Transcriptional roles of nuclear factor kappa B and nuclear factor-interleukin-6 in the tumor necrosis factor alpha-dependent induction of cyclooxygenase-2 in MC3T3-E1 cells.
When a mouse osteoblastic cell line MC3T3-E1 was cultured in the presence of tumor necrosis factor alpha (TNF alpha), the release of prostaglandin E2 and the cyclooxygenase activity increased in a dose- and time-dependent manner. The increase of the enzyme activity was attributed mostly to the induction of cyclooxygenase-2 rather than cyclooxygenase-1 as judged by the inhibitory effect of NS398, Western blotting, and Northern blotting. In this system we attempted to elucidate the transcriptional regulation of the cyclooxygenase-2 gene. As examined by the luciferase assay, two positive regulatory regions (-186 to -131 and -512 to -385 base pairs) were found in the 5'-flanking promoter region of the mouse cyclooxygenase-2 gene in the TNF alpha-stimulated cells. The former included putative NF-IL6 (C/EBP beta) and AP2 elements, and the latter contained the NF kappa B motif. A DNA probe including the NF-IL6 and AP2 sites gave positive bands upon electrophoretic mobility shift assay using the nuclear extracts of MC3T3-E1 cells. The bands were supershifted by the addition of anti-NF-IL6 antibody but not by anti-AP2 antibody. A probe including the NF kappa B site also gave positive bands, which were supershifted by anti-NF kappa B p50 and p65 antibodies. Furthermore, when the motif of NF-IL6 or NF kappa B or both was subjected to point mutation, the luciferase activity was markedly reduced. These data suggested a potential role of both NF-IL6 and NF kappa B in the induction of cyclooxygenase-2 by TNF alpha.
Department of Biochemistry, University of Tokushima, School of Medicine, Japan.
K
K
Yamamoto
T
T
Arakawa
N
N
Ueda
S
S
Yamamoto
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004790:Enzyme Induction; D007527:Isoenzymes / Q000096:biosynthesis; D051379:Mice; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides; D011401:Promoter Regions, Genetic; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis* / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid; D014158:Transcription, Genetic; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D007527:Isoenzymes; D016328:NF-kappa B; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D014409:Tumor Necrosis Factor-alpha; D011451:Prostaglandin-Endoperoxide Synthases