Characterization of c-Jun N-terminal kinase (JNK) gene reveals involvement of immune defense against Vibrio splendidus infection in Apostichopus japonicus.

ABSTRACT

The c-Jun N-terminal kinase (JNK) constitutes an evolutionarily conserved family of serine/threonine protein kinases, pivotal in regulating various physiological processes in vertebrates, encompassing apoptosis and antibacterial immunity. Nevertheless, the involvement of JNK in the innate immune response remains largely unexplored in pathogen-induced echinoderms. We isolated and characterized the JNK gene from Apostichopus japonicus (AjJNK) in our investigation. The full-length cDNA sequences of AjJNK spanned 1806 bp, comprising a 1299 bp open reading frame (ORF) encoding 432 amino acids, a 274 bp 5'-untranslated region (UTR), and a 233 bp 3'-UTR. Structural analysis revealed the presence of a classical S_TKc domain (37-335 amino acids) within AjJNK and contains several putative immune-related transcription factor-binding sites, including Elk-1, NF-κB, AP-1, and STAT5. Spatial expression analysis indicated ubiquitous expression of AjJNK across all examined tissues, with the highest expression noted in coelomocytes. The mRNA, protein, and phosphorylation levels of AjJNK were obviously induced in coelomocytes upon V. splendidus challenge and lipopolysaccharide stimulation. Immunofluorescence analysis demonstrated predominant cytoplasmic localization of AjJNK in coelomocytes with subsequent nuclear translocation following the V. splendidus challenge in vivo. Moreover, siRNA-mediated knockdown of AjJNK led to a significant increase in intracellular bacterial load, as well as elevated levels of Ajcaspase 3 and coelomocyte apoptosis post V. splendidus infection. Furthermore, the phosphorylation levels of AjJNK inhibited by its specific inhibitor SP600125 and also significantly suppressed the expression of Ajcaspase 3 and coelomocyte apoptosis during pathogen infection. Collectively, these data underscored the pivotal role of AjJNK in immune defense, specifically in the regulation of coelomocyte apoptosis in V. splendidus-challenged A. japonicus.