Transcriptional regulation by lovastatin and 25-hydroxycholesterol in HepG2 cells and molecular cloning and expression of the cDNA for the human hepatic squalene synthase.

Primers, based on the cDNA nucleotide sequences for rat hepatic squalene synthase (EC 2.5.1.21) (McKenzie, T.L., Jiang, G., Straubhaar, J.R., Conrad, D., and Shechter, I. (1992) J. Biol. Chem. 267, 21368-21374), were synthesized and used for the amplification and sequencing of a 1672-base pair (bp) cDNA for the human hepatic squalene synthase (HSS) from human hepatic RNA. An open reading frame of 1251 bp encoding 417 amino acids (M(r) = 48,200) was detected for HSS. We have constructed a pHSS 1286 expression vector by molecular cloning of a 1286-bp cDNA, that includes sequences of the entire coding region for HSS, into pBluescript. Expression in Escherichia coli of a functional, full-length HSS was confirmed by immunoblot analysis and enzymatic activity. Northern blot analyses of poly(A+) RNA obtained from the human hepatoma cell 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 grown in the presence of 25-hydroxycholesterol or lovastatin. The ratio between the level of HSS mRNA in cells grown in the absence and presence of 5 micrograms/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 presence of 5 micrograms/ml lovastatin in lipid-depleted or full serum, respectively. These studies show that HSS exhibit a relatively high level of transcriptional regulation in response to 25-hydroxycholesterol regardless of the presence of cholesterol in the growth media.