TRANSCRIPTIONAL REGULATION BY LOVASTATIN AND 25-HYDROXYCHOLESTEROL INHEPG2 CELLS AND MOLECULAR-CLONING AND EXPRESSION OF THE CDNA FOR THE HUMAN HEPATIC SQUALENE SYNTHASE

Primers, bared on the cDNA nucleotide sequences for rat hepatic squale ne synthase (EC 2.5.1.21) (McKenzie, T. L., Jiang, G., Straubhaar, J. R., Conrad, D., and Shechter, I. (1992) J. Biol. Chem. 267, 21368-2137 4), were synthesized and used for the amplification and sequencing of a 1672-base pair (bp) cDNA for the human hepatic squalene synthase (HS S) from human hepatic RNA. An open reading frame of 1251 bp encoding 4 17 amino acids (M(r) = 48,200) was detected for HSS. We have construct ed a pHSS1286 expression vector by molecular cloning of a 1286-bp cDNA , that includes sequences of the entire coding region for HSS, into pB luescript. Expression in Escherichia coli of a functional, full-length HSS was confirmed by immunoblot analysis and enzymatic activity. Nort hern blot analyses of poly(A+) RNA obtained from the human hepatoma ce ll line HepG2 show three distinct size classes of mRNA for HSS. 1.4-, 1.6- and 2.1-kilobase mRNA were observed. The relative abundance is in the order 1.6 > 1.4 > 2.1 and did not change when the cells were grow n in the presence of 25-hydroxycholesterol or lovastatin. The ratio be tween the level of HSS mRNA in cells grown in the absence and presence of 5 mug/ml 25-hydroxycholesterol varies between 8- and 16-fold. This lowering of the mRNA level was observed when the cells were grown in 10% of either full serum or lipid-depleted serum. A 2.7- and 4.0-fold increase of HSS mRNA was observed when HepG2 cells were grown in the p resence of 5 mug/ml lovastatin in lipid-depleted or full serum, respec tively. These studies show that HSS exhibit a relatively high level of transcriptional regulation in response to 25-hydroxycholesterol regar dless of the presence of cholesterol in the growth media.