Characterization of the Human Lung CYP2F1 Gene and Identification of a Novel Lung-specific Binding Motif

Carr, Brian A.; Wan, Jie; Hines, Ronald N.; Yost, Garold S.

doi:10.1074/jbc.m300319200

Cited by 43 publications

(24 citation statements)

References 45 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…2, was similar in the two cell types. This result contrasts with the finding of a study using a different gene, CYP2F1, a gene that is also lung-selective; there, promoter constructs were active in BEAS-2B cells but silent in HepG2 cells (Carr et al, 2003). In the present study, the 134-bp and 216-bp CYP2A13 promoter constructs were the most active; for the other constructs, activities decreased as the length of the promoter molpharm.aspetjournals.org fragment increased.…”

Section: Resultscontrasting

confidence: 56%

Transcriptional Regulation of Human CYP2A13 Expression in the Respiratory Tract by CCAAT/Enhancer Binding Protein and Epigenetic Modulation

2006

View full text Add to dashboard Cite

CYP2A13, which is highly active in the metabolic activation of tobacco-specific nitrosamines, is selectively expressed in the respiratory tract, in which it is believed to play an important role in chemical carcinogenesis. The aim of this study was to determine the basis for tissue-specific regulation of CYP2A13 gene expression. We have shown that expression of CYP2A3, the rat homolog of CYP2A13, is regulated by nuclear factor I (NFI) in a tissue-specific manner. In the present study, we found that the transcriptional regulation of human CYP2A13 gene involves CCAAT/enhancer binding protein (C/EBP) transcription factors instead of NFI. DNase I footprinting and gel-shift assays with human lung nuclear extract identified two DNA elements bound by C/EBP. Reporter gene assays using a 216-base pair CYP2A13 promoter fragment confirmed the activation of CYP2A13 by transfected C/EBP factors, and results from chromatin immunoprecipitation assays indicated that C/EBP is associated with CYP2A13 promoter in vivo in the olfactory mucosa of CYP2A13-transgenic mice. In NCI-H441 human lung cancer cells, we discovered that CYP2A13 expression can be induced by a combined treatment with 5-aza-2Ј-deoxycytosine, a DNA demethylation agent, and trichostatin, a histone deacetylation inhibitor. In 5-aza-2Ј-deoxycytosine/trichostatin-treated NCI-H441 cells, overexpression of C/EBP␦, a lung-enriched C/EBP, led to additional increases in CYP2A13 expression, whereas C/EBP␦ knockdown by small interference RNA suppressed CYP2A13 expression, findings that confirm a role for C/EBP in CYP2A13 regulation. Our findings pave the way for further studies of the regulation of the CYP2A13 gene, particularly the gene's potential suppression by airway inflammation, and the role of epigenetic modulation in the gene's tissue-selective expression.

show abstract

Section: Resultscontrasting

confidence: 56%

Transcriptional Regulation of Human CYP2A13 Expression in the Respiratory Tract by CCAAT/Enhancer Binding Protein and Epigenetic Modulation

2006

View full text Add to dashboard Cite

show abstract

“…The preferential expression of CYP2A13 and CYP2F1 transgene mRNAs in the respiratory tract is comparable to the respiratory tract-predominant expression reported previously for CYP2A13 (Su et al, 2000;Wong et al, 2005) and CYP2F1 (Carr et al, 2003) transcripts in humans. It was reported that CYP2A13 is expressed at appreciable levels in human bladders (Nakajima et al, 2006).…”

Section: Discussionsupporting

confidence: 57%

“…CYP2F1, which is the least well characterized of the three human P450s, is expressed primarily in the respiratory tract (Carlson, 2008;Zhang and Ding, 2008;Weems et al, 2010). Little CYP2F1 mRNA expression was detected in other tissues (Carr et al, 2003). Studies using mammalian cells containing low levels of heterologously expressed CYP2F1 suggested that CYP2F1 is active toward several pulmonary toxicants, including naphthalene, styrene, 3-methylindole, and benzene (Nakajima et al, 1994;Lanza et al, 1999;Powley and Carlson, 2000); however, heterologous expression of CYP2F1 in nonmammalian systems yielded nonfunctional P450 proteins (e.g., Baldwin et al, 2005).…”

Section: Introductionmentioning

confidence: 99%

Generation and Characterization of aCYP2A13/2B6/2F1-Transgenic Mouse Model

Wei

Lei

et al. 2012

Drug Metab Dispos

View full text Add to dashboard Cite

ABSTRACT:CYP2A13, CYP2B6, and CYP2F1, which are encoded by neighboring cytochrome P450 genes on human chromosome 19, are active in the metabolic activation of many drugs, respiratory toxicants, and chemical carcinogens. To facilitate studies on the regulation and function of these human genes, we have generated a CYP2A13/2B6/2F1-transgenic (TG) mouse model (all *1 alleles). Homozygous transgenic mice are normal with respect to gross morphological features, development, and fertility. The tissue distribution of transgenic mRNA expression agreed well with the known respiratory tract-selective expression of CYP2A13 and CYP2F1 and hepatic expression of CYP2B6 in humans. CYP2A13 protein was detected through immunoblot analyses in the nasal mucosa (NM) (ϳ100 pmol/mg of microsomal protein; similar to the level of mouse CYP2A5) and the lung (ϳ0.2 pmol/mg of microsomal protein) but not in the liver of the TG mice. CYP2F1 protein, which could not be separated from mouse CYP2F2 in immunoblot analyses, was readily detected in the NM and lung but not the liver of TG/Cyp2f2-null mice, at levels 10-and 40-fold, respectively, lower than that of mouse CYP2F2 in the TG mice. CYP2B6 protein was detected in the liver (ϳ0.2 pmol/mg of microsomal protein) but not the NM or lung (with a detection limit of 0.04 pmol/mg of microsomal protein) of the TG mice. At least one transgenic protein (CYP2A13) seems to be active, because the NM of the TG mice had greater in vitro and in vivo activities in bioactivation of a CYP2A13 substrate, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (a lung carcinogen), than did the NM of wild-type mice.

show abstract

“…Electrophoretic Mobility Shift Assay. Nuclear extracts from A549 or HepG2 cells and human lung tissues were prepared as described previously (Carr et al, 2003). EMSA was performed using the gel-shift assay system from Promega essentially as described by the manufacturer.…”

Section: Nested Deletionsmentioning

confidence: 99%

“…The protein/DNA complexes and unbound probes were separated by nondenaturing gel electrophoresis using 4% polyacrylamide gels (1-8 V/length in centimeters) and detected by autoradiography. Double-stranded oligonucleotides comprising the consensus binding sequences for AP1, NFB, Sp1, OCT1, and the 2F1-LSF (Carr et al, 2003) were included (175-fold molar excess) as nonspecific competitors in the gel shift assay system (Promega). Sequence-specific competitive oligonucleotides (1-11, Table 3) were generated by mutating five base pairs at a time, using the conversion of AϾC and TϾG; competitors M9 to M55 (Table 3) contain single base pair mutations corresponding to the distance 5Ј-to 3Ј within the 57-bp insertion; and E-box knockout competitors were created by converting the consensus sequence (CACCTG) to a sequence that is not known to bind any transcription factor (AAAAT).…”

Section: Repression Of Cyp3a4 Transcription In Lung 517mentioning

confidence: 99%

Transcription Factor Binding to a Putative Double E-Box Motif Represses CYP3A4 Expression in Human Lung Cells

Biggs¹,

Wan²,

Cutler³

et al. 2007

Mol Pharmacol

Self Cite

View full text Add to dashboard Cite

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 5Ј 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 (Ϫ71 to Ϫ127) 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 32 P-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, ␦EF1, E2A, HEB, etc.) failed to identify this factor as a previously characterized trans-acting double E-box binding protein. These results demonstrated that the 5Ј-upstream region of CYP3A4 contains an active putative double E-box repressor motif, not present in the 5Ј-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.

show abstract

Characterization of the Human Lung CYP2F1 Gene and Identification of a Novel Lung-specific Binding Motif

Cited by 43 publications

References 45 publications

Transcriptional Regulation of Human CYP2A13 Expression in the Respiratory Tract by CCAAT/Enhancer Binding Protein and Epigenetic Modulation

Transcriptional Regulation of Human CYP2A13 Expression in the Respiratory Tract by CCAAT/Enhancer Binding Protein and Epigenetic Modulation

Generation and Characterization of aCYP2A13/2B6/2F1-Transgenic Mouse Model

Transcription Factor Binding to a Putative Double E-Box Motif Represses CYP3A4 Expression in Human Lung Cells

Contact Info

Product

Resources

About