Inhibition of lipopolysaccharide-stimulated NO production by a novel synthetic compound CYL-4d in RAW 264.7 macrophages involving the blockade of MEK4/JNK/AP-1 pathway.
In the present study, a novel synthetic compound 4-(2-(cyclohex-2-enylidene)hydrazinyl)quinolin-2(1H)-one (CYL-4d) was found to inhibit lipopolysaccharide (LPS)-induced nitric oxide (NO) production without affecting cell viability or enzyme activity of expressed inducible NO synthase (iNOS) in RAW 264.7 macrophages. CYL-4d exhibited parallel inhibition of LPS-induced expression of iNOS protein, iNOS mRNA and iNOS promoter activity in the same concentration range. LPS-induced activator protein-1 (AP-1) DNA binding, AP-1-dependent reporter gene activity and c-Jun nuclear translocation were all markedly inhibited by CYL-4d with similar efficacy, whereas CYL-4d produced a weak inhibition of nuclear factor-kappaB (NF-kappaB) DNA binding, NF-kappaB-dependent reporter gene activity and p65 nuclear translocation without affecting inhibitory factor-kappa B alpha (I kappa B alpha) degradation. CYL-4d had no effect on the LPS-induced phosphorylation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK) and its upstream activator MAPK kinase (MEK) 3, whereas it significantly attenuated the phosphorylation of c-Jun, c-Jun NH(2)-terminal kinase (JNK) and its upstream activator MEK4 in a parallel concentration-dependent manner. Other Toll-like receptors (TLRs) ligands (peptidoglycans, double-stranded RNA, and oligonucleotide containing unmethylated CpG motifs)-induced iNOS protein expression were also inhibited by CYL-4d. Furthermore, the NO production from BV-2 microglial cells as well as rat alveolar macrophages in response to LPS was diminished by CYL-4d. These results indicate that the blockade of NO production by CYL-4d in LPS-stimulated RAW 264.7 cells is attributed mainly to interference in the MEK4-JNK-AP-1 signaling pathway. CYL-4d inhibition of NO production is not restricted to TLR4 activation and immortalized macrophage-like cells.
Lin
Meng-Wei
MW
Graduate Institute of Pharmaceutical Chemistry, China Medical University, Taichung, Taiwan, ROC.
Tsao
Lo-Ti
LT
Chang
Ling-Chu
LC
Chen
Ye-Long
YL
Huang
Li-Jiau
LJ
Kuo
Sheng-Chu
SC
Tzeng
Cherng-Chyi
CC
Lee
Miau-Rong
MR
Wang
Jih-Pyang
JP
D000818:Animals; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000494:pharmacology; D048670:MAP Kinase Kinase 4 / Q000378:metabolism*; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism; D016676:Macrophages, Alveolar / Q000187:drug effects / Q000378:metabolism; D017628:Microglia / Q000187:drug effects / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D015363:Quinolones / Q000138:chemical synthesis / Q000494:pharmacology*; D012333:RNA, Messenger / Q000187:drug effects / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D018808:Transcription Factor AP-1 / Q000378:metabolism*
C520372:4-(2-(cyclohex-2-enylidene)hydrazinyl)quinolin-2(1H)-one; D008070:Lipopolysaccharides; D016328:NF-kappa B; D015363:Quinolones; D012333:RNA, Messenger; D018808:Transcription Factor AP-1; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D048670:MAP Kinase Kinase 4