Studies On Activation Of The Receptor For Advanced Glycation End Prodcuts Via Histone

The receptor for advanced glycation end products(RAGE)is a member of the immunoglobulin superfamily.RAGE was named because it was found as the receptor for advanced glycation end prodects(AGEs).When RAGE binds to its ligands,the downstream signaling pathways(including proinflammatory responces)are activated.Histones are structural components of chromatin in eukaryotic cells,while extracellular histones could participate in various inflammatory reactions such as peritonitis and pancreatitis.Thus,extracellular histones are considered as biomarkers of various diseases.Previous studies showed that the phagocytosis of histone modified macromoleculars was mediated by RAGE;however,there is no report showing that histones are ligands of RAGE.Thus,the present study aims at studying whether histones can act as ligands of RAGE and the interaction leads to induction of pro-inflammatory responces.Histones are known to be presented on the surface of apoptotic cells.Thus,we further examined whether phagocytosis of apoptotic cells was mediated by histone-RAGE interaction.The results are as follows:(1)Soluble histones are endocytosed in human cultured cells by RAGE.Confocal microscopy and flow cytometry were used to analyze whether soluble histone molecules were internalized in a human cultured cell line HEK293 T.Microscopy analyses showed that histone molecules were co-localized with lysosomes in wild-type,but not in RAGE-knockout cells.Similar results were obtained with flow cytometry analysis.Histone molecules interact with DNA to form nucleosomes in cells.We found that internalization of histones was enhanced by binding to DNA.(2)Histone molecules bind to RAGE.To explore direct interaction between histones and RAGE,co-immunoprecipitation experiment was performed in HEK293 T cells.We found that histone molecules were precipitated with RAGE.Their interaction was further verified with an in vitro binding assay by using purified RAGE and histone molecules.Binding of DNA to histones can improve the affinity between histones and RAGE.(3)Histone molecules can activate the RAGE downstream signaling pathway.Reactive oxygen species(ROS)levels are known to be elevated by activation of RAGE.It was found that histones could increase the levels of ROS in wild-type HEK293 T cells,while ROS levels were not increased in RAGE-knockout cells.DNA-histone complex could increase the levels of ROS more than histones alone.Furthermore,inflammatory cytokines in murine macrophage cell line RAW264.7 were induced by histone molecules;this effect was also improved when DNA is attached to histones.In RAGE-knockout cells,histones and DNA-histone complexs did not induce secretion of inflammatory cytokines.This result suggests that RAGE-mediated inflammation process may affect extracellular histone-related diseases.(4)Histones on the surface of apoptotic cells can act as an "eat me" signal to induce RAGEmediated phagocytosis.RAGE can induce phagocytosis of apoptotic cells in HEK293 T cells.However,this process was inhibited when apoptotic cells were treated with proteinase or an anti-histone antibody.These treatments did not affect internalization of apoptotic cells in RAGE-knockout cells.Thus,histone molecules serve as signaling molecules for apoptotic cells to induce RAGE-mediated phagocytosis.