RESUMEN
The metabolism of 25-hydroxyvitamin D(3) was studied with a crude mitochondrial cytochrome P450 extract from pig kidney and with recombinant human CYP27A1 (mitochondrial vitamin D(3) 25-hydroxylase) and porcine CYP2D25 (microsomal vitamin D(3) 25-hydroxylase). The kidney mitochondrial cytochrome P450 catalyzed the formation of 1alpha,25-dihydroxyvitamin D(3), 24,25-dihydroxyvitamin D(3) and 25,27-dihydroxyvitamin D(3). An additional metabolite that was separated from the other hydroxylated products on HPLC was also formed. The formation of this 25-hydroxyvitamin D(3) metabolite was dependent on NADPH and the mitochondrial electron transferring protein components. A monoclonal antibody directed against purified pig liver CYP27A1 immunoprecipitated the 1alpha- and 27-hydroxylase activities towards 25-hydroxyvitamin D(3) as well as the formation of the unknown metabolite. These results together with substrate inhibition experiments indicate that CYP27A1 is responsible for the formation of the unknown 25-hydroxyvitamin D(3) metabolite in kidney. Recombinant human CYP27A1 was found to convert 25-hydroxyvitamin D(3) into 1alpha,25-dihydroxyvitamin D(3), 25,27-dihydroxyvitamin D(3) and a major metabolite with the same retention time on HPLC as that formed by kidney mitochondrial cytochrome P450. Gas chromatography-mass spectrometry (GC-MS) analysis of the unknown enzymatic product revealed it to be a triol different from other known hydroxylated 25-hydroxyvitamin D(3) metabolites such as 1alpha,25-, 23,25-, 24,25-, 25,26- or 25,27-dihydroxyvitamin D(3). The product had the mass spectrometic properties expected for 4beta,25-dihydroxyvitamin D(3). Recombinant porcine CYP2D25 converted 25-hydroxyvitamin D(3) into 1alpha,25-dihydroxyvitamin D(3) and 25,26-dihydroxyvitamin D(3). It can be concluded that both CYP27A1 and CYP2D25 are able to carry out multiple hydroxylations of 25-hydroxyvitamin D(3).
Asunto(s)
Calcifediol/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Microsomas Hepáticos/enzimología , Mitocondrias/enzimología , Adrenodoxina/metabolismo , Animales , Anticuerpos Monoclonales/metabolismo , Línea Celular Transformada , Sistema Enzimático del Citocromo P-450/aislamiento & purificación , Ferredoxina-NADP Reductasa/metabolismo , Humanos , Hidroxilación , Riñón/citología , Cinética , Mitocondrias Hepáticas/enzimología , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Porcinos , Factores de TiempoRESUMEN
OBJECTIVES: The present study tested the hypothesis that treatment with human recombinant immunoglobulin G1 (IgG1) antibodies against a specific oxidized low-density lipoprotein (oxLDL) epitope will induce regression of existing atherosclerotic lesions in LDL receptor-deficient mice expressing apolipoprotein B-100 (apoB-100) (Apobec-1(-/-)/LDLR(-/-)). BACKGROUND: Oxidized LDL plays an essential role in the pathogenesis of atherosclerosis. We previously showed that an antibody against oxLDL reduces progression of atherosclerosis in mice. METHODS: Apobec-1(-/-)/LDLR(-/-) mice were fed a high-fat diet until they were 24 weeks and were subsequently transferred to chow. Starting at 25 weeks, mice were given 3 weekly injections of either of 2 recombinant human IgG1 antibodies (IEI-E3 or 2D03) against a malondialdehyde-modified apoB-100 peptide sequence. RESULTS: At 25 weeks, atherosclerotic lesions covered 10.3 +/- 3.7% of the descending aorta. Transfer to chow diet resulted in a modest regression of atherosclerosis over a 5-week period (8.28 +/- 4.36%; p = NS). Antibody treatment induced additional regression of atherosclerosis by 50% (2D03; p = 0.001) and 36% (IEI-E3; p = 0.004) compared with control IgG1. The 2D03 treatment also reduced plaque inflammation, enhanced plaque expression of the adenosine triphosphate-binding cassette transporter A1, and inhibited expression of monocyte chemoattractant protein-1 in cultured monocytes. CONCLUSIONS: Human IgG1 against a specific oxLDL epitope can induce rapid and substantial regression of atherosclerotic lesions, possibly by stimulating lipid efflux and inhibiting macrophage recruitment. These recombinant human antibodies could represent a novel strategy for rapid regression/stabilization of atherosclerotic lesions.
Asunto(s)
Apolipoproteína B-100/inmunología , Aterosclerosis/tratamiento farmacológico , Inmunoglobulina G/uso terapéutico , Factores Inmunológicos/uso terapéutico , Fragmentos de Péptidos/inmunología , Desaminasas APOBEC-1 , Transportador 1 de Casete de Unión a ATP , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Aterosclerosis/metabolismo , Aterosclerosis/patología , Citidina Desaminasa/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Receptores de LDL Oxidadas/fisiología , Proteínas Recombinantes/uso terapéuticoRESUMEN
The nucleotide sequence data reported in this paper will appear in EMBL Nucleotide Sequence Database under the accession number AJ 544720. The mitochondrial sterol 27-hydroxylase (CYP27A1) is a multifunctional cytochrome P450 enzyme that catalyses important hydroxylations in the biosynthesis of bile acids and bioactivation of vitamin D(3). Previous results [Babiker, Andersson, Lund, Xiu, Deeb, Reshef, Leitersdorf, Diczfalusy and Bj örkhem (1997) J. Biol. Chem. 272, 26253-26261] suggest that CYP27A1 plays an important role in cholesterol homoeostasis and affects atherogenesis. In the present study, the regulation of the human CYP27A1 gene by growth hormone (GH), insulin-like growth factor-1 (IGF-1), dexamethasone, thyroid hormones and PMA was studied. HepG2 cells were transfected transiently with luciferase reporter gene constructs containing DNA fragments flanking the 5'-region of the human CYP27A1 gene. GH, IGF-1 and dexamethasone increased the promoter activity by 2-3-fold, whereas thyroxine (T(4)) and PMA repressed the activity significantly when measured with luciferase activity expressed in the cells. The endogenous CYP27A1 enzyme activity in the cells was stimulated by GH, IGF-1 and dexamethasone, whereas T(4) and PMA inhibited the activity. Experiments with progressive deletion/luciferase reporter gene constructs indicated that the response elements for GH may be localized in a region upstream to position -1094 bp. The putative response elements for dexamethasone were mapped to positions between -792 and -1095 bp. The -451 bp fragment of the human CYP27A1 gene was found to confer the activation by IGF-1, and the inhibition by T(4) and PMA. Results of the present study suggest that CYP27A1 is regulated in human cells by hormones and signal-transduction pathways.
Asunto(s)
Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regiones Promotoras Genéticas , Esteroide Hidroxilasas/genética , Esteroide Hidroxilasas/metabolismo , Transcripción Genética , Antineoplásicos Hormonales/farmacología , Secuencia de Bases , Sitios de Unión , Carcinógenos/farmacología , Carcinoma Hepatocelular/genética , Línea Celular , Colestanotriol 26-Monooxigenasa , Dexametasona/farmacología , Represión Enzimática/efectos de los fármacos , Hormona del Crecimiento/farmacología , Humanos , Factor I del Crecimiento Similar a la Insulina/farmacología , Neoplasias Hepáticas/genética , Luciferasas/metabolismo , Datos de Secuencia Molecular , Plásmidos , Elementos de Respuesta , Eliminación de Secuencia , Acetato de Tetradecanoilforbol/farmacología , Tiroxina/farmacología , TransfecciónRESUMEN
It has been reported that there is a coordinate regulation of sterol 27-hydroxylase (CYP27A1) and cholesterol 7alpha-hydroxylase (CYP7A1) in rats. Thus, the levels of the mRNA corresponding to these two enzymes were found to change in the same direction in rat liver and in isolated rat hepatocytes. In contrast, other groups have not seen such regulation of CYP27A1 in rabbit liver or in rat liver when using an activity assay. In the present work, the effect of bile acid treatment on human CYP27A1/luciferase reporter activity was studied in a transient transfection assay in human liver-derived HepG2 cells. Neither the endogenous 27-hydroxylase activity nor the CYP27A1/luciferase reporter activity were down-regulated by treatment of HepG2 cells with chenodeoxycholic acid or taurochenodeoxycholic acid. We also measured CYP27A1 mRNA and CYP7A1 mRNA in liver of humans subjected to treatment with chenodeoxycholic acid, ursodeoxycholic acid, hydroxymethylglutaryl (HMG)-CoA reductase inhibitor and a combination of HMG-CoA reductase inhibitor and cholestyramine. There was a 60-fold variation in the levels of CYP7A1 mRNA but only a 5-fold variation in the levels of CYP27A1 mRNA. There was no correlation between the two mRNA species. It is concluded that, in humans, there is little or no coordinate regulation of CYP7A1 and CYP27A1 at the transcriptional level, and that CYP27A1 is not subject to a negative feedback control by bile acids. The results underline that marked species differences may exist in mechanisms for control of synthesis of bile acids and cholesterol homeostasis.