The evolutionary ancient family of NF-κB (nuclear factor kappa-light chain-enhancer of activated B cells)/Rel transcription factors is activated by environmental and endogenous cues including viral and bacterial pathogen-associated molecula r patterns (PAMPs) or cytokines. The activated NF-κB pathways play a major role in immune and stress responses. The inhibitors of NF-κB (IκB) regulate the activity of NF-κB through a dynamic interplay with their antagonists, IκB kinases (IKK). In mammals, t he family of NF-κB inhibitors comprises nine members with different or mutual affinities for the various combinations of NF-κB/Rel dimers. a is still lacking. This study provides the first characterisation of the entire NF-κB- inhibitor family in a salmonid fish (rainbow trout, Oncorhynchus mykiss).
Material & methods
The different NF-κB inhibitors in rainbow trout (Oncorhynchus mykiss ) were identified using the NCBI-database. We conducted structural analyses using phylogeny, synteny and 3D-modelling of proteins to characterize the canonical iκbα and iκbε proteins, the nuclear iκbδ and iκbz proteins and bcl3. Additionally, comprehensive iκb overexpression studies in CHSE-214 cells were performed for functional analyses of iκbα and iκbε.
In this study, six nfkbia , two nfkbie , two nfkbid , two nfkbiz and two bcl3 genes in rainbow trout were identified. The sequence identity of ohnologous nfkbi-encoded iκb protein s from rainbow trout ranges from 82% to 100%. The two pairs of the iκbα ohnologs a1/a2 versus b1/b2 share about 60% identity. However, the comparison of the iκbα paralogs a1/a2 or b1/b2 versus c1/c2 revealed a sequence identity below 30%. A phylogenetic analysis across the amino-acid sequences of all IκB proteins from human and fishes revealed that this pair of ohnologous iκbα sequences (c1 and c2) cluster with the human IκBβ factor, while the other two pairs of iκbα ohnologs (a1 and a2 as well as b1 and b2) cluster with the human IκBα factor.
Functional analysis revealed significantly higher transcript levels of nfkbia-a, compared to the transcript levels of nfkbia-b and nfkbia-c, in immune tissues and immune-cell fractions. Also, the expression of nfkbie-a1 was significantly higher than for nfkbie-a2 in different immune tissues . With regard to tissue-specific expression patterns, the levels of nfkbia-a and nfkbie were significantly higher in immune-relevant tissues including head kidney, gill and spleen, but there was (almost) no differential expression of other nfkbi genes between tissues. Confocal imaging indicated a distinct localisation of iκbα and iκbε constructs in salmonid fish cell model. The concentration of iκbα was higher in the cytoplasm compared to the nucleus, whereas iκbε factor seem to be evenly distribut ed across cytoplasm and nucleus (Figure 1).
The overexpression of iκbα and iκbε robustly reduced the basal NF-κ B activity down to a 0.09-fold and 0.06-fold, respectively , compared to the non-transfected controls (Figure 2). Stimulation of the non-transfected CHSE-214 cells with the fungal cell-wall com ponent zymosan doubled the NF-κB activity (2.0-fold ) compared to the basal state. Increasing amounts of the overexpressed iκbα and iκbε factors from rainbow trout lowered this stimulated nf-κb activity in a dose-dependent fashion compared to the non-transfected cells.
Furthermore, s timulation with zymosan increased the transcript levels of characteristic inflammatory markers such as il1b and cxcl8 , but also . Nevertheless, the transcript levels of the induced immune genes in non-transfected cells versus cells expressing iκbα or iκbε were not significantly different after stimulation with zymosan.
Discussion and conclusion
In this study, we describe the iκb family in rainbow trout which comprises each multiple gene copies coding for iκbα, iκbε, iκbδ, iκbz and bcl3 . We compared canonical iκbα and iκbε proteins, the nuclear iκbδ and iκbz proteins and bcl3 and identified various structural differences. Our comprehensive overexpression studies in fish cells confirmed a NF-κB-regulation potential of the iκb factors investigated and reveal the first functional results on iκbε in lower vertebrates.