Dual mechanisms of CYP3A protein regulation by proinflammatory cytokine stimulation in primary hepatocyte cultures.
Whereas many cytochrome P450 enzymes are transcriptionally suppressed by inflammatory stimuli, down-regulation of CYP2B protein by the inflammatory cytokine interleukin (IL)-1beta 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-1beta 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-1beta 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-1beta 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.
Lee
Choon-Myung
CM
Department of Pharmacology, Emory University School of Medicine, 5119 Rollins Research Center, 1510 Clifton Road, Atlanta, GA 30322, USA.
Pohl
Jan
J
Morgan
Edward T
ET
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D051544:Cytochrome P-450 CYP3A / Q000235:genetics / Q000378:metabolism*; D016207:Cytokines / Q000378:metabolism*; D017931:DNA Primers; D015536:Down-Regulation; D022781:Hepatocytes / Q000378:metabolism*; D018836:Inflammation Mediators / Q000378:metabolism*; D008297:Male; D009569:Nitric Oxide / Q000378:metabolism; D010634:Phenobarbital / Q000494:pharmacology; D040901:Proteomics; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction
D016207:Cytokines; D017931:DNA Primers; D018836:Inflammation Mediators; D012333:RNA, Messenger; D009569:Nitric Oxide; D051544:Cytochrome P-450 CYP3A; D010634:Phenobarbital