Li Shuo, Wang Nan, Zhang Tongtong, Feng Yu, Wang Liyan, Sun Jinsheng
Tianjin Key Laboratory of Animal and Plant Resistance, College of Life Sciences, Tianjin Normal University, 393 West Binshui Road, Xiqing District, Tianjin, 300387, China.
Tianjin Key Laboratory of Animal and Plant Resistance, College of Life Sciences, Tianjin Normal University, 393 West Binshui Road, Xiqing District, Tianjin, 300387, China.
Fish Shellfish Immunol. 2020 Nov;106:181-189. doi: 10.1016/j.fsi.2020.07.066. Epub 2020 Aug 6.
Extracellular ATP (eATP) is a potent singling molecule in activation of fish innate immunity while the molecular determinants for eATP release in fish were not completely understood. Connexin32 (Cx32) is a member of gap junction protein family that plays important immunological functions in mammals. However, the immune relevance of Cx32 and its role in ATP release in fish has not been investigated. Here, we identified, characterized three Cx32 isoform genes (Cx32.2, Cx32.2x and Cx32.7) from the Japanese flounder Paralichthys olivaceus, and investigated their role in inflammation-induced ATP release in fish. Expression analysis revealed that even though all the three Cx32 genes are constitutively expressed in all examined Japanese flounder tissues, Cx32.2 and Cx32.2x are dominantly expressed in liver, and Cx32.7 is highly expressed in intestine and head kidney macrophages. In addition, we showed that gene expression of all the three Cx32 isoforms was modulated by cAMP stimulation and inflammatory challenges. Furthermore, we revealed that Cx32 expression was upregulated in TNF-alpha overexpressed Japanese flounder FG-9307 cells. Moreover, overexpression of the three Cx32 isoforms significantly reduced the gene expression level of LPS-induced pro-inflammatory cytokine IL-8 and TNF-alpha, indicating that Cx32 is involved in modulating inflammatory response in fish. Finally, we showed that inflammation-induced ATP release was significantly increased in Cx32-overexpressed Japanese flounder FG-9307 cells, and this increased ATP release could be attenuated by pre-incubation with gap junction protein blocker carbenoxolone. Taken together, we for the first time reported the involvement of Cx32 in fish immunity. Our findings suggested that in addition to Cx43 and pannexin1 channels, Cx32 also plays a role in inflammation-induced ATP release in fish.
细胞外ATP（eATP）是鱼类先天免疫激活过程中的一种强效信号分子，然而鱼类中eATP释放的分子决定因素尚未完全明确。连接蛋白32（Cx32）是间隙连接蛋白家族的成员，在哺乳动物中发挥重要的免疫功能。然而，Cx32在鱼类中的免疫相关性及其在ATP释放中的作用尚未得到研究。在此，我们从牙鲆（Paralichthys olivaceus）中鉴定并表征了三个Cx32亚型基因（Cx32.2、Cx32.2x和Cx32.7），并研究了它们在鱼类炎症诱导的ATP释放中的作用。表达分析表明，尽管这三个Cx32基因在所有检测的牙鲆组织中均组成性表达，但Cx32.2和Cx32.2x在肝脏中占主导表达，而Cx32.7在肠道和头肾巨噬细胞中高表达。此外，我们表明这三个Cx32亚型的基因表达受到cAMP刺激和炎症刺激的调节。此外，我们发现Cx32表达在肿瘤坏死因子-α（TNF-α）过表达的牙鲆FG-9307细胞中上调。此外，这三个Cx32亚型的过表达显著降低了脂多糖（LPS）诱导的促炎细胞因子白细胞介素-8（IL-8）和TNF-α的基因表达水平，表明Cx32参与调节鱼类的炎症反应。最后，我们表明在Cx32过表达的牙鲆FG-9307细胞中，炎症诱导的ATP释放显著增加，并且这种增加的ATP释放可以通过与间隙连接蛋白阻滞剂羧苄青霉素预孵育而减弱。综上所述，我们首次报道了Cx32参与鱼类免疫。我们的研究结果表明，除了Cx43和泛连接蛋白1通道外，Cx32在鱼类炎症诱导的ATP释放中也发挥作用。
