摘要
Interleukin-6 （IL-6） is a pleiotropic, pro-inflammatory cytokine produced by various types of cells, including macrophages. Within the IL-6 gene promoter region, the signature binding motif of CBFI/Su（H）/Lag-1 （CSL）, a key DNA-binding protein in the Notch signaling pathway, was identified and found to overlap with a consensus nuclear factor （NF）-κB-binding site. Notch signaling is highly conserved and is involved in the regulation of biological functions in immune cells. In this study, we investigated the role of Notch signaling in the regulation of the IL-6transcript in murine macrophages. The upregulation of Notch1 protein levels and the appearance of cleaved Notch1 （Va11744） correlated well with the increased IL-6 mRNA expression levels in murine primary bone marrow-derived macrophages （BMMφ） after activation by lipopolysaccharide （LPS） together with interferon-gamma （IFN-γ）. Treatment of BMMφ with the γ-secretase inhibitor IL-CHO to suppress the transduction of Notch signaling resulted in a partial decrease in the level of IL-6mRNA and the amount of I L-6 protein produced. In contrast, the overexpression of a constitutively activated intracellular Notch I protein （N^IC） in the RAW264.7 macrophage-like cell line resulted in significantly higher IL-6transcript expression levels than in cells transfected with the empty vector control. The NF-κB inhibitor completely abrogated IL-6 mRNA expression induced by the overexpression of N^IC. Chromatin immunoprecipitation （CHIP） using an anti-Notch1 antibody demonstrated that Notch 1 is associated with the IL-6promoter in RAW264.7 cells activated by LPS/IFN-γ but not in unstimulated cells. Taken together, these results strongly suggest that Notch 1 positively regulates IL-6 expression via NF-κB in activated macrophages.
Interleukin-6 （IL-6） is a pleiotropic, pro-inflammatory cytokine produced by various types of cells, including macrophages. Within the IL-6 gene promoter region, the signature binding motif of CBFI/Su（H）/Lag-1 （CSL）, a key DNA-binding protein in the Notch signaling pathway, was identified and found to overlap with a consensus nuclear factor （NF）-κB-binding site. Notch signaling is highly conserved and is involved in the regulation of biological functions in immune cells. In this study, we investigated the role of Notch signaling in the regulation of the IL-6transcript in murine macrophages. The upregulation of Notch1 protein levels and the appearance of cleaved Notch1 （Va11744） correlated well with the increased IL-6 mRNA expression levels in murine primary bone marrow-derived macrophages （BMMφ） after activation by lipopolysaccharide （LPS） together with interferon-gamma （IFN-γ）. Treatment of BMMφ with the γ-secretase inhibitor IL-CHO to suppress the transduction of Notch signaling resulted in a partial decrease in the level of IL-6mRNA and the amount of I L-6 protein produced. In contrast, the overexpression of a constitutively activated intracellular Notch I protein （N^IC） in the RAW264.7 macrophage-like cell line resulted in significantly higher IL-6transcript expression levels than in cells transfected with the empty vector control. The NF-κB inhibitor completely abrogated IL-6 mRNA expression induced by the overexpression of N^IC. Chromatin immunoprecipitation （CHIP） using an anti-Notch1 antibody demonstrated that Notch 1 is associated with the IL-6promoter in RAW264.7 cells activated by LPS/IFN-γ but not in unstimulated cells. Taken together, these results strongly suggest that Notch 1 positively regulates IL-6 expression via NF-κB in activated macrophages.
