OSBPL2介导RhoA/ROCK2信号通路调控听觉细胞肌动蛋白细胞骨架

OSBPL2 mediates RhoA/ROCK2 signaling pathway,regulating actin cytoskeleton in auditory cells

目的:探索氧化固醇结合蛋白样蛋白2(oxysterol binding protein-like 2,OSBPL2)对听觉细胞肌动蛋白骨架形态和功能的影响及其相关分子机制。方法:采用OSBPL2基因敲除(Osbpl2-KO)HEI-OC1细胞探讨OSBPL2缺陷对听觉细胞肌动蛋白细胞骨架形态和功能的影响;扫描电镜观察Osbpl2-KO小鼠毛细胞静纤毛形态;Western blot实验检测HEI-OC1细胞和耳蜗内Rho/ROCK信号通路关键效应因子Rho激酶2(Rho-associated coiled-coil-containing protein kinase 2,ROCK2)、Lim激酶1(LIM domain kinase 1,LIMK1)、肌动蛋白解聚因子/丝切蛋白1(actin depolymerizing factor/cofilin 1,ADF/CFL-1)的表达水平。结果:Osbpl2-KO HEI-OC1细胞微丝骨架中纤维肌动蛋白(F-actin)的分布明显减少,细胞外周微刺突和丝状伪足明显减少,细胞迁移能力明显减弱;扫描电镜下观察发现Osbpl2敲除导致小鼠耳蜗内毛细胞静纤毛变短且排列紊乱;Western blot检测结果表明,在HEI-OC1细胞和小鼠耳蜗中OSBPL2-KO均导致RhoA/ROCK2信号通路的抑制。结论:在听觉细胞中OSBPL2介导的RhoA/ROCK2信号通路在维持肌动蛋白细胞骨架形态和功能的过程中发挥重要的调控作用。

Objective:To explore the effect of oxysterol⁃binding protein⁃like 2(OSBPL2)on the actin cytoskeleton in auditory cells and its related molecular mechanisms.Methods:OSBPl2 knockout(Osbpl2⁃KO)HEI⁃OC1 cells were used to investigate the effect of OSBPL2 deficiency on the the actin cytoskeleton in auditory cells.The stereocilia onmouse hair cells were observed by scanning electron microscopy.Western blot was used to detect the expression of Rho⁃associated coiled⁃coil⁃containing protein kinase 2(ROCK2),Lim domain kinase 1(LIMK1)and actin depolymerizing factor 1(ADF/CFL⁃1)in HEI⁃OC1 cells and mouse cochleae.Results:In Osbpl2⁃KO HEI⁃OC1 cells,the distribution of F⁃actin in the microfilament skeleton was down⁃regulated,the pericellular microspikes and filopodia were noticeably reduced,and the cell migration ability was significantly weakened.In vivo,OSBPL2 deficiency resulted in shortened and disordered stereocilia on mouse inner hair cells.Western blot assay showed that OSBPL2 deficiency led to the inhibition of RhoA/ROCK2 signaling pathway in HEI⁃OC1 cells and mouse cochleae.Conclusion:OSBPL2 mediats RhoA/ROCK2 signaling pathway in auditory cells and plays an important role in maintaining the morphology and function of actin cytoskeleton.