The role of nuclear transport system in cell senescence

背景：最近来自各种实验系统的数据表明，细胞衰老的诱导与RAN调节的核运输系统NTS的显著变化(主要是下调)有关。在老年供者来源的成纤维细胞中，Importinα1、CAS/Cse1和RanBP1的水平降低，核输入减少(1)，在体外复制衰老的人二倍体成纤维细胞(HDF)中，NTS的许多组分的mRNA表达显著降低(2)。在老化的线虫和大鼠中，由于核祖细胞外周成分合成减少，核祖细胞恶化并失去其分子门控功能，允许细胞质内容物不受控制地进入细胞核(3)。另一方面，在两个不同的层粘连蛋白A突变体的表达所致的细胞早衰过程中，三种不同的NTR(exportin1、Transportin、Importinα/ImportinβComplex)携带的核转运货物的动力学降低。有趣的是，尽管层蛋白A突变导致Hutchison-Gilford Progeria综合征(HGPS)或限制性皮肤病(RD)所导致的衰老表型的严重程度不同，但在表达HGPs和RD的细胞中，核转运能力的下降是相似的。从这些研究中可以看出，NTS的下降是包括生理老化在内的各种刺激引起的细胞衰老的共同过程。另一方面，在肿瘤和肿瘤来源的组织培养细胞中，RAN水平升高，许多RAN调节的有丝分裂纺锤体组装因子(SAF)被认为参与促进癌细胞有丝分裂。因此，RAN和NTS在细胞衰老过程中的功能变化似乎与在癌细胞中发现的相反。我们推测，在衰老细胞中发现的一些NTS的变化是必需的，并且可能足以诱导和/或维持细胞衰老。我们的研究旨在解决这一假设。进展：利用复制诱导的正常人成纤维细胞细胞衰老模型，我们检测了NTS的许多成分的蛋白水平和细胞定位。与基因芯片的mRNA表达数据一致，我们发现NTS的许多但不是所有成分都经历了显著的下调，部分但不是全部的定位发生了变化。用Rango FRET传感器进行的FliM/FRET测量表明，与细胞核中Importinβ浓度增加一致，RAN调节的Importinβ货物跨核膜的梯度显著中断。最近，我们建立了RasV12癌基因表达或携带Progerin突变的lamin A诱导细胞衰老的体外模型。与复制诱导的细胞衰老相似，我们检测到这些细胞中RanBP1和Importinα1的表达显著减少。实验正在进行中，将检验RanBP1、RAN和Importin Alpha 1水平在RAS或Progerin诱导的细胞衰老中的作用。结论：该项目处于相对早期的阶段，我们能够建立我们的实验细胞模型、分析方法和试剂，以满足计划研究的需要。我们的初步结果表明，尽管RanGTP梯度在衰老细胞中保持不变，但由于RanBP1和RanGAP水平的降低以及Importinβ核定位的增加，其在核运输中的功能似乎发生了显著变化。我们现在正在过渡到该项目的第二阶段，在该阶段我们将重点关注RanBP1、Importin Alpha1和RAN在衰老诱导中的作用。1.Pujol，G.，H.Soderqvist和A.Radu.2002年。人类成纤维细胞中与年龄相关的核蛋白输入减少。生物化学生物物理通讯294：354-358。2.Kim，S.Y.，S.J.Ryu，H.J.Ahn，H.R.Choi，H.T.Kang，S.C.Park。2010年。核质转运基因表达下调与衰老相关的功能核屏障。生物化学生物物理通讯391：28-32.3.D‘Angelo，M.A.，M.Raices，S.H.Panowski和M.W.Hetzer。2009年。随着年龄的增长，核孔复合体的退化会导致有丝分裂后细胞核完整性的丧失。电话136：284-295。4.布希，A.，T.基尔，W.M.Heupel，M.Wehnert和S.Hubner。2009年。在表达导致核包膜病变的Lamin A突变体的细胞中，核蛋白进口减少。EXP单元格：315：2373-2385。

Background: Recent data from variety of experimental systems indicate that the induction of cell senescence is associated with significant changes (mostly downregulation) of Ran-regulated nuclear transport system, NTS. Decreased levels of importin alpha 1, CAS/Cse1, and RanBP1, and decreased nuclear import were found in fibroblasts derived from old human donors (1), and strongly decreased mRNA expression of many components of NTS was found in human diploid fibroblasts (HDF) undergoing replicative senescence in vitro (2). As a consequence of decreased synthesis of peripheral components of NPCs, in aging C. elegans and rats, NPCs deteriorate and lose their molecular gating function, allowing uncontrolled passage of cytoplasmic contents into nuclei (3). On the other hand, in cell undergoing premature senescence induced by the expression of two different mutants of lamin A, the kinetics of nuclear transport cargos carried by three different NTRs (exportin1, transportin, importin alpha/importin beta complex) was decreased (4). Interestingly, despite a different severity of senescence phenotypes induced by mutations in lamin A leading to Hutchison-Gilford Progeria Syndrom (HGPS) or restrictive dermopathy (RD), the decline of nuclear transport competency was similar in both HGPs- and RD- expressing cells (4). The emerging picture from these studies is that the decline of NTS is a process common to cell senescence induced by various stimuli, including physiological aging. On the other hand, Ran levels are increased in tumors and cancer-derived tissue culture cells and many Ran-regulated mitotic spindle assembly factors (SAFs) are known for their involvement in promoting cancer cell mitosis. Thus, the changes of Ran and NTS function in cell senescence appear to be the opposite of those found in cancer cells. We hypothesize that some of the changes of NTS found in senescent cells are required and perhaps sufficient for the induction and/or maintenance of cell senescence. Our research is designed to address this hypothesis. Advance: Using replication-induced in vitro model of cell senescence in normal human fibroblasts, we examined the protein levels and cellular localization of many components of NTS. Consistent with microarray data on mRNA expression, we found that many but not all components of NTS underwent significant downregulation and some, but not all, change of localization. Consistent with increased concentration of importin beta in the nucleus, FLIM/FRET measurements with Rango FRET sensors indicated significant disruption of Ran-regulated gradient of importin beta cargos across nuclear envelope. More recently, we developed in vitro models for cell senescence induced by the expression of RasV12 oncogene or lamin A carrying Progerin mutation. Similar to replication-induced cell senescence, we detected significant decrease of RanBP1 and importin alpha 1 in those cells. Experiments are in progress which will examine the role of RanBP1, Ran, and importin alpha 1 levels in the induction of cell senescence induced by Ras or Progerin. Conclusion: This project is in relatively early stages in which we were able to establish our experimental cell models, assays and reagents required for the planned research. Our preliminary results indicate that although RanGTP gradient is maintained in senescent cells, its functions in nuclear transport appears to be significantly changes due to decrease of RanBP1 and RanGAP levels and increased nuclear localization of importin beta. We are now transiting to the second phase of the project in which we focus on the role of namely RanBP1, importin alpha1 and Ran levels in senescence induction. 1. Pujol, G., H. Soderqvist, and A. Radu. 2002. Age-associated reduction of nuclear protein import in human fibroblasts. Biochem Biophys Res Commun 294:354-358. 2. Kim, S. Y., S. J. Ryu, H. J. Ahn, H. R. Choi, H. T. Kang, and S. C. Park. 2010. Senescence-related functional nuclear barrier by down-regulation of nucleo-cytoplasmic trafficking gene expression. Biochem Biophys Res Commun 391:28-32. 3. D'Angelo, M. A., M. Raices, S. H. Panowski, and M. W. Hetzer. 2009. Age-dependent deterioration of nuclear pore complexes causes a loss of nuclear integrity in postmitotic cells. Cell 136:284-295. 4. Busch, A., T. Kiel, W. M. Heupel, M. Wehnert, and S. Hubner. 2009. Nuclear protein import is reduced in cells expressing nuclear envelopathy-causing lamin A mutants. Exp Cell Res 315:2373-2385.