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) 是激活鱼类先天免疫的有效单分子，但鱼类 eATP 释放的分子决定因素尚不完全清楚。 Connexin32 (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 的免疫相关性及其在鱼类 ATP 释放中的作用尚未得到研究。在这里，我们从日本牙鲆牙鲆中鉴定、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。表达分析显示，尽管所有三个Cx32基因在所有检查的日本牙鲆组织中均组成型表达，但Cx32.2和Cx32.2x主要在肝脏中表达，而Cx32.7在肠和头肾巨噬细胞中高表达。此外，我们发现所有三种Cx32亚型的基因表达均受到 cAMP 刺激和炎症挑战的调节。此外，我们发现在 TNF-α 过表达的日本牙鲆 FG-9307 细胞中， Cx32表达上调。此外，三种Cx32亚型的过表达显着降低了LPS诱导的促炎细胞因子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，我们发现在 Cx32 过表达的日本牙鲆 FG-9307 细胞中，炎症诱导的 ATP 释放显着增加，并且这种增加的 ATP 释放可以通过与间隙连接蛋白阻断剂甘诺索龙预孵育来减弱。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 pannexin1 通道外，Cx32 也在鱼类炎症诱导的 ATP 释放中发挥作用。