The DNA damage response induces inflammation and senescence by inhibiting autophagy of GATA4

Abstract

Cellular senescence is a terminal stress-activated program controlled by the p53 and p16(INK4a) tumor suppressor proteins. A striking feature of senescence is the senescence-associated secretory phenotype (SASP), a pro-inflammatory response linked to tumor promotion and aging. We have identified the transcription factor GATA4 as a senescence and SASP regulator. GATA4 is stabilized in cells undergoing senescence and is required for the SASP. Normally, GATA4 is degraded by p62-mediated selective autophagy, but this regulation is suppressed during senescence, thereby stabilizing GATA4. GATA4 in turn activates the transcription factor NF-κB to initiate the SASP and facilitate senescence. GATA4 activation depends on the DNA damage response regulators ATM and ATR, but not on p53 or p16(INK4a). GATA4 accumulates in multiple tissues, including the aging brain, and could contribute to aging and its associated inflammation.

Fig. 1. GATA4 regulates cellular senescence

(A) Left: IMR90 cells expressing the miR-146a GFP reporter were infected with either vector control or GATA4-expressing viruses, and GFP fluorescence was measured by fluorescence-activated cell sorter (FACS) (AFU, arbitrary fluorescence units). Data are shown as percentage of maximum expression (i.e., the number of cells in each bin divided by the number of cells in the bin that contains the largest number of cells) for normalization. Right: Median fluorescence intensity (MFI) was computed and normalized to vector control. Data are mean ± SEM. (B) Left: IMR90 cells expressing the miR-146a GFP reporter and transfected with the indicated siRNAs were exposed to IR (12 Gy), and GFP fluorescence was measured by FACS; cont denotes the firefly luciferase siRNA control. Center: MFI was computed and normalized to control siRNA (−IR); data are mean ± SEM. Right: Immunoblotting analysis shows the efficiency of GATA4 depletion. (C) BJ cells carrying either a Dox-inducible (Tet-On) vector expressing GATA4 (Tet-GATA4) or an empty vector (Tet-Vector) were grown with or without Dox, and SA-β-Gal staining (left) and BrdU incorporation (right) were analyzed. Data are mean ± SEM. (D) BJ cells carrying either a vector expressing a control shRNA targeting firefly luciferase or a GATA4 shRNA were exposed to IR (8 Gy), and 7 days later immunoblotting analysis (left) and SA-β-Gal staining (right) were performed. Data are mean ± SEM; one-way analysis of variance (ANOVA) was used for the statistical analysis. (E) Replicative senescence was assessed by cell growth analysis of BJ cells expressing either control or GATA4 shRNAs. Left: Population doubling analysis Right: Quantification of SA-β-Gal staining. Data are mean ± SEM; one-way ANOVA was used for the statistical analysis. Data are representative of four (A) or three [(B) to (E)] independent experiments.

Fig. 2. Selective autophagy degrades GATA4 in a p62-dependent manner to prevent senescence

(A) Western blot showing abundance of GATA4 protein during IR-induced senescence (top), oncogene (RAS^V12)–induced senescence (middle left), or replicative senescence (middle right); abundance of GATA4 mRNA during IR-induced senescence is shown at the bottom. PD denotes population doubling. Relative abundance of GATA4 mRNA is expressed as change with respect to expression in cells without IR treatment. (B) GATA4 protein stability was examined in the presence of cycloheximide (CHX) in proliferating (−IR) or IR-induced senescent cells [+IR, 7 days after exposure to IR (12 Gy)]. Shown are representative immunoblots (top) and quantification from three independent experiments (bottom). Data are mean ± SEM. (C) IMR90 cells were treated with the proteasomal inhibitor MG-132 for the indicated times, and proteins were analyzed by Western blotting. p21 served as a positive control for MG-132. (D) IMR90 cells were treated with the indicated autophagy inhibitory agents for the indicated times, and proteins were analyzed by immunoblotting with the indicated antibodies. Shown are representative immunoblots (left) and quantification from four independent experiments (right, 24 hours of treatment). Data are mean ± SEM; one-way ANOVA was used for the statistical analysis. (E and F) IMR90 cells were transfected with the indicated siRNAs, and proteins were analyzed by Western blotting 79 hours after transfection; cont denotes firefly luciferase siRNA. (G) Immunoblotting of Flag-GATA4 immunoprecipitates from proliferating (−IR) or IR-induced senescent IMR90 cells [+IR, 7 days after exposure to IR (12 Gy)]. Six days after IR, cells were treated with either dimethyl sulfoxide (DMSO) or Baf A1 for 24 hours before sampling to block GATA4 and p62 degradation. (H) IMR90 cells expressing the indicated Dox-inducible shRNAs were treated with Dox, and SA-β-Gal staining was performed (−Dox, 16 days off Dox; +Dox, 16 days on Dox; wash, 12 days on Dox then 4 days off Dox); cont denotes the control shRNA targeting firefly luciferase. (I) IMR90 cells carrying a Dox-inducible vector expressing an ATG7 shRNA and either control shRNA or GATA4 shRNA were treated with Dox, and immunoblotting analysis (top), SA-β-Gal staining (middle), and BrdU incorporation (bottom) were performed. Labels are as in (H); data are mean ± SEM.Two-way ANOVA was used for the statistical analysis. Data are representative of three [(A), (B), (E)], four (D), or two [(C), (F), (G), (H), (I)] independent experiments.

Fig. 3. Gene expression profiling reveals that GATA4 controls the SASP

(A) Left: mRNA expression profiling from IMR90 cells carrying a Dox-inducible vector expressing GATA4 untreated or treated with Dox identified genes that were significantly up-regulated (green, more than 200% expression of control, Q < 0.05) or down-regulated (red, less than 50% expression of control, Q < 0.05) upon GATA4 expression for 2.5 days. Right: GO term analysis of the up-regulated (green) or down-regulated (red) genes. Data are from three independent experiments. (B) Overlap between GATA4-regulated genes (GATA4-regulated set) and replicative senescence–regulated genes (Senescent set) for genes with more than 200% expression or less than 50% expression of control. The χ² test was used for statistical analysis. (C) IMR90 cells bearing a Dox-inducible vector expressing GATA4 or an empty vector control were untreated or treated with Dox for 2 days, and abundance of mRNAs for the indicated genes was quantified by RT-qPCR. Relative abundance of the indicated mRNAs is expressed as change with respect to expression in cells with the empty vector control without Dox treatment. (D) IMR90 cells transfected with the indicated siRNAs were exposed to IR (12 Gy) and incubated for 7 days. Abundance of mRNA was quantified as in (C). Relative abundance of the indicated mRNAs is expressed as change with respect to expression in cells transfected with control siRNA without IR treatment. Data are representative of four (C) or three (D) independent experiments.

Fig. 4. GATA4 regulates NF-κB

(A) IMR90 cells were transfected with the indicated siRNAs; 2 days after transfection, cells were untreated or treated with Dox for 2 days to induce GATA4. mRNA abundance was quantified by qPCR. Relative abundance of the indicated mRNA is expressed as change with respect to expression in cells transfected with control siRNA without GATA4 induction. (B) Immunocytochemistry of nuclear NF-κB/RELA accumulation (left) or immunoblotting (center) in IMR90 cells expressing GATA4. Immunoblotting analysis (right) was performed in IMR90 cells transfected with the indicated siRNAs during IR-induced senescence. (C) IMR90 cells expressing GATA4 were treated with Dox for 2 days, and abundance of mRNA for the indicated genes was quantified by RT-qPCR. Relative abundance of the indicated mRNA is expressed as change with respect to expression in cells without Dox treatment. Inset: Immunoblotting analysis for TRAF3IP2. (D) IMR90 cells were transfected with the indicated siRNAs; 1 day later, GATA4 was induced for 2 days and the abundance of the indicated proteins (left) or mRNAs (right) was analyzed. Relative abundance of the indicated mRNA is expressed as change with respect to expression in cells transfected with control siRNA without GATA4 induction. (E) One day after exposure to IR (12 Gy), IMR90 cells were transfected with the indicated siRNAs. Three days after transfection, TRAF3IP2 driven by the TRE promoter was induced for 5 days with Dox. Cells were retransfected with the indicated siRNAs 1 day after TRAF3IP2 induction to reinforce depletion. Abundance of mRNA was analyzed using RT-qPCR, and relative abundance of the indicated mRNAs is expressed as change with respect to expression in cells transfected with control siRNA without IR and TRAF3IP2 induction; cont refers to the firefly luciferase siRNA control. (F) One day after transfection of the indicated siRNAs,GATA4 was induced for 3.5 days and cells were quantified for SA-β-Gal staining.Two-way ANOVAwas used for the statistical analysis. Data are representative of three [(A), (B), (D), (F)] or two [(C), (E)] independent experiments.

Fig. 5. The GATA4 pathway functions independently of the p53 and p16 pathways and is regulated by the DDR kinases ATM and ATR

(A) BJ cells expressing either control or p53 shRNAs were treated with Dox for 4 days to induce GATA4 driven by the TRE promoter. Immunoblotting analysis (left) and SA-β-Gal staining (right) were performed. Data are mean ± SEM; cont refers to the control shRNA targeting firefly luciferase. (B) BJ cells expressing either HPV E6 or E6 and E7 were treated with Dox for 3.5 days to induce GATA4 driven by the Tet promoter, and SA-β-Gal staining was performed. Data are mean ± SEM. (C) Abundance of the GATA4 protein was examined in control (−IR) or IR-treated [+IR, 7 days after exposure to IR (12 Gy)] cells expressing either HPV E6 or E6 and E7. (D) Top: BJ and IMR90 cells were treated with 10 mM nutlin-3 for 7 days. Media were refreshed every 2 days. Bottom: p16 driven by the TRE promoter was induced for either 4 or 7 days. Media were refreshed every 2 days. Abundance of the indicated proteins was analyzed by Western blotting. (E) IMR90 cells were pretreated with 10 mM nutlin-3 for 7 days and nutlin-3 was washed out before exposure to IR (12 Gy). Abundance of the indicated protein was analyzed by Western blotting 7 days after IR. (F) IMR90 cells were pretreated with caffeine (ATM and ATR inhibitor, 1 mM) (left) or ATM inhibitor (ku55933, 10 mM), ATR inhibitor (VE-821, 10 mM), or both (right) for 1 hour before exposure to IR (12 Gy). Media were refreshed every 2 days for 7 days, and abundance of the indicated proteins was analyzed by Western blotting. Data are representative of three [(A), (E), (F)] or two [(B), (C), (D)] independent experiments. (G) Model of how GATA4 links autophagy and the DDR to SASP and cellular senescence. See text for details.

Fig. 6. GATA4 accumulates during mouse aging, human aging, and mouse IR-induced senescence

(A) Mouse embryonic fibroblasts (MEFs) were exposed to IR (12 Gy), and Western blotting (left) and immunofluorescence (center) were performed for GATA4 protein. MEF cells expressing either control or GATA4 shRNA were exposed to IR (12 Gy); after 7 days, abundance of mRNA for the indicated genes was quantified by RT-qPCR (right). Relative abundance of the indicated mRNA is expressed as change with respect to expression in cells without IR treatment. Data are representative of two independent experiments. (B) Left: Liver and skin tissues were collected from control (−IR) and IR-treated [+IR, 3 months after total body irradiation (TBI) of 7 Gy] C57BL/6 mice and analyzed by Western blotting. Right: Densitometric analysis was performed to determine the GATA4/GAPDH ratio. Data are mean ± SEM. (C) Left: Liver tissues were taken from young (6-month-old) and old (22-month-old) C57BL/6 mice and analyzed by Western blotting. Right: Densitometric analysis as in (B). Data are mean ± SEM. (D) Left: Abundance of GATA4 protein in extracts of prefrontal cortex (PFC) from young and aged individuals were analyzed by Western blotting. Age in years and sex (F = female, M = male) for each sample are given. Right: Densitometric analysis as in (B). Data are mean ± SEM. In (B) to (D), each lane represents an individual case. (E) Left: Confocal immunofluorescence labeling for GATA4 (red), p16 (green), GFAP (white), and DNA (DAPI, blue) in the prefrontal cortex of representative young and aged cases. Upper right: Quantitative analysis of immunofluorescence for oligodendrocytes (young, n = 8; aged, n = 8), pyramidal neurons (young, n = 8; aged, n = 8), and astrocytes (young, n = 5; aged, n = 6). AFU values are shown as mean ± SEM. Lower right: Correlation analysis of abundance of GATA4 and p16 protein. Each point represents an individual cell (oligodendrocytes, n = 633; pyramidal neurons, n = 505; astrocytes, n = 411). Spearman correlation coefficient (R) and P value are shown for cells from aged individuals.