The Novel KLF4/BIG1 Regulates LPS-mediated Neuro-inflammation and Migration in BV2 Cells via PI3K/Akt/NF-kB Signaling Pathway. (15th April 2022) - You, Zhijun
Highlights: BIG1 promotes LPS-mediated inflammatory cytokines production. BIG1 facilitated LPS-mediated BV2 cell migration. Activation of PI3K/Akt pathway reversed the protective roles of BIG1 silencing in inflammation and migration. KLF4 transcriptionally regulated BIG1 expression in BV2 cells. Ectopic expression of BIG1 partly reversed the biological activities of KLF4. Abstract: Excessive microglia activation occurred in many neurodegenerative diseases. Brefeldin A-inhibited guanine nucleotide-exchange protein 1 (BIG1, ARFGEF1) is involved in cell migration and neurite growth. In the present study, we aimed to explore the effects and potential mechanisms of BIG1 in LPS-mediated neuro-inflammation and migration in BV2 cells. Loss-of-function and gain-of-function experiments were performed. Inflammatory cytokines (TNF-α, IL-1β, IL-6, IL-10) mRNA levels and concentrations were analyzed by Quantitative Real-time PCR (RT-qPCR) and ELISA kits. The NO concentration was tested by ELISA kit. iNOS and COX-2 mRNA and protein levels were measured by RT-qPCR and western blot. Cell migration was determined by transwell assay. The results demonstrated that BIG1 silencing reduced TNF-α, IL-1β, and IL-6 expression, while increased IL-10 expression. The NO production, iNOS and COX-2 expression were clearly inhibited by BIG1 knockdown in the presence of LPS. Furthermore, ablation of BIG1 attenuated the migration capacity of BV2 cells. Overexpression of BIG1 displayed the opposite trends. Moreover, we found BIG1 suppression inhibited PI3K/Akt/NF-κB pathway activation. 740Y-P, an agonist of PI3K, abolished the roles of BIG1 silencing in neuro-inflammation and migration. Additionally, ChIP-qPCR and Dual-luciferase reporter assay determined that KLF4 binds to the promoter of BIG1, western blot analysis demonstrated that KLF4 could regulate BIG1 positively. In addition, we observed that BIG1 overexpression partly rescued the biological activities of KLF4 silencing in neuro-inflammation and migration in LPS-stimulated BV2 cells. Taken together, BIG1 was mediated by KLF4 regulated LPS-mediated neuro-inflammation and migration in BV2 cells via PI3K/Akt/NF-kB signaling pathway.