Abstract
In cells of the innate immune system, pathological increases in intracellular cAMP attenuate immune responses and contribute to infections by bacteria such as Bacillus anthracis. In this work, cAMP from B. anthracis edema toxin (ET) is found to activate the Notch signaling pathway in both mouse macrophages and human monocytes. ET as well as a cell-permeable activator of PKA induce Notch target genes (HES1, HEY1, IL2RA, and IL7R) and are able to significantly enhance the induction of these Notch target genes by a Toll-like receptor ligand. Elevated cAMP also resulted in increased levels of Groucho/transducin-like enhancer of Split (TLE) and led to increased amounts of a transcriptional repressor complex consisting of TLE and the Notch target Hes1. To address the mechanism used by ET to activate Notch signaling, components of Notch signaling were examined, and results revealed that ET increased levels of recombinant recognition sequence binding protein at the Jκ site (RBP-J), a DNA binding protein and principal transcriptional regulator of Notch signaling. Overexpression studies indicated that RBP-J was sufficient to activate Notch signaling and potentiate LPS-induced Notch signaling. Further examination of the mechanism used by ET to activate Notch signaling revealed that C/EBP β, a transcription factor activated by cAMP, helped activate Notch signaling and up-regulated RBP-J. These studies demonstrate that cAMP activates Notch signaling and increases the expression of TLE, which could be an important mechanism utilized by cAMP to suppress immune responses.
Highlights
Many bacterial pathogens, such as Bacillus anthracis, increase cAMP in monocytes, leading to disruption of immune responses
In an effort to identify and understand signaling pathways impacted by cAMP in macrophages, we evaluated a panel of gene reporters in macrophages and found that the Notch signaling pathway was modified by edema toxin (ET)
Increased cAMP in Macrophages/Monocytes Activates Notch-induced Gene Expression—In an effort to identify and understand signaling pathways impacted by cAMP generated by ET, we screened a selection of RAW 264.7 cell lines stably transduced with luciferase-based gene reporters
Summary
Many bacterial pathogens, such as Bacillus anthracis, increase cAMP in monocytes, leading to disruption of immune responses. Of the Notch target genes, the most important and best characterized are the families of basic helix-loop-helix proteins, hairy and enhancer of split (Hes) and hairy and enhancer of split with YRPW motif (Hey) [12] These DNA binding proteins function as transcriptional repressors and are the primary effectors of Notch signaling. Notch signaling has emerged as a critical signaling pathway in macrophages [13,14,15,16,17] In these types of cells, Notch signaling combines with Toll-like receptor (TLR) signaling to induce Notch target genes such as Hes and Hey [13]. Further studies reveal mechanisms used by ET to activate Notch signaling and to enhance the formation of the Hes transcription repressor complex
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