The Role Of C1QBP In Chemotaxis Of Monocytes/macrophages

Objectives:Monocyte/macrophage chemotaxis is an important part of the immune response, inflammation, tumor metastasis. Our previous studies have shown that PKC is a key molecule regulating monocyte/macrophage chemotaxis. A Mass spectroscopy analysis revealed that C1QBP is a novel partner of PKCζ. Our previous study showed that the application of small RNA interference silencing of C1QBP suppressed CSF-1induced monocyte/macrophage chemotaxis. Accordingly, we inferred that C1QBP may regulate PKCζ to modulate the chemotaxis of monocyte/macrophage. In this study, we knockdown the expression of C1QBP, and to explore the effect on monocyte/macrophage migration and adhesion, initially identified the related molecular mechanism. This study showed the mechanism of C1QBP, interact with PKCζ regulating monocyte/macrophage chemotaxis, is of great significance to explore the pathogenesis of inflammatory diseases and the prevention and treatment of tumor.Methods:1. Co-immunoprecipitation assays were used to examine the interaction between C1QBP and PKCζ in monocytes/macrophages.2. Immunofluorescence were used to observe the co-localization between C1QBP and PKCζ in monocytes/macrophages.3. We designed StealthTM RNA targeted against human/mouse C1QBP to attenuate C1QBP expression in monocytes/macrophages, which were identified by Western Blotting analysis.4. Lenti-virus mediated RNAi technology was used to establish C1QBP stable knockdown mouse monocyte/macrophage cell line RAW264.7, which was identified by Western Blotting analysis.5. Chemotaxis assay and F-actin polymerization assay were used to demonstrate the influence of knockdown C1QBP on the chemotaxis ability of human monocyte/macrophage cell line THP-1. 6. Chemotaxis assay and adhesion assay were used to demonstrate the influence of knockdown C1QBP on the C1q、CSF-1、MCP-1induced chemotaxis ability of mouse monocyte/macrophage cell line RAW264.7.7. Western Blotting analysis was used to detect the activation of PKC C and signaling pathway protein upon stimulation with C1q、CSF-1、MCP-1after knockdown C1QBP. Further identified the molecular mechanism of C1QBP in monocyte/macrophage chemotaxis.Results:1. Co-immunoprecipitation assays revealed CIQBP interacted with PKCζ.2. Confocal microscopy showed CIQBP and PKCζ, located in the cytoplasm and cytomembrane, co-located in the cytoplasm.3. After transfected monocytes/macrophages by StealthTMNA, Western Blotting analysis revealed a severe decrease in protein level.4. Established C1QBP stable knockdown mouse monocyte/macrophage cell line RAW264.7by lenti-virus mediated RNAi, the expression of C1QBP significantly decreased.5. C1QBP-reduced THP-1cells showed decreased chemotaxis ability compared with control cells(P<0.01).6. Disruption of CIQBP expression can decreased Clq and CSF-1induced monocyte/macrophage chemotaxis, Clq, CSF-1, MCP-1induced adhesion(.P<0.05).7. Knockdown of C1QBP blocked Clq and CSF-1induced integrinβ1and cofilin phosphorylation severely, while had no effect on MCP-1induced.Conclusions:1. C1QBP was involved monocyte/macrophage chemotaxis and actin polymerization through the regulation of PKCζ phosphorylation and activation.2. C1QBP participated in CSF-1and C1q-induced monocyte/macrophage chemotaxis and CSF-1, MCP-1, C1q-induced adhesion.3. C1QBP exerts its functions in monocyte/macrophage chemotaxis and adhesion through chemokines-induced PKCζ and downstream signal protein intergrin β, cofilin phosphorylation.4. C1QBP is an important signal molecular involved in chemokines-induced chemotaxis of monocyte/macrophage.