Abstract
A stable HepG2 cell line harboring a human cholesterol 7 alpha-hydroxylase (CYP7A) minigene/luciferase reporter gene construct was selected for studying transcriptional regulation of CYP7A gene promoter. Insulin and phorbol 12-myristate-13-acetate (PMA) strongly repressed the promoter activity as measured with luciferase activity expressed in the cells. The promoter activity of the 5' progressive deletion/luciferase reporter gene constructs was studied in a transient transfection assay in HepG2 cells. PMA represses the promoter activity and the response elements were localized in the -184/-151 and -134/-81 regions. Insulin also represses the promoter activity and response element was mapped in the -298/-81 region. Surprisingly, glucocorticoid receptor (GR) strongly inhibited promoter activity in the presence of dexamethasone, and response elements were localized in the -298/-151 and the -150/+24 regions. Thyroid hormone receptor also repressed promoter activity and response elements were localized in the -150/+24 and upstream regions. Cotransfection of CYP7A chimeric constructs with an expression vector carrying liver-enriched transcription factor HNF3 alpha stimulated the reporter gene activity, but cotransfection with GR plasmid interfered with the HNF3 alpha-stimulated activity possibly through competition for binding to overlapping GR/HNF3 binding sites. Thus, human cholesterol 7 alpha-hydroxylase gene promoter is strongly repressed by insulin, PMA, and steroid/thyroid hormones and results in the low level of cholesterol 7 alpha-hydroxylase expression in the human liver.
Highlights
A stable HepG2 cell line harboring a human cholesterol 7a-hydroxylase (CYP7A) minigene/luciferase reporter gene construct was selected for studying transcriptional regulation of cholesterol 7a-hydroxylasegene (CYP7A) gene promoter
The expression and regulation of cholesterol 7a-hydroxylase mRNAs in HepG2 cells by bile acids, hormones and cholesterol have been reported by several laboratories [2, 14, 15].We found that rat CYP7A gene promoter is stimulated by glucocorticoid, CAMP,and retinoic acids, and repressed by bile acids, insulin and phorbol esters [16]
The rationale for choosing this segment of C W 7 A gene for the design of minigene construct are: the Hind I11 restriction site is conserved in rat, human, hamster, and mouse CYP7A gene;the sequencesfrom transcriptionstart site to Hind I11 site are highly conserved among different species; most of the response elements conferring regulation by physiological regulators are found in this upstream region; and introns may increase transcriptional efficiency of eukaryotic genes [23]
Summary
A stable HepG2 cell line harboring a human cholesterol 7a-hydroxylase (CYP7A) minigene/luciferase reporter gene construct was selected for studying transcriptional regulation of CYP7A gene promoter. HepG2 cells synthesize and secrete normal primary bile acids [14] and have been adopted as a model for studying transcriptional regulation of cholesterol 7 a - h ~ droxylase gene by transient transfection assay [15, 16].
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