Abstract
Cytochrome P450 3A4 (CYP3A4) metabolizes more than 50% of prescribed drugs. The expression of CYP3A4 changes during liver development and may be affected by the administration of some drugs. Alternative mRNA transcripts occur in more than 90% of human genes and are frequently observed in cells responding to developmental and environmental signals. Different mRNA transcripts may encode functionally distinct proteins or contribute to variability of mRNA stability or protein translation efficiency. The purpose of this study was to examine expression of alternative CYP3A4 mRNA transcripts in hepatocytes in response to developmental signals and drugs. cDNA cloning and RNA sequencing (RNA-Seq) were used to identify CYP3A4 mRNA transcripts. Three transcripts were found in HepaRG cells and liver tissues: one represented a canonical mRNA with full-length 3′-untranslated region (UTR), one had a shorter 3′-UTR, and one contained partial intron-6 retention. The alternative mRNA transcripts were validated by either rapid amplification of cDNA 3′-end or endpoint polymerase chain reaction (PCR). Quantification of the transcripts by RNA-Seq and real time quantitative PCR revealed that the CYP3A4 transcript with shorter 3′-UTR was preferentially expressed in developed livers, differentiated hepatocytes, and in rifampicin- and phenobarbital-induced hepatocytes. The CYP3A4 transcript with shorter 3′-UTR was more stable and produced more protein compared with the CYP3A4 transcript with canonical 3′-UTR. We conclude that the 3′-end processing of CYP3A4 contributes to the quantitative regulation of CYP3A4 gene expression through alternative polyadenylation, which may serve as a regulatory mechanism explaining changes of CYP3A4 expression and activity during hepatocyte differentiation and liver development and in response to drug induction.
Footnotes
↵ The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
This study was supported by the National Institutes of Health National Institute of General Medical Sciences [Grant 1R01-GM087376–01A1] (to X.Z.); the National Institutes of Health National Center for Research Resources [Grant 5P20-RR021940] (to X.Z.); and the National Institutes of Health National Institute for Environmental Health Sciences [Grant 1R01-ES019487–01A1] (to X.Z.) and [Grant 1R01-ES10855] (to J.S.L.). The Liver Tissue Cell Distribution System (Kostrubsky et al., 1999) from the University of Pittsburgh was funded by the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases (Contract N01-DK7-0004/HHSN26700700004C).
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
-
ABBREVIATIONS:
- 3′RACE
- rapid amplification of cDNA 3′ end
- 3′-UTR
- 3′-untranslated region
- bp
- base pair(s)
- DMSO
- dimethyl sulfoxide
- FPKM
- fragments per kilobase of exon per 106 fragments mapped
- miRNA
- microRNA
- NCBI
- National Center for Biotechnology Information
- PB
- phenobarbital
- PCR
- polymerase chain reaction
- PHH
- primary human hepatocyte
- PXR
- pregnane X receptor
- qPCR
- quantitative polymerase chain reaction
- RFP
- red fluorescent protein
- RIF
- rifampicin
- RNA-Seq
- RNA sequencing
- RQI
- RNA quality indicator values
- RT-qPCR
- real time quantitative polymerase chain reaction
- svRNA
- small vault RNA
- UCSC
- University of California Santa Cruz
- UTR
- untranslated region.
- Received June 22, 2011.
- Accepted October 13, 2011.
- Copyright © 2012 The American Society for Pharmacology and Experimental Therapeutics
MolPharm articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|