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. Copyright 2000 Academic Press.