Characterization of SiIκB confirms the existence of a conserved IκB–NF-κB regulatory mechanism in sea urchin Strongylocentrotus intermedius

Abstract

The large-scale death of cultured sea urchins due to pathogenic infections has profoundly affected the aquaculture sector. However, the exact details of the innate immune mechanisms in these organisms are unclear and associated research remains limited. The transcription factor nuclear factor-kappa B (NF-κB) plays a key role in the immune process of animals, thereby significantly influencing cellular regulation, development, and diverse biological functions. The inhibitor of NF-κB (IκB) protein interacts directly with the transcription factor to suppress its activity. However, this regulatory process has not been fully explored in sea urchins. In this study, a newly identified IκB gene (designated SiIκB) from the sea urchin Strongylocentrotus intermedius was extensively characterized. SiIκB comprises a 1140 bp open reading frame, producing a 379-amino-acid protein that contains six conserved ankyrin repeats. Phylogenetic analysis indicated that SiIκB grouped with IκB proteins from other echinoderms. The expression of SiIκB mRNA in various tissues was assessed using the quantitative real-time polymerase chain reaction, identifying the highest expression levels in the intestine and coelomocytes. Moreover, the SiIκB protein was located primarily in the cytoplasm of transfected eukaryotic cells. Lipopolysaccharide and polyinosinic: polycytidylic acid stimulation triggered a substantial increase in SiIκB gene expression, which showed a pattern of initial suppression followed by upregulation. The interaction between SiIκB and SiRel, part of the sea urchin NF-κB family, was verified through co-immunoprecipitation experiments. Furthermore, dual-luciferase reporter assays showed that SiIκB suppressed several SiRel-activated reporter genes (AP-1, IFN-α/γ, and IL-6) in a dose-dependent manner. These findings indicate that SiIκB is essential for regulating SiRel activity, therefore facilitating sea urchin innate immunity. This study advances our theoretical understanding of echinoderm immunity and provides a foundation for the development of disease-resistant sea urchins.