The role of nuclear transport system in cell senescence

Division Of Basic Sciences - Nci

Investigators

Abstract

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.