Abstract
Two vital enzymes of the CYP3A subfamily, CYP3A4 and CYP3A5, are differentially expressed in the human lung. However, the molecular mechanisms that regulate tissue-selective expression of the genes are poorly understood. The ability of the 5′ upstream promoter region of these two genes to drive luciferase reporter activities in human lung A549 cells was dramatically different. The CYP3A5 promoter region activated luciferase gene expression by 10-fold over the promoterless construct, whereas the CYP3A4 promoter did not drive expression. Sequence comparisons of the promoters identified a 57-base pair insertion in the CYP3A4 promoter region (–71 to –127) that was absent in the CYP3A5 promoter. Deletion of the 57-bp motif from CYP3A4 or insertion into the CYP3A5 promoter, showed that this motif represses CYP3A4 expression in lung. EMSA analysis using nuclear extracts from either A549 cells or human lung tissues showed two specific protein/DNA complexes formed with the 32P-labeled CYP3A4 57-bp oligonucleotide. EMSA analyses identified two E-box motifs as the minimal specific cis-elements. Supershift assays with antibodies directed against known double- or single-E-box binding factors (TAL1, δEF1, E2A, HEB, etc.) failed to identify this factor as a previously characterized trans-acting double E-box binding protein. These results demonstrated that the 5′-upstream region of CYP3A4 contains an active putative double E-box repressor motif, not present in the 5′-upstream region of the CYP3A5 gene, that attenuates CYP3A4 expression in the human lung. We believe that this is the first documented case in which a cytochrome P450 gene is actively repressed in a tissue-specific manner.
Footnotes
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This research was supported by the National Heart, Lung, and Blood Institute (grant HL60143), a National Institutes of Health Minority Graduate Research Assistance Supplement, and the Colgate-Palmolive/SOT Award for Student Research Training in Alternative Methods.
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
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doi:10.1124/mol.106.033795.
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ABBREVIATIONS: P450, cytochrome P450; PCR, polymerase chain reaction; EMSA, electrophoretic mobility shift assay; kb, kilobase pair(s); bp, base pair(s); NHBE, normal human bronchial epithelial cells; AP1, activator protein-1; NFκB, nuclear factor κB; C/EBP, CCAAT/enhancer-binding protein; ANOVA, analysis of variance; LSD, least significant difference; BTE, basic transcriptional element; NF-Y, nuclear factor Y; Sp1, specificity protein 1; LSF, lung-specific factor; A549, human lung adenocarcinoma A549 cells.
- Received December 27, 2006.
- Accepted June 4, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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