Inhibitory effect of E3330, a novel quinone derivative able to suppress tumor necrosis factor-alpha generation, on activation of nuclear factor-kappa B.
(2E)-3-[5-(2,3-Dimethoxy-6-methyl-1,4-benzoquinoyl)]-2-nonyl-2- propenoic acid (E3330), is a novel agent with hepatoprotective activity. We report the effect of E3330 on transcriptional activation of tumor necrosis factor (TNF)-alpha gene and on nuclear factor (NF)-kappa B activation. Nuclear run-on experiments showed that E3330 decreases transcriptional activation of TNF-alpha gene induced by lipopolysaccharide (LPS) stimulation in human peripheral monocytes. To investigate the inhibitory mechanisms, we constructed a secreted-type placental alkaline phosphatase (PLAP) reporter gene whose transcription is controlled by a 1.4-kb human TNF-alpha promoter. A stable transformant of the PLAP reporter gene derived from human monocytic cell line showed very little activity on the promoter before stimulation, whereas LPS stimulation led to a dramatic increase in PLAP activity. E3330 inhibited this induced promoter activity in a dose-dependent manner. There are four putative NF-kappa B binding sites (kappa B-1, kappa B-2, kappa B-3, kappa B-4) in human TNF-alpha promoter. By using mutated promoter-PLAP plasmids, we established that these NF-kappa B sites were necessary for induction of TNF-alpha transcription on stimulation with LPS. A gel retardation experiment with synthetic double-stranded oligonucleotides showed that activated NF-kappa B consisting of p50/p65 heterodimer bound to all four putative NF-kappa B DNA probes, suggesting that all four putative NF-kappa B recognition sites play an important role in inducible TNF-alpha expression. E3330 decreased activated NF-kappa B in nuclei, suggesting that E3330 inhibits NF-kappa B activation and/or translocation of the nuclei. Western blotting analysis with anti-I kappa B-alpha antibody indicated that E3330 inhibited degradation of I kappa B-alpha, which is an inhibitory protein of NF-kappa B, in LPS-stimulated monocytes. E3330 may suppress the production of active oxygen species serving as common messengers to activate NF-kappa B.
Goto
M
M
Tsukuba Research Laboratories, Eisai Co., Ltd., Ibaraki, Japan.
Yamada
K
K
Katayama
K
K
Tanaka
I
I
D000469:Alkaline Phosphatase / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D016227:Benzoquinones / Q000494:pharmacology*; D002460:Cell Line; D017931:DNA Primers / Q000737:chemistry; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009000:Monocytes / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism*; D010766:Phosphorylation; D010920:Placenta / Q000201:enzymology; D011401:Promoter Regions, Genetic; D011422:Propionates / Q000494:pharmacology*; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D013481:Superoxides / Q000378:metabolism; D051841:Transcription Factor RelB; D014157:Transcription Factors; D014158:Transcription, Genetic / Q000187:drug effects; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors*
D016227:Benzoquinones; D017931:DNA Primers; D016328:NF-kappa B; D011422:Propionates; D011518:Proto-Oncogene Proteins; C497593:RELB protein, human; D012333:RNA, Messenger; C497594:Relb protein, mouse; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; D013481:Superoxides; C075569:E 3330; D051841:Transcription Factor RelB; D000469:Alkaline Phosphatase