Human WRN is an intrinsic inhibitor of progerin, abnormal splicing product of lamin A

Werner syndrome (WRN) is a rare progressive genetic disorder, caused by functional defects in WRN protein and RecQ4L DNA helicase. Acceleration of the aging process is initiated at puberty and the expected life span is approximately the late 50 s. However, a Wrn-deficient mouse model does not show premature aging phenotypes or a short life span, implying that aging processes differ greatly between humans and mice. Gene expression analysis of WRN cells reveals very similar results to gene expression analysis of Hutchinson Gilford progeria syndrome (HGPS) cells, suggesting that these human progeroid syndromes share a common pathological mechanism. Here we show that WRN cells also express progerin, an abnormal variant of the lamin A protein. In addition, we reveal that duplicated sequences of human WRN (hWRN) from exon 9 to exon 10, which differ from the sequence of mouse WRN (mWRN), are a natural inhibitor of progerin. Overexpression of hWRN reduced progerin expression and aging features in HGPS cells. Furthermore, the elimination of progerin by siRNA or a progerin-inhibitor (SLC-D011 also called progerinin) can ameliorate senescence phenotypes in WRN fibroblasts and cardiomyocytes, derived from WRN-iPSCs. These results suggest that progerin, which easily accumulates under WRN-deficient conditions, can lead to premature aging in WRN and that this effect can be prevented by SLC-D011.


WRN shows a similar gene expression profile to HGPS.
To investigate our hypothesis, we first performed microarray to examine the gene expression profile of fibroblasts from HGPS subjects, WRN subjects, and an unaffected old-aged subject (N81; 81 years old) compared to fibroblasts from an unaffected young-aged subject (N9; 9 years old). We found that a large portion of gene sets were commonly upregulated (blue boxes;  S1B). This result is consistent with our and others' previous reports that senescence blocks cell cycle progression and induces cell adhesion [32][33][34][35] . We confirmed the alteration of several genes including LMNA, IL-8, CENP-E, and Rad51 by RT-PCR (Fig. S1C). This result is consistent with our and other previous reports that fibroblasts derived from prematurely aged and naturally aged subjects exhibit cellular senescence such as blocked cell cycle progression and induced cell adhesion 32 . Six specific gene sets were altered only in HGPS (Fig. S2A), 24 specific elements only in WRN cells (Fig. S2B), and 51 elements only in N81 fibroblasts (Fig. S2C) compared to N9 fibroblasts. We also analyzed the commonly regulated gene sets in WRN and HGPS cells. Compared to N9 fibroblasts, 881 genes were altered in both HGPS and WRN cells ( Fig. 1D and Fig. S2D and S2E) and their functions were categorized into nine elements (Fig. 1E), which were mainly involved in cell cycle progression. We also confirmed by western blot assay that common alterations occurred in HGPS and WRN cells (Fig. 1F). Fluorescence staining of Ki67 showed that the proliferation of HGPS and WRN cells was reduced ( Fig. S3A and S3B). Conversely, fluorescence staining of paxillin and phalloidin showed that cell adhesion and cell size were increased in both HGPS and WRN cells compared to N9 fibroblasts ( Fig. S3C and S3D). Furthermore, we examined the basal level of γ-H2A.X foci in N9, HGPS, and WRN fibroblasts by immunofluorescence assay. HGPS and WRN fibroblasts showed a slightly higher level of γ-H2A.X expression than N9 fibroblasts (Fig. S3E). Senescence-associated β-galactosidase (SA-β-gal) activity was increased in HGPS and WRN cells (Fig. S3F). We also counted each cell line for 5 days to compare the rate of cell propagation and the doubling time of the population. The cell propagation of N9 fibroblasts was much higher than that of HGPS and WRN cells. The density of N9 fibroblasts doubled within 24 h, while HGPS and WRN cells took more than 3 days to double. In particular, WRN cells took a longer time than HGPS cells to double (Fig. S3G). These results indicate that HGPS and WRN cells (and to a lesser extent N81 cells) have very similar cellular senescence phenotypes.
Fibroblasts derived from patients with WRN express progerin. Since normal cells of healthy aged people can produce progerin 36 , we examined the expression of progerin in WRN cells. RT-PCR analysis showed a small amount of progerin in WRN cells and N81 fibroblasts ( Fig. 2A and Fig. S1C). To determine whether the lower band was progerin, we performed cloning and sequencing of this product and confirmed a perfect  www.nature.com/scientificreports/ match with progerin ( Fig. S4A). However, these WRN cells did not possess genetic mutations in the genomic DNA ( Fig. S4B and S4C). These results indicate that progerin can be expressed by an alternative splicing process without a HGPS-related mutation in WRN cells. Additionally, we detected progerin expression by IF staining ( Fig. 2B) and found that, its level was much lower in WRN cells than in HGPS cells (Fig. 2C). To examine whether the expression of progerin is related to the senescence phenotype of WRN, we eliminated progerin using siRNA and measured the expression of H3K9me3, which is decreased in senescent cells [37][38][39][40][41][42] . Elimination of progerin obviously induced H3K9me3 expression in WRN cells but did not affect the expression of lamin A (Fig. 2D,E, Fig. S5A and S5C). To confirm that progerin is one of the triggers inducing premature aging in WRN, we crossbred heterozygous Lmna G609G mice with Wrn-deficient mouse to generate a mouse model expressing progerin in Wrn-deficient background (Fig. S5D). However, unlike humans, Wrn-deficiency alone does not cause premature aging phenotypes in mice 18,21 and progerin is not naturally expressed in the mouse model 43,44 . Interestingly, heterozygous Lmna G609G mice did not show any differences in pathophysiological phenotypes including body weight and lifespan with or without the mouse Wrn gene (Fig. 2F,G). To explore why the Wrn-deficient mouse model does not exhibit premature aging phenotypes, we analyzed and compared the WRN sequences of humans and mice. We found that human WRN possesses repeated regions (Fig. 2H), due to exon duplication (in fact, exon 9 and exon 10 share the same DNA sequence encoding the same amino acid sequence in humans; Fig. S6A and S6B). In contrast, mice do not have consistent duplicated sequences like those in humans. The exon 9 sequence of mouse WRN is not duplicated ( Fig. 2F and Fig. S6A). We hypothesized that the duplicated sequence of human WRN has an important role in the aging process. Thus we generated recombinant proteins consisting of unduplicated sequences (WRN-R1) or duplicated sequences (WRN-R2; Fig. S6C). WRN-R1 is a peptide of the exon 9 sequence of human WRN that mimics the unduplicated exon 9 sequence of mouse WRN. WRN-R2 is a peptide that replaces the duplicated exon 9 sequence of human WRN.

Human WRN rescues the senescence phenotypes in WRN cells.
To investigate whether the duplicated region of human WRN affects premature aging, we first delivered the recombinant proteins into WRN cells for 24 h and observed the changing phenotypes. WRN-R2 induced the expression of H3K9me3 and reduced the expression of p16INK4A in WRN cells (Fig. 3A,B, Fig. S7A to S7C). The expression of Ki67 was increased in WRN-R2-positive WRN cells ( Fig. S7D and S7E). Although WRN-R1 also partially reduced the expression of p16INK4A (Fig. S7B), it did not differ significantly from the control group in general (Fig. 3A,B, Fig. S7A, S7D, and S7E). Next, we checked the rescue effect of human WRN on premature aging by using DNA vectors. We transferred DNA vectors expressing human WRN (hWRN) and mouse WRN (mWRN) in WRN cells. The overexpression of hWRN, but not mWRN, increased KI67 and H3K9me3 expression ( Fig. 3C and Fig. S8A to S8E). Conversely, the average number of 53BP1 foci and the levels of γ-H2A.X foci were decreased after transfection with the human WRN expression vector ( Fig. S8F to S8J). These results strongly suggest that only hWRN is involved in the aging process and explain why Wrn-deficient mice do not show aging features.

Human WRN reduces progerin expression and rescues the senescence phenotypes in HGPS cells.
To investigate the effect of hWRN on progerin expression and premature aging phenotypes, we also conducted experiments in HGPS cells. As seen in WRN cells, WRN-R2 also increased the expression of H3K9me3 and Ki67 in HGPS cells (Fig. 3D,E and Fig. S9A to S9C). However, WRN-R1 had only a slight effect on the expression of Ki67 (Fig. S9C). These results confirmed again that the repeated region of hWRN might be related to the inhibition of aging. Therefore, we next tested the effect of hWRN on HGPS cells by using hWRN and mWRN expression vectors. Overexpression of hWRN, but not mWRN, induced H3K9me3 and Ki67 expression in HGPS cells (Fig. 3F,G and Fig. S9D and S9E). Moreover, hWRN reduced the expression of progerin and p16INK4A and increased the levels of Rad51 and cyclin B1 in HGPS cells (Fig. 3G). The basal levels of DNA damage-related genes, including 53BP1 and γ-H2A.X were reduced in hWRN-positive cells ( Fig. S9F to S9J). We   www.nature.com/scientificreports/ also observed that SA-β-gal activity was reduced 48 h after transferring the hWRN expression vector (Fig. S9K). These results suggest that hWRN, especially its duplicated region, is closely related to progerin-induced senescence. To address this, we first tested the interaction between WRN and wild-type (WT) lamin A or progerin. By an immunoprecipitation (IP) assay using an anti-WRN antibody, we observed the interaction of WRN with progerin as well as WT-lamin A. However, the interaction between WRN and progerin seemed to be stronger than the interaction between WRN and WT-lamin A (Fig. S10A). To confirm their interaction, we performed a GST-pulldown assay using recombinant progerin proteins and found that hWRN interacted strongly with progerin ( Fig. 4A). WRN-R2 showed a much stronger binding affinity than WRN-R1 with progerin ( Fig. 4B). Moreover, WRN-R2 blocked the interaction between lamin A and progerin (Fig. 4C). Considering our previous finding that progerin induces premature aging by abnormal binding with lamin A 32 , this result suggests that hWRN is a natural inhibitor of progerin and a protector against progerin-induced senescence by inhibiting progerin from binding to wild-type lamin A.

An inhibitor of progerin (SLC-D011) can ameliorate the senescence phenotypes in WRN cells.
We predicted that if premature aging syndrome caused by hWRN deficiency is affected by progerin, then a progerin inhibitor could have a favorable effect on WRN cells. To test this hypothesis, we treated WRN fibroblasts with a progerin inhibitor, SLC-D011 (D011 also called progerinin; an optimized chemical version of JH4 32 ). WRN cells showed abnormal nuclear morphology (deformed nuclei; Fig. 4D and Fig. S10B). Treatment with D011 normalized the nuclear morphology of WRN cells ( Fig. 4D and Fig. S10B), promoted cell proliferation (Fig. 4E), and reduced SA-β-gal activity (Fig. S10C). The expression of H3K9me3 was induced by D011 treatment (Fig. 4F,G and Fig. S10D). We also observed increased of CENP1 and Rad51 expression and decreased IL-8 expression after treatment with D011 at the transcriptional level ( Fig. 4G and Fig. S10E). Next, we performed an IP assay to directly measure progerin expression in WRN cells. Although it was extremely difficult to detect progerin expression in WRN cells at the protein level, we observed that treatment with D011 could suppress progerin expression in WRN cells, similar to HGPS cells ( Fig. 4H and Fig. S11A). We also confirmed the reduction in progerin expression and consistent induction of H3K9me3 expression after treatment with D011 by IF assay (Fig. S11B to S11D). In fact, cardiovascular disease is one of the symptoms and main causes of death in WRN. Therefore, we tried to explore the anti-senescence effect of D011 in WRN cardiomyocytes. We generated iPSCs from WRN fibroblasts and differentiated them into cardiac muscle cells for 29 days (Fig. S12A to S12D). The expression of H3K9me3 and cyclin B1 in WRN iPSC-derived cardiac muscle cells was induced after treatment with D011 ( Fig. 4I and Fig. S12E to S12G). These results strongly suggest that the progerin inhibitor D011 has potential as a treatment for patients with WRN.

Discussion
In this study, we found that progerin is expressed in fibroblasts derived from patients with Werner syndrome (WRN) without genetic mutation as much as in fibroblasts derived from a person with healthy normal aging (N81) and suggested that progerin could be an influential factor to induce premature senescence in WRN patients. We confirmed that WRN cells have a very similar gene expression profile to HGPS cells and a partially similar profile to fibroblasts from an unaffected aged person (N81, Fig. 1A,B,D). Genes related to the cell cycle, DNA replication, DNA repair, and histone segregation were commonly downregulated in HGPS, WRN, and N81 fibroblasts compared to fibroblasts derived from a normal young person (N9, Fig. 1C), while cell adhesionrelated genes were upregulated in senescence models (Fig. S1B). Based on gene ontology, we tested senescencerelated markers by western blotting, RT-PCR, and IF assays ( Fig. 1F and Fig. S3). The cell proliferation (KI67) and doubling time (cell counts for 5 days) of HGPS and WRN cells were reduced compared with those of N9 fibroblasts. The expression of cell cycle-related genes (cyclin B1 and cdc25c), DNA repair-related genes (BRCA1 and Rad51), and H3K9me3 was also reduced in both HGPS and WRN cells. H3K9me3 is necessary for proper chromosome segregation 45,46 , and the expression of H3K9me3 is well known as a proliferation marker in HGPS studies [37][38][39][40][41][42] . A WRN stem cell model also showed a reduction in H3K9me3 under WRN-deficient conditions 38 . It seems that the reduction in H3K9me3 in HGPS and WRN cells is related to the downregulation of chromosome segregation in gene ontology analysis (Fig. 1C). Conversely, the cell size and cell adhesion were increased. In addition, the basal expression of γ-H2A.X and p16INK4A was also increased in HGPS and WRN cells compared  www.nature.com/scientificreports/ to N9 fibroblasts. Overall, as seen in our previous study 32 , the characteristics of the cellular senescence phenotype commonly appear in HGPS and WRN cells. Additionally, we observed that a small amount of progerin was expressed in WRN cells and N81 fibroblasts. Normal healthy people can produce progerin and the amount of progerin increases with age 29,30 . We observed that the amount of progerin in WRN cells was approximately the same as in N81 fibroblasts, even though the subjects who provided the WRN cells were in their 20 s and 30 s. We thought that the accumulation of progerin would be faster in WRN-deficient conditions than normal aging conditions. However, when we crossbred transgenic Lmna G609G progeroid mice with Wrn-deficient mice to generate a mouse model expressing progerin under Wrn-deficient conditions, the pathological phenotypes and lifespan of Lmna G609G progeroid mice did not differ depending on the presence or absence of the Wrn gene. In fact, Wrn-deficient mice have almost same physiological phenotypes and lifespan as wild-type mice 21 . We concluded that the function of mWRN is not related to progerin expression since the mouse does not naturally produce progerin 43,44 . Therefore, we expected sequence differences between hWRN and mWRN. To test this hypothesis, we compared the sequences of WRN genes in humans and mice. In addition to other minor differences, the most distinct difference was hWRN possesses repeated regions (exon 9 and exon 10 encode the same amino acid sequence) but mWRN does not. We hypothesized that duplication in hWRN has a role in the senescence process. To confirm our hypothesis, we generated a recombinant protein consisting of a duplicated sequence (WRN-R2) and also generated an unduplicated sequence (WRN-R1) as a negative control. However, the delivery of recombinant protein into primary fibroblasts could not last more than 24 h because cells easily became contaminated or deteriorated after transfection. Nevertheless, the duplicated peptide, WRN-R2, rescued several cellular senescence phenotypes in WRN cells. This duplicated region was also effective in HGPS cells. However, the unduplicated peptide WRN-R1 had little or no effect on progeroid cells. We also observed that senescence phenotypes were ameliorated after transfection with vectors expressing full-length hWRN and mWRN for 48 h.
Overexpression of hWRN induced cell proliferation and H3K9me3 expression but reduced DNA damage in both WRN and HGPS cells. In particular, hWRN, but not mWRN, reduced progerin expression and SA-β-Gal activity in HGPS cells. We concluded that hWRN, especially its duplicated regions, are closely related to progerin-induced senescence and examined the interaction of hWRN and progerin. We found that hWRN bound strongly to progerin and inhibited progerin from binding to wild-type lamin A. It seems that the duplicated region of hWRN is important for this binding and thus affects the aging process. Based on the preceding results, we proposed that hWRN might be a natural inhibitor of progerin, and for patients with WRN, premature aging would be caused by progerin accumulation. Therefore, we thought that inhibiting progerin in WRN cells could suppress aging phenotypes, and confirmed this effect by treatment siRNA and with a progerin-inhibitor (SLC-D011). SLC-D011,  www.nature.com/scientificreports/ also called progerinin (a modified chemical version of JH4 32 ), is an inhibitor of the interaction between progerin and lamin A. In our previous study, we discovered that progerin strongly binds to lamin A leading to nuclear deformation and aging processes in HGPS 47 . We observed that progerin was stabilized by interaction with lamin A and developed the binding inhibitor SLC-D011, which inhibited progerin from binding to wild-type lamin A and suppressed the expression of progerin in HGPS. Although WRN protein is also expressed in HGPS, it is thought that the amount of WRN protein is insufficient to prevent progerin accumulation because the level of progerin in HGPS is very high. However, SLC-D011 can substantially decrease progerin levels in HGPS and improve premature aging phenotypes. Likewise, this binding inhibitor of progerin ameliorated the aging features of WRN cells in this study. Furthermore, the main cause of death in WRN is cardiovascular disease or cancer 2,48 . Therefore, we treated cardiomyocytes derived from WRN iPSCs with SLC-D011. The expression of H3K9me3 and cyclin B was also induced in cardiac muscle cells after treatment with SLC-D011.
In this study, we suggest that progerin can be involved in causing premature aging in WRN and that human WRN protein is a natural inhibitor of progerin (Fig. 5). In addition, we revealed that the repeated regions of hWRN are important in this aging process. In contrast, it is assumed that mWRN is not related to this aging process because progerin is not naturally expressed in mice, and there is no duplication in mWRN. Therefore, the Wrn-deficient mouse model does not show human-like premature aging phenotypes, and the phenotypes of the Lmna G609G progeroid mouse model are not affected by the presence or absence of mWRN. Based on these results, we are not sure but expect that expressing the duplicated peptide of WRN in Lmna G609G progeroid mice will affect the premature senescence phenotypes of the mouse model. Therefore, we are currently in the process of generating the corresponding mouse model, which will be discussed in our next paper. Moreover, SLD-D011 (progerinin) has favorable effects in WRN cells, so this compound could be a plausible drug candidate for patients with WRN in the future.

Materials and methods
Animal experiments. All methods performed in this study were approved by the Institutional Review    Immunoblotting. Immunoblotting assays were designed under protocols approved by the PNU IRB in accordance with relevant guidelines. Radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1% NP-40, 0.1% SDS, and 10% sodium deoxycholate) was used for protein extraction from cells. After heat-inactivation in sample buffer, proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. Blotted membranes were blocked with 3% skim milk for 1 h and incubated overnight with specific primary antibodies, followed by incubation with horseradish peroxidase-conjugated goat anti-mouse, goat anti-rabbit, or mouse anti-goat IgG secondary antibodies (Pierce, Thermo Fisher Scientific, Inc., Rockford, IL, USA). Signals were