Sp1 is an essential transcription factor for LPS-induced tissue factor expression in THP-1 monocytic cells, and nobiletin represses the expression through inhibition of NF-kappaB, AP-1, and Sp1 activation.
Nobiletin is a citrus polymethoxylated flavonoid extracted from Citrus depressa, and has several reported biological effects. In this study, we investigated the effect of nobiletin on bacterial lipopolysaccharide (LPS)-induced expression of tissue factor (TF), a trigger protein for the blood coagulation cascade, and studied the possible mechanism of TF transcriptional regulation. THP-1 monocytic cells stimulated with LPS showed an increased expression of both TF protein and mRNA levels. However, pretreatment with nobiletin resulted in inhibition of LPS-induced expression of both TF protein and mRNA in a dose-dependent manner. Electrophoretic mobility shift assays revealed that binding of nuclear proteins from LPS-stimulated THP-1 cells to the NF-kappaB or AP-1 binding motif was increased as compared to non-stimulated control cells. Such increased binding activities were significantly reduced by pretreatment with nobiletin. Binding activity of nuclear proteins to the Sp1 binding motif was observed irrespective of LPS stimulation, but Sp1 activation was inhibited by nobiletin treatment of the cells. Treatment of THP-1 cells with Sp1-specific small interfering RNA (Sp1 siRNA) abolished the ability of LPS to induce TF activity. A similar reduction in the level of TF mRNA was also observed upon treatment of cells with Sp1 siRNA. These studies reveal that constitutive Sp1 activation is an essential event for transcriptional activation of TF, and nobiletin prevents LPS-induced TF expression by inhibiting NF-kappaB, AP-1, and Sp1 activation.
Hirata
Yuki
Y
Department of Molecular and Cellular Pathophysiology, Showa Pharmaceutical University, Higashi Tamagawa Gakuen, Machida, Tokyo 194-8543, Japan.
Masuda
Yutaka
Y
Kakutani
Hideki
H
Higuchi
Toshiyuki
T
Takada
Kimihiko
K
Ito
Akira
A
Nakagawa
Yoshio
Y
Ishii
Hidemi
H
D000818:Animals; D002478:Cells, Cultured; D002957:Citrus; D047309:Flavones / Q000302:isolation & purification / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology; D009000:Monocytes / Q000187:drug effects / Q000378:metabolism*; D016328:NF-kappa B / Q000037:antagonists & inhibitors* / Q000378:metabolism; D010936:Plant Extracts / Q000302:isolation & purification / Q000494:pharmacology; D011817:Rabbits; D016329:Sp1 Transcription Factor / Q000037:antagonists & inhibitors* / Q000378:metabolism; D013925:Thromboplastin / Q000096:biosynthesis* / Q000235:genetics; D018808:Transcription Factor AP-1 / Q000037:antagonists & inhibitors* / Q000378:metabolism
D047309:Flavones; D008070:Lipopolysaccharides; D016328:NF-kappa B; D010936:Plant Extracts; D016329:Sp1 Transcription Factor; D018808:Transcription Factor AP-1; D013925:Thromboplastin; C008661:nobiletin