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.
摘要：
c-JunN末端激酶（JNK）构成了一个进化保守的丝氨酸/苏氨酸蛋白激酶家族，在调节脊椎动物的各种生理过程中至关重要，包括细胞凋亡和抗菌免疫。然而，在病原体诱导的棘皮动物中,JNK在先天免疫应答中的参与在很大程度上仍未被研究.在我们的研究中，我们从刺参（AjJNK）中分离并鉴定了JNK基因。AjJNK的全长cDNA序列跨越1806bp,包含一个1299bp的开放阅读框（ORF），编码432个氨基酸，274bp的5'非翻译区（UTR），和233bp3'-UTR。结构分析显示AjJNK中存在一个经典的S_TKc结构域（37-335个氨基酸），并包含几个假定的免疫相关转录因子结合位点。包括Elk-1,NF-κB,AP-1和STAT5。空间表达分析表明AjJNK在所有检查的组织中普遍存在，在腔体细胞中表达最高。mRNA，蛋白质，脾弧菌攻击和脂多糖刺激后，明显诱导腔体细胞中AjJNK的磷酸化水平。免疫荧光分析表明AjJNK在腔体细胞中的主要细胞质定位，随后在体内脾弧菌攻击后发生核易位。此外，siRNA介导的AjJNK敲除导致细胞内细菌负荷的显着增加，以及脾弧菌感染后Ajcaspase3水平升高和腔体细胞凋亡。此外，其特异性抑制剂SP600125抑制的AjJNK磷酸化水平显著抑制了病原体感染过程中Ajcaspase3的表达和心肌细胞凋亡。总的来说，这些数据强调了AjJNK在免疫防御中的关键作用，特别是在脾弧菌攻击的日本血吸虫的腔体细胞凋亡的调节中。
