Interaction between ROS dependent DNA damage, mitochondria and p38 MAPK underlies senescence of human adult stem cells

Abstract

Human endometrium-derived mesenchymal stem cells (hMESCs) enter the premature senescence under sublethal oxidative stress, however underlying mechanism remains unknown. Here, we showed that exogenous H2O2 induces a rapid phosphorylation and co-localization of ATM, H2A.X, 53BP1 leading to DNA damage response (DDR) activation. DDR was accompanied with nuclear translocation of p-p53 followed by up-regulation of p21Waf1 and the permanent hypophosphorylation of pRb. Additionally, the increased p38MAPK/MAPKAPK-2 activation persisted in H2O2-treated cells. We suggest that both p53/p21/pRb and p38MAPK/MAPKAPK-2 pathways are responsible for establishing an irreversible cell cycle arrest that is typical of senescence. The process of further stabilization of senescence required prolonged DDR signaling activation that was provided by the permanent ROS production which in turn was regulated by both p38MAPK and the increased functional mitochondria. To reverse senescence, the pharmacological inhibition of p38MAPK was performed. Cell treatment with SB203580 was sufficient to recover partially senescence phenotype, to block the ROS elevation, to decrease the mitochondrial function, and finally to rescue proliferation. Thus, suppression of the p38MAPK pathway resulted in a partial prevention of H2O2-induced senescence of hMESCs. The current study is the first to reveal the molecular mechanism of the premature senescence of hMESCs in response to oxidative stress.

Figure 1. The dynamics of H₂O₂ penetration into hMESCs

(A) Intracellular ROS levels upon application of 200 µM H₂O₂. Cells were loaded with DCF, each trace represents the response of one cell. Three (N = 3) experiments were performed. (B) Fluorescent images taken at different stages (indicated by dashed lines) of the experiment illustrated in (A). Scale bar is 200 µm and valid for all images. (C) Intracellular ROS levels measured by FACS after staining with the fluorescent probe DCF. Values are means ± SD of three independent experiments. **p<0.005, ***p<0.001 versus control.

Figure 2. A rapid DDR activation in H₂O₂-treated hMESCs

Immunofluorescence analysis of ATM (A), 53BP1 (B) activation and γH2AX foci formation (C) performed at indicated time points after H₂O₂ addition. DAPI was used as nuclear stain (blue). Representative photomicrographs of the staining are made at original magnification X100. Scale bar is 25 µm.

Figure 3. Formation of DNA damage foci containing pATM, γH2AX, p53BP1 in H₂O₂-treated hMESCs

Immunofluorescent analysis with the use of specific antibodies against pATM, γH2AX, p53BP1 revealed co-localization of pATM with either γH2AX (A) or p53BP1 (B) in 60 min after beginning of H₂O₂ treatment. DAPI was used as nuclear stain (blue). Images are taken at magnification X100. Scale bar is 20 µm and valid for all images.

Figure 4. H₂O₂-induced activation of the p53/p21/pRb pathway in hMESCs

(A) H₂O₂-induced ATM phosphorylation tested by Western blotting. (B) Phosphorylation status of p53 in both untreated and H₂O₂-treated cells examined by Western blotting. (C) Nuclear accumulation of pp53 and co-localization pp53 with pATM revealed by immunofluorescence. Original magnification, X100. Scale bar, 20 µm. (D) The expression levels of p21 mRNA. (E) The expression levels of p21 protein. (F) Abrogation of Rb phosphorylation in H₂O₂-treated hMESCs examined by Western blotting. GAPDH and β-actin were used as loading controls. Representative results of three independent experiments are shown.

Figure 5. Permanent ROS generation and prolonged DDR activation

(A) Intracellular ROS levels and (C) cellular peroxide levels detected at indicated time points by FACS analysis after staining with either DCF or DHR123, respectively. (M ± SD, N = 3, *p<0.05, **p<0.005, ***p<0.001, versus control). Representative DCF (B) or DHR123 (D) fluorescent images of control (Ctr) and treated hMESCs. Scale bar is 200 µm and valid for all images. (E) Co-localization of pATM with either p53BP1 or (F) γH2AX in 5 days after H₂O₂ treatment. DAPI was used as nuclear stain (blue). Representative photomicrographs of the staining are shown. Images are taken at magnification X100. Scale bar, 20 µm.

Figure 6. Persistent increase in the number of functional mitochondria in H₂O₂-treated hMESCs

(A) FACS analysis of mitochondrial mass in control (Ctr) and H₂O₂-treated hMESCs stained with NAO. Values are means ± SD of three independent experiments (*p<0.05, **p<0.005, versus control). (B) Representative NAO confocal fluorescence images of control (Ctr) and treated hMESCs. (C) Rho123 fluorescence in hMESCs at the indicated time points after H₂O₂ treatment as measured by FACS. (M ± SD, N = 3, *p<0.05, **p<0.005, versus control). (D) Rho123 fluorescent images of control (Ctr) and H₂O₂-treated hMESCs. Micrographs are representative for three experiments. Scale bar is 200 µm and valid for all images.

Figure 7

(A) Immunoblot analysis of p38 phosphorylation during 1 h H₂O₂ treatment. (B) SB had no effect on long-term p38 phosphorylation as detected by Western blot. (C) Selective inhibition of p38 activity abolished phosphorylation of MAPKAPK-2 during the whole observation period. Representative results of three independent experiments are shown. GAPDH was used as loading control.

Figure 8

(A) SB rescued the cell proliferation of H₂O₂-treated hMESCs. Cell number was determined daily by FACS (M ± SD, N = 3, **p<0.005, ***p<0.001, versus control, §p<0.05, versus H₂O₂-treated cells). (B) SB partially prevented H₂O₂-induced increase of cell size. Forward scatter (FS) reflects the average cell size. M ± SD, N = 3, *p<0.05, **p<0.005, versus control, §p<0.05, §§<0.005, versus H2O2-treated cells. (C) Inhibition of p38 activity had no effect on long-term activation of pp53 or (D) p21. (E) An impact of inhibition of p38 kinase activity on pRb phosphorylation status.

Figure 9. SB diminished ROS production and mitochondrial function in senescent hMESCs

(A) DCF, (B) DHR123, (C) Rho123, and (D) NAO fluorescence in control (Ctr), H₂O₂-treated and (SB+H₂O₂)-treated hMESCs at indicated time points as measured by flow cytometry (M ± SD, N = 3, *p<0.05, ***p<0.001, versus control, §p<0.05, §§§<0.001, versus H₂O₂-treated cells).

Figure 10. A pathway interaction scheme displaying the proposed molecular mechanism of premature sense-cence of hMESCs under oxidative stress