Intracellular distribution of NFkappaB decoy and its inhibitory effect on TNFalpha production by LPS stimulated RAW 264.7 cells.
Nuclear factor kappa B (NFkappaB) is a transcriptional factor for the expression of many cytokines that are involved in the pathogenesis of inflammatory diseases. Unstimulated NFkappaB sequestered in the cytoplasm bound to inhibitory proteins is called IkappaBs. Many activators of NFkappaB cause degradation of IkappaB proteins and free NFkappaB can enter the nucleus and induce gene expression. In this study, we analyzed the relationship between the intracellular distribution and pharmacological effect of NFkappaB decoy in RAW 264.7 cells. Most of the fluorescent labeled NFkappaB decoy was observed in the cytoplasm both with or without cationic transfection without LPS stimulation. Furthermore, under LPS stimulation, most of NFkappaB decoy was also observed in the cytoplasm. However, NFkappaB decoy effectively inhibited the production of TNFalpha in RAW 264.7 cells. The inhibitory effect of TNFalpha production by NFkappaB decoy transfected by cationic liposomes was much stronger than that by naked NFkappaB decoy, because the amount of cellular association of NFkappaB transfected by cationic liposome decoy was 7 times higher than that of naked NFkappaB decoy. This information is of great value for the design of NFkappaB decoy carrier systems.
Higuchi
Yuriko
Y
Department of Drug Delivery Research, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.
Kawakami
Shigeru
S
Nishikawa
Makiya
M
Yamashita
Fumiyoshi
F
Hashida
Mitsuru
M
D000818:Animals; D002460:Cell Line; D007124:Immunoenzyme Techniques; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008081:Liposomes; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D018613:Microscopy, Confocal; D016328:NF-kappa B / Q000235:genetics*; D010766:Phosphorylation; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis*
D008070:Lipopolysaccharides; D008081:Liposomes; D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha