Regulation of IL-6 expression by notch signaling in murine macrophages

Abstract

Notch receptors and ligands are families of proteins involved in signal transduction of Notch signaling pathway. Interaction between Notch ligands and their receptors triggers cleavage of Notch receptor by gamma secretase which leads to signal transduction. Notch signaling plays important roles in development of metazoan cells, including immune cells. For signaling transduction, Notch receptors bind to CSL, a DNA- protein, in the nucleus and induce expression of target genes. In macrophages which are innate immune cells, Notch signaling regulates antigen presentation capacity and inflammatory responses such as secretion of pro-inflammatory cytokines including IL-6. IL-6 is a multifunctional cytokine secreted by various cell types, including macrophages. IL-6 functions in immune regulation and inflammation. Previous studies and our preliminary search identified the consensus CSL binding site in the promoter region of IL-6 and CSL was shown to negatively regulate IL-6 expression, but the role of Notch signaling in IL-6 expression in macrophages remained unresolved. Therefore, this study aimed at investigating the role of Notch signaling in regulation of IL-6 expression in murine macrophages. Our results showed that activated macrophages with LPS and interferon gamma induced IL-6 expression in correlation with the upregulation of Notch1 expression. Inhibition of the Notch signaling in macrophages using IL-CHO, a gamma secretase inhibitor, significantly decreased IL-6 expression. Macrophages transiently transfected with truncated constitutively active Notch1 resulted in dramatic increase in both IL-6 mRNA and secreted IL-6. When chromatin immunoprecipitation assay was used to prove whether Notch1 binds to the promoter of IL-6, only RNA polymerase II was found to bind to such promoter. The result for Notch1 binding was inconclusive because of the non-specific binding. Taken together, the results obtained in this study demonstrated that Notch signaling pathway plays an important role in IL-6 expression in macrophages. This finding may lead to a drug design targeting diseases with aberrant IL-6 secretion through Notch signaling.