title: Transcriptional regulation by lovastatin and 25-hydroxycholesterol in HepG2 cells and molecular cloning and expression of the cDNA for the human hepatic squalene synthase.
abstract: 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.
authors.affiliation: Eleanor Roosevelt Institute, Denver, Colorado 80206.
authors.forename: G
authors.initials: G
authors.lastname: Jiang
authors.forename: T L
authors.initials: TL
authors.lastname: McKenzie
authors.forename: D G
authors.initials: DG
authors.lastname: Conrad
authors.forename: I
authors.initials: I
authors.lastname: Shechter
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D006528:Carcinoma, Hepatocellular; D002455:Cell Division; D003001:Cloning, Molecular; D004273:DNA, Neoplasm; D013186:Farnesyl-Diphosphate Farnesyltransferase / Q000096:biosynthesis* / Q000235:genetics; 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; D008099:Liver / Q000201:enzymology*; D008113:Liver Neoplasms; D008148:Lovastatin / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction; D012313:RNA / Q000302:isolation & purification / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D051381:Rats; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic / Q000187:drug effects; D014407:Tumor Cells, Cultured
chemical_list: D004273:DNA, Neoplasm; D006888:Hydroxycholesterols; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D012313:RNA; C007997:25-hydroxycholesterol; D008148:Lovastatin; D006903:Hydroxymethylglutaryl CoA Reductases; D013186:Farnesyl-Diphosphate Farnesyltransferase