NLS-RARα通过与importin α1/β1(KPNA2/KPNB1)结合入核并抑制U937细胞分化

NLS-RARα transports into the nucleus by binding to importin α1/β2(KPNA2/KPNB1) and inhibits the differentiation of U937 cells

目的探究核定位信号-维甲酸受体α(NLS-RARα)异常的核定位对细胞分化的抑制作用以及其核转运机制。方法过表达带有血凝素(HA)标签的NLS-RARα慢病毒和空载体慢病毒转染HEK293T细胞和U937细胞,设为NLS-RARα过表达组(NR组)和阴性对照组(NC组)。提取HEK293T细胞与U937细胞NC组和NR组细胞核和细胞质蛋白,Western blot法检测NLS-RARα的定位。同时采用免疫荧光细胞化学染色检测NLS-RARα的定位情况。1α,25-二羟维生素D3(1,25D3)诱导U937细胞分化,实时定量PCR,Western blot法检测细胞CD11b、CCAAT/增强子结合蛋白β(CEBPβ)在mRNA和蛋白水平,采用质谱和免疫共沉淀技术(CoIP)筛选与NLS-RARα相作用的转运蛋白,并且通过小干扰RNA(siRNA)转染验证其对NLS-RARα的核定位作用。结果NLS-RARα主要位于细胞核中。与对照组相比,过表达NLS-RARα组CD11b和CEBPβ的增加减少,说明NLS-RARα对1,25D3诱导的细胞分化有抑制作用。质谱和CoIP结果发现核转运蛋白α2/输入蛋白α1(KPNA2/importinα1)和核转运蛋白β1/输入蛋白β1(KPNB1/importinβ1)与NLS-RARα有相互作用,敲低KPNA2/KPNB1则抑制NLS-RARα入核。结论NLS-RARα通过结合KPNA2/KPNB1入核,抑制U937细胞分化。

Objective To investigate the inhibitory effect of abnormal nuclear localization of the nuclear localization signal-retinoic acid receptorα(NLS-RARα)on cell differentiation and its mechanism of nuclear transport.Methods Over-expression of HA-NLS-RARαand empty vector in HEK293T cells and U937 cells were achieved through a lentivirus vector and were assigned as NLS-RARαover-expression(NR)group and negative control(NC)group.Extracted nucleoproteins and cytosolic proteins of NC and NR groups of HEK293T cells and U937 cells were detected by Western blot analysis in order to demonstrate the localization of NLS-RARα.Meanwhile,immunofluorescence assay was performed to explore the localization of NLS-RARα.The real-time quantitative PCR and Western blot analysis were used to detect difference in the mRNA and protein expression of CD11b and CEBPβin the NR cells treated with 1,25-dihydroxyvitamin D3(1,25D3)compared with NC cells treated with 1,25D3.Mass spectrometric analysis and co-immunoprecipitation were conducted to screen the transport proteins which were associated with NLS-RARα,which was followed by the verification of nuclear accumulation of NLS-RARαby the transfection of transport protein small interfering RNA.Results Western blot assay and immunofluorescence showed that NLS-RARαwas mainly located in the nucleus.And the qRT-PCR analysis and western blot assay showed a significant decrease in the mRNA and protein expression of CD11b and CEBPβin the NR group compared with the NC group.It demonstrated that NLS-RARαinhibited cell differentiation.Mass spectrometric analysis and COIP demonstrated that KPNA2(importinα1)and KPNB1(importinβ1)interacted with NLS-RARα,and the knockdown of KPNA2/KPNB1 inhibited the nuclear accumulation of NLS-RARα.Conclusion Abnormal localization of NLS-RARαinhibits cell differentiation via binding to KPNA2 and KPNB1 into the nucleus.