Mitochondria-to-nucleus retrograde signaling drives formation of cytoplasmic chromatin and inflammation in senescence

Abstract

Cellular senescence is a potent tumor suppressor mechanism but also contributes to aging and aging-related diseases. Senescence is characterized by a stable cell cycle arrest and a complex proinflammatory secretome, termed the senescence-associated secretory phenotype (SASP). We recently discovered that cytoplasmic chromatin fragments (CCFs), extruded from the nucleus of senescent cells, trigger the SASP through activation of the innate immunity cytosolic DNA sensing cGAS-STING pathway. However, the upstream signaling events that instigate CCF formation remain unknown. Here, we show that dysfunctional mitochondria, linked to down-regulation of nuclear-encoded mitochondrial oxidative phosphorylation genes, trigger a ROS-JNK retrograde signaling pathway that drives CCF formation and hence the SASP. JNK links to 53BP1, a nuclear protein that negatively regulates DNA double-strand break (DSB) end resection and CCF formation. Importantly, we show that low-dose HDAC inhibitors restore expression of most nuclear-encoded mitochondrial oxidative phosphorylation genes, improve mitochondrial function, and suppress CCFs and the SASP in senescent cells. In mouse models, HDAC inhibitors also suppress oxidative stress, CCF, inflammation, and tissue damage caused by senescence-inducing irradiation and/or acetaminophen-induced mitochondria dysfunction. Overall, our findings outline an extended mitochondria-to-nucleus retrograde signaling pathway that initiates formation of CCF during senescence and is a potential target for drug-based interventions to inhibit the proaging SASP.

Keywords: cytoplasmic chromatin; inflammation; mitochondria; senescence.

Figure 1.

Mitochondria are required for formation of CCFs. (A) Heat map of RNA-seq analysis of nuclear-encoded mitochondrial genes belonging to complexes I, II (CII), III (CIII), IV (CIV), and V (CV) in proliferating (Prolif) and senescent (IR) IMR90 cells 10 d after IR, from n = 3 independent experiments. The color intensity represents the log₂ fold change, where red indicates highly and blue lowly expressed. (B) Ten days after IR, proliferating (Prolif) and senescent (IR) IMR90 cells were incubated with TMRE (100 nM) and Mitotracker Green (MTG; 100 nM) followed by flow cytometric analysis. The ratio of TMRE and MTG fluorescence intensity was used to represent mitochondrial membrane potential (MMP). (C) Mitochondrial reactive oxygen species (mROS) were measured by flow cytometry using MitoSOX (5 μM) in proliferating (Prolif) and senescent (IR) IMR90 cells 10 d after IR. For raw data, see Supplemental Figures S6 and S7. (D) Expression of anti-ROS gene SOD2 was determined by RT-qPCR in proliferating (Prolif) and senescent (IR) IMR90 cells 10 d after IR. Data shown in B–D are mean ± SEM of n = 3 independent experiments. P-value was calculated by unpaired two-tailed Student's t-test. (***) P < 0.001; (*) P < 0.05. (E) Scheme summarizing the experimental design of Parkin-directed mitophagy experiments in F–K. (F) The expression of mitochondrial proteins from the different mitochondrial complexes NDUFB8 (complex I), SDHA (complex II), and UQCR2 (complex III) and for the mitochondrial outer membrane marker Tom20 were analyzed by immunoblotting in senescent (IR) control (C) and Parkin (P)-expressing IMR90 cells treated with or without CCCP. (G) Representative fluorescent images of Tom20 staining in senescent (IR) Parkin-expressing cells treated with or without CCCP. Scale bar, 20 μm. (H) Representative confocal immunofluorescence images of proliferating (Prolif) and senescent (IR) Parkin-expressing cells stained for DAPI (blue), anti-γH2AX (green). CCFs are indicated by arrows and quantified. Scale bar, 5 μm. Graph shows mean ± SEM of n = 3 independent experiments. P-value was calculated by unpaired two-tailed Student's t-test. (****) P < 0.0001. (I) The expression of the indicated SASP components was monitored by RT-qPCR in proliferating (Prolif) and senescent (IR) Parkin-expressing cells. Data are mean ± SEM of n = 3 independent experiments. P-value was calculated by unpaired two-tailed Student's t-test. (***) P < 0.001; (**) P < 0.01. (J) Heat map of RNA-seq analysis of SASP genes in the depicted IMR90 cells from n = 3 independent experiments. The color intensity represents the Z-score of the mean FPKM expression, where red indicates highly and blue lowly expressed. (K) Lysates from proliferating (Prolif) and senescent (IR) of control (C) and Parkin-expressing (P) cells were probed for the expression of IL8 by immunoblotting.

Figure 2.

ROS promote formation of CCFs and expression of the SASP. (A) Mitochondrial reactive oxygen species (mROS) were measured by flow cytometry using MitoSOX (5 μM) in proliferating and senescent (IR) of control and Parkin-expressing cells with or without CCCP treatment 10 d after IR. For raw data, see Supplemental Figure S7. (B) Proliferating IMR90 cells were exposed to exogenous ROS (H₂O₂, 600 μM) for 2 h. Quantification of mitochondrial reactive oxygen species (mROS) was assessed by flow cytometry using MitoSOX (5 μM) at day 4. For raw data, see Supplemental Figure S7. (C–E) Proliferating IMR90 cells exposed to exogenous ROS (H₂O₂, 600 μM) and harvested at the indicated times were analyzed for the depicted proteins by immunoblotting (C), quantified for CCFs (D), and analyzed for the expression of SASP components by RT-qPCR (E). Data shown in A, B, D, and E are mean ± SEM of n = 3 independent experiments. P-value was calculated by unpaired two-tailed Student's t-test or one-way ANOVA coupled with Dunnett's test. (****) P < 0.0001; (***) P < 0.001; (**) P < 0.01; (*) P < 0.05; (NS) not significant. (F–I) Proliferating IMR90 cells treated with MitoPQ (40 μM) for 8 d were analyzed for mitochondrial superoxide production by flow cytometry using MitoSOX (5 μM) (for raw data, see Supplemental Figure S7) (F), probed for the indicated proteins by immunoblotting (G), quantified for CCFs (H), and assessed for expression of the indicated SASP genes by RT-qPCR (I). For F, H, and I, data are mean ± SEM of n = 3 independent experiments. P-value was calculated by unpaired two-tailed Student's t-test. (***) P < 0.001; (**) P < 0.01; (*) P < 0.05. (J–N) IMR90 cells were treated with 100 nM MitoQ and then irradiated to initiate senescence. Cells were harvested after 10 d of irradiation. (J) Quantification of mitochondrial reactive oxygen species (mROS) by flow cytometry using MitoSOX (5 μM) in proliferating and senescent (IR) with or without MitoQ. For raw data, see Supplemental Figure S7. (K) Formation of CCFs was monitored by immunofluorescence, imaged, and quantified in proliferating and senescent (IR) IMR90 cells treated with or without MitoQ. Representative fluorescent images are shown. CCFs are indicated by arrows. Scale bar, 8 μm. (L) Expression of the selected SASP genes was measured by RT-qPCR in proliferating and senescent (IR) IMR90 cells treated with or without MitoQ. Mean ± SEM of n = 3 independent experiments are shown in J–L. P-value was calculated by unpaired two-tailed Student's t-test. (****) P < 0.001; (***) P < 0.001; (**) P < 0.01; (*) P < 0.05. (M) The effect of MitoQ on cell proliferation was assessed by EdU incorporation. Representative images and quantification from two independent experiments are shown. Scale bar, 15 μm. (N) Lysates from proliferating and senescent (IR) IMR90 cells with or without MitoQ were analyzed for the indicated proteins by immunoblotting.

Figure 3.

JNK mediates formation of CCFs and expression of the SASP. (A) Proliferating (Prolif) and senescent (IR) IMR90 cells harvested at the indicated times were analyzed for ph-JNK and JNK by immunoblotting. (B–H) IMR90 cells were treated with JNK inhibitor (JNKi, SP600125, 20 μM) and then irradiated to induce senescence. Cells were harvested 10 d after IR. (B) Immunoblot analysis of the indicated proteins in proliferating (Prolif) and senescent (IR) IMR90 cells with or without JNKi. (C) Proliferating (Prolif) and senescent cells (IR) with or without JNKi were imaged and quantified for CCF formation. CCFs are indicated by arrows. Scale bar, 8 μm. (D) Expression of the SASP measured by RT-qPCR in the depicted IMR90 cells. (E) Principal component analysis scatter plot showing the global gene expression of the cohorts used in the NanoString analysis. (F) Heat map of SASP genes included in the NanoString analysis in proliferating (Prolif) and senescent (IR) with or without JNKi. Mean of three independent replicates is shown. The color intensity represents the Z-score of the mean FPKM expression. (G) Immunoblot analysis of IL8 in proliferating and senescent (IR) IMR90 cells in the absence or presence of JNKi. (H) Determination of mitochondrial reactive oxygen species (mROS) by flow cytometry using MitoSOX (5 μM) in proliferating (Prolif) and senescent (IR) IMR90 cells, in the absence or presence of JNKi. The percentage of positive cells is shown. For raw data, see Supplemental Figure S7. Data shown in C,D, and H are mean ± SEM of three independent experiments. P-value was calculated by unpaired two-tailed Student's t-test. (***) P < 0.001; (**) P < 0.01; (*) P < 0.05. (I–K) Proliferating IMR90 cells were infected with a nontargeting control shRNA (sh-NTC) or with shRNA against JNK1/2 (sh-JNK1/2), followed by mock (Prolif) or ionizing irradiation (IR). Ten days after IR, cells were assessed for expression of JNK by immunoblotting (I) or percentage of CCF-positive cells by immunofluorescence (J) or monitored for the expression of the indicated SASP genes by RT-qPCR (K). Data shown in J and K are mean ± SEM of three independent experiments. Statistical significance was calculated using unpaired two-tailed Student's t-test. (**) P < 0.01; (*) P < 0.05.

Figure 4.

53BP1 controls formation of CCFs in the nucleus. (A) Confocal fluorescent images of senescent (IR) IMR90 cells stained for DAPI (blue), γH2AX (green), and 53BP1(red) 10 d after IR. CCFs are indicated with arrows. Scale bar, 10 μm. (B) Immunoblot analysis for 53BP1 and JNK after immunoprecipitation of 53BP1 from proliferating (Prolif) and senescent (IR) IMR90 cells 10 d after IR. One representative experiment out of four experiments is shown. (C–E) Proliferating IMR90 cells were infected with a nontargeting control shRNA (sh-NTC) or with shRNA against 53BP1 (sh-53BP1), followed by mock (Prolif) or ionizing irradiation (IR). Ten days after IR, cells were analyzed for expression of 53BP1 by immunoblotting (C), stained and quantified for formation of CCF (indicated by arrows) (D), or examined for the expression of SASP components by RT-qPCR (E). Scale bar, 10 μm. Mean ± SEM of three independent experiments is shown in D and E. Statistical significance was calculated using unpaired two-tailed Student's t-test. (*) P < 0.05; (NS) not significant. (F–I) IMR90 cells were infected with lentivirus directing expression of 53BP1 or control, induced to senesce by irradiation (IR) or control (Prolif). Ten days after IR, cells were analyzed for the depicted proteins by immunoblotting (F), stained and quantified for CCFs (indicated by arrows) (G), assessed for the indicated SASP genes by RT-qPCR (H), or probed for the expression of IL8 by immunoblotting (I). Scale bar, 6 μm. Data shown in G and H represent mean ± SEM of three independent experiments. P-values were calculated by unpaired two-tailed Student's t-test. (****) P < 0.0001; (***) P < 0.001; (**) P < 0.01.

Figure 5.

HDAC inhibitors improve mitochondria function and suppress formation of CCFs and expression of the SASP. (A) Scheme of experimental design in B–M. (B) Proliferating or senescent (IR) IMR90 cells treated with TSA at indicated dose range (20–50–100 nM) were analyzed for H4K16ac and H4 by immunoblotting. (C) The effect of TSA (100 nM) on cell proliferation was assessed by EdU incorporation. Representative fluorescent images and quantification are shown. Scale bar, 10 μm. (D) Lysates from proliferating (Prolif) and senescent (IR) IMR90 cells with or without TSA (100 nM) were analyzed for the indicated proteins by immunoblotting. (E) Relative fold change in nuclear-encoded oxidative phosphorylation genes (KEGG hsa00190; subunits of complexes I–V) was represented in a histogram with bin size of 0.1 log₂ fold change units. Counts in each bin were normalized to total number of nuclear-encoded mitochondrial genes detected. (F) The expression of the indicated nuclear-encoded mitochondrial genes was analyzed by RT-qPCR in senescent cells (IR) treated with or without TSA (100 nM). (G) Proliferating (Prolif) and senescent (IR) cells treated with or without TSA (100 nM) were analyzed for the indicated proteins by immunoblotting. (H) Proliferating (Prolif) and senescent (IR) cells treated with or without TSA were incubated with TMRE (100 nM) and Mitotracker Green (MTG; 100 nM) and analyzed by flow cytometry. The ratio of TMRE and MTG fluorescence intensity was used to represent mitochondrial membrane potential (MMP). (I) Senescent (IR) IMR90 cells with or without TSA (100 nM) were incubated with MitoSOX (5 μM) and analyzed by flow cytometry for mitochondrial reactive oxygen species. Percentage of positive cells is shown. For raw data, see Supplemental Figure S8. (J) Expression of ROS-responsive gene SOD2 was determined by RT-qPCR in proliferating (Prolif) and senescent (IR) IMR90 cells with or without TSA (100 nM). (K,L) Proliferating (Prolif) and senescent (IR) cells treated with or without TSA (100 nM) were imaged by confocal microscope and quantified for formation of γH2AX-positive CCFs (indicated by arrows) (K) or monitored for the expression of indicated SASP genes by RT-qPCR (L). Scale bar, 8 μm. Data shown in C, F, and H–L are mean ± SEM of three independent experiments. Statistical analysis was performed with unpaired two-tailed Student's t-test. (****) P < 0.0001; (***) P < 0.001; (**) P < 0.01; (*) P < 0.05. (M) Heat map of RNA-seq analysis of SASP genes in the indicated IMR90 cells from n = 3 independent experiments. Color intensity represents the Z-score of mean FPKM expression.

Figure 6.

Effects of HDAC inhibitors on other pathways. (A) Proliferating IMR90 cells were transfected with chromatin fragments and treated with TSA as indicated. On day 4, conditioned medium (CM) was collected and analyzed for IL8 by immunoblotting. (B) Proliferating (Prolif) and senescent (IR) cells with or without TSA as in Figure 5A were immunostained for γH2AX. Shown is the percentage of cells with more than three intranuclear γH2AX foci. Data are mean ± SEM of three independent experiments. Statistical analysis was performed with unpaired two-tailed Student's t-test. (**) P < 0.01; (NS) not significant. (C) Proliferating and senescent (IR) IMR90 cells treated as in Figure 5A were analyzed for phospho-p38 (p-p38) and p38 by immunoblotting. (D) Immunoblot for phospho-S6 (p-S6) and S6 in proliferating and senescent (IR) cells 10 d after IR. (E,F) IMR90 cells were treated with TSA, irradiated to induce senescence, and incubated with recombinant IL1α (r-IL1α) as indicated. (E) Cell lysates were analyzed for the indicated proteins by immunoblotting 10 d after IR. (F) Conditioned medium (CM) was collected and probed for IL8 by immunoblotting 10 d after IR.

Figure 7.

HDAC inhibitors suppress inflammation in vivo. (A) Scheme of experimental design in B–D. C57BL/6 mice were pretreated with vehicle (HPO-β-CD) or SAHA (0.1 g/L) in drinking water, subjected to irradiation (IR, 4Gy), and administrated vehicle (HPO-β-CD) or SAHA in drinking water for seven more days. Seven days after IR, livers were harvested. (B,C) Livers were lysed and analyzed for H4K16ac, H4, H3K9ac, and H3 by immunoblotting. (D) Livers were stained for γH2AX and IL1α and quantified. Representative immunohistochemistry images are shown. N = 5 mice. Data are the mean ± SEM. Statistical significance was calculated using unpaired two-tailed Student's t-test. (*) P < 0.05; (NS) not significant. Scale bar, 20 μm. (E) Livers from mice treated as indicated were harvested 48 h after APAP administration, stained for γH2AX and IL1α. CCFs are indicated by arrows. The percentage of cells positive for CCFs and IL1α is shown. Representative immunohistochemistry images are shown. N = 5 mice for DMSO; n = 8 for APAP DMSO and APAP TSA. Scale bar, 20 μm. Data represent the mean ± SEM. Statistical analysis was performed with unpaired two-tailed Student's t-test. (****) P < 0.0001; (*) P < 0.05. (F) Livers from mice treated as indicated were harvested 48 h after APAP administration and stained for γH2AX and quantified. Representative immunohistochemistry images are shown. N = 5 mice for DMSO; n = 8 for APAP DMSO and APAP TSA. Scale bar, 20 μm. Data represent the mean ± SEM. Statistical analysis was performed with unpaired two-tailed Student's t-test. (****) P < 0.0001. (G,H) Livers were harvested 14 h after APAP injection, lysed, and subjected to RNA-seq analysis. (G) Heat map of RNA-seq analysis showing all genes that are significantly up-regulated in APAP DMSO versus untreated mice and significantly down-regulated in APAP TSA versus APAP DMSO. N = 4 mice per group. (H) Heat map of RNA-seq analysis of NRF2 target genes in mice treated as indicated, 14 h after APAP administration. N = 4 mice for cohort. Color intensities are Z-scores of the mean FPKM expression. (I) RT-qPCR analysis of NRF2 target genes in the liver of mice treated as indicated, 14 h after APAP administration. N = 10 mice for group. Data represent the mean ± SEM. Statistical analysis was performed with unpaired two-tailed Student's t-test. (**) P < 0.01; (*) P < 0.05.