Abstract
The human pregnane X receptor (hPXR) regulates the expression of CYP3A4, which plays a vital role in hepatic drug metabolism and has considerably reduced expression levels in proliferating hepatocytes. We have recently shown that cyclin-dependent kinase 2 (CDK2) negatively regulates hPXR-mediated CYP3A4 gene expression. CDK2 can be dephosphorylated and inactivated by protein phosphatase type 2C beta isoform long (PP2Cβl), a unique phosphatase that was originally cloned from human liver. In this study, we sought to determine whether PP2Cβl is involved in regulating hPXR's transactivation activity and whether PP2Cβl affects CDK2 regulation of this activity in HepG2 liver carcinoma cells. In transactivation assays, transiently coexpressed PP2Cβl significantly enhanced the hPXR-mediated CYP3A4 promoter activity and decreased the inhibitory effect of CDK2 on hPXR transactivation activity. In addition, shRNA-mediated down-regulation of endogenous PP2Cβl promoted cell proliferation, inhibited the interaction of hPXR with steroid receptor coactivator-1, and attenuated the hPXR transcriptional activity. The levels of PP2Cβl did not affect hPXR expression. Our results show for the first time that PP2Cβl is essential for hPXR activity and can positively regulate this activity by counteracting the inhibitory effect of CDK2. Our results implicate a novel and important role for PP2Cβl in regulating hPXR activity and CYP3A4 expression by inhibiting or desensitizing signaling pathways that negatively regulate the function of pregnane X receptor in liver cells and are consistent with the notion that both the activity of hPXR and the expression of CYP3A4 are regulated in a cell cycle-dependent and cell proliferation-dependent manner.
Footnotes
This work was supported in part by the National Institutes of Health National Institute of General Medical Sciences [Grant GM086415] (to T.C.); the National Institutes of Health National Cancer Institute [Grant P30-CA027165]; the American Lebanese Syrian Associated Charities; and St. Jude Children's Research Hospital.
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
doi:10.1124/dmd.110.032128.
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ABBREVIATIONS:
- hPXR
- human pregnane X receptor
- P450
- cytochrome P450
- PXR
- pregnane X receptor
- NR
- nuclear receptor
- CDK
- cyclin-dependent kinase
- PP2Cβl
- protein phosphatase type 2C beta isoform long
- DMSO
- dimethyl sulfoxide
- shRNA
- short hairpin RNA
- PCR
- polymerase chain reaction
- RT-CES
- real-time cell-based electronic sensing
- RLA
- relative luciferase activity
- SRC-1
- steroid receptor coactivator-1
- 18S rRNA
- 18S small subunit ribosomal RNA
- Ct
- threshold cycle
- NF-κB
- nuclear factor-κB
- IKKβ
- IκB kinase beta.
- Received January 7, 2010.
- Accepted June 9, 2010.
- Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics
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