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

As a member of inhibitory kappa B family (I kappa B) family, I kappa B alpha is best-characterized and plays a central negative feedback regulator of NF-kappa B pathway in mammals, but the information about I kappa B alpha in the regulation of immune responses is still limited in teleost fishes. In the present study, the full-length cDNA of an I kappa B alpha homologue, AjI kappa B alpha , 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 AjI kappa B alpha GFP fusion protein and the induction of AjI kappa B alpha alone or co-expression with Japanese eel IKK alpha (AjIKK alpha) in the activation of NF-kappa B, type I IFN and AP1 performed using Dual-Glo luciferase assay system were also detected. Sequence comparison analysis revealed that AjI kappa B alpha 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 AjI kappa B alpha is similar to that of human I kappa B alpha. qRT-PCR analysis revealed a broad expression for AjI kappa B alpha 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 AjI kappa B alpha 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 AjI kappa B alpha 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 AjI kappa B alpha expression significantly decrease the activation of NF-kappa B, AP1, and IFN beta-responsive promoters in HEK293 cells and EPC cells, but also robustly inhibited the activity of these three promoters in HEK293 cells or NF-kappa B and AP1-responsive promoters in EPC cells induced by AjIKK alpha. Additionally, subcellular localization studies showed that AjI kappa B alpha was evenly distributed in the cytoplasm and nucleus both in HEK293 cells and EPC cells under natural state. AjI kappa B alpha 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 AjI kappa B alpha was observed in the cytoplasm of EPC cells upon the stimulation of poly I:C. These results collectively indicated that AjI kappa B alpha 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-kappa B, AP1, and type I IFN signaling pathways.