Abstract
Whereas many cytochrome P450 enzymes are transcriptionally suppressed by inflammatory stimuli, down-regulation of CYP2B protein by the inflammatory cytokine interleukin (IL)-1β is nitric oxide (NO)-dependent and occurs via polyubiquitination and proteasomal degradation. Here, we used iTRAQ proteomic analysis to search for other proteins that are potentially down-regulated by cellular NO in cultured rat hepatocytes, and we identified CYP3A1 as one such protein. Therefore, we examined whether CYP3A proteins, like CYP2B, undergo NO- and proteasome-dependent degradation in response to cytokine treatment of rat hepatocytes. In cultured rat hepatocytes treated with phenobarbital, IL-1β stimulation failed to down-regulate CYP3A1 mRNA within 24 h of treatment, whereas CYP3A protein was down-regulated to 40% of control within 6 h, showing the post-transcriptional down-regulation of CYP3A1 protein. The down-regulation of CYP3A after 9 h of stimulation by IL-1β was attenuated by inhibitors of NO synthase (NOS) and of the proteasome, showing NO- and proteasome-dependent down-regulation at earlier time points. However, the down-regulation of CYP3A evoked by IL-1β measured 24 h after stimulation was not affected by the inhibition of NOS or by proteasomal inhibitors, showing that CYP3A1 down-regulation at later time points is NO- and proteasome-independent. IL-6, which did not evoke NO production nor affect CYP3A1 mRNA within 24 h, produced a delayed proteasome-independent down-regulation as well. Taken together, these observations show a novel dual mode of post-transcriptional CYP3A down-regulation by cytokines: NO- and proteasome-dependent at earlier time points and NO- and proteasome-independent at later times.
Footnotes
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This work was supported in part by the National Institutes of Health National Institute of General Medical Sciences [Grant GM069971]; the National Institutes of Health National Institute of Environmental Health Sciences [Grant T32-ES01287]; and the National Institutes of Health National Center for Research Resources [Grants RR02878, RR12878, RR13948].
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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.108.026187.
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ABBREVIATIONS: P450, cytochrome P450; NO, nitric oxide; NOS, NO synthase; NFκB, nuclear factor κB; LPS, lipopolysaccharide; IL, interleukin; NMA, NG-methyl-l-arginine; PB, phenobarbital; l-NIL, l-N6-(1-iminoethyl)lysine; NOC-18, (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate; GSNO, N-(N-l-γ-glutamyl-S-nitroso-l-cysteinyl)-glycine; l-NAME, Nω-nitro-l-arginine methyl ester hydrochloride; Dex, dexamethasone; DDEP, 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine; PAGE, polyacrylamide gel electrophoresis; MS/MS, tandem mass spectrometry; PCR, polymerase chain reaction; RT-PCR, reverse transcription-polymerase chain reaction; Ct, cycle threshold; NOx, nitrate plus nitrite; IRP2, iron regulatory protein 2; ANOVA, analysis of variance.
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↵ The online version of this article (available at http://dmd.aspetjournals.org) contains supplemental material.
- Received December 12, 2008.
- Accepted January 23, 2009.
- U.S. Government work not protected by U.S. copyright.
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