敲减钙离子/钙调蛋白依赖性激酶Ⅳ抑制干扰素-γ诱导C2C12细胞免疫分子的表达
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摘要
目的探究敲减钙离子/钙调蛋白依赖性激酶Ⅳ(Ca MKⅣ)基因对炎性培养条件下C2C12细胞免疫分子表达的影响。方法采用慢病毒基因干扰技术敲减C2C12细胞的Ca MKⅣ基因,以2%马血清将C2C12细胞分化成肌管,以干扰素-γ(IFN-γ)刺激C2C12细胞,采用Real-time PCR和Western blotting检测Ca MKⅣ的表达,采用Western blotting和免疫荧光检测免疫分子主要组织相容性复合体(MHC) classⅠ(H-2Kb)、MHC classⅡ(H2-Ea)、Toll样受体3(TLR3)的表达,采用Real-time检测肌细胞分泌型促炎性细胞因子白细胞介素(IL)-1β、IL-6、肿瘤坏死因子(TNF)-α、巨噬细胞炎性蛋白(MIP)-1α和单核细胞趋化因子(MCP)-1的基因表达。结果 IFN-γ可诱导成肌干细胞以及分化肌管表达或上调表达免疫分子MHC-Ⅰ、MHC-Ⅱ、TLR3,同时上调C2C12细胞促炎性细胞因子IL-1β、IL-6、TNF-α、MIP-1α和MCP-1的基因表达量;敲减Ca MKⅣ基因导致上述上调趋势被抑制。结论敲减Ca MKⅣ基因可有效抑制IFN-γ诱导的免疫分子的表达,可能提示Ca MKⅣ参与调节肌细胞的免疫学特性。
Objective Whether calcium/calmodulin-dependent protein kinase Ⅳ( Ca MKⅣ) plays a role in regulating immunologic features of muscle cells in inflammatory environment,remains mostly unknown. In this study,we investigated the influence of Ca MKⅣ on the immunological characteristics of myoblasts and myotubes received interferon( IFN)-γ stimulation. Methods To investigate the effects of Ca MKⅣ on immune characteristics of C2 C12 myoblasts and differentiated myotubes,which are firstly knocked down endogenous Ca MKⅣ gene and then treated with IFN-γ. Real-time PCR and Western blotting were performed to analyze the expression of Ca MKⅣ. 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-2 Kb,MHC-Ⅱ molecule H2-Ea,TLR3 significantly up-regulated in myoblasts and in differentiated myotubes. In striking contrast,Ca MK Ⅳ 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 C2 C12 cells( especially in myotubes) were markedly down-regulated by Ca MKⅣ knocking off. Conclusion Knockdown of Ca MKⅣ gene can effectively inhibit the expression of IFN-γ-induced immune molecules,suggesting that Ca MKⅣ is involved in regulating the immunological properties of muscle cells.
Objective Whether calcium/calmodulin-dependent protein kinase Ⅳ( Ca MKⅣ) plays a role in regulating immunologic features of muscle cells in inflammatory environment,remains mostly unknown. In this study,we investigated the influence of Ca MKⅣ on the immunological characteristics of myoblasts and myotubes received interferon( IFN)-γ stimulation. Methods To investigate the effects of Ca MKⅣ on immune characteristics of C2 C12 myoblasts and differentiated myotubes,which are firstly knocked down endogenous Ca MKⅣ gene and then treated with IFN-γ. Real-time PCR and Western blotting were performed to analyze the expression of Ca MKⅣ. 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-2 Kb,MHC-Ⅱ molecule H2-Ea,TLR3 significantly up-regulated in myoblasts and in differentiated myotubes. In striking contrast,Ca MK Ⅳ 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 C2 C12 cells( especially in myotubes) were markedly down-regulated by Ca MKⅣ knocking off. Conclusion Knockdown of Ca MKⅣ gene can effectively inhibit the expression of IFN-γ-induced immune molecules,suggesting that Ca MKⅣ is involved in regulating the immunological properties of muscle cells.
引文
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[10]Iwasa K,Kato-Motozaki Y,Furukawa Y,et al.Up-regulation of MHC classⅠand classⅡin the skeletal muscles of myasthenia gravis[J].J Neuroimmunol,2010,225(1-2):171-174.
[11]Wiendl H,Hohlfeld R,Kieseier BC.Immunobiology of muscle:advances in understanding an immunological microenvironment[J].Trends Immunol,2005,26(7):373-380.
[12]Garlepp MJ,Chen W,Tabarias H,et al.Antigen processing and presentation by a murine myoblast cell line[J].Clin Exp Immunol,1995,102(3):614-619.
[13]Lightfoot AP,Cooper RG.The role of myokines in muscle health and disease[J].Curr Opin Rheumatol,2016,28(6):661-666.
[14]Ma P,Zhu R,Liu XH,et al Notexin effects on the survival and proliferation of CD8+T cell in the injuried-muscle draining lymph nodes[J].Chinese Journal of Clinical Anatomy,2014,3 296-299.(in Chinese)马平,术蓉,刘幸卉,等.Notexin对小鼠体内CD8+T淋巴细胞活性的干扰作用[J].中国临床解剖学杂志,2014,(3):296-299.
[2]Naz H,Islam A,Ahmad F,et al.Calcium/calmodulin-dependent protein kinaseⅣ:a multifunctional enzyme and potential therapeutic target[J].Prog Biophys Mol Biol,2016,121(1):54-65.
[3]Koga T,Kawakami A.The role of CaMK4 in immune responses[J].Mod Rheumatol,2018,28(2):211-214.
[4]Illario M,Giardino-Torchia ML,Sankar U,et al.Calmodulindependent kinaseⅣlinks Toll-like receptor 4 signaling with survival pathway of activated dendritic cells[J].Blood,2008,111(2):723-731.
[5]Zhu R,Cao YY,Shi DD,et al Immune features of mouse myoblast and myotubes cultured in vitro inflammatory enviroment[J].Immunological Jouranl,2016,32(06):501-506.(in Chinese)术蓉,曹永英,史丹丹,等.体外炎性环境下小鼠成肌细胞/分化肌管的免疫特性研究[J].免疫学杂志,2016,32(06):501-506.
[6]Ding M,Huang T,Zhu R,et al.Immunological behavior analysis of muscle cells under IFN-γstimulation in vitro and in vivo[J].Anat Rec(Hoboken),2018,301(9):1551-1563.
[7]Ba MA,Surina J,Singer CA,et al.Knockdown of subunit 3 of the COP9 signalosome inhibits C2C12 myoblast differentiation via NF-Kappa B signaling pathway[J].BMC Pharmacol Toxicol,2017,18(1):47.
[8]Racioppi L,Means AR.Calcium/calmodulin-dependent kinaseⅣin immune and inflammatory responses:novel routes for an ancient traveller[J].Trends Immunol,2008,29(12):600-607.
[9]Ichinose K,Juang YT,Crispín JC,et al.Suppression of autoimmunity and organ pathology in lupus-prone mice upon inhibition of calcium/calmodulin-dependent protein kinase typeⅣ[J].Arthritis Rheum,2011,63(2):523-529.
[10]Iwasa K,Kato-Motozaki Y,Furukawa Y,et al.Up-regulation of MHC classⅠand classⅡin the skeletal muscles of myasthenia gravis[J].J Neuroimmunol,2010,225(1-2):171-174.
[11]Wiendl H,Hohlfeld R,Kieseier BC.Immunobiology of muscle:advances in understanding an immunological microenvironment[J].Trends Immunol,2005,26(7):373-380.
[12]Garlepp MJ,Chen W,Tabarias H,et al.Antigen processing and presentation by a murine myoblast cell line[J].Clin Exp Immunol,1995,102(3):614-619.
[13]Lightfoot AP,Cooper RG.The role of myokines in muscle health and disease[J].Curr Opin Rheumatol,2016,28(6):661-666.
[14]Ma P,Zhu R,Liu XH,et al Notexin effects on the survival and proliferation of CD8+T cell in the injuried-muscle draining lymph nodes[J].Chinese Journal of Clinical Anatomy,2014,3 296-299.(in Chinese)马平,术蓉,刘幸卉,等.Notexin对小鼠体内CD8+T淋巴细胞活性的干扰作用[J].中国临床解剖学杂志,2014,(3):296-299.