School of Life Sciences, Key Laboratory for Animal Genetic Diversity and Evolution of High Education in Yunnan Province, Yunnan University, Kunming, Yunnan, PR China.
PLoS One. 2013 Jul 30;8(7):e70456. doi: 10.1371/journal.pone.0070456. Print 2013.
Gap junctions formed by two hemichannels from two neighboring cells are cell-to-cell communication channels; hemichannels are communication channels between intracellular and extracellular environments. Hemichannels are hexameric proteins formed by connexins, pannexins, innexins and vinnexins. Innexin-hemichannels (innexons) exist in the lepidopteran cell surface, but their component innexins and functions have not been reported. Recent studies by others have demonstrated that hemichannels, connexons and pannexons from vertebrates serve as regulators of apoptosis via inactivating the PI3K/Akt signaling pathway. Here, the apoptogenic properties of innexons are demonstrated using two innexin cDNAs, Spli-inx2 and Spli-inx3, which were isolated from hemocytes of lepidopteran Spodoptera litura. Alignment analysis revealed that these two genes belong to a conserved innexin family, as they contain the insect signature YYQWV motif at the beginning of the second transmembrane domain. Immunofluorescence showed that two fusion proteins, Inx2-V5 and Inx3-V5, were localized predominantly in the cell membrane, cytoplasm and also nuclei. Ectopic expression in Sf9 cells and over-expression of Inx2 and Inx3 in Spli221 cells promoted apoptosis. In the Spli221 cells, apoptotic cells presented remarkable membrane blebbing. This study also showed that Sf9 and Spli221 cells undergo low level apoptosis under normal culture conditions, but not Hi5 cells. In Hi5 stable cell lines, biotinylation was used to isolate surface proteins and confirm Inx2 and Inx3 localization in the cell membrane and also further data showed that Hi5 cells may activate the PI3K signaling pathway via phosphorylating molecular Akt downstream. This result suggests that innexon-promoted apoptosis may be involving the PI3K/Akt signaling pathway. These findings will facilitate further examinations of the apoptotic regulation by the PI3K/Akt signaling pathway and comparative studies of innexons, connexons, pannexons, and vinnexons.
间隙连接由两个相邻细胞的两个半通道形成，是细胞间通讯通道；半通道是细胞内和细胞外环境之间的通讯通道。半通道由连接蛋白、pannexin、nexin 和 vinnexin 组成六聚体蛋白。nexin-半通道（nexin）存在于鳞翅目细胞表面，但尚未报道其组成 nexin 和功能。最近的研究表明，脊椎动物的半通道、连接蛋白和 pannexin 作为凋亡的调节剂，通过使 PI3K/Akt 信号通路失活来起作用。本文通过从鳞翅目昆虫 Spodoptera litura 的血细胞中分离出的两个连接蛋白 cDNA（Spli-inx2 和 Spli-inx3），证明了 nexin 的促凋亡特性。序列分析表明，这两个基因属于保守的 nexin 家族，因为它们在第二个跨膜域的开头含有昆虫特征性的 YYQWV 基序。免疫荧光显示两种融合蛋白 Inx2-V5 和 Inx3-V5 主要定位于细胞膜、细胞质和细胞核。Sf9 细胞中的异位表达和 Spli221 细胞中 Inx2 和 Inx3 的过表达促进了细胞凋亡。在 Spli221 细胞中，凋亡细胞表现出明显的细胞膜起泡。本研究还表明，Sf9 和 Spli221 细胞在正常培养条件下会发生低水平的凋亡，但 Hi5 细胞不会。在 Hi5 稳定细胞系中，使用生物素化来分离表面蛋白，并证实 Inx2 和 Inx3 定位于细胞膜上，进一步的数据表明，Hi5 细胞可能通过磷酸化下游分子 Akt 激活 PI3K 信号通路。这一结果表明，nexin 促进的凋亡可能涉及 PI3K/Akt 信号通路。这些发现将有助于进一步研究 PI3K/Akt 信号通路对凋亡的调节作用，并对 nexin、connexin、pannexin 和 vinnexin 进行比较研究。
