p21 maintains senescent cell viability under persistent DNA damage response by restraining JNK and caspase signaling

Abstract

Cellular senescence is a permanent state of cell cycle arrest that protects the organism from tumorigenesis and regulates tissue integrity upon damage and during tissue remodeling. However, accumulation of senescent cells in tissues during aging contributes to age-related pathologies. A deeper understanding of the mechanisms regulating the viability of senescent cells is therefore required. Here, we show that the CDK inhibitor p21 (CDKN1A) maintains the viability of DNA damage-induced senescent cells. Upon p21 knockdown, senescent cells acquired multiple DNA lesions that activated ataxia telangiectasia mutated (ATM) and nuclear factor (NF)-κB kinase, leading to decreased cell survival. NF-κB activation induced TNF-α secretion and JNK activation to mediate death of senescent cells in a caspase- and JNK-dependent manner. Notably, p21 knockout in mice eliminated liver senescent stellate cells and alleviated liver fibrosis and collagen production. These findings define a novel pathway that regulates senescent cell viability and fibrosis.

Keywords: JNK; DNA damage response; apoptosis; cellular senescence; p21(CDKN1A).

Figure 1. p21 maintains the viability of DNA damage‐induced senescent cells

1. Normal human IMR‐90 fibroblasts were induced to senesce by DNA damage (DNA damage‐induced senescent (DIS)) or by expression of Hras^V12 (oncogene‐induced senescent (OIS)). Proliferating IMR‐90 (growing, G) and empty vector‐transfected (V) cells served as respective controls. The cells were transduced with siRNAs targeting p21 (sip21) or control siRNAs (siCtrl). Histograms indicate the percentages of surviving senescent cells compared with G or V controls. Western blots show p21 protein levels in the corresponding samples.

2. Mouse embryonic fibroblasts (MEF; G and DIS) were transduced with siRNAs targeting p21 or control siRNA, and cell survival was evaluated. Western blots show p21 protein levels in the corresponding samples.

3. BJ human fibroblasts (DIS and G cells) were transduced with siRNAs targeting p21 or control siRNA, and cell survival was determined 4 days following the transduction. Western blots show p21 protein levels in the corresponding samples.

4. Mouse embryonic fibroblasts (MEF) from wild‐type (WT) or p21 knockout mice (p21^−/−) were treated with etoposide to induce DNA damage‐induced senescence. Survival of DIS cells was determined by quantification of the remaining adherent cells 5 days following etoposide washout.

5. BJ DIS fibroblasts were transduced with siRNAs targeting p21 and cell survival was determined at the indicated time points.

Data information: Data are presented as mean ± SEM of three repeats, performed in triplicates. Data were analyzed using Student's t‐test. *P < 0.05, ***P < 0.0005.

Figure 2. Gene‐expression profiles of growing and senescent BJ cells after p21 knockdown

BJ human fibroblasts (proliferating, G; and DNA damage‐induced senescent, DIS) were transduced with either siRNA targeting p21 (sip21) or control siRNA (siCtrl). Cells were harvested and analyzed by Affymetrix PrimeView microarrays (3 replicates).

1. Results are presented as K‐means clustering of the microarray data. Probe sets whose abundance was above the mean are shown in red, those below the mean in blue, and those equivalent to the mean in green.

2. Principal component analysis (PCA) scatterplot. Points are colored according to cell type (G, red; DIS, blue). Squares and triangles are drawn for sip21 and siCtrl siRNA groups, respectively.

3. Venn diagram showing the distribution of shared genes among G and DIS cells after p21 knockdown.

4. Enrichment analysis from the WikiPathways database identified pathways affected in 1,545 genes that were uniquely changed in DIS cells after p21 knockdown.

5. K‐means clustering of the 1,545 genes that were uniquely changed in DIS BJ cells after p21 knockdown. Probe sets whose abundance was above the mean are shown in red, and those below the mean in blue.

6. mRNA expression levels relative to controls of p21, COL1A1, CDK‐1, cyclin A2, Smad‐7, andTNF‐α genes after transduction with sip21 or control siRNA in G and DIS BJ cells. Data are presented as means ± SEM of three repeats, each performed in triplicate. Data were analyzed using Student's t‐test. *P < 0.05, **P < 0.005, ***P < 0.0005.

Figure 3. p21 regulates the DNA damage response in senescent cells

1. BJ human fibroblasts (proliferating, G; and DNA damage‐induced senescent, DIS) were transduced with either siRNA targeting p21 (sip21) or control siRNA (siCtrl). Representative immunoblots for p‐ATM, p‐CHK2, γH2AX, p53, and p‐p53(Ser 15) of at least two independent experiments are shown.

2. Immunofluorescence analysis of p‐ATM in G and DIS cells transduced with sip21 or siCtrl; representative images of at least two independent experiments are shown. Arrows indicate positive staining.

3. Quantification of the total numbers of p‐ATM foci per cell in (B).

4. Immunofluorescence analysis of γH2AX and p53BP1 in G and DIS BJ cells transduced with sip21 or siCtrl; representative images of at least two independent experiments are shown.

5. Quantification of total γH2AX foci per cell.

6. Quantification of total p53BP1 foci per cell.

7. ImageStreamX analysis. Distribution of numbers of γH2AX foci in G and DIS cells transduced with sip21 or siCtrl 3 days after siRNA washout. DIS cells stained with the secondary antibodies only and DAPI served as a negative control.

8. Comet assay. G and DIS BJ cells were transduced with sip21 or siCtrl. 72 h post‐siRNA washout cells were tested for DNA damage using the comet assay. Olive tail moment values were calculated using ImageJ. Each specimen was evaluated in triplicate. At least 50 cells were analyzed per replica and the quantification is shown on the right panel. Representative DNA tails are shown on the left.

9. Immunofluorescence analysis of BrdU incorporation in G and DIS cells transduced with sip21 or siCtrl, 24 h post‐siRNA washout; representative images of at least two independent experiments are shown.

10. Quantification of BrdU‐positive G and DIS cells.

11. DAPI staining of DIS cells transduced with sip21 or siCtrl 4 days after siRNA washout.

12. SubG1 cell cycle analysis of DIS cells transduced with sip21 or siCtrl 3 days after siRNA washout; representative histograms of three independent experiment are shown.

Data information: Data were analyzed using Student's t‐test. *P < 0.05. **P < 0.005, ***P < 0.0005. Data represent mean ± SEM (n = 3).Source data are available online for this figure.

Figure 4. Death of DIS cells after p21 knockdown ensued by TNF‐α secretion

1. Survival of DIS BJ cells transduced with siRNAs targeting p21, p53 or their combinations, as indicated. Data are presented as means ± SEM of 3 repeats, each performed in triplicate. Western blots of p21, p53, cleaved PARP, and cleaved caspase‐3 after siRNA treatment.

2. Survival of DIS BJ cells transduced with siRNAs targeting p21, pRb or their combinations, as indicated. Data are presented as means ± SEM of 3 repeats, each performed in triplicate. Western blots depict p21 and pRb protein levels following siRNA treatment.

3. Western blot analysis of p21, p65, p‐p65, JNK, p‐JNK, cleaved PARP, and cleaved caspase‐3 proteins 4 days after sip21 or siCtrl treatment of G and DIS cells; representative blots of at least two independent experiments are depicted.

4. mRNA expression levels of TNF‐α and IL‐8 in DIS cells at the indicated times after sip21 or siCtrl treatment. Data are presented as means ± SEM of three repeats.

5. Survival of DIS BJ cells transduced with siRNAs targeting p21, p65 or their combinations, as indicated, 4 days after siRNA washout. Western blots depict levels of p21, p65, JNK, p‐JNK, cleaved PARP, and cleaved caspase‐3 following siRNA treatment. Data are presented as means ± SEM of three repeats, each performed in triplicate.

6. mRNA expression levels of TNF‐α relative to control following treatment with siRNAs targeting p21, p65, or their combinations. Data are presented as means ± SEM of three repeats.

Data information: Data were analyzed using Student's t‐test. *P < 0.05, **P < 0.005; ***P < 0.0005. n.s, not significant. Source data are available online for this figure.

Figure 5. Death of DIS cells after p21 knockdown is caspase‐ and JNK‐dependent

1. Survival of DIS BJ cells transduced with sip21 or siCtrl, with or without incubation with pan‐caspase inhibitor QVD. Western blots depict levels of p21, cleaved PARP and cleaved caspase‐3 after the siRNA treatment.

2. Survival of DIS BJ cells transduced with sip21 or siCtrl, with or without incubation with the JNK inhibitor SP600125 (JNKi). Western blots depict levels of p21, JNK, p‐JNK, cleaved PARP and cleaved caspase‐3 after JNKi treatment.

3. Survival of DIS BJ cells transduced with sip21 or siCtrl, with or without co‐incubation with QVD and JNKi. Treatment with the two inhibitors rescues the cells from sip21‐induced decrease in cell viability.

4. Survival of DIS BJ cells transduced with siRNAs targeting p21, JNK1 or their combinations, as indicated. Western blots depict levels of p21, JNK, p‐JNK, cleaved PARP and cleaved caspase‐3 following siRNA treatment.

5. Survival of DIS BJ cells transduced with sip21 or siCtrl, with or without transduction of siJNK1 and incubation QVD. Western blots depict levels of p21, JNK, p‐JNK, cleaved PARP and cleaved caspase‐3 after treatments.

Data information: For all the survival plots, data are presented as means ± SEM of 3 repeats, each performed in triplicate. Data were analyzed using Student's t‐test. *P < 0.05. **P < 0.005. n.s, not significant. Source data are available online for this figure.

Figure 6. p21 knockout alleviates liver fibrosis

1. A–C

   Liver sections from wild‐type (WT) or p21‐knockout mice (p21^−/−) treated with CCl₄ (n = 10 from each group) or vehicle (oil; n = 3 (WT) and n = 4 (p21^−/−)) were stained with SA‐β‐gal (A) cleaved caspase‐3 (B) and Sirius Red (C).

2. D

   Quantification of SA‐β‐gal staining of sections presented in (A).

3. E

   Quantification of Sirius Red staining presented in (C).

4. F

   Relative expression levels of mRNA of p21, the HSC marker α‐SMA (ACTA2), and the fibrosis markers COL1A1 (collagen, type I, alpha 1) and TGF‐β1 in WT and p21^−/− fibrotic mouse livers.

5. G

   Relative expression levels of mRNA in p21 and COL1A1 in BJ DIS cells after transduction with sip21 and siCtrl at the indicated time points.

6. H

   Western blot analysis of p21 and collagen‐1 (COL1) proteins after sip21 and siCtrl treatment of G and DIS cells.

7. I

   Sirius Red staining of DIS cells after transduction with sip21 and siCtrl.

8. J

   Quantification of Sirius Red staining presented in (H).

Data information: Data were analyzed using Student's t‐test. *P < 0.05. **P < 0.005. ***P < 0.0005. Data represent mean ± SEM (n = 3). Source data are available online for this figure.