Inducible nitric-oxide synthase is regulated by the proteasome degradation pathway.
Inducible nitric-oxide synthase (iNOS) is responsible for nitric oxide (NO) synthesis from l-arginine in response to inflammatory mediators. To determine the degradation pathway of iNOS, human epithelial kidney HEK293 cells with stable expression of human iNOS were incubated in the presence of various degradation pathway inhibitors. Treatment with the proteasomal inhibitors lactacystin, MG132, and N-acetyl-l-leucinyl-l-leucinyl-l-norleucinal resulted in the accumulation of iNOS, indicating that these inhibitors blocked its degradation. Moreover, proteasomal inhibition blocked iNOS degradation in a dose- and time-dependent manner as well as when NO synthesis was inhibited by N(omega)-nitro-l-arginine methyl ester. Furthermore, proteasomal inhibition blocked the degradation of an iNOS splice variant that lacked the capacity to dimerize and of an iNOS mutant that lacks l-arginine binding ability, suggesting that iNOS is targeted by proteasomes, notwithstanding its capacity to produce NO, dimerize, or bind the substrate. In contrast to proteasomal inhibitors, the calpain inhibitor calpastatin and the lysosomal inhibitors trans-epoxysuccinyl-l-leucylamido-4-guanidino butane, leupeptin, pepstatin-A, chloroquine, and NH(4)Cl 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. Lactacystin added prior to cytokine stimulation prevented iNOS induction by blocking the degradation of the NF-kappaB inhibitor IkappaB-alpha, thus preventing activation of NF-kappaB. In contrast, lactacystin added 48 h after iNOS induction led to the accumulation of iNOS. Similarly, in murine macrophage cell line RAW 264.7, lactacystin blocked iNOS degradation when added 48 h after iNOS induction by lipopolysaccharide. These data identify the proteasome as the primary degradation pathway for iNOS.
Musial
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Department of Medicine, Baylor College of Medicine, Houston, Texas 77030, USA.
Eissa
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D000111:Acetylcysteine / Q000031:analogs & derivatives* / Q000494:pharmacology; D017398:Alternative Splicing; D000818:Animals; D002154:Calpain / Q000037:antagonists & inhibitors; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000502:physiology*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D016207:Cytokines / Q000494:pharmacology; D019281:Dimerization; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D006801:Humans; D008247:Lysosomes / Q000187:drug effects; D051379:Mice; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000502:physiology*; D009154:Mutation; D009569:Nitric Oxide / Q000096:biosynthesis; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D046988:Proteasome Endopeptidase Complex; D011499:Protein Processing, Post-Translational; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D014158:Transcription, Genetic
D015853:Cysteine Proteinase Inhibitors; D016207:Cytokines; D004791:Enzyme Inhibitors; D009097:Multienzyme Complexes; D015842:Serine Proteinase Inhibitors; C067713:lactacystin; D009569:Nitric Oxide; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D002154:Calpain; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D000111:Acetylcysteine