Abstract:

Objective Whether calcium/calmodulin-dependent protein kinase Ⅳ (CaMKⅣ) plays a role in regulating immunologic features of muscle cells in inflammatory environment, remains mostly unknown. In this study, we investigated the influence of CaMKⅣ on the immunological characteristics of myoblasts and myotubes received interferon(IFN)-γ stimulation. Methods To investigate the effects of CaMKⅣ on immune characteristics of C2C12 myoblasts and differentiated myotubes, which are firstly knocked down endogenous CaMKⅣ gene and then treated with IFN-γ. Real-time PCR and Western blotting were performed to analyze the expression of CaMKⅣ. Western blotting and immunofluorescence were performed to analyze the expression of major histocompatibility complex(MHC) class-Ⅰ,MHC class-Ⅱ,Toll-like receptor3(TLR3). The expression of interleukin(IL)-1β,IL-6,tumor necrosis factor(TNF)-α,macrophage inflamator protein(MIP)-1α and monocyte chemoattractant protein(MCP)-1 were measured by Real-time PCR. Results Under IFN-γ induced pro-inflammatory milieu, MHC-Ⅰ molecule H-2K^b, MHC-Ⅱ molecule H2-Ea, TLR3 significantly up-regulated in myoblasts and in differentiated myotubes. In striking contrast, CaMKⅣ inhibition in myoblasts and myotubes led to expression suppression of the above molecules. As well, gene levels of pro-inflammatory IL-1β, IL-6, TNF-α, MIP-1α and MCP-1 in C2C12 cells (especially in myotubes) were markedly down-regulated by CaMKⅣ knocking off. Conclusion Knockdown of CaMKⅣ gene can effectively inhibit the expression of IFN-γ-induced immune molecules, suggesting that CaMKⅣ is involved in regulating the immunological properties of muscle cells.

Key words: Calcium/calmodulin-dependent protein kinase Ⅳ, Immune molecules, C2C12 cell, shRNA transfection, Real-time PCR, Western blotting, Immunofluorescence