Oxidative stress impairs the Nur77-Sirt1 axis resulting in a decline in organism homeostasis during aging - PubMed (original) (raw)

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

Yang Yu et al. Aging Cell. 2023 May.

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.

Keywords: Nur77; Sirt1; aging; kidney injury; oxidative stress.

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Conflict of interest statement

The authors have declared that no conflicts of interest exist.

Figures

FIGURE 1

Nur77 deficiency accelerates the aging process in multiple organs. (a) Expression of Nur77, Sirt1, and p53 in the liver, kidney, and peri‐adipose tissue of aged mice. (b) Morphology of 5‐ and 15‐month‐old WT and Nr4a1 −/− mice. (c) Survival curves for WT (n = 30) and Nr4a1 −/− mice (n = 30). (d) 53BP1 staining and β‐galactosidase staining in the liver, kidney, and peri‐adipose tissue of 5‐ and 15‐month‐old WT and Nr4a1 −/− mice. Scale bar: 100 μm. The data were analyzed by two‐way ANOVA followed by a multiple comparisons test. The results are plotted as the mean ± standard error. **p ≤ 0.01. ATM, ataxia‐telangiectasia mutated protein; Bax, B‐cell lymphoma 2‐associated X‐protein; Chk2, checkpoint kinase 2; β‐GAL, β‐galactosidase; DCFH‐DA, 2′,7′‐dichlorofluorescein diacetate; DNA, deoxyribonucleic acid; γH2AX, phosphorylated histone 2AX; H2O2, hydrogen peroxide; MEFs, mouse embryonic fibroblasts; NAC, _N_‐acetyl‐

‐cysteine; Nur77, nuclear hormone receptor 77; OS, serum extracted from aged mice; p53, tumor protein p53; Sirt1, sirtuin 1 protein; YS, serum extracted from young mice; WT, wild‐type.

FIGURE 2

Nur77 attenuates cellular senescence by preventing the overactivation of the DNA damage response. (a) The expression of DNA damage response pathway factors (γH2AX, ATM, Chk2, p53, p21, p16) in shScramble and sh_Nr4a1_ HEK‐293T cells under H2O2 stimulation. n = 3 independent experiments. (b) β‐Galactosidase staining of shScramble and sh_Nr4a1_ HEK‐293T cells under H2O2 stimulation. (c) The expression of Sirt1, p53, and Bax in shScramble and sh_Nr4a1_ HEK‐293T cells under H2O2 stimulation. n = 3 independent experiments. (d) The expression of DNA damage response pathway factors (γH2AX, ATM, Chk2, p53, p21, p16) in Scramble and Flag‐Nr4a1 HEK‐293T cells under H2O2 stimulation. n = 3 independent experiments. (e) β‐Galactosidase staining of Scramble and Flag‐Nr4a1 HEK‐293T cells under H2O2 stimulation. (f) The expression of Sirt1, p53, and Bax in Scramble and Flag‐Nr4a1 HEK‐293T cells under H2O2 stimulation. n = 3 independent experiments. (g) The expression of Sirt1, p53, Bax, p21, and p16 in WT and Nr4a1 −/− mouse embryonic fibroblasts (MEFs) treated with serum from aged mice (OS) or young mice (YS) and _N_‐acetyl‐

‐cysteine (NAC). n = 2 independent experiments. (h) Reactive oxygen species (ROS) levels in WT and Nr4a1 −/− MEFs treated with serum from OS or YS and analyzed by flow cytometry. n = 3 independent experiments. (i) The apoptosis rates of WT and Nr4a1 −/− MEFs treated with serum from OS or YS and analyzed by flow cytometry. 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; DCFH‐DA, 2′,7′‐dichlorofluorescin diacetate; NAC, _N_‐acetyl‐

‐cysteine; OS, serum extracted from aged mice; YS, serum extracted from young mice.

FIGURE 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 H2O2 stimulation. n = 3 independent experiments. (b) β‐Galactosidase staining of Flag‐Nr4a1 HEK‐293T cells in the presence or absence of Sirt1 under H2O2 stimulation. (c) The effects of Sirt1 overexpression on p53, p21, and p16 in sh_Nr4a1_ HEK‐293T cells under H2O2 stimulation. n = 3 independent experiments. (d) β‐Galactosidase staining of sh_Nr4a1_ HEK‐293T cells overexpressing Sirt1 under H2O2 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; H2O2, 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 4

Nur77 enhances Sirt1 homeostasis via negative transcriptional regulation of MDM2. (a) The expression of MDM2 and Sirt1 in Scramble and Flag‐Nr4a1 HEK‐293T cells under H2O2 stimulation. n = 3 independent experiments. (b) The expression of MDM2 and Sirt1 in shScramble and sh_Nr4a1_ HEK‐293T cells under H2O2 stimulation. n = 3 independent experiments. (c) MDM2 mRNA expression in the presence or absence of Nur77. n = 6 independent experiments. (d) Dual‐luciferase reporter assay results of HEK‐293T cells expressing Nur77 and a luciferase reporter containing wild‐type (WT) or mutant (MUT) MDM2. n = 5 independent experiments. (e) Chromatin immunoprecipitation (ChIP) assays performed on HEK‐293T cells with an antibody against Nur77 or IgG as a control. n = 5 independent experiments. (f) The effects of silencing MDM2 on Sirt1 expression in sh_Nr4a1_ HEK‐293T cells treated with 100 μg/mL cycloheximide. n = 3 independent experiments. (g) Co‐IP analysis of the interaction between MDM2 and Sirt1 in sh_Nr4a1_ HEK‐293T cells under H2O2 stimulation. n = 2 independent experiments. (h) Analysis of the ubiquitination of Sirt1 in the presence or absence of Nur77 or MDM2. n = 2 independent experiments. (i) Sirt1 expression in the presence or absence of Nur77 or MDM2. n = 2 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. ChIP, chromatin immunoprecipitation; CHX, cycloheximide; Co‐IP, co‐immunoprecipitation; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; GFP, green fluorescent protein; HA, human influenza hemagglutinin; H2O2, hydrogen peroxide; IB, immunoblot; IgG, immunoglobin G; IP, immunoprecipitation; MDM2, murine double minute 2 protein; mRNA, messenger ribonucleic acid; MUT, mutant; Nur77, nuclear hormone receptor 77; Sirt1, sirtuin 2 protein; Ub, ubiquitin; WCL, whole cell lysate.

FIGURE 5

Nur77 deficiency stabilizes p53 protein expression in response to oxidative stress. (a) Co‐IP analysis of the interaction between MDM2 and p53 in sh_Nr4a1_ HEK‐293T cells under H2O2 stimulation. n = 2 independent experiments. (b) Co‐IP analysis of the interaction between p53 and Sirt1 in sh_Nr4a1_ HEK‐293T cells under H2O2 stimulation. n = 2 independent experiments. (c) Co‐IP analysis of the interaction between MDM2 and p53 in sh_Nr4a1_ HEK‐293T cells with or without Sirt1 overexpression under H2O2 stimulation. n = 2 independent experiments. (d) The effects of Sirt1 overexpression on p53 expression in sh_Nr4a1_ HEK‐293T cells treated with 100 μg/mL cycloheximide. n = 2 independent experiments. (e) Co‐IP analysis of the interaction between p53 and Chk2 in sh_Nr4a1_ HEK‐293T cells under H2O2 stimulation. n = 2 independent experiments. (f) Co‐IP analysis of the interaction between MDM2 and p53 in sh_Nr4a1_ HEK‐293T cells with or without Chk2 silencing under H2O2 stimulation. n = 2 independent experiments. (g) The effects of silencing Chk2 on p53 expression in sh_Nr4a1_ HEK‐293T cells treated with 100 μg/mL cycloheximide. n = 2 independent experiments. CHX, cycloheximide; Chk2, checkpoint kinase 2; Co‐IP, co‐immunoprecipitation; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; HA, human influenza hemagglutinin; H2O2, hydrogen peroxide; IB, immunoblot; IP, immunoprecipitation; MDM2, murine double minute 2 protein; Nur77, nuclear hormone receptor 77; Sirt1, sirtuin 2 protein; WCL, whole cell lysate.

FIGURE 6

Nur77 deficiency accelerates podocyte senescence and glomerular damage. (a) The expression of Sirt1, MDM2, γH2AX, p53, p21, p16, and podocyte markers (nephrin, synaptopodin) in shScramble and sh_Nr4a1_ podocytes under H2O2 stimulation. n = 2 independent experiments. (b) The expression of Sirt1, MDM2, γH2AX, p53, p21, p16, and podocyte markers (nephrin, synaptopodin) in scramble and Flag‐Nr4a1 podocytes under H2O2 stimulation. n = 2 independent experiments. (c) The expression of Sirt1, MDM2, p53, p21, and Bax in kidney tissue of 5‐ and 15‐month‐old WT and Nr4a1 −/− mice. n = 2 independent experiments. (d) Sirt1 and MDM2 mRNA expression in kidney tissue of 5‐ and 15‐month‐old WT and Nr4a1 −/− mice. n = 6 independent experiments. (e) Immunofluorescence staining of nephrin, synaptopodin, and desmin in the glomeruli of 5‐ and 15‐month‐old WT and Nr4a1 −/− mice. The data were analyzed by two‐way ANOVA followed by a multiple comparisons test. The results are plotted as the mean ± standard error. **p ≤ 0.01. MO, month‐old.

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Oxidative stress impairs the Nur77-Sirt1 axis resulting in a decline in organism homeostasis during aging - PubMed (original) (raw)