Identification of IκBα in Japanese eel Anguilla japonica that impairs the IKKα-dependent activation of NF-κB, AP1, and type I IFN signaling pathways

Abstract

As a member of inhibitory κB family (IκB) family, IκBα is best-characterized and plays a central negative feedback regulator of NF-κB pathway in mammals, but the information about IκBα in the regulation of immune responses is still limited in teleost fishes. In the present study, the full-length cDNA of an IκBα homologue, AjIκBα, was cloned by 5' and 3' SMART RACE from Japanese eel, and its characteristics of expression in response to various PAMPs and A. hydrophila infection were investigated both in vivo and in vitro using quantitative real-time polymerase chain reaction (qRT-PCR). In addition, the subcellular localization of AjIκBα GFP fusion protein and the induction of AjIκBα alone or co-expression with Japanese eel IKKα (AjIKKα) in the activation of NF-κB, type I IFN and AP1 performed using Dual-Glo luciferase assay system were also detected. Sequence comparison analysis revealed that AjIκBα has typical conserved domains, including the N-terminal conserved degradation motif, the ankyrin repeats, and the C-terminal PEST domain. The predicted three-dimensional structure of AjIκBα is similar to that of human IκBα. qRT-PCR analysis revealed a broad expression for AjIκBα in a wide range of tissues, with the highest expression in the spleen, followed by intestine, liver, gills, skin, kidney, and with a lower expression in the heart and muscle. The AjIκBα expressions in the kidney, spleen, and especially in liver were significantly induced following injection with Gram-negative bacterial component LPS, the viral mimic poly I:C and Aeromonas hydrophila infection. In vitro, the AjIκBα transcripts of Japanese eel liver cells were significantly enhanced by the treatment of LPS, poly I:C, or the stimulation of different concentration of Aeromonas hydrophil. Luciferase assays demonstrated that not only could the AjIκBα expression significantly decrease the activation of NF-κB, AP1, and IFNβ-responsive promoters in HEK293 cells and EPC cells, but also robustly inhibited the activity of these three promoters in HEK293 cells or NF-κB and AP1-responsive promoters in EPC cells induced by AjIKKα. Additionally, subcellular localization studies showed that AjIκBα was evenly distributed in the cytoplasm and nucleus both in HEK293 cells and EPC cells under natural state. AjIκBα was found to aggregate into spots in the cytoplasm and nucleus stimulated by LPS or mostly aggregate into nucleus with the treatment of poly I:C in HEK293 cells, whereas the elevated expression of AjIκBα was observed in the cytoplasm of EPC cells upon the stimulation of poly I:C. These results collectively indicated that AjIκBα function as an important negative regulation in innate immunity of host against antibacterial and antiviral infection likely via the inhibition of the activation of NF-κB, AP1, and type I IFN signaling pathways.

Keywords: AP-1; Anguilla japonica; IKKα; IκBα; NF-κB; Subcelluar localization; Type I IFN; mRNA expression.