PKCβI And ERK1/2Involve DC-SIGN-mediated Signaling Pathway Triggered By Fucose-type DC-SIGN Ligand

Recently, lots of research showed that a variety of pathogens and some tumor cells target C-type lectin molecules DC-SIGN which on the surface of dendritic cells (DC), triggering specific cellular signaling pathways, and suppressing the function of DC antigen presented, which result in immune tolerance. Ligand for DC-SIGN mainly includes the mannose and fucose type, which induce different signaling pathways. Although mannose signaling pathways has been studied deeply, the signaling pathways which triggered by fucose is still unclear.In this study, followed by construction of eukaryotic expression vector of DC-SIGN and LSP1, we developed U937-DC-SIGN-LSP1stable transformed cell lines. At last, the signaling pathways triggered by fucose ligands for DC-SIGN have been studied preliminarily.DC-SIGN and LSP1gene was amplified by PCR respectively for pWPXLd-DC-SIGN and pWPXLd-LSP1eukaryotic expression vector construction. After verification by PCR, enzyme digestion and sequence analysis, pWPXLd-DC-SIGN and pWPXLd-LSP1were transformed into U937cell to generate U937-DC-SIGN-LSP1cell lines. FACS and Western Blot detection showed that U937-DC-SIGN-LSP1cell lines can express DC-SIGN and LSP1, which can be used to signaling pathway research. In research of signaling pathway, we detected binding of LSP1to cytoplasm tail of DC-SIGN by Co-IP. For inducing DC-SIGN mediated signaling, Lex, as the ligand for DC-SIGN, was added into culture. After10min, phosphorylation signal of PKCβI and ERK1/2was enchanced significantly under the condition of Lex stimulation, indicating that PKCβI and ERK1/2involved in Lex signaling pathway. After20min, phosphorylation signal of PKCβI was stable, but phosphorylation signal of ERK1/2was decreased, indicating that PKCβI mediated signal was sustained whereas ERK1/2mediated signal was instantaneous.So far as, we have known the majority of ligand for DC-SIGN on the tumor cells surface is fucose type. Study on fucose type ligand triggering signaling pathway, which mediated by DC-SIGN will help to reveal the molecular mechanisms of tumor immunologic tolerance development.