title: Transcription activation of mouse mammary tumor virus-chloramphenicol acetyltransferase: a model to study the metabolism of cortisol.
abstract: The human 11 beta-hydroxysteroid dehydrogenase (h11 beta-HSD) inactivates the active corticosteroid cortisol to its inactive metabolite cortisone. We have developed transactivation analyses of the reporter chimeric gene mouse mammary tumor virus-chloramphenicol acetyltransferase (MMTV-CAT) to study the catalytic activity of h11 beta-HSD introduced by cotransfection into receptor and 11 beta-HSD deficient CV-1 cells. Assay of 11 beta-HSD expressed in CV-1 cells by cotransfection showed that the catalyzed dehydrogenation of cortisol to cortisone was 2-fold higher in the presence of NADP. The reductase activity was dependent on the coenzyme NADPH. The addition of increasing concentrations of the inhibitor carbenoxolone (CBX) in the incubates blocked the enzyme activity in a dose dependent fashion. In CV-1 cells cotransfected with expression vectors of either human glucocorticoid (hGR1-777) or mineralocorticoid (hMR1-984) and the reporter plasmid MMTV-CAT, dexamethasone (DEX), aldosterone (ALDO), cortisol, and corticosterone induction of CAT activity was dose dependent. Cotransfection of CV-1 cells transfected with 10 micrograms of 11 beta-HSD expression vector reduced the transactivation of MMTV-CAT by hGR or hMR in the presence of either cortisol or corticosterone to basal values. The concomitant addition of 100 nM cortisone and 1 microM NADPH to these transfectants elevated CAT activity. These data show that transactivation analyses can be used to study the 11 beta-HSD-catalyzed regulation of corticosteroid levels, which triggers physiological processes and in certain cases provides an alternative to animal experimentation.
authors.affiliation: Medical Research Council Group in Molecular Endocrinology, CHUL Research Center, Québec, Canada.
authors.forename: N
authors.initials: N
authors.lastname: Warriar
authors.forename: N
authors.initials: N
authors.lastname: Pagé
authors.forename: M V
authors.initials: MV
authors.lastname: Govindan
mesh_terms: D042842:11-beta-Hydroxysteroid Dehydrogenases; D002229:Carbenoxolone / Q000494:pharmacology; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics* / Q000378:metabolism; D003348:Cortisone / Q000378:metabolism; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006801:Humans; D006854:Hydrocortisone / Q000378:metabolism*; D006913:Hydroxysteroid Dehydrogenases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D008324:Mammary Tumor Virus, Mouse / Q000235:genetics*; D009249:NADP / Q000494:pharmacology; D011965:Receptors, Glucocorticoid / Q000235:genetics / Q000502:physiology; D018161:Receptors, Mineralocorticoid / Q000235:genetics / Q000502:physiology; D011993:Recombinant Fusion Proteins; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection
chemical_list: D011965:Receptors, Glucocorticoid; D018161:Receptors, Mineralocorticoid; D011993:Recombinant Fusion Proteins; D009249:NADP; D006913:Hydroxysteroid Dehydrogenases; D042842:11-beta-Hydroxysteroid Dehydrogenases; D015500:Chloramphenicol O-Acetyltransferase; D002229:Carbenoxolone; D003348:Cortisone; D006854:Hydrocortisone