Abstract
Cytochrome P450 (P450) 2D2 (CYP2D2) enzyme is known to metabolize the majority of typical substrates of the human CYP2D6 enzyme, which is the most extensively characterized polymorphic drug-metabolizing enzyme. Despite its impact on drug metabolism in rats, the transcriptional regulation of CYP2D2 remains to be elucidated. We clarified the molecular mechanism of CYP2D2 gene expression. The CYP2D2 gene was positively regulated by the poly(C)-binding protein heterogeneous nuclear ribonucleoprotein K (hnRNP K) through a transcriptional regulatory element located in the 5′-flanking region from –94 to –113. To date, nothing is known about the potential role of hnRNP K in P450 gene regulation. Thus, this is the first report that hnRNP K protein is involved in CYP2D2 gene regulation. Furthermore, we elucidated the genetic basis of the extremely low expression of CYP2D2 mRNA in Dark Agouti (DA) rats. Because of its relatively low abundance, DA rats have been frequently used for the study of CYP2D substrate metabolism as the animal model of the poor metabolizer phenotype for CYP2D6 compared with Sprague-Dawley rats as an extensive metabolizer phenotype. We found a single substitution within the transcriptional regulatory element of the CYP2D2 gene in DA rats. The mutation was detected in the polypyrimidine sequence that is the preferred binding site for hnRNP K protein. The mutation within the transcriptional regulatory element attenuated the binding of hnRNP K protein. In conclusion, decreased recruitment of hnRNP K protein to the mutated sequence causes the low expression of CYP2D2 mRNA in DA rats.
Footnotes
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This work was supported in part by the Japanese Ministry of Education, Science, Sports, Culture, and Technology [Grants 19671001, 19208028]; and the Japan Society for the Promotion of Science [Grant 18.4513].
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.109.027284.
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ABBREVIATIONS: P450, cytochrome P450; PM, poor metabolizer; EM, extensive metabolizer; DA, Dark Agouti; SD, Sprague-Dawley; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; hnRNP K, heterogeneous nuclear ribonucleoprotein K; SNP, single nucleotide polymorphism; Sp1, specificity protein 1; C/EBP, CCAAT/enhancer binding protein; GATA, GATA binding protein; STAT, signal transducers and activators of transcription; bp, base pair; PCR, polymerase chain reaction; EMSA, electrophoresis mobility shift assay; TFA, trifluoroacetic acid; ChIP, chromatin immunoprecipitation; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; kb, kilobase.
- Accepted April 30, 2009.
- Received February 23, 2009.
- The American Society for Pharmacology and Experimental Therapeutics
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