TWO INNEXINS OF Spodoptera litura INFLUENCES HEMICHANNEL AND GAP JUNCTION FUNCTIONS IN CELLULAR IMMUNE RESPONSES

Abstract

Insect cellular immune responses include encapsulation, nodule formation, and phagocytosis. Hemichannels and gap junctions are involved in these cellular actions. Innexins (Inxs: analogous to the vertebrate connexins) form hemichannels and gap junctions, but the molecular mechanisms underlying their biology is still unclear. In this article, we reported a steady-state level of Inxs (SpliInxs) in hemocytes of Spodoptera litura, which formed nonfunctional hemichannels on the cell surface to maintain normal metabolism. We also reported that two innnexins (SpliInx2 and SpliInx3) were expressed significantly higher in hemocytes compared to other tissues, suggesting that they play important roles in hemocytes. Amino acid analysis found that two cysteine residues in two extracellular loops provided the capability for SpliInx2 and SpliInx3 hemichannels to dock into gap junctions. Western blotting demonstrated that both extracellular and intracellular loops of SpliInx3 and the extracellular loops of SpliInx2 might undergo posttranslational modification during the formation of a steady-state hemichannel. During hemichannel formation, SpliInx2 presented as one isoform, while SpliInx3 presented as three isoforms. These results provide fundamental knowledge for further study of how steady-state levels of SpliInxs are dynamically adjusted to perform cellular immune responses under immune challenge.

Unexpected link between insect innexins and apoptosis of HeLa cells

“…Briefly, the total proteins were isolated and the expression of specific proteins among them was determined by blotting against their respective antibodies: anti‐FLAG rabbit antibody (1:2000) from Sangon Biotech (catalog no. AB10005, Shanghai, China); anti‐SpliInx2EL1(110) rabbit antibody (1:500), which specifically recognizes the SpliInx2 extracellular loop1 sequence SHVDGQDEVKYHKYYQ (Pang et al., ); anti‐Akt (Phospho‐Ser473) rabbit antibody (1:2000) from Abmart (catalog no. P30159, Shanghai, China; Liu et al., ); anti‐caspase 3 (1:2000) rabbit antibody from Beyotime (catalog no.…”

“…Real‐time quantitative PCR (qRT‐PCR) was performed as previously described (Pang et al., ). Briefly, total RNA was isolated from HCT116 and HeLa cells by using the RNAiso Plus kit (catalog no.…”

“…Gap junction proteins modulate cell death and survival, which are known to be partially channel‐dependent processes in both invertebrates and vertebrates (Chekeni et al., ; Decrock et al., ; Liu et al., ; Qu et al., ). Innexins (Inxs) and connexins (Cxs) can form gap junctions and hemichannels (Bauer, Lehmann, Martini, Eckardt, & Hoch, ; Decrock et al., ; Kameritsch, Khandoga, Pohl, & Pogoda, ; Pang et al., ). In contrast, pannexins (Panxs) can only form hemichannels (Scemes, Spray, & Meda, ).…”

“…Under immune challenges such as a polydnavirus infection, the steady‐state levels of Inx2 and Inx3 in immunocytes are disrupted via the downregulation of their mRNA expression. Overexpression of endogenous Inxs can restore this steady state in insect cell lines and results in the activation of PI3K signaling pathway (Li et al., ; Pang et al., ). In contrast, overexpression of Inxs may lead to apoptosis in insect cell lines that maintain a low level of cleaved caspase 3 via inactivation of PI3K signaling (Liu et al., ).…”

“…These reBac‐Nte‐Inxs‐infected cells with closed Inxs accumulate phosphate ions, which are known to stimulate apoptotic signaling and mediate the activation of caspase 3 dependent cell death via the loss of mitochondrial function (Adams, Mansfield, Perlot, & Shapiro, ; Guo et al., ; Zhang et al., ). Inxs are highly expressed in hemocytes and adipose tissue, which are the major tissues responsible for the innate immune response in the lepidopteran Spodoptera litura (Pang et al., ). While vertebrates possess both adaptive immunity and innate immunity, it is innate immunity that plays a critical role in the antitumor effects of cancer therapy and manifests cancer immunoediting activity in the absence of adaptive immunity (O'Sullivan et al., ).…”