title: Two CCAAT/enhancer binding protein sites in the cytochrome P4503A1 locus. Potential role in the glucocorticoid response.
abstract: Induction of CYP3A genes by the ligand-activated pregnane-X-receptor (PXR) involves the interaction of other as yet unidentified liver transcription factors. Here we show that the CYP3A1 promoter contains two active sites controlled by the CCAAT/enhancer-binding protein alpha (C/EBPalpha), previously shown to regulate a number of liver stress response genes. We have identified two functional C/EBP binding sites at the CYP3A1 promoter that confer luciferase activity to C/EBPalpha cotransfected CHO cells. When inserted upstream of a thymidine kinase promoter, oligonucleotides corresponding to these elements (-350/-311 and -628/-608), increase reporter gene expression when cotransfected with a C/EBPalpha expression vector. Point mutations in the most conserved nucleotides in either element prevent binding of C/EBPalpha and abolish transactivation of the CYP3A1 promoter. Moreover, we demonstrate that C/EBPalpha accumulates in the rat liver nuclei in response to dexamethasone, and that under these conditions C/EBPalpha binds to the CYP3A1 promoter elements. Our results suggest a correlation between transcription of C/EBPalpha, nuclear protein function and induction of CYP3A1 by dexamethasone in the liver. They also support the notion that C/EBPalpha participates in the up-regulation of the CYP3A1 gene in response to synthetic glucocorticoids.
authors.affiliation: Molecular Biology Unit, Faculty of Pharmacy, University of Lisbon, Portugal.
authors.forename: Elsa
authors.initials: E
authors.lastname: Rodrigues
authors.forename: Marie-José
authors.initials: MJ
authors.lastname: Vilarem
authors.forename: Vera
authors.initials: V
authors.lastname: Ribeiro
authors.forename: Patrick
authors.initials: P
authors.lastname: Maurel
authors.forename: Maria C
authors.initials: MC
authors.lastname: Lechner
mesh_terms: D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000378:metabolism*; D016466:CHO Cells; D019556:COS Cells; D002522:Chlorocebus aethiops; D006224:Cricetinae; D051544:Cytochrome P-450 CYP3A; D024202:Electrophoretic Mobility Shift Assay; D005786:Gene Expression Regulation; D005938:Glucocorticoids / Q000008:administration & dosage / Q000494:pharmacology; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017208:Rats, Wistar; D011993:Recombinant Fusion Proteins / Q000502:physiology; D020218:Response Elements; D014162:Transfection
chemical_list: D022763:CCAAT-Enhancer-Binding Protein-alpha; D005938:Glucocorticoids; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D001189:Aryl Hydrocarbon Hydroxylases; C465138:Cyp3a23-3a1 protein, rat; D051544:Cytochrome P-450 CYP3A