Posttranslational regulation of CYP2J2 by nitric oxide

Nitric oxide (NO) is an essential signaling molecule in the body, regulating numerous biological processes. Beside its physiological roles, NO affects drug metabolism by modulating the activity and/or expression of cytochrome P450 enzymes. Previously, our lab showed that NO generation caused by inflammatory stimuli results in CYP2B6 degradation via the ubiquitin-proteasome pathway. In the current study, we tested the NO-mediated regulation of CYP2J2 that metabolizes arachidonic acids to bioactive epoxyeicosatrienoic acids, as well as therapeutic drugs such as astemizole and ebastine. To investigate the effects of NO on CYP2J2 expression and activity, Huh7 cells stably transduced with CYP2J2 with a C-terminal V5 tag were treated with dipropylenetriamine-NONOate (DPTA), a NO donor. The level of CYP2J2 proteins were decreased in a time- and concentration-dependent manner, and the activity was also rapidly inhibited. However, mRNA expression was not altered and the protein synthesis inhibitor cycloheximide did not attenuate DPTA-mediated downregulation of CYP2J2. Removal of DPTA from the culture media quickly restored the activity of remaining CYP2J2, and no further CYP2J2 degradation occurred. To determine the mechanism of CYP2J2 down-regulation by NO, cells were treated with DPTA in the presence or absence of protease inhibitors including proteasomal, lysosomal and calpain inhibitors. Remarkably, the down-regulation of CYP2J2 by NO was attenuated by calpeptin, a calpain inhibitor. However, other calpain inhibitors or calcium chelator show no inhibitory effects on the degradation. The proteasome inhibitor bortezomib showed small but significant restoration of CYP2J2 levels although stimulated ubiquitination of CYP2J2 was not detected. In conclusion, these data suggest that NO regulates CYP2J2 posttranslationally and NO-evoked CYP2J2 degradation undergoes ubiquitin-independent proteasomal degradation pathway unlike CYP2B6.