Faculty Bibliography

Human endothelium obtained from both the aorta and the pulmonary artery has been evaluated for the presence of the messenger RNA coding for the expression of sterol 27-hydroxylase. Unique oligomers were designed to detect the mRNA by reverse transcription followed by the polymerase chain reaction. The amplified product was sequenced and was found to be identical to the published sequence for nucleotides 491 to 802 of the human sterol 27-hydroxylase cDNA. Northern blot analysis confirmed the presence of 27-hydroxylase mRNA in pulmonary artery and aortic endothelium. As part of these studies, enzymatic activity was assayed in cultured arterial endothelium using cholesterol as a substrate and isotope ratio gas-liquid chromatography-mass spectrometry to identify the metabolites, 27-hydroxycholesterol and 3 beta-hydroxy-5-cholestenoic acid, in the medium. Localization of sterol 27-hydroxylase to vascular endothelium indicates intracellular production of the biologically active metabolite 27-hydroxycholesterol

The report of a novel cytochrome P450 enzyme in mouse hippocampus (cyp7b) with close homology to cholesterol 7 alpha-hydroxylase led us to determine the substrate specificity with respect to 27-hydroxycholesterol, known to be a potent inhibitor of cholesterol synthesis. Transfection of 293/T-cells with PcDNA3.1(+)-mcyp7b was followed by metabolism of 2.5 microM 27-hydroxycholesterol to the 7 alpha-hydroxy intermediate, cholest-5-ene,3 beta,7 alpha,27-triol, with complete loss of down-regulation of cholesterol synthesis. Addition of 5 microM and 10 microM concentrations of the triol to HepG2 and CHO cells, respectively, also did not reduce cholesterol synthesis. The contrast between the biologic effect on cholesterol synthesis by these two C27 hydroxysterols and the wide tissue distribution of both cholesterol 27-hydroxylase and hydroxysterol 7 alpha-hydroxylase implies local regulatory effects prior to their further catabolism in the liver to chenodeoxycholic and cholic acids

The cytochrome P450 2B subfamily in the rat contains an estimated eight to eleven members at the genomic level. Synthesis in the liver of the prototypic forms P450 2B1 and P450 2B2 is dramatically induced by phenobarbital. The 1.9-kb mRNA for P450 2B3, a third member of the P450 2B subfamily, is constitutively present in rat liver but is not inducible by phenobarbital. We have now cloned and sequenced exonic sequences corresponding to the entire 2B3 mRNA and determined their exon-intron structure, which is identical to that of CYP2B1/CYP2B2 and other CYP2B genes. A putative CYP2B3 transcription start site was identified and CYP2B3 5'- and 3'-flanking sequences were compared to those of CYP2B1 and CYP2B2. CYP2B3, like CYP2B1 and CYP2B2, has a modified TATA box preceding the transcription start site and lacks the canonical polyadenylation signal preceding the poly(A) site. A 2B3 expression vector, pMT2-2B3, directed the synthesis in COS-1 cells of an approximately 50-kD protein detectable on Western blots with a polyclonal antibody and with one of four monoclonal antibodies raised against 2B1 but not with a polyclonal antibody raised against P450 PB6. The 2B3 protein migrated with a slightly higher electrophoretic mobility than 2B1 and comigrated with a protein detected by anti-2B1 antibodies in liver microsomes from untreated rats. The results indicate that a 2B3-like protein is present in rat liver and that it is distinct from P450 PB6 and other known constitutive rat hepatic P450s

Sterol 27-hydroxylase activity in bovine aortic endothelial (BAE) cells in culture has been compared with that in HepG2 cells and in Chinese hamster ovary (CHO) cells using identical culture conditions. The total enzyme activity of BAE cells (3.0 nmol/72 h per mg cell protein) was comparable with that of HepG2 cells (4.0 nmol/72 h per mg protein) and both values were significantly greater than that in CHO cells (0.002 nmol/72 h per mg protein). The enzyme was identified in the mitochondria extracted from BAE cells by Western blotting using an antibody of proven specificity, and its metabolites 27-hydroxycholesterol and 3 beta-hydroxy-5-cholestenoic acid were identified by mass spectrum analysis. The presence of the enzyme in endothelium provides a mechanism for preventing accumulation of intracellular cholesterol by initiating a pathway of bile acid synthesis different from that initiated by 7 alpha-hydroxylation of cholesterol in the liver

NAD(P)H: quinone oxidoreductase (NQO1) is believed to be protective against cancer and toxicity caused by exposure to quinones and their metabolic precursors. This enzyme catalyzes the two-electron reduction of compounds, compared with one-electron reduction mediated by NADPH: cytochrome-P450 oxidoreductase which produces toxic and mutagenic free radicals. Recently we cloned and sequenced the cDNA encoding human 2.3,7,8-tetrachlorodibenzo-p-dioxin (dioxin)-inducible cytosolic NQO1 [Jaiswal et al. (1988) J. Biol. Chem. 263, 13572-13578] and provided preliminary evidence that this enzyme may correspond to diaphorase 4, an enzymatic activity present in various tissues that catalyzes the reduction of a variety of quinones by both NADH and NADPH [Edwards et al. (1980) Biochem. J. 187, 429-436]. In the present report we characterize the catalytic properties of the protein encoded by the NQO1 cDNA. The enzyme was synthesized in monkey kidney COS-1 cells transfected with a pMT2-based expression plasmid containing the NQO1 cDNA. Western blot analysis of the transfected cells using an antibody against rat liver cytosolic NQO1 revealed a 31-kDa band that was not detected in nontransfected cells. This band corresponded to a polypeptide with the same electrophoretic mobility as the endogenous NQO1 protein detected in the human hepatoblastoma (Hep-G2) cells with the same antibody. The immunoreactive protein detected in human Hep-G2 cells was induced approximately fourfold by exposure of the cultures to dioxin, an increase commensurate with the increased in quinone oxidoreductase activity. These studies suggest that the protein encoded by NQO1 cDNA is indeed similar, if not identical, to the dioxin-inducible protein band detected in human Hep-G2 cells. Further characterization of the product of NQO1 cDNA, which was present at approximately 20-30-fold higher levels in transfected COS cells than the endogenous product in uninduced human Hep-G2 cells indicated that it had very high capacity (greater than 1000-fold over background) to catalyze the reduction of 2.6-dichloroindophenol and menadione. Besides these two commonly used substrates for quinone reductase, the expressed NQO1 protein also effectively metabolized 2,6-dimethylbenzoquinone, methylene blue, p-benzoquinone, 1,4-naphthoquinone, 2-methyl-1,4-benzoquinone, with the latter being the most potent electron acceptor at 50 microM concentration of the substrate