Cellular activation, expression analysis and functional characterization of grass carp IκBα: Evidence for its involvement in fish NF-κB signaling pathway

• Grass carp IκBα (gcIκBα) is isolated and characterized.
• LPS and PHA enhance gcIκBα gene expression in periphery blood lymphocytes (PBLs).
• LPS but not PHA induce gcIκBα phosphorylation and protein degradation in PBLs.
• Overexpression of gcIκBα could abolish basal and LPS-stimulated NF-κB activity.

IκBα is a well-known member of the inhibitors of kappa B (IκB) family that controls NF-κB signaling by blocking NF-κB translocation from cytoplasm to nucleus. In the present study, an IκBα homologue was identified from grass carp (gcIκBα), showing the structural characteristics of IκB family. Moreover, mRNA expression of this molecule in grass carp periphery blood lymphocytes (PBLs) was enhanced significantly by both LPS and PHA in a time- and dose-dependent manner, indicating the involvement of gcIκBα in fish immune response. Further analysis demonstrated that LPS but not PHA induced gcIκBα phosphorylation and protein degradation in PBLs, implying different signaling pathways mediated by LPS and PHA in gcIκBα expression regulation in grass carp PBLs. In particular, the time-dependent oscillation of gcIκBα phosphorylation and total protein levels induced by LPS is in accordance with the characteristics of mammalian IκBα phosphorylation followed by protein degradation during NF-κB activation. In support of this notion, overexpression of gcIκBα was able to block both basal and LPS-stimulated NF-κB activity in grass carp kidney cell line, indicating the negatively regulatory role of gcIκBα in NF-κB activity as seen in mammals. Therefore, our results not only reveal a dynamic variation of NF-κB activity based on the activation and expression of IκBα for the first time, but also provide the direct evidence for the involvement of IκBα in NF-κB signaling in fish immune cells.