Oxidative stress impairs the Nur77‐Sirt1 axis resulting in a decline in organism homeostasis during aging

Abstract Sirt1 is an NAD+‐dependent deacetylase that protects against premature aging and cell senescence. Aging accompanied by oxidative stress leads to a decrease in Sirt1 levels and activity, but the regulatory mechanism that connects these events remains unclear. Here, we reported that Nur77, which shares similar biological pathways with Sirt1, was also decreased with age in multiple organs. Our in vivo and in vitro results revealed that Nur77 and Sirt1 decreased during aging and oxidative stress‐induced cell senescence. Deletion of Nr4a1 shortened the lifespan and accelerated the aging process in multiple mouse tissues. Overexpression of Nr4a1 protected the Sirt1 protein from proteasomal degradation through negative transcriptional regulation of the E3 ligase MDM2. Our results showed that Nur77 deficiency markedly aggravated aging‐related nephropathy and elucidated a key role for Nur77 in the stabilization of Sirt1 homeostasis during renal aging. We proposed a model wherein a reduction of Nur77 in response to oxidative stress promotes Sirt1 protein degradation through MDM2, which triggers cell senescence. This creates additional oxidative stress and provides positive feedback for premature aging by further decreasing Nur77 expression. Our findings reveal the mechanism by which oxidative stress reduces Sirt1 expression during aging and offers an attractive therapeutic strategy for targeting aging and homeostasis in organisms.


| INTRODUC TI ON
Aging is a biological phenomenon in which the structure and function of organisms decline with increasing age (Lopez-Otin et al., 2013).
The Sir2 protein and its homologs belong to the sirtuin family of protein deacetylases and are collectively known to extend the lifespan in various species (Imai & Guarente, 2014). Of all the sirtuins, Sirt1 is the most extensively studied member. Sirt1 deacetylates key histone residues of multiple protein targets, including p53, forkhead box O 1 and 3 (FoxO1/3), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), and nuclear factor kappa B (NF-κB) (Bi et al., 2019;Gomes et al., 2013;Mouchiroud et al., 2013;Wellman et al., 2017). By affecting transcriptional activation, Sirt1 is involved in the regulation of a broad range of vital aging-related biological pathways, including DNA repair and apoptosis, cell stress responses, and glucose and insulin homeostasis (Meng et al., 2020).
Furthermore, Sirt1 declines with age, which disrupts the homeostasis of multiple organs and accelerates the aging process (Donato et al., 2011). However, the mechanism of the reduction in Sirt1 during aging in mammalian systems has remained unclear.
Another factor that shows an intrinsic relationship with aging is the overproduction of reactive oxygen species (ROS) (Szilard, 1959).
During the aging process, multiple age-related organ dysfunctions are associated with ROS accumulation. High levels of ROS hamper the repair of damaged nuclear and mitochondrial DNA at multiple steps and contribute to genomic instability . There are mutual effects between oxidative stress and Sirt1 during the aging process. For example, moderate overexpression of Sirt1 protects against oxidative stress by inducing the major intracellular antioxidant catalase (Alcendor et al., 2007). Conversely, an increase in ROS can damage the protein expression and enzymatic activity of Sirt1 (Salminen et al., 2013). An increase in oxidative stress and a reduction in Sirt1 protein have been shown to occur in senescent cells, but the mechanisms that regulate the crosstalk between them during aging are still unclear.
The orphan nuclear hormone receptor Nur77 (also called TR3) has been implicated in similar biological pathways involved in aging.
Nur77 belongs to the NR4A subgroup of nuclear hormone receptors and has emerged as an important regulator of the inflammatory response, metabolic homeostasis, and oxidative stress .
In macrophages, Nur77 helps to repress the transcription of proinflammatory factors, leading to a systemic decrease in the inflammatory response in elderly mice (Koenis et al., 2018). The overexpression of Nur77 in melanoma cells prevents ROS accumulation by binding to mitochondrial trifunctional protein β subunit (TPβ) to avoid NADPH depletion and maintain GSH levels .
Exactly how Nur77 is connected with other pathways and molecular mechanisms involved in aging is not understood.
Here, we investigated the internal regulatory mechanism that links increasing ROS levels and decreasing Sirt1 protein levels during aging. We found that Nur77 declined during aging and in response to ROS stimulation, which led to the loss of Sirt1 via MDM2-mediated proteasomal degradation. These events increased p53 stability and activation, cell senescence, and ROS accumulation, which in turn further downregulated the expression of Nur77 and Sirt1 and accelerated the aging process.

| Nur77 deficiency accelerates the aging process in multiple organs
To determine the regulatory mechanism that affects multiple organs during aging, we examined differentially expressed genes in three different naturally aged tissues in NCBI GEO datasets. We detected 461 genes ( Figure S1a) that were mainly regulated by transcription factors such as chromodomain-helicase-DNA-binding protein 7 (Chd7), p53, and signal transducer and activator of transcription 3 (Stat3) Two genes were identified from the intersection of differentially expressed genes in the abovementioned aging-related signaling pathways: Igf1r and Nr4a1 ( Figure S1d). The relationship between insulin-like growth factor-1 (Igf)/Igf1r signaling and aging has been extensively studied (Kim & Lee, 2019;Lee & Kim, 2018;Narasimhan et al., 2009). Although the results of different studies have been controversial, inhibiting this signaling pathway has been shown to extend the lifespan of several species. By contrast, Nr4a1 encodes the Nur77 protein, and its association with longevity and aging is still unknown.
We verified Nur77 expression in the aforementioned tissues and several other tissues in naturally aged mice. Nur77 and the longevity protein Sirt1 were significantly decreased, while the aging-associated protein p53 was significantly increased in aged tissues, including liver, kidney, peri-adipose, lung, and brain tissue (Figure 1a, Figure S1e-g), suggesting a potential role for Nur77 in the aging process.
To investigate the effects of Nur77 on aging, we established a natural aging model in wild-type (WT) and Nr4a1 −/− mice ( Figure S2a).
The pathologic phenotypes of 15-month-old Nr4a1 −/− mice showed more obvious alopecia, intervertebral disk degeneration, and greater abdominal circumferences than WT mice of the same age, which may be due to the role of Nur77 in lipid-lowering ( Figure 1b, Figure S2b).
Nur77 deficiency caused a rough decline in the median lifespan compared with that of WT mice (Figure 1c). Impaired glucose tolerance and increased serum lipid levels, including triglycerides (TGs) and total cholesterol (T-CHO), were more evident in 15-month-old Nr4a1 −/− mice than in WT mice of the same age ( Figure S2c-f).

| Nur77 attenuates cell senescence by preventing overactivation of the DNA damage response
Oxidative stress is an upstream pathogenic event that leads to cell senescence and tissue aging. Since mice lacking Nur77 showed elevated ROS levels and DNA damage in vivo, we tested the effect of Nur77 on cell senescence via the DNA damage response. Nr4a1 We also performed relevant verifications in primary cells. We used extracted serum from old mice and young mice and adminis- When MEFs were treated with the antioxidants NAC and OS, no obvious alterations in p53, p21, p16, Bax, or Sirt1 were observed in the presence of serum from young mice (YS) (Figure 2g). These results showed that Nur77 attenuates cell senescence by preventing overactivation of the oxidative stress-induced DNA damage response.
As Nur77 belongs to the nuclear receptor family, we first tested whether Nur77 was involved in the transcriptional regulation of

| Nur77 enhances Sirt1 homeostasis via negative transcriptional regulation of MDM2
We then investigated the mechanism by which Nur77 enhances the homeostasis of the Sirt1 protein. Along with Sirt1, Nur77 belongs to the group of nuclear and cytoplasmic shuttling proteins. However, we found no evidence of an interaction between Nur77 and the Sirt1 protein, suggesting that Nur77 did not stabilize Sirt1 in a direct manner ( Figure S5). We further investigated whether Nur77 regulates the proteasomal degradation of Sirt1 through its E3 ligase. We reanalyzed the renal transcriptome data of Nr4a1 −/− rats in NCBI GEO datasets and found that the top pathways associated with differentially expressed gene enrichment included the proteasome pathway (GO: 0043161) and apoptosis pathway (GO: 0097190) ( Figure S6a).
The E3 ligase MDM2 was one of the differentially expressed genes in these two pathways ( Figure S6b). In addition, MDM2 ranked second among the major E3 ligases predicted by the UbiBrowser website for Sirt1 ( Figure S6c). Indeed, we found that Nur77 negatively regulated the expression of MDM2 at both the mRNA and protein levels (Figure 4a-c). We used the Jasper website to predict whether Nur77 could regulate MDM2 transcription, and the results showed that there were 16 Nur77-binding regions upstream of the MDM2 promoter (Table S1). Two highly rated binding sequences were selected and verified by luciferase reporter and chromatin immuno-

| Nur77 deficiency stabilizes p53 protein expression in response to oxidative stress
The MDM2 is a classical E3 ligase of p53. We further investigated whether Nur77 deficiency affected p53 expression in response to oxidative stress. Enhanced acetylation of p53 is widely believed to be closely related to its stability and activation in response to cellular stress (Barlev et al., 2001;Knights et al., 2006;Luo et al., 2000;Zhao et al., 2008). C-terminal acetylation-deficient p53-6KR knock-in mice have reduced p53-dependent gene expression after DNA damage (Feng et al., 2005). Moreover, DNA damage-induced phosphorylation of p53 by the kinases ATM/Chk2 at Ser20 disrupts the p53-MDM2 interaction, which stabilizes F I G U R E 3 Nur77 attenuates oxidative stress-induced cell senescence by enhancing the homeostasis of Sirt1. (a) The effects of Nur77 on p53, p21, and p16 in the presence or absence of Sirt1 in HEK-293T cells under H 2 O 2 stimulation. n = 3 independent experiments. (b) β-Galactosidase staining of Flag-Nr4a1 HEK-293T cells in the presence or absence of Sirt1 under H 2 O 2 stimulation. (c) The effects of Sirt1 overexpression on p53, p21, and p16 in shNr4a1 HEK-293T cells under H 2 O 2 stimulation. n = 3 independent experiments. (d) β-Galactosidase staining of shNr4a1 HEK-293T cells overexpressing Sirt1 under H 2 O 2 stimulation. (e) p53, p21, p16, and γh2AX expression levels in Sirt1 −/− HEK-293T cells with or without Nur77 overexpression and rescued with WT or 363HY Sirt1. n = 2 independent experiments. (f) Sirt1 mRNA expression in the presence or absence of Nur77. n = 6 independent experiments. (g) Sirt1 levels in the presence or absence of Nur77 and 50 μM Z-Leu-Leu-Leu-al (MG132) for 4 h or 20 μM chloroquine for 10 h. n = 3 independent experiments. (h) Sirt1 expression in the presence or absence of Nur77 and 100 μg/mL cycloheximide. n = 3 independent experiments. The data were analyzed by two-way ANOVA followed by a multiple comparisons test. The results are plotted as the mean ± standard deviation (n ≤ 6) or standard error (n > 6). **p ≤ 0.01. β-GAL, β-galactosidase; CHX, cycloheximide; CQ, chloroquine; γH2AX, phosphorylated histone 2AX; H 2 O 2 , hydrogen peroxide; MG132, carbobenzoxy-Leu-Leu-leucinal; mRNA, messenger ribonucleic acid; Nur77, nuclear hormone receptor 77; p53, tumor protein p53; Sirt1, sirtuin 1 protein; MG132, Z-Leu-Leu-Leu-al; WCL, whole cell lysate.  Figure S7). This finding suggested that the activation and stabilization of p53 in shNr4a1 HEK-293T cells were partially dependent on its phosphorylation at Ser20. We also examined whether the increase in p53 stability when Nr4a1 was knocked down was dependent on the ATM/Chk2 pathway. Nur77 deficiency increased p53-Chk2 binding, which was responsible for the phosphorylation of p53 at Ser20 (Figure 5e). Silencing Chk2 increased MDM2 binding to p53 in Nr4a1-knockdown HEK-293T cells (Figure 5f), which shortened the half-life of p53 (Figure 5g). These results showed that Nur77 deficiency could stimulate an increase in p53 acetylation at Lys382 and phosphorylation at Ser20 by downregulating Sirt1 and activating the DNA damage response, thus stabilizing p53 expression.
as determined by TdT-mediated dUTP nick end labeling (TUNEL) staining ( Figure S8g). Nur77 deficiency aggravated podocyte senescence and apoptosis in the kidney, and Nur77 expression in renal compartments exhibited dichotomous associations with aging nephropathy.

| Resveratrol rescues Nur77 deficiency-induced kidney damage in aged mice
To identify whether Nur77 deficiency-mediated Sirt1 degradation was the key process in aging nephropathy, we treated aged WT

| DISCUSS ION
The classic longevity protein Sirt1 gradually decreases during the natural aging process, but the underlying regulatory mechanism remains unclear. Our findings revealed that the loss of Nur77 with age augmented the DNA damage response and cell senescence and accelerated the aging process in several mouse tissues. We revealed that Nur77 stabilized the Sirt1 protein by reducing its degradation by the proteasome through negative transcriptional regulation of its E3 ligase MDM2. In addition, we confirmed the compelling role of Nur77 in protecting against aging nephropathy via Sirt1. However, these findings need to be validated in other aging diseases to clarify the general role of Nur77-stabilized Sirt1 against aging.
Nur77 is a nuclear receptor and a negative regulator of inflammatory factor production and fatty acid synthesis (Hedrick et al., 2019;Hu et al., 2017;Li et al., 2018;Yang et al., 2020). In addition, Nur77 has been reported to protect TPβ (a key rate-limiting enzyme for fatty acid oxidation) against oxidation, thereby maintaining normal fatty acid metabolic processes and preventing an increase in ROS . Although Nur77 can regulate a variety of agingrelated stimuli, the relationship between Nur77 and aging has not been revealed. We found that the Nur77 protein was reduced in multiple aged tissues and cells with oxidative stress-induced senescence. Nur77 deficiency resulted in ROS accumulation, an enhanced DNA damage response, and increased the senescence markers β-galactosidase, p53, p21, and p16 in multiple tissues. These phenotypes are consistent with the regulatory role of Nur77 in oxidative stress and lipid metabolism. Fifteen-month-old Nr4a1 −/− mice exhibited more obvious alopecia, intervertebral disk degeneration, and greater abdominal circumference than WT mice. Mice lacking Nr4a1 also had significantly shorter lifespans. Accordingly, Nur77 deficiency that occurs with age accelerates the aging process.
Sirt1 is involved in the regulation of various important senescence-related biological processes, including inhibiting inflammation, the DNA damage response, and cellular apoptosis (Bi et al., 2019;Gomes et al., 2013;Wellman et al., 2017). It is important to clarify the mechanism of the age-related decline in Sirt1 to delay the aging process. Recent studies have shown no obvious alterations in Sirt1 mRNA in DNA damage-induced senescence .
Nur77 and Sirt1 share many similarities in the regulation of agingrelated biological pathways. We found that Nur77 ameliorated the DNA damage response and cell senescence via the deacetylation function of Sirt1. Similar to previous results, our results showed that Nur77 did not affect Sirt1 mRNA expression under oxidative stress.
Our findings suggested that Nur77 indirectly protected the Sirt1 protein against proteasomal degradation by regulating its E3 ligase MDM2. Wu et al.  presented evidence that Nur77 inhibited MDM2 expression at the mRNA level. Our results further confirmed that Nur77 could bind directly to the promoter of MDM2 and suppress its transcription. Silencing MDM2 reversed the decrease in Sirt1 expression, the increase in the DNA damage response, and the cell senescence markers p21 and p16 caused by the decline in Nur77 under stress conditions. It has been suggested that Nur77 downregulates p53 transcriptional activity by blocking its acetylation . Based on our findings, we hypothesized that this might be related to Nur77-mediated stabilization of Sirt1 expression, which affects the transcriptional activity of p53.
It is well known that MDM2 is the primary E3 ligase of p53 (Haupt et al., 1997;Peng et al., 2015). In the present study, the activation of MDM2 by Nur77 deficiency selectively degraded Sirt1 but not p53 under oxidative stress. The enhancement of p53 acetylation and phosphorylation levels strongly correlates with protein stabilization and activation in response to cellular stress.
Mutations of lysine residues 370, 372, 373, 381, 382, and 386 to arginine residues in p53 (6KR p53 mutant) result in resistance to MDM2-induced degradation (Rodriguez et al., 2000). Among them, the acetylation of p53 at lysine 373/382 induces the expression of p21 (Zhao, Lu, et al., 2006). Sirt1-mediated deacetylation of p53 represses p53-mediated cell growth arrest and apoptosis in response to DNA damage and oxidative stress (Luo et al., 2001). Therefore, the reduction in Sirt1 caused by Nur77 deficiency with age led to the acetylation of p53 at Lys382, reduced the recognition of p53 by MDM2, and caused an increase in the cell-cycle arrest protein p21. The phosphorylation of p53 at serines 15, 20, and 37 impairs the binding of p53-MDM2 to inhibit p53-dependent transactivation (Shieh et al., 1997). Our results showed that Nur77 deficiencyinduced oxidative stress and the DNA damage response, which in turn stimulated p53 phosphorylation. This also explained why p53 was stable in Nr4a1-deficient tissues and cells in response to oxidative stress. These events constituted a positive feedback mechanism that accelerates the aging process: the oxidative stress-induced reduction in Nur77 disrupts the homeostasis of the Sirt1 protein through MDM2, resulting in cell senescence and reactive oxygen production, which in turn further decreases the expression of Nur77 and accelerates the aging process.
Sirt1 is widely expressed in tubular cells and podocytes in the kidney (Zhong et al., 2018). Podocytes maintain the glomerular filtration barrier, and initial glomerular injury affects podocytes, which are important target cells for the progression of aging nephropathy (Nagata, 2016). Podocyte-specific deletion of Sirt1 results in ROS accumulation, inflammatory response, and podocyte loss in the kidney (Chuang et al., 2017;Hong et al., 2018). We further examined the effects of Nur77 in renal aging, which may be representative of the general role of Nur77 in aging diseases. Our results revealed that the expression of Nur77 in the kidney decreased gradually during the natural aging process. Sirt1 was significantly reduced in the kidneys in the context of Nur77 deficiency, corresponding to the activation of senescence signals. Nur77 deficiency aggravated aging-related morphological changes and functional damage to the kidney. Activating Sirt1 or Nur77 helped improve Nur77 deficiency-aggravated aging nephropathy. Therefore, Nur77 may be a new therapeutic target to combat aging-related nephropathy.
Collectively, our study showed for the first time that Nur77 was an important target in combating the aging process. Nur77 is also an important upstream regulator that maintains Sirt1 protein homeostasis during the aging process. Preventing the reduction in Nur77 with age by pharmacological targeting during late adulthood may be a novel approach for the treatment of aging diseases.

| Mouse experiments
All animal experiments were approved by the Animal Ethics Committee of China Medical University (CMU2019277). Wildtype (WT) and Nr4a1-targeted mutant (No: 006187; Nr4a1 −/− ) mice with a C57BL/6J background were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). Nr4a1-targeted mutant mice were produced with a neomycin cassette introduced to exon 2 of murine Nur77 to block the transcription of both the DNA-binding domain (DBD) and ligand-binding domain (LBD) (Lee et al., 1995). All mice were housed in a temperature-and climate-controlled barrier system (23 ± 2°C and 45%-60% relative humidity, 12 h cycle of light and darkness) and fed regular rodent chow. Naturally aging mice were divided into four groups for analysis (5-, 8-, 15-, 18-month-old).

| Histological staining of tissues and analysis
photographed using an Olympus IX-71 fluorescence microscope and quantified using ImageJ.

| Transmission electron microscopy (TEM)
For transmission electron microscopy, sample handling and detection were performed by Wuhan Servicebio Technology. Tissues were collected and fixed with 2.5% glutaraldehyde at 4°C. The sections were washed with PBS and fixed in 1% osmium tetroxide at room temperature for 2 h. The specimens were then dehydrated using a series of ascending ethanol gradients and 100% acetone.
After being dehydrated, the sections were embedded in Pon 812 resin overnight at 37°C using acetone as a transitional solvent. The ultrathin sections were stained with 2% saturated uranyl acetate and lead citrate. Glomerular basement membrane (GBM) thickness, foot process width, and the number of foot processes per μm of GBM and TEM images were analyzed using ImageJ.

| RNA-sequencing analysis
The RNA-sequencing data were downloaded and referenced to the Gene Expression Omnibus under accession numbers GSE54714, GSE6591, GSE8150, and GSE25905. A log fold change (log 2 FC) > 1.5 and an adjusted p value < 0.05 were set as the thresholds for the identification of differentially expressed genes. Bioinformatic analyses using Metascape pathway analysis (Tripathi et al., 2015) and Ingenuity Pathways Analysis (Kramer et al., 2014) were carried out to determine molecular functions and upstream signaling pathways.

| Co-immunoprecipitation (Co-IP) and western blot analysis
Co-IP and western blot analysis were performed as previously described (Fu et al., 2020). The different primary antibodies used are listed in Table S2.

| Real-time polymerase chain reaction (PCR)
RNA was isolated from podocytes using an RNeasy Plus Mini Kit (Qiagen). Copy DNA was prepared using a PrimeScript™ RT reagent kit (TaKaRa) followed by quantitative real-time PCR using SYBR Green (TaKaRa). Relative quantitation was carried out using 2 −ΔΔCT .
The RT-PCR primers are listed in Table S3.
At 30 h post-transfection, the cells were harvested and subjected to co-immunoprecipitation (Co-IP) analysis.

| Flow cytometry
Apoptosis assessments were made based on allophycocyanin (APC) Annexin V and propidium iodide (PI) staining (Thermo Fisher). Cells were harvested followed by staining with APC Annexin V and PI according to the manufacturer's instructions.  Table S4.

| Chromatin immunoprecipitation (ChIP)
HEK-293T cells were crosslinked with 1% formaldehyde (final concentration) for 10 min by inverting the flasks at room temperature and quenched with 0.125 M glycine for 5 min. The cell pellets were washed repeatedly in PBS and then stored at −80°C. The pellets were lysed in a lysis buffer (50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 0.1% sodium deoxycholate, 1% Triton X-100, and complemented with a protease inhibitor cocktail) for 10 min. After centrifugation, the supernatant was discarded, and the pellet was lysed in a lysis buffer and subjected to sonication. The sheared chromatin was incubated with Nur77 primary antibodies or IgG bound to Pierce™ Protein A/G Agarose Beads (Thermo Fisher) overnight, followed by elution and reverse cross-linking at 65°C overnight. A TE buffer (10 mM Tris-HCl, 1 mM EDTA) was added to DNA elution buffer followed by RNase treatment (0.5 mg/mL) at 37°C for 30 min and proteinase K treatment (0.3 mg/mL) at 51°C for 1 h, and the DNA was subsequently isolated and purified. The RT-PCR primers are listed in Table S5.

| Statistical analysis
Data (n > 6) are expressed as mean ± standard error (SE), and additional data (n ≤ 6) are expressed as mean ± standard deviation (SD).
An unpaired two-tailed Student's t test and the Mann-Whitney test were used for comparisons between two groups. One-way ANOVA coupled with the Tukey's multiple comparison test or two-way ANOVA coupled with Sidak's multiple comparisons tests were used for comparisons of more than two groups. A value of p < 0.05 was considered significant. The number of replicates for each experiment is indicated in the figure legends. was responsible for the bioinformatic analysis. All authors discussed the results and reviewed the manuscript.

CO N FLI C T O F I NTER E S T S TATEM ENT
The authors have declared that no conflicts of interest exist.

DATA AVA I L A B I L I T Y S TAT E M E N T
The RNA-sequencing data were referenced in the Gene Expression Omnibus under accession numbers GSE54714, GSE6591, GSE8150, and GSE25905. All other data supporting the findings of this study are available from the corresponding author upon reasonable request.