Inducible nitric-oxide synthase is regulated by the proteasome degradationpathway

Inducible nitric-oxide synthase (iNOS) is responsible for nitric oxide (NO) synthesis from L-arginine in response to inflammatory mediators. To determ ine the degradation pathway of iNOS, human epithelial kidney HEK293 cells w ith stable expression of human iNOS were incubated in the presence of vario us degradation pathway inhibitors. Treatment with the proteasomal inhibitor s lactacystin, MG132, and N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal resu lted in the accumulation of iNOS, indicating that these inhibitors blocked its degradation. Moreover proteasomal inhibition blocked iNOS degradation i n a dose- and time-dependent manner as well as when NO synthesis was inhibi ted by N-omega-nitro-L-arginine methyl ester. Furthermore, proteasomal inhi bition blocked the degradation of an iNOS splice variant that lacked the ca pacity to dimerize and of an iNOS mutant that lacks L-arginine binding abil ity, suggesting that iNOS is targeted by proteasomes, notwithstanding its c apacity to produce NO, dimerize, or bind the substrate. In contrast to prot easomal inhibitors, the calpain inhibitor calpastatin and the lysosomal inh ibitors transepoxysuccinyl-L-leucylamido-4-guanidino butane, leupeptin, pep statin-A chloroquine, and NH4Cl did not lead to significant accumulation of iNOS. Interestingly, when cytokines were used to induce iNOS in RT4 human epithelial cells, the effect of proteasomal inhibition was dichotomous. Lac tacystin added prior to cytokine stimulation prevented iNOS induction by bl ocking the degradation of the NF-KB inhibitor I kappaB-alpha, thus preventi ng activation of NF-kappaB. In contrast, lactacystin added 48 h after iNOS induction led to the accumulation of iNOS. Similarly in murine macrophage c ell line RAW 264.7, lactacystin blocked iNOS degradation when added 48 h af ter MOS induction by Lipopolysaccharide. These data identify the proteasome as the primary degradation pathway for iNOS.