TY - JOUR
T1 - Differential induction of JE/MCP-1 in subclones from a murine macrophage cell line, raw 264.7
T2 - Role of -B-3 binding protein
AU - Ueno, Masaya
AU - Sonoda, Yoshiko
AU - Funakoshi, Megumi
AU - Mukaida, Naofumi
AU - Nose, Kiyoshi
AU - Kasahara, Tadashi
PY - 2000/3
Y1 - 2000/3
N2 - 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-α or 1α/lβ. In electrophoretic mobility shift assays (EMSA), there were no significant differences in DNA binding to the NF-κB-consensus sequence and interferon regulatory factor (IRF)-1,2 binding sequences. However, significantly higher binding activity to the NF-κB-like sequence (κB-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 κB-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.
AB - 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-α or 1α/lβ. In electrophoretic mobility shift assays (EMSA), there were no significant differences in DNA binding to the NF-κB-consensus sequence and interferon regulatory factor (IRF)-1,2 binding sequences. However, significantly higher binding activity to the NF-κB-like sequence (κB-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 κB-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.
KW - Chemokine
KW - IRF-2
KW - JE/MCP-1
KW - LPS
KW - NF-κB
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U2 - 10.1006/cyto.1999.0544
DO - 10.1006/cyto.1999.0544
M3 - Article
C2 - 10704247
AN - SCOPUS:0033938394
VL - 12
SP - 207
EP - 219
JO - Cytokine
JF - Cytokine
SN - 1043-4666
IS - 3
ER -