Differential induction of JE/MCP-1 in subclones from a murine macrophage cell line, RAW 264.7: role of kappaB-3 binding protein.
The JE/MCP-1 gene is an immediate-early gene, and its product is a CC chemokine that attracts monocytes, basophils and T lymphocytes. JE/MCP-1 gene expression is induced by various inflammatory stimuli, but its transcriptional mechanism is not fully understood. To address this question, we obtained two subclones from a parental RAW264.7 cell line, one subline with low JE/MCP-1-producing capacity (named RAW.c11) and the other with high JE/MCP-1-producing capacity (named RAW.c25), in response to lipopolysaccharide (LPS). These subclones have no significant differences in CD14 expression, nitric oxide production, or production of other cytokines, including TNF-alpha or IL-1alpha/beta. In electrophoretic mobility shift assays (EMSA), there were no significant differences in DNA binding to the NF-kappaB-consensus sequence and interferon regulatory factor (IRF)-1,2 binding sequences. However, significantly higher binding activity to the NF-kappaB-like sequence (kappaB-3), which is located in the promoter region of the JE/MCP-1 gene, was shown by a high producer subclone than by a low producer subclone. Transient transfection analysis using deletion mutants of a 0.5-kb region from -467 to +59 identified an LPS-responsive region in a kappaB-3 site (from -169 to -132) in the high producer subclone. Mutation of this site markedly reduced sensitivity to LPS in the high producer subclone. These data suggest that a yet undefined nuclear factor may be involved in differential JE/MCP-1 gene transcription.
Ueno
M
M
Department of Biochemistry, Kyoritsu College of Pharmacy, Tokyo, Japan.
Sonoda
Y
Y
Funakoshi
M
M
Mukaida
N
N
Nose
K
K
Kasahara
T
T
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000096:biosynthesis / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004586:Electrophoresis; D005786:Gene Expression Regulation; D050856:Interferon Regulatory Factor-1; D007375:Interleukin-1 / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis / Q000378:metabolism
D018932:Chemokine CCL2; D004268:DNA-Binding Proteins; D050856:Interferon Regulatory Factor-1; D007375:Interleukin-1; C494258:Irf1 protein, mouse; D016328:NF-kappa B; D010750:Phosphoproteins; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide; D013755:Tetradecanoylphorbol Acetate