Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Ministry of Education, Shanghai Ocean University, Shanghai, China.
International Research Center for Marine Biosciences at Shanghai Ocean University, Ministry of Science and Technology, Shanghai, China.
Front Immunol. 2022 Apr 19;13:870679. doi: 10.3389/fimmu.2022.870679. eCollection 2022.
Many immunological diseases can be treated by regulating neurobehavior, in which extracellular ATP is a vital member of endogenous danger-associated molecular pattern signaling molecule that plays a crucial part in innate neuro-related immunity. It is actively released through pannexin (Panx) and connexin (Cx) hemichannels from activated or stressed cells during inflammation, injury, or apoptosis. In addition to participating in ATP release, Panxs and Cxs also have crucial immune functions. In this study, pannexin1, three connexin32 isoforms and connexin43 were identified and characterized in spotted sea bass (), which were named Panx1, Cx32.2, Cx32.3, Cx32.7, and Cx43. Their similar topological structures were discovered by sequence analysis: a relatively unconserved C-terminal region and four highly conserved transmembrane (TM) domains, and so on. Each extracellular (ECL) region of Panx1 has two conserved cysteine residues. Unlike Panx1, each ECL region of Cx32 and Cx43 contains three conserved cysteine residues, forming two conserved motifs: CXCXC motif in ECL1 and CXCXC motif in ECL2. Furthermore, Panx1 and Cx43 share similar genomic organization and synteny with their counterparts in selected vertebrates. Cx32 and CX43 were located in the same locus in fish, but diverged into two loci from amphibian. Moreover, despite varying expression levels, the identified genes were constitutively expressed in all examined tissues. All genes were upregulated by PAMP [lipopolysaccharide and poly(I:C)] stimulation or bacterial infection and , but they were downregulated in the brain at 6 or 12 h after stimulation. Especially, the three Cx32 isoforms and Cx43 were upregulated by ATP stimulation in primary head kidney leukocytes; however, downregulation of Cx32.3 and Cx43 expression were noted at 12 h. Conversely, ATP treatment inhibited the expression of Panx1. Importantly, we showed that the spotted sea bass Panx1, Cx43, and Cx32 were localized on the cellular membrane and involved in inflammation-induced ATP release. Taken together, our results demonstrated that Panx1, Cx32, and Cx43 are important neuro-related immune response genes involved in inflammation-induced ATP release.
许多免疫性疾病可以通过调节神经行为来治疗，其中细胞外 ATP 是内源性危险相关分子模式信号分子的重要成员，在先天神经相关免疫中起着关键作用。在炎症、损伤或细胞凋亡过程中，细胞外 ATP 通过激活或应激细胞中的连接蛋白（Panx）和缝隙连接蛋白（Cx）半通道被主动释放。除了参与 ATP 释放外，Panxs 和 Cxs 还具有重要的免疫功能。在这项研究中，鉴定并表征了点带石斑鱼中的 pannexin1、三种 connexin32 同工型和 connexin43，分别命名为 Panx1、Cx32.2、Cx32.3、Cx32.7 和 Cx43。通过序列分析发现它们具有相似的拓扑结构：相对保守的 C 端区域和四个高度保守的跨膜（TM）结构域等。Panx1 的每个细胞外（ECL）区域都有两个保守的半胱氨酸残基。与 Panx1 不同，Cx32 和 Cx43 的每个 ECL 区域都含有三个保守的半胱氨酸残基，形成两个保守的基序：ECL1 中的 CXCXC 基序和 ECL2 中的 CXCXC 基序。此外，Panx1 和 Cx43 与所选脊椎动物中的对应物具有相似的基因组组织和基因同线性。Cx32 和 CX43 在鱼类中位于相同的基因座上，但从两栖动物分化为两个基因座。此外，尽管表达水平不同，但鉴定的基因在所有检测的组织中均持续表达。所有基因均受 PAMP [脂多糖和多聚（I：C）]刺激或细菌感染而上调[31,32]，但在刺激后 6 或 12 小时在大脑中下调。特别是，三种 Cx32 同工型和 Cx43 在原头肾白细胞中受 ATP 刺激而上调；然而，在 12 小时时观察到 Cx32.3 和 Cx43 表达下调。相反，ATP 处理抑制 Panx1 的表达。重要的是，我们表明斑点石斑鱼 Panx1、Cx43 和 Cx32 定位于细胞膜上，并参与炎症诱导的 ATP 释放。综上所述，我们的研究结果表明，Panx1、Cx32 和 Cx43 是参与炎症诱导的 ATP 释放的重要神经相关免疫反应基因。
