A novel cis-acting element in a liver cytochrome P450 3A gene confers synergistic induction by glucocorticoids plus antiglucocorticoids.
The induction by dexamethasone of rat liver CYP3A1 differs from classical glucocorticoid gene regulation in part because both glucocorticoids and antiglucocorticoids such as pregnenolone 16 alpha-carbonitrile (PCN) induce CYP3A1 through transcriptional gene activation. In the present study, we transiently expressed in primary cultures of rat hepatocytes plasmids consisting of CYP3A1 5'-flanking sequences fused to a chloramphenicol acetyltransferase reporter plasmid. Deletional analysis identified a 78-base pair (bp) element located approximately 135 bp upstream of the transcriptional start site that was inducible by treatment of the cultures with dexamethasone or PCN and was induced synergistically by dexamethasone plus PCN. Nuclear extract from control rat liver protected two regions within the 78-bp sequence against digestion with DNase I. The same two regions were protected when nuclear extracts from dexamethasone-treated animals were used. Analysis of both of the "footprints" (FP1 and FP2) failed to reveal a classical sequence for the glucocorticoid-responsive element. A 33-bp element that includes FP1 sequences inserted into the chloramphenicol acetyltransferase reporter plasmid and transiently expressed in rat hepatocytes conferred a profile of dexamethasone and PCN induction similar to that of the 78-bp element. However, an Escherichia coli expressed glucocorticoid receptor protein failed to protect sequences within FP1 in DNase I footprinting experiments and failed to change its mobility in gel shift assays. Moreover, as judged by the gel shift assay, the specific protein binding to this fragment was the same whether nuclear extracts from the liver of untreated or dexamethasone-treated rats were used. We conclude that the activation of CYP3A1 gene transcription by glucocorticoids may involve proteins already bound to the controlling element in the CYP3A1 gene through a mechanism in which GR in the presence of hormone does not bind directly to CYP3A1 DNA.
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D003907:Dexamethasone / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D008099:Liver / Q000166:cytology / Q000201:enzymology*; D008297:Male; D006899:Mixed Function Oxygenases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011285:Pregnenolone Carbonitrile / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015534:Trans-Activators / Q000378:metabolism; D015533:Transcriptional Activation
D009838:Oligodeoxyribonucleotides; D015534:Trans-Activators; D011285:Pregnenolone Carbonitrile; D003907:Dexamethasone; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; D015500:Chloramphenicol O-Acetyltransferase