Unexpected link between insect innexins and apoptosis of HeLa cells.

Little is known about how mammalian cells respond to the expression of innexins (Inxs), which are known to mediate cell-to-cell communication that causes apoptosis in the cells of the insect Spodoptera litura. The mammalian expression system, p3xFLAG tag protein, containing the CMV promoter, allowed us to construct two C-terminally elongated innexins (Cte-Inxs), SpliInx2 (Inx2-FLAG), and SpliInx3 (Inx3-FLAG), which were predicted to have the same secondary topological structures as the native SpliInx2 and SpliInx3. Here, we found that only the mRNAs of the two Cte-Inxs were expressed under the control of the CMV promoter in HeLa cells. Unexpectedly, mRNA expression of the two Cte-Inxs enhanced apoptosis of HeLa cells. The two Cte-Inx mRNAs were associated with a significant decrease in Akt phosphorylation in HeLa cells undergoing apoptosis. Furthermore, Inx3-FLAG mRNA expression in nonapoptotic HCT116 cells was also associated with a significant decrease in the levels of phosphorylated Akt. Intriguingly, expression of the mRNAs of the two Cte-Inxs did not activate caspase 3, but it markedly reduced Bid levels in HeLa cells undergoing apoptosis. These results suggest that mRNA expression of the two Cte-Inxs may activate a Bid-dependent apoptotic pathway in HeLa cells. Our study demonstrates that invertebrate gap junction mRNAs can function in vertebrate cancer cells as tumor suppressors.

Bracovirus-mediated innexin hemichannel closure in cell disassembly.

Cell-cell communication is necessary for cellular immune response. Hemichannel closure disrupts communication between intracellular and extracellular environments during polydnavirus-induced immunosuppression in invertebrates. However, the effects of hemichannel closure on cellular immune response are unclear. Here, we examined apoptotic body formation triggered by hemichannel closure in hemocytes of Spodoptera litura infected with bracovirus from the parasitic wasp, Microplitis bicoloratus. We showed that Microplitis bicoloratus bracovirus (MbBV) induced apoptotic cell disassembly, accompanied by hemichannel closure. Hemocyte apoptotic body formation was caused by the dysregulation of the innexins (Inxs), Inx1, Inx2, Inx3, and Inx4, during the MbBV-mediated inhibition of pI3K/AKT signaling and activation of caspase-3, which cleaved gap junction Inx proteins. Our results showed that hemichannel opening or closure in response to various stimuli, which induces the modulation of Inx levels, could inhibit or activate apoptotic body formation, respectively. Therefore, the "hemichannel open and close" model may regulate the cellular immune response.

Microplitis bicoloratus bracovirus modulates innate immune suppression through the eIF4E-eIF4A axis in the insect Spodoptera litura.

Eukaryotic initiation factor 4E (eIF4E) is regulated during the innate immune response. However, its translational regulation under innate immune suppression remains largely unexplored. Microplitis bicoloratus bracovirus (MbBV), a symbiotic virus harbored by the parasitoid wasp, Microplitis bicoloratus, suppresses innate immunity in parasitized Spodoptera litura. Here, we generated eIF4E dsRNA and used it to silence the eIF4E gene of S. litura, resulting in a hallmark immunosuppressive phenotype characterized by increased apoptosis of hemocytes and retardation of head capsule width development. In response to natural parasitism, loss of eIF4E function was associated with similar immunosuppression, and we detected no significant differences between the response to parasitism and treatment with eIF4E RNAi. Under MbBV infection, eIF4E overexpression significantly suppressed MbBV-induced increase in apoptosis and suppressed apoptosis to the same extent as co-expression of both eIF4E and eIF4A. There were no significant differences between MbBV-infected and uninfected larvae in which eIF4E was overexpressed. More importantly, in the eIF4E RNAi strain, eIF4A RNAi did not increase apoptosis. Collectively, our results indicate that eIF4E plays a nodal role in the MbBV-suppressed innate immune response via the eIF4E-eIF4A axis.

A secreted-Cu/Zn superoxide dismutase from Microplitis bicoloratus reduces reactive oxygen species triggered by symbiotic bracovirus.

In the parasitoid/polydnavirus/host system, polydnaviruses protect larva development in the host hemocoel by suppressing the host immune response. However, the negative effects on the parasitoid and the strategy of the parasitoid to deal with this disadvantage are still unknown. Microplitis bicoloratus bracovirus induces granulocyte apoptosis to suppress immune responses, resulting in an apoptotic haemolymph environment in which immature M. bicoloratus larva develop. Here, we determined the transcriptional profiles of immature M. bicoloratus across five time-points throughout the immature developmental process from egg to third instar. Dynamic gene expression pattern analysis revealed clear rapid changes in gene expression characteristic of each developmental stage, indicating faster sequential unambiguous functional division during development. Combined with the proteome of the host haemolymph, immature parasitoids likely secreted a Cu/Zn superoxide dismutase to reduce reactive oxygen species generation by symbiotic bracovirus. These data established a basis for further studies of parasitoid/host interactions and identified a novel positive self-protection mechanism for the parasitoid.