Rapamycin down-regulates inducible nitric oxide synthase by inducing proteasomal degradation.
We investigated the effect of rapamycin, a specific inhibitor of the mammalian serine/threonine kinase, mammalian target of rapamycin (mTOR), on the expression of inducible nitric oxide synthase (iNOS) in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Pretreatment of cells with rapamycin significantly inhibited LPS-induced nitrite production and the expression of iNOS protein in a dose-dependent manner. However, LPS-induced mRNA expression of iNOS and its concomitant activation of nuclear factor (NF)-kappaB remained unchanged by rapamycin. Intriguingly, LPS-induced nitrite production and iNOS protein expression were partially blocked at nanomolar concentrations of rapamycin, whereas phosphorylation of both p70 S6 kinase and 4E-BP1 was completely abolished. The suppression of LPS-induced iNOS expression by rapamycin was reversed by the protease inhibitor lactacystin. Furthermore, rapamycin treatment stimulated 20S proteasome activity, which was slightly elevated by LPS. Taken together, our findings strongly suggest that rapamycin down-regulates LPS-induced iNOS protein expression via proteasomal activation, as well as through inhibition of the mTOR signaling pathway.
Jin
Hye Kyoung
HK
Department of Biochemistry and Molecular Biology, College of Pharmacy, Sungkyunkwan University, Suwon, Korea.
Ahn
Seong Hoon
SH
Yoon
Jong Woo
JW
Park
Jong Woo
JW
Lee
Eun Kyung
EK
Yoo
Jeong Soo
JS
Lee
Jae Cheol
JC
Choi
Wahn Soo
WS
Han
Jeung-Whan
JW
D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D024202:Electrophoretic Mobility Shift Assay; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D051379:Mice; D009569:Nitric Oxide / Q000096:biosynthesis; D052247:Nitric Oxide Synthase Type II / Q000037:antagonists & inhibitors* / Q000096:biosynthesis; D009573:Nitrites / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D061988:Proteasome Inhibitors; D011494:Protein Kinases / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000378:metabolism; D020123:Sirolimus / Q000494:pharmacology*; D058570:TOR Serine-Threonine Kinases
D008070:Lipopolysaccharides; D009573:Nitrites; D061988:Proteasome Inhibitors; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D011494:Protein Kinases; C546843:mTOR protein, mouse; D038762:Ribosomal Protein S6 Kinases, 70-kDa; D058570:TOR Serine-Threonine Kinases; D046988:Proteasome Endopeptidase Complex; D020123:Sirolimus