IκB kinase α-1 and-2 regulate cytokine expression in the orange-spotted grouper (Epinephelus coioides)

I kappa B kinase (IKK) is the core regulator of the nuclear factor-kappa B (NF-kappa B) pathway, which is involved in cellular development and proliferation, as well as the inflammatory response. IKK alpha is an important subunit of the IKK complex. In this study, two IKK alpha s (EcIKK alpha-1 and -2) were characterized in E. coioides. Similar to IKK alpha of other species, EcIKK alpha-1 and -2 contained a kinase domain, a leucine zipper, a helix-loop-helix domain and a beta NF-kappa B essential modulator-binding domain. Sequence alignment indicated that EcIKK alpha-1 and -2 shared high degrees of sequence identity with IKKs from other species (about 63%-96%). EcIKK alpha-1 and -2 are widely expressed in all tissues, but have different expression profiles in normal groupers. Additionally, EcIKK alpha-1 and -2 responded rapidly to Cryptocaryon irritans infection at the local infection site (i.e., gill tissue), but there was no significant change in EcIKK alpha-2 expression. In GS cells, EcIKK alpha-1 was uniformly distributed in the cytoplasm, while EcIKK alpha 2 was observed uniformly both in the cytoplasm and nucleus. Both EcIKK alpha-1 and -2 were found to activate NF-kappa B, but the luciferase activity of EcIKK alpha-2 was twice that of EcIKK alpha-1. In addition, EcIKK alpha-1 and -2 can regulate the expression of immune-related cytokines (IL-1 beta,IL-6, IL-8, IL-12 [p35 subunit], and TNF-alpha). These findings should prove helpful to further elucidate the innate immunity function of IKK alpha in fish.