Structure and functions of human oxysterol 7α-hydroxylase cDNAs and gene CYP7B1

Abstract

Oxysterol 7 ␣ -hydroxylase has broad substrate specificity for sterol metabolites and may be involved in many metabolic processes including bile acid synthesis and neurosteroid metabolism. The cloned human oxysterol 7 ␣hydroxylase (CYP7B1) cDNA encodes a polypeptide of 506 amino acid residues that shares 40% sequence identity to human cholesterol 7 ␣ -hydroxylase (CYP7A1), the rate-limiting enzyme in the conversion of cholesterol to bile acids in the liver. In contrast to the liver-specific expression of CYP7A1, CYP7B1 mRNA transcripts were detected in human tissues involved in steroid genesis (brain, testes, ovary, and prostate) and in bile acid synthesis (liver) and reabsorption (colon, kidney, and small intestine). The human oxysterol 7 ␣ -hydroxylase transiently expressed in 293/T cells was able to catalyze 7 ␣ -hydroxylation of 27-hydroxycholesterol and dehydroepiandrosterone (DHEA). The human CYP7A1 and CYP7B1 both contain six exons and five introns. However, CYP7B1 spans at least 65 kb of the genome and is about 6-fold longer than CYP7A1. The transcription start site ( ؉ 1) was localized 204 bp upstream of the initiation codon. No TATA box-like sequence was found near the transcription start site. Transient transfection assays of CYP7B1 promoter/luciferase reporter constructs in HepG2 cells revealed that the promoter was highly active. The 5 upstream region from nt Ϫ 83 to ؉ 189 is the core promoter of the gene. -

Expression of key enzymes in bile acid biosynthesis during development: CYP7B1-mediated activities show tissue-specific differences

“…The ability of a human CYP7B1 cDNA probe to hybridize with a pig liver RNA transcript also strongly indicates that a protein belonging to the CYP7B1 subfamily is expressed in pig liver. The size of the porcine CYP7B1 transcript was considerably larger than the CYP7A1 and CYP27A1 transcripts, which is also the case with human CYP7B1 RNA (15).…”

“…Catrin Furster and Kjell Wikvall, Uppsala. First-strand cDNA synthesis was conducted using 1 g of pig liver RNA and Reverse Transcription System (Promega) with 0.5 g Oligo(dT) 15 primer according to the manufacturer's instructions. PCR was performed with 30 pmol of each primer and 2.5 units of Taq polymerase, in a total volume of 50 l (reaction buffer: 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 3 mM MgCl 2 , 0.2 mM dNTP).…”

“…Whereas CYP7A1 is found only in the liver, CYP7B1 is expressed also in many extrahepatic tissues (15). The role of CYP7B1 in extrahepatic tissues remains uncertain.…”

“…The role of CYP7B1 in extrahepatic tissues remains uncertain. In addition to oxysterols, CYP7B1 catalyzes 7 ␣ -hydroxylation of dehydroepiandrosterone and pregnenolone (15,16). Dehydroepiandrosterone and pregnenolone as well as their 7 ␣ -hydroxylated derivatives are considered to have partly unclear functions in various organs such as the brain (16).…”

“…cDNA encoding CYP7B1 has been isolated from mouse and human (15)(16)(17) but the enzyme has not been isolated or characterized from tissues of these species. In previous reports, we described the properties of a purified CYP en-zyme fraction, isolated from pig liver, with the same enzymatic activities as those of recombinantly expressed murine CYP7B1 (12)(13)(14).…”