State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products, School of Marine Sciences, Ningbo University, Ningbo, 315211, PR China.
Yantai Marine Economic Research Institute, Yantai, 265503, PR China.
Fish Shellfish Immunol. 2024 Oct;153:109804. doi: 10.1016/j.fsi.2024.109804. Epub 2024 Aug 3.
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.
c-Jun N-末端激酶（JNK）构成了一个进化上保守的丝氨酸/苏氨酸蛋白激酶家族，在调节脊椎动物的各种生理过程中起着关键作用，包括细胞凋亡和抗菌免疫。然而，JNK 在病原体诱导的棘皮动物固有免疫反应中的参与在很大程度上仍未得到探索。我们在研究中从刺参（Apostichopus japonicus）中分离并鉴定了 JNK 基因。AjJNK 的全长 cDNA 序列长 1806bp，包含一个 1299bp 的开放阅读框（ORF），编码 432 个氨基酸、一个 274bp 的 5'-非翻译区（UTR）和一个 233bp 的 3'-UTR。结构分析表明，AjJNK 内存在一个经典的 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 在所有检测到的组织中均有广泛表达，其中在体腔细胞中的表达最高。在受到灿烂弧菌刺激和脂多糖刺激后，体腔细胞中 AjJNK 的 mRNA、蛋白质和磷酸化水平明显上调。免疫荧光分析表明，在体内受到灿烂弧菌刺激后，AjJNK 在体腔细胞中主要定位于细胞质，随后发生核转位。此外，用 AjJNK 的特异性抑制剂 SP600125 介导的 AjJNK 敲低导致体内感染灿烂弧菌后细胞内细菌负荷显著增加，以及 Ajcaspase 3 和体腔细胞凋亡水平升高。此外，其特异性抑制剂 SP600125 抑制 AjJNK 的磷酸化水平，也显著抑制病原体感染过程中 Ajcaspase 3 和体腔细胞凋亡的表达。综上所述，这些数据强调了 AjJNK 在免疫防御中的关键作用，特别是在调节灿烂弧菌刺激的刺参体腔细胞凋亡中的作用。
