A feedback loop involving FREP and NF-κB regulates the immune response of sea cucumber Apostichopus japonicus

Abstract

Our previous work indicated that fibrinogen-related protein (AjFREP) from sea cucumber (Apostichopus japonicus) plays important roles in innate immunity. To understand AjFREP expression regulation in A. japonicus, we cloned the AjFREP promoter and characterized the putative transcription-factor binding motifs. The AjFREP promoter region spans 1365bp, containing several transcription-factor binding sites. The full-length sequence and all truncated fragments exhibited high promoter activity in HEK-293 cells. Luciferase activity significantly increased for P1(−1365/+16) after LPS exposure, suggesting that the promoter responded to LPS. We also found that two potential NF-κB binding sites were involved in the promoter region, and co-transfection assay demonstrated that the first binding site was necessary for AjFREP transcription. The expression of AjNF-κB/Rel after AjFREP knock-down followed by LPS injection in vivo was further investigated. We found that AjNF-κB/Rel transcript significantly decreased after silencing AjFREP with LPS challenge compared with the control at 4 and 8h, suggesting that activated AjFREP in turn affected the NF-κB pathway under immune responses. These results provided novel insights into the activation mechanism of AjFREP, i.e., that selectively changing AjFREP expression may prevent pathogen infection in A. japonicus.