www.nature.com/emm ARTICLE OPEN Disruption of nucleocytoplasmic trafficking as a cellular senescence driver Ji-Hwan Park1,14, Sung Jin Ryu2,13,14, Byung Ju Kim3,13,14, Hyun-Ji Cho3, Chi Hyun Park4, Hyo Jei Claudia Choi2, Eun-Jin Jang3, Eun Jae Yang5, Jeong-A Hwang5, Seung-Hwa Woo5, Jun Hyung Lee5, Ji Hwan Park5, Kyung-Mi Choi6, Young-Yon Kwon6, 6 7 3 3 8 9 10 5 ✉ Cheol-Koo Lee , Joon✉ Tae Park , Sung✉ Chun Cho , Yun-Il Lee , Sung✉ Bae Lee , Jeong A. Han , Kyung A. Cho , Min-Sik Kim , Daehee Hwang11 , Young-Sam Lee3,5 and Sang Chul Park3,12 © The Author(s) 2021 Senescent cells exhibit a reduced response to intrinsic and extrinsic stimuli. This diminished reaction may be explained by the disrupted transmission of nuclear signals. However, this hypothesis requires more evidence before it can be accepted as a mechanism of cellular senescence. A proteomic analysis of the cytoplasmic and nuclear fractions obtained from young and senescent cells revealed disruption of nucleocytoplasmic trafficking (NCT) as an essential feature of replicative senescence (RS) at the global level. Blocking NCT either chemically or genetically induced the acquisition of an RS-like senescence phenotype, named nuclear barrier-induced senescence (NBIS). A transcriptome analysis revealed that, among various types of cellular senescence, NBIS exhibited a gene expression pattern most similar to that of RS. Core proteomic and transcriptomic patterns common to both RS and NBIS included upregulation of the endocytosis-lysosome network and downregulation of NCT in senescent cells, patterns also observed in an aging yeast model. These results imply coordinated aging-dependent reduction in the transmission of extrinsic signals to the nucleus and in the nucleus-to-cytoplasm supply of proteins/RNAs. We further showed that the aging-associated decrease in Sp1 transcription factor expression was critical for the downregulation of NCT. Our results suggest that NBIS is a modality of cellular senescence that may represent the nature of physiological aging in eukaryotes. Experimental & Molecular Medicine (2021) 53:1092–1108; https://doi.org/10.1038/s12276-021-00643-6 INTRODUCTION An increasing amount of evidence has indicated a novel Cellular senescence is characterized by the arrest of cell proliferation, senescence-associated feature, i.e., resistance to intrinsic and extrinsic which can be induced by various intrinsic and extrinsic stress factors; signals7–9. This resistance can be explained by a novel concept, i.e., according to these factors, cellular senescence can be categorized as dysfunctional transmission of signals between the cytoplasm and replicative senescence (RS), which is caused by long-term cell nucleus10–13. Under normal conditions, nucleocytoplasmic trafficking division; oncogene-induced senescence (OIS); DNA damage-induced (NCT) proteins14 select signals that can pass through the nuclear senescence (DDIS); and oxidative stress-induced senescence (OSIS). membrane through a process called selective permeability during All these types of senescence share common senescent hallmarks, signal transduction. For example, NCT is activated by the Ran protein such as permanent growth arrest, genomic instability, senescence- gradient across a nuclear pore13. Disruption of the Ran gradient is associated β-galactosidase (SA-β-gal) expression, the senescence- frequently observed in senescent cells, including DDIS models15,and associated secretory phenotype, and heterochromatin foci forma- in fibroblasts of patients with Hutchinson-Gilford progeria syn- tion1 in addition to common transcriptomic signatures of cellular drome16. Correspondingly, depletion of the Ran protein in mouse senescence2. However, several differential senescence-associated embryonic fibroblasts accelerates cellular senescence17.Agedyeast features unique to these individual modalities lead to the question cells also show decreased transport across the nuclear membrane18. of the nature of physiological aging. The shared and distinctive Moreover, the nuclear entry of Sp1, a key transcription factor that features of the individual cellular senescence models have been induces the expression of NCT-related genes, is decreased in extensively reviewed3–6. senescent cells19. Furthermore, secondary mitotic and apoptotic 1Korea Bioinformation Center, Korea Research Institute of Bioscience & Biotechnology, Daejeon 34141, Republic of Korea. 2Samsung Advanced Institute of Technology, Samsung Electronics Co., Ltd., Suwon 16677, Republic of Korea. 3Present address: Well Aging Research Center, Division of Biotechnology, DGIST, Daegu 42988, Republic of Korea. 4Department of Computer Science and Engineering, Kangwon National University, Chuncheon 24341, Republic of Korea. 5Department of New Biology, DGIST, Daegu 42988, Republic of Korea. 6Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul 02841, Republic of Korea. 7Division of Life Sciences, College of Life Sciences and Bioengineering, Incheon National University, Incheon 22012, Republic of Korea. 8Department of Brain & Cognitive Science, DGIST, Daegu 42988, Republic of Korea. 9Department of Biochemistry and Molecular Biology, Kangwon National University School of Medicine, Chuncheon 24341, Republic of Korea. 10Department of Biochemistry, Chonnam National University, Medical School, Gwangju 61469, Republic of Korea. 11Department of Biological Sciences, Seoul National University, Seoul 08826, Republic of Korea. 12The Future Life & Society Research Center, Advanced Institute of Aging Science, Chonnam National University, Gwangju 61469, Republic of Korea. 13Present address: UBLBio Corporation, Suwon 16679, ✉ Republic of Korea. 14These authors contributed equally: Ji-Hwan Park, Sung Jin Ryu, Byung Ju Kim. email: [email protected]; [email protected]; [email protected]; scpark@snu. ac.kr Received: 13 October 2020 Revised: 20 April 2021 Accepted: 10 May 2021 Published online: 29 June 2021 J.-H. Park et al. 1093 a Young Old LMB-treated d (n=3) (n=3) (n=3) Nucleus Cytoplasm Nucleus Cell cycle Cytoplasm DNA replication Cell cycle DNA repair Tryptic digestion chromosome organization Spliceosome Peptide samples RNA processing RNA processing & 10-plex TMT labeling RNA transport transport ribosome assembly TMT labeled peptides (2 sets) Ribosome biogenesis in eukaryotes Universal nuclear export Nucleocytoplasmic Young Old LMB-treated reference nuclear import trafficking 126 127N 127C 128N 128C 129N 129C 130N 130C 131 Nucleus integrin-mediated signaling pathway (set 1) endocytosis Endocytic & Cytoplasm lysosome network (set 2) Lysosome mitochondrial transport Mitochondrial organization & mitochondrion organization transport Fatty acid metabolism Glucose & fatty acid mid-pH reverse phase mRP metabolism (mRP) fractionation Glycolysis / Gluconeogenesis immune response-activating signal transduction 24 mRP peptide fractions innate immune response Immune response, Intracellular signaling cytokine-mediated signaling pathway Nucleus Cytoplasm small GTPase mediated LC-MS/MS signal transduction 1234567890();,: Easy-nLC & Q-Exactive Ras protein signal transduction 24 LC-MS/MS dataset 24 LC-MS/MS dataset 6 006 (Nucleus, Set 1) (Cytoplasm, Set 2) Z-score Up Down Z-score b e Nucleus Cytoplasm HDFs 16 16 p7 p23 p33 p42 p47 p53 (DT, day) 1 1.5 5 8 10 14 kDa - 70 12 Down Up 12 Down Up Imp α1 (389) (336) (213) (270) value) 8 8 - 100 P Imp β ( 10 -log 4 4 CRM1 - 100 - 50 0 0 RCC1 -6-4-20246-6 -4 -2 0 2 4 6 - 25 log2-fold-change log2-fold-change RanBP1 c Up Down pS10H3 - 15 Nucleus Cytoplasm Nucleus Cytoplasm (336) (270) (389) (213) p53 - 50 p16 - 15 311 25 245 375 14 199 Actin - 40 signals, such as signals transmitted through the p-ERK, p-P38, and p- functional concept formed on the basis of dysregulated NCT proteins Jun kinase signaling pathways, are trapped in the cytoplasm of in senescent cells. This barrier disrupts the selective permeability of senescent cells8,19–23. Considering the findings showing that NCT is signals passing through the nuclear membrane, which is actively disrupted during cellular senescence, we previously proposed a modulated by intact NCT proteins. This nuclear barrier hypothesis nuclear barrier hypothesis of aging12,24. The nuclear barrier is a was originally based on the expression of several molecular markers; Experimental & Molecular Medicine (2021) 53:1092 – 1108 J.-H. Park et al. 1094 Fig. 1 Proteomic analysis reveals the presence of a nuclear barrier in senescent cells. a Overall scheme of proteomic analysis of nuclear and cytoplasmic fractions of young, senescent (RS), and leptomycin B-treated (LMB-NBIS) HDFs. Three biological replicates (n = 3) were analyzed. Sets 1 and 2, nuclear and cytoplasmic fractions, respectively; tandem mass tag (TMT) 126, 127N, and 127C for young HDFs; TMT 128N, 128C, and 129N for RS; and TMT 129C, 130N, and 130C for LMB-NBIS. b Volcano plots showing upregulated (red) and downregulated (blue) proteins in the nucleus (left) and cytoplasm (right). c Relationships between upregulated (left) or downregulated (right) proteins in the nucleus and cytoplasm of RS. d Cellular processes (GOBPs) significantly (P < 0.1) enriched by upregulated (red) or downregulated (blue) genes in RS. The Z- score indicates –N-1(P), where P is the enrichment P value determined with DAVID software and N−1(·) is the inverse normal distribution. e Immunoblot of NCT-related proteins using whole-cell lysates of HDFs at different passages during senescence. Doubling times (DTs) corresponding to passage numbers are shown.