HcCnAα regulates NF-κB signaling in Hyriopsis cumingii by interacting with HcIKK and facilitating IκB phosphorylation

Calcineurin (CN), a serine/threonine protein phosphatase regulated by Ca²⁺ and calmodulin, plays a crucial role in the immune response of bivalves. In this study, we examined the effects of gene silencing of the CN subunit (HcCnAα) in Hyriopsis cumingii on the expression of genes associated with the NF-κB signaling pathway, as well as the phosphorylation of the inhibitor of NF-κB (IκB), through RNA interference (RNAi), quantitative PCR (qPCR), and Western blot (WB) analyses. The IκB kinase (HcIKK) and B-cell CLL/lymphoma 10 (HcBcl10) genes of H. cumingii were cloned using rapid amplification of cDNA ends, and protein interactions with HcCnAα were investigated through yeast two-hybrid assays. The results demonstrated that RNAi-mediated knockdown of HcCnAα significantly reduced the expression of IKK, p65, and downstream immune-related genes in the NF-κB signaling pathway-Lys, The, Def, α2-M, TNF-α, and IL-17-all showing significant decreases (P < 0.05). Additionally, phosphorylation of IκB was inhibited. These findings suggest that HcCnAα plays a regulatory role in the NF-κB signaling pathway, influencing the expression of downstream immune response-related genes and defense proteins. Furthermore, yeast two-hybrid assay results indicated a direct protein-protein interaction between HcIKK and HcCnAα, while no interaction was observed between HcBcl10 and HcCnAα. This study elucidates the specific molecular mechanisms by which HcCnAα regulates the immune response in H. cumingii, providing a foundational basis for further exploration of immune regulation mechanisms and the development of disease prevention strategies in bivalves.