DRG2 Accelerates Senescence via Negative Regulation of SIRT1 in Human Diploid Fibroblasts

Accumulating evidence suggests that developmentally regulated GTP-binding protein 2 (DRG2), an evolutionarily conserved GTP-binding protein, plays an important role in regulating cell growth, inflammation, and mitochondria dynamics. However, the effect of DRG2 in aging remains unclear. In this study, we found that endogenous DRG2 protein expression is upregulated in oxidative stress-induced premature senescence models and tissues of aged mice. Ectopic expression of DRG2 significantly promoted senescence-associated β-galactosidase (SA-β-gal) activity and inhibited cell growth, concomitant with increase in levels of acetyl (ac)-p53 (Lys382), ac-nuclear factor-kB (NF-κB) p65 (Lys310), p21Waf1/Cip1, and p16Ink4a and a decrease in cyclin D1. In this process, reactive oxygen species (ROS) and phosphorylation of H2A histone family member X (H2A.X), forming γ-H2A.X, were enhanced. Mechanistically, ectopic expression of DRG2 downregulated Sirtuin-1 (SIRT1), resulting in augmented acetylation of p53 and NF-κB p65. Additionally, DRG2 knockdown significantly abolished oxidative stress-induced premature senescence. Our results provide a possible molecular mechanism for investigation of cellular senescence and aging regulated by DRG2.


Introduction
Aging is one of the biggest risk factors for the development of various diseases, including dementia, chronic respiratory diseases, angiocardiopathy, infection, and cancer [1]. At cellular and molecular levels, senescent cells play important roles in tissue deterioration and disorganization and in organ dysfunction. Reduction of senescent cell levels is related to a significant decrease in the incidence of agingassociated ailments, such as cardiovascular diseases [2,3]. However, augmentation of senescent cells suppresses the development of cancer [4]. Cellular senescence was described first by Hayflick in the 1960s as an irreversible process of cell cycle arrest [5]. Cellular senescence exists spontaneously in vivo and in vitro and can be induced in vitro when cells are exposed to oxidative stress, such as hydrogen peroxide (H 2 O 2 ) [5][6][7]. Senescent cells show several dramatic changes, including upregulation of structural proteins that allow enlarged and flattened cell morphology, increased senescence-associated β-galactosidase (SA-β-gal) activity, and secretion of proinflammatory cytokines [1]. The senescence process is accompanied by abundant accumulation of reactive oxygen species (ROS), which can result in severe damage to DNA, protein, and lipids [8].
Sirtuin-1 (SIRT1) is a nicotinamide adenine dinucleotide-(NAD+-) dependent class III histone deacetylase that takes part in numerous vital signaling pathways, such as those of DNA damage, apoptosis, mitochondrial biogenesis, cellular senescence, and inflammation [9]. Dysregulation of SIRT1 activity participates in aging-related diseases, including Alzheimer's disease, cardiovascular disease, neurodegeneration, obesity, and metabolic disorders [10]. SIRT1 expression is downregulated in chronic inflammatory conditions and the aging process, both of which involve oxidative stress [11]. SIRT1 localizes in the nucleus and deacetylate histones and nonhistone proteins, such as p53 and p65 (also known as RelA), a subunit of nuclear factor-kB (NF-κB) [12][13][14][15]. SIRT1 deacetylates p53 and antagonizes p53-induced cellular senescence in response to DNA damage or oxidative stress, and SIRT1-deficient mouse embryonic fibroblasts (MEFs) exhibit hyperacetylation following DNA damage [13,16]. SIRT1 limits inflammation by deacetylating p53 and p65, whereas inhibition of SIRT1 downregulates deacetylation and promotes activation of p53 and p65, leading to increased proinflammatory gene expression [14]. These observations suggest modulation of SIRT1 expression level as a potential treatment for aging and aging-associated diseases.
The small GTPase superfamily, one of the GTP-binding protein superfamilies, regulates many processes in eukaryotic cells such as signal transduction, cell proliferation, cytoskeletal organization, and intracellular membrane trafficking [17]. Recently, researchers found a novel subfamily of the GTPase superfamily, developmentally regulated GTPbinding proteins (DRGs), which has two closely related proteins, DRG1 and DRG2 [18][19][20]. DRG2 has been reported to control cell growth and differentiation, regulate mitochondrial morphology, and modulate inflammatory response [21][22][23][24]. Thus, we hypothesized that DRG2 affects the modulation of cellular senescence and aged tissue.
Here, we report the effect of DRG2 expression induced by H 2 O 2 exposure or pcDNA-hDRG2 transfection in the WI-38 cell line. To elucidate the mechanism of DRG2 in cellular senescence, we explored the relationship between DRG2 and SIRT1. We further examined the effect of DRG2 knockdown on oxidative stress-induced premature senescence. Furthermore, we examined DRG2 expression in several organ tissues from C57BL/6 aged mice in vivo.

Western Blot Analyses and Immunoprecipitation (IP).
The cells were harvested and resuspended in cold RIPA buffer (50 mM Tris-HCl buffer, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 2 mM phenylmethanesulfonyl fluoride, 5 mM protease inhibitor cocktail, and 1 mM Na orthovanadate). Protein concentration was quantified using a Bio-Rad proteins assay kit. Western blot analysis was performed as previously described [26]. For IP analyses, after sonication, whole cell lysates were precleared by preclearing with recombinant protein G agarose (15920-010; Invitrogen) slurry. The protein samples were added to 5 μl SIRT1 antibody, 5 μl DRG2 antibody, and 5 μl normal IgG, respectively, and incubated overnight at 4°C using a rotator. Recombinant protein G (rProtein G) agarose (15920010; Invitrogen) was added to capture the immunocomplex for 4 h at 4°C with mixing. The immunocomplex was subjected to western blot analyses.

MTS Assay for Cell Proliferation.
Cell viability was determined using a commercially available kit named CellTiter 96 ® AQ ueous One Solution Cell Proliferation Assay kit (G3580; Promega). The assay was performed according to the manufacturer's instructions.
2.6. Plasmids, shRNA, and Transfections. The pcDNA6-V5/hDRG2 (human DRG2), pEGFP-N1/DRG2, and PLKO/ShDRG2 plasmids were obtained from the Department of Biological Sciences, Ulsan University (Republic of Korea). The pcDNATM4/his-Max A/hSIRT1 (human SIRT1) was obtained from the Department of Biological Sciences, Wonkwang University (Republic of Korea). These plasmid constructs have been described previously [24,27]. Cells were transfected using Lipofectamine 2000 (11668-019; Invitrogen) according to the manufacturer's protocol (Invitrogen, Carlsbad, CA, USA). After transfection, the medium was changed, and the cells were used in other experiments. 2.8. SIRT1 Deacetylase Activity Assay. SIRT1 deacetylase activity was measured using a commercial kit (CS1040; Sigma-Aldrich) following the manufacturer's protocol [28]. The fluorescence signal was measured at excitation/emission wavelengths of 355/460 nm using a SpectraMax M3 instrument. Finally, the SIRT1 deacetylase activity was calculated using a standard curve.

Reverse Transcription-Quantitative Polymerase Chain
Reaction (RT-qPCR). Total RNA was extracted by easy-BLUE™ kit, according to the manufacture's protocol. RNA concentration was read using a SpectraMax® ABS Microplate Reader (Molecular Devices, San Jose, CA, USA). According to the manufacture's protocol, cDNA was synthesized with total RNA using ReverTra Ace® qPCR RT kit (TOYOBO, FSQ-101). The cDNA was mixed with IL-6 primer (Hs00174131_m1; Applied Biosystems; Thermo Fisher Scientific, Inc.) and then performed using Applied Biosystems™ StepOne™ Real-Time PCR System (LS4376357, Thermo Fisher Scientific, Inc.). Cycling conditions were performed as follows: preparation at 50°C for 2 min, denaturation at 95°C for 10 min, followed by 40 cycles at 95°C for 10 sec, and at 60°C for 30 sec. The data were analyzed using StepOne™ software (version 2.3; Applied Biosystems; Thermo Fisher Scientific, Inc.).
2.11. Animals. C57BL/6 male mice were obtained from the Central Laboratory Animal Inc. (Seoul, Korea). The animals were kept in a 12 h light/12 h dark cycle at 23 ± 1°C for 8 weeks or 24 months with free access to food and water. All animal studies were performed according to protocols approved by the Animal Care Committee of Wonkwang University (WKU15-18).

Immunofluorescent Staining In Vivo.
Immunofluorescence assays for DRG2 were performed on paraffinembedded muscle, heart, and liver tissue sections. The sections were incubated in different concentrations of ethanol and washed in PBS. The sections were boiled in an antigen retrieval buffer (10 mM sodium citrate buffer, pH 6.0, 0.5 ml Tween 20) for 10 min and washed in PBS. The sections were incubated in 0.3% H 2 O 2 for 10 min at RT and blocked with 5% Normal Goat Serum for 1.5 h at RT. The sections then were incubated with primary antibodies against DRG2 (1 : 250 dilution in 5% Normal Goat Serum) overnight at 4°C, followed by the fluorescence-labeled secondary antibody Alexa Fluor 568 goat anti-rabbit (1 : 1,000). Nuclei were stained with DAPI (1 : 2,000) for 5 min at RT. The sections were mounted on glass slides and viewed on an Olympus FluoView 1000 confocal laser scanning system.

Statistical Analysis.
All results were expressed as the mean ± standard error of the mean (SEM). One-way analysis of variance (ANOVA) analysis (R software) was used for data comparisons among groups. Values of P < 0:05 were considered statistically significant. The experiments were repeated three times under the same conditions.

DRG2 Expression Is Upregulated in Oxidative Stress-Induced Premature Senescence in WI-38 Cells.
Hydrogen peroxide (H 2 O 2 ) is a well-known oxidative stress trigger for inducing cellular premature senescence [29,30]. To build an oxidative stress-induced senescence model, cells were treated with 200 μM H 2 O 2 and were examined at various time points. We found that cell proliferation was significantly inhibited after 72 h and 96 h of H 2 O 2 exposure (Figure 1(a)). The quantity of SA-β-gal stain-positive cells (blue) increased in a time-dependent manner with senescence-associated morphologic transformation to an enlarged and flattened shape (Figures 1(b) and 1(c)). The induction of SA-β-gal-positive cell staining reached a peak at 200 μM H 2 O 2 at 72 h. In addition, the phosphorylation level of H2A.X (γ-H2A.X), a DNA damage maker that usually accompanies cellular senescence [1], dramatically increased under fluorescence microscopy compared to that of the control group after H 2 O 2 exposure (Figures 1(d) and 1(e)). Moreover, our further observations indicated that acetylation of p53 (Lys382) and p21 Cip1/WAF1 , two hallmarks of cellular senescence, augmented in a time-dependent manner after H 2 O 2 exposure (Figure 1(f)). Parallelly, the protein level of DRG2 also increased gradually in WI-38 cells treated with 200 μM H 2 O 2 (Figure 1(f)). We speculated that DRG2 may correlate with H 2 O 2 -induced cellular premature senescence.

DRG2 Expression Accelerates Premature Senescence in WI-38 Cells.
To examine whether DRG2 overexpression promotes cellular premature senescence, different amounts of DRG2 expression plasmid (pcDNA/pcDNA-hDRG2) were transfected into WI-38 cells (Figure 2(a)). Ectopic  ). Previous studies have reported that DNA damage and ROS accumulation are main effectors for acceleration of cellular premature senescence [31]. Therefore, we investigated whether DRG2 expression enhances alterations of DNA damage and ROS levels. As shown in Figures 2(f) and 2(i), DRG2 upregulation promoted ROS production maximally up to 14.74% at 400 ng of pcDNA-hDRG2 transfection. Also, we found upregulation of γ-H2A.X (red fluorescence) by overexpression of DRG2 (green fluorescence) (Figures 2(g) and 2(j)).

DRG2 Aggravates Oxidative Stress-Induced Premature
Senescence by Suppressing SIRT1 Deacetylase Activity for p53. To clarify the molecular mechanism of how DRG2 drives cellular senescence, we focused on the dramatic increase in representative molecular markers (ac-p53) in cellular senescence. We considered whether DRG2 can control the function of SIRT1 because the SIRT1 plays a pivotal role in the regulation of cellular senescence by controlling p53 acetylation [15] and upregulation of SIRT1 or chemical activators for SIRT1 leads to the reduction of p53 acetylation [13,28,32]. To evaluate a role for DRG2-related signaling of SIRT1 in cellular senescence, we examined whether DRG2 can inhibit the deacetylase activity of SIRT1. Consistent with previous reports, augmentation of SIRT1 by pcDNA-hSIRT1 transfection abolished p53 acetylation mediated by H 2 O 2 ( Figure S1). Reversely, we transfected various amounts of the pcDNA-hDRG2 plasmid to examine the role of DRG2 on SIRT1 function. Our results showed that DRG2 overexpression reversed the effect of SIRT1 on p53 acetylation in a dose-dependent manner (Figures 3(a) and 3(b)). In our previous study, we found that 80 μM of 6,4 ′ -dihydroxy-7-methoxyflavanone (DMF) could effectively protect human diploid fibroblasts against H 2 O 2 -induced senescence by producing SIRT1 and inhibiting ac-p53 [32]. However, here, we found that DRG2 overexpression by pcDNA-hDRG2 plasmid reversed the effect of endogenous SIRT1 on p53 acetylation in a dose-dependent manner ( Figure S2A and S2B). To confirm the effect of DRG2 on SIRT1 function, we measured SIRT1 deacetylase activity after overexpression of DRG2. Our data revealed that SIRT1 deacetylase activity was enhanced in SIRT1 overexpressed or DMF treated WI-38 cells, but DRG2 overexpression inhibited the suppressive effect of SIRT1 on p53 acetylation activity (Figure 3(c) and S2C). Moreover, the effect of cellular senescence, which was estimated by senescent-specific morphological changes and SA-β-gal activity in the same setting, indicated that DRG2 overexpression neutralizes the protective role of SIRT1 against H 2 O 2 -induced cellular senescence (Figures 3(d) and 3(e), S2D and S2E). Next, we investigated the cellular localization of SIRT1 and DRG2 using confocal microscopy analysis. WI-38 cells were treated with or without H 2 O 2 as a control. In the control group, SIRT1 was uniformly located in the nucleus (green fluorescent, Figure 3  Expression of DRG2 (green) and p-H2A.X (red) was assessed by immunofluorescence staining, along with nuclear counterstaining using DAPI (blue). Scale bar, 10 μm. Results shown were quantitated using Image J software. Data are presented as the mean ± SEM value for each treatment. Similar results were obtained from three independent experiments. * P < 0:05 versus control. † P < 0:05 versus H 2 O 2 -treated cells. 6 Oxidative Medicine and Cellular Longevity

DRG2 Aggravates Oxidative Stress-Induced Premature
Senescence by Suppressing SIRT1 Deacetylase Activity for NF-κB p65. NF-κB plays an important role in the inflammatory response, including enhancing the transcription of proinflammatory cytokines, which are closely associated with age-related diseases [33]. As DRG2 accelerated cellular premature senescence, we examined the effect of DRG2 on NF-κB activation. DRG2 overexpression induced the expression of ac-NF-κB p65 (Lys310) in a dose-dependent manner (Figure 4(a)). The level of acetylated NF-κB p65 (Lys310) peaked at 14-fold that of the control at 400 ng of pcDNA-hDRG2 transfection (Figure 4(c)). Previous studies have shown that SIRT1 overexpression decreases acetylation of the RelA/p65 subunit of NF-κB, followed by suppression of
3.6. DRG2 In Aged Muscle, Heart, and Liver. To investigate the changes of DRG2 expression in young mice (8 weeks old) and aged mice (24 months old), we measured the expression level of DRG2 in the muscle, heart, and liver obtained from young and aged mice. Immunofluorescence (IF) staining for DRG2 was performed, and its relative expression was quantified (Figure 6(a)). DRG2 (red colour) was markedly increased in the aged mice. To confirm the above finding, the DRG2 protein expression was assessed by western blotting (Figure 6(b)). Consistently, DRG2 proteins increased in the aged mice tissues. These data indicate that the expression level of DRG2 in aged mice is much higher than that in young mice, and this in vivo observation strongly supports the proposed mechanism.

Discussion
In this study, we showed that DRG2 is overexpressed in an H 2 O 2 -induced cellular senescence model, and that it regulates SIRT1 activity in an antiparallel manner in the cellular senescence process. This regulation plays a crucial role in balancing the acetyl modifications of p53 and NF-κB p65 to switch on or off cellular senescence. Accordingly, the ectopic expression of DRG2 increases acetylation of p53 (Lys382) and NF-κB p65 (Lys310), resulting in failure to upregulate SIRT1 expression and activity, and abrogate the protective effect of SIRT1 against H 2 O 2 -induced senescence ( Figure 7). Moreover, DRG2 is upregulated in muscle, heart, and liver of aged mice in vivo.
Oxidative stress theory in aged was described first by Denhan Harman and is one of the most accepted hypotheses of molecular-level studies for aging [36][37][38]. Accumulation of chronic oxidative stress is produced by all cells of aerobic organisms owing to an imbalance between oxidant and antioxidant systems [39]. H 2 O 2 has been used extensively as an inducer of oxidative stress in in vitro models [29,30,32]. We investigated DRG2 protein expression during H 2 O 2induced senescence in WI-38. Our results showed that Expression of p-H2A.X (red) was assessed by immunofluorescence staining, along with nuclear counterstaining using DAPI (blue). Scale bar, 10 μm. Results shown were quantitated using Image J software. (g) Expression of SIRT1, ac-p53 (Lys382), p53, p21 WAF1/Cip1 , p16 Ink4α , cyclin D1, and β-actin was analyzed by western blotting. (h) SIRT1 was quantified by densitometry based on immunoblot images. β-Actin was used as a loading control. Data are presented as the mean ± SEM value for each treatment. Similar results were obtained from three independent experiments. * P < 0:05 versus control. † P < 0:05 versus H 2 O 2 -treated cells. 12 Oxidative Medicine and Cellular Longevity DRG2 was upregulated after H 2 O 2 exposure. Moreover, DRG2 was upregulated consistently in aged tissue from naturally aged mice. Senescent cells exhibit an enlarged and flattened morphology, SA-β-gal activity, cell proliferation inhibition, ROS production, and alterations in expression of certain genes [1]. Therefore, we explored the role of DRG2 in the cellular senescence process. DRG2 overexpression by pcDNA-hDRG2 plasmid transfection strongly triggered inhibition of cell growth via upregulation of p53, p21 WAF1/Cip1 , and p16 Ink4α and downregulation of cyclin D1 with increasing SA-β-gal-positive signals, ROS, and γ-H2A.X in WI-38 cells. Although a previous study concluded that DRG2 knockdown substantially reduces growth speed but upregulates p21   Figure 6: Immunofluorescence staining of DRG2 in aged mice. C57BL/6 male mice were sacrificed at 8 weeks old (Y 1/2 : young 1/2) or 24 months old (O 1/2 : old 1/2), and the muscle, heart, and liver were collected. (a) Expression of DRG2 (red) in sections was assessed by immunofluorescence staining, along with nuclear counterstaining using DAPI (blue). Results shown were quantitated using Image J software. (b) Expression of DRG2 and β-actin was analyzed by western blotting. The proteins were quantified by densitometry based on immunoblot images. β-Actin was used as a loading control. Data are presented as the mean ± SEM value for three mice in each group. Similar results were obtained from three independent experiments. * P < 0:05 versus control. Scale bar, 20 μm. 13 Oxidative Medicine and Cellular Longevity protein level in HeLa cells [40], the role of DRG2 should be reconsidered because it can be induced in an H 2 O 2 -induced senescence model in WI-38 cells. Consistent with these reports, DRG2 overexpression suppresses cell growth in human T cells and reduces sensitivity to nocodazolestimulated apoptosis [21,22]. SIRT1 is a longevity-related gene that plays an important role in the regulation of inflammation and cellular senescence [11]. Previous studies have shown that SIRTdeficient cells exhibit hyperacetylation of p53 after DNA damage, but SIRT1-overexpressed cells sufficiently deacetylate p53 and block PML/p53-induced senescence [13,16]. Han et al. reported that peroxisome proliferator-activated receptor-γ (PPARγ), a ligand-regulated modular nuclear receptor, directly interacts with SIRT1 and inhibits SIRT1 activity in cellular senescence [41]. Ko et al. reported that DRG2 interacts with PPARγ in antigen presenting cells, and this process enhances PPARγ activity [24]. Thus, we chose to consider the role of DRG2 on SIRT1 in the senes-cence process. We found that DRG2 expression by pcDNA-hDRG2 decreases SIRT1 protein level and deacetylase activity and increases acetylation of p53. We also observed that DRG2 and SIRT1 colocalize in the nucleus, although we could not show a direct interaction between them by biochemical experiments. SIRT1 reduction leads to acetylation of NF-κB p65 and forked box O (FOXO3), as well as modification of histones H3 and H4, resulting in the expression of pro-inflammatory, antioxidant, prosenescent, and proapoptotic genes that are involved in inflammation, oxidative stress, and premature cellular senescence [11,42]. Our data indicate that DRG2 expression induces NF-κB p65 acetylation by suppression of SIRT1 expression and activity.
Interestingly, we also found that DRG2 knockdown strongly reduced senescence markers that respond to oxidative stress including p53, p21 WAF1/Cip1 , p16 Ink4α , γ-H2A.X, and SA-β-gal activity under the condition induced cellular senescence by H 2 O 2 . Consistent with this, our observation

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Oxidative Medicine and Cellular Longevity shows that DRG2 level increased in naturally aged mouse tissues as well as the cells induced cellular senescence by H 2 O 2 . In this study, we propose that elevated level of DRG2 could induce premature aging and aging-related diseases.

Conclusion
In summary, this study demonstrated a previously unknown role for DRG2 in cellular senescence. DRG2 overexpression promoted premature senescence in normal cells and downregulated SIRT1 expression (Figure 7). In addition, we showed that downregulated DRG2 expression abolished oxidative stress-induced senescence. Consider that aging is a vital risk factor for aging-related diseases, and our study provides a possible new therapeutic strategy.

Data Availability
The data used to support the finding of this study are available from the corresponding author upon request.

Conflicts of Interest
The authors declare no conflict of interest.