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. 2023 Aug 15;42(16):e111133.
doi: 10.15252/embj.2022111133. Epub 2023 Jul 11.

Cellular senescence induction leads to progressive cell death via the INK4a-RB pathway in naked mole-rats

Yoshimi Kawamura  1   2   3 , Kaori Oka  1   2 , Takashi Semba  4 , Mayuko Takamori  1   2 , Yuki Sugiura  5 , Riyo Yamasaki  1 , Yusuke Suzuki  1 , Takeshi Chujo  6 , Mari Nagase  1 , Yuki Oiwa  1   2   7 , Shusuke Fujioka  1   2 , Sayuri Homma  8 , Yuki Yamamura  1 , Shingo Miyawaki  2   9 , Minoru Narita  8   10 , Takaichi Fukuda  11 , Yusuke Sakai  12 , Takatsugu Ishimoto  4   13 , Kazuhito Tomizawa  6   14 , Makoto Suematsu  5   15 , Takuya Yamamoto  16   17   18 , Hidemasa Bono  19   20 , Hideyuki Okano #  3 , Kyoko Miura #  1   2   3   14
Affiliations

Cellular senescence induction leads to progressive cell death via the INK4a-RB pathway in naked mole-rats

Yoshimi Kawamura et al. EMBO J. .

Abstract

Naked mole-rats (NMRs) have exceptional longevity and are resistant to age-related physiological decline and diseases. Given the role of cellular senescence in aging, we postulated that NMRs possess unidentified species-specific mechanisms to prevent senescent cell accumulation. Here, we show that upon induction of cellular senescence, NMR fibroblasts underwent delayed and progressive cell death that required activation of the INK4a-retinoblastoma protein (RB) pathway (termed "INK4a-RB cell death"), a phenomenon not observed in mouse fibroblasts. Naked mole-rat fibroblasts uniquely accumulated serotonin and were inherently vulnerable to hydrogen peroxide (H2 O2 ). After activation of the INK4a-RB pathway, NMR fibroblasts increased monoamine oxidase levels, leading to serotonin oxidization and H2 O2 production, which resulted in increased intracellular oxidative damage and cell death activation. In the NMR lung, induction of cellular senescence caused delayed, progressive cell death mediated by monoamine oxidase activation, thereby preventing senescent cell accumulation, consistent with in vitro results. The present findings indicate that INK4a-RB cell death likely functions as a natural senolytic mechanism in NMRs, providing an evolutionary rationale for senescent cell removal as a strategy to resist aging.

Keywords: INK4a; cell death; cellular senescence; monoamine oxidase; naked mole-rat.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1. Induction of cellular senescence by DNA damage leads to progressive cell death, a delayed response after upregulation of INK4a in NMR fibroblasts
  1. A

    Adult naked mole‐rats (NMRs).

  2. B

    Scheme for doxorubicin (DXR) treatment and time‐course analysis.

  3. C

    Cell morphology and SA‐β‐Gal activity of mouse or NMR fibroblasts 12 or 21 days after DXR treatment. Arrowheads indicate dying cells. The number in the upper left corner indicates Hoechst‐positive nuclei. Scale bar, 100 μm.

  4. D–H

    Time‐course analysis of mouse or NMR fibroblasts after DXR treatment: quantification of SA‐β‐Gal‐positive cells (%) (D); quantification of BrdU‐positive cells (%) (E); qRT‐PCR analysis of the expression of INK4a (F) or p21 (G) normalized to ACTB mRNA levels; quantification of Annexin V‐positive cells (%) (Annexin V+/PI− as early apoptotic and Annexin V+/PI+ double‐positive as late apoptotic) (H).

  5. I

    Scheme for DXR treatment after INK4a knockdown in NMR fibroblasts.

  6. J, K

    qRT‐PCR analysis of the expression of INK4a (J) and quantification of Annexin V‐positive cells (%) (K) in shINK4a‐transduced NMR fibroblasts at 21 days after DXR treatment.

Data information: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant. One‐way ANOVA followed by Dunnett's multiple comparison test for (D–H, J, and K). Data are expressed as the mean ± SD from n = 3 biological replicates.
Figure 2
Figure 2. INK4a transduction leads to progressive cell death in NMR cells
  1. A

    Scheme for INK4a transduction.

  2. B

    qRT‐PCR analysis of the expression of INK4a at 12 days after INK4a or mock transduction, normalized to ACTB mRNA levels.

  3. C

    Cell morphology and SA‐β‐Gal activity of mouse or NMR fibroblasts 12 days after INK4a transduction. Arrowheads indicate dying cells. The number in the upper left corner indicates Hoechst‐positive nuclei. Scale bar, 100 μm.

  4. D, E

    Quantification of SA‐β‐Gal‐positive cells (%) (D) and BrdU‐positive cells (%) (E) at 12 days after INK4a transduction.

  5. F

    Western blot analysis of RB in mouse or NMR fibroblasts at 12 days after INK4a transduction (p, phospho‐specific antibody). ACTIN was used as a loading control. The arrowhead indicates a nonspecific band.

  6. G

    Quantification of Annexin V/PI‐positive cells (%) (Annexin V+/PI− as early apoptotic and Annexin V+/PI+ double‐positive as late apoptotic) at 2, 12, or 20 days after INK4a transduction.

  7. H

    Scheme for SA‐β‐Gal staining of adherent and floating cells.

  8. I

    SA‐β‐Gal activity in the adherent living cell population and floating dead cell population in mouse or NMR fibroblast cultures at 12 days after INK4a transduction. Scale bar, 100 μm.

  9. J

    Quantification of SA‐β‐Gal‐positive cells in the adherent living cell population and floating dead cell population after the same treatment as in (I).

Data information: *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant. Unpaired t‐test versus control for (B, D, E, day 2 in G, and J). One‐way ANOVA followed by Dunnett's multiple comparison test for (day 12–20 in G). Data are expressed as the mean ± SD from n = 3 biological replicates. Source data are available online for this figure.
Figure 3
Figure 3. Upregulation of INK4a results in progressive cell death via RB, independent of p53 in NMR fibroblasts
  1. A

    Scheme for transduction of INK4a and different forms of SV40 Large T antigen (LT, LTΔ, and LTK1).

  2. B

    RT‐PCR analysis of SV40 Large T expression in NMR‐fibroblasts transduced with different forms of SV40 Large T antigen (LT, LTΔ, and LTK1) and INK4a.

  3. C, D

    Quantification of SA‐β‐Gal‐positive cells (%) (C) and Annexin V‐positive cells (%) (Annexin V+/PI− as early apoptotic and Annexin V+/PI+ double‐positive as late apoptotic) (D) in NMR‐fibroblasts transduced with different forms of SV40 Large T antigen (LT, LTΔ, and LTK1) and INK4a. OE; overexpression.

  4. E

    Scheme for doxorubicin (DXR) treatment following transduction of different forms of SV40 Large T antigen (LT, LTΔ, and LTK1).

  5. F

    Quantification of SA‐β‐Gal‐positive cells (%) at 21 days after DXR treatment in NMR‐fibroblasts transduced with different forms of SV40 Large T antigen (LT, LTΔ, and LTK1).

  6. G

    Quantification of Annexin V‐positive cells after the same treatment as in (F). Annexin V+/PI− cells were counted as early apoptotic cells and Annexin V+/PI− cells as late apoptotic cells.

Data information: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant. One‐way ANOVA followed by Dunnett's multiple comparison test. Data are expressed as the mean ± SD from n = 3 biological replicates. Source data are available online for this figure.
Figure 4
Figure 4. INK4a‐transduced NMR cells activate serotonin metabolism that produce hydrogen peroxide

  1. Venn diagram showing the differentially expressed genes (DEGs, > 1.5‐fold in INK4a‐transduced fibroblasts) identified from comparisons of mock‐transduced and INK4a‐transduced NMR fibroblasts 12 days after transduction. Data were obtained from n = 3 biological replicates.

  2. Top 20 enriched gene ontology (GO) terms and KEGG pathways obtained using Metascape analysis of the 165 common genes in (A).

  3. Quantification of PI‐positive cells in NMR and mouse fibroblasts 18 h after 6 h of treatment with the indicated doses of hydrogen peroxide (H2O2). Data are expressed as the mean ± SD from n = 3 biological replicates.

  4. Levels of serotonin and 5‐hydroxyindoleacetic acid (5‐HIAA) measured by liquid chromatography–tandem mass spectrometry (LC–MS/MS) in mouse or NMR fibroblasts at 12 days after INK4a transduction. Data are expressed as the mean ± SD from two technical replicates for each fibroblasts culture (n = 3 biological replicates).

Data information: *P < 0.05; **P < 0.01; ****P < 0.0001; ns, not significant. Two‐way ANOVA followed by Sidak's multiple comparisons test for (C). Unpaired t‐test versus control for (D).
Figure EV1
Figure EV1. Continuous cultivation after contact inhibition results in progressive cell death in NMR fibroblasts through INK4a upregulation
  1. A

    Scheme for continuous cultivation after induction of contact inhibition (CI).

  2. B, C

    Cell morphology (B) and SA‐β‐Gal staining (C) in mouse or NMR fibroblasts at 14 or 28 days after induction of CI. Scale bar, 100 μm. The number in the upper left corner indicates Hoechst‐positive nuclei.

  3. D, E

    Quantification of SA‐β‐Gal‐positive cells (%) (D), and BrdU‐positive cells (%) (E) at 14 or 28 days after induction of CI.

  4. F

    qRT‐PCR analysis of the expression of INK4a in mouse or NMR fibroblasts at 14 or 28 days after induction of CI, normalized to ACTB mRNA levels.

  5. G

    Quantification of Annexin V‐positive (early apoptotic; Annexin V+/PI− and late apoptotic; Annexin V+/PI+ double‐positive) cells (%) at 14 or 28 days after induction of CI.

  6. H, I

    qRT‐PCR analysis of the expression of INK4a normalized to ACTB mRNA levels (H), and quantification of Annexin V‐positive (early apoptotic; Annexin V+/PI− and late apoptotic; Annexin V+/PI+ double‐positive) cells (%) (I) in shINK4a‐transduced NMR‐fibroblasts at 21 days after induction of CI.

  7. J

    Mouse fibroblasts were passaged 28 days after induction of CI and subjected to cell proliferation analysis.

  8. K

    NMR fibroblasts were passaged 28 days after induction of CI and subjected to cell proliferation analysis.

Data information: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant. One‐way ANOVA followed by Dunnett's multiple comparison test for (D–I). Data are expressed as the mean ± SD from n = 3 biological replicates except for (G) (n = 5).
Figure 5
Figure 5. Activation of monoamine oxidases plays a crucial role in the induction of INK4a‐RB cell death in NMR cells

  1. Quantification of reactive oxygen species (ROS) using 2′,7′‐dihydrodichlorofluorescin diacetate (DCFH‐DA) in mock‐ or INK4a‐transduced NMR fibroblasts at 12 days after transduction. Data are expressed as the mean ± SD from n = 3 biological replicates.

  2. Left, representative images of lipid peroxidation (green) staining in NMR fibroblasts at 12 days after INK4a‐transduction. Scale bar, 50 μm. Right, quantification of signal intensity of lipid peroxidation staining. Data are expressed as the mean ± SD from n = 3 biological replicates.

  3. Quantification of PI‐positive cells in NMR fibroblasts treated for 24 h with N‐acetyl L‐cysteine (NAC), Trolox, or Tempol at 20 days after INK4a transduction (%). Data are expressed as the mean ± SD from n = 6 biological replicates except for Trolox and Tempol (n = 4).

  4. Western blot of monoamine oxidase (MAO)‐A and MAO‐B in mouse or NMR fibroblasts at 20 days after INK4a transduction. ACTIN was used as a loading control. Numbers below the gel images indicate quantification of MAO‐A or ‐B/ACTIN intensity (n = 3 average).

  5. Quantification of ROS using DCFH‐DA in INK4a‐transduced NMR fibroblasts at 20 days after transduction. Cells were treated with each inhibitor for 24 h prior to analysis; Clorgyline (MAO‐A inhibitor, 10 μM), Rasagiline (MAO‐B inhibitor, 10 μM), Phenelzine (inhibitor of both MAO‐A and MAO‐B, 50 μM). Data are expressed as the mean ± SD from n = 4 biological replicates.

  6. Quantification of PI‐positive cells in NMR fibroblasts treated for 24 h with Clorgyline, Rasagiline, or Phenelzine at 20 days after INK4a transduction (%). Data are expressed as the mean ± SD from n = 3 biological replicates except for phenelzine (n = 5).

  7. Western blot of MAO‐A and MAO‐B in mouse or NMR fibroblasts at 21 days after doxorubicin (DXR) treatment. ACTIN was used as a loading control. Numbers below the gel images indicate quantification of MAO‐A or ‐B/ACTIN intensity (n = 3 average).

  8. Quantification of PI‐positive cells in NMR fibroblasts treated for 24 h with the indicated dose of Phenelzine (Phe) at 21 days after DXR treatment (%). Data are expressed as the mean ± SD from n = 3 biological replicates.

Data information: *P < 0.05; **P < 0.01; ****P < 0.0001; ns, not significant; Unpaired t‐test versus control for (A, B, NAC of C, and F). One‐way ANOVA followed by Dunnett's multiple comparison test for (Trolox and Tempol of C, E, and H). Source data are available online for this figure.
Figure 6
Figure 6. Induction of cellular senescence leads to less accumulation of senescent cells and causes delayed, progressive cell death in NMR lungs
  1. A

    Left, young NMR (one‐year‐old). Right, middle‐aged NMR (15‐year‐old).

  2. B

    qRT‐PCR analysis of INK4a expression in the skin, muscle, and white adipose tissue (WAT) of 6‐week‐old mice (young; 6 w), 1‐year‐old mice (middle‐aged; 1 y), 1‐year‐old NMRs (young; 1 y), and 15‐year‐old NMRs (middle‐aged; 15 y).

  3. C

    Scheme for bleomycin (Bleo) treatment.

  4. D

    SA‐β‐Gal activity (SA‐β‐Gal, blue; nuclei, green) and TUNEL staining (TUNEL, red; nuclei, blue) in lungs of mice or NMRs at 2, 7, 14, and 21 days after Bleo administration.

  5. E–G

    Time‐course analysis of mice or NMR lungs after Bleo administration: quantification of SA‐β‐Gal‐positive cells (%) (E); qRT‐PCR analysis of the expression of INK4a normalized to ACTB mRNA levels (F); quantification of TUNEL‐positive cells (%) (G).

Data information: Scale bar, 50 μm. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant. Unpaired t‐test versus young for (B). One‐way ANOVA followed by Dunnett's multiple comparison test for (E–G). Data are expressed as the mean ± SD from n = 3 biological replicates except for NMR skin in (B) (n = 4).
Figure 7
Figure 7. Induction of cellular senescence leads to delayed, progressive cell death via MAO activation and the resulting suppression of senescent cell accumulation in the NMR lungs
  1. A

    Scheme for bleomycin (Bleo) treatment and additional phenelzine (Phe) treatment. Mouse or NMR lungs were treated with Phe for 5 days starting at 16 days after Bleo administration.

  2. B

    SA‐β‐Gal activity (SA‐β‐Gal, blue; nuclei, green) and TUNEL staining (TUNEL, red; nuclei, blue) in lungs of mice or NMRs at 21 days after Bleo administration, with or without Phe are shown.

  3. C–E

    Quantification of SA‐β‐Gal‐positive cells (%) (C), qRT‐PCR analysis of INK4a expression normalized to ACTB mRNA levels (D), quantification of TUNEL‐positive cells (%) (E) in mouse or NMR lungs at 21 days after Bleo administration, with or without Phe.

Data information: Scale bar, 50 μm. *P < 0.05; ns, not significant. Unpaired t‐test versus Phe‐ for (C–E). Data are expressed as the mean ± SD from n = 3 biological replicates.
Figure 8
Figure 8. The molecular mechanism of cell death upon cellular senescence induction in NMR
Graphical illustration showing the main findings of this study. NMR fibroblasts uniquely accumulate serotonin and are inherently vulnerable to hydrogen peroxide (H2O2). Upon induction of cellular senescence, the activated INK4a‐RB pathway causes NMR fibroblasts to increase monoamine oxidase (MAO) levels, leading to serotonin oxidization and H2O2 production, resulting in increased oxidative damage and activation of cell death.
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References

    1. Al‐Mohanna MA, Manogaran PS, Al‐Mukhalafi Z, Al‐Hussein A, Aboussekhra KA (2004) The tumor suppressor p16INK4a gene is a regulator of apoptosis induced by ultraviolet light and cisplatin. Oncogene 23: 201–212 - PubMed
    1. Andziak B, O'Connor TP, Buffenstein R (2005) Antioxidants do not explain the disparate longevity between mice and the longest‐living rodent, the naked mole‐rat. Mech Ageing Dev 126: 1206–1212 - PubMed
    1. Andziak B, O'Connor TP, Qi W, DeWaal EM, Pierce A, Chaudhuri AR, Van Remmen H, Buffenstein R (2006) High oxidative damage levels in the longest‐living rodent, the naked mole‐rat. Aging Cell 5: 463–471 - PubMed
    1. Aoshiba K, Tsuji T, Nagai A (2003) Bleomycin induces cellular senescence in alveolar epithelial cells. Eur Respir J 22: 436–443 - PubMed
    1. Azpurua J, Ke Z, Chen IX, Zhang Q, Ermolenko DN, Zhang ZD, Gorbunova V, Seluanov A (2013) Naked mole‐rat has increased translational fidelity compared with the mouse, as well as a unique 28S ribosomal RNA cleavage. Proc Natl Acad Sci U S A 110: 17350–17355 - PMC - PubMed

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