bile acid synthesis 13 in cultured hepatoma cells, [14][15][16] experimental animals, 10,13,17,18 and humans. 19,20 Therefore, an increased intestinal formation of secondary bile acids during expansion of the primary bile acid pool could result in a decreased hepatic formation of primary bile acids.…”

Bile acid synthesis in primary cultures of rat and human hepatocytes

“…Trihydroxycoprostanoic acid is formed due to impaired oxidation of C 27 sterols. [16][17][18] HepG2 cells may also be different from normal human hepatocytes with regard to their conjugation of bile acids. 19 Primary human hepatocytes cultured under appropriate conditions have been shown to maintain specialized hepatic functions such as albumin secretion and cytochrome P450 expression (Liddle C et al, unpublished data).…”

Decreased Expression of Cholesterol 7α-Hydroxylase and Altered Bile Acid Metabolism in Apobec-1−/− Mice Lead to Increased Gallstone Susceptibility

“…We have previously demonstrated that APO-BEC-1 binds AU-rich RNA templates, particularly those containing the high affinity consensus sequence (UUUN(A/U)U) embedded within its canonical target ApoB RNA as well as others, including c-Myc and Cox-2, and demonstrated that APO-BEC-1 binding to these RNA templates also modulates mRNA stability (33,36). Other work has established a strong precedent for posttranscriptional regulation of Cyp7a1 mRNA, and several authors have pointed out the short half-life (ϳ30 min to 1 h) of Cyp7a1 mRNA both in cell culture (42) and in vivo in rat (43)(44)(45) and hamster liver (46). In particular, the 3Ј-UTR of murine (as well as human and rat) Cyp7a1 contains several AUUUA motifs, which are characteristic of short lived mRNAs (37,38,47).…”