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. 2017 Oct;16(5):1094-1103.
doi: 10.1111/acel.12639. Epub 2017 Jul 9.

Cooperation between p21 and Akt is required for p53-dependent cellular senescence

Young Yeon Kim  1   2 , Hye Jin Jee  1   2 , Jee-Hyun Um  1   2 , Young Mi Kim  1   2 , Sun Sik Bae  3 , Jeanho Yun  1   2
Affiliations

Cooperation between p21 and Akt is required for p53-dependent cellular senescence

Young Yeon Kim et al. Aging Cell. 2017 Oct.

Abstract

Cellular senescence has been implicated in normal aging, tissue homeostasis, and tumor suppression. Although p53 has been shown to be a central mediator of cellular senescence, the signaling pathway by which it induces senescence remains incompletely understood. In this study, we have shown that both Akt and p21 are required to induce cellular senescence in response to p53 expression. In a p53-induced senescence model, we found that Akt activation was essential for inducing a cellular senescence phenotype. Surprisingly, Akt inhibition did not abolish p53-induced cell cycle arrest, but it suppressed the increase in intracellular reactive oxygen species (ROS) levels. The results of the cell cycle and morphological analysis suggest that p53 induced quiescence, not senescence, following Akt inhibition. Conversely, the inhibition of p21 induction abolished cell cycle arrest but did not affect the p53-induced increase in ROS levels. Additionally, p21 and Akt separately controlled cell cycle arrest and ROS levels, respectively, during H-Ras-induced senescence in human normal fibroblasts. The mechanistic analysis revealed that Akt increased ROS levels through NOX4 induction, and increased Akt-dependent NF-κB binding to the NOX4 promoter is responsible for NOX4 induction upon p53 expression. We further showed that Akt activation upon p53 expression is mediated by mammalian target of rapamycin complex 2. In addition, p53-mediated IL6 and IL8 induction was abrogated by Akt inhibition, suggesting that Akt activation is also required for the senescence-associated secretory phenotype. Collectively, these results suggest that p53 simultaneously controls multiple pathways to induce cellular senescence through p21 and Akt.

Keywords: Akt; NOX4; p53; reactive oxygen species; senescence.

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Figures

Figure 1
Figure 1
Akt activation is required for p53‐induced senescence. (A–C) EJ cells were infected with a p53 adenovirus at 100 MOI. SA‐β‐gal staining was examined after 6 days, and a representative image is shown (A). EJ cells were harvested at the indicated time points, and cell lysates were subjected to Western blot analysis using the indicated antibodies (B). The levels of Akt pS473 and pT308 from three replicate experiments were calculated and plotted, along with the percentage of SA‐β‐gal‐positive cells (C). (D–F) EJ cells were infected with a p53 adenovirus and treated with LY294002 (20 μm) for the indicated durations. The schematic diagram depicts the LY294002 treatment procedure (LY) (D). EJ cells were harvested at the indicated time points and subjected to Western blot analysis using the indicated antibodies (E). SA‐β‐gal staining was performed after 6 days (F). (G) EJ cells were treated with the Akt inhibitor IV (1.2 μm) from days 1 to 2 after infection with a p53 adenovirus. SA‐β‐gal staining was examined at the indicated time points. (H–I) EJ cells expressing an Akt (shAkt) or control (shNC) shRNA were infected with a p53 adenovirus. EJ cells were harvested at the indicated time points and subjected to Western blot analysis using the indicated antibodies (H). SA‐β‐gal staining was performed after 6 days (I). *P <0.05; **P < 0.01 by Student's t‐test.
Figure 2
Figure 2
Inhibition of Akt abolished ROS induction but not cell cycle arrest upon p53 expression. (A–F) EJ cells were treated with LY294002 (20 μm) from days 0 to 2 (LY 0–2 d) after infection with a p53 adenovirus. The percentage of cells in the S‐phase was examined using an EPICS XL cytometer (A). BrdU incorporation was assessed using a BrdU staining kit (Invitrogen, Carlsbad, CA, USA) (B). Intracellular ROS levels were measured with an EPICS XL cytometer after the staining of cells with 2′,7′‐dichlorodihydrofluorescein diacetate (DCF‐DA) (50 μm) (C). Cell numbers were determined at the indicated time points by trypan blue exclusion (D). SA‐β‐gal staining was examined after 6 days, and a representative image is shown (E). The percentage of cells in the G0 phase was examined by Pyronin Y/Hoechst 33342 staining (F). *P < 0.05 by Student's t‐test. NS, not significant.
Figure 3
Figure 3
Inhibition of p21 abolished cell cycle arrest but not ROS induction upon p53 expression. (A–F) EJ cells expressing either a p21 shRNA (shp21) or nontargeting shRNA (shNC) were infected with a p53 adenovirus. EJ cells were harvested at the indicated time points and subjected to Western blot analysis using the indicated antibodies (A). SA‐β‐gal staining was performed after 6 days (B), and a representative image is shown (D). Cell numbers were determined at the indicated time points by trypan blue exclusion (D). The percentage of cells in the S‐phase was examined using an EPICS XL cytometer (E). Intracellular ROS levels were measured at the indicated time points using an EPICS XL cytometer after the staining of cells with DCF‐DA (50 μm) (F). *P < 0.05 by Student's t‐test. NS, not significant.
Figure 4
Figure 4
Akt and p21 are independently regulated in H‐Ras‐induced senescence. (A‐G) WI‐38 cells were infected with an H‐RasV12 retrovirus and treated with LY294002 from days 0 to 2 (LY 0–2 d). The experimental procedure for the induction of H‐Ras‐mediated senescence and LY294002 (LY) treatment (A). WI‐38 cells were harvested at the indicated time points and subjected to Western blot analysis using the indicated antibodies (B). SA‐β‐gal staining was examined at the indicated time points (C). Intracellular ROS levels were measured at the indicated time points using an EPICS XL cytometer following the staining of cells with DCF‐DA (50 μm) (D). A representative image of SA‐β‐gal staining captured on the 6th day is shown (E). Cell numbers were determined at the indicated time points by trypan blue exclusion (F). The percentage of cells in the S‐phase was examined using an EPICS XL cytometer (G). (H–L) WI‐38 cells expressing either a p21 (shp21) or nontargeting (shNC) shRNA were infected with an H‐RasV12 retrovirus. SA‐β‐gal staining was performed at the indicated time points (H). Cell numbers were determined at the indicated time points by trypan blue exclusion (I). A representative image of SA‐β‐gal staining captured on the 6th day is shown (J). Intracellular ROS levels were measured at the indicated time points using an EPICS XL cytometer following the staining of cells with DCF‐DA (50 μm) (K). The percentage of cells in the S‐phase was examined using an EPICS XL cytometer (L). *P < 0.05; **P < 0.01 by Student's t‐test. NS, not significant.
Figure 5
Figure 5
NOX4 mediates the Akt‐dependent increase in ROS levels following the induction of p53 expression. (A) The expression of NADPH oxidases was examined in EJ and H1299 cells using semi‐quantitative RT–PCR with the indicated primers. (B–C) EJ cells were treated with LY294002 (20 μm) for 2 days (LY 0–2 d). (B) or with the Akt inhibitor IV (1.2 μm) from days 1 to 2 (C) after p53 adenovirus infection. Total RNA samples were harvested after 2 days, and the NOX4 mRNA level was determined using real‐time RT–PCR. (D–F) EJ cells were treated with 1 μm VAS2870 (VAS) from days 0 to 2 after infection with a p53 adenovirus. SA‐β‐gal staining was performed after 6 days (D). The intracellular ROS levels (E) and the percentage of cells in the S‐phase (F) were examined. (G–I) EJ cells expressing either a NOX4 (shNOX4) or nontargeting (shNC) shRNA were infected with a p53 adenovirus. SA‐β‐gal staining was performed after 6 days (G). The intracellular ROS levels (H) and the percentage of cells in the S‐phase (I) were determined. (J) EJ cells were treated with Bay11‐7082 (Bay11, 5 μm) from days 1 to 2 after infection with a p53 adenovirus. The NOX4 mRNA level was determined using real‐time RT–PCR. (K–L) ChIP assay for NF‐κB binding to the NOX4 promoter. EJ cells were treated with Bay11‐7082 (5 μm) from days 1 to 2 (K), LY294002 (20 μm) for 2 days, or the Akt inhibitor IV (1.2 μm) from days 1 to 2 (L) after p53 adenovirus infection. The ChIP assay was performed using either the NF‐κB p65 antibody or IgG as a control. *P < 0.05; **P < 0.01 by Student's t‐test. NS, not significant.
Figure 6
Figure 6
Akt activation is mediated by mTOR and is also required for the SASP. (A) EJ cells were infected with the p53 adenovirus and treated either rapamycin (Rapa, 100 nm) or Torin1 (50 nm) for 2 days before harvested. (B) EJ cells expressing Raptor (shRap), Rictor (shRic), or control (shNC) shRNAs were infected with a p53 adenovirus and harvested 2 days later. Cell lysates were subjected to Western blot analysis using the indicated antibodies. (C–E) Real‐time PCR analysis for IL6 and IL8 mRNA after Akt inhibition. (F–H) ELISA for IL6 and IL‐8 secretion after Akt inhibition. EJ cells were treated with LY294002 (20 μm) for 2 days after p53 adenovirus infection and harvested on day 4 (C, F). EJ cells expressing Akt (shAkt) or control (shNC) shRNAs were infected with the p53 adenovirus and harvested on day 4 (D, G). WI‐38 cells were treated with LY294002 (20 μm) for 2 days after H‐RasV12 retrovirus infection and harvested (E, H). Total RNA samples were subjected to real‐time PCR, and the supernatants were analyzed for the secretory levels of IL6/IL8 by ELISA. (I) A model depicting the cooperative activity between Akt and p21 in inducing cellular senescence. **P < 0.01 by Student's t‐test.
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