Transcriptional regulation of cytochrome P450 4B1 (CYP4B1) gene expression

Abstract

CYP4B1, a member of the cytochrome P450 superfamily, constitutes one of the predominant cytochrome P450 activities in the human lung. CYP4B1 plays a role in the bioactivation of some pulmonary toxicants and may be relevant to developmental processes of the human lung. Significant tissue, cell, and species-specific differences are known to exist in the expression and catalytic properties of CYP4B1. The CYP4B1 gene exhibits high levels of constitutive expression in the human pulmonary cell line H322 derived from Clara cells, but little or no constitutive expression in other human pulmonary cell lines (including A549, H358, EKVX, Hopkins 62, and Hopkins 92), human liver tissue, or the human hepatoma cell line, HepG2. In H322 cells, the CYP4B1 gene also exhibits inducible expression in response to glucocorticoids. This induction has been shown to require active transcription, the involvement of glucocorticoid receptors, and new protein synthesis. Such findings prompted us to isolate and characterize the promoter of the human CYP4B1 gene and examine the transcriptional regulation of the CYP4B1 gene with regard to cell type. By ribonuclease protection assay (RPA) and 5prime-rapid amplification of cDNA ends (5prime-RACE), a single transcriptional start site of the 4B1 promoter was localized. Nucleotide sequencing of 2.89kb of the 5prime-flanking region, exon 1, and part of intron 1 of the CYP4B1 gene revealed the presence of a number of consensus elements including two TATA boxes, multiple GREs, NF-1, CAAT, and AP-1 sites, etc. To identify functional DNA elements regulating transcription of the human CYP4B1 gene, transient transfection experiments using deletion mutants from the 5prime-flanking region of the CYP4B1 gene were conducted in H322, A549, and HepG2 cells. The NF-1 (bp-179 to bp-157) and CAAT (bp-74 to bp-58) consensus elements were identified as essential to the constitutive expression of the CYP4B1 gene in H322 and A549 cells, while a lack of significant reporter gene activity was demonstrated in HepG2 cells. The synthetic glucocorticoid, dexamethasone, had no significant effect on reporter gene activity of all of the 4B1 5prime-deletion mutants tested in H322 cells, suggesting that the dexamethasone-mediated increase in 4B1mRNA observed by Northern analysis may be due to 4B1mRNA stabilization. In addition, Northern analysis of total RNA isolated from some human lung tissue samples revealed the presence of two 4B1mRNAs. Using reverse transcription-polymerase chain reaction (RTPCR) techniques, the difference in size of the mRNAs was attributed to deletions in the 3prime-untranslated region of the smaller 4B1mRNA. Finally, we demonstrated that the large interindividual variation observed in 4B1mRNA expression between human lung tissue samples is not the result of genetic polymorphism in the 5prime-flanking region of the CYP4B1 gene.