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 cel ls was studied and compared with that of two other sterol-responsive g enes, those for the low density lipoprotein (LDL) receptor and 3-hydro xy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. In culture medium containing 10% fetal bovine serum (complete medium) for up to 24 h, th e mRNA for cholesterol 7 alpha-hydroxylase gradually increased to 2-fo ld 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-f old) and HMG-CoA reductase (6-fold). Surprisingly, the mRNA level for cholesterol 7 alpha-hydroxylase also increased 5-fold at 8 h and 4-fol d at 24 h compared with the time 0 control. The addition of beta-migra ting very low density lipoprotein (beta-VLDL) (40 mug/ml) and 25-hydro xycholesterol (5 mug/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 r eturned to base line by 24 h. In contrast, beta-VLDL increased cholest erol 7 alpha-hydroxylase mRNA level above the serum-free control withi n 8 h (+32%), and this was sustained for 24 h (+47%). There was a slig ht induction of cholesterol 7 alpha-hydroxylase mRNA levels by 25-hydr oxycholesterol 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-hyd roxylase mRNA levels by beta-VLDL or 25-hydroxycholesterol when the ce lls were grown in complete medium. As determined by nuclear run-on ass ay, the increase in the transcriptional rate of the cholesterol 7 alph a-hydroxylase gene in cells grown in serum-free medium (3.9-fold of th e rate in complete medium) and incubated with beta-VLDL (+68% above se rum-free control) at 8 h, was comparable with the increase in mRNA lev els (3.5-fold and +32%, respectively). When bile salts were added to s erum-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 conjugate s did not. The chenodeoxycholate effect was dose-dependent and was app arent within 2 h. It occurred at the level of transcription, as judged by decreased nuclear run-on. These bile salts did not appear to effec t cholesterol efflux from the cells, their viability, the rate of tran scription of several housekeeping genes or albumin secretion. These re sults demonstrate that Hep-G2 cells will be useful for elucidating the molecular events in the regulation of the cholesterol 7 alpha-hydroxy lase gene in a human liver system. In Hep-G2 cells grown in the absenc e of serum, the cholesterol 7 alpha-hydroxylase gene responds to stero ls, hormones, and bile salts.