Differential induction of JE/MCP-1 in subclones from a murine macrophage cell line, raw 264.7: Role of -B-3 binding protein

Masaya Ueno, Yoshiko Sonoda, Megumi Funakoshi, Naofumi Mukaida, Kiyoshi Nose, Tadashi Kasahara

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)207-219
Number of pages13
JournalCytokine
Volume12
Issue number3
DOIs
Publication statusPublished - 2000 Mar

Keywords

  • Chemokine
  • IRF-2
  • JE/MCP-1
  • LPS
  • NF-κB

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology
  • Biochemistry
  • Hematology
  • Molecular Biology

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