Study On The Role Of PAK4 In Japanese Encephalitis Virus-Induced Inflammatory Response In Glial Cells

Japanese encephalitis virus(JEV) is an amphixenosis and insect-borne pathogen. It can target to central nervous system and cause Japanese encephalitis(JE), which can do seriously harm to both animals and human beings. “Inflammatory storm” in central nervous system is the main pathogenic factor of JEV, but its molecular mechanism is remaining to be studied on. P21-activated kinase(PAK) is a kind of protein kinase which is rich in serine/ threonine, and is thought to be an important downstream effector of small G protein Cdc42 and Rac1, plays an important role in signal transduction, gene transcription and cell apoptosis. ERK is a downstream media of PAK4, and its phosphorylation can be mediated by PAK4 and then activate the signaling pathway of NF-κB. That indicates that PAK4 is probably a protein that relates to inflammation. In this study, we are working on PAK4 protein in glial cells and try to clarify its function in JEV-mediated inflammatory response.1. Effects of JEV infection on the expression and phosphorylation of PAK4We investigated pak4 expression at the levels of protein and Phosphorylation using Western-blot. In JEV-infected U251 cells(human astrocytes), we found that JEV infection upregulates PAK4 at phosphorylation levels, but doesn’t upregulate PAK4 at protein levels.2. The effect of pak4 on JEV-mediated inflammationUsing pak4-specific siRNA, we silenced pak4 which resulted in inhibition of JEV mediated inflammation. Thus, PAK4 has a positive regulatory role of JEV mediated inflammation.3. The molecular mechanism of PAK4 regμlating JEV-mediated inflammationWe inhibited the expression of PAK4 in U251 cells and then infected the cells with JEV and found that, the inhibition of the expression of PAK4 can downregulates JEV-mediated ERK phosphorylation. At the same time, we also tested P38 MAPK, another member of MAPK family. Results showed that, after the inhibition of the expression of PAK4, JEV-mediated P38 MAPK phosphorylation can also be downregulated significantly. Furthermore, we investigated regulation of PAK4 in JEV-mediated NF-κB and AP-1 transcriptional activity, we found that PAK4 siRNA can significantly inhibits the nuclear entry of NF-κB and AP-1 phosphorylation mediated by JEV. Above results show that, PAK4 can promote JEV mediated inflammation via activating ERK/P38 MAPK and the downstream NF-κB/AP-1 signaling pathway.It is the first time that we found that JEV can upregulate the level of phosphorylation modification of PAK4 in U251 cells and PAK4 can promote JEV-mediated inflammatory response by activating MAPK- NF-κB/AP-1 signaling pathway. This offers a new scientific evidence for demonstrating the molecular mechanism of JEV-mediated neural inflammatory response, and a new clue for the research about JE treatment targets.