The anti-inflammatory carbazole, LCY-2-CHO, inhibits lipopolysaccharide-induced inflammatory mediator expression through inhibition of the p38 mitogen-activated protein kinase signaling pathway in macrophages.
1. The present study was undertaken to investigate the anti-inflammatory effects of a synthetic compound, LCY-2-CHO, on the expression of inducible nitric oxide synthase (iNOS), COX-2, and TNF-alpha in murine RAW264.7 macrophages. 2. Within 1-30 microm, LCY-2-CHO concentration-dependently inhibited lipopolysaccharide (LPS)-induced nitric oxide (NO), prostaglandin E(2) (PGE(2)), and tumor necrosis factor-alpha (TNF-alpha) formation, with IC(50) values of 2.3, 1, and 0.8 microm, respectively. Accompanying inhibition of LPS-induced iNOS, cyclooxygenase-2 (COX-2), and pro-TNF-alpha proteins was observed. 3. Reverse transcription-polymerase chain reaction (RT-PCR) and promoter analyses indicated that iNOS expression was inhibited at the transcriptional level (IC(50)=2.3 microm), that inhibition of COX-2 expression only partially depended on gene transcription (IC(50)=7.6 microm), and that TNF-alpha transcription was unaffected. 4. Transcriptional assays revealed that activation of AP-1, but not NF-kappaB, was concomitantly blocked by LCY-2-CHO. Our results showed that LCY-2-CHO was capable of interfering with post-transcriptional regulation, altering the stability of COX-2 and TNF-alpha mRNAs. 5. Since the 3'-untranslated region (3' UTR) of both COX-2 and TNF-alpha mRNA contains a p38 mitogen-activated protein kinase (MAPK)-regulated element involved in mRNA stability, we assessed the effect of LCY-2-CHO on p38 MAPK. Our data clearly indicated an inhibition (IC(50)=1.7 microm) of LPS-mediated p38 MAPK activity, but not of extracellular signal-regulated kinase (ERK) or c-Jun N-terminal kinase (JNK) activity. However, kinase assays ruled out a direct inhibition of p38 MAPK action. The selective p38 MAPK inhibitor, SB203580, inhibited the promoter activities of iNOS and COX-2 rather than that of TNF-alpha. 6. In conclusion, LCY-2-CHO downregulates inflammatory iNOS, COX-2, and TNF-alpha gene expression in macrophages through interfering with p38 MAPK and AP-1 activation.
Ho
Feng-Ming
FM
Department of Internal Medicine, Tao-Yuan General Hospital, Department of Health, the Executive Yuan, Taiwan.
Lai
Chih-Chang
CC
Huang
Li-Jiau
LJ
Kuo
Tsun Cheng
TC
Chao
Chien M
CM
Lin
Wan-Wan
WW
D000818:Animals; D000893:Anti-Inflammatory Agents / Q000494:pharmacology*; D002227:Carbazoles / Q000494:pharmacology*; D002478:Cells, Cultured; D051546:Cyclooxygenase 2; D015232:Dinoprostone / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D015870:Gene Expression / Q000187:drug effects; D007249:Inflammation / Q000139:chemically induced / Q000235:genetics; D008070:Lipopolysaccharides; D008264:Macrophages / Q000187:drug effects* / Q000201:enzymology; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism; D009569:Nitric Oxide / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics; D052247:Nitric Oxide Synthase Type II; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis; D011485:Protein Binding; D012333:RNA, Messenger / Q000096:biosynthesis; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*
C499102:9-(2-chlorobenzyl)-9H-carbazole-3-carbaldehyde; D000893:Anti-Inflammatory Agents; D002227:Carbazoles; D008070:Lipopolysaccharides; D016328:NF-kappa B; D012333:RNA, Messenger; D018808:Transcription Factor AP-1; D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D051546:Cyclooxygenase 2; D011451:Prostaglandin-Endoperoxide Synthases; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D015232:Dinoprostone