Abstract
Hepatocyte growth factor (HGF), an antimitogenic factor for HepG2 cells, increased mRNA and protein levels of UGT1A1 and CYP2B6, as well as the endogenous cyclin-dependent kinase (CDK) inhibitors p16, p21, and p27 in HepG2 cells but not in HuH6, Caco2, or MCF7 cells. Treatment with 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene (U0126) (an extracellular signal-regulated kinase inhibitor) suppressed the HGF-induced expression of UGT1A1 and CYP2B6, as well as p16, p21, and p27 in HepG2 cells. The CDK inhibitor roscovitine also enhanced the expression of UGT1A1, CYP2B6, and CYP3A4. Transfection of anti-CDK2 siRNA led to elevated levels of UGT1A1, CYP2B6, and CYP3A4 in HepG2 and SW480 cells, whereas anti-CDK4 small interfering RNA (siRNA) did not significantly enhance the expression of these enzymes. In fact, CDK2 activity was decreased in HGF-treated HepG2 cells. In cells arrested in S phase by a thymidine block and then released into a synchronous cell cycle, there was a clear dissociation among the activation of CDK2 and the expression of UGT1A1, CYP2B6, and CYP3A4. Furthermore, the induction of CYP3A4 but not UGT1A1 or CYP2B6 mRNA expression by roscovitine was repressed in pregnane X receptor (PXR) siRNA-transfected HepG2 cells. Transfection with constitutive androstane receptor siRNA or PXR siRNA in HepG2 cells did not repress the HGF-stimulated expression of UGT1A1 mRNA. Taken together, our results show that the expression of UGT1A1 and CYP2B6 is negatively regulated through a CDK2 signaling pathway linked to cell cycle progression in HepG2 and SW480 cells, the mechanism of which may differ from that of CYP3A4 expression through PXR phosphorylated by CDK2.
Footnotes
This work was supported in part by the 21st Century Center of Excellence (COE21) Program, Global COE, a grant-in-aid for Scientific Research [Grants 19590070, 19590151, and 21590170]; and Cooperation of Innovative Technology from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
doi:10.1124/dmd.109.029785
↵ The online version of this article (available at http://dmd.aspetjournals.org) contains supplemental material.
-
- CAR
- constitutive androstane receptor
- PXR
- pregnane X receptor
- PB
- phenobarbital
- GR
- glucocorticoid receptor
- AhR
- aryl hydrocarbon receptor
- P450
- cytochrome P450
- EGF
- epidermal growth factor
- TCPOBOP
- 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene
- ERK
- extracellular signal-regulated kinase
- HGF
- hepatocyte growth factor
- U0126
- 1,4-diamino-2,3-dicyano-1,4-bis(phenylthio)butadiene
- CDK
- cyclin-dependent kinase
- U0124
- 1,4-diamino-2,3-dicyano-1,4-bis(aminomethylthio)butadiene
- LY294002
- 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride
- SB203580
- 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole
- SP600125
- anthra[1,9-cd]pyrazole-6 (2H)-one
- SU9516
- 3-[1-(3H-imidazol-4-yl)-meth-(Z)-ylidene]-5-methoxy-1,3-dihydro-indol-2-one
- PDGF
- platelet-derived growth factor
- DMEM
- Dulbecco's modified Eagle's medium
- FCS
- fetal calf serum
- siRNA
- small interfering RNA
- PCR
- polymerase chain reaction
- RXR
- retinoid X receptor
- CPR
- NADPH-cytochrome P450 reductase
- PI3
- phosphatidylinositol 3
- JNK
- c-Jun NH2-terminal kinase
- bp
- base pair
- WY-14643
- 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthioacetic acid
- BSA
- bovine serum albumin.
- Received August 3, 2009.
- Accepted September 28, 2009.
- Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics
DMD 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.
|